The role of hydrogen sulfide, substance P and Kupffer

i

The role of hydrogen sulfide, substance P and Kupffer cells on

inflammation and liver sinusoidal endothelial cells in sepsis

Ravinder Reddy Gaddam

A thesis submitted for the degree of

Doctor of Philosophy

Department of Pathology,

University of Otago, Christchurch

May 2017

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Abstract

Sepsis is life-threatening organ dysfunction caused by a dysregulated host response to

infection. The inflammatory response is an integral part of sepsis and leads to the systemic

inflammatory response syndrome (SIRS) and multiple organ failure. The overall objective of

this thesis was to investigate whether hydrogen sulfide (H2S), substance P (SP) and Kupffer

cells modulate the inflammatory response, organ damage and liver sinusoidal endothelial

cells (LSECs) fenestrations in sepsis.

H2S is a key mediator of inflammation and recent studies have implicated H2S in the

pathogenesis of sepsis. However, these studies have limitations due to the disadvantages of

the H2S-synthesising enzyme inhibitor and H2S donors used to conduct the experiments.

Gene deletion technology offers a definitive approach to investigate the role of H2S in sepsis.

The aim of this thesis was to investigate the potential role of endogenous H2S synthesised

through cystathionine-γ-lyase (CSE) using CSE knockout (CSE KO) mice in caecal-ligation and

puncture (CLP)-induced sepsis. This thesis also aimed to examine the underlying mechanisms

by which CSE-derived H2S regulates inflammation and to determine the interaction between

H2S and SP in regulating the inflammatory response in sepsis.

Kupffer cells are tissue-resident macrophages in the liver that play an important role in

inflammation associated with infection. The studies described in this thesis investigated the

potential roles of Kupffer cells on liver and lung injury, inflammation and the systemic

inflammatory response in sepsis using gadolinium chloride (GdCl3) to inactivate these cells.

LSECs are specialised fenestrated endothelial cells in the liver that undergo structural

alteration during inflammation and infection. The structural alterations in LSEC fenestrae

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following CLP-induced sepsis were examined and the effect of GdCl3, CSE gene deletion and

PPTA gene deletion (PPTA, a SP encoding gene) were determined.

The final aim was to investigate the alteration of circulatory H2S and SP levels and their

association with the inflammatory response in patients with sepsis compared to non-septic

patients with similar disease severity and organ dysfunction admitted to the hospital

Intensive Care Unit (ICU).

Following CLP-induced sepsis in mice, increased expression of liver and lung CSE (liver: ~1.98

fold; lung: ~2.49 fold), increased liver H2S-synthesising activity (~1.27 fold) and plasma H2S

levels (~1.45 fold) were observed. Mice deficient in the CSE gene showed significantly

reduced sepsis-associated tissue (liver and lung) myeloperoxidase (MPO) activity, tissue

(liver and lung) and circulatory levels of cytokines (TNF-α, IL-6 and IL-1β) and chemokines

(MCP-1 and MIP-2α), and histological changes in the liver and lung. In addition, mechanistic

studies revealed that the proinflammatory role of CSE-derived H2S was mediated by the

activation of the ERK1/2-NF-B p65 signalling pathway. SP and NK-1R expression have been

shown to play an essential role in sepsis-associated liver and lung injury. Mice with CSE gene

deletion had significantly reduced tissue (liver and lung) and circulatory SP levels (liver: ~0.50

fold; lung: ~0.42 fold; plasma: ~0.61 fold) and tissue (liver and lung) NK-1R expression (liver:

~1.11 fold; lung: ~0.93 fold). This study showed that CSE-derived H2S in sepsis could

upregulate SP and NK-1R expression, thereby contributing to liver and lung injury and

inflammation.

Examination of the effect of GdCl3 on the inflammatory response and organ injury following

induction of sepsis showed there was protection against injury in the liver, as there was

reduced MPO activity, cytokine (TNF-α, IL-6 and IL-1β) and chemokine (MCP-1 and MIP-2α)

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levels and histological changes in the liver. In contrast, administration of GdCl3 failed to

reduce lung injury and inflammation (as there was no change in MPO activity, cytokine and

chemokine levels and histological changes) and the systemic inflammatory response (as

evidenced by no change in circulatory cytokines and chemokines) in sepsis.

Study of LSEC fenestrae following induction of sepsis revealed that CLP-induced sepsis was

associated with defenestration (decreased diameter, frequency and porosity) and gaps

formation in LSEC fenestrae (~9 fold). Mice with CSE gene deletion, PPTA gene deletion and

mice treated with GdCl3 showed less defenestration (increased diameter, frequency and

porosity) and fewer gaps (~0.16 fold) in LSEC fenestrae following sepsis.

Studies of septic patients admitted to the ICU showed higher circulatory levels of H2S and SP

compared to non-septic patients, which correlated with the inflammatory response in septic

patients.

In conclusion, the results presented in this thesis have shown that the CSE-derived H2S, SP

and Kupffer cells all play a key role in modulating inflammation, associated organ damage

and LSEC fenestrae in experimental sepsis. This thesis has also shown that higher circulatory

levels of H2S and SP are associated with inflammatory response in septic patients and are

consistent with results from experimental sepsis, suggesting that CSE-derived H2S and SP

play an important role in the inflammatory process of sepsis in both experimental and human

sepsis. This study contributes to a better understanding of the pathogenesis of sepsis and

highlights novel potential approaches to the treatment of sepsis.

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Acknowledgements

I am pleased to acknowledge and thank my supervisor Professor Madhav Bhatia whose

direction and assistance has been wonderful throughout this PhD. Many thanks go to my co-

supervisor Professor Stephen Chambers for his assistance and support throughout this PhD.

Thanks go to my associate supervisor Emeritus Professor Robin Fraser for his guidance,

enthusiasm and appreciation for my research. Special thanks go to my collaborators

Professor David Le Couteur and Dr. Victoria Cogger at ANZAC Research Institute, University

of Sydney, Australia for providing training on scanning electron microscopy (SEM) and to

finish SEM studies, Dr. Isao Ishii at Department of Biochemistry, Graduate School of

Pharmaceutical Sciences, Keio University, Japan for providing cystathionine-gamma-lyase

(CSE) knockout mice and Professor Allan Basbaum at University of California, San Francisco,

United States for providing preprotachykinin (PPTA) knock out mice.

I acknowledge all the members of the Inflammation Research Group, especially Dr. Abel

Damien Ang and Dr. Alireza Badiei for their experimental and technical support and Dr.

Piyush Jha for his friendship.

I acknowledge Free Radical Research Group members, Dr. Rufus Turner for his experimental

assistance with LC-MS and Dr. Heather Parker and Masuma Zawari for their assistance on

proofreading this thesis.

I acknowledge Professor Peter George at Canterbury Health Laboratories, Christchurch, New

Zealand for his assistance with ALT and AST enzyme activity assays.

I am very grateful for the funding support from the Canterbury Medical Research Foundation

(CMRF), Maurice and Phyllis Paykel Trust (MPPT) and the University of Otago.

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I acknowledge Dr. John Pearson, Biostatistician at Department of Population Health,

University of Otago, Christchurch and Ms. Ma Yi, Biostatistician at Canterbury District Health

Board, Christchurch for their statistical advice.

Thank you to the all the staff of the Department of Pathology in particular Prof. Martin

Kennedy, Head of the Department and administration staff (Linda Kerr, Alice Milnes and Lisa

McLaughlin) for their wonderful support during my study.

Finally, I really have to thank my family members who recognised my interest and

encouraged to pursue my career in the field of research. Thanks to my friends, and to

everyone who has supported and encouraged me in different ways.

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Declaration of the candidate

All the results and light and electron microscopy photographs presented in this thesis are my

original work unless otherwise acknowledged. None of the work of this thesis has been used

for obtaining any other degrees

Ravinder Reddy Gaddam

viii

Dedication

I dedicate this thesis to my family and friends for their unconditional love, caring and support

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Publications

Publications pertaining to the work in this thesis

1. Gaddam R R, Fraser R, Badiei A, Chambers S, Cogger V, Le Couteur D, Bhatia M

Differential effects of kupffer cell inactivation on inflammation and the liver sieve

following caecal-ligation and puncture induced sepsis in mice. Shock. 2017; 47(4):

480-490.

2. Gaddam R R, Fraser R, Badiei A, Chambers S, Cogger V, Le Couteur D, Ishii I, Bhatia

M. Cystathionine-Gamma-Lyase Gene Deletion Protects Mice Against Inflammation

and Liver Sieve Injury Following Polymicrobial Sepsis. PLOS ONE. 2016; 11(8):

e0160521.

Other Publications

1) Singh P, Reid K, Gaddam R R, Bhatia M, Smith S, Jacob A, Chambers P. Effect of choline

chloride premedication on xylazine-induced hypoxaemia in sheep. Veterinary

Anesthesia and Analgesia. 2017 (in press).

2) Badiei A, Chambers ST, Gaddam R R, Bhatia M. Cystathionine-gamma-lyase gene

silencing with siRNA in monocytes/macrophages attenuates inflammation in Cecal

Ligation and Puncture-induced sepsis in the mouse. Journal of Biosciences. 2016;

41(1):87-95.

3) Badiei A, Chambers ST, Gaddam R R, Fraser R, Bhatia M. Cystathionine-gamma-lyase

gene silencing with siRNA in monocytes/macrophages protects mice against acute

pancreatitis. Applied Microbiology and Biotechnology; 2015; 100(1):337-46.

4) Gaddam R R, Ang AD, Badiei A, Chambers ST, Bhatia M. Alteration of the renin-

angiotensin system in caerulein induced acute pancreatitis in the mouse.

Pancreatology.2015; 15(6):647-53.

5) Gaddam R R, Chambers S, and Bhatia M: ACE and ACE2 in inflammation: A tale of two

enzymes. Inflammation and Allergy & Drug-Targets, 2014; 13 (4): 224-34.

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Manuscripts in preparation and submitted publications

1) Gaddam R R, Chambers S, Shaw G M, Bhatia M. Circulatory hydrogen sulfide and

substance P levels in patients with sepsis admitted to the intensive care unit (in

communication).

2) Gaddam R R, Fraser R, Chamber S, Cogger V, Le Couteur D, Bhatia M. Substance P

mediated regulation of inflammatory response and liver sieve injury in caecal-ligation

and puncture-induced sepsis in mice (in communication).

Abstracts from conference presentations

1) Fraser R, Gaddam RR, Bhatia M, Dobbs BR. Jamieson, HA, Cogger VC, Warren, A. Le

Couteur DG. ANITSCHKOW’S EXPANDING LEGACY: Porosity of the “liver sieve”

influences atherosclerosis and many other maladies. Symposium of the International

Atherosclerosis Society, 2016, At St. Petersburg, Russia.

2) Ravinder R. Gaddam, Alireza Badiei, Victoria Cogger, David Le Couteur, Isao Ishii,

Robin Fraser, Madhav Bhatia. The effect of hydrogen sulfide through cystathionine-

gamma-lyase on inflammation and liver sinusoidal endothelial cells in polymicrobial

sepsis in mice. 18th International Symposium on Cells of the Hepatic Sinusoids

(ISCHS), 2015, Asilomer, California, USA.

3) Gaddam R R, A. Badiei, S.T. Chambers, I. Ishii, M. Bhatia. The effect of cystathionine-

gamma-lyase gene deletion on the renin-angiotensin system in caerulein-induced

acute pancreatitis in Mice. 47th American pancreatic Association (APA) meeting,

2015, San Diego, California, USA.

4) Madhav Bhatia, Alireza Badiei, Gaddam R R, Robin Fraser, Stephen Chambers.

Inhibition of hydrogen sulfide synthesis by gene silencing protects mice against

caerulein-induced acute pancreatitis. 47th European Pancreatic Club (EPC) meeting,

2015, Barcelona, Spain.

5) Alireza Badiei, Ravinder Gaddam, Stephen Chambers, Robin Fraser and Madhav

Bhatia. Inhibition of hydrogen sulfide production through gene silencing attenuates

inflammation in a mouse model of caerulein-induced acute pancreatitis.

International Conference on Innate Immunity 2015, Barcelona, Spain.

6) Robin Fraser, Madhav Bhatia, Ravinder R. Gaddam, Hamish A. Jamieson, Roy T.

Wade, Elizabeth J. Latimer-Hill, David G. LeCouteur. Arsenic containing water from

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Bangladeshi Wells correlates with Cardiovascular Disease. Exploratory Fracking

extends internationally the presence of ground-water arsenical contamination.

Arsenical drinking water by blocking the “Liver Sieve” explains dyslipidaemia and

Heart Disease? Otago International Health Research Network (OIHRN) Conference,

2014, Dunedin, New Zealand.

7) Ravinder R. Gaddam, Abel Damien Ang, Alireza Badiei, Stephen Chambers, Madhav

Bhatia. Alteration of metalloprotease enzymes of the renin-angiotensin system in

acute pancreatitis in a mouse model. Research Forum on learning different research

languages, 2014, Dunedin, New Zealand.

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Table of contents

Abstract……. .......................................................................................................... ii

Acknowledgments ................................................................................................ v

Declaration. ......................................................................................................... vii

Dedication.. ........................................................................................................ viii

Publications .......................................................................................................... ix

Table of contents ................................................................................................. xii

List of figures ..................................................................................................... xvii

List of tables ........................................................................................................ xix

List of abbreviations ............................................................................................. xx

1 Introduction………………………………………………………………………………………………1

1.1 General introduction ........................................................................... ……..1

1.2 Sepsis ........................................................................................................ 2

1.2.1 Definition and epidemiology ........................................................................ 2

1.2.2 Animal models of sepsis ............................................................................... 3

1.2.3 Pathophysiology of sepsis ............................................................................. 6

1.2.3.1 The nuclear factor-B (NF-B) transcription factor ..................................... 8

1.2.3.1.1 The role of NF-B in sepsis ........................................................................... 9

1.2.3.2 Role of immune cells in sepsis .................................................................... 11

1.2.3.3 Role of cytokines in sepsis .......................................................................... 11

1.2.3.4 Role of chemokines in sepsis ...................................................................... 13

1.3 Hydrogen sulfide (H2S) ............................................................................. 15

1.3.1 Physical and chemical properties of H2S .................................................... 15

1.3.2 Enzymatic and non-enzymatic pathways for H2S production .................... 16

1.3.3 H2S catabolism and mode of action ........................................................... 17

1.3.4 Physiological role of H2S ............................................................................. 18

1.3.5 Pathological effects of H2S .......................................................................... 20

1.3.6 Role of H2S in inflammation ........................................................................ 20

1.3.6.1 Role of H2S in sepsis .................................................................................... 23

1.4 Substance P (SP) ....................................................................................... 26

1.4.1 Biosynthesis and physiological functions of SP .......................................... 26

1.4.2 The role of SP in sepsis ................................................................................. 27

1.5 Liver and liver sinusoidal endothelial cells ................................................ 28

1.5.1 Anatomy and function of the liver ............................................................. 28

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1.5.2 The liver sinusoid ........................................................................................ 30

1.5.3 Kupffer cells ................................................................................................ 31

1.5.3.1 Kupffer cells role in infection and inflammation ............................................ 32

1.5.4 Liver sinusoidal endothelial cells (LSECs) .................................................... 35

1.5.4.1 Structure and functions of LSECs .................................................................... 35

1.5.4.2 Alteration of LSEC fenestration ...................................................................... 36

1.5.4.3 Infection, inflammation and LSEC fenestrae .................................................... 37

1.6 Research rationale, hypothesis and objectives .......................................... 38

1.6.1 Hypothesis .................................................................................................. 41

1.6.2 Objectives ................................................................................................... 42

Significance .............................................................................................. 44

2 Materials and methods……………………………………………………………………………46

2.1 Materials ................................................................................................. 46

2.2 Buffers and solutions ................................................................................ 47

2.3 Mice ......................................................................................................... 51

2.4 Induction of polymicrobial sepsis in mice .................................................. 52

2.5 Western blotting ...................................................................................... 53

2.6 H2S-synthesising activity assay ................................................................. 54

2.7 Plasma H2S measurement ......................................................................... 54

2.8 Tissue myeloperoxidase activity measurement ......................................... 55

2.9 Morphological examination of liver and lung damage ............................... 56

2.10 Measurement of sulfur amino acid homocysteine using HILIC-MS/MS ...... 56

2.11 Preparation of nuclear extract and determination of NF-B p65 activation ............................................................................................. .57

2.12 Cytokine and chemokine measurement by enzyme-linked immunosorbent assay ..................................................................................................... 58

2.13 Measurement of SP levels ........................................................................ 59

2.14 Measurement of procalcitonin levels ........................................................ 60

2.15 Scanning electron microscopy .................................................................. 61

2.15.1 Liver perfusion and fixation (primary fixation) ........................................... 61

2.15.2 Processing of tissue blocks (secondary fixation) ........................................ 62

2.15.3 Tissue preparation for scanning electron microscopy ............................... 62

2.15.4 Tissue examination with scanning electron microscope ............................ 62

2.16 Measurement of plasma ALT and AST activity levels ................................. 63

2.17 Statistical analysis ................................................................................... 63

3 Effect of CSE gene deletion on leukocyte infiltration and organ damage following caecal-ligation and puncture-induced sepsis in mice………………..64

3.1 Introduction ............................................................................................. 64

3.2 Aims......................................................................................................... 67

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3.3 Experimental approach ............................................................................ 67

3.4 Results ..................................................................................................... 68

3.4.1 CSE protein expression in WT and CSE KO mice following CLP-induced sepsis .......................................................................................................... .68

3.4.2 H2S-synthesising activity and H2S levels in WT and CSE KO mice following CLP-induced sepsis ...................................................................................... 69

3.4.3 Measurement of liver and lungs leukocyte infiltration in WT and CSE KO mice following CLP-induced sepsis... .......................................................... 70

3.4.4 Liver and lung injury in WT and CSE KO mice following CLP-induced sepsis... ........................................................................................................ 71

3.4.5 Alteration of homocysteine levels in WT and CSE KO mice following CLP-induced sepsis... .......................................................................................... 74

3.5 Discussion ................................................................................................ 75

4 Effect of CSE gene deletion on proinflammatory cytokines, and chemokines and the ERK1/2-NF-B p65 pathway following caecal-ligation and puncture-induced sepsis in mice……………………………………………………………………………..79

4.1 Introduction ............................................................................................. 79

4.2 Aims......................................................................................................... 81


4.4 Results ..................................................................................................... 82

4.4.1 Phosphorylation of ERK1/2 in WT and CSE KO mice following CLP-induced sepsis ........................................................................................................... 82

4.4.2 Effect of CSE gene deletion on liver and lungs NF-B p65 activation following CLP-induced sepsis ..................................................................... 84

4.4.3 Effect of CSE gene deletion on liver proinflammatory mediators following CLP-induced sepsis ...................................................................................... 85

4.4.4 Effect of CSE gene deletion on lungs proinflammatory mediators following CLP-induced sepsis ...................................................................................... 87

4.4.5 Effect of CSE gene deletion on circulatory proinflammatory mediators following CLP-induced sepsis ...................................................................... 89

4.5 Discussion ................................................................................................ 91

5 Effect of CSE gene deletion on substance P and neurokinin 1 receptor following caecal-ligation and puncture-induced sepsis in mice……………..…93

5.1 Introduction ............................................................................................. 93

5.2 Aims......................................................................................................... 95


5.4 Results ..................................................................................................... 96

5.4.1 CSE gene deletion affects SP levels following CLP-induced sepsis ............. 96

5.4.2 Effect of CSE gene deletion on NK-1R protein expression following CLP-induced sepsis. ............................................................................................ 97

5.5 Discussion ................................................................................................ 99

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6 Effect of Kupffer cell inactivation by gadolinium chloride on inflammation following caecal-ligation and puncture-induced sepsis in mice..…………….102

6.1 Introduction ........................................................................................... 102

6.2 Aims....................................................................................................... 106

6.3 Experimental approach .......................................................................... 106

6.4 Results ................................................................................................... 107

6.4.1 Effect of GdCl3 pretreatment on tissue leukocyte infiltration following CLP-induced sepsis ........................................................................................... 107

6.4.2 Effect of GdCl3 pretreatment on liver and lungs damage following CLP-induced sepsis ........................................................................................... 108

6.4.3 Effect of GdCl3 pretreatment on plasma ALT and AST activity levels following CLP-induced sepsis .................................................................... 111

6.4.4 Effect of GdCl3 pretreatment on liver proinflammatory mediators following CLP-induced sepsis .................................................................... 112

6.4.5 Effect of GdCl3 pretreatment on lungs proinflammatory mediators following CLP-induced sepsis .................................................................... 114

6.4.6 Effect of GdCl3 pretreatment on circulatory proinflammatory mediators following CLP-induced sepsis .................................................................... 116

6.5 Discussion .............................................................................................. 118

7 Effect of caecal-ligation and puncture-induced sepsis on liver sinusoidal endothelial cell fenestration (liver sieve)…………………………………………….…122

7.1 Introduction ........................................................................................... 122

7.2 Aims....................................................................................................... 124


7.4 Results ................................................................................................... 125

7.4.1 Effect of CLP-induced sepsis and GdCl3 pretreatment on LSEC fenestration. ............................................................................................. 125

7.4.2 Alteration of LSEC fenestration in WT and CSE KO mice following CLP-induced sepsis.. ......................................................................................... 127

7.4.3 Alteration of LSEC fenestration in WT and PPTA KO mice following CLP-induced sepsis.. ......................................................................................... 129

7.5 Discussion .............................................................................................. 131

8 Circulatory hydrogen sulfide and substance P levels in patients with sepsis admitted to the intensive care unit…………………………………………………….…137

8.1 Introduction ........................................................................................... 137

8.2 Aims....................................................................................................... 139


8.3.1 Design and subject. ................................................................................... 139

8.3.2 Variables recorded.. .................................................................................. 140

8.3.3 Blood sampling and laboratory assays.. ................................................... 141

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8.4.4 Statistical analysis.. ................................................................................... 141

8.4 Results ................................................................................................... 142

8.4.1 Demographic characteristics of patients .................................................. 142

8.4.2 Comparison of disease severity and organ dysfunction between septic and non-septic patients ................................................................................... 144

8.4.3 Plasma H2S and SP levels in septic patients compared to non-septic patients ..................................................................................................... 145

8.4.4 Plasma PCT, IL-6, CRP and blood lactate levels in septic patients compared to non-septic patients ............................................................................... 146

8.5 Discussion .............................................................................................. 149

9 General discussion, conclusion and future perspectives ………………………..154

9.1 General discussion ................................................................................. 154

9.2 Conclusions and future perspectives ....................................................... 158

10 References……………………………………………………………..…………………………….163

11 Appendix………………………………………………………………………………………………193

11.1 Summary of primary diagnosis, complications and comorbidities of patients with non-sepsis admitted to the ICU during the 2015-16 study period .. 193

11.2 Summary of primary diagnosis, complications and comorbidities of patients with sepsis admitted to the ICU during the 2015-16 study period ......... 194

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List of figures

Figure 1.1 Schematic representation of the pathogenesis of sepsis .................................................... 7

Figure 1.2 Schematic representation of endogenous H2S generation through enzymatic and non-enzymatic pathways ....................................................................................................... 17

Figure 1.3 Possible physiological functions of H2S .............................................................................. 19

Figure 1.4 Segmental anatomy of the liver. ........................................................................................ 29

Figure 1.5 Diagrammatic representation of the classic lobule and liver acinus ................................. 30

Figure 1.6 Role of Kupffer cells in sepsis. ............................................................................................ 33

Figure 1.7 Schematic representation of hypothesis............................................................................ 42

Figure 3.1 CSE protein expression in WT and CSE KO mice following CLP-induced sepsis ................. 68

Figure 3.2 Liver H2S-synthesising activity and H2S levels in WT and CSE KO mice following CLP-induced sepsis ............................................................................................................................... 70

Figure 3.3 Leukocyte infiltration in the liver and lungs of WT and CSE KO mice following CLP-induced sepsis ............................................................................................................................... 71

Figure 3.4 Morphological changes in the liver and lungs of WT and CSE KO mice following CLP-induced sepsis ............................................................................................................................... 72

Figure 3.5 Alteration of homocysteine levels in WT and CSE KO mice following CLP-induced sepsis .............................................................................................................................. .74

Figure 4.1 Phosphorylation of ERK1/2 in WT and CSE KO mice following CLP-induced sepsis. .......... 83

Figure 4.2 Activation of NF-B p65 in WT and CSE KO mice following CLP-induced sepsis ............... 84

Figure 4.3 Alteration of liver cytokine and chemokine levels in WT and CSE KO mice following CLP-induced sepsis ................................................................................................................. 86

Figure 4.4 Alteration of lungs cytokine and chemokine levels in WT and CSE KO mice following CLP-induced sepsis ................................................................................................................. 88

Figure 4.5 Alteration of plasma cytokine and chemokine levels in WT and CSE KO mice following CLP-induced sepsis ................................................................................................................. 90

Figure 5.1 SP levels in WT and CSE KO mice following CLP-induced sepsis ........................................ 97

Figure 5.2 NK-1R protein expression in WT and CSE KO mice following CLP-induced sepsis ............. 98

Figure 6.1 Effect of GdCl3 administration on MPO activity following CLP-induced sepsis ................ 107

Figure 6.2 Morphological changes in the liver and lungs following CLP-induced sepsis and the effect of GdCl3 pretreatment .................................................................................................. 109

Figure 6.3 Alteration of plasma ALT and AST activity levels following CLP-induced sepsis and the effect of GdCl3 pretreatment .................................................................................................. 112

Figure 6.4 Effect of GdCl3 pretreatment on protein expression levels of cytokines and chemokines in the liver from mice with CLP-induced sepsis ................................................................ 113

Figure 6.5 Effect of GdCl3 pretreatment on protein expression levels of cytokines and chemokines in the lungs from mice with CLP-induced sepsis. ............................................................. 115

Figure 6.6 Effect of GdCl3 pretreatment on protein expression levels of cytokines and chemokines in plasma from mice with CLP-induced sepsis .................................................................. 117

Figure 7.1 Effect of GdCl3 pretreatment on LSEC defenestration in mice following CLP-induced sepsis, assessed using scanning electron microscopy (SEM) ................................................... 126

Figure 7.2 Effect of CLP-induced sepsis on LSEC fenestration in WT and CSE KO mice, assessed using scanning electron microscopy (SEM) ............................................................................ 128

xviii

Figure 7.3 Effect of CLP-induced sepsis on LSEC fenestration in WT and PPTA KO mice, assessed using scanning electron microscopy (SEM) ............................................................................ 130

Figure 8.1 Plasma levels of H2S and SP in septic patients ................................................................. 145

Figure 8.2 Plasma levels of PCT, IL-6, CRP and blood lactate in septic patients ............................... 147

Figure 9.1 Schematic summary of the role of CSE-derived H2S, SP and Kupffer cells in sepsis ........ 159

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i) List of tables

Table 1.1 Sequential [sepsis-related] organ failure assessment score ................................................................. 3

Table 1.2 Summary of studies investigating the role of H2S in animal models of disease/injury ....................... 22

Table 2.1 List of different buffers/solutions and their preparation used in different assays and experiments. 48

Table 7.1 Effect of GdCl3 pretreatment on LSEC defenestration in mice following CLP-induced sepsis, assessed using scanning electron microscopy (SEM) ................................................................................ 127

Table 7.2 Effect of CLP-induced sepsis on LSEC fenestration in WT and CSE KO mice, assessed using scanning electron microscopy (SEM). ........................................................................................................ 129

Table 7.3 Effect of CLP-induced sepsis on LSEC fenestration in WT and PTTA KO mice, assessed using scanning electron microscopy (SEM). ........................................................................................................ 131

Table 8.1 Characteristics of patients with non-sepsis and sepsis admitted to medical intensive care unit (ICU) during the 2015-16 study period. ............................................................................................... 143

Table 8.2 Disease severity and organ dysfunction scores and mortality in septic and non-septic patients admitted to medical intensive care unit (ICU) during the 2015-16 study period ....................... 144

Table 8.3 Plasma H2S and SP levels in septic patients ....................................................................................... 146

Table 8.4 Plasma PCT, IL-6, CRP and blood lactate levels in septic patients ..................................................... 148

Table 11.1 Summary of primary diagnosis, complications and comorbidities of patients with non-sepsis admitted to the ICU during the 2015-16 study period ............................................................... 194

Table 11.2 Summary of primary diagnosis, complications and comorbidities of patients with sepsis admitted to the ICU during the 2015-16 study period ................................................................................... 195

xx

List of abbreviations

ALI Acute lung injury

ALT Alanine transaminase

ANOVA Analysis of variance

APACHE Acute physiology and chronic health evaluation

APS Ammonium persulfate

ARDS Acute respiratory distress syndrome

AST Aspartate aminotransferase

BHMT Betaine-homocysteine methyltransferase

BSA Bovine serum albumin

CAT Cysteine aminotransferase

CBS Cystathionine--synthase

CHOP C/EBP homologous protein 10

CKD Chronic kidney disease

CLP Caecal-ligation and puncture

CO Carbon monoxide

COX-2 Cyclooxygenase-2

CRP C-reactive protein

CSE Cystathionine--lyase

CSE KO CSE knockout

DAO D-amino acid oxidase

DTT Dithiothreitol

EDTA Ethylenediaminetetraacetic acid

EIA Enzyme immunoassay

ELISA Enzyme linked immunosorbent assay

ERK1/2 Extracellular signal regulated kinase 1/2

FD Fenestration diameter

FeCl3 Ferric chloride

GAPDH Glyceraldehyde 3-phosphate dehydrogenase

GdCl3 Gadolinium chloride

HCl Hydrochloric acid

HMDS Hexamethyldisilazane

HMGB1 High mobility group box 1

H2O2 Hydrogen peroxide

HOCl Hypochlorous acid

HPRT Hypoxanthine-guanine phosphoribosyltransferase

H2SO4 Sulfuric acid

H2S Hydrogen sulfide

ICU Intensive care unit

IL-1 Interleukin-1

IL-6 Interleukin-6

IL-10 Interleukin-10



xxi

IB Inhibitory B

iNOS Inducible nitric oxide synthase

KATP Potassium ATP

KCl Potassium chloride

KH2PO4 Potassium dihydrogen phosphate

LPS Lipopolysaccharide

LSECs Liver sinusoidal endothelial cells

MAP Mean arterial pressure

MAPK Mitogen activated protein kinase

MCP-1 Monocyte chemoattractant protein-1

MIP-2 Macrophage inflammatory protein-2

miR-21 MicroRNA-21

MODS Multiple organ damage syndrome

MPO Myeloperoxidase

3-MST 3-mercaptopyruvate sulfurtransferase

MTR Methionine synthase

NaCl Sodium chloride

NADPH Nicotinamide adenine dinucleotide phosphate

NaHS Sodium hydrosulfide

NaOH Sodium hydroxide

Na2HPO4 Disodium hydrogen phosphate

NaH2PO4 Sodium dihydrogen phosphate

Na2S Sodium sulfide

NEM 2-mercaptoethanol, N-ethyl maleimide

NF-B Nuclear factor-B

NK-1R Neurokinin-1 receptor

NMDA N-methyl-D-aspartate

NO Nitric oxide

PAG DL-propargylglycine

PAMPs Pathogen associated molecular patterns

PCT Procalcitonin

PD-L1 Programmed death ligand-1

PI3K Phosphatidylinositol-4,5-bisphosphate 3-kinase

PKA Protein kinase A

PLP Pyridoxal-5’-phosphate

PPTA Preprotachykinin A

PRRs Pattern recognition receptors

RNS Reactive nitrogen species

ROS Reactive oxygen species

SAPS Simplified acute physiology score

SDS Sodium dodecyl sulfate

SEM Scanning electron microscopy

SIRS Systemic inflammatory response syndrome

SOFA Sequential [sepsis-related]-organ failure assessment

SP Substance P

SP1 Specificity protein 1

xxii

TCA Trichloroacetic acid

TEMED Tetramethylethylenediamine

TFA Trifluoroacetic acid

TLRs Toll-like receptors

TNF- Tumour necrosis factor-

TNFRs TNF receptors

TRPV1 Transient receptor potential vanilloid 1

VEGF Vascular endothelial growth factor

xxiii

1

Chapter 1

Introduction

1.1 General introduction

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host

response to infection (1). The inflammatory response is an integral part of sepsis that leads

to systemic inflammatory response syndrome (SIRS) and multiple organ failure. This literature

review summarises the pathophysiology of sepsis, describing the mediators and cells involved

as well as the underlying mechanisms and multiple organ failure during sepsis.

Hydrogen sulfide (H2S) and substance P (SP) are known mediators of inflammation in sepsis.

This review is focused on the physiological roles of H2S and SP and their pathological

significance in sepsis. Kupffer cells are tissue resident macrophages in the liver. They are the

primary responders to infection and act by engulfing pathogenic microorganisms and

subsequently releasing a series of inflammatory mediators. This review discusses the

pathophysiological role of Kupffer cells in inflammation and sepsis. Liver sinusoidal

endothelial cells (LSECs) are specialised fenestrated endothelial cells in the liver and play a

key role in the transfer of substrates from sinusoidal blood to the hepatic parenchyma

through space of Disse. They are known to undergo structural alteration in different

physiological and pathological conditions. This review also encompasses the physiological role

of LSEC fenestrae and its alteration in different pathophysiological conditions including sepsis.

Finally, based on the existing knowledge of H2S, SP, Kupffer cells and LSECs in sepsis, this

literature review discusses the rationale and objectives presented in this thesis.

2

1.2 Sepsis

1.2.1 Definition and epidemiology

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host

response to infection. In lay terms, sepsis is a life-threatening condition that arises when the

body’s response to an infection injures its own tissues and organs (1, 2). Septic shock is a

subset of sepsis in which underlying circulatory, cellular and metabolic abnormalities are

associated with a greater risk of mortality than with sepsis alone (2). The definitions of sepsis

and septic shock have been unified considerably by the recommendations of the European

Society of Intensive Care Medicine and the Society of Critical Care Medicine in 2016 (1, 2).

Based on their recommendations, organ dysfunction during sepsis can be identified by the

Sequential [Sepsis-related] Organ Failure Assessment (SOFA) Score (summarised in Table 1.1)

(1-3). Despite advances in care, sepsis remains a major health problem worldwide and reports

on incidence are increasing. This is likely to reflect ageing of the population, high rates of

comorbidities that increase susceptibility to infection, and increasing numbers of

immunocompromised patients such as those with malignancies, organ transplants or HIV

infection (4-7). For instance, in the United States, sepsis incidence rates increased from 359

cases per 100,000 population in 2003 to 535 cases per 100,000 population in 2009 (49%

increase), accounting for more than $20 billion in costs and 5.2% of the total United States

hospital costs in 2011 (4, 6, 8). Although the true incidence is unknown, conservative

estimates indicate that sepsis is a leading cause of mortality and critical illness worldwide and

in-hospital mortality rates remain high at 25-30% (4, 6). The scale and seriousness of this

problem requires innovative approaches to management.

3

Table 1.1 Sequential [Sepsis-related] Organ Failure Assessment Score (adopted from Singer et al.

2016) (1).

1.2.2 Animal models of sepsis

A number of animal models have been developed and modified to study the pathophysiology

of sepsis (9, 10). They include exogenous administration of endotoxin lipopolysaccharide

(LPS), alteration of the animal’s endogenous barrier to normal bacterial flora (caecal-ligation

and puncture, CLP) and exogenous administration of various pathogens (intravenous (i.v.)

infusion of live bacteria, administration of faecal matter into the peritoneal cavity and the

placement of infectious foreign material into the soft tissue of extremity). All of these models

System Score 0 1 2 3 4

Respiration

PaO2/FiO2, mmHg (kPa)

≥400 (53.3) <400 (53.3)

<300 (40) <200 (26.7) with respiratory support

<100 (13.3) with respiratory support

Coagulation

Platelets, x103/µL ≥150 <150 <100 <50 <20

Liver

Bilirubin, mg/dL (µmol/L)

<1.2 (20) 1.2-1.9 (20-32)

2.0-5.9 (33-101)

6.0-11.9 (102-204)

>12.0 (204)

Cardiovascular

MAP (mm Hg) or Catecholamine (µg/kg/min)

MAP>70 mm Hg

MAP<70 mm Hg

Dopamine <5 or dobutamine (any dose)

Dopamine 5.1-15 or epinephrine ≤0.1 or norepinephrine ≤0.1

Dopamine >15 or epinephrine >0.1 or norepinephrine >0.1

Central Nervous System

Glasgow Coma Scale Scores

15 13-14 10-12 6-9 <6

Renal

Creatinine, mg/dL (µmol/L)

<1.2 (110) 1.2-1.9 (110-170)

2.0-3.4 (171-299)

3.5-4.9 (300-440)

>5.0 (440)

Urine output, mL/d)

<500 <200

Abbreviations FiO2, fraction of inspired oxygen; MAP, mean arterial pressure; PaO2, partial pressure of oxygen

4

have contributed significantly to our understanding of mechanisms of sepsis pathophysiology.

Although these models replicate many features of human sepsis, injection of LPS and caecal-

ligation and puncture surgery are the most commonly used animal models to study sepsis.

Even though many researchers use administration of endotoxin LPS to study sepsis, the

clinical features of human sepsis are quite different from LPS-induced endotoxaemia in mice

and rats (10, 11). The kinetics and magnitude of hemodynamic changes and peritoneal and

systemic cytokinemia in LPS-induced endotoxaemia do not accurately mimic the

hemodynamic changes and cytokine levels of human sepsis (12, 13). For example, the

hemodynamic response observed during LPS-induced endotoxaemia in mice is a

hypodynamic response, as opposed to the hyperdynamic response observed in septic

patients (9, 10). Changes in the serum cytokine profile are transient and much greater in

magnitude than those observed in septic patients (12). Many anti-cytokine clinical trials based

on promising results from this model have turned out to be unsuccessful (13, 14).

Furthermore, LPS injection in rodents causes suppressed gluconeogenesis and subsequent

hypoglycaemia, whereas the opposite results occur in patients with sepsis (10). Injection of

LPS in this model also causes activation of the innate immune system, which can have

deleterious effects; therefore, any intervention that blunts the inflammatory response is likely

to be beneficial. In contrast, sepsis in patients is triggered by an infectious process and the

immunological responses to microbial challenge that can have both beneficial and deleterious

effects. For these reasons, LPS injection is currently considered to be a suitable model for

endotoxic shock but not for sepsis.

On the other hand, sepsis in the CLP model causes a persistent endogenous release of bacteria

from the perforated caecum into the abdominal cavity, resulting in bacterial peritonitis that

5

successfully mimics human sepsis (9-11). A major advantage of this model is that the initial

hyperdynamic cardiovascular changes such as cardiac output and systemic vascular

resistance, accurately reflect the response seen in human sepsis (15, 16). Another major

advantage of this model is that it reproduces the relative magnitude of the release of serum

cytokines and chemokines observed in human sepsis (12, 17, 18). In addition, the CLP model

has the advantage of inducing sepsis of varied severity, which is used for investigating both

acute and chronic sepsis. However, it is important to use a consistent protocol in this model

to obtain reliable and reproducible results, as the length of the caecum ligated, size of the

needle used and number of punctures determine the outcome of resulting sepsis.

Furthermore, in experiments using small animals (mice and rats), an increased sample size

can resolve the problem of variability that can sometimes be seen in this model.

In summary, different animal models can be used to study different aspects of sepsis and the

choice of animal model depends on the type of pathological change to be studied in sepsis.

For instance, the CLP model is good for studying hemodynamic and metabolic changes and

biochemical, immune and inflammatory responses in sepsis, whereas i.v. infusion of live

bacteria may be suitable for observing the blood clearance kinetics of bacteria. Although LPS-

induced endotoxaemia is to some extent similar to human sepsis, the dissimilarities in

hemodynamic and inflammatory changes between these two conditions make the LPS

injection model less relevant to the study of human sepsis.

1.2.3 Pathophysiology of sepsis

The pathogenesis of sepsis is a complex and multifactorial disease and so far no single

mediator, system, pathway or pathogen has been reported to drive this disease. Despite

extensive basic and clinical studies, the pathophysiology of sepsis is still poorly understood.

6

Sepsis is most commonly associated with bacterial infections but may also be caused by

viruses and fungal infections. Although gram-positive pathogens have been documented to

be most common cause of sepsis, there is now evidence that sepsis caused by gram-negative

bacteria occurs with equal frequency in some settings (19, 20). Staphylococcus aureus,

Streptococcus pneumonia and Escherichia coli are the most common causes of sepsis, while

Pseudomonas aeruginosa is reported to be the most lethal (21, 22). However, many cases of

sepsis are attributed to mixed infection and polymicrobial origin (23).

Innate and adaptive immune systems preserve host integrity against invading pathogens

during sepsis. The interaction between the host immune system and molecular components

of invading pathogens is crucial for initiating immune events during sepsis (24). Immune cells

such as monocytes/macrophages, neutrophils and natural killer cells recognise biochemical

patterns displayed by pathogens and trigger active responses, either directly or indirectly by

activating intracellular signalling pathways (25, 26). Immune cells utilise pattern recognition

receptors (PRRs) to recognise highly conserved pathogen associated molecular patterns

(PAMPs) such as bacterial cell wall components (lipopolysaccharide, LPS and peptidoglycans).

Toll-like receptors (TLRs) are a family of pattern-recognition receptors present on mammalian

macrophages, dendritic cells and other cells that initiate innate immune responses (27). To

date, at least 11 human TLRs have been identified; each is known to detect specific PAMP and

have a specific intracellular signalling pathway. Of these, TLR-4s and TLR-2s have been widely

studied in sepsis. The peptidoglycan from gram-positive bacteria and the LPS from gram-

negative bacteria bind to TLR-2 and TLR-4, respectively (27). Once activated, TLRs trigger a

cascade of signals such as mitogen-activated protein kinase (MAPK) and phosphatidylinositol-

4, 5-bisphostate 3-kinase (PI3K) activation and engage a sequence of cytoplasmic interactions

7

that result in activation of transcription factor nuclear factor-B (NF-B) and transcription of

targeted proinflammatory genes. The resulting release of mediators such as proinflammatory

cytokines and chemokines, adhesion molecules and lipid mediators as well as generation of

reactive oxygen and nitrogen species (ROS and RNS) and complement-activation products can

lead to a systemic inflammatory response, multiple organ failure and death.

Figure 1.1 Schematic representation of the pathogenesis of sepsis. During sepsis, bacteria and their

components, including LPS and peptidoglycan, activate pattern recognition receptors (PRRs such as

TLR2 and TLR4, respectively) present on the surface of immune cells (27). Activation of these receptors

can lead to the recruitment of adapter proteins at the receptor site and subsequently stimulate

intracellular signalling pathways such as MAP kinases (MAPK) (25, 26). Activation of MAPK such as

ERK1/2 can lead to the activation of IB kinases (IKK), which in turn degrades IB and allows

translocation of NF-B subunits into the nucleus. Binding of NF-B subunits to the promoter region of

the DNA can lead to the transcription and release of proinflammatory mediators such as cytokines,

chemokines and adhesion molecules (28, 29). Activation of TLRs can also lead to the generation of

reactive oxygen species (ROS) through NADPH oxidase (30, 31). Together, these inflammatory

mediators and ROS can cause significant organ dysfunction and systemic inflammatory response

during sepsis.

8

The inflammatory response is an integral part of sepsis. A finely-tuned balance between the

immune and inflammatory systems, and between the pro- and anti-inflammatory networks

is crucial for maintaining the balance between protective and tissue-damaging responses

during sepsis (24, 32). The initial hyperinflammatory response, a result of an overwhelming

immune response to infection, leads to imbalance between pro- and anti-inflammatory

pathways during sepsis (33). The subsequent anti-inflammatory response serves as a negative

feedback to downregulate proinflammatory mediators and to modulate their effects, thereby

restoring homeostasis. Although anti-inflammatory mediators counterbalance pro-

inflammatory substances, prolonged and excessive production of these mediators leads to

immune dysregulation and may cause increased host susceptibility to concurrent infections,

subsequently, and a range of clinical sequelae (24).

The present chapter focuses mainly on the proinflammatory pathways such as NF-B and

MAPK signalling, the role of immune cells, and proinflammatory mediators such as cytokines

and chemokines in pathophysiology of sepsis.

1.2.3.1 The nuclear factor-κB (NF-B) transcription factor

NF-B is a transcription factor belonging to the Rel protein family (34). NF-B is formed by

various homo- and hetero-dimeric combinations of Rel family proteins such as c-Rel, RelA

(p65), RelB, NF-B1 (p50 and its precursor, p105) and NF-B2 (p52 and its precursor, p100)

(34, 35). The combination of the p50/p65 heterodimer is the most predominant form of NF-

B (36). In normal resting cells, NF-B is bound to inhibitory B (IB) proteins such as IBα,

IBβ and IBε in the cytoplasm. IB proteins interact with NF-B through multiple ankyrin

repeats that inhibit its DNA binding activity. Of these, IBα blocks the nuclear translocation

9

of p65 whereas IBβ masks both p65 and p50. In addition, IBα provides a negative feedback

mechanism for the termination of the NF-B response (35, 37-39).

1.2.3.1.1 The role of NF-B in sepsis

NF-B is an important transcription factor involved in regulating immune and inflammatory

responses to infection. Activation of NF-B plays a central role in the pathophysiology of

sepsis. A greater level of NF-B activity is associated with a higher rate of mortality and poorer

clinical outcomes in septic patients. For example, it has been shown that NF-B activity is

significantly higher in non-surviving septic patients than in surviving septic patients during

their illness (40, 41). A significant increase in NF-B activity is also observed in the alveolar

macrophages of patients with septic lung injury (42). Inhibition of NF-B activation prevents

multiple organ injury and improves survival rates in rodent models of sepsis (43-47). For

example, mice deficient in NF-B-dependent genes are resistant to the development of septic

shock and death from sepsis (48-50). More importantly, blockade of the NF-B pathway

corrects septic abnormalities. For example, inhibition of NF-B activation restores systemic

hypotension, ameliorates myocardial dysfunction and vascular derangement, diminishes

intravascular coagulation, inhibits multiple inflammatory gene expression, reduces tissue

neutrophil influx and prevents microvascular endothelial leakage in the rat model of sepsis

(47, 51, 52).

Extracellular signal-regulated kinases (ERKs), a family of serine/threonine protein kinases

belonging to the MAPKs, play a key role in NF-B activation (53). Two isoforms of ERKs, p44

MAPK (ERK1) and p42 MAPK (ERK2) are known and both are important in the NF-B activation

process. Following stimulation of TLRs by bacteria and their components, a series of adaptor

10

proteins are recruited to the receptor site and activate ERK1/2. Subsequently, the

coordination and interaction of the ERK1/2 and NF-B cascade is important in regulating the

transcription of inflammatory mediators (28, 29). For example, ERK1/2 activation has been

shown to be an important temporal regulator of NF-B and, subsequently, NF-B-dependent

gene expression (54). Specific inhibitors of ERK1/2 have been shown to inhibit NF-B

activation or to suppress the expressions of proinflammatory genes following sepsis (44).

NF-B activation is a central event in regulating cytokine response and subsequent

chemokines and adhesion molecules in sepsis. The upstream signalling pathways that activate

NF-B proteins result in its translocation to the nucleus, where it binds to the consensus

sequence in the promoter or enhancer regions of NF-B target genes and induces

transcription of genes for cytokines, chemokines and adhesion molecules. Excessive

generation and release of these mediators by activation of NF-B in sepsis leads to an

extensive activation of the inflammatory cascade and widespread organ injury (34, 35). For

example, elevation of inflammatory mediators during sepsis has been shown to depend on

NF-B activation, while inhibition of NF-B activation decreases production of inflammatory

cytokines, chemokines and adhesion molecules, ameliorating disease severity during

endotoxaemia and sepsis (43, 44, 52, 55, 56). These results suggest that NF-B is an important

signalling cascade that plays a central role in the pathophysiology of sepsis.

1.2.3.2 Role of immune cells in sepsis

Immune cells are the first cellular responders to invading organisms and are therefore vital to

host response. Among the immune cells, macrophages and neutrophils are key cells in the

pathogenesis of sepsis. Macrophages, after being stimulated by pathogenic substances, can

11

augment the release of classic proinflammatory cytokines such as tumour necrosis factor-α

(TNF-α), interleukin-1 (IL-1) and interleukin-6 (IL-6), and in addition release an array of other

cytokines, including interleukin-12 (IL-12), interleukin-15 (IL-15) and interleukin-18 (IL-18) and

other small molecules (57, 58). The high mobility group B1 (HMGB1) has been identified as a

cytokine-like product of macrophages that appears much later after LPS stimulation and may

represent a more tractable target for intervention (59). This hyperinflammatory state in sepsis

is called systemic inflammatory response syndrome (SIRS) and is associated with an enhanced

release of chemokines and expression of adhesion molecules that lead to a major infiltration

of neutrophils and monocytes into the body organs. Activated neutrophils and macrophages

also generate large amount of ROS, including hydrogen peroxide (H2O2), hypochlorous acid

(HOCl) and other hydroxyl radical via nicotinamide adenine dinucleotide phosphate (NADPH)

oxidases and myeloperoxidase, and RNS such as nitric oxide and peroxynitrite anions (30, 31).

Although the activated immune cells initially promote clearance of bacteria, their products

can bring both reversible and irreversible changes in proteins and DNA, resulting in

diminished biochemical functions that subsequently contribute to organ injury and death in

sepsis (60).

1.2.3.3 Role of cytokines in sepsis

Cytokines are a group of soluble, low molecular weight glycoproteins that are synthesised and

released by many types of cells (mainly monocytes, macrophages and neutrophils and also

from lymphocytes, mast cells and fibroblasts) in response to tissue damage. They are

important regulators for host defence, tissue repair and other homeostasis functions and are

often secreted when bacteria or bacteria-derived components are exposed to the host

immune response.

12

Cytokines are the key elements in the pathogenesis of sepsis and the “cytokine storm”

(overwhelming release of the cytokines) is thought to be responsible for triggering

inflammation in sepsis. The major proinflammatory cytokines that stimulate systemic

inflammation are TNF-α, IL-1 and IL-6 (58). Activation of TLRs by pathogens trigger the

production and release of “early” cytokines such as TNF-α and IL-1. These are released during

the first 30-90 mins after exposure to LPS and work synergistically to release secondary

cytokines such as IL-6 and other lipid mediators and ROS. These mediators signal endothelial

cells to upregulate chemokines and adhesion molecules and begin the recruitment of

neutrophils and other inflammatory cells to the site of infection (61, 62). In addition, these

mediators signal the release of anti-inflammatory cytokines such as interleukin-4 (IL-4),

interleukin-10 (IL-10) and interleukin-13 (IL-13), which may counter-regulate the

inflammatory response and play a role in tissue repair (49). Although cytokines are crucial for

host defence, overwhelming levels of cytokines can result in the systemic inflammatory

response syndrome (SIRS) and multiple organ dysfunction in sepsis. Therefore, cytokines are

considered to play a key role in the pathogenesis of sepsis.

Among all cytokines, TNF-α is one of the most important and a primary mediator of

inflammation in the pathogenesis of sepsis (63, 64). Through type I and II TNF receptors

(TNFRs), TNF-α increases NF-B-mediated transcription of proinflammatory mediators and

coordinates with other cytokines such as IL-1 and IL-6 to initiate acute phase response in

sepsis. For example, TNF-α, together with IL-1, exert profound effects upon the endothelium,

vasculature and coagulation cascade during sepsis (65, 66). The importance of

proinflammatory cytokines has been reported in both experimental and human sepsis.

Animal studies have demonstrated that concentrations of TNF-α in plasma were significantly

13

increased in severe sepsis and in animals that subsequently died (67). Administration of a

combination of TNF-α and IL-1 acted synergistically to cause increased toxicity in rabbits (65,

66). Patients with fulminant meningococcal septicaemia are also reported to have high

plasma levels of TNF-α (68). Experiments conducted with anti-TNF-α antibodies have shown

decreased levels of other cytokines and improved survival in animal models of septic shock

(69, 70). In baboons infused with live Escherichia coli, anti-TNF-α almost reduced circulating

IL-1β, IL-6 and IL-8 (71, 72) levels to the normal. In contrast, the role of IL-6 in the

pathophysiology of sepsis remains unclear. In animal models of sepsis, blockage of IL-6 has

produced inconsistent protection (61). In addition, infusion of IL-6 into human volunteers and

experimental animals has not induced a sepsis-like state (73). However, compared to other

cytokines, IL-6 has been found to be a better predictor of severity and outcome in human

sepsis and the development of multiple organ failure in patients with sepsis (74).

1.2.3.4 Role of chemokines in sepsis

Chemokines are a large family of 8-14 kDa-inducible cytokines that play a crucial role in

trafficking and recruiting anti-bacterial leukocytes to the primary sites of the immune

response (75). To date, more than 40 chemokines have been identified and are classified into

four subfamilies (C, CC, CXC and CX3C chemokines) depending on the relative position of

cysteine residues (76, 77). Of these, CC and CXC chemokines are the most widely studied

peptides in inflammation and sepsis (78).

It has been demonstrated that exposure to various pathogens results in a substantial increase

in chemokines in both humans and animals. For example, circulatory levels of CXCL8 (IL-8)

were significantly elevated in patients with bacterial sepsis (79). Similarly, high plasma levels

of CXCL9 (a monokine induced by interferon-γ, MIG), CCL2 (monocyte chemoattractant

14

protein-1, MCP-1), CCL3 (macrophage inflammatory protein-1α, MIP-1α), CCL4 (MIP-1β) and

CCL8 (MCP-2) were also observed during sepsis (57). Although chemokines are crucial for host

defence against invading pathogens, overexpression of these chemokines may play an

important role in the inflammatory response and organ damage in sepsis. For example, MIP-

2, a CXC chemokine released by macrophages in sepsis, binds to CXCR1 and CXCR2 on

neutrophils and has been associated with neutrophil-induced organ injury and death rates in

CLP-induced sepsis (80). Neutralising the effects of CXCR2 stimulation in murine peritonitis

resulted in an attenuated neutrophil response with less organ injury and improved survival

(81).

Although cytokines are considered to play a key role in the pathogenesis of sepsis and

neutralising these mediators has shown protection in experimental sepsis models, clinical

studies based on inhibiting the activity of cytokines have failed to improve the outcome in

sepsis patients. Despite the encouraging results in experimental sepsis models, monoclonal

antibodies targeted against TNF-α (82-84), soluble TNF-α receptors (85), IL-1 receptor

antagonists (86-88) and soluble IL-1 receptors have failed to decrease mortality in phase II

and III clinical studies (89, 90). The improved outcome of anti-cytokine therapies in septic

animals compared with humans is thought to be a result of a short therapeutic window period

for reversing the events of lethal sepsis in animals (91). Moreover, the ineffectiveness of anti-

cytokine therapies in patients arises as a result of a prolonged immunosuppressive state at

later time points of sepsis development. Furthermore, although several experimental and

clinical studies have shown the importance of chemokines and their receptors in the

pathophysiology of sepsis, clinical approach based on chemokines failed to prove as a

therapeutic targets.

15

To understand the complex role of inflammatory mediators in sepsis, there is a need to study

newly characterised mediators. In addition, studying novel mediators will improve our

understanding of their role in signal transduction, cross-talk, and synergistic and

immunomodulating during sepsis. Recent research has shown that hydrogen sulfide (H2S), a

gasotransmitter, plays an important role in the inflammatory process in experimental disease

models of sepsis. Further understanding the inflammatory role of H2S and its interaction with

other inflammatory mediators will help to design novel therapeutic targets for sepsis

treatment.

1.3 Hydrogen sulfide (H2S)

H2S has been known for decades as a toxic gas with a strong characteristic odor of rotten

eggs. Recent research has shown that H2S is generated endogenously in many tissues and

exerts various physiological functions. It has been proposed as a third physiological

gasotransmitter, following nitric oxide (NO) and carbon monoxide (CO).

1.3.1 Physical and chemical properties of H2S

H2S is a colorless, flammable gas with a strong odor of rotten eggs or the obnoxious odor of a

blocked sewer. It is a weak acid (pKa 6.96). At physiological conditions (pH 7.4), approximately

one third of H2S remains in a non-dissociated form and two thirds dissociate into H+ and HS-

(hydrosulfide anion), which may subsequently dissociate into H+ and S2-. Since the latter

reaction only takes place at high pH, the level of S2- in vivo is very low. At present it is unknown,

whether the biological effects of H2S are mediated directly by H2S, HS-, or whether a

combination of both species is required.

16

1.3.2 Enzymatic and non-enzymatic pathways for H2S production

H2S is generated endogenously by both enzymatic and non-enzymatic pathways. Through an

enzymatic pathway, many mammalian tissues produce H2S from sulfur amino acids such as

homocysteine and L-cysteine, during their metabolism. This reaction is catalysed by three

enzymes, cystathionine-γ-lyase (CSE, EC 4.4.1.1), cystathionine-β-synthase (CBS, EC 4.2.1.22)

and 3-mercaptopyruvate sulfurtransferase (3-MST, EC 2.8.1.2), along with cysteine

aminotransferase (CAT, EC 2.6.1.3) (92-94). Of these, CSE and CBS are the major H2S-

synthesising enzymes and their activity is dependent on the availability of pyridoxal-5’-

phosphate (PLP), an active form of vitamin B6, which acts as a co-factor for CSE and CBS.

Although CBS and CSE are widely expressed in cells and tissues, CSE is the predominant H2S-

synthesising enzyme in the peripheral organs and vascular system, whereas CBS is mainly

distributed in the central nervous system. As the end product of the CSE- and CBS-catalysed

cysteine metabolism, H2S exerts a negative feedback effect on the activity of these two

enzymes. It has been found recently that H2S is also produced from D-cysteine by the enzymes

D-amino acid oxidase (DAO) and 3-MST. This pathway is mainly localised within the

cerebellum and kidney (95).

H2S is also produced from non-enzymatic pathways from stored sulfur compounds; however,

this is physiologically much less significant. Two forms of sulfur stores in cells have been

identified: acid-labile sulfur and bound sulfane-sulfur (96, 97). Under acidic conditions (low

pH), H2S is released from acid-labile sulfur compounds such as iron-sulfur clusters (97). In the

presence of reducing agents or under reducing conditions, H2S is also produced from bound

sulfane-sulfur such as protein persulfide and polysulfides (98) (Figure 1.2).

17

Figure 1.2 Schematic representation of endogenous H2S generation through enzymatic and non-

enzymatic pathways. The enzymatic source of H2S involves the action of transsulfuration pathway

enzymes cystathionine--lyase (CSE) (EC 4.4.1.1), cystathionine--synthase (CBS) (EC 4.2.1.22) and 3-

mercaptopyruvate sulfurtransferase (3-MST) (EC 2.8.1.2). Both CSE and CBS catalyse the synthesis of

H2S by utilising homocysteine and L-cysteine as substrates (99, 100), whereas 3-MST utilises 3-

mercaptopyruvate (generated by cysteine aminotransferase (CAT) from L-cysteine and D-amino acid

oxidase (DAO) from D-cysteine) to generate H2S (95, 101). H2S is also generated from stored sulfur

pools such as acid-labile sulfur and sulfane-sulfur through the non-enzymatic pathway. H2S is released

from iron-sulfur clusters of non-heme sulfur proteins under acidic conditions (97) and from sulfane-

sulfur of persulfide and polysulfide with the help of reducing agents (98).

1.3.3 H2S catabolism and mode of action

The major catabolic pathway of H2S is via a series of redox conversions in the mitochondria

that ultimately leads to the formation of thiosulfate. This pathway is mediated by three

enzymes, sulfidequinoneoxido-reductase (SQR), and sulfur dioxygenase and sulfur

transferase. Apart from this catabolic pathway, H2S has recently been shown to

physiologically react with other molecules owing to the nucleophilic capacity of the

18

hydrosulfide ion (HS-) (99). Currently, there are three general forms of hydrogen sulfide

interaction within the biological milieu reported under physiological conditions; as a

reductant (100), through protein s-sulfhydration (327) and interaction with metal ion

containing molecules (101). The discovery of these interactions have also led to the

elucidation of the direct targets and mechanisms by which H2S exerts its observed

physiological effects.

1.3.4 Physiological role of H2S

H2S is thought to perform a wide range of physiological functions, including neuromodulation

and neuroprotection (102), vasorelaxation (103), cytoprotection and anti-oxidation (104,

105), angiogenesis (106), cellular energy production and metabolism (107, 108). H2S also

regulates cellular processes such as proliferation, migration and apoptosis (106).

Functionally, by acting on ATP-dependent potassium (KATP) channels, endogenous H2S can

hyperpolarise cell membranes, vascular smooth muscle cells, gastrointestinal smooth muscle

cells, cardiomyocytes, neurons and pancreatic-β cells, thereby regulating vascular tone,

intestinal contractility, myocardial contractility, neurotransmission and insulin secretion,

respectively (103, 109). Despite a direct vasodilator effect on vascular smooth muscles, H2S

can modulate nitric oxide (NO)-mediated vasodilation (110). H2S is a strong reducing agent

(anti-oxidant) and reacts with ROS and RNS, limiting their toxic effects and resulting in the

protection of proteins and lipids from ROS/RNS-mediated damage (111). H2S produced by 3-

MST can scavenge ROS in mitochondria and protect cells from oxidative stress (104). Further,

H2S plays a role in the angiogenesis process. The angiogenic effects of H2S are mediated

through the activation of the vascular endothelial growth factor (VEGF) (112). In addition to

serving as a signalling molecule, H2S participates in mitochondrial function and cellular

19

bioenergetics. It also acts as a mitochondrial electron donor, which results in the stimulation

of ATP synthesis (113).

In the central nervous system, H2S acts as a neuromodulator. It enhances the sensitivity of N-

methyl-D-aspartate (NMDA) receptors to glutamate, thereby promoting hippocampal long-

term potentiation (learning and memory) as well as phosphorylation of these receptors by

protein kinase A (PKA) (105). H2S also evokes calcium (Ca+2) waves in astrocytes and microglia

cells, which play an important role in the regulation of brain pH levels, neurotransmitter levels

and neuronal excitability (114). Additionally, H2S donor NaHS is linked to the inhibition of

apoptosis, a decrease of oedema formation in the brain and amelioration of cognitive

dysfunction (115) (Figure 1.3).

Figure 1.3 Possible physiological functions of H2S. Illustration of the current known physiological

effects of H2S. H2S performs wide range of physiological functions in both central nervous system and

peripheral organs, including neuromodulation (102), vasorelaxation (103), cytoprotection, antioxidant

(104, 105), energy production and metabolism (107, 108) and other cellular processes (106).

20

1.3.5 Pathological effects of H2S

Despite physiological functions, the pathological roles of H2S have been demonstrated in a

number of diseases in both the central nervous system and the peripheral organs. In the

central nervous system, H2S has a proven neuromodulatory role in the protection of neurons

from oxidative stress (116) and cytotoxicity (111). H2S promotes glutamate-mediated

transmission via NMDA receptors, which might also have implications for neurodegenerative

diseases in which excessive activation of NMDA receptors is involved (117). On the other

hand, in peripheral organs H2S has been reported to be deficient in various animal models of

arterial and pulmonary hypertension (118), myocardial injury (119), gastric mucosal injury

(120), colitis (121) and cirrhosis (122). Exogenous H2S-releasing drugs attenuate

cardiovascular dysfunction (123) and repair the damage of gastrointestinal mucosa (124). In

addition, H2S scavenges RNS, peroxynitrite (ONO2-), oxygen free radicals and lipid

peroxidation, resulting in cardiovascular protection and neuroprotection (111, 125). In

contrast, increased levels of H2S have also been reported in various animal models of

inflammatory diseases such as sepsis (43, 44, 126), endotoxaemia (127) and acute pancreatitis

(128, 129). Therefore, it is clear that endogenous H2S plays an important role in the

pathophysiology of different diseases in both the central nervous system and peripheral

organs.

1.3.6 Role of H2S in inflammation

Numerous studies have proposed that H2S plays a key role in the pathogenesis of

inflammatory diseases such as acute pancreatitis (128-132), inflammatory bowel disease

(IBD) (133-136), hindpaw oedema (137), burn injuries (138), endotoxaemia (139) and sepsis

(44, 126, 140, 141). The role exerted by H2S in inflammation remains controversial because it

21

has been reported to have both pro- and anti-inflammatory effects. Previous research has

indicated that H2S is likely to be an endogenous regulator of inflammatory response in various

inflammatory diseases. Table 1.2 lists the pro- and anti-inflammatory role of H2S in animal

models of inflammatory disease. The mixed results of both pro- and anti-inflammatory effects

of H2S appear to be due to differences between studies such as variation in the choice of H2S

donor or inhibitor, route of administration and dosage regime, as well as chosen method of

inducing disease or injury.

Although H2S has wide implications in different inflammatory disease conditions, this chapter

is primarily focused on the inflammatory role of H2S in the pathophysiology of sepsis.

22

Model of disease/injury Species Treatment/approach Type of inflammatory

response

Reference

H2S inhibitor H2S donor

CLP-induced sepsis Mouse SiRNA (7 mg/kg) i.v. Pro-inflammatory (126)

CLP-induced sepsis Mouse PAG (50 mg/kg) i.p. NaHS (180 µmol/kg) i.p.

Pro-inflammatory (142-144)

LPS-induced endotoxaemia 10 mg/kg i.p.

Mouse PAG (50 mg/kg) i.p. Pro-inflammatory (145)

LPS-induced endotoxaemia ~4 µg/kg i.p.

Mouse PAG (113 mg/kg) s.c.

NaHS (30 µmol/kg) s.c.

Pro-inflammatory (146)


Rat PAG (50 mg/kg) i.p. Pro-inflammatory (147)

Burns injury (30% exposure for 8s)

Mouse PAG (50 mg/kg) i.p. NaHS (180 µmol/kg) i.p.


Caerulein-induced pancreatitis; 50 µg/kg/hr, 10 hours, i.p.

Mouse SiRNA (7 mg/kg) i.v. Pro-inflammatory (149)


Mouse CSE gene deletion Pro-inflammatory (129)


Mouse PAG (100 mg/kg) i.p.

Pro-inflammatory (150, 151)

Sodium taurocholate-induced pancreatitis

Rat PAG (80 mg/kg) i.p. NaHS (28 µmol/kg) i.p.


Haemorrhagic Shock Rat PAG (50 mg/kg) Pro-inflammatory (153)

Carrageenan-induced hind paw oedema 150 μL, 2% (w/v) intraplantar injection

Rat PAG (50 mg/kg) i.p. Pro-inflammatory (154)

Carrageenan-induced hind paw oedema 150 μL, 1% (w/v) intraplantar injection

Rat PHE-4i (2, 5, and 10 mg/kg) i.p.


23

CLP-induced sepsis Mouse PAG (50 mg/kg) i.p. NaHS/Laws (10-100 µmol/kg) s.c.

Anti-inflammatory (156)

CLP-induced sepsis Mouse NaHS (100 µmol/kg) s.c.



Rat S-Diclofenac i.p.


LPS-induced endotoxaemia 4 mg/kg i.v.

Rat GYY4137 (50 mg/kg) i.v.


Burns injury (40% exposure for 10s)

Mouse NaHS (36 µmol/kg) s.c.


Caerulein-induced pancreatitis ; 50 µg/kg/hr, 10 hours, i.p.

Mouse ACS15 (15 mg/kg) i.p.


L-arginine-induced pancreatitis 250 mg/100g i.p.

Rat PAG (50 mg/kg) i.p. NaHS (5, 10, 20 or 100 mg/kg) i.p.


Renal ischaemia reperfusion, 45min

Rat PAG (50 mg/kg) i.p. Anti-inflammatory (163)


Rat NaHS (0.15 µmol) i.p.



Pig Na2S (1.28 µmol/10min) i.v.


Haemorrhagic shock Rat PAG (50 mg/kg) i.v. NaHS (3.57 µmol/kg) i.v.


Carrageenan-induced knee joint arthritis 10 µL, 2% i.a.

Rat Na2S (5 nmol/joint) i.a.


Carrageenan-induced knee joint synovitis

Rat PAG (0.47 µg/joint) i.a.

Laws (3.6 µmol/joint) i.a.


Carrageenan-induced hind paw oedema 150 µL, 2% w/v, intraplantar injection

Rat S-Diclofenac (11.8-47.2 µmol/kg) i.p.


Colitis Rat PAG (50 mg/kg/) i.p.

NaHS (1.7 mg/kg) Laws (12.1 mg/kg) i.c.


Myocardial ischaemia reperfusion injury

Rat NaHS (54 µmol/kg) i.v.


24

Table 1.2 Summary of studies investigating the role of hydrogen sulfide (H2S) in animal models of

disease/injury. This table shows reported role of H2S in different inflammatory experimental disease

animal models and injury. Studies used different approaches to modulate endogenous H2S levels

results are mixed, showing both pro- and anti-inflammatory effects of H2S even in similar models of

disease. However, there appear to be differences between studies such as variations in the choice of

H2S donor or inhibitor, route of administration and dosage regime, as well as chosen method of

inducing disease or injury. (i.p., intraperitoneal; s.c., subcutaneous; i.v., intravenous; i.a.,

intraarticular; i.c., intracolonic; PAG, DL-propargylglycine; NaHS, sodium hydrogen sulfide; and Na2S,

disodium sulfide).

1.3.6.1 Role of H2S in sepsis

Understanding the role of H2S in sepsis and septic shock is particularly important due to the

high mortality rates associated with these conditions. Several early studies have confirmed

the inflammatory role of H2S in sepsis. Two animal models of sepsis have been used to study

the role of H2S: administration of the LPS endotoxin and CLP-induced sepsis.

Early studies used the LPS-induced endotoxaemia model to study the role of H2S in sepsis.

Injection of LPS resulted in an overproduction of endogenous H2S, as shown by a marked

increase in plasma H2S concentrations, CSE activity and CSE mRNA levels in liver and kidney

tissues associated with an increased inflammatory response, and by multiple organ damage

(127, 172, 173). Administration of NaHS aggravated endotoxaemia-induced leukocyte

infiltration of tissues and multiple organ damage, whereas these symptoms were alleviated

by pretreatment with CSE inhibitor DL-propargylglycine (PAG) (174). In addition, formation of

H2S in endotoxaemia was greatly inhibited by NO-releasing flurbiprofen, highlighting the

potential interaction between NO and H2S in endotoxaemia (175). Together, these results

suggested that H2S had a proinflammatory role in LPS-induced endotoxaemia. In contrast,

other studies have reported H2S has an anti-inflammatory role. For example, studies with H2S

donors S-diclofenac and GYY4137 have reported decreased leukocyte infiltration, cytokine

25

and eicosanoid generation, and NF-B activation in LPS-induced endotoxaemia (158, 176). The

discrepancy between different studies might be due to differences in the use of H2S donors

and inhibitors, the route of administration and dosage regime. Furthermore, irrespective of

the pro- or anti-inflammatory role of H2S, LPS-induced endotoxaemia does not mimic the

cytokine profiles or hemodynamic changes of human sepsis (9).

To avoid the limitations of LPS-induced endotoxaemia as a model for sepsis in humans (9),

the inflammatory role of H2S was studied using another animal disease model: CLP-induced

sepsis. In recent years, Bhatia and colleagues have shown that significantly increased

expression of CSE and H2S levels are associated with leukocyte infiltration and organ injury in

CLP-induced sepsis (126, 140, 177). H2S is also overproduced in the vascular tissues of rats

with experimental shock induced by CLP-induced sepsis (178). Similarly, a preliminary study

in patients with septic shock showed there was a significant increase in plasma H2S levels

compared with healthy controls (172). Conversely, administration of the CSE inhibitor PAG

decreased plasma H2S levels and inhibited leukocyte infiltration, liver and lung injury, and

improved survival following CLP-induced sepsis (44, 140). A recent study used siRNA to silence

CSE gene-protected mice against sepsis-induced leukocyte infiltration and liver and lung

damage (126). Elevated levels of proinflammatory cytokines and chemokines correlated with

increased CSE expression, activity and H2S synthesis in CLP-induced sepsis (43, 126, 173), and

the proinflammatory cytokine and chemokine response was further augmented when H2S

donor NaHS was administered to mice with sepsis (43, 44). Treatment with PAG attenuated

activation of ERK1/2 and NF-B p65 and subsequent cytokine and chemokine production

following CLP-induced sepsis (43, 44). Together, these results suggested that the temporal

increase in CSE expression and H2S synthesis during sepsis correlated with the occurrence of

26

phosphorylation of ERK1/2 and activation of NF-B p65 and subsequently regulated

generation of proinflammatory cytokines and chemokines.

In contrast to the proinflammatory role, other studies have reported the protective effect of

H2S in CLP-induced sepsis. For example, it has been reported that administration of H2S

donors such as NaHS and Lawesson’s reagent improved neutrophil migration and survival rate

through a mechanism involving the activation of KATP channels in CLP-induced sepsis (156).

Similarly, another study using NaHS reported increased survival rates in mice by inhibition of

the C/EBP homologous protein 10 (CHOP) following CLP-induced sepsis (157).

Together, these studies have shown the inflammatory role of H2S in sepsis. Irrespective of the

type of sepsis disease model, H2S has been reported to have both pro- and anti-inflammatory

effects in LPS-induced endotoxaemia and CLP-induced sepsis. These mixed results showing

both the pro- and anti-inflammatory effects of H2S are appear to be due to the use of different

pharmacological donors and inhibitors of H2S. Dosage regime, route and time of

administration of H2S donors and inhibitors may activate or inhibit different signalling

pathways, which in turn produce either a pro- or anti-inflammatory effect in sepsis. Newer

and more promising alternative tools are required to further investigate the complex

inflammatory role of H2S in sepsis.

1.4 Substance P (SP)

1.4.1 Biosynthesis and physiological functions of SP

SP is an 11 amino acid neuropeptide belonging to the tachykinin family and encoded by the

preprotachykinin-A (PPTA) gene (179-181). SP shares tachykinins’ common carboxyl-terminal

sequence Phe-X-Gly-Leu-Met-NH2, which is essential to interacting with its specific receptors

27

and producing biological actions (182). SP is widely distributed in the central nervous system

and is released from nerve endings in several peripheral tissues, including the entire length

of the gastrointestinal tract and the pancreas (183-186). Although SP has been described as a

peptide of neuronal origin, studies on rodents have demonstrated that it is produced by

inflammatory cells such as macrophages (187), eosinophils (188) and dendritic cells (189). The

biological actions of SP are mainly mediated through a family of rhodopsin-like G-protein-

coupled receptors, of which neurokinin-1 receptor (NK-1R) has the highest affinity for SP

(190). The most widely known roles of SP are in nociception and neurogenic inflammation

(191, 192). However, the diverse expression of NK-1R suggests that SP also elicits local

vasodilation and increases microvascular permeability and plasma extravasation, thereby

enhancing the delivery and accumulation of leukocytes in inflamed tissue (193, 194).

1.4.2 The role of SP in sepsis

SP acts as a proinflammatory mediator in sepsis and studies in both experimental and clinical

sepsis have provided evidence of its proinflammatory role. For example, in both LPS-induced

endotoxaemia and CLP-induced sepsis SP levels were significantly increased in plasma and

lungs and were associated with lung injury (195-200). Similarly, in patients with postoperative

sepsis, circulatory levels of SP (related to the lethal outcome of sepsis) were significantly

elevated (201-203). Intervention studies using NK-1R antagonists and mice deficient in the

PPTA gene were protected against endotoxaemia and sepsis-induced lung injury (196, 198,

200). These data suggest that SP has a proinflammatory role in sepsis and associated organ

damage.

An increased inflammatory response and organ injury through H2S-mediated activation of SP

and NK-1R has also been reported in sepsis. H2S upregulates the generation of SP and

28

activation of NK-1R in CLP-induced sepsis (141), both of which are further augmented by

administration of NaHS (204). Conversely, administration of PAG reduced SP levels and NK-

1R following sepsis (141, 205, 206). However, the mechanisms of H2S-mediated regulation of

SP production and NK-1R receptor activation remain to be elucidated.

1.5 Liver and liver sinusoidal endothelial cells

1.5.1 Anatomy and function of the liver

The liver is the largest organ in the body and is responsible for multiple dynamic functions. As

the principal organ for maintaining systemic homeostasis, the liver has a major role in the

metabolism, synthesis, storage, detoxification and secretion of numerous substrates (207).

The liver is also vital in maintaining and establishing immune functions (208). The liver

synthesises plasma proteins such as albumin and is involved in the metabolism of lipoproteins

and bilirubin, together with bile formation and secretion. It also plays a key role in nutrient

storage and the processing and regulation of blood glucose (209).

Anatomically, the liver is located in the upper right quadrant of the abdominal cavity. An adult

human liver weighs approximately two kilograms and comprises 2 to 5% of total body weight.

The liver is functionally divided into mainly four lobes: left, right, median or quadrate and

caudate, which are further subdivided into eight segments by divisions of the right, middle

and left hepatic veins, with each segment receiving blood from its own portal pedicle.

Uniquely, the liver receives its blood supply from two main sources: the portal vein and

hepatic artery. Seventy percent of its blood is from the portal vein, which carries

deoxygenated blood from the splanchnic circulation system while the remaining 30% comes

via the hepatic artery, which carries oxygenated blood (210) (Figure 1.4).

29

Figure 1.4 Segmental anatomy of the liver (adapted from Siriwardena et al., 2014). Anatomically,

the liver divided into eight segments by divisions of the right, middle and left hepatic veins, with each

segment receiving blood from its own portal pedicle (210).

The functional units of the liver are described as interconnecting lobules. Lobules are

hexagonal in shape and contain a central vein surrounded by repeating branches of the portal

triad (portal vein, bile ducts and hepatic artery). Depending on the area’s proximity to the

central vein and portal triads, each lobule is classified into three anatomical zones: periportal

or portal zone, midzonal or midlobular zone and centrilobular or central zone. In addition to

the classical lobular delineation, the functional units of the liver are classified as acini, each of

which are divided into three zones according to the level of oxygenation and nutrient

exposure of the cells. Zone 1 contains cells receiving the most oxygenated and nutrient-rich

blood (located closest to the portal triad), while zones 2 and 3 respectively contain cells that

are exposed to less oxygenated and less nutrient-rich blood (remote from arteriolar blood

30

and located in the microcirculatory periphery of the acinus) (211) . The zones are depicted in

Figure 1.5.

Figure 1.5 Diagrammatic representation of the classic lobule and liver acinus (adopted from Cattley

et al., 2002). Depending on the area’s proximity to the central vein and portal triads, each

lobule is classified into three anatomical zones: periportal zone (close to the portal triad),

midzonal zone and centrilobular zone (close to the central vein). The functional units of the

liver are classified as acini, each of which are divided into three zones according to the level of

oxygenation and nutrient exposure of the cells. Zone 1 exposed to most oxygenated and

nutrient-rich blood (close to the portal triad), zone 2 and zone 3 exposed to less oxygenated

and less nutrient-rich blood (close to the central vein) (211).

1.5.2 The liver sinusoid

The liver sinusoid is a unique and highly specialised capillary blood vessel. These channels

have an average diameter of approximately 7 to 10 µm and connect the portal venous system

with the terminal hepatic veins in the centre of the hepatic lobules (212). Blood flow is

relatively slow in the liver sinusoids and allows for prolonged contact with the hepatocytes.

31

The smaller diameter and slow blood flow facilitate metabolic exchange between afferent

blood and the hepatic parenchyma, which may play an important role in immune surveillance

and cellular interactions. Flow of arterial and venous blood is regulated by contractions of

sphincters in the walls of the sinusoids (213). The hepatocytes, arranged in single cell plates,

are separated by sinusoids. The space between the sinusoidal wall and the hepatocytes is

called space of Disse. Therefore, through its basolateral surface and the space of Disse, each

hepatocyte has access to the blood and its content (214).

Four types of cells constitute the liver sinusoid: liver sinusoidal endothelial cells (LSECs),

Kupffer cells, stellate cells (also known as Ito cells or fat storage cells) and pit cells, each with

specific morphology and function. LSECs constitute approximately 20% of liver cells, Kupffer

cells 15% and stellate cells 5%. Depending on the disease process, each cell type can undergo

morphologic or quantitative changes (215, 216). This chapter mainly focuses on Kupffer cells

and LSECs and their role in the inflammation associated with infection.

1.5.3 Kupffer cells

Kupffer cells are the resident macrophages in the liver sinusoid. They differentiate from yolk

sac cells and represent 80-90% of the body’s resident macrophages (217-219). Kupffer cells

are mainly located in the lumen of the liver sinusoids, usually in close proximity to endothelial

cells, and play a key role in host defence, homeostasis and regulation of metabolic functions

in the liver (218). The major function of Kupffer cells is to clear particulate and foreign

materials from portal circulation. Substrates including microorganisms, endotoxins, old and

foreign cells, complement components, immune complexes and collagen fragments, are

phagocytosed and degraded by the Kupffer cells (220).

32

1.5.3.1 Kupffer cells role in infection and inflammation

Kupffer cells play an important role in the clearance of pathogenic substances carried to the

liver through portal circulation and regulate immune and inflammatory responses. Pathogens

or endotoxins enter the liver through portal circulation are phagocytosed by Kupffer cells,

which activate specific membrane receptors (such as TLR4) present on their surface (221).

TLR4 associates with CD14 on the surface of Kupffer cells, which then mediate endotoxin-

induced signal transduction and release proinflammatory mediators. Kupffer cells primarily

produce TNF-α, but also other cytokines such as IL-6 and IL-1β, as well as NO and ROS

(including superoxide) to modulate inflammatory and immune responses. Although these

cells and cellular responses are necessary to combating infection and endotoxin, persistently

high or overwhelming activation may result in an uncontrolled proinflammatory cascade,

leading to a systemic inflammatory response and potentially to multiple organ failure (222-

224) (Figure 1.6).

The role of Kupffer cells during infection and inflammation has been studied using

experimental disease models of LPS-induced endotoxaemia (221, 222, 225, 226) and CLP-

induced sepsis (220, 227-230). Inactivation of Kupffer cells using gadolinium chloride (GdCl3)

was shown to protect against LPS-induced liver injury and inflammation (222, 223, 231).

Increased superoxide production and hepatic expression of TNF-α, IL-1β and IL-6 in response

to LPS-induced Kupffer cell activation was decreased by GdCl3 (231-234). Similarly, inhibition

of Kupffer cell phagocytosis by GdCl3 attenuated sepsis-associated hepatic dysfunction by

reducing the expression of cyclooxygenase-2 (COX-2) and modulation of inflammatory

response. At the same time, Kupffer cells released the anti-inflammatory cytokine IL-10 to

overcome the inflammatory response. For example, the inflammatory response to

33

endotoxaemia is downregulated by the local release of IL-10 from Kupffer cells (235). These

results suggest that Kupffer cells play a crucial in regulating inflammatory response associated

with infection and endotoxaemia.

Figure 1.6 Role of Kupffer cells in sepsis. Steps involved in the Kupffer cell-mediated inflammatory

response during infection: 1) Entering of bacteria and their components into the gastrointestinal tract

(GIT). 2) Translocation of bacteria and their components from the GIT into the liver via portal

circulation. 3) Activation of Kupffer cells. 4) Release of cytokines and chemokines from activated

Kupffer cells. 5) Migration of chemokines into systemic circulation. 6) Migration of neutrophils from

circulation into the liver. 7) Activation of neutrophils and transformation of monocytes to

macrophages. 8) Engulfment of bacteria and their components by macrophages and neutrophils. 9)

Release of cytokines and chemokines from activated neutrophils and macrophages. 10) Migration of

cytokines and chemokines into systemic circulation. 11) Increased inflammatory mediators in

circulation leading to a systemic inflammatory response. 12) Multiple organ failure (222-224).

34

The role of Kupffer cells in multiple organ injury (particularly lung injury) and systemic

inflammatory response was also investigated using different experimental inflammatory

disease models. For example, it has been shown that treatment with GdCl3 attenuates LPS-

induced pulmonary injury by decreased activation of caspase-3 (236). Similarly, ozone-

induced pulmonary injury was abrogated by pretreatment with GdCl3 (237). In contrast,

pretreatment with GdCl3 increased leukocyte infiltration into the lungs as well as chemokine

levels in LPS-induced lung alveolitis and endotoxaemia (223, 234). Furthermore, pretreatment

with GdCl3 decreased expression of anti-inflammatory cytokine IL-10 in CLP-induced

peritonitis (230). Despite the local effects on liver and lung injury, mice pretreated with GdCl3

showed significant increases in mortality in CLP-induced sepsis (227). Conversely, rats

pretreated with GdCl3 showed decreases in mortality by suppression of superoxide

production associated with endotoxaemia (226, 238). Additionally, both GdCl3 and clodronate

liposomes have been shown to lower serum levels of TNF-α, IL-6 and IL-1β, which were

increased following LPS-induced endotoxaemia and sepsis (220, 225, 228, 239). However,

treatment with GdCl3 had no effect on elevated serum TNF-α levels in other disease models

(222, 226, 240).

The results of previous studies on the effect of GdCl3 on liver and lung injury and the systemic

inflammatory response indicate that there is currently a poor understanding of the role of

Kupffer cells in different disease models of endotoxaemia and sepsis. This discrepancy might

be due to the use of different experimental disease models and observations of injury or

inflammation in only one organ rather than multiple organ systems. Studying the effect of

GdCl3 on multiple organ injury or inflammation using a single disease model would help to

contribute a better understanding of the role of Kupffer cells in sepsis.

35

1.5.4 Liver sinusoidal endothelial cells (LSECs)

1.5.4.1 Structure and functions of LSECs

LSECs are specialised endothelial cells lining the hepatocytes that form a barrier between the

sinusoidal blood and hepatic parenchyma, from which they are separated by space of Disse.

LSECs represents approximately 15-20% of liver cells but only 3% of the total liver volume.

Unlike vascular endothelial cells, LSECs do not have an organised basement membrane (both

diaphragm and underlying basal lamina) and are fenestrated. LSECs fenestrations are pores

that range in size from 50 to 250 nm in diameter. Most LSECs fenestrations are aggregated

into groups of 10-100 called sieve plates, which together with the subendothelial space of

Disse (containing an extracellular matrix) constitute the liver sieve (241). These fenestrations

are surrounded by a dense filamentous ring of actin (fenestration-associated cytoskeleton),

while the sieve plates are surrounded by a network of microtubules. The fenestrated

sinusoidal endothelium acts as a dynamic filter to exchange fluids, solutes and substrates

between the sinusoidal blood and the space of Disse, sieving substrates on the basis of size

(242, 243). The sinusoidal endothelium also acts as a scavenger system: endothelial scavenger

receptors are responsible for clearing blood of physiological waste products such as oxidised

low density proteins, matrix components and advanced glycation end products from normal

cell turnover (244).

LSEC porosity is determined by fenestration frequency (F/µm2) and fenestration diameter

(FD). The natural porosity of the liver sinusoid increases from the portal triad (zone 1) towards

the central vein (zone 3) owing to slight increases in fenestration frequency and perhaps also

an increase in fenestration diameter. It has been demonstrated that there are approximately

36

double the number of sieve plates and fenestrations per sieve plate in the pericentral

sinusoids than in the periportal sinusoids (216).

1.5.4.2 Alteration of LSEC fenestration

LSEC fenestrations are dynamic structures and alteration in their size and number can affect

hepatic trafficking of lipoproteins and other endogenous substrates across the sinusoidal

lumen and space of Disse, influencing liver function. Loss of fenestrae is called defenestration

and can be due to reductions in fenestration size and/or fenestration number. LSEC

fenestration diameter and frequency can vary between and within species (reviewed by

Wisse et al.) (245). For example, New Zealand white rabbits have smaller fenestrations than

rats and more fenestrations than chickens (246-248). Changes in fenestration diameter and

number are also observed in response to various biological mediators such as hormones

(serotonin) (249), neurotransmitters (adrenaline and acetylcholine) and growth factors

(vascular endothelial growth factors) (250). LSEC fenestrations are inducible structures and

cytoskeleton plays an important role in the formation and maintenance of fenestrae and liver

sieve; agents that alter sieve plate structure and porosity induce changes in cytoskeleton and

vice versa. For example, actin disrupting agents such as cytochalasin B (251), jasplakinolides,

swinolide A and latrunculin A misakinolide A (252), each with their own distinct actin-

disrupting properties alter LSEC fenestration diameter and frequency.

Fenestration diameter and frequency can also be altered by a variety of processes affecting

liver function. Several studies have investigated the effects of acute and chronic exposure of

different substances and conditions on LSEC fenestrae and how these changes affect liver

function. For example, acute and medium exposure to alcohol in rats in vivo or in isolated

LSECs in vitro is associated with increased LSEC fenestration diameter, frequency and

37

porosity. In contrast, with chronic long-term alcohol intake, humans and mice display LSEC

defenestration (253-255). Drugs such as pantethine increase LSEC porosity, whereas nicotine

decreases fenestrae diameter (256). In addition, the carcinogen dimethylnitrosamine (257)

and surfactant poloxamer-407 reduce LSEC porosity by decreasing fenestration frequency,

which leads to defenestration. Pathological conditions such as liver cirrhosis and fibrosis

(258), oxidative stress (259, 260), endotoxaemia, infection, inflammation (261, 262) and more

recently, normal ageing, have also been associated with loss of LSEC fenestrae.

1.5.4.3 Infection, inflammation and LSEC fenestrae

LSEC fenestrae are often altered in liver diseases associated with infection and inflammation

and their lesions may contribute to the downstream functional impairment of parenchymal

cells. Inflammatory mediators released from Kupffer cells and LSECs play a role in the loss of

LSEC fenestrations. Excessive stimulation of Kupffer cells and LSECs during infection or

endotoxaemia leads to secretion of an array of mediators including proinflammatory

cytokines and biologically-active free radicals (oxygen and nitrogen radicals), which lead to

LSEC damage and hepatocyte injury (235, 261-263). For example, it has been shown that LPS

induces defenestration in LSECs by the release of proinflammatory mediators from activated

LSECs and Kupffer cells (235, 261). Recent evidence has shown that interaction between

programmed death (PD-1) molecule of Kupffer cells and programmed death ligand-1 (PD-L1)

of LSECs potentiate LSEC injury in sepsis (263). These results suggest that inflammatory

mediators released from Kupffer cells and LSECs play a crucial role in the alteration of LSEC

fenestrae.

38

1.6 Research rationale, hypothesis and objectives

Sepsis and septic shock are common and serious medical problems in severely ill patients and

leading causes of morbidity and mortality in medical and surgical intensive care units. The

inflammatory response is an integral part of sepsis and the systemic inflammatory response

to bacterial infection during sepsis is characterised by coagulatory, hemodynamic and

metabolic changes that contribute to the progression to septic shock, MODS and even death.

Although antibiotic treatment may efficiently eradicate the infection, it does not specifically

reverse systemic inflammation and its sequelae. In addition, clinical approaches based on the

cytokines (inhibition of cytokines) have failed to improve the outcome in septic patients. A

better understanding of the mechanisms of sepsis and its sequelae may identify novel targets

for the development of new and effective therapies.

Recent research has shown that H2S acts as a mediator of the inflammatory process in

experimental disease models of sepsis. Although previous studies have indicated the

importance of H2S in sepsis using PAG, pharmacological inhibitor of the CSE enzyme, and H2S

donors such as NaHS, Na2S and GYY4137, these drugs have limitations to use for investigating

the role of H2S in sepsis (43, 44, 140, 141, 174, 177, 205, 206). For example, PAG has non-

specific actions, including alteration of homocysteine metabolism (264) and inhibition of AST

(265) and ALT (266) enzyme activities, which are unrelated to CSE enzyme inhibition. Similarly,

the therapeutic potential of H2S donors are limited due to difficulties in obtaining precisely-

controlled concentrations and the possible toxic impact of excess H2S (133, 172, 176, 267). In

addition, the use of these drugs in different investigations have reported both pro- and anti-

inflammatory roles of H2S in sepsis. These contradictory results showing both pro- and anti-

inflammatory effects of H2S appear to be due to the use of different pharmacological donors

39

and inhibitors of H2S. Newer and more promising alternative tools are required to further

investigate the complex inflammatory role of H2S in sepsis.

The gene deletion approach overcomes the disadvantages of H2S inhibitors and donors and

offers a definitive method for investigating the role of H2S in sepsis. Hence, the first part of

this thesis (Chapters 3 to 5) uses mice deficient in the CSE gene to study the potential role of

H2S in CLP-induced sepsis, examines the underlying mechanisms by which H2S regulates

inflammation and explores interaction between H2S and SP in regulating inflammatory

response in sepsis.

Kupffer cells are tissue-resident macrophages in the liver. They play a crucial role in the

clearance of invading pathogenic substances from portal circulation and subsequent

inflammatory response during sepsis. Numerous efforts have been made to elucidate the role

of Kupffer cells in liver and lung injury, inflammation and systemic inflammatory response in

different experimental disease models using GdCl3 and clodronate liposomes (220-222, 225-

230). However, there are many divergent findings among the published studies. These

discrepancies might be due to the use of different experimental disease models and the

observation of injury or inflammation in only single organs rather than multiple organ

systems. Studying the effect of GdCl3 on multiple organ injury or inflammation using a single

experimental disease model of sepsis that closely mimics human sepsis would help contribute

a better understanding of the role of Kupffer cells in sepsis. Hence, the second part of this

thesis (Chapter 6) uses GdCl3 to explore the role of Kupffer cells in liver and lung injury,

inflammation and systemic inflammatory response in CLP-induced sepsis.

Liver sinusoidal endothelial cells (LSECs) play an important role in the exchange of

endogenous substrates between sinusoidal blood and hepatic parenchyma. Although the

40

physiological and pathological significance of LSEC fenestrae has been increasingly

recognised, understanding the structural changes in LSEC fenestrae during sepsis is still at

early stage. Studying structural alteration of LSEC fenestrae will help contribute to our

understanding of the systemic complications associated with sepsis. Previous studies have

demonstrated that inflammatory mediators, particularly TNF-α, play a key role in the

structural changes of LSEC fenestrae following sepsis; however, these studies were confined

to LPS-induced endotoxaemia (235, 261, 262). Therefore, to further explore structural

alterations in LSEC fenestrae it is necessary to use an animal model of sepsis that mimics

human sepsis. Furthermore, the roles of H2S and SP (as a mediators of inflammation) and

Kupffer cells (as key resident macrophages) in the modulation of LSEC fenestrae during sepsis

have not yet been elucidated. Therefore, the third part of this thesis (Chapter 7) explores

structural alterations in LSEC fenestrae following sepsis and examines the effects of Kupffer

cell inactivator GdCl3, as well as gene deletion of H2S-synthesising enzyme CSE and SP

encoding PPTA on sepsis-induced structural alterations in LSEC fenestrae.

The fatal outcome in sepsis is mainly due to an overwhelming inflammatory response and

subsequent multiple organ failure. However, clinical approaches based on inflammatory

cytokines (inhibition of cytokines) have failed to reduce fatal outcomes in septic patients.

Studying the roles of novel inflammatory mediators such as H2S and SP will help contribute to

our understanding of the underlying mechanisms of the inflammatory process in human

sepsis with the aim of improving the outcome of sepsis. Previous studies using different

experimental sepsis models have shown the importance of H2S in sepsis (43, 44, 140, 141,

174, 177, 205, 206); however, the importance of H2S in patients with sepsis remains to be

elucidated. In addition, circulatory SP levels have been assessed in septic patients; however,

41

these studies mainly focused on the comparison of septic patients with healthy controls and

septic patients who survived compared to those who died. Furthermore, results contradict

each other (201-203). It is clear that healthy controls are very different physiologically from

patients who are acutely unwell, and acutely unwell non-septic patients experience a wide

variety of activated stress responses that may modulate the response to sepsis. Studying the

role of H2S and SP in human sepsis (associated with infection) in comparison to patients with

similar disease severity and organ dysfunction from non-infectious complications is therefore

important in the understanding precise role of H2S and SP in sepsis; however, this remains to

be elucidated. The final part of this thesis (Chapter 8) therefore explores the alteration of

circulatory H2S and SP in septic patients and their possible association with inflammatory

response in septic patients compared to severely ill patients with non-infectious

complications admitted to the hospital ICU.

1.6.1 Hypothesis

The hypothesis of the present thesis was to determine whether CSE-derived H2S, SP and

Kupffer cells modulate the inflammatory response, organ injury and LSECs fenestration in CLP-

induced sepsis. To investigate this, mice deficient in the CSE gene (H2S-synthesising enzyme),

mice deficient in the PPTA gene (SP encoding gene), and mice treated with GdCl3 (Kupffer cell

inhibitor) were used. Furthermore, the thesis was to determine whether alteration of

circulatory H2S and SP levels correlate with the inflammatory response in septic patients. The

schematic representation of thesis hypothesis depicted in Figure 1.7.

42

Figure 1.7 Schematic representation of hypothesis.

1.6.2 Objectives

The objective of this study was to investigate the role of H2S, SP and Kupffer cells on the

inflammatory response, organ injury and LSEC fenestration following CLP-induced sepsis.

Further, these studies aimed to investigate possible associations between circulatory H2S and

SP levels and the inflammatory response in septic patients.

More specifically, the study sought to examine:

1) The role of H2S on systemic inflammation and organ injury in CLP-induced sepsis.

2) The underlying mechanisms by which H2S regulates the inflammatory response in CLP-

induced sepsis.

3) The role of H2S in regulating the production and release of SP and the activation of NK-

1R in CLP-induced sepsis.

4) The role of Kupffer cells on CLP sepsis-induced organ injury, inflammation and

systemic inflammatory response.

43

5) The role Kupffer cells, H2S and SP on CLP sepsis-induced structural changes in LSEC

fenestrae.

6) To determine alteration in circulatory H2S and SP levels and their association with the

inflammatory response in septic patients admitted to the ICU.

44

Significance

The first part of the present study (Chapters 3 to 5) used mice deficient in the gene for the

H2S-synthesising enzyme CSE to study the role of CSE-derived H2S in sepsis. To the best of my

knowledge, this is the first time the role of CSE-derived H2S has been studied using CSE KO

mice in CLP-induced sepsis. Previous research has investigated the role of H2S in sepsis using

CSE enzyme inhibitor and H2S donors; however, pharmacological inhibitor and donors have

limitations to their use for investigating the role of H2S in sepsis. For example, PAG has non-

specific actions such as alteration of homocysteine metabolism and inhibition of AST and ALT

enzyme activities, which are unrelated to CSE enzyme inhibition. Similarly, the therapeutic

potential of H2S donors such as NaHS and Na2S are limited due to difficulties in obtaining

precisely-controlled concentrations and possible toxic impact of excess H2S. In addition,

dosage regime, route and time of administration of H2S donors and inhibitors activate or

inhibit different signalling pathways, which in turn produce either a pro- or anti-inflammatory

effect in sepsis. Gene deletion technology overcomes the disadvantages of these inhibitors

and donors and offers a definitive approach to investigate the role of CSE-derived H2S in

sepsis. The results of this study provide evidence that contribute to our understanding of the

potential roles of endogenous CSE-derived H2S in CLP-induced sepsis and the underlying

mechanisms by which endogenous H2S regulates inflammation and organ injury in sepsis.

Furthermore, the results presented in these chapters show the interaction between H2S and

SP in regulating the inflammatory response in sepsis.

The second part of the present study (Chapter 6) used GdCl3 to investigate the role of Kupffer

cells in sepsis. This is the first study showing the effect of GdCl3 on CLP sepsis-induced liver

and lung injury, inflammation and the systemic inflammatory response. Kupffer cells play an

45

important role in inflammation associated with infection; however, the role of Kupffer cells

on the systemic inflammatory response and multiple organ failure in CLP-induced sepsis is

unknown. This study helps to elucidate the role of Kupffer cells in liver and lung injury,

inflammation and the systemic inflammatory response in CLP-induced sepsis.

The third part of the present study (Chapter 7) used three different approaches (Kupffer cell

inactivation, CSE gene deletion and PPTA gene deletion) to study the alterations in LSEC

fenestrae in sepsis. To the best of my knowledge, this is the first study to report structural

alterations in LSEC fenestrae following CLP-induced sepsis and the effect of GdCl3, CSE gene

deletion and PPTA gene deletion on CLP sepsis-induced structural changes in LSEC fenestrae.

LSEC fenestrae play a key role in the transfer of endogenous substrates and mediators

between the sinusoidal blood and the space of Disse. Inflammation associated with infection

causes structural changes in LSEC fenestrae; however, the alteration of LSEC fenestrae during

inflammation associated with sepsis was unknown. This study demonstrates the structural

alterations in LSEC fenestrae following CLP-induced sepsis and the role of Kupffer cells, CSE

or CSE-derived H2S and SP on CLP sepsis-induced alterations in LSEC fenestrae.

Finally, the present study investigated the alteration in circulatory H2S and SP levels and their

association with inflammatory response in septic patients as compared to non-septic patients

with similar disease severity and organ dysfunction admitted to the ICU (Chapter 8). The

results of this study have increased our understanding of the role of H2S and SP during the

inflammatory response in septic patients. They have also shed light on the correlation

between experimental sepsis and human sepsis with regard to the proinflammatory role of

H2S and SP.

46

Chapter 2

Materials and methods

2.1 Materials

The DuoSet® Enzyme-linked Immunosorbent Assay (ELISA) Development System (for TNF-α,

IL-6, IL-1β, MCP-1 and MIP-2α) and Substrate Reagent Pack (stabilised tetramethylbenzidine

and hydrogen peroxide mixture) were purchased from R&D Systems (Minneapolis, USA). The

SP Enzyme Immunoassay (EIA) kit was purchased from Peninsula Laboratories International,

Inc. (California, USA). The TransAmTM NF-B p65 transcription factor ELISA kit (Active Motif)

was purchased from Australian Biosearch (Wangarra, Australia). The clone 4E1-1B7

monoclonal antibody against CSE was purchased from Abnova (Taipei, Taiwan). The rabbit

monoclonal antibody against phospho-p44/42 MAPK (ERK1/2) and rabbit monoclonal anti-

p44/42 MAPK (pERK1/2) antibody were purchased from Cell Signalling (Boston, USA). The

rabbit polyclonal antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH),

rabbit polyclonal IgG antibody against hypoxanthine phosphoribosyl transferase 1 (HPRT) and

goat anti-mouse IgG-HRP conjugated-secondary antibody were purchased from Santa Cruz

Biotechnology, Inc. (Texas, USA). The rabbit polyclonal antibody against NK-1R was purchased

from Abcam (Victoria, Australia). Prestained recombinant protein molecular weight markers,

30% acrylamide/bis solution and the DC protein assay kit were obtained from Bio-rad

(California, USA). Halt Protease Inhibitor Cocktail and Halt Phosphatase Inhibitor Cocktail

were purchased from Thermo Scientific Pierce Protein Biology (Rockford, USA). Nitrocellulose

blotting membrane and filter paper (Whatman™ 3030-861) were obtained from GE

Healthcare (Little Chalfont, UK). The chemiluminescence detection kit was purchased from

Perkin Elmer (Massachusetts, USA). Glutaraldehyde, Grade I (25% in H2O), zinc acetate,

47

hexadecyltrimethylammonium bromide (hDMAB), calcium chloride anhydrous (CaCl2),

paraformaldehyde (powder, 95%), trichloroacetic acid (TCA), sodium cacodylate trihydrate,

gadolinium chloride hexahydrate (GdCl3.6H2O), ammonium persulphate (APS), glycerol, N’N’-

tetramethylethylenediamine (TEMED), Tween20, PLP, hexamethyldisilazane (HMDS), L-

cysteine, sulfuric acid (H2SO4), hydrochloric acid (HCl), Tris base, sodium dodecyl sulfate (SDS),

ethylenediaminetetraacetic acid (EDTA), N,N-dimethyl-p-phenylenediamine sulfate (NNDM),

ferric chloride (FeCl3), 2-mercaptoethanol, N-ethyl maleimide (NEM), sodium chloride (NaCl),

potassium chloride (KCl), sodium hydroxide (NaOH), sodium deoxycholate, sodium

dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4), potassium

dihydrogen phosphate (KH2PO4) and glycine were purchased from Sigma-Aldrich (St Louis,

USA). Sodium pentobarbital (60 mg/kg; 150 mg/kg), buprenorphine (Temgesic, 0.2mg/kg) and

isoflurane were obtained from Christchurch Animal Research Area (CARA) and the

commercial mice diet was obtained from Envigo (Cambridgeshire, UK).

2.2 Buffers and solutions

Table 2.1 List of different buffers/solutions and their preparation used in different assays and

experiments.

Buffers/solutions

Preparation

Phosphate-buffered saline

(PBS)

PBS was prepared by dissolving 8 g NaCl, 0.2 g KCl, 2.84 g

Na2HPO4 and 0.2 g KH2PO4 in 1 L ultrapure water (provided in

the facility at University of Otago-Christchurch).

Tris-buffered saline (TBS)

TBS was prepared by dissolving 2.4 g Tris base and 8 g NaCl in

900 mL ultrapure water. pH was adjusted to 7.6 with either

48

NaOH or HCl and the final volume was adjusted to 1 L with

ultrapure water.

Tris-buffered saline-tween

(TBST)

TBST was prepared by adding 1 mL Tween20 to 1 L of TBS.

Transfer buffer Transfer buffer for western blotting was prepared by

dissolving 3.02 g Tris base and 14.4 g glycine in 500 mL of

ultrapure water. To this, 100 mL of methanol was added and

made up to 1 L with ultrapure water.

Running buffer Upper tank buffer was prepared by dissolving 30 g Tris base,

144 g glycine and 10 g SDS in ultrapure water; final volume

was adjusted to 1 L with ultrapure water.

Lower tank buffer was prepared by dissolving 20 g Tris base in

ultrapure water; final volume was adjusted to 1 L with

ultrapure water.

Western blotting blocking solution

Western blotting blocking solution was prepared by dissolving

2.5 g milk powder in 50 mL TBST.

1.5 M Tris (pH 6.8, stock

buffer for stacking gel)

118.20 g Tris-HCl and 90.82 g Tris base were dissolved in 700

mL ultrapure water and pH was adjusted to 6.8 either with

NaOH or HCl. The volume was adjusted to 1L with ultrapure

water (solution cooled to room temperature before making

the final pH adjustment).

1.5 M Tris (pH 8.8, stock

buffer for stacking gel)

41.19 g Tris-HCl and 150 g Tris base were dissolved in 700 mL

ultrapure water and pH was adjusted to 8.8 either with NaOH

49

or HCl. The volume was adjusted to 1L with ultrapure water

(solution cooled to room temperature before making the final

pH adjustment).

Protein loading buffer (3x) To prepare 45 mL of stock, 4.5 g of SDS was dissolved in 20 mL

of ultrapure water and then 6.25 mL 1M Tris solution (pH

adjusted to 6.8), 15 g sucrose, 1.5 mL of 100 mM EDTA and 6

mg bromophenol blue were added and stirred to dissolve

before the addition of 7.2 mL 2-mercaptoethanol; final

volume was adjusted to 45 mL with ultrapure water.

10% stacking gel 10% stacking gel was prepared by adding 1.67 mL of 30% Bis-

acrylamide to a mixture of 1.25 mL Tris-HCl buffer (1.5 M, pH

8.8), 1.98 mL ultrapure water, 50 µL of 10% (w/v) APS and 50

µL of 10% (w/v) SDS. 5 µL TEMED was added immediately

prior to pouring the gel to initiate polymerisation.

5% resolving gel To prepare 5% resolving gel, 0.83 mL of 30% Bis-acrylamide

solution was added to a mixture of 0.63 mL Tris-HCl buffer (1

M, pH 6.8), 3.4 mL ultrapure water and 50 µL of 10% (w/v)

APS, 50 µL of 10% (w/v) SDS. 5 µL TEMED was added

immediately prior to pouring the gel to initiate

polymerisation.

Radioimmunoprecipitation

assay (RIPA) buffer

RIPA buffer was prepared by dissolving 10 mM Tris-Cl (5 mL)

(pH 8.0), 1 mM EDTA (186 mg) (EDTA), 140 mM NaCl (500 mg),

1% Triton X-100 (5 mL), 0.1% sodium deoxycholate, 0.1% SDS

50

(500 mg), 0.9 % NP-40 (4.5 mL) and halt protease inhibitor

cocktail (1:100) in ultrapure water; final volume was adjusted

to 500 mL with ultrapure water.

Sodium phosphate buffer

(pH 7.4)

Sodium phosphate buffer (1 L) was prepared by combining

774 mL NaH2PO4 and 226 mL Na2HPO4. pH was adjusted to

7.4.

Neutral phosphate-

buffered formalin (10%)

To prepare 1L of 10% neutral phosphate-buffered formalin,

100 mL of 37% stock formalin was added to 900 mL ultrapure

water and then neutralised by dissolving 4 g NaH2PO4 and 6.5

g Na2HPO4 into the solution.

Electron Microscopy (EM)

fixative

To prepare 100 mL EM fixative, 8 g paraformaldehyde was

dissolved in 25 mL ultrapure water by heating (65°C). 10 mL

of 25% glutaraldehyde, 50 mL of 0.2 M sodium cacodylate

buffer, 2 g sucrose and 2 mL of 2 mM CaCl2 were dissolved in

the solution. pH was adjusted to 7.4 either with NaOH or HCl

and the final volume was adjusted to 100 mL with ultrapure

water.

0.2 M Sodium cacodylate

buffer

700 mL of 0.2 M sodium cacodylate buffer was prepared by

dissolving 22.9 g sodium cacodylate trihydrate then adding

25.2 mL of 0.2 M HCl in 674.8 mL ultrapure water.

0.1 M Sodium cacodylate

buffer

1 L of 0.1 M sodium cacodylate buffer was prepared by

combining equal volumes of 0.2 M sodium cacodylate buffer

(500 mL) and ultrapure water (500 mL).

51

250 mM L-cysteine 250 mM L-cysteine was prepared by dissolving 30 mg L-

cysteine in 1 mL of 20 mM pH 7.4 sodium phosphate buffer.

2 N H2SO4 100 mL of 2 N H2SO4 was prepared by adding 5.5 mL

concentrated H2SO4 to 94.5 mL ultrapure water.

30 mM FeCl3 30 mM FeCl3 was prepared by dissolving 4.86 mg FeCl3 in 1 mL

of 1.2 M HCl.

18 mM PLP 18 mM PLP was prepared by dissolving 5 mg PLP in 1 mL of 20

mM Na2HPO4.

20 mM NNDM 20 mM NNDM was prepared by dissolving 4.18 mg NNDM in

1 mL of 7.2 M HCl.

2.3 Mice

All mice were maintained in the Christchurch Animal Research Area (CARA) under specific

pathogen-free (SPF) conditions and were allowed free access to water and sterilised

commercial mouse diet. Experiments were performed using male mice aged between 8 and

10 weeks old (weight 25-30 g). C57BL/6J, CSE KO (on a C57BL/6J background), BALB/c and

PPTA KO (on a BALB/c background) mice were obtained from the CARA. PPTA KO mice were

a gift from Prof. Allan Basbaum, University of California, San Francisco, USA. CSE KO mice

generated by crossing CSE heterozygous mice and were a generous gift from Dr. Isao Ishii,

Graduate School of Pharmaceutical Sciences, Keio University, Japan. All experiments were

approved by the Animal Ethics Committee of the University of Otago-Christchurch (protocol

number C3/13) and were performed according to established university guidelines.

52

2.4 Induction of polymicrobial sepsis in mice

Caecal-ligation and puncture (CLP)-induced sepsis was induced according to a previously

described protocol with minor modifications (268). Mice were randomly assigned to either a

control (sham) or experimental group (CLP sepsis). Mice were lightly anesthetised with

inhaled isoflurane (2% isoflurane in 1 L/min O2) and maintained with isoflurane during surgery

(1.5% isoflurane in 1 L/min O2). Buprenorphine (Temgesic, 0.2 mg/kg, subcutaneously, s.c.)

was administered 45 mins prior to surgery for analgesia. Sterile surgical techniques were used

to perform the CLP operation. After shaving the abdominal fur, a topical disinfectant was

applied. Thereafter, a small midline incision was made through the skin and peritoneum of

the abdomen to expose the caecum. The caecal appendage was ligated with silkam 5.0 thread

at 8-10 mm from the tip of the caecum without occluding the bowel passage then perforated

in two-evenly spaced locations at the distal end of the caecum with a 22 gauge (22G) sized

needle. Following this, a small amount of stool was squeezed out through both holes. Finally,

the bowel was repositioned and the abdomen was sutured with sterile permilene 5.0 thread.

Mice in the sham operation group underwent the same procedure without caecal-ligation

and puncture.

Gadolinium chloride (GdCl3, 10 mg/kg), a selective inhibitor of Kupffer cell activation or saline

was administered before the CLP or sham operation (prophylactic). Eight hours after the

operation, mice were euthanised by an intraperitoneal (i.p.) injection of a lethal dose of

sodium pentobarbital (150 mg/kg). Blood samples were withdrawn from the right ventricle

using heparinised syringes and centrifuged (1,000 g for 5 mins at 4°C). Thereafter, plasma was

aspirated and stored at -80°C. Samples of liver and lung tissues were stored at -80°C for

subsequent measurement.

53

2.5 Western blotting

Liver and lung tissue lysates were prepared by homogenising ~50 mg of tissue with a Labserv

homogeniser in ice-cold RIPA buffer supplemented with a halt protease inhibitor cocktail (and

halt phosphatase inhibitor cocktail for p-ERK1/2). The resulting homogenates were incubated

at 4°C for 30 mins and centrifuged (10,000 g for 10 mins at 4°C). The clear lysates

(supernatants) were collected and measured for protein content using the Bio-rad DC protein

assay and bovine serum albumin (BSA) protein standards as reference. Samples were run on

a 10% SDS-PAGE gel under reducing conditions with an equal loading of 15 µg of protein from

each sample per well. Gels were run (using the running buffer) at a constant 200 V until the

gel front had run out, then transferred (using transfer buffer) onto a 0.45 m nitrocellulose

membrane and run at a constant 100 V for 1 h using a Bio-rad system. Membranes were then

rinsed in TBST and blocked with 5% non-fat dry milk prepared in TBST. Blots were cut at the

37 kDa Mw marking and each portion of the membrane was probed for CSE (~42 kDa;

1:1,000), anti-ERK1/2 and anti-p-ERK1/2 (~44/42 kDa; 1:2,000), NK-1R (~46 kDa; 1:1,000), and

GAPDH (~35 kDa; 1:2,000) or HPRT (~23 kDa; 1:1,000) in blocking buffer overnight at 4°C.

Blots were then washed with TBST three times for 10 mins each then probed with the

corresponding HRP-conjugated secondary antibody (1:10,000) for 2 h at room temperature.

Blots were then washed with TBST three times for 10 mins each with a final TBS rinse for 5

mins. Blots were incubated with a chemiluminescence substrate for 1 min and visualised on

a chemi-doc system (Uvitec, UK). Bands were semi-quantitated using Alliance 4.2 software

and compared for relative intensities. Results were expressed as fold increases over the

control.

54

2.6 H2S-synthesising activity assay

H2S-synthesising activity in the liver and lung homogenates were measured with a modified

protocol based on the method described previously (269). Liver and lung tissues (~50 mg

each) were homogenised using a Labserv homogeniser in 20 mM ice-cold sodium phosphate

buffer (pH 7.4) with a protease inhibitor cocktail. The reaction mixture contained tissue

homogenate (230 µL) in 20 mM sodium phosphate buffer (pH 7.4), L-cysteine (10 µL, 250 mM)

and PLP (10 µL, 18 mM). The reaction was performed in tightly parafilm-sealed microfuge

tubes and initiated by transferring the tubes from ice to a shaking water bath at 37°C. After

incubation for 30 mins, 1% w/v zinc acetate (125 µL) was injected to trap evolved H2S.

Subsequently, a mixture of NNDM (20 mM) in 7.2 M HCl and FeCl3 (30 mM) in 1.2 M HCl (133

µL, in 1:1 ratio) was injected. Samples were left to incubate at room temperature in the dark

for 20 mins. Following this, 10% v/v TCA (25 µL) was added to denature the protein and stop

the reaction. After centrifugation (20,000 g for 10 mins at 4°C), the absorbance of the clear

supernatant (200 µL) was measured in a 96-well microplate using a spectrophotometer

(Varioskan flash, Thermo Fisher Scientific) at 670 nm. The H2S concentration was calculated

against a calibration curve of sodium sulfide (Na2S). Results were then corrected for the

protein content of the tissue sample determined by the Bio-rad DC protein assay and

expressed as nmole H2S formed per mg protein.

2.7. Plasma H2S measurement

Plasma H2S levels were measured with a modified protocol based on the method described

previously (270). The reaction mixture contained plasma (100 µL), 20 mM sodium phosphate

buffer (pH 8.5) (100 µL), L-cysteine (10 µL, 250 mM), PLP (10 µL, 18 mM), 1% w/v zinc acetate

(100 µL) and a mixture of NNDM (20 mM) in 7.2 M HCl and FeCl3 (30 mM) in 1.2 M HCl (80 µL,

55

in 1:1 ratio). Samples were left to incubate at room temperature in the dark for 20 mins.

Following this, 10% v/v TCA (120 µL) was added to denature the protein and stop the reaction.

After centrifugation (7700 g for 5 mins at 4°C), the absorbance of the clear supernatant (150

µL) was measured in a 96-well microplate using a spectrophotometer (Varioskan flash,

Thermo Fisher Scientific) at 670 nm. The H2S concentration was calculated against a

calibration curve of Na2S. Results were expressed as µmole H2S formed per mL plasma.

2.8 Tissue myeloperoxidase activity measurement

Leukocyte sequestration in liver and lung tissues was quantified by measuring tissue MPO

activity as described previously with minor modifications (129). Tissue samples were thawed

and homogenised using a Labserv homogeniser in ice-cold 20 mM sodium phosphate buffer

(pH 7.4) (~50 mg/mL) supplemented with a protease inhibitor cocktail. Homogenates were

then centrifuged (10,000 g for 10 mins at 4°C) and the resulting pellet resuspended in 50 mM

phosphate buffer (pH 6.0) containing 0.5% w/v hDMAB. The suspension was subjected to

three cycles of freezing and thawing and further disrupted by sonication on ice (20% power,

80% pulse for 40 seconds). Samples were then centrifuged (10,000 g for 5 mins at 4°C) and

the supernatant was used for the MPO activity assay. The reaction mixture consisted of

supernatant (50 μL), stabilised tetramethylbenzidine and hydrogen peroxide mixture (reagent

volume: 100 μL). This mixture was incubated at room temperature for ~10 mins for the colour

to develop and the reaction was terminated with 50 μL of 2 N H2SO4. Absorbance was

measured using a spectrophotometer (Varioskan flash, Thermo Fisher Scientific) at 450 nm

with a 570 nm correction. This absorbance was then corrected for the protein content of the

tissue sample using results from the Bio-rad DC protein assay. The results were expressed as

fold increases over the control.

56

2.9 Morphological examination of liver and lung damage

Samples of liver and lung were fixed in 10% v/v neutral phosphate-buffered formalin for 24 h

at room temperature without rotation and subsequently impregnated in paraffin wax before

being sectioned to 4-5 m slices and mounted onto microscopic slides. Liver and lung tissue

sections were dehydrated through a graded ethanol series (100% to 80%) and stained by the

Christchurch Anatomical Pathology Department with hematoxylin and eosin. Three sections

were prepared for each liver and lung sample and were examined by light microscopy using

a Carl-Zeiss Microscope (Axiocam, Germany) (objective lens magnification of ×20; eyepiece

magnification of ×10). Ten random images (Imager.Z1) from each section of liver and lung

were taken and assessed for liver and lung pathology. Liver images were assessed for capsular

inflammation and lobular necrotic damage using modified Knodell Histology Activity Index

(HAI) of blinded scoring system of liver injury (271, 272), while lung sections were assessed

for leukocyte infiltration and alveolar wall thickening using blinded lung injury scoring system

suggested by American Thoracic Society guidelines (273).

2.10 Measurement of sulfur amino acid homocysteine using HILIC-MS/MS

A hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS/MS)

technique was used to measure homocysteine levels with a modified protocol based on the

method described previously (274).

For measurement of homocysteine, liver and lung tissues (~50 mg each) were homogenised

using a Labserv homogeniser in 1 mL sodium phosphate buffer (pH 7.4) with a protease

inhibitor cocktail and centrifuged (1,000 g for 10 mins at 4°C). Supernatants were collected in

separate tubes and protein content was measured using the Bio-rad DC protein assay and

57

normalised by diluting with sodium phosphate buffer (pH 7.4). Protein-corrected liver and

lung supernatants and plasma were treated with an addition of 9-fold excess of acetonitrile

to sample (20 µL of the sample with 180 µL of acetonitrile added). The samples were left in

the freezer (-20°C) for 30 mins then centrifuged (1,000 g for 10 mins at 4°C) to collect the

supernatants. These supernatants were then injected onto the HILIC-MS/MS for

measurement of homocysteine.

Chromatography was performed using a Dionex (Sunnyvale, USA) 3000 HPLC. Separation was

achieved using a Luna HILIC 100 x 2 mm, 3 micron column (Phenomenex, Torrance, USA). The

sample (20 µL) was injected with a gradient of two solvents (solvent A, ammonium formate

20 mM, pH 4.2 and solvent B, acetonitrile). Initial conditions were 85% B/15% A at 0.2 mL/min

flow rate. This was held for 8 mins followed by a gradient of 50/50 over 2.5 mins before re-

equilibration. The column was held at 40°C and samples were kept at 4°C.

Effluent from the HPLC was directed to a Sciex (Framingham, USA) 4000 QTrap mass

spectrometer after the first 3 mins. Detection was performed using electrospray ionisation in

positive mode with multiple reaction monitoring, with needle spray at 4.5 kV, detector

temperature at 40°C and collisional energy of 24. Homocysteine was monitored using 136.2

to 90 m/z and this analyte was also confirmed using secondary ions. Data analysis was

performed using Analyst 1.6 (Sciex, California, USA) and homocysteine levels were expressed

as mass intensity in arbitrary units (A.U.).

2.11 Preparation of nuclear extract and determination of NF-κB p65 activation

Nuclear extracts from liver and lung tissues were prepared using a Nuclear Extraction Kit as

described by the manufacturer’s protocol (Active Motif, Tokyo, Japan). Liver and lung

homogenates were prepared by homogenising ~50 mg tissue in 200 µL ice-cold Complete

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Lysis Buffer (10 mM dithiothreitol (DTT) (0.2 µL), lysis buffer (178 µL), protease inhibitor

cocktail (2 µL) and phosphatase inhibitor cocktail (20 µL)) on ice using a Labserv homogeniser

and incubated for 30 mins at 4°C. Tissue homogenates were centrifuged (10, 000 g, 10 mins

at 4°C) and supernatant/nuclear extract was measured for protein content using the Bio-rad

DC protein assay. The protein-corrected nuclear extracts were used to measure NF-B p65

activation.

To monitor NF-B p65 activation in liver and lung nuclear extracts, a TransAM NF-B p65

Transcription Factor Assay Kit was used following the manufacturer’s instructions and as

described previously (275). The kit consists of a 96-well plate, onto which oligonucleotide

containing the NF-B consensus site (5’-GGGACTTTCC-3’) is bound. The active form of NF-B

p65 in the nuclear extracts (20 µg) specifically binds to this consensus site and is recognised

by a primary antibody specific for the activated form of p65 of NF-B. A HRP-conjugated

secondary antibody provides the basis for the colorimetric quantification. The absorbance of

the resulting solution was measured 2 mins later (450 nm with a reference wavelength of 655

nm) using a spectrophotometer (Spectramax, Molecular Devices, USA). The wild-type

consensus oligonucleotide is provided as a competitor for NF-B p65 binding to monitor the

specificity of the assay. Results were expressed as fold increases over the control group.

2.12 Cytokine and chemokine measurement by enzyme-linked immunosorbent

assay

Liver, lungs and plasma TNF-α, IL-6, IL-1β, MCP-1 and MIP-2α were measured using ELISA Duo

Set kits from R&D Systems according to the manufacturer’s protocol. Liver and lung

homogenates were prepared by homogenising ~50 mg tissue in 1 mL 20 mM sodium

phosphate buffer (pH 7.4) supplemented with a protease inhibitor cocktail on ice using a

59

Labserv homogeniser. Homogenates were centrifuged (10,000 g for 10 mins at 4°C) and 100

µL of the supernatant (or 100 µL of plasma) was used for each assay. Each kit consisted of a

capture and biotin-conjugated detection antibody pair, standards and streptavidin-HRP

conjugate. ELISA specific plates (Corning, USA) were first coated with capture antibody in PBS

overnight at room temperature. Plates were then aspirated, washed with PBST (0.05% w/v)

and blocked with 300 L of BSA (1% w/v) for 1 h at room temperature, followed by washing

and addition of 100 L samples or standards. After 2 h incubation, plates were decanted,

washed and 100 L of biotin-conjugated detection antibody was added. After 2 h incubation,

plates were decanted, washed and 100 L of streptavidin-conjugated HRP was added. After

a 20 min incubation, plates were decanted, washed and 100 L of substrate reagent was

added. Plates were incubated at room temperature for up to 20 mins to allow the colour to

develop and the reaction was terminated by addition of 50 L 2 N H2SO4. Plates were read at

450 nm on a spectrophotometer (Spectramax, Molecular Devices, USA) with a 570 nm

correction. Cytokine and chemokine levels were corrected for total protein content measured

using the Bio-rad DC protein assay and expressed as pg or ng per mg protein in tissue samples

or ng per mL in plasma.

2.13 Measurement of SP levels

SP levels in liver and lung tissues and plasma were measured using the peptide EIA kit from

Peninsula Laboratories according to the manufacturer’s protocol and as described previously

(204, 275). Liver and lung tissues (~50 mg of each) were homogenised by adding 1 mL ice-cold

SP assay buffer (for plasma, an equal amount of assay buffer was added) on ice using a Labserv

homogeniser. The homogenates were centrifuged (17,000 g for 20 mins at 4°C) and

supernatants were collected. SP levels were extracted using C18 cartridge columns (Bachem)

60

as described in the manufacturer’s protocol. The C18 columns were equilibrated using Buffer

A (1% trifluoroacetic acid, TFA) and thereafter, samples were loaded onto the equilibrated C18

columns. The adsorbed peptide was eluted with Buffer B (60% acetonitrile, 1% TFA and 39%

distilled water). The samples were freeze-dried and reconstituted in the SP assay buffer.

SP content in the samples was determined with an EIA kit according to the manufacturer's

instructions. Each kit consisted of a capture and biotin-conjugated detection antibody pair,

standards and streptavidin-HRP conjugate. Standards or samples (50 µL of each) and 25 µL

antiserum were added to precoated (with captured antibody and blocked with 1% BSA) EIA

plates and incubated for 1 h at room temperature. Thereafter, 25 µL biotin-conjugated

detection antibody was added. After 2 h incubation, plates were decanted, washed with 300

µL EIA buffer and 100 µL of streptavidin-conjugated HRP was added. After a 20 min

incubation, plates were aspirated, washed with 300 µL EIA buffer and 100 µL of TMB solution

was added. Plates were incubated at room temperature for 30-60 mins to allow the colour to

develop and the reaction was terminated by addition of 100 µL 2 N H2SO4. Plates were read

at 450 nm on a spectrophotometer (Spectramax, Molecular Devices, USA) with a 570 nm

correction. Results were then corrected for the protein content of the tissue samples and

were expressed as ng per mg protein or ng per 100 µL plasma.

2.14 Measurement of procalcitonin levels

Plasma procalcitonin (PCT) was measured using the ELISA DuoSet kit from R&D Systems

according to the manufacturer’s protocol. Each kit consisted of a capture and biotin-

conjugated detection antibody pair, standards and streptavidin-HRP conjugate. ELISA-specific

plates (Corning, USA) were first coated with capture antibody in PBS overnight at room

61

temperature. Plates were then aspirated, washed with PBST (0.05% w/v) and blocked with

300 L of BSA (1% w/v) for 1 h at room temperature followed by washing and addition of 100

L plasma or standards. After 2 h incubation, plates were decanted, washed and 100 L of

biotin-conjugated detection antibody was added. After 2 h incubation, plates were decanted,

washed and 100 L of streptavidin conjugated-HRP was added. After a 20 min incubation,

plates were decanted, washed and 100 L of substrate reagent was added. Plates were

incubated at room temperature for up to 20 mins to allow the colour to develop and the

reaction was terminated by addition of 50 L 2 N H2SO4. Plates were read at 450 nm on a

spectrophotometer (Spectramax, Molecular Devices, USA) with a 570 nm correction. PCT

concentration was expressed as pg per mL in plasma.

2.15 Scanning electron microscopy

2.15.1 Liver perfusion and fixation (primary fixation)

Sample preparation for scanning electron microscopy (SEM) was performed as described

previously (276). Eight hours after sham or CLP operation, mice were anaesthetised with a

single i.p. injection of sodium pentobarbital (80 mg/kg). A large midline laparotomy incision

was made and liver and portal vein were exposed. The portal vein was cannulated with a 22G

sized intravenous (i.v.) cannula. Liver perfusion was commenced first with approximately 5-

20 mL PBS (pH 7.4; warmed to 37°C) at a pressure of 15 cm height. The vena cava was severed

before commencing perfusion to minimise outflow resistance and high perfusion pressures.

PBS was then replaced with EM fixative and perfused for approximately 5 mins until the liver

was hardened and very pale. Following adequate fixation, the liver was excised, weighed and

placed in a small amount of fresh EM fixative. Random samples of liver were cut into 1-2 mm3

62

blocks using a sharp scalpel and blocks were immersed in fresh EM fixative for approximately

24-72 h at 4°C (post-fixation).

2.15.2 Processing of tissue blocks (secondary fixation)

After post-fixation, the liver blocks were washed for 5 mins, three times in 0.1 M sodium

cacodylate buffer and fixed in 2% osmium tetroxide in 0.1 M sodium cacodylate buffer for 2

h to impregnate the tissue with a heavy metal for interaction with the electron beam.

Specimens were again washed for 5 mins, three times in 0.1 M sodium cacodylate buffer.

Specimens were gradually dehydrated in an increasing ethanol gradient: 50% ethanol (twice

for 10 mins), 70% ethanol (twice for 10 mins), 90% ethanol (twice for 10 mins), 95% ethanol

(twice for 10 mins) and finally 100% dehydrated ethanol (four times for 5 mins).

2.15.3 Tissue preparation for scanning electron microscopy

Processing for SEM involved the complete drying of the tissue. All the ethanol was replaced

with HMDS and samples were incubated for 10 mins then left for overnight in a fume hood to

allow residual HMDS to be evaporated. The liver specimen blocks were mounted onto SEM

slug mounts under the dissecting microscope then coated with a thin layer of platinum using

the sputter coater to increase the electrical conductivity.

2.15.4 Tissue examination with scanning electron microscope

Randomly selected blocks of liver were examined with a JSM6380 SEM (JEOL, Japan) at 15 kV

acceleration voltage. Examination at low magnification was performed to assess successful

fixation and tissue drying. At least ten images of the sinusoidal endothelial cells from each

mounted slug were taken at 15,000× magnification for visualisation of endothelial lumen

surface and fenestrations. Fenestration number and diameter were measured, together with

63

total area assessed, using Image J (NIH). These data were used to calculate the porosity (the

percentage of the LSEC surface area covered with fenestrations) (276).

2.16. Measurement of plasma ALT and AST activity levels

Eight hours after CLP or a sham operation, mice were sacrificed and blood samples were

withdrawn from the right ventricle using heparinised syringes. Plasma was collected following

centrifugation (1,000g for 5 mins at 4°C) and the ALT and AST activity levels were measured

following established protocols at Canterbury Health Laboratories, Christchurch, New

Zealand. The results of ALT and AST activity levels were expressed in U/L.

2.17 Statistical analysis

Data are presented as mean + S.E.M. Statistical analysis was performed using GraphPad Prism

Software version 6.07. (GraphPad Software Incorporated, La Jolla, CA, USA). All experimental

data were analysed for Gaussian or Normal distribution using the Shapiro-Wilk test. One-way

Analysis of Variance (ANOVA) with post-hoc Tukey’s test was performed with the Gaussian

distribution data and a non-parametric Kruskal-Wallis test was performed with the data that

were not followed Gaussian distribution to compare multiple groups. A P-value of less than

0.05 was considered as a statistically significant.

64

Chapter 3

Effect of CSE gene deletion on leukocyte infiltration and organ damage following

caecal-ligation and puncture-induced sepsis in mice

3.1 Introduction

Hydrogen sulfide (H2S) has been well known for several decades as a toxic gas with the smell

of rotten eggs (277, 278). However, it is also generated endogenously through the

transsulfuration pathway catalysed by CBS and CSE enzymes (279). The sulfur amino acids

such as homocysteine and cysteine, are the intermediate precursors for H2S synthesis through

CBS and CSE enzymes (280). Of these two enzymes, CSE is the predominant H2S-synthesising

enzyme, with significant biological functions in the peripheral organs (281).

In recent years, H2S has been identified as a mediator of inflammation (43, 44, 173, 174, 177,

277, 282). H2S synthesised through CSE acts as a proinflammatory mediator in various

inflammatory conditions including, endotoxaemia (127, 172), haemorrhagic shock (282) and

sepsis (126, 140, 177). Studies on endotoxaemia-induced by LPS injection showed an increase

in H2S synthesis and has been associated with inflammation, leukocyte infiltration and

multiple organ damage (127, 172, 173). However, LPS-induced endotoxaemia does not

accurately reflect the cytokine profiles and hemodynamic changes of human sepsis (9).

Alternatively, another disease model, CLP-induced sepsis has been used to study the

pathogenesis of sepsis that mimics the cytokine profiles and hemodynamic changes of human

sepsis and has been shown to promote significant inflammatory response (283). Recent

studies have shown that increased CSE expression and H2S levels are associated with

leukocyte infiltration and organ injury following CLP-induced sepsis (126, 140, 177). For

example, it has been shown that treatment with CSE enzyme inhibitor PAG inhibits leukocyte

65

infiltration and liver and lung injury following CLP-induced sepsis (44, 140). These studies

suggest the significance of H2S in the inflammatory process in sepsis.

The sulfur amino acid homocysteine plays an important role in immune function and

inflammation, as well as being one of the intermediate precursors of H2S synthesis (280, 284).

Homocysteine acts as a potential proinflammatory compound that enhances the production

of specific cytokines and contributes to the pathogenesis of inflammatory diseases (285, 286).

Alteration of homocysteine levels has been reported in various inflammatory diseases such

as cardiovascular diseases (287-289), diabetes (290), cirrhosis (291), rheumatoid arthritis

(284), chronic kidney damage (CKD) (292) and sepsis (293, 294). For example, it has been

demonstrated that higher circulating concentrations of TNF-α are significantly correlated with

hyperhomocysteinemia in atherosclerosis (289). Recent studies have shown that

homocysteine also induces chemokines such as MCP-1 and MIP-2α in mesangial cells and that

the induction of these inflammatory mediators is linked to the adverse outcomes in patients

with cardiovascular disease and CKD (295-298). Furthermore, it has been shown that an early

increase in plasma homocysteine levels is associated with poor outcomes in patients with

sepsis (293, 294, 299). Elevated levels of homocysteine have also been observed in peripheral

blood nuclear cells when exposed to inflammatory stimuli in vitro (300, 301). Based on

previous research, homocysteine has been implicated in promoting inflammation in several

different conditions including sepsis.

The interaction between H2S and homocysteine has been reported in various pathological

conditions. Although homocysteine is one of the precursors of H2S synthesis and high levels

of homocysteine seem to promote H2S generation, it has been reported that increased

peripheral blood levels of homocysteine (or hyperhomocysteinemia) are associated with

66

decreased CSE expression and H2S synthesis (302, 303). For example, it has been shown that

CSE expression and H2S production are attenuated in the renal cortical tissue of

hyperhomocysteinemia mice (292). In addition, Li et al. have shown decreased CSE expression

and H2S production in macrophages exposed to homocysteine in vitro (264). Furthermore,

mouse glomerular mesangial cells stimulated with homocysteine increase MCP-1 and MIP-2α

production while endogenous H2S production is decreased (303). Conversely, treatment with

H2S in conjunction with CSE cDNA over-expression markedly reduced homocysteine-mediated

upregulation of MCP-1 and MIP-2α in mesangial cells (303). Similarly, H2S prevents renal

damage and regulates inflammation associated with hyperhomocysteinemia in

glomerulosclerosis mice (292, 304). Together, these previous studies suggest counter-

regulatory interactions occur between homocysteine, CSE expression and H2S production

during inflammation in different pathological conditions.

Although the inflammatory role of H2S has been studied in sepsis using the CSE inhibitor PAG,

these studies have limitations due to there being possible actions of PAG that are unrelated

to CSE; this has led to criticism of studies where PAG has been used. For example, PAG has

non-specific actions such as alteration of homocysteine metabolism (264) and inhibition of

AST (265) and ALT (266) enzyme activities, which are unrelated to CSE enzyme inhibition. In

addition, previous studies using H2S inhibitors and donors have reported the anti-

inflammatory effect of H2S in sepsis (305, 306). It is clear that newer and more promising

alternative tools are required to further investigate the complex biological roles of H2S in

sepsis. Gene deletion technology offers a definitive approach to investigate the roles of H2S

in sepsis. In addition, alteration in homocysteine levels and its impact on H2S synthesis remain

unknown in CLP-induced sepsis.

67

The objective of the present study was to determine whether CSE gene deletion modulates

homocysteine and H2S synthesis and consequently liver and lung injury in sepsis.

3.2 Aims

The present study aimed to determine the effect of H2S-synthesising enzyme CSE gene

deletion on leukocyte infiltration (as evidenced by MPO activity), homocysteine levels, H2S

synthesis, and liver and lung injury (evidenced by the amount of oedema and necrosis in the

tissues). To investigate this, an animal model of CLP-induced sepsis was used in mice deficient

in the CSE gene and CSE protein expression, H2S-synthesising activity, H2S levels and

homocysteine levels were measured. Disease severity was assessed by leukocyte infiltration

and liver and lung damage.

3.3 Experimental approach

Thirty two WT (C57BL/6J) and CSE KO (on a C57BL/6J background) (males aged between 8-10

weeks; 25-30 g) mice were randomly divided into control (WT Sham and CSE KO Sham) and

experimental (WT Sepsis and CSE KO Sepsis) groups, with eight mice in each group (n=8).

Sepsis was induced by CLP as described in Section 2.4 and 8 h after CLP or sham operation,

mice were euthanised by an i.p. injection of pentobarbital (150 mg/kg). Thereafter, blood was

aspirated, plasma separated and stored at -80°C. Liver and lung tissues were processed and

stored at -80°C. Subsequently, CSE protein expression (Section 2.5), H2S-synthesising activity

(Section 2.6), MPO activity (Section 2.8), liver and lung injury (Section 2.9) and homocysteine

levels (Section 2.10) were measured by the methods described in the Material and Methods

of Chapter 2. Statistical analysis was performed as described in Section 2.17.

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3.4 Results

3.4.1 CSE protein expression in WT and CSE KO mice following CLP-induced sepsis

69

Figure 3.1 CSE protein expression in WT and CSE KO mice following CLP-induced sepsis. WT and CSE

KO mice underwent CLP or sham operation. Sham operated mice acted as controls. Eight hours after

CLP or sham operation, mice were sacrificed and liver and lung tissues were processed to measure CSE

protein expression in the liver (A and B) and lungs (C and D) by western blotting. After densitometry

analysis, the data were expressed as ratios of CSE to GAPDH (plotted as the relative-fold increase of

CSE expression over the sham control). For western blot results, each lane represents a separate

animal. The blots showed representative of all mice in each group with similar results. There was a

significant increase in CSE protein expression in the liver and lungs following CLP-induced sepsis

compared to the sham group in WT mice. There was no CSE protein expression in the liver and lungs of

CSE KO mice. Data represent mean ± S.E.M. (n=8). The significance of differences among groups was

evaluated by ANOVA with post-hoc Tukey’s test. Statistical significance was assigned as **P<0.01 and

***P<0.001.

CLP-induced sepsis resulted in a significant increase of CSE protein expression in both liver

and lung compared to WT mice that underwent sham operation (liver: 1.98-fold increase

(P<0.001); lung: 2.49-fold increase (P<0.01)). As expected, CSE protein expression did not

occur at detectable levels in the liver and lung tissues of CSE KO mice, irrespective of sham

and CLP operation (Figure 3.1).

3.4.2 H2S-synthesising activity and H2S levels in WT and CSE KO mice following CLP-induced

sepsis

Following CLP-induced sepsis, there was a significant increase in liver H2S-synthesising activity

and plasma H2S levels compared to WT mice that underwent a sham operation (H2S

synthesising activity, 35.64 ± 6.88 nmole/mg vs 27.90 ± 4.33 nmole/mg, P<0.05; and H2S

levels, 3.90 ± 0.31 µmole/mL vs 2.68 ± 0.28 µmole/mL). Mice deficient in the CSE gene

showed significantly lower liver H2S-synthesising activity and plasma H2S levels compared to

WT mice following CLP-induced sepsis (6.10 ± 3.25 nmole/mg vs 35.64 ± 6.88 nmole/mg,

P<0.0001; and H2S levels, 1.72 ± 0.19 µmole/mL vs 3.90 ± 0.31 µmole/mL). These results

suggest that CSE is the major H2S-synthesising enzyme present in the liver. The experiments

70

in this study failed to detect any measurable levels of H2S-synthesising activity in lungs using

the method described in Section 2.6 (Figure 3.2).

Figure 3.2 Liver H2S-synthesising activity and H2S levels in WT and CSE KO mice following CLP-induced

sepsis. WT and CSE KO mice underwent CLP or sham operation. Sham operated mice acted as controls.

Eight hours after CLP and sham operation, mice were sacrificed and liver tissue and blood were

processed to measure H2S-synthesising activity and H2S levels, respectively using spectrophotometric

assay. H2S-synthesising activity and H2S levels increased following CLP-induced sepsis compared to

sham control in WT mice. CSE KO mice showed lower H2S-synthesising activity and H2S levels compared

to WT mice following sepsis. Data represent mean ± S.E.M. (n=8). The significance of differences among

groups was evaluated by ANOVA with post-hoc Tukey’s test. Statistical significance was assigned as

*P<0.05, **P<0.01 and ****P<0.0001.

3.4.3 Measurement of liver and lung leukocyte infiltration in WT and CSE KO mice following

CLP-induced sepsis

Leukocyte infiltration was assessed by measuring liver and lung MPO activity, as an increase

in MPO activity reflects leukocyte infiltration into these organs. WT mice with CLP-induced

sepsis showed significantly higher MPO activity in the liver and lungs compared to sham

operated control mice, with mean fold increases of 1.62 (P<0.05; liver) and 1.91 (P<0.01;

lung), respectively. The CLP-induced sepsis CSE KO mice showed significantly lower MPO

71

activity in the liver and lungs compared to WT sepsis mice, with mean fold decreases of 0.91

vs 1.62 (P<0.01; liver) and 1.00 vs 1.91 (P<0.01; lung), respectively (Figure 3.3).

Figure 3.3 Leukocyte infiltration in the liver and lungs of WT and CSE KO mice following CLP-induced

sepsis. WT and CSE KO mice underwent CLP or sham operation. Sham operated mice acted as controls.

Eight hours after CLP or sham operation, mice were sacrificed and liver and lung tissues were processed

to measure MPO activity in the liver (A) and lungs (B) using spectrophotometric assay. MPO activity

increased significantly in the liver and lungs of WT mice following CLP-induced sepsis compared to

sham control. CSE KO mice showed significantly less MPO activity in the liver and lungs compared to

WT mice following CLP-induced sepsis. Data represent mean ± S.E.M. (n=8). The significance of

differences among groups was evaluated by ANOVA with post-hoc Tukey’s test. Statistical significance

was assigned as *P<0.05 and **P<0.01.

3.4.4 Liver and lung injury in WT and CSE KO mice following CLP-induced sepsis

Both WT and CSE KO mice showed the typical effects of injury on the liver and lungs following

CLP-induced sepsis. Liver sections stained with hematoxylin and eosin from WT mice after CLP

operation were characterised by capsular inflammation, slight hepatocyte necrosis and

marginated, pavemented and transmigrated neutrophils that could be easily observed

compared to tissues from sham operated control and CSE KO sepsis (and sham operated)

mice. Lung sections from the WT mice after CLP operation exhibited characteristic signs of

72

lung injury, including interstitial oedema, alveolar thickening, severe leukocyte infiltration in

the interstititum and alveoli compared to CSE KO sepsis mice. Normal lung histoarchitecture

was observed in sham operated WT and CSE KO mice (Figure 3.4).

73

Figure 3.4 Morphological changes in the liver and lungs of WT and CSE KO mice following CLP-

induced sepsis. WT and CSE KO mice underwent CLP or sham operation. Sham operated mice acted as

controls. Eight hours after CLP or sham operation, mice were sacrificed and liver and lung tissues were

processed for histological examination using hematoxylin and eosin staining. Haematoxylin (purple)

stains nucleic acids (cell nucleus), while eosin (pink) stains protein non-specifically (cell cytoplasm and

extracellular matrix). Liver images were assessed for capsular inflammation and lobular necrotic

damage using modified Knodell Histology Activity Index (HAI) of blinded scoring system of liver injury

(271, 272), while lung sections were assessed for leukocyte infiltration and alveolar wall thickening

74

using blinded lung injury scoring system suggested by American Thoracic Society guidelines (273).

Representative liver (A) and lung (B) sections show capsular inflammation and focal necrosis in the

liver and leukocyte infiltration and alveolar thickening in the lungs as a result of CLP-induced sepsis in

WT mice. CSE KO mice showed less capsular inflammation and focal necrosis in liver and reduced

leukocyte infiltration and alveolar thickening in lungs following CLP-induced sepsis. Data represent

mean ± S.E.M. (n=8). The significance of differences between groups was evaluated by non-parametric

Kruskal–Wallis test. Statistical significance was assigned as *P<0.05 and **P<0.01.

3.4.5 Alteration of homocysteine levels in WT and CSE KO mice following CLP-induced sepsis

Figure 3.5 Alteration of homocysteine levels in WT and CSE KO mice following CLP-induced sepsis.

WT and CSE KO mice underwent CLP or sham operation. Sham operated mice acted as controls. Eight

hours after CLP or sham operation, mice were sacrificed and liver and lung tissues and plasma were

processed to measure homocysteine levels in the liver (A), lungs (B) and plasma (C) using HILIC-MS/MS.

WT mice with CLP-induced sepsis showed lower liver and plasma levels of the homocysteine compared

to sham control group. Mice with CSE gene deletion showed significantly lower homocysteine levels in

the liver and plasma compared to WT mice, which were further lowered in sepsis mice compared to

the sham operated group in CSE KO mice. There was no significant difference in lung homocysteine

levels between sepsis and sham groups of both WT and CSE KO mice and between WT and CSE KO

mice. Data represent mean ± S.E.M. (n=8). The significance of differences among groups was evaluated

by ANOVA with post-hoc Tukey’s test. Statistical significance was assigned as *P<0.05, **P<0.01 and

****P<0.0001.

75

WT mice with CLP-induced sepsis showed lower liver and plasma levels of homocysteine

compared to the sham control group (liver: 0.58 ± 0.09 vs 0.85 ± 0.10; plasma: 0.33 ± 0.05 vs

0.79 ± 0.09; P<0.0001). Mice with CSE gene deletion showed significantly lower homocysteine

levels in the liver and plasma compared to WT mice, which were further lowered in sepsis

mice compared to sham group in CSE KO mice. There was no significant difference in lung

homocysteine levels between sepsis and sham operated groups of both WT and CSE KO mice

and between WT and CSE KO mice (Figure 3.5).

3.5 Discussion

The present study demonstrated an important role of the H2S-synthesising enzyme CSE on

leukocyte infiltration and liver and lung injury following CLP-induced sepsis. Mice with CLP-

induced sepsis showed increased liver and lung injury that could be characterised both

biochemically (leukocyte infiltration, as evidenced by MPO activity) and histologically

(hematoxylin and eosin stained sections, evidenced by oedema and necrosis). Increased CSE

protein expression, H2S-synthesising activity and H2S levels are correlated with MPO activity

and liver and lung injury in CLP-induced sepsis. The results in the CSE KO mice showed less

H2S-synthesising activity and H2S levels, decreased MPO activity, and liver and lung damage

following CLP-induced sepsis. These results confirm the previous studies on endogenous H2S

synthesis through CSE on organ injury and inflammation in sepsis (43, 44, 174, 177, 277). For

example, prophylactic and therapeutic administration of PAG protected mice against sepsis-

induced inflammation characterised by a decrease in liver and lung MPO activity (44, 140,

177). Of even greater significance, PAG pre-treatment and post-treatment improved the

sepsis-associated multiple organ damage, including liver and lung injury (177). A recent study

has used siRNA to silence CSE gene protected mice against sepsis-induced leukocyte

76

infiltration and liver and lung damage (126). Together, these results suggest that the CSE-

derived H2S regulates leukocyte infiltration and liver and lung injury following CLP-induced

sepsis.

The present study also demonstrated changes in homocysteine levels following CLP-induced

sepsis. Interestingly, results showed decreased levels of homocysteine in the liver and plasma

following CLP-induced sepsis in mice. These results contradict previously published research

on the role of homocysteine during inflammatory conditions such as glomerulosclerosis (296),

atherosclerosis (289), rheumatoid arthritis (284) and sepsis (294). For example, it has been

reported that increased total homocysteine concentrations are observed in patients with

sepsis (294). In contrast, another study has shown that homocysteine levels are not

significantly altered in mechanically ventilated patients with severe sepsis (293). In the

present study, the decreased levels of homocysteine in the liver and plasma are most likely

due to increased CSE expression following CLP-induced sepsis. This notion is supported by

previous research where elevated homocysteine levels were inhibited by increased CSE

expression (292, 303, 304). However, lung homocysteine levels were not altered despite

increased lung CSE protein expression following CLP-induced sepsis. The mechanisms

involving differential effects of CLP-induced sepsis on homocysteine levels in the lungs, liver

and plasma therefore remain unclear.

An altered homocysteine interaction with the CSE-H2S synthesis transsulfuration pathway in

mice deficient in the CSE gene following CLP-induced sepsis has also been demonstrated in

the present study. Liver and circulatory homocysteine levels were significantly lower in mice

deficient in the CSE gene compared to WT mice both with sepsis and after the sham

operation. Furthermore, CSE KO sepsis mice showed significantly lower liver and circulatory

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homocysteine levels compared to CSE KO sham control mice. These results suggest that

homocysteine may undergo metabolism via alternative enzymatic pathways such as

methionine synthase (MTR) and betaine-homocysteine methyltransferase (BHMT) in mice

deficient in the CSE gene (307). In addition, the decrease in homocysteine levels following

sepsis in the CSE KO mice may be due to a decrease in the activities of the alternative enzymes

(MTR and BHMT) involved in the homocysteine metabolism (307). In contrast, CSE gene

deletion has no effect on lung homocysteine levels. The mechanisms involving differential

regulation of homocysteine levels in different organs in mice deficient in the CSE gene

following CLP-induced sepsis remain unknown.

CLP-induced sepsis is, of course, multifactorial and numerous mediators other than CSE-

derived H2S are involved. CSE gene deletion partly reversed the pathological progression of

sepsis. Based on the results, the present study suggests that CSE-derived H2S is one of the

pivotal factors in determining the severity of organ damage in sepsis. At the same time, some

limitations of the current study need mention. This study is limited to only one time point (8

h post-CLP) and represents changes that occur at that time point only. In addition, the

mechanisms by which sepsis upregulates CSE expression and subsequent H2S synthesis were

not elucidated. A recent study has demonstrated the potential roles of microRNA-21 (miR-21)

in specificity protein-1 (SP1) mediated regulation of CSE gene expression in smooth muscle

cells (308, 309). Whether sepsis modifies these elements in the CSE promoter region and

therefore raise the expression of CSE is yet to be investigated. Furthermore, this study has

not investigated CLP sepsis- and CSE gene deletion-induced discrepancies in the regulation of

homocysteine in different organs.

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In conclusion, CSE-derived H2S plays a role in the regulation of leukocyte infiltration into

tissues and liver and lung injury following CLP-induced sepsis. Although homocysteine is a

precursor for H2S synthesis through the CSE transsulfuration pathway, it does not play any

role in leukocyte infiltration or organ damage associated with sepsis. The present study

contributes to our understanding of the role of CSE-derived H2S in the regulation of leukocyte

infiltration and liver and lung injury following CLP-induced sepsis.

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Chapter 4

Effect of CSE gene deletion on proinflammatory cytokines, and chemokines and

the ERK1/2-NF-B p65 pathway following caecal-ligation and puncture-induced

sepsis in mice

4.1 Introduction

Cystathionine--lyase (CSE) is the predominant H2S-synthesising enzyme with significant

biological functions (279). Upregulation of CSE expression and associated H2S synthesis has

been demonstrated in various inflammatory diseases including acute pancreatitis (129, 131,

132, 149), inflammatory bowel disease (133), arthritis (270, 310, 311), airway inflammation

(312), hind-paw oedema (137, 174), burn injuries (313, 314), endotoxaemia (127, 172),

haemorrhagic shock (282) and CLP-induced sepsis (43, 44, 126, 140, 177, 315). Increased CSE

expression and H2S synthesis have also been reported in immune response cells such as

macrophages when stimulated with LPS (316-318). Previous research indicates that CSE-

derived H2S contributes to the inflammatory response and regulates the severity of multiple

organ injury following endotoxaemia and sepsis. For example, it has been shown that

endotoxaemia and sepsis are associated with increased CSE expression and activity and in

turn H2S overproduction (43, 44, 127, 172, 177). Intervention studies using PAG have shown

this compound to exhibit anti-inflammatory activity and protect mice against sepsis-

associated multiple organ injury, supporting a role for H2S in sepsis (140, 177).

In an experimental animal model of sepsis and human sepsis, a great number of bacterial

compounds initiate excessive production and release of proinflammatory cytokines (TNF-α,

IL-6 and IL-1β) and chemokines (MCP-1 and MIP-2α) (319-321). Elevated levels of

proinflammatory cytokines and chemokines correlate with increased CSE expression, as well

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as activity and H2S synthesis in CLP-induced sepsis and LPS-induced endotoxaemia (43, 126,

173). Hence, it is necessary to understand the possible mechanisms through which CSE or

CSE-derived H2S modulate the development of inflammation and affect multiple organ injury

in CLP-induced sepsis.

Proinflammatory mediators particularly TNF-α activate various MAPK signalling networks,

thereby regulating NF-B activation and subsequent transcriptional regulation of

proinflammatory genes in many inflammatory diseases (55, 322). Notably, the activation of

ERK has been shown to be an essential temporal regulator of NF-B activity (323). ERK1/2

induces NF-B activation by stimulating ERK1/2 downstream MAPK-activated protein kinases

(MKs) such as ribosomal S6 kinase-1 and mitogen- and stress-activated protein kinase-1 (324,

325). Early research investigated the possible interactions between CSE or CSE-derived H2S

and ERK1/2-NF-B signalling in various in vitro and in vivo inflammatory disease models. For

example, H2S has been shown to mediate the activation of ERK1/2 when RAW264.7

macrophages are stimulated with LPS (316). In human macrophages, H2S was found to amplify

the phosphorylation of ERK1/2 and subsequent activation of NF-B, proinflammatory

cytokines and chemokines (326). Recent studies in mice have shown that CSE gene deletion

or silencing of the CSE gene with siRNA inhibits activation of NF-B and ERK1/2-NF-B in

caerulein-induced acute pancreatitis and LPS-stimulated macrophages, respectively (129,

317). Similarly, the role of endogenous H2S in mediating inflammation is supported by results

that show the inhibition of CSE using the inhibitor PAG in CLP-induced sepsis in mice.

Treatment with PAG attenuates activation of ERK1/2 and NF-B p65 following CLP-induced

sepsis (43, 44); however, these studies are limited due to a lack of specific evidence of CSE

enzyme inhibition by PAG to target H2S biosynthesis (264-266). A recent study using siRNA to

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silence the CSE gene found mice were protected against sepsis-induced liver and lung injury

as a result of a decrease in proinflammatory cytokines and chemokines (126). However, this

study did not investigate the mechanism through which CSE or CSE-derived H2S mediates the

decrease in proinflammatory mediators of inflammation and organ injury in sepsis.


proinflammatory cytokines and chemokines through a mechanism involving ERK1/2-NF-B

p65 signalling in CLP-induced sepsis in mice.

4.2 Aims

Chapter 3 has shown that deletion of the CSE gene protects mice against leukocyte infiltration

and liver and lung injury following CLP-induced sepsis. The present study aimed to determine

the effect of CSE gene deletion on the activation of ERK1/2 (p-ERK1/2) and NF-B p65

translocation and the subsequent effect on proinflammatory cytokine (TNF-α, IL-6 and IL-1β)

and chemokine (MCP-1 and MIP-2α) generation following CLP-induced sepsis in mice.




experimental (WT Sepsis and CSE KO Sepsis) groups with eight mice in each group (n=8).

Sepsis was induced by CLP as described in Section 2.4 and 8 h after CLP or sham operation the


aspirated and plasma separated and stored at -80°C. Samples of liver and lungs were

processed and stored at -80°C. Subsequently, p-ERK1/2 and ERK1/2 protein expression

(Section 2.5), NF-B p65 activation (Section 2.11), and cytokine and chemokine levels (Section

82

2.12) were measured by the methods described in the Materials and Methods of Chapter 2.

Statistical analysis was performed as described in Section 2.17.

4.4 Results

4.4.1 Phosphorylation of ERK1/2 in WT and CSE KO mice following CLP-induced sepsis

The western blot showed that CLP-induced sepsis significantly increased the phosphorylation

of ERK1/2 in the liver and lungs compared to the sham control in WT mice (liver: p-ERK1, 2.10

± 0.34-fold increase (P<0.01), p-ERK2, 2.37 ± 0.42-fold increase (P<0.05); lung: p-ERK1, 3.17 ±

0.64-fold increase (P<0.001), p-ERK2, 2.58 ± 0.59-fold increase (P<0.01)). Mice with CSE gene

deletion showed lower ERK1/2 phosphorylation in the liver and lungs following sepsis

compared to their WT counterparts (liver: p-ERK1, 1.00 ± 0.00-fold less (P<0.01), p-ERK2, 0.79

± 0.21-fold less (P<0.05); lung: p-ERK1, 1.83 ± 0.12-fold less (P<0.05), p-ERK2, 1.37 ± 0.13-fold

less (P<0.01)) and there was no significant difference between sham and CLP-induced sepsis

in CSE KO mice (Figure 4.1).

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Figure 4.1 Phosphorylation of ERK1/2 in WT and CSE KO mice following CLP-induced sepsis. WT and

CSE KO mice underwent CLP or sham operation. Sham operated mice acted as controls. Eight hours

after CLP or sham operation mice were sacrificed and liver and lung tissues were processed to measure

phospho-ERK1/2 (p-ERK1/2) and total ERK1/2 protein expression in the liver (A and B) and lungs (C and

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D) using western blotting. After analysis by densitometry, the data were expressed as the ratio of p-

ERK1/2 to total ERK1/2 (plotted as the relative-fold increase of p-ERK1/2 expression over the sham

control). For western blot results, each lane represents a separate animal. The blots shown are

representative of all animals in each group with similar results. There was a significant increase in

phosphorylation of ERK1/2 in the liver and lungs following CLP-induced sepsis compared to sham

control in WT mice. CSE KO mice showed significantly lower phosphorylation of ERK1/2 in the liver and

lungs compared to WT mice following CLP-induced sepsis. Data represent mean ± S.E.M. (n=8). The

significance of differences among groups was evaluated by ANOVA with post-hoc Tukey’s test.

Statistical significance was assigned as *P<0.05, **P<0.01 and ***P<0.001.

4.4.2 Effect of CSE gene deletion on liver and lungs NF-B p65 activation following CLP-

induced sepsis

Figure 4.2 Activation of NF-B p65 in WT and CSE KO mice following CLP-induced sepsis. WT and CSE

KO mice underwent CLP or sham operation. Sham operated mice acted as controls. Eight hours after

CLP or sham operation, mice were sacrificed and liver and lung tissues were processed to measure NF-

B p65 activity in the nuclear extracts of liver and lungs using an NF-B p65 immuno assay. CLP-

induced sepsis resulted in a significant increase in NF-B p65 activity compared to sham control in WT

mice. CSE KO mice showed significantly lower NF-B p65 activation compared to WT mice following

CLP-induced sepsis. Data represent mean ± S.E.M. (n=8). The significance of differences among groups

was evaluated by ANOVA with post-hoc Tukey’s test. Statistical significance was assigned as **P<0.01

and ***P<0.001.

As expected, the activity of NF-B p65 in the nuclear extracts of liver and lung tissues

increased significantly after CLP-induced sepsis compared to sham control in WT mice (liver:

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2.08 ± 0.75-fold increase (P<0.001); lung: 2.18 ± 0.59-fold increase (P<0.01)). CSE KO mice

with CLP operation showed significantly lower NF-B p65 activation in comparison to their

respective WT mice (liver: 0.92 ± 0.14-fold less (P<0.001); lung: 1.02 ± 0.25-fold less (P<0.01)).

There was no significant difference between sham and CLP-induced sepsis CSE KO mice

(Figure 4.2).

4.4.3 Effect of CSE gene deletion on liver proinflammatory mediators following CLP-induced

sepsis

The elevated production of proinflammatory mediators is a feature of sepsis. WT mice with

CLP-induced sepsis showed significantly higher levels of cytokines (TNF-α, IL-6 and Il-1β) and

chemokines (MCP-1 and MIP-2α) in liver samples compared to mice with the sham operation

(TNF-α: 7.11 ± 2.50 ng/mg vs 4.72 ± 0.93 ng/mg (P<0.05); IL-6: 0.61 ± 0.25 ng/mg vs 0.35 ±

0.07 ng/mg (P<0.05); IL-1β: 0.82 ± 0.22 ng/mg vs 0.3 ± 0.07 ng/mg (P<0.0001); MCP-1: 550.51

± 129.70 ng/mg vs 274.35 ± 36.67 ng/mg (P<0.0001); and MIP-2α: 1.73 ± 0.45 ng/mg vs 0.82

± 0.05 ng/mg (P<0.0001)). IL-1β and MCP-1 levels in CSE KO mice were significantly elevated

following CLP-induced sepsis; however, the extent of these increases was significantly lower

in the CSE KO mice compared to WT mice (TNF-α: 3.57 ± 0.76 ng/mg vs 7.11 ± 2.50 ng/mg

(P<0.01); IL-6: 0.27 ± 0.07 ng/mg vs 0.61 ± 0.25 ng/mg (P<0.01); IL-1β: 0.61 ± 0.12 ng/mg vs

0.82 ± 0.22 ng/mg (P<0.05); MCP-1: 347.47 ± 106.29 ng/mg vs 550.51 ± 129.70 ng/mg

(P<0.01); and MIP-2α: 0.93 ± 0.37 ng/mg vs 1.73 ± 0.45 ng/mg (P<0.001)) (Figure 4.3).

86

Figure 4.3 Alteration of liver cytokine and chemokine levels in WT and CSE KO mice following CLP-


controls. Eight hours after CLP or sham operation, mice were sacrificed and liver tissue was processed

to measure cytokine (TNF-α (A), IL-6 (B) and (IL-1β (C)) and chemokine (MCP-1 (D) and MIP-2α (E))

levels using ELISA. CLP-induced sepsis resulted in a significant increase in liver proinflammatory

cytokine and chemokine levels. There was a significant reduction in all these mediators in CSE KO mice

compared to WT mice following CLP-induced sepsis. Data represent mean ± S.E.M. (n=8). The


Statistical significance was assigned as *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.

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4.4.4 Effect of CSE gene deletion on lungs proinflammatory mediators following CLP-

induced sepsis

Substantial elevations in lungs cytokine (TNF-α, IL-6 and IL-1β) and chemokine (MCP-1 and

MIP-2α) concentrations were seen following CLP-induced sepsis animals compared to sham

operation in WT mice (TNF-α: 1.70 ± 0.51 ng/mg vs 1.10 ± 0.28 ng/mg (P<0.05); IL-6: 0.72 ±

0.15 ng/mg vs 0.37 ± 0.15 ng/mg (P<0.001); IL-1β: 0.95 ± 0.22 ng/mg vs 0.23 ± 0.10 ng/mg

(P<0.001); MCP-1: 1880.37 ± 937.36 ng/mg vs 622.6 5± 202.17 ng/mg (P<0.001); and MIP-2α:

1.37 ± 0.40 ng/mg vs 0.77 ± 0.35 ng/mg (P<0.01)). Mice with CLP sepsis and CSE gene deletion

showed significantly lower levels of these cytokines and chemokines compared to WT mice

with CLP sepsis (TNF-α: 1.00 ± 0.24 ng/mg vs 1.70 ± 0.51 ng/mg (P<0.01); IL-6: 0.50 ± 0.12

ng/mg vs 0.72 ± 0.15 ng/mg (P<0.05); IL-1β: 0.65 ± 0.16 ng/mg vs 0.95 ± 0.22 ng/mg (P<0.05);

MCP-1: 919.17 ± 352.87 ng/mg vs 1880.37 ± 937.36 ng/mg (P<0.05) and MIP-2α: 0.90 ± 0.36

ng/mg vs 1.37 ± 0.40 ng/mg (P<0.05)). However, levels of IL-6 and IL-1β in CSE KO mice with

CLP-induced sepsis were significantly higher compared to KO mice that had undergone sham

operation (Figure 4.4).

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Figure 4.4 Alteration of lungs cytokine and chemokine levels in WT and CSE KO mice following CLP-


controls. Eight hours after CLP or sham operation, mice were sacrificed and lung tissues were processed

to measure cytokine (TNF-α (A), IL-6 (B) and (IL-1β (C)) and chemokine (MCP-1 (D) and MIP-2α (E))

levels using ELISA. CLP-induced sepsis resulted in a significant increase in lung proinflammatory

cytokine and chemokine levels. There was a significant reduction in all these mediators in CSE KO mice

compared to WT mice following CLP-induced sepsis. Data represent mean ± S.E.M. (n=8). The


Statistical significance was assigned as *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.

89

4.4.5 Effect of CSE gene deletion on circulatory proinflammatory mediators following CLP-

induced sepsis

WT mice with CLP-induced sepsis showed significantly increased circulatory proinflammatory

cytokine (TNF-α, IL-6 and IL-1β) and chemokine (MCP-1 and MIP-2α) levels compared with

sham control animals (TNF-α: 0.17 ± 0.04 ng/mL vs 0.10 ± 0.02 ng/mL (P<0.01); IL-6: 0.07 ±

0.03 ng/mL vs 0.01 ± 0.006 ng/mL (P<0.0001); IL-1β: 0.15 ± 0.03 ng/mL vs 0.048 ± 0.041 ng/mL

(P<0.0001); MCP-1: 917.52 ± 203.20 ng/mL vs 210.72 ± 57.84 ng/mL (P<0.0001); and MIP-2α:

0.13 ± 0.06 ng/mL vs 0.03 ± 0.01 ng/mL (P<0.0001)). Mice deficient in the CSE gene with CLP-

induced sepsis also showed significantly higher levels of cytokines and chemokines, with the

exception of TNF-α and MIP-2α, which were not significantly elevated. However, the extent

of these increases was significantly lower in the CSE KO mice compared to WT mice following

CLP-induced sepsis (TNF-α: 0.10 ± 0.02 ng/mL vs 0.17 ± 0.04 ng/mL (P<0.01); IL-6: 0.03 ± 0.01

ng/mL vs 0.07 ± 0.03 ng/mL (P<0.001); IL-1β: 0.09 ± 0.04 ng/mL vs 0.15 ± 0.03 ng/mL (P<0.01);

MCP-1 639.23 ± 224.03 ng/mL vs : 917.52 ± 203.20 ng/mL (P<0.01); and MIP-2α: 0.06 ± 0.01

vs 0.13 ± 0.06 ng/mL (P<0.01)) (Figure 4.5).

90

Figure 4.5 Alteration of plasma cytokine and chemokine levels in WT and CSE KO mice following CLP-


controls. Eight hours after CLP or sham operation, mice were sacrificed and plasma was harvested to

measure cytokine (TNF-α (A), IL-6 (B) and (IL-1β (C)) and chemokine (MCP-1 (D) and MIP-2α (E)) levels

using ELISA. CLP-induced sepsis resulted in a significant increase in plasma proinflammatory cytokine

and chemokine levels. There was a significant reduction in all these mediators in CSE KO mice compared

to WT mice following CLP-induced sepsis. Data represent mean ± S.E.M. (n=8). The significance of


was assigned as **P<0.01, ***P<0.001 and ****P<0.0001.

91

4.5 Discussion

The study described here in Chapter 3 has provided convincing evidence that CSE-derived H2S

regulates leukocyte infiltration into the tissues and subsequently liver and lung injury

following CLP-induced sepsis in mice. The results of the present study demonstrate that mice

deficient in the CSE gene have decreased proinflammatory cytokine and chemokine levels

following CLP-induced sepsis. Further, the results in CSE KO mice show that CSE gene deletion

not only decreased phosphorylation of ERK1/2 but also inhibited NF-B p65 DNA binding

activity in the liver and lungs following CLP-induced sepsis. As a result, proinflammatory

cytokine and chemokine levels are decreased in the liver, lungs and circulatory system. There

is little published information on the importance of the H2S in inflammation and the

mechanism by which CSE-derived H2S alters the inflammatory response in sepsis using CSE

inhibitor PAG (43, 44, 140, 174, 177). For example, it has been shown that inhibition of CSE

with PAG significantly decreases ERK1/2 phosphorylation, NF-B activation and subsequent

cytokine (TNF-α, IL-6 and IL-1β) and chemokine (MCP-1 and MIP-2α) production in the liver

and lungs (43, 44). However, the use of PAG for investigating the role of H2S in sepsis has

limitations, due to its other, non-specific actions such as alteration of homocysteine

metabolism and inhibition of ALT and AST enzyme activities (264-266). However, despite the

non-specific actions of PAG, the results of previous studies are in agreement with the present

study using CSE KO mice. These results together suggest that a temporal increase in CSE

protein expression and subsequent H2S synthesis during sepsis correlates with the occurrence

of phosphorylation of ERK1/2 and activation of NF-B p65 and subsequently regulates

generation of the proinflammatory cytokines and chemokines.

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It is important to note that CSE gene deletion did not completely inhibit ERK1/2

phosphorylation, NF-B p65 activation or proinflammatory mediator production, which

suggests that mediators other than CSE-derived H2S are involved in the activation of the

ERK1/2-NF-B p65 pathway in CLP-induced sepsis. At the same time, some limitations of the

current study need mention. This study is limited to one time point (8 h post-CLP) and

represents changes that occur at that time point only. In addition, although the present study

indicates that CSE-derived H2S regulates the inflammatory response via the ERK1/2-NF-B

p65 signalling cascade, it is difficult to establish from the present experimental data how CSE-

derived H2S directly or indirectly regulates NF-B p65 activation. A recent study found that

H2S activates NF-B by sulfhydrating the cysteine residues or that other sites of the p65

subunit of NF-B may elicit the transcriptional activation of proinflammatory genes (327);

however, this has not been investigated in the present study. Furthermore, H2S may also

induce NF-B activation through mechanisms other than ERK1/2 phosphorylation. For

example, it has been shown that H2S induces a dose-dependent increase in intracellular

calcium ions (Ca+2) and protein kinase A (PKA), which facilitate NF-B translocation into the

nucleus where it binds to DNA in microglial cells (114). These mechanisms are yet to be

investigated in CLP-induced sepsis.

In summary, the results of the present study demonstrated that the CSE-derived H2S regulates

production and release of proinflammatory mediators and exacerbates the systemic

inflammatory response in sepsis through a mechanism involving NF-B p65 via increased

activation or phosphorylation of ERK1/2. The present study contributes to our understanding

of the precise mechanism underlying the proinflammatory role of H2S in CLP-induced sepsis.

93

Chapter 5

Effect of CSE gene deletion on substance P and neurokinin 1 receptor following

caecal-ligation and puncture-induced sepsis in mice

5.1 Introduction

Substance P (SP) is an 11 amino acid immunoregulatory peptide, a member of the tachykinin

family, encoded by the preprotachykinin A (PPTA) gene (199, 328). Although SP is a peptide

primarily of neuronal origin, animal studies in rodents have demonstrated its production in

non-neuronal tissues and inflammatory cells (329). The biological actions of SP are primarily

mediated through NK-1R, a G-protein coupled receptor (198). SP mediated-activation of NK-

1R on effector cells elicits local vasodilation and increases endothelial microvascular

permeability and plasma extravasation, thereby promoting local inflow of inflammatory and

immune cells (195). SP has also been implicated in inducing the release of proinflammatory

mediators and stimulating the chemotaxis of lymphocytes, monocytes and neutrophils (197).

By promoting vasodilation, extravasation, leukocyte chemotaxis and cytokine release, SP acts

as an important mediator of inflammation in many inflammatory diseases, including

inflammatory bowel disease (330), airway inflammation, arthritis (331, 332), acute

pancreatitis (131, 333), sepsis (199, 203), endotoxaemia (200, 334) and burn injuries (335).

Various preclinical and clinical studies have studied the importance of SP in sepsis. In one

study, circulatory levels of SP were reported to decrease in patients with sepsis and septic

shock (202). In contrast, other clinical studies have shown that high serum levels of SP are

related to the lethal outcome of sepsis and that these increased SP levels act as a predictive

indicator of mortality during the late course of sepsis and septic shock (201, 203). Previous

studies have shown the proinflammatory role of SP in sepsis and endotoxaemia in rodents.

94

For example, CLP-induced sepsis is associated with an increase in plasma and pulmonary

levels of SP and genetic deletion of the PPTA has been shown to convey significant protection

against inflammation, organ injury and mortality in mice, improving survival rate (197, 199).

In addition to PPTA gene deletion, treatment with the NK-1R antagonist modulates the lung

injury and inflammatory response in mice against CLP-induced sepsis (195, 198). Similarly,

LPS-induced neutrophil adherence in vitro (334) and endotoxaemia-induced inflammation

and multiple organ injury in mice (200) are found to stimulate the release of SP, while PPTA

gene deletion protected mice against inflammation and organ damage (200). These studies

suggest that SP plays an important role in sepsis and associated organ injury. Although

elevated SP levels have been found in sepsis and associated organ injury, to date the

underlying mechanisms by which the release and production of SP are regulated remain

unclear.

Several in vitro and in vivo studies have suggested that H2S is involved in regulating the release

of SP, leading to the development and exacerbation of inflammation in sepsis. For example,

pulmonary defense against inhaled H2S gas was modified by pretreatment with capsaicin,

which is known to deplete SP local sensory nerves (336). In isolated rat urinary bladders, H2S

has been reported to stimulate the release of tachykinins and in turn activate NK-1R and NK-

2R through capsaicin-sensitive afferent neurons (337, 338). Similarly, in another study H2S

provoked the tachykinin-mediated neurogenic inflammatory response in guinea pig airways

(339). However, these studies investigated the role and physiological significance of H2S in the

lungs or isolated tissues using either H2S donor NaHS or H2S gas, both of which are unrelated

to the inflammatory role in sepsis.

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Previous studies have demonstrated that H2S donor NaHS increases the release of SP and that

in turn, SP promotes inflammation and lung injury through activation of NK-1R in mice (204).

In animal models of sepsis, it has been shown that H2S upregulates the generation of SP, which

contributes to lung inflammation and injury via activation of the NK-1R. This provides some

evidence for the role of SP in H2S-induced lung inflammation (141). In addition, H2S regulates

systemic inflammation and multiple organ failure via transient receptor potential vanilloid

type 1 (TRPV1)-mediated enhancement of SP production and activation of the ERK1/2-NF-B

pathway in CLP-induced sepsis (205, 206). However, these previous studies used CSE inhibitor

PAG or H2S donors such as NaHS and Na2S to investigate the interaction between CSE-derived

H2S and SP production in sepsis; their limitations in investigating the role of H2S in sepsis have

been discussed in Chapter 1 (Section 1.6).


SP and NK-1R in CLP-induced sepsis in mice.

5.2 Aims

The results in Chapter 4 have shown that deletion of the CSE gene protects mice against

increased inflammatory response through activated ERK1/2-NF-B p65 signalling following

CLP-induced sepsis. The present study aimed to determine the effect of CSE gene deletion on

SP production and NK-1R protein expression following CLP-induced sepsis.




experimental (WT Sepsis and CSE KO Sepsis) groups, with eight mice in each group (n=8).

96

Sepsis was induced by CLP as described in Section 2.4 and 8 h after CLP or sham operation


aspirated, plasma separated and stored at -80°C. Samples of liver and lungs were processed

and stored at -80°C. Subsequently, SP levels (Section 2.13) and NK-1R protein expression

(Section 2.5) were measured by the methods described in the Materials and Methods of

Chapter 2. Statistical analysis was performed as described in Section 2.17.

5.4 Results

5.4.1 CSE gene deletion affects SP levels following CLP-induced sepsis

Induction of sepsis by CLP in WT mice resulted in a significant increase in the liver, lungs and

plasma levels of SP compared to the sham control (liver: 0.91 ± 0.14 ng/mg vs 0.18 ± 0.01

ng/mg (P<0.001); lungs: 0.68 ± 0.17 ng/mg vs 0.21 ± 0.01 ng/mg (P<0.01); plasma: 0.72 ± 0.11

ng/mL vs 0.21 ± 0.02 ng/mL (P<0.0001)). Next, the effect of CSE gene deletion on the levels

of SP following sepsis was examined. As shown in Figure 5.1, mice lacking the CSE gene had

significantly lower liver, lungs and plasma levels of SP compared to WT mice following

induction of CLP sepsis (liver: 0.91 ± 0.14 ng/mg vs 0.46 ± 0.10 ng/mg; lungs: 0.68 ± 0.17 ng/mg

vs 0.29 ± 0.03 ng/mg; plasma: 0.72 ± 0.11 ng/mL vs 0.44 ± 0.04 ng/mL) (P<0.05) (Figure 5.1).

97

Figure 5.1 SP levels in WT and CSE KO mice following CLP-induced sepsis. WT and CSE KO mice

underwent CLP or sham operation. Sham operated mice served as controls. 8 h after CLP or sham

operation, mice were sacrificed and SP levels in liver (A), lungs (B) and plasma (C) were measured using

a SP immunoassay. SP levels were significantly increased in WT mice following CLP-induced sepsis

compared to sham control. CSE KO mice had significantly lower SP levels compared to WT mice

following CLP-induced sepsis. Data represent mean ± S.E.M. (n=8). The significance of differences

among groups was evaluated by ANOVA with post-hoc Tukey’s test. Statistical significance was

assigned as *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.

5.4.2 Effect of CSE gene deletion on NK-1R protein expression following CLP-induced sepsis

Protein expression levels of NK-1R in the liver and lungs were investigated using western blot.

Densitometry analysis of western blots showed that liver and lungs NK-1R protein expression

increased significantly following CLP-induced sepsis compared to sham control in WT mice

98

99

Figure 5.2. NK-1R protein expression in WT and CSE KO mice following CLP-induced sepsis. WT and

CSE KO mice underwent CLP or sham operation. Sham operated mice served as controls. 8 h after CLP

or sham operation, mice were sacrificed. The NK-1R protein expression in liver (A and B) and lungs (C

and D) were measured by western blot. After analysis by densitometry, the data were expressed as

ratios of NK-1R to GAPDH (plotted as the relative fold increases of NK-1R expression over the sham

control). For western blot results, each lane represents a separate animal. The blots shown are

representative of all animals in each group. There was a significant increase in NK-1R expression in the

liver and lungs of WT mice following CLP-induced sepsis compared to sham control. CSE KO mice

showed significantly decreased NK-1R protein expression in the liver and lungs compared to WT mice

following CLP-induced sepsis. Data represent mean ± S.E.M. (n=8). The significance of differences

among groups was evaluated by ANOVA with post-hoc Tukey’s test. Statistical significance was

assigned as *P<0.05.

(liver: 1.40 ± 0.01-fold increase (P<0.05); lungs: 8.68 ± 1.97-fold increase (P<0.05)). CSE KO

mice showed significantly decreased NK-1R protein expression following CLP sepsis compared

to the WT sepsis mice (liver: 1.11 ± 0.01-fold decrease (P<0.05); lungs: 0.93 ± 0.26-fold

decrease (P<0.05)) (Figure 5.2).

5.5 Discussion

Previous studies in Chapter 3 and 4 showed the importance of CSE-derived H2S on organ injury

and the inflammatory response following CLP-induced sepsis. In this chapter, the role of

endogenous CSE-derived H2S on the upregulation of SP levels and NK-1R expression following

CLP-induced sepsis is described. SP has been proposed as a key mediator of inflammation in

sepsis and endotoxaemia and associated organ injury (195, 197-199, 201, 334). In the present

study, a significant increase in plasma and tissue (liver and lung) SP levels and subsequent NK-

1R expression (in liver and lungs) following CLP-induced sepsis was shown. Gene deletion of

the CSE enzyme attenuated the observed increase in levels of SP and NK-1R protein

expression in sepsis compared to their WT counterparts, suggesting that H2S synthesised

through CSE-facilitated the production of SP production and subsequent NK-1R activation in

CLP-induced sepsis. The findings of the present chapter are consistent with earlier

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observations with pharmacological inhibitor of CSE such as PAG (141, 205, 206) and H2S donor

such as NaHS (204); these results reinforce the essential role of CSE-derived H2S on SP-

mediated inflammation and organ injury in sepsis. Of course, CLP-induced sepsis is

multifactorial and numerous mediators other than CSE-derived H2S are involved. Deletion of

the CSE gene moderately decreased SP levels and subsequently cytokines and chemokines in

sepsis (as shown in Chapter 4).

Endogenous H2S appears to play an important role in regulating the severity of inflammation

and organ damage in sepsis (43, 44, 140). Thus, it is of interest to determine whether

endogenous H2S synthesised through CSE is correlated to SP in sepsis. The H2S donor NaHS

induced the contractile response by provoking the release of SP both in rat gall bladders and

isolated guinea pig airways and this was totally suppressed with capsaicin or a combination

of NK-1R and NK-2R antagonists (337-339). Administration of NaHS to normal mice caused a

significant rise in circulatory levels of SP in a dose-dependent manner (204). Furthermore,

other studies have previously shown that inhibiting CSE using PAG pre- or post-treatment

significantly decreased the level of PPTA gene expression and SP in lungs, whereas exogenous

H2S (NaHS) magnified the pulmonary levels of SP in CLP-induced sepsis (141, 205, 206). On

the other hand, CSE activity and H2S synthesis in mice genetically deficient in the PPTA gene

were similar to WT mice in sepsis, indicating that the absence of SP has no effect on the level

of H2S (141). The experimental results in this chapter consistently show that CSE gene deletion

significantly decreases levels of SP in the liver, lungs and plasma of CLP-induced sepsis mice.

This indicates that CSE-derived H2S is located upstream of SP and plays an important role in

regulating the production and release of SP in sepsis. In addition, the experiments in this

chapter tested the relationship between CSE and NK-1R, which also increased following

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sepsis. Mice deficient in the CSE gene showed significantly decreased NK-1R protein

expression in the liver and lungs. This indicates that CSE-derived H2S stimulates liver and lung

inflammation via SP is mainly mediated by NK-1R.

The experimental findings of the present study indicate that the increased CSE-mediated H2S

synthesis (shown in Chapter 3) in sepsis upregulates SP production. Although the present

study suggests that CSE-derived H2S modulates the production of SP and activation of NK-1R,

the mechanism in this process remains unclear. Previous studies have suggested that H2S may

regulate the release of SP in guinea pig airways and rat urinary bladders via TRPV1 on sensory

nerve endings (337-339). In addition, inhibition of sensory nerves with capsaicin or

pretreatment with capsazepine (a TRPV1 antagonist) protected mice from H2S-induced lung

inflammation (205). Similarly, previous studies have also shown that the depletion of SP by

genetically removing the PPTA gene and blocking the effect of SP by pretreatment with the

NK-1R inhibitor not only alleviates lung damage caused by sepsis but also decreases lung

microvascular permeability impaired by exogenous H2S (195, 198, 199). The precise

mechanism by which CSE-derived H2S elicits its effect on SP and thereby contributes to sepsis

and associated inflammation and organ injury remains to be investigated.

In conclusion, the present findings show that CSE-derived H2S plays an important role in

regulating increased SP and NK-1R expression associated with CLP-induced sepsis in mice.

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Chapter 6

Effect of Kupffer cell inactivation by gadolinium chloride on inflammation

following caecal-ligation and puncture-induced sepsis in mice

6.1 Introduction

Kupffer cells are resident liver macrophages, derived prenatally from yolk sac cells and

maintained into adulthood without replenishment from blood monocytes (217-219). Kupffer

cells play essential roles in tissue homeostasis, and remodelling and regulation of metabolic

functions in the liver (218). As highly phagocytic cells, they are primarily responsible for

sequestering pathogenic substances, including bacteria and LPS bacterial endotoxins, from

the portal circulation (220). Upon activation, Kupffer cells are known to release biologically-

active substances such as reactive oxygen and nitrogen radicals, peptide mediators,

eicosanoids and proinflammatory cytokines (TNF-α, IL-6 and IL-1β) and chemokines (MCP-1

and MIP-2α). The released mediators help recruit other inflammatory cells such as

neutrophils and monocytes from circulation to the liver to overcome an ongoing bacterial or

endotoxin challenge (221). Although these cells and cellular responses are necessary to

combat infection and endotoxins, persistently high or overwhelming activation may result in

an uncontrolled initiation of the proinflammatory cascade, leading to a systemic

inflammatory response and potentially to multiple organ dysfunction syndrome (MODS) (222-

224).

Gadolinium chloride (GdCl3) is a lanthanide that is commonly used to evaluate the functional

roles of Kupffer cells in several processes (220, 232). While the exact mechanism of action of

GdCl3 is not yet clear, it has been proposed that it inhibits Kupffer cell phagocytosis by

competitive blockade of K type Ca+2 channels and thereby inhibits phagocytosis at their

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surface attachment and engulfment phases. This results in reduced Kupffer cell activation and

subsequently Kupffer cell number (225, 232). Blockading of Kupffer cell phagocytosis by GdCl3

has been used to study Kupffer cell function both in vivo and in vitro.

The role of Kupffer cells has been studied in various experimental animal disease models using

GdCl3, including hepatic ischaemia-reperfusion injury (240), bile duct ligation-induced

obstructive jaundice (224), LPS-induced endotoxaemia (221, 222, 225, 226) and CLP-induced

sepsis (220, 227-230). Much evidence has been accumulated on the protective effect of

Kupffer cell inactivation against liver injury and inflammation. Treatment with GdCl3 reduces

elevated serum ALT and AST enzyme activities following endotoxaemia (222, 223, 231),

trauma (229), sepsis (220) and radiation-induced liver injury (233). Activation of Kupffer cells

with stimulants such as LPS produces ROS and proinflammatory cytokines (particularly TNF-

α), resulting in oxidative stress and inflammation, indicating that activation of Kupffer cells

could be responsible for liver damage. Increased superoxide production and hepatic

expression of TNF-α, IL-1β and IL-6 in response to LPS-induced Kupffer cell activation is

decreased by GdCl3 (231-234). Similarly, experimental evidence has shown that inhibition of

Kupffer cell phagocytosis by GdCl3 attenuates sepsis-associated hepatocellular and hepatic

drug-metabolising dysfunction by reducing the expression of cyclooxygenase-2 (COX-2) (229)

and modulation of the inflammatory response (220), respectively. A recent study has also

demonstrated that enhanced susceptibility of jaundiced animals to endotoxaemia was

reduced with GdCl3 pretreatment (224). Conversely, as a part of the counter-regulatory

mechanism, Kupffer cells release the anti-inflammatory cytokine IL-10 to overcome the

inflammatory response of activated Kupffer cells. The inflammatory response to

endotoxaemia is downregulated by local release of IL-10 from Kupffer cells (235). However,

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in contrast to previous reports some studies show Kupffer cells eliminate LPS and other agents

primarily from the portal circulation and protect the hepatocytes from exposure to high levels

of LPS and other oxidative stresses. For example, inactivation of Kupffer cells with GdCl3 has

led to elevated serum ALT and AST enzyme activities in rats with obstructive jaundice exposed

to endotoxaemia (224). Similarly, treatment with clodronate liposomes, another Kupffer cell

inhibitor, and GdCl3 had no effect on leukocyte MPO activity in the liver following LPS-induced

endotoxaemia (223, 239). Furthermore, in vitro Kupffer cells isolated from GdCl3-treated

animals produced more superoxide and TNF-α compared with control cells (232). These

conflicting results could be due to several factors such as the difference between the

pathology of disease models studied, the dosage regime of GdCl3 and euthanasia time points.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are the first steps in

the development of multiple organ failure in patients with sepsis (230, 236). There have been

significant efforts to understand lung injury in endotoxaemia and sepsis models and GdCl3

treatment. However, there is still a poor understanding of the mechanisms by which GdCl3

modulates lung injury or the role of Kupffer cells in causing lung injury. For example,

treatment with GdCl3 abrogates ozone-induced pulmonary injury by modulation of

inflammatory mediator production (237). It has also been shown that GdCl3 attenuates LPS-

induced pulmonary apoptosis by decreased activation of caspase-3; however, this had no

effect on the increased mRNA expression of proinflammatory cytokines such as TNF-α, IL-6

and IL-1β (236). Conversely, GdCl3 has been shown to predispose rats to lung alveolitis and

lead to increased leukocyte MPO activity in LPS-induced endotoxaemia (223). In parallel,

increased and decreased expression of MCP-1 and IL-10 in the lungs has been reported

following treatment with GdCl3 in endotoxaemia (234) and CLP-induced peritonitis (230),

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respectively. These results on the effect of GdCl3 on lung injury indicate that there is currently

a poor understanding of its role in lung injury in different disease models of endotoxaemia

and sepsis. Furthermore, it remains to be elucidated whether inactivation of Kupffer cells by

GdCl3 can inhibit lung injury and inflammation following CLP-induced sepsis.

Despite the local effects of GdCl3 on liver and lung injury, Kupffer cell blockade increases the

circulatory appearance of bacteria and toxins and leads to impaired host defense and poor

survival. In vivo, mice pretreated with GdCl3 have shown a significant increase in mortality,

despite apparent improvement in systemic inflammation in CLP-induced sepsis (227).

Conversely, rats pretreated with GdCl3 have shown improved mortality by suppression of

superoxide production associated with endotoxaemia (226, 238), hepatic ischaemia-

reperfusion injury when exposed to endotoxins (240) and when hepatectomised rats were

given endotoxins (222). Furthermore, both GdCl3 and clodronate liposomes have been shown

to lower serum levels of TNF-α, IL-6 and IL-1β, which were increased following LPS-induced

endotoxaemia and sepsis (220, 225, 228, 239). However, treatment with GdCl3 has no effect

on elevated serum TNF-α levels in other disease models (222, 226, 240).

All previous investigations have used the approach of Kupffer cell inactivation to study the

role of these cells in different animal disease models of liver and lung inflammation, and the

chemical tool for these studies was either GdCl3 or clodronate liposomes. However, previous

studies have focused either on the local (liver or lung inflammation) or the systemic (systemic

inflammatory response and mortality) effects of Kupffer cell inactivation; none have looked

into both local and systemic effects in one disease model. In addition, the role of Kupffer cells

on liver and lung inflammation and systemic inflammatory response, by focusing on

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proinflammatory cytokines and chemokines profiles remain to be elucidated in CLP-induced

sepsis model.

The objective of the present study was to determine whether Kupffer cell inactivation by

GdCl3 protects against liver and lung injury, inflammation and the systemic inflammatory

response in CLP-induced sepsis in mice.

6.2 Aims

The present study aimed to determine the effect of GdCl3 pretreatment on leukocyte

infiltration, ALT and AST activity, and proinflammatory cytokine and chemokine levels on liver

and lung injury and systemic inflammatory response following CLP-induced sepsis.


Thirty two C57BL/6J (males aged between 8-10 weeks; 25-30 g) mice were randomly divided

into control (Sham and Sham + GdCl3) and experimental (Sepsis and Sepsis + GdCl3) groups,

with eight mice in each group (n=8). GdCl3 was administered (10 mg/kg; i.v., tail vein) before

the CLP and sham operations and saline-treated CLP and sham mice acted as vehicle controls.

Sepsis was induced by CLP as described in Section 2.4 and 8 h after CLP or a sham operation,


aspirated, plasma separated and stored at -80oC. Samples of liver and lungs were processed

and stored at -80oC. Subsequently, MPO activity (Section 2.8), liver and lung injury (Section

2.9), ALT and AST enzyme activity levels (Section 2.16) and proinflammatory cytokine (TNF-α,

IL-6, and IL-1β) and chemokine (MCP-1 and MIP-2α) levels (Section 2.12) were measured by

the methods described in the Material and Methods of Chapter 2. Statistical analysis was

performed as described in Section 2.17.

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6.4 Results

6.4.1 Effect of GdCl3 pretreatment on tissue leukocyte infiltration following CLP-induced

sepsis

Mice with CLP-induced sepsis showed significantly more MPO activity in the liver and lung

compared to sham control mice, with a mean fold increase of 1.62-(P<0.05, liver) and 3.63-

fold (P<0.01, lung), respectively, demonstrating leukocyte infiltration occurs following CLP-

induced sepsis. Pretreatment of mice with GdCl3 before induction of sepsis decreased MPO

activity in the liver compared to sepsis mice without GdCl3 pretreatment, with a mean fold

increase over the sham control of 0.86 compared to 1.62 (P<0.05).

Figure 6.1 Effect of GdCl3 administration on MPO activity following CLP-induced sepsis. Mice with

CLP-induced sepsis or sham operation were randomly given GdCl3 (10 mg/kg, i.v.) or saline before the

CLP or sham operation. Eight hours after CLP or sham operation, mice were sacrificed and MPO activity

in liver (A) and lungs (B) was measured using a spectrophotometric assay. MPO activity increased

significantly in the liver and lungs following CLP-induced sepsis compared to the sham control. Mice

administered with GdCl3 showed significantly decreased mean MPO activity in the liver following

sepsis, though there was no effect on the mean lung MPO activity. Instead, GdCl3 administered sepsis

mice showed increased mean lung MPO activity compared to sepsis mice without GdCl3. Data

represent mean ± S.E.M. (n=8). The significance of differences between groups was evaluated by

ANOVA with post-hoc Tukey’s test. Statistical significance was assigned as *P<0.05, **P<0.01 and

****P<0.0001.

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In contrast, lung MPO activity was significantly increased in sepsis mice pretreated with GdCl3

compared to sepsis mice without GdCl3 pretreatment, with a mean fold increase over the

sham control of 7.01 compared to 3.63 (P<0.0001) (Figure 1).

6.4.2 Effect of GdCl3 pretreatment on liver and lungs damage following CLP-induced sepsis

Mice with CLP-induced sepsis showed the effects of the injury on the liver and lungs. Liver

sections stained with hematoxylin and eosin from mice after CLP operation were

characterised by capsular inflammation and focal necrotic damage that could be easily

observed when compared to sham control sections. Pretreatment of sepsis mice with GdCl3

showed less capsular inflammation and focal necrotic damage in the liver compared to sepsis

mice without GdCl3 pretreatment. Lung sections from mice after CLP operation exhibited

characteristic signs of lung injury, which included leukocyte infiltration and alveolar

thickening in the interstitium compared to the sham operated mice. In contrast to the liver,

GdCl3 pretreatment had no significant effect on leukocyte infiltration and alveolar thickening

in the lungs. However, normal liver and lungs histoarchitecture was observed in the sham

operated mice with and without GdCl3 pretreatment (Figure 6.2).

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110

Figure 6.2 Morphological changes in the liver and lungs following CLP-induced sepsis and the effect

of GdCl3 pretreatment. In mice, GdCl3 (10 mg/kg, i.v.) or saline was administered before the CLP or a

sham operation. Eight hours after CLP or sham operation, mice were sacrificed and liver and lung

tissues were processed for histological examination using hematoxylin and eosin staining.

Haematoxylin (purple) stains nucleic acids (cell nucleus), while eosin (pink) stains protein non-

specifically (cell cytoplasm and extracellular matrix). Liver images were assessed for capsular

inflammation and lobular necrotic damage using modified Knodell Histology Activity Index (HAI) of

blinded scoring system of liver injury (271, 272) whereas lung sections were assessed for leukocyte

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infiltration and alveolar wall thickening using blinded lung injury scoring system suggested by

American Thoracic Society guidelines (273). Representative liver (A) and lung (B) sections show

capsular inflammation and focal necrosis in the liver and leukocyte infiltration and alveolar thickening

in the lungs as a result of CLP-induced sepsis. Pretreatment with GdCl3 significantly reduced capsular

inflammation and focal necrosis in liver but failed to reduce leukocyte infiltration and alveolar

thickening in lungs following CLP-induced sepsis. Data represent mean ± S.E.M. (n=8). The significance

of differences between groups was evaluated by non-parametric Kruskal–Wallis test. Statistical

significance was assigned as *P<0.05, **P<0.01 and ***P<0.001.

6.4.3 Effect of GdCl3 pretreatment on plasma ALT and AST activity levels following CLP-

induced sepsis

Increased levels of ALT and AST activity in plasma is an indication of liver damage. CLP-induced

sepsis in mice was associated with significantly higher levels of plasma ALT and AST activity

compared to sham control mice (ALT: 48.54 ± 5.86 U/L vs 25.17 ± 2.04 U/L, P<0.001; and AST:

194.7 ± 21.62 U/L vs 77.75 ± 5.35 U/L, P<0.0001). Pretreatment of sepsis mice with GdCl3

significantly reduced plasma ALT and AST activity levels compared to sepsis mice without

GdCl3 pretreatment (ALT: 26.65 ± 1.54 U/L vs 48.54 ± 5.86 U/L, P<0.01; and AST: 119.0 ± 9.51

vs U/L 194.7 ± 21.62 U/L, P<0.01). There was no significant difference between GdCl3

pretreated sham and GdCl3 sepsis mice and sham mice without GdCl3 pretreatment (Figure

6.3).

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Figure 6.3 Alteration of plasma ALT and AST activity levels following CLP-induced sepsis and the

effect of GdCl3 pretreatment. In mice, GdCl3 (10 mg/kg, i.v.) or saline was administered before the CLP

or sham operation. Eight hours after CLP or sham operation, mice were sacrificed and ALT (A) and AST

(B) activity levels in plasma were measured using the enzymatic method. CLP-induced sepsis resulted

in a significant increase in plasma ALT and AST activity levels. There was a significant reduction in

plasma ALT and AST activity levels in sepsis mice pretreated with GdCl3 compared to sepsis mice

without GdCl3 pretreatment. Data represent mean ± S.E.M. (n=8). The significance of differences

between groups was evaluated by ANOVA with post-hoc Tukey’s test. Statistical significance was

assigned as **P<0.01, ***P<0.001 and ****P<0.0001.

6.4.4 Effect of GdCl3 pretreatment on liver proinflammatory mediators following CLP-

induced sepsis

As expected, mice with CLP-induced sepsis showed significantly higher liver levels of cytokines

(TNF-α, IL-6 and IL-1β) and chemokines (MCP-1 and MIP-2α) compared to control mice with

a sham operation (TNF-α: 107.5 ± 9.49 ng/mg vs 51.62 ± 13.49 ng/mg (P<0.001); IL-6: 3.68 ±

0.41 ng/mg vs 3.07 ± 0.46 ng/mg (P<0.05); IL-1β: 0.64 ± 0.09 ng/mg vs 0.33 ± 0.03 ng/mg

(P<0.01); MCP-1: 1685.0 ± 128.0 ng/mg vs 784.1 ± 66.15 ng/mg (P<0.001); and MIP-2α: 4.99

± 0.58 ng/mg vs 3.08 ± 0.20 ng/mg (P<0.05)). Sham operations produce a degree of

inflammation as the pretreatment with GdCl3 reduced cytokine and chemokine levels in the

sham operated mice. Sepsis mice pretreated with GdCl3 had lower liver levels of

proinflammatory cytokines and chemokines, suggesting a protective effect of GdCl3 against

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Figure 6.4 Effect of GdCl3 pretreatment on protein expression levels of cytokines and chemokines in

the liver from mice with CLP-induced sepsis. Mice with CLP-induced sepsis or a sham operation were

randomly given GdCl3 (10 mg/kg, i.v.) or saline before the operation. Eight hours after CLP or sham

operation, mice were sacrificed and cytokine (TNF-α (A), IL-6 (B) and (IL-1β (C)) and chemokine (MCP-

1 (D) and MIP-2α (E)) levels were measured in the liver by ELISA. CLP-induced sepsis resulted in a

significant increase in liver proinflammatory cytokines and chemokines. There was a significant

reduction in all these mediators in GdCl3-pretreated mice following CLP-induced sepsis. Data represent

mean ± S.E.M. (n=8). The significance of differences between groups was evaluated by ANOVA with

post-hoc Tukey’s test. Statistical significance was assigned as *P<0.05, **P<0.01, ***P<0.001 and

****P<0.0001.

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sepsis-induced liver inflammation (TNF-α: 21.44 ± 2.40 ng/mg vs 107.5 ± 9.49 ng/mg

(P<0.0001); IL-6: 1.53 ± 0.11 ng/mg vs 3.68 ± 0.41 ng/mg (P<0.0001); IL-1β: 0.27 ± 0.06 ng/mg

vs 0.64 ± 0.09 ng/mg (P<0.001); MCP-1: 1352.0 ± 125.1 ng/mg vs 1685.0 ± 128.0 ng/mg; and

MIP-2α: 1.75 ± 0.11 ng/mg vs 4.99 ± 0.58 ng/mg (P<0.0001)) (Figure 6.4).

6.4.5 Effect of GdCl3 pretreatment on lungs proinflammatory mediators following CLP-

induced sepsis

A substantial elevation of lungs cytokines (TNF-α, IL-6 and IL-1β) and chemokines (MCP-1 and

MIP-2α) was seen in CLP-induced sepsis mice compared to sham operated mice (TNF-α: 18.86

± 1.69 ng/mg vs 5.66 ± 0.32 ng/mg (P<0.01); IL-6: 1.0 ± 0.152 ng/mg vs 0.48 ± 0.06 ng/mg

(P<0.01); IL-1β: 0.51 ± 0.11 ng/mg vs 0.18 ± 0.04 ng/mg (P<0.01); MCP-1: 524.0 ± 90.39 ng/mg

vs 142.5 ± 18.85 ng/mg (P<0.01); and MIP-2α: 1.37 ± 0.07 ng/mg vs 0.77 ± 0.13 ng/mg

(P<0.01)). Pretreatment with GdCl3 failed to decrease levels of proinflammatory cytokines and

chemokines in the lungs after CLP-induced sepsis. Moreover, lungs TNF-α levels were

significantly increased in GdCl3 pretreated sepsis mice compared to the sepsis mice without

GdCl3 pretreatment (TNF-α: 30.07 ± 2.83 ng/mg vs 18.86 ± 1.69 ng/mg (P<0.01); IL-6: 1.32 ±

0.11 ng/mg vs 1.0 ± 0.152 ng/mg; IL-1β: 0.37 ± 0.01 ng/mg vs 0.51 ± 0.11 ng/mg; MCP-1: 648.1

± 121.3 ng/mg vs 524.0 ± 90.39 ng/mg and MIP-2α: 1.36 ± 0.11 ng/mg vs 1.37 ± 0.07 ng/mg))

(Figure 6.5).

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Figure 6.5 Effect of GdCl3 administration on the levels of cytokines and chemokines in lungs from mice with CLP-induced sepsis. Mice with CLP-induced sepsis or a sham operation were randomly given GdCl3 (10 mg/kg, i.v.) or saline before the operation. Eight hours after CLP or sham operation, mice were sacrificed and cytokine (TNF-α (A), IL-6 (B) and (IL-1β (C)) and chemokine (MCP-1 (D) and MIP-2α (E)) levels were measured in lungs by ELISA. CLP-induced sepsis mice showed significant increases in lungs proinflammatory cytokines and chemokines. Pretreatment with GdCl3 had no effect on the increased levels of lungs cytokines and chemokines following CLP-induced sepsis. Instead, mean TNF-α levels were significantly increased compared to sepsis mice without GdCl3 pretreatment. Data represent mean ± S.E.M. (n=8). The significance of differences between groups was evaluated by ANOVA with post-hoc Tukey’s test. Statistical significance was assigned as *P<0.05, **P<0.01 and ***P<0.001.

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6.4.6 Effect of GdCl3 pretreatment on circulatory proinflammatory mediators following CLP-

induced sepsis

Higher level of circulatory proinflammatory cytokines and chemokines are a feature of sepsis.

After a CLP operation mice had significantly increased levels of circulatory proinflammatory

cytokines (TNF-α, IL-6 and IL-1β) and chemokines (MCP-1 and MIP-2α) compared to sham

control mice (TNF-α: 2.02 ± 0.14 ng/mL vs 0.57 ± 0.03 ng/mL (P<0.0001); IL-6: 0.07 ± 0.01

ng/mL vs 0.01 ± 0.002 ng/mL (P<0.01); IL-1β: 0.15 ± 0.01 ng/mL vs 0.048 ± 0.01 ng/mL

(P<0.01); MCP-1: 917.52 ± 71.84 ng/mL vs 210.72 ± 20.45 ng/mL (P<0.0001); and MIP-2α: 0.13

± 0.02 ng/mL vs 0.03 ± 0.01 ng/mL (P<0.0001)). Sepsis mice pretreated with GdCl3 failed to

decrease the levels of circulatory proinflammatory cytokines and chemokines. Instead,

plasma IL-1β levels were significantly increased in GdCl3-pretreated sepsis mice compared to

sepsis mice without GdCl3 pretreatment (TNF-α: 2.29 ± 0.17 ng/mL vs 2.02 ± 0.14 ng/mL; IL-

6: 0.08 ± 0.01 ng/mL vs 0.07 ± 0.01 ng/mL; IL-1β: 0.24 ± 0.03 ng/mL vs 0.15 ± 0.01 ng/mL

(P<0.05); MCP-1: 853.0 ± 204.1 ng/mL vs : 917.52 ± 71.84 ng/mL; and MIP-2α: 0.13 ± 0.03 vs

0.13 ± 0.02 ng/mL) (Figure 6.6).

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Figure 6.6 Effect of GdCl3 administration on cytokine and chemokine levels in plasma from mice with

CLP-induced sepsis. Mice with CLP-induced sepsis or sham operation were randomly given GdCl3 (10

mg/kg, i.v.) or saline before the operation. Eight hours after CLP or sham operation, mice were

sacrificed and cytokine (TNF-α (A), IL-6 (B) and (IL-1β (C)) and chemokine (MCP-1 (D) and MIP-2α (E))

levels were measured in plasma by ELISA. CLP-induced sepsis mice showed a significant increase in

mean plasma proinflammatory cytokines and chemokines. GdCl3 pretreatment did not decrease

plasma cytokine and chemokine levels following CLP-induced sepsis. Instead, IL-1β levels were

significantly increased compared to sepsis mice without GdCl3 pretreatment. Data represent mean ±

S.E.M. (n=8). The significance of differences among groups was evaluated by ANOVA with post-hoc

Tukey’s test. Statistical significance was assigned as *P<0.05, **P<0.01, ***P<0.001 and

****P<0.0001.

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6.5 Discussion

The results of the present study have shown that Kupffer cells play a key role in CLP-induced

sepsis, particularly the effects on the liver. The main findings of this study are: (1) increased

liver injury and inflammation as a result of CLP-induced sepsis in mice was attenuated by

pretreatment with GdCl3; and (2) pretreatment with GdCl3 failed to protect against CLP-

induced lung injury, inflammation and the systemic inflammatory response in mice.

Kupffer cells are primary macrophages in the liver. They regulate blood detoxification and

maintain a functional immune response to exogenous infectious challenge. Activation of

Kupffer cells during endotoxaemia or bacterial infection has been shown to cause liver injury

and inflammation during sepsis (220, 228). Proinflammatory cytokines and chemokines

secreted by activated Kupffer cells mediate liver injury and inflammation in sepsis and

inactivation of Kupffer cells can provide a survival advantage in sepsis (226). The results of the

present study demonstrated that pretreatment with GdCl3 resulted in decreased liver

inflammation, as evidenced by reduced leukocyte MPO activity, proinflammatory cytokines

such as TNF-α, IL-6 and IL-1β, and chemokines such as MCP-1 and MIP-2α. Histological

examination of the liver sections showed that pretreatment with GdCl3 reduced capsular

inflammation and focal necrosis following sepsis. These results are consistent with previous

reports in animal models of endotoxaemia and sepsis (220, 224, 231, 233, 240). For example,

it has been shown that treatment with GdCl3 protects animals against liver injury by reducing

superoxide production and TNF-α levels following LPS-induced endotoxaemia and sepsis (220,

231, 232). Furthermore, in the present study, sepsis mice pretreated with GdCl3 showed

reduced levels of plasma ALT and AST activity. In contrast, previous studies have reported

that treatment with GdCl3 elevated plasma ALT and AST levels in obstructive jaundice mice

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exposed to endotoxin (224), while others have reported no such increase in liver enzyme

levels (220, 234). The conflicting results between this study and other investigations might be

due to differences in the animal disease model and the dosage regime of GdCl3. The results

in this chapter support the hypothesis that inactivation of Kupffer cells by GdCl3 can be

protective against liver injury and inflammation in CLP-induced sepsis.

Under conditions of sepsis and septic shock, the respiratory system is the most vulnerable

organ system, and ALI leading to ARDS is the first step in the development of multiple organ

failure (340). Although there have been sustained efforts to understand lung injury during

endotoxaemia, and sepsis and GdCl3 treatment, the role of GdCl3 in lung injury is still poorly

understood. The findings of the present study show that despite improvement of liver injury

and inflammation by GdCl3 in CLP-induced sepsis, pretreatment of sepsis mice with GdCl3 did

not decrease lung injury or inflammation. In addition, GdCl3 pretreatment was shown to

increase leukocyte MPO activity in the lungs of mice with CLP-induced sepsis. Moreover,

histological examination of the lung sections showed GdCl3 pretreatment failed to reduce

sepsis induced-leukocyte infiltration and alveolar congestion. It is reasonable to assume that

in vivo exposure to GdCl3 inhibits the uptake of bacteria or endotoxins by Kupffer cells and

that this could result in increased exposure of extrahepatic organs such as the lungs to

infection or endotoxins. Pulmonary cells such as alveolar macrophages can produce

proinflammatory cytokines and chemokines during sepsis, and it is plausible that production

of these mediators increased when removal of bacteria or endotoxins by Kupffer cells was

impaired. Increased lung injury might be due to increased migration of leukocytes, particularly

neutrophils, into the alveolar space as a result of increased amounts of cytokines and

chemokines released from activated alveolar macrophages during sepsis in GdCl3-treated

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mice. These findings suggest that sepsis-induced lung injury and inflammation are

independent of the direct effect of GdCl3 on Kupffer cells in the liver. Functional differences

between alveolar macrophages and Kupffer cells have been reported in studies that clearly

indicate that significant heterogeneity exists between these two cell types (217, 219).

Although GdCl3 selectively inhibits Kupffer cell activation, it has been shown that GdCl3

pretreatment alters the responses of alveolar macrophages to ozone exposure; however, the

mechanism is not known (237). Therefore, the possibility cannot be ruled out that there are

independent effects of GdCl3 on alveolar macrophages after CLP-induced sepsis. For example,

it has been reported that pretreatment with GdCl3 predisposes lungs to alveolitis in

endotoxaemic rats (223). Conversely, other studies have shown that GdCl3 treatment

prevents endotoxaemia and sepsis-induced lung injury by a mechanism that involves

inhibiting production of proinflammatory mediators and caspase-3 and by increasing IL-10

from Kupffer cells, suggesting an interaction between Kupffer cells and alveolar macrophages

in lung injury during endotoxaemia and sepsis (230, 234, 236). Further studies are required to

gain a better understanding of the role of GdCl3 in lung injury during sepsis.

The results of the present study also showed that circulatory cytokine and chemokine levels

did not differ between GdCl3-treated and non-treated sepsis mice, suggesting circulatory

proinflammatory mediators may not accurately reflect those that are secreted by Kupffer

cells. The results of the present study are supported by previous reports, which indicated that

serum levels of TNF-α and IL-6 are not affected by GdCl3 in hepatic-reperfusion-induced injury

exposed to endotoxin and LPS-induced endotoxaemia (222, 226, 240). In contrast, previous

experiments using both GdCl3 and clodronate liposomes have shown that inactivation of

Kupffer cells lowered serum levels of TNF-α, IL-6 and IL-1β following LPS-induced

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endotoxaemia and sepsis (220, 225, 228, 239). The conflicting results between this study and

other studies might be due to differences in the animal disease model and the dosage regime

of GdCl3. However, it is reasonable to assume that, as in the lungs, in vivo exposure to GdCl3

inhibits the uptake of bacteria or endotoxin by Kupffer cells, and that this could result in

increased exposure of circulatory monocytes and neutrophils to infection or endotoxin.

Hence, inhibition of Kupffer cells by GdCl3 could potentially increase circulatory levels of

inflammatory mediators produced by activated monocytes and neutrophils. Therefore, the

results of the present study suggest that the increased circulatory levels of cytokines and

chemokines found during sepsis are not directly responsible for the liver injury and similarly

that the protective effect of GdCl3 against liver damage and inflammation is not due to the

inhibition of circulatory proinflammatory mediators.

At the same time, the present study has some limitations. This study is limited by short

duration of the end endpoint (8 h after CLP-induced sepsis). In addition, the present study did

not investigate survival rates, which would help to understand the differential effects of GdCl3

in CLP-idncued sepsis. Also, endpoints such as alveolar thickening may take time to evolve

and may commence sometime after liver injury has occurred; hence, an 8 h time point may

be too early to detect effects that may evolve later.

In conclusion, the experimental results described in this chapter have shown that mice

pretreated with GdCl3 have decreased liver injury and inflammation following CLP-induced

sepsis. However, pretreatment with GdCl3 failed to protect mice against sepsis-induced lung

injury, inflammation and the systemic inflammatory response. Therefore, it is concluded that

most likely GdCl3 prevents liver injury and inflammation due to a reduced local effect of

cytokine and chemokine on hepatocytes from Kupffer cells.

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Chapter 7

Effect of caecal-ligation and puncture-induced sepsis on liver sinusoidal

endothelial cell fenestration (liver sieve)

7.1 Introduction

Liver sinusoidal endothelial cells (LSECs) are fenestrated endothelial cells that act as a

permeable barrier between sinusoidal blood and hepatic parenchyma (341). The LSECs

fenestrations are pores which range in size from 50-250 nm in diameter and form liver sieve

plates (342). The structural integrity of the fenestrated liver sinusoidal endothelium is

essential for the maintenance of a normal exchange of fluids, solutes, particles and

metabolites between sinusoidal blood and hepatic parenchymal cells (343). The diameter and

number of the fenestrae vary from species to species and within single individuals of a species

under the influence of various physiological and pharmacological circumstances. For example,

in comparison with the rat, rabbits have smaller fenestrae and chicken have fewer fenestrae

(246-248). In addition, differences in fenestrae diameter and frequency vary from one lobe to

another and within different regions of a lobe. For example, differences in fenestrae diameter

and frequency in peripheral and centrilobular zones have been demonstrated in the rat liver

(216). Changes in fenestration diameter and number are also observed in response to a

variety of biological mediators such as growth factors (VEGF) (250), hormones (serotonin)

(249) and neurotransmitters (acetylcholine and adrenaline), drugs (pantethine, nicotine)

(256), toxins (cytochalasin B (251), latrunculin A, misakinolide (252), antimycin A (344),

thioacetamide (258), dimethylnitrosamine (257) and carbon tetrachloride (345)) and diseases

such as alcohol abuse associated liver injury (253-255), cirrhosis (258), oxidative stress (259,

260), inflammation, infection and endotoxaemia (261, 262)). Impairment of substrate

123

exchange through the alteration of LSEC fenestrae is a major contributor to hepatic

dysfunction and leads to liver and systemic diseases.

Sepsis involves a systemic inflammatory response to infection and is the leading cause of

death in intensive care patients (346, 347). During sepsis, both Kupffer cells and LSECs are the

first responders to invading bacteria and endotoxins (220, 228, 235, 240). Excessive

stimulation of Kupffer cells during infection or endotoxaemia leads to secretion of an array of

mediators, including proinflammatory cytokines (TNF-α, IL-6 and IL-1β), arachidonate

derivatives (thromboxanes and leukotrienes) and biologically-active free radicals (oxygen and

nitrogen radicals, among others), which lead to LSEC damage and hepatocyte injury (235, 261-

263). LSECs also have the capacity to produce proinflammatory cytokines which are increased

when LSECs stimulated with LPS (235). For example, it has been shown that LPS induces LSEC

defenestration by the release of proinflammatory mediators from activated LSECs and

Kupffer cells (235, 261). Recent evidence has shown that Kupffer cells potentiate LSEC injury

by ligating programmed death ligand-1 (PD-L1) in sepsis (263). In contrast, Kupffer cells

protect LSECs by decreasing Fas-mediated apoptosis in sepsis. Nevertheless, CLP sepsis-

induced structural changes in LSEC fenestrae and the role of Kupffer cell inactivation on these

changes remain to be elucidated.

The role of H2S and SP has been well-documented in the animal models of endotoxaemia and

sepsis (44, 140, 177, 197, 198, 348-350). H2S synthesised through CSE acts as a

proinflammatory mediator in LPS-induced endotoxaemia and CLP-induced sepsis and is

associated with liver injury and inflammation (43, 44, 126, 140, 177, 317). A mechanism of

CSE-derived H2S-mediated inflammation involves activation of ERK1/2 and NF-B p65

signalling and subsequent transcription of proinflammatory mediators (particularly TNF-α)

124

(43, 44) (also shown in Chapter 4). In addition, SP and subsequent activation of NK-1R are

under the control of H2S and it has been shown that SP acts as the upstream regulator of CSE

or CSE-derived H2S-mediated inflammation (141) (also shown in Chapter 5). However, as an

important regulators of inflammation, the role of neither H2S nor SP on LSEC fenestrae

following CLP-induced sepsis is known.

The objective of the present study was to determine whether sepsis alters LSEC fenestrae and

if so, to investigate the potential roles of Kupffer cell, CSE-derived H2S and SP on sepsis-altered

LSEC fenestrae.

7.2 Aims

The previous chapters (Chapters 3 to 5) have shown the importance of CSE-derived H2S

signalling on inflammation and organ injury following CLP-induced sepsis. These chapters

focused on the mechanisms involved in CSE-derived H2S-mediated inflammation and

interaction with SP and NK-1R following sepsis. In addition, in Chapter 6 the role of GdCl3 on

sepsis-associated liver and lung injury, inflammation and systemic inflammatory response

was investigated.

The present study aimed to examine the structural changes in LSEC fenestrae (diameter,

frequency, porosity and gaps formation) induced by CLP-induced sepsis and the effect of

GdCl3, CSE gene deletion and PPTA gene deletion on CLP sepsis-induced structural changes in

LSEC fenestrae.


Three sets of experiments were performed. In the first experiment, sixteen C57BL/6J (males

aged between 8-10 weeks; 25-30 g) mice were randomly divided into control (Sham operation

125

and Sham operation + GdCl3) and experimental (Sepsis and Sepsis + GdCl3) groups, with four

mice in each group (n=4). GdCl3 was administered (10 mg/kg; i.v., tail vein) before the CLP and

sham operations and saline-treated CLP and sham mice acted as vehicle controls.

In the second experiment, sixteen WT (C57BL/6J) and CSE KO (on a C57BL/6J background)

(males aged between 8-10 weeks; 25-30 g) mice were randomly divided into control (WT

Sham operation and CSE KO Sham operation) and experimental (WT Sepsis and CSE KO Sepsis)

groups, with four mice in each group (n=4).

In the third experiment, sixteen WT (BALB/c) and PPTA KO (on a BALB/c background) (males

aged between 8-10 weeks; 25-30 g) mice were randomly divided into control (WT Sham

operation and PPTA KO Sham operation) and experimental (WT Sepsis and PPTA KO Sepsis)

groups, with four mice in each group (n=4).

In all these experiments, sepsis was induced by CLP as described in Section 2.4 and 8 h after

CLP or sham operation mice were anaesthtised by an i.p. injection of pentobarbital (80

mg/kg). Liver perfusion was performed and perfusion-fixed liver sections were processed for

scanning electron microscopy, as described in Section 2.15. Statistical analysis was performed

as described in Section 2.17.

7.4 Results

7.4.1 Effect of CLP-induced sepsis and GdCl3 pretreatment on LSEC fenestration

Changes in LSEC fenestration was assessed by scanning electron micrographs and showed

LSEC defenestration following CLP-induced sepsis, as evidenced by decreased frequency, and

porosity and more gaps formation in the LSECs compared to sham control (gap area: 0.18 ±

0.01 nm2/µm2 vs 0.02 ± 0.01 nm2/µm2). Sepsis mice pretreated with GdCl3 showed

126

significantly less defenestration (increased frequency and porosity) and fewer gaps formation

compared to mice without GdCl3 pretreatment (gap area: 0.07 ± 0.01 nm2/µm2 vs 0.18 ± 0.01

nm2/µm2) (Figure 7.1 and Table 7.1).

*

* *

*

127

Figure 7.1 Effect of GdCl3 pretreatment on LSEC defenestration following CLP-induced sepsis in mice,

assessed using scanning electron microscopy (SEM). Mice with CLP-induced sepsis or sham operation

were randomly given GdCl3 (10 mg/kg, i.v.) or saline before the CLP or sham operation. Eight hours

after CLP or sham operation, liver perfusion was performed through the cannulation of portal vein

using a fixative buffer (2.5% glutaraldehyde in cacodylate buffer). Perfusion-fixed liver sections were

processed to measure changes in LSEC fenestration using SEM. (A) Representative images of LSEC

micrographs and (B) average gap area in LSEC fenestrae. CLP-induced sepsis resulted in a significant

increase of LSEC injury, as evidenced by gaps formation. Mice pretreated with GdCl3 showed fewer

gaps formation compared to mice without GdCl3 following CLP-induced sepsis. Data represent mean ±

S.E.M. (n=4). The significance of differences among groups was evaluated by ANOVA with post-hoc

Tukey’s test. Statistical significance was assigned as *P<0.05.

Table 7.1 Effect of GdCl3 pretreatment on LSEC fenestration in mice following CLP-induced sepsis,

assessed using scanning electron microscopy (SEM).

Mice with CLP-induced sepsis or sham operation were randomly given GdCl3 (10 mg/kg, i.v.) or saline

before the CLP or sham operation. Eight hours after CLP or sham operation, liver perfusion was

performed through cannulation of portal vein using a fixative buffer (2.5% glutaraldehyde in

cacodylate buffer). Perfusion-fixed liver sections were processed to measure changes in LSEC

fenestration using SEM. CLP-induced sepsis resulted in a significant decrease in frequency and porosity

in LSEC fenestrae. There was a significant increase (except diameter) of frequency and porosity of LSEC

fenestrae in GdCl3 pretreated mice compared to mice without GdCl3 pretreatment following sepsis.

Data represent mean ± S.E.M. (n=4). The significance of differences among groups was evaluated by

ANOVA with post-hoc Tukey’s test. Statistical significance was assigned as **P<0.01 and ***P<0.001

vs sham and ##P<0.01 vs sepsis.

7.4.2 Alteration of LSEC fenestration in WT and CSE KO mice following CLP-induced sepsis.

Scanning electron micrographs showed that CLP-induced sepsis was associated with

defenestration (decreased diameter, frequency and porosity) and gaps formation in LSEC

Treatment group Diameter of fenestrae (nm)

Number of fenestrae/µm2

Porosity (%)

Sham

Sham + GdCl3

Sepsis

Sepsis + GdCl3

137.63 ± 6.46

126.37 ± 7.72

123.45 ± 3.13

119.51 ± 5.42

8.42 ± 0.50

9.18 ± 0.29

6.56 ± 0.28**

9.19 ± 0.71###

12.51 ± 0.59

12.23 ± 0.27

8.40 ± 0.67***

10.93 ± 0.98##

128

fenestrae compared to sham control in WT mice (gap area: 0.18 ± 0.01 nm2/µm2 vs 0.02 ±

0.01 nm2/µm2). Sepsis mice deficient in the CSE gene showed less defenestration (increased

diameter, frequency and porosity) and fewer gaps formation than WT sepsis mice (gap area:

0.03 ± 0.01 nm2/µm2 vs 0.18 ± 0.01 nm2/µm2) (Figure 7.2 and Table 7.2).

*

*

*

*

*

129

Figure 7.2 Effect of CLP-induced sepsis on LSEC fenestration in WT and CSE KO mice, assessed using

scanning electron microscopy (SEM). WT and CSE KO mice underwent CLP or sham operation. Sham

operated mice acted as controls. Eight hours after CLP or sham operation, liver perfusion was

performed through the cannulation of portal vein using a fixative buffer (2.5% glutaraldehyde in

cacodylate buffer). Perfusion-fixed liver sections were processed to measure LSEC injury using SEM. (A)

Representative images of LSEC micrographs and (B) average gap area in LSEC fenestrae. CLP-induced

sepsis resulted in a significant increase of LSEC injury, as evidenced by gaps formation in WT mice. CSE

KO mice showed significantly fewer gaps formation compared to WT mice following CLP-induced

sepsis. Data represent mean ± S.E.M. (n=4). The significance of differences among groups was

evaluated by ANOVA with post-hoc Tukey’s test. Statistical significance was assigned as ****P<0.0001.

Table 7.2 Effect of CLP-induced sepsis on LSEC fenestration in WT and CSE KO mice, assessed using

scanning electron microscopy (SEM).

WT and CSE KO mice underwent CLP or sham operation. Sham operated mice acted as controls. Eight

hours after CLP or sham operation, liver perfusion was performed through the cannulation of portal

vein using a fixative buffer (2.5% glutaraldehyde in cacodylate buffer). Perfusion-fixed liver sections

were processed to measure changes in LSEC fenestration using SEM. CLP-induced sepsis resulted in a

significant decrease of diameter, frequency and porosity in LSEC fenestrae of WT mice. There was a

significant increase (except diameter) of frequency and porosity in LSEC fenestrae of CSE KO mice

compared to WT mice following sepsis. Data represent mean ± S.E.M. (n=4). The significance of


was assigned as *P<0.05 and **P<0.01 vs WT sham and #P<0.05 and ##P<0.01 vs WT sepsis.

7.4.3 Alteration of LSEC fenestration in WT and PPTA KO mice following CLP-induced sepsis

Scanning electron micrographs show that CLP-induced sepsis was associated with

defenestration (decreased frequency and porosity) and gaps formation in LSEC fenestrae

compared to sham operated WT mice (gap area: 0.12 ± 0.02 nm2/µm2 vs 0.06 ± 0.01

nm2/µm2). Sepsis mice deficient in the PPTA gene showed less defenestration (increased

Group Diameter of fenestrae (nm)


Porosity (%)

WT Sham

CSE KO Sham

WT Sepsis

CSE KO Sepsis

137.63 ± 6.46

118.51 ± 3.07

123.45 ± 3.13*

125.91 ± 5.42

8.42 ± 0.50

8.44 ± 1.00

6.56 ± 0.28*

8.92 ± 0.68#

12.51 ± 0.59

10.49 ± 0.66

8.40 ± .67**

11.27 ± 1.14##

130

frequency and porosity) and fewer gaps formation than WT sepsis mice (gap area: 0.09 ± 0.01

nm2/µm2 vs 0.12 ± 0.02 nm2/µm2) (Figure 7.3 and Table 7.3).

*

* * *

*

*

*

*

131

Figure 7.3 Effect of CLP-induced sepsis on LSEC fenestration in WT and PPTA KO mice, assessed using

scanning electron microscopy (SEM). WT and PPTA KO mice underwent CLP or sham operation. Sham

operated mice acted as controls. Eight hours after CLP or sham operation, liver perfusion was

performed through the cannulation of portal vein using a fixative buffer (2.5% glutaraldehyde in

cacodylate buffer). Perfusion-fixed liver sections were processed to measure LSECs injury using SEM.

(A) Representative images of LSEC micrographs and (B) average gap area in LSEC fenestrae. CLP-

induced sepsis resulted in a significant increase of LSEC injury as evidenced by gaps formation in WT

mice. PPTA KO mice showed fewer gaps formation in LSEC fenestrae compared to WT mice following

CLP-induced sepsis. Data represent mean ± S.E.M. (n=3-4). The significance of differences among


*P<0.05.

Table 7.3 Effect of CLP-induced sepsis on LSEC fenestration in WT and PPTA KO mice, assessed using

scanning electron microscopy (SEM).

WT and PPTA KO mice underwent CLP or sham operation. Sham operated mice acted as controls. Eight

hours after CLP or sham operation, liver perfusion was performed through cannulation of portal vein

using a fixative buffer (2.5% glutaraldehyde in cacodylate buffer). Perfusion-fixed liver sections were

processed to measure for changes in LSEC fenestration using SEM. CLP-induced sepsis resulted in a

significant decrease in frequency and porosity in LSEC fenestrae of WT mice. PPTA KO mice showed an

increase (not significant) of frequency and porosity in LSEC fenestrae compared to WT mice following

CLP-induced sepsis. Data represent mean ± S.E.M. (n=3-4). The significance of differences among


*P<0.05 and **P<0.01 vs WT sham.

7.5 Discussion

The present study demonstrates that increased inflammatory response in CLP-induced sepsis

causes structural changes in LSEC fenestrae. Results of Chapter 4 and 6 have demonstrated

the role of proinflammatory cytokines and chemokines in CLP-induced sepsis. The results

Group Diameter of fenestrae (nm)


Porosity (%)

WT Sham

PPTA KO Sham

WT Sepsis

PPTA KO Sepsis

149.39 ± 12.40

135.83 ± 7.83

143.91 ± 9.04

144.68 ± 8.39

10.63 ± 1.54

11.60 ± 2.56

6.40 ± 2.49*

9.05 ± 0.94

18.44 ± 2.18

17.64 ± 3.28

11.05 ± 3.23**

15.71 ± 2.19

132

from the three experiments here show defenestration in LSECs, i.e. decreased diameter,

frequency and porosity of LSEC fenestrae following CLP-induced sepsis. These structural

changes in LSEC fenestrae are comparable with earlier observations from different

laboratories. For example, it has previously been reported that increased inflammatory

response associated with endotoxaemia and sepsis causes structural changes in LSEC

fenestrae, i.e. defenestration of LSECs (228, 235, 259, 260, 351). In vivo experiments of LPS-

induced endotoxaemia or pyocyanin challenge, a virulence factor of Pseudomonas

aeruginosa-induced liver injury are associated with defenestration in LSEC fenestrae (262,

352). Similarly, in vitro experiments with isolated LSECs exposed to pyocyanin or LPS resulted

in a decrease in porosity, LSEC fenestrae enlargement and sieve plate disruption (352, 353).

Together, the results of the present study suggest that inflammatory response associated

with sepsis causes defenestration in LSEC fenestrae.

For the first time, the experimental data of the present study demonstrates the formation of

gaps in LSEC fenestrae following CLP-induced sepsis. Gaps are large defects in LSEC fenestrae

(usually more than 250 nm in diameter) that are associated with LSEC injury. Mice with CLP-

induced sepsis showed more gaps formation in LSEC fenestrae than sham control mice.

Increased formation of gaps in LSEC fenestrae might be due to increased inflammatory

mediators and oxidative stress in sepsis. The concept of gaps formation has been described

by previous studies using different experimental disease models during oxidative stress and

endotoxaemia; however, the mechanism of their formation remains unclear. Fraser et al.

reported the formation of large gaps in the liver sinusoidal endothelium following high

perfusion pressure of the liver (354). Furthermore, large gaps formation has previously been

seen in LSEC fenestrae following hepatic venous outflow obstruction, irradiation and oxidative

133

stress; these gaps occur as a result of loss or degradation of sieve plates (259, 355, 356). Most

gaps in the endothelium that occur after oxidative stress are of similar size distribution as the

sieve plates (259). Similarly, the experiments in this study show consistency in gaps formation

following CLP-induced sepsis. The results of the present study demonstrate the significance

of gaps formation in LSEC fenestrae, which are mainly due to increased inflammation and

oxidative stress following CLP-induced sepsis.

Based on the CLP sepsis-induced defenestration and gaps formation in LSEC fenestrae, the

first approach of the present study was to investigate the role of Kupffer cell-secreted

proinflammatory mediators on the alteration of LSEC fenestrae following CLP-induced sepsis

in mice. To prove this, the present study used the chemical compound GdCl3, which

inactivates Kupffer cells, to see the effect on CLP sepsis-induced defenestration and gaps

formation in LSEC fenestrae. The results of Chapter 6 demonstrated that GdCl3 pretreatment

decreases proinflammatory mediators and subsequently reduces liver injury following sepsis.

In the present study, GdCl3 pretreatment resulted in less defenestration and fewer gaps

formation in LSEC fenestrae following CLP-induced sepsis. The findings are consistent with

previously published reports on the GdCl3 effect on LSEC defenestration using different

experimental models including LPS-induced endotoxaemia in rats or in vitro exposure to the

pseudomonas toxin pyocyanin (223, 262, 351, 357-359). Some previous evidence suggests

that released TNF-α from activated Kupffer cells causes defenestration in LSEC fenestrae

during infection and endotoxaemia (262, 360). However, these previous studies have no

mention of the significance of gaps formation in LSEC fenestrae. The results of the present

study suggest that Kupffer cells signalling to LSECs contribute to the regulation of fenestration

and gaps formation.

134

The second approach was to use mice deficient in the CSE or PPTA genes separately to study

the role of H2S or SP on CLP sepsis-induced defenestration and gaps formation in LSEC

fenestrae. The results of the present experiments with CSE gene deficient mice and PPTA gene

deficient mice show less defenestration and fewer gaps formation in LSEC fenestrae following

CLP-induced sepsis. Previous studies have shown that CSE-derived H2S acts as an

inflammatory mediator in sepsis and endotoxaemia by increasing proinflammatory cytokines

(TNF-α, IL-6 and IL-1β) and chemokines (MCP-1 and MIP-2α) (43, 126, 173, 177, 317). It has

also been shown that SP acts as an upstream regulator of CSE-derived H2S-mediated

inflammation in sepsis (141). In addition, the experimental results in Chapter 4 demonstrated

that mice deficient in the CSE gene regulate liver injury and inflammation following CLP-

induced sepsis. Mice with a CSE deficient gene have also shown decreased SP production

following CLP-induced sepsis, suggesting that SP acts as an upstream regulator of CSE-derived

H2S-mediated inflammatory response (shown in Chapter 5). As a regulators of inflammation,

H2S and SP play a role in the structural changes observed in LSEC fenestrae. For the first time,

the results of the present study indicate that increased inflammatory mediators through CSE-

derived H2S and SP following CLP-induced sepsis play a key role in the structural changes and

gaps formation in LSEC fenestrae.

Elucidating the mechanisms involved in the regulation and formation of LSEC fenestrations is

important in understanding the underlying pathology of liver diseases. Several previous in

vivo and in vitro studies have demonstrated the mechanisms of LSEC defenestration

associated with different experimental disease models of liver injury. For example, the

mechanism of alcohol-induced liver injury and defenestration of LSECs involves deposition of

collagen, laminin, fibronectin and other connective tissue matrices in the space of Disse (254,

135

255, 361-363). This is due to activation (transformation) of fat storage cells (Ito cells) to

produce collagen with secondary changes in the endothelial cells by ethanol or one of its

metabolites (361, 363). Ageing, a natural process of the body, also increases the thickness of

LSECs with a reduction in the number of LSEC fenestrae (364-366). Pseudocapillarisation and

the formation of basement membrane with perisinusoidal fibrosis and central vein fibrosis

are involved in the defenestration process of LSECs with ageing (365, 367, 368). Other studies

have identified the involvement of actin cytoskeleton in the fenestration and defenestration

process of LSECs (251, 252, 341, 344, 369-374). Various actin cytoskeleton disruptors such as

cytochalasin B, latrunculin A, antimycin A, misakinolide and dihydrohalichondramide cause a

significant increase in the size and frequency of fenestrae, with a subsequent loss of liver sieve

plates (252, 343, 344, 371). These previous studies have established the importance of

cytoskeleton and membrane rafts in the dynamics of LSEC fenestrae. In addition to the

structural changes in the LSECs, abnormal function of LSECs has been reported during

experimental sepsis. LSECs undergo Fas-mediated apoptosis through activation of gp130

expression and by ligating programmed cell death ligand 1 (PD-L1) (263, 375). In both these

experiments, Kupffer cells were the key macrophages of LSEC injury during sepsis.

The present results show that released proinflammatory mediators following sepsis cause

LSEC defenestration and that Kupffer cells, CSE-derived H2S and the SP play an important role

in regulating fenestral structures in LSECs. Nevertheless, the present experiments did not

investigate the mechanisms of defenestration in LSEC fenestrae following CLP-induced sepsis.

Furthermore, this study is limited by the short duration of the endpoint (8 h post-CLP). It is

important to study the time dependent changes in LSEC fenestrae for longer periods using

136

the same animal disease model for better understanding of defenestration process in LSECs;

however, this has not been investigated in the present study.

In conclusion, for the first time the present study has demonstrated that the CSE-derived H2S,

SP and Kupffer cells play a role in the regulation of defenestration and gaps formation in LSEC

fenestrae following CLP-induced sepsis. The present study indicates the importance of H2S,

SP and Kupffer cells in the structural maintenance of LSEC fenestrae under the

pathophysiological conditions of sepsis.

137

Chapter 8

Circulatory hydrogen sulfide and substance P levels in patients with sepsis

admitted to the intensive care unit

8.1 Introduction

Sepsis is life-threatening organ dysfunction that arises from the body’s response to infectious

stimuli (1). Despite advances in antimicrobial therapy and supportive care, sepsis remains a

leading cause of death in critically ill patients in the ICU and arises from an overwhelming

inflammatory response and multiple organ injury (23, 376). For instance, in the United States,

sepsis incidence rates increased from 359 cases per 100,000 population in 2003 to 535 cases

per 100,000 population in 2009 (a 49% increase), accounting for more than $20 billion (5.2%)

of total United States hospital costs in 2011 (4, 6, 8). Although antibiotic treatment may

efficiently eradicate the infection, it does not specifically reverse the systemic inflammation

and its sequelae. A better understanding of the pathogenesis of sepsis is central to identifying

novel targets for new therapeutic interventions designed to reverse systemic inflammation

and its sequelae.

H2S and SP act as proinflammatory mediators in both infectious and non-infectious

inflammatory disease models such as sepsis (43, 197), acute pancreatitis (129, 377) and burn

injuries (313, 378). For example, increased H2S-synthesising activity and plasma H2S levels

were observed in CLP-induced sepsis and caerulein-induced acute pancreatitis (132, 177).

Treatment with H2S donors such as NaHS and Na2S increased systemic inflammatory

response, whereas the CSE inhibitor PAG protected against organ injury and inflammatory

response in sepsis and acute pancreatitis (43, 44, 140, 177). Similarly, increased upregulation

of SP and NK-1R activation have been reported in sepsis and acute pancreatitis. Treatment

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with NK-1R antagonist and gene deletion of PPTA showed protection against organ injury,

inflammation and systemic inflammatory response following sepsis and acute pancreatitis

(195, 197). These results suggest that H2S and SP have a proinflammatory role in inflammatory

diseases.

Although H2S and SP are known proinflammatory mediators in experimental sepsis and other

inflammatory disease models, their role in human sepsis has been poorly understood. One

study has reported higher serum H2S levels in septic patients compared to non-septic and

healthy subjects; however, this study was confined to a very small number of patients (n=5)

(172). Similarly, previous studies have also reported circulatory SP levels in septic patients

(201-203). For example, Arnalich et al. observed lower SP levels in plasma of both septic and

septic shock patients (associated with infection) compared with healthy volunteers for all

time points analysed (onset, 12 h and 24 h) (202). In contrast, Beer et al. observed higher

serum SP levels in post-operative septic patients than in patients without post-operative

sepsis, with similar malignant disease on all days analysed. However, when survivors and non-

survivors were compared, higher SP levels were only associated with lethal outcome during

the late course of sepsis (203). In contrast, a recent study by Lorente et al. found that surviving

septic patients showed higher serum SP levels than nonsurvivors (201). These studies, with

small numbers of septic patients in the case of H2S, and controversial reports with SP, have

clouded our understanding of the role of H2S and SP in septic patients. In addition, healthy

controls are very different physiologically from patients who are acutely unwell. Acutely

unwell non-septic patients experience a wide variety of activated stress responses that may

modulate the response to sepsis. Therefore, studying role of H2S and SP in human sepsis

(associated with infection) in comparison to patients with similar disease severity and organ

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dysfunction from non-infectious complications is important in understanding the precise role

of H2S and SP in sepsis.

The objective of this study was to determine the circulatory H2S and SP levels in septic patients

compared to patients with similar disease severity and organ dysfunction complications from

non-infectious aetiology admitted to the ICU.

8.2 Aims

The aims of this study was to determine:

1) The alteration in plasma H2S and SP levels in septic patients compared to non-septic

patients in the acute phase of the illness; and

2) The association between plasma H2S and SP levels and the inflammatory response in

septic patients compared to non-septic patients.


8.3.1 Design and subjects

The study was approved by the Health and Disability Ethics Committee (HDEC), Wellington,

New Zealand (protocol number 15/STH/36). This study was conducted on intensive care

patients at Christchurch Hospital, New Zealand. Informed consent was obtained from patients

in all cases. Where patients were unable to give consent, provisional consent was obtained

from family members or friends and consent was reviewed with the patient when they

recovered. The selection criteria was broadly based on those used for the Australasian

Resuscitation in Sepsis Evaluation-Randomised Controlled Trial (ARISE-RCT) (379). All patients

older than 18 years admitted to the ICU were included in the study, except those not expected

to survive 72 h or when consent could not be obtained. Patients with suspected or confirmed

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infection, systemic inflammatory response syndrome, evidence of either refractory

hypotension or hypoperfusion and whose first dose of IV antimicrobial therapy commenced

prior to randomisation were included in the sepsis cohort. Patients admitted to the general

ICU without sepsis or other known inflammatory conditions with matched age, gender,

severity of illness and organ dysfunction were included in the non-septic cohort. Patients aged

below 18 years and with a contraindication to blood products, hemodynamic instability due

to active bleeding, pregnancy (confirmed or suspected) and in-patient transfer from another

acute health care facility were excluded from this study. Two independent investigators

assessed patients for study eligibility according to the described criteria and collected data

throughout the study period.

All patients with sepsis and non-sepsis were treated by standard supportive treatment, fluid

resuscitation, vasoactive drugs, medical and technological interventions for organ

dysfunction or failure, and empirical antibiotic therapy according to hospital practice

guidelines. Relevant clinical details were taken from hospital records and the interpretation

discussed with Professor Geoffrey Shaw and Professor Stephen Chambers. Professor Geoff

Shaw is an intensive care specialist with clinical and research interests in the area of sepsis

and sepsis biomarkers and participated in study design, patient recruitment, and analysis of

clinical data, and in dissemination of results. Professor Stephen Chambers is a physician

researcher with interests in the area of infectious diseases and participated in study design,

data analysis and dissemination of results.

8.3.2 Variables recorded

The following variables were recorded for each patient: age and sex, type of infection, Acute

Physiology and Chronic Health Evaluation (APACHE) II and III Scores, Simplified Acute

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Physiology Score (SAPS) II, while failure of organs and severity of MODS were evaluated by a

Sequential (Sepsis-Related) Organ Failure Assessment (SOFA) Score. APACHE II and III and

SAPS II were determined on the day of ICU admission whereas SOFA scores were determined

daily on observation from day 0 to day 4 (1, 380).

8.3.3 Blood sampling and laboratory assays

Blood samples were collected from all ICU patients from the time of admission to 96 h, with

collections at different time intervals (0 h, 12 h, 24 h, 48 h, 72 h and 96 h). Collection of blood

samples were stopped if the patient was discharged from ICU to another hospital ward in

between blood collection time points.

Blood samples from septic and non-septic patients were drawn through venipuncture,

collected in a plastic tube containing EDTA, then immediately placed on ice. Assays for blood

lactate and C-reactive protein (CRP) were performed at Canterbury Health Laboratory (CHL),

Christchurch, New Zealand. Remaining blood samples were centrifuged at 1,000xg for 10 mins

at 4oC and plasma was stored at -80oC for further analysis. Plasma H2S (Section 2.7), SP

(Section 2.13), IL-6 (Section 2.12) and procalcitonin (PCT) (Section 2.14) levels were measured

by the methods described in the Materials and Methods (Chapter 2).

8.3.4 Statistical analysis

All the data were analysed for Gaussian or Normal distribution using the Shapiro-Wilk test.

Demographics were presented as means with a 95% confidence interval. Non-parametric

Wilcoxon Rank Sum test was used to compare the medians of septic and non-septic groups

(rather than means) when normality assumption was violated. When normality assumptions

held, ordinary t-test was carried out to assess the statistical difference in means. A Repeated

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Measures two-way ANOVA (RM-two-way ANOVA) was applied to compare two groups at

different time points. P-values less than 0.05 indicated a significant difference between two

groups. All statistical analysis were performed using SAS 9.3 and GraphPad Prism 6.07.

8.4 Results

8.4.1 Demographic characteristics of patients

A total of thirty seven (n=37) patients were recruited for this study. Of these, fourteen

patients (n=14) were non-septic and twenty three (n=23) were septic. There was no significant

difference in the mean age of either of the two groups; however, a male predominance was

seen in both groups. A summary of the major reasons for admission to ICU and infecting

organisms identified are shown in Table 8.1. Detailed tables, including the major presenting

diagnosis, complications and co-morbidities, are included as tables in the appendix to this

thesis.

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Table 8.1 Characteristics of patients with non-sepsis and sepsis admitted to medical intensive care

unit (ICU) during the 2015-16 study period.

* Polymicrobial infection denotes when more than one organism was identified from a normally sterile

site. Ten of these patients had peritonitis from intra-abdominal pathology, one Fournier’s gangrene

and one with infection of ischium. ** No patient was not identified with any organisms.

Characteristic Non-sepsis Sepsis

Demographics Number of patients (N) N=14 N=23

Age in years (Mean ± SD), (95% CI) 64.06 ± 2.73 (59.56-68.56)

63.92 ± 2.96 (58.11-69.73)

Male 10 12

Female 4 11

Major reasons for admission to ICU

Post-coronary artery by-pass grafting 4

Post-repair of aortic dissection 2

Persistent seizures causing multiple fractures and soft tissue injury

2

Post-surgery aortic stenosis 1

Cardiac arrest out of hospital causing brain injury 1

Myocardial infarction 1

Congestive heart failure 1

Failed renal transplant 1

Car crash causing multi-trauma 1

Peritonitis of multiple causes 10

Community-acquired pneumonia 6

Fournier’s gangrene 1

Necrotizing pancreatitis 1

Pancreatitis, cholecystitis 1

Septic arthritis 1

Osteomyelitis and soft tissue infection 1

Pyelonephritis with obstructed outflow tract 1

Post-coronary artery by-pass surgery infection 1

Type of microorganism causing sepsis Polymicrobial sepsis* N=12

Pseudomonas aeruginosa N=4

Candida albicans N=1

Cytomegalovirus N=1

Escherichia coli N=1

Klebsiella pneumoniae N=1

Legionella sp. N=1

Pantoea agglomerans N=1

No organism identified** N=2

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8.4.2 Comparison of disease severity and organ dysfunction between septic and non-septic

patients

Patient disease severity and organ dysfunction of both septic and non-septic patients are

shown in Table 8.2. The APACHE II and III and SAPS II scores calculated at the time of ICU

admission and SOFA scores (from day 0 to day 4 of ICU admission) were not statistically

different between septic and non-septic patients. SOFA scores gradually decreased from day

0 to day 4 in both septic and non-septic groups. Seven of the total thirty seven (7/37) patients

died (four from the non-sepsis group and three from the sepsis group) during the observation

period in the ICU.

Table 8.2 Disease severity, organ dysfunction scores and mortality in septic and non-septic patients

admitted to the medical intensive care unit (ICU) during the 2015-16 study period.

Fourteen non-septic patients and twenty three septic patients were included in this study. There was

no significant difference in APACHE II and III, SAPS II and SOFA scores between septic and non-septic

patients. SOFA scores gradually decreased from day 0 to day 4 in both septic and non-septic groups. A

total of seven patients (four from the non-septic group and three from the septic group) died during

the observation period in the ICU. Results were expressed as mean ± S.E.M (n=14 with non-septic

patients and n=23 with septic patients). Statistical significance between two groups was evaluated by

ordinary t-test.

Characteristic Non-sepsis Sepsis

Disease severity and organ dysfunction scores (Mean ± S.E.M)

APACHE II (Day 0) 21 ± 1.70 19.04 ±1.23

APACHE III (Day 0) 77.76 ± 5.61 76.32 ± 5.44

SAPS II (Day 0) 46.47 ± 3.02 45.26 ± 3.28

SOFA (Day 0) 10.47 ± 0.68 9.04 ± 0.52

SOFA (Day 1) 9.93 ± 0.94 7.94 ± 0.69

SOFA (Day 2) 9.15 ± 0.93 7.13 ± 0.85

SOFA (Day 3) 8.50 ± 1.04 5.80 ± 0.83

SOFA (Day 4) 7.78 ± 1.49 6.77 ± 1.46

Mortality (30 day mortality)

Total patients N=14 N=23

Died 4 3

Alive 10 20

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8.4.3 Plasma H2S and SP levels in septic patients compared to non-septic patients

Plasma levels of H2S and SP were measured in both septic and non-septic patients at various

time points (from 0 h to 96 h). Septic patients showed higher H2S and SP levels compared to

non-septic patients at every time point, except for H2S at 96 h. Plasma H2S levels were

significantly higher at 12 h (1.45 vs 0.75; p<0.05) and 24 h (1.11 vs 0.72; p<0.01), whereas

plasma SP levels were only significant at 48 h (0.55 vs 0.31; p<0.05) compared to non-septic

patients.

Figure 8.1 Plasma levels of H2S and SP in septic patients. Blood samples were withdrawn from

intensive care septic and non-septic patients at different time intervals (0 h, 12 h, 24 h, 48 h, 72 h and

96 h). Plasma was aspirated and H2S (A) and SP (B) levels were measured using spectrophotometric

and SP immunoassay, respectively. Septic patients showed higher circulatory H2S and SP levels

compared to non-septic patients, with differences in H2S levels being significant at 12 h and 24 h and

with differences in SP levels being significant at 48 h. Graphs represent median with interquartile range

(n=14 with non-septic patients and n=23 with septic patients). Repeated Measures two way ANOVA

was used to plot the graphs and statistical significance between two groups at different time points

was evaluated by non-parametric Wilcoxon Rank Sum test. Statistical significance was assigned as

*P<0.05 and **P<0.01.

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Table 8.3 Plasma H2S and SP levels in septic patients.

Time Parameter Non-sepsis Sepsis P-value

0 hours H2S (µM/mL) 1.01 (0.19 – 2.65) 1.06 (0.31 – 8.20) 0.5148

SP (ng/mL) 0.19 (0.02 – 1.42) 0.58 (0.00 – 10.31) 0.2739

12 hours H2S (µM/mL) 0.75 (0.22 – 1.49) 1.45 (0.31 – 11.52) 0.0215

SP (ng/mL) 0.26 (0.13 – 1.24) 0.55 (0.00 – 10.79) 0.6915

24 hours H2S (µM/mL) 0.72 (0.17 – 0.91) 1.11 (0.24 – 3.70) 0.0056

SP (ng/mL) 0.43 (0.08 – 1.21) 0.53 (0.02 – 11.00) 0.5773

48 hours H2S (µM/mL) 0.61 (0.10 – 2.42) 1.16 (0.21 – 23.07) 0.1184

SP (ng/mL) 0.31 (0.01 – 1.41) 0.55 (0.17 – 18.39) 0.0473

72 hours H2S (µM/mL) 0.52 (0.03 – 1.82) 1.16 (0.35 – 10.31) 0.0806

SP (ng/mL) 0.29 (0.10 – 1.27) 0.88 (0.04 – 12.35) 0.1098

96 hours H2S (µM/mL) 1.72 (0.17 – 4.11) 1.19 (0.55 – 4.14) 0.9247

SP (ng/mL) 0.35 (0.17 – 1.15) 0.87 (0.02 – 2.36) 0.1200

Blood samples were withdrawn from intensive care septic and non-septic patients at different time

intervals (0 h, 12 h, 24 h, 48 h, 72 h and 96 h). Plasma was aspirated and H2S and SP levels were

measured using spectrophotometric and SP immunoassay, respectively. Septic patients showed higher

circulatory H2S and SP levels compared to non-septic patients, with differences in H2S levels being

significant at 12 h and 24 h and with differences in SP levels being significant at 48 h. Statistical

significance between two groups at different time points was evaluated by nonparametric Wilcoxon

Rank Sum test. Results were expressed as median with range. (n=14 with non-septic patients and n=23

with septic patients). P-values less than 0.05 were considered statistically significant.

8.4.4 Plasma PCT, IL-6, CRP and blood lactate levels in septic patients compared to non-

septic patients.

Septic patients showed higher plasma PCT, IL-6 and CRP levels at all time points except, at 96

h in the case of PCT and IL-6. Although septic patients showed higher plasma PCT, IL-6 and

CRP levels, these were not significant at all the time points when compared to non-septic

patients. Plasma PCT levels in septic patients were significantly higher at 0 h (1.45 vs 0.75;

p<0.05), whereas IL-6 levels were significantly higher at all time points except 96 h (0 h, 522.35

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vs 144.05, p<0.0001; 12 h, 281.8 vs 94.26, p<0.001; 24 h, 198.7 vs 77.54, p<0.001; 48 h, 147.15

vs 53.135, p<0.001; and 72 h, 103.4 vs 42.93, p<0.01) compared to non-septic patients.

Figure 8.2 Plasma levels of PCT, IL-6, CRP and blood lactate in septic patients. Blood samples were

withdrawn from intensive care septic and non-septic patients at different time intervals (from day 0 to

day 4 at 0 h, 12 h, 24 h, 48 h, 72 h and 96 h). Plasma was aspirated and PCT (A) and IL-6 (B) levels were

measured by immunoassay, while CRP (C) and lactate (D) levels were measured by spectrophotometric

assay. Septic patients showed higher circulatory PCT and IL-6 levels compared to non-septic patients,

with significance at 0 h for PCT and at 0 h, 12 h, 24 h, 48 h and 72 h for IL-6. Septic patients showed

higher circulatory CRP levels compared to non-septic patients, with significance at day 0, day 1 and

day 2. There was no difference between septic and non-septic patient blood lactate levels except at

day 3 when being significantly higher levels were seen in septic patients. Graphs represent median with

interquartile range (n=14 with non-septic patients and n=23 with septic patients). Repeated Measures

two way ANOVA was used to plot the graphs and statistical significance between two groups at

different time points was evaluated by non-parametric Wilcoxon Rank Sum test. Statistical significance

was assigned as *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.

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In the case of CRP, septic patients showed significantly higher CRP values at day 0 (302.5 vs

83, p<0.001), day 1 (339 vs 165, p<0.001) and day 2 (284 vs 195, p<0.05). In contrast, there

was no difference in blood lactate levels between septic and non-septic patients, except at

day 3 (1.1 vs 0.6, p<0.01) when they were significantly higher in septic patients.

Table 8.4 Plasma PCT, IL-6, and CRP and blood lactate levels in septic patients.

Time Parameter Non-sepsis Sepsis P-value

0 hours

PCT (pg/mL) 120.95 (66.86-145.2) 371.4 (72.93 – 4644.38) 0.0472

IL-6 (pg/mL) 114.05 (9.31 – 403.3) 522.35 (119 – 8441.4) <0.0001

CRP (mg/L) 83 (17 – 303) 302.5 (70 – 460) 0.0004

Blood Lactate (mmol/L) 1.85 (0.4 – 6.0) 2.1 (0.4 – 6.5) 0.4686

12 hours

PCT (pg/mL) 121.5 (81.66 – 157.8) 414.5 (34.59 – 4953.19) 0.0513

IL-6 (pg/mL) 94.265 (8.5 – 677.6) 281.8 (39.17 - 3965) 0.0010

CRP (mg/L) - - -

Blood Lactate (mmol/L) - - -

24 hours

PCT (pg/mL) 93.01 (39.86 – 141.3) 437 (28.21 – 5208.51) 0.0513

IL-6 (pg/mL) 77.54 (8.64 – 219) 198.7 (59.37 – 1334) 0.0004

CRP (mg/L) 165 (14 – 336) 339 (99 – 450) 0.0005


48 hours

PCT (pg/mL) - - -

IL-6 (pg/mL) 53.135 (11.92-135.2) 147.15 (25.17 – 945.20) 0.0004

CRP (mg/L) 195 (7 – 313) 284 (111 – 497) 0.0187


72 hours

PCT (pg/mL) - - -

IL-6 (pg/mL) 42.93 (18.47 – 83.33) 103.4 (29.42 – 1088.2) 0.0041

CRP (mg/L) 163 (18 – 306) 179 (57 – 374) 0.2465


96 hours

PCT (pg/mL) - - -

IL-6 (pg/mL) 48.69 (17.2 – 290.5) 91.3 (19.36 – 310.9) 0.1278

CRP (mg/L) 166.5 (81 – 262) 165 (40 – 377) 0.9646


Blood samples were withdrawn from intensive care septic and non-septic patients at different time

intervals (from day 0 to day 4 at 0 h, 12 h, 24 h, 48 h, 72 h and 96 h). Plasma was aspirated and PCT

and IL-6 levels were measured by immunoassay, while CRP and lactate levels were measured by

spectrophotometric assay. Septic patients showed higher circulatory PCT and IL-6 levels compared to

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non-septic patients, with significance at 0 h for PCT and at 0 h, 12 h, 24 h, 48 h and 72 h for IL-6. Septic

patients showed higher circulatory CRP levels compared to non-septic patients, with significance at

day 0, day 1 and day 2. There was no difference between septic and non-septic patient blood lactate

levels, except at day 3 when significantly higher lactate levels were seen in septic patients. Statistical

significance between two groups at different time points was evaluated by non-parametric Wilcoxon

Rank Sum test. Results were expressed as median with range. (n=14 with non-septic patients and n=23

with septic patients). P-values less than 0.05 were considered as statistically significant.

8.5 Discussion

The studies described in Chapters 3 to 5 showed the proinflammatory role of CSE-derived H2S

and the importance of H2S-mediated upregulation of SP in CLP-induced sepsis. In this chapter,

the alteration of plasma H2S and SP levels in septic patients was described. The most relevant

and new finding of this study shows that septic patients have higher circulatory H2S and SP

levels than patients with similar disease severity and organ dysfunction from non-infectious

complications. This study also suggests that there is a correlation of initial plasma H2S and SP

concentrations and the inflammatory response in septic patients at the time of admission to

the ICU.

This is the first report to study alterations in plasma H2S levels in septic patients. The results

of the present study demonstrate that septic patients have higher plasma H2S levels

compared to non-septic patients without infectious complications, with peak values at 48 h.

Circulatory SP levels followed a similar pattern to H2S levels: higher in septic patients than

non-septic patients, with peak values at 48 h. Previously, circulatory SP levels have been

assessed in septic patients; however, these studies mainly focused on comparison of septic

patients with healthy controls, survival with non-survival septic patients and reported results

contradict one another (201-203). For example, Arnalich et al. and Mpouzika et al. observed

lower SP levels in septic patients than in control subjects (202). In contrast, Beer et al. found

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higher serum SP levels in post-operative septic patients than control subjects and during the

final phase of lethal sepsis (203). A recent study by Lorente et al. showed higher serum SP

levels in survivor septic patients than non-survivors (201). None of these studies observed the

association of SP concentrations in septic patients compared to patients with similar non-

infectious complications. This study shows, for the first time, that higher circulatory H2S and

SP levels are associated with sepsis in a patient cohort with severe clinical conditions requiring

admission to intensive care.

Inflammation is an essential host defense mechanism required for protection against

pathogenic microorganisms; however, an excessive inflammatory response is harmful to the

host. In sepsis, there is a loss of homeostatic control over the inflammatory response

mounted against an infectious agent, resulting in a proinflammatory, procoagulant state that

can result in organ dysfunction and even death. The present study demonstrates that as

mediators of inflammation, elevated plasma H2S and SP levels at initial time points are

important for the host defensive mechanism to eradicate infections. However, overwhelming

increases in circulatory H2S and SP levels exacerbate the systemic inflammatory response that

leads to organ damage. Different experiments using mouse models (of CLP-induced sepsis)

confirm the detrimental role of H2S and SP in sepsis (43, 126, 140, 177, 195, 197, 381).

Increased tissue H2S-synthesising activity, plasma H2S levels and tissue and plasma SP levels

were found following CLP-induced sepsis (177, 197). Administration of H2S donors such as

NaHS and Na2S showed increased liver and lung inflammation and systemic inflammation

whereas treatment with PAG and an NK-1R antagonist showed protection against CLP sepsis-

induced organ injury and inflammation (44, 140, 195). The results with CSE KO mice described

in Chapters 3 and 4 and previous studies with PPTA KO mice showed protection against CLP

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sepsis-induced organ injury and inflammation (199, 200). Together, the results of the present

study suggest that alteration in circulatory H2S and SP levels in septic patients are consistent

with results from experimental sepsis studies and that these mediators play an important role

in the inflammatory process of sepsis in both experimental and human sepsis.

The present study documented the severity of the disease and multiple organ dysfunction in

both septic and non-septic patients at presentation to the ICU and over the course of the

admission. As expected, there was no significant difference in disease severity and organ

dysfunction scores such as APACHE II and III, SAPS II and SOFA between these two groups and

as expected. SOFA scores gradually decreased from day 0 to day 4 as the patients improved.

These results demonstrate that a severely disordered physiological response and organ

dysfunction alone are not associated with changes in circulatory levels of H2S and SP but, are

specific to the inflammatory response. Therefore, non-septic patients served as an

appropriate control group.

Altered plasma H2S and SP levels are positively correlated with PCT and CRP levels in septic

patients. CRP is an acute reactant protein commonly used to detect inflammation caused by

different aetiologies, including sepsis (382, 383). On the other hand, it is well known that

elevated levels of plasma PCT positively correlate with severity of infection. Over the last

decade, PCT has grown increasingly popular as a diagnostic marker for bacterial infections

and, it has been suggested, is more helpful than CRP (384, 385). Various recent studies also

confirm the significant relationship between elevated plasma PCT levels and inflammatory

response in sepsis (386-390). Results of the present study show that septic patients have

higher plasma levels of both CRP and PCT, with peak values at day 1 (24 h) and 48 h,

respectively. Although septic patients have higher plasma CRP levels, altered patterns of CRP

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levels were not closely correlated with plasma H2S and SP levels; alternatively, PCT levels

follow H2S and SP levels more closely. Plasma H2S, SP and PCT levels gradually increased from

0 h to reach peak values at 48 h, then decreased thereafter to 96 h. These results suggest that

in septic patients elevated circulatory PCT levels are more strongly correlated with H2S and SP

levels than are CRP levels. These results confirm that higher circulatory H2S and SP levels are

more strongly induced in septic patients than in patients with non-infectious complications.

The present study also demonstrated alterations in plasma IL-6 levels, which correlated with

plasma H2S and SP levels in septic patients. IL-6 serves as both a marker and mediator for

severity of sepsis and is a predictor of early-phase acute response in septic patients. Several

reports indicate that plasma IL-6 may be used as a diagnostic marker for the presence of

bacteremia (391-393). The results of the present study showed higher plasma IL-6 levels in

septic patients compared to non-septic patients at the time of admission (0 h) to the ICU.

However, IL-6 levels gradually decreased from 0 h to 96 h and at 96 h there was no significant

difference between septic and non-septic patients. Although septic patients showed higher

plasma IL-6 levels, there was no correlation between IL-6 and altered H2S and SP levels. In

contrast to IL-6, H2S and SP levels gradually increased from 0 h to 48 h, then decreased

towards 96 h. Together, these results suggest that IL-6 may be used clinically as an early stage

predictor of sepsis, while H2S and SP become elevated at a later stage (within 48 h) of the

inflammatory response in septic patients.

The alteration of plasma H2S and SP levels in septic patients followed two phases. In the first

48 h of the ICU admission, plasma H2S and SP levels gradually increased from 0 h, reaching

peak values at 48 h. During the second phase, between 48 h and 96 h, levels gradually

decreased and at 96 h there was no difference. In addition, an increase in H2S levels preceded

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increases in SP levels, as shown in previous studies on experimental sepsis (141). Based on

these observations, it is reasonable to assume that during the early stages of infection or

sepsis, elevated H2S and SP levels from activated immune cells are essential to achieve

adequate host defense against invading pathogens. At later stages of infection, resolution of

inflammation and restoration of normal tissue function are critical events following the

clearance of the infectious agent. The pattern of decreased PCT and CRP levels and SOFA

scores, along with H2S and SP levels, was consistent with the resolution of inflammation and

restoration of normal tissue function at later stages (after 48 h) of sepsis.

This study was not powered to find which concentrations of H2S and SP would predict the

onset or prognosis of sepsis; both the small number of patients and the heterogeneous nature

of patients admitted to intensive care did not allow for such interpretation. Although septic

patients showed higher circulatory H2S and SP levels and a positive correlation with sepsis,

these mediators were not significant at all time points. In addition, the initial height of H2S

and SP concentrations (0 h to 48 h) did not correlate with the later course of the disease (48

h to 96 h). Therefore, further studies with a larger sample size, that would allow subset

analysis of separate causes of sepsis such as pneumonia or abdominal sepsis, are required to

predict significance and prognostic importance of H2S and SP in human sepsis.

In conclusion, for the first time, this study shows that higher circulatory H2S and SP levels are

associated with the inflammatory response in septic patients. The results show that H2S and

SP are important in clinical sepsis and that increases in H2S levels precede those in SP levels,

as they are in experimental sepsis. Therefore, alteration in circulatory H2S and SP levels may

be of pathological significance in septic patients and research into modifications in H2S and

SP levels could be of interest as a therapeutic potential in these patients.

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Chapter 9

General discussion, conclusions and future perspectives

9.1 General discussion

The objective of this study was to investigate the role of the endogenous H2S, SP and Kupffer

cells on the inflammatory response, organ injury and LSEC fenestration following CLP-induced

sepsis. Further, this thesis investigated alteration of circulatory H2S and SP levels and their

possible association with the inflammatory response in septic patients.

The first part of the thesis (Chapters 3 to 5) examined whether endogenous CSE-derived H2S

would affect the inflammatory response and organ injury following sepsis. These chapters

also examined the mechanisms by which CSE-derived H2S regulates inflammation and

interaction between H2S and SP in sepsis. The intensity of systemic inflammation was

evaluated by tissue MPO activity, a marker of leukocyte infiltration, while the severity of

MODS was evaluated by histology. It was found (Chapter 3) that CLP-induced sepsis resulted

in a significant increase in CSE protein expression in liver and lung tissues, H2S-synthesising

activity in the liver and H2S levels in the plasma suggesting increased endogenous H2S

synthesis by CSE following CLP-induced sepsis. For the first time, the present study revealed

that mice with CSE gene deletion have less sepsis-associated systemic inflammation and

organ injury (liver and lung oedema and necrosis). Although previous findings using the CSE

inhibitor PAG have showed protection against CLP sepsis-induced systemic inflammation and

organ injury, these studies have limitations due to the non-specific actions of PAG such as

inhibition of ALT and AST enzyme activities that are unrelated to CSE enzyme inhibition (140,

174, 177, 265, 266, 277). In addition, dosage regime, route and time of administration of H2S

155

donors and inhibitors activate or inhibit different signalling pathways, which in turn produce

either a pro- or anti-inflammatory effect in sepsis. The results in Chapter 3 have demonstrated

that CSE-derived H2S plays an important role in regulating leukocyte infiltration and

associated liver and lung injury.

To further elucidate the role of CSE-derived H2S in sepsis, the association between cytokine

and chemokine expression and the involvement of ERK1/2-NF-B signalling with increased

endogenous H2S in sepsis was investigated (Chapter 4). It was found that mice with CSE gene

deletion had lower proinflammatory cytokine (TNF-α, IL-6 and IL-1β) and chemokine (MCP-1

and MIP-2α) generation following sepsis, which is consistent with data on tissue MPO activity

and liver and lung organ injury. In addition, mice deficient in the CSE gene not only had less

ERK1/2 phosphorylation, but also reduced NF-B p65 activation. These results demonstrated

for the first time that temporal regulation of CSE expression, H2S-synthesising activity and H2S

levels correlate with the activation of ERK1/2-NF-B p65 signalling. Although previous studies

using PAG and H2S donors such as Na2S, NaHS and GYY4137 have investigated the

mechanisms involved in the H2S-mediated inflammatory response in sepsis, these studies

suffered due to the limitations of PAG and H2S donors, as described in Section 1.6 (Chapter 1)

(43, 44, 129, 133, 172, 176, 265-267, 313, 317). Together, these findings suggest that

increased CSE-derived H2S in sepsis may participate in the phosphorylation of ERK1/2 and

thereby promote activation of NF-B p65, leading to increased production of

proinflammatory mediators and exacerbating systemic inflammation and multiple organ

damage.

Further investigation then focused on the relationship between H2S and SP in sepsis (Chapter

5). Mice with CSE gene deletion showed less SP production and NK-1R protein expression

156

following sepsis. These results are consistent with previous studies with PAG (141) and

suggest a potential linkage between CSE-derived H2S and the production of SP and

subsequent activation of NK-1R in sepsis.

Together, results from the first part of the thesis (Chapters 3 to 5) suggest that increased CSE-

derived H2S regulates inflammatory response and organ damage, both directly and indirectly,

following sepsis. Through activation of the ERK1/2-NF-B p65 signalling pathway and

subsequent release of proinflammatory cytokines and chemokines, H2S directly regulates

inflammation and organ damage following sepsis. Indirectly, H2S increases proinflammatory

cytokine and chemokine generation by upregulating SP production and subsequent NK-1R

activation following CLP-induced sepsis.

The second part of the thesis (Chapter 6) explored the role of Kupffer cells in sepsis-associated

liver and lung injury, inflammation and the systemic inflammatory response. This is the first

study to report the significance of Kupffer cell inactivation on multiple organ damage and

systemic inflammatory response following CLP-induced sepsis. GdCl3 pretreatment resulted

in protection against sepsis-induced liver injury and inflammation as evidenced by a decrease

in liver MPO activity, liver necrosis and oedema and liver proinflammatory cytokines and

chemokines. In contrast, GdCl3 pretreatment failed to protect against sepsis-induced lung

injury and inflammation, and the systemic inflammatory response. However, previous

research has reported results contradictory to the present experiments; this could be due to

differences in the experimental animal disease model and dosage regime of GdCl3 (220, 223-

225, 228, 237-239). The results from the present experiments suggest that GdCl3

pretreatment selectively reduces liver injury and inflammation associated with CLP-induced

sepsis.

157

The results from the first two parts of the thesis (Chapters 3 to 6) suggest the significance of

both CSE-derived H2S and Kupffer cells in the inflammatory process during sepsis. Although

Kupffer cells are crucial in trafficking pathogenic substances and subsequent local and

systemic inflammatory response, inactivation of Kupffer cells by GdCl3 selectively protects

mice against liver inflammation and injury, whereas CSE gene deletion protects against liver

and lung inflammation, injury and the systemic inflammatory response in sepsis.

The third part of the thesis (Chapter 7) examined structural changes in LSEC fenestrae

following CLP-induced sepsis and the effect of GdCl3, CSE gene deletion and PPTA gene

deletion on CLP sepsis-induced structural changes in LSEC fenestrae. This is the first study to

report the importance of H2S, SP and Kupffer cells on CLP sepsis-induced structural changes

in LSEC fenestrae. Mice with CLP-induced sepsis showed defenestration (decreased diameter,

frequency and porosity) and increased formation of gaps in LSEC fenestrae. These results are

consistent with previous studies, which showed the release of proinflammatory mediators

and the inflammatory response are associated with infection and endotoxaemia leading to

defenestration in LSECs (235, 261, 262, 352, 353, 394, 395). In this study, mice pretreated

with GdCl3, CSE gene deletion and PPTA gene deletion showed less defenestration and fewer

gaps formation in LSEC fenestrae following sepsis. Together, these results suggest the

importance of inflammatory mediators released by activation of Kupffer cells, H2S and SP

during sepsis as they may play a role in the regulation of LSEC fenestrae.

The final part of this thesis (Chapter 8) examined the alteration of circulatory H2S and SP levels

in patients with sepsis admitted to the ICU. The is the first study to report an alteration in

circulatory H2S levels in septic patients and the possible association between circulatory H2S

and SP levels and the inflammatory response as compared to patients with similar disease

158

severity and organ dysfunction from non-infectious complications. The results showed that

septic patients have higher circulatory H2S and SP levels and that these are associated with

the inflammatory response in septic patients. Furthermore, these results shed light on the

correlation between experimental sepsis and human sepsis with regard to the

proinflammatory role of H2S and SP.

9.2 Conclusions and future perspectives

The work of this thesis shows the importance of the CSE-derived H2S, SP and Kupffer cells in

mediating the regulation of the inflammatory response, organ injury and structural changes

in LSEC fenestrae in sepsis. Increased CSE-derived H2S may upregulate the systemic

inflammatory response and thus contribute to organ damage in sepsis. For instance, it may

provoke the production of proinflammatory mediators (cytokines and chemokines) as well as

leukocyte trafficking via the activation of the ERK1/2-NF-B p65 pathway. It may also

stimulate the generation of SP and subsequent NK-1R activation, which aggravate liver and

lung inflammation and injury. Furthermore, increased CSE-derived H2S and SP may cause

defenestration and the formation of gaps in LSEC fenestrae. As a result, inhibition of the

proinflammatory effect of the CSE-derived H2S through a CSE gene knockout approach seems

to protect animals against the sepsis-associated inflammatory response, organ injury and

defenestration in LSECs. Furthermore, inactivation of Kupffer cells using GdCl3 may protect

against liver injury, inflammation and LSEC defenestration in sepsis.

This thesis also shows the significance of circulatory H2S and SP on the inflammatory response

in septic patients. Higher circulatory H2S and SP levels may be associated with the

inflammatory response in septic patients. The schematic summary of the thesis conclusions

159

depicted in Figure 9.1

Figure 9.1 Schematic summary of the role of CSE-derived H2S, SP and Kupffer cells in sepsis. Sepsis

results in increased CSE protein expression, H2S-synthesising activity and H2S levels. Increased

endogenous H2S through CSE stimulates the activation of ER1/2, which subsequently activates the NF-

B. As a result, the transcription of genes for proinflammatory mediators is upregulated. The amplified

production of cytokines and chemokines, together with the augmented leukocyte infiltration,

aggravates the systemic inflammatory response in sepsis, exacerbating sepsis-associated multiple

organ failure. The CSE-derived H2S can also provoke the production of SP and NK-1R expression, which

worsen systemic inflammatory response and organ injury by leukocyte chemotaxis and cytokine

release. Sepsis also leads to the increased activation of Kupffer cells, which leads to the production of

proinflammatory cytokines and chemokines, causing liver and lung injury, inflammation and systemic

inflammatory response in sepsis. Increased H2S and SP production and Kupffer cell activation also cause

liver sieve injury by increasing defenestration and gaps formation in LSEC fenestrae.

The important contributions of this study are that it:

Demonstrates the proinflammatory role of the CSE-derived H2S in CLP-induced sepsis

160

and associated organ injury;

Presents mechanisms by which CSE-derived H2S modulates the inflammatory

response and may contribute to a better understanding of the proinflammatory role

of the H2S in inflammatory diseases;

Demonstrates that Kupffer cells are important in modulating hepatic injury following

various potentially hepatotoxic insults but it appears that they have little effect on

other organs, e.g., lung;

Demonstrates for the first time the role of Kupffer cells, CSE-derived H2S and SP on

CLP sepsis-induced structural changes in LSEC fenestrae; and

Demonstrates for the first time the alteration of circulatory H2S levels in septic

patients and a possible association between altered circulatory H2S and SP levels and

inflammatory response in septic patients.

As research in this area is still at an early stage, several key aspects are recommended for

exploration in future studies:

The work presented in this study was limited to only one time point (8 h post-CLP) and

represents changes that occur at that point only. Studying time-dependent changes in

the inflammatory response, organ injury and structural changes in LSEC fenestrae for

longer periods (at multiple time points) using same CLP model would lead to a better

understanding of the roles of CSE-derived H2S, SP and Kupffer cells in sepsis.

The mechanisms by which CLP sepsis-induced upregulation of CSE is not known. A

recent study has demonstrated the potential role of microRNA (miR-21) in specificity

protein-1 (SP1)-mediated regulation of CSE gene expression in smooth muscle cells

161

(309); however, further experiments need to be performed using CLP sepsis model to

determine whether sepsis modifies these elements in the CSE promoter region and

therefore increases the expression.

The mechanisms involved in CSE-derived H2S-mediated activation of ERK-1/2 and NF-

B p65 and SP remain unknown. Recently, Sen et al. have reported that H2S activates

NF-B by sulfhydrating the cysteine residues or other sites of the p65 subunit of NF-

B and thereby elicits the transcriptional activation of proinflammatory genes (327).

Furthermore, H2S may induce NF-B activation through mechanisms other than

ERK1/2 phosphorylation. For example, it has been shown that H2S induces a dose-

dependent increase in intracellular calcium ions (Ca+2) and PKA, which facilitate NF-B

translocation into the nucleus where it binds to DNA in microglial cells (114). These

mechanisms are yet to be investigated in CLP-induced sepsis. In addition, it is not clear

that whether pathophysiological action of H2S are mainly due to its direct effect or

substances derived from H2S. This is a current challenge in this field because of

complexity of H2S signalling and needs to be further investigated.

The role of GdCl3 in differential regulation of inflammation and organ injury in sepsis

are unclear. Further investigations to understand the mechanisms in differential

regulation of Kupffer cell mediated inflammation and multiple organ injury are

required.

The mechanisms by which CSE-derived H2S, SP and Kupffer cells mediate structural

changes in LSEC fenestrae in sepsis are unknown. One possible mechanism is

structural alteration in cytoskeleton of LSEC fenestrae by H2S and SP and the

mediators released upon activation of the Kupffer cells; however, this needs further

162

investigation.

The present study showed that higher circulatory levels of H2S and SP in patients with

sepsis admitted to the ICU; however, this study was limited by the small number of

patients recruited into the study. Further investigations with large number of patients

and using specific H2S inhibitors and donors and SP activators are required to

understand their therapeutic implications in septic patients.

163

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11 Appendix

11.1 Summary of primary diagnosis, complications and comorbidities of patients

with non-sepsis admitted to the ICU during the 2015-16 study period.

Non-Sepsis cohort

Primary diagnoses Complications Comorbidities

1 Failed living donor renal transplant

Pulseless electrical activity cardiac arrest, Fast atrial fibrillation, Bowel Ileus and Wound dehiscence

End stage renal failure, hypertension, subarachnoid haemorrhage, ventriculoperitoneal (VP)-shunt, gout, asthma and hyperparathyroidism

1 Car crash Multi-trauma Nil

2 Cardiac arrest out of hospital

Traumatic brain injury and subarachnoid haemorrhage

Splenic lymphoma, diverticular disease and prostate cancer

4 Myocardial infarction Refractory ventricular tachycardia

Nil

5 Congestive heart failure Hypotension Susac syndrome, non-Hodgkin’s lymphoma, gout, autologous bone marrow transplantation and chronic renal impairment

6 Post-coronary artery by-pass grafting

Hypotension Hypertension and goitre


Hypotension Myocardial infarction, atrial fibrillation, hypercholesterolemia and ischaemic cardiac myopathy


Asystolic cardiac arrest Hypertension, hyperlipidaemia and high Body Mass Index (BMI)


Required re-exploration of surgical site

Ischaemic heart disease, type 2 diabetes, obesity and hyperlipidaemia

10 Post-type A aortic dissection-repair

Ventricular tachycardia, cardiac arrest, multifocal cerebral infarcts and right sided hemiplegia

Hypertension, pyelonephritis and high BMI

11 Post-surgery for aortic stenosis

Hypotension Hypertension, hypercholesterolemia, myocardial infarction, hypothyroidism and cerebrovascular accident

12 Post-surgery for Type A aortic dissection

Hypotension Prostate cancer and myocardial infarction

13 Glioblastoma with seizures

Aspiration lung injury Hypertension

14 Astrocytoma causing status epilepticus

Multi-trauma Hypertension

195

11.2 Summary of primary diagnosis, complications and comorbidities of patients

with sepsis admitted to the ICU during the 2015-16 study period.

Sepsis-cohort

S. no Primary diagnoses Complications Comorbidities

1 Perforated appendix Peritonitis Prior sleeve gastrectomy surgery

2 Perforated bowel Peritonitis and left pneumothorax

Atrial fibrillation, ischemic heart disease and dyslipidaemia

3 Sigmoid perforation and Hartmann’s procedure

Faecal peritonitis Chronic obstructive pulmonary disease (COPD) and hypertension

4 Sigmoid tumour and Hartmann’s procedure

Peritonitis Myocardial infarction, ischaemic heart disease, hypertension and dyslipidaemia

5 Perforated diverticulum, emergency Hartmann’s procedure

Peritonitis Alport syndrome and hypertension

6 Perforation sigmoid colon and post laparoscopic hernia repair

Peritonitis and candidaemia

Cervical cancer

7 Post laparotomy for perforated colon

Peritonitis and scrotal abscess

Hypertension

8 Perforated diverticulitis

Peritonitis Obstructive sleep apnea, obesity, previous pancreatitis, hypertension, cerebrovascular accident, atrial fibrillation and type 2 diabetes mellitus

9 Resection of ischaemic small bowel and loop ileostomy

Peritonitis Previous Roux en Y for Candy Cane syndrome, Sphincter of Oddi dysfunction, diverticulosis, malnutrition and bipolar disorder

10

11 Fourniers gangrene Type 2 diabetes

12 Pancreatitis Cholecystitis and hepatobiliary sepsis

Ischaemic heart disease, heart failure, hypercholesterolemia, diverticulosis and chronic obstructive pulmonary disease

13 Necrotising pancreatitis Previous trans urethral prostatectomy and lumber disc prolapse

14 Osteomyelitis and soft tissue infection left ischial tuberosity.

Type I respiratory failure

T8 paralgia, biventricular pacemaker, cerebrovascular accident, obstructive sleep apnea obesity, melanoma hyperlipidaemia, colostomy, nissen fundoplication, depression and cholecystectomy

15 Post-operative coronary artery bypass grafting

Candidaemia Ischaemic heart disease, COPD; chronic kidney disease and left ventricular ejection fraction (LVEF) <25%

16 Pelvic ureteric junction obstruction of kidney

Pyelonephritis Renal calculi and glomerulonephritis

17 Septic arthritis knee Pneumonia Chronic kidney impairment

18 Community-acquired pneumonia

Empyema B cell lymphoproliferative disorder and gout

196


Nil


Rheumatoid on methotrexate and hypercholesterolemia


Myocardial infarction and atrial fibrillation


Lung abscess Nil


Pulmonary oedema Ischaemia heart disease, Coronary artery bypass grafting (CABG), obstructive sleep apnoea, COPD and increased BMI

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The role of hydrogen sulfide, substance P and Kupffer