CIRCULATING MONOCYTE ENDOTOXIN TOLERANCE IN ACUTE LIVER FAILURE: ROLE

OF HEPATICALLY DERIVED IL-10CG. Antoniades1,2, L. Taams3, M. Paris3, M.S. Longhi2, I. Carey2, M. Bruce2, G. Auzinger2, W. Bernal2, W. Jassem2, A. Quaglia2, N.

Heaton2, D. Vergani2, J. Wendon2 and M. Thursz1

1Hepatology Centre, Imperial College London, 2Institute of Liver Studies, Kings College Hospital, Denmark Hill, 3CMCBI, King's College London,

London, UK.

Infection and ALF

• Occurs frequently in patients with ALF (Karevllas et al, Crit Care’08)

• SIRS and secondary infection important contributor to mortality (Rolando et al Hepatology ‘00; Vaquero et al Gastro ’03)

progression of encephalopathy, mutliple organ failure

• Mechanisms conferring susceptibility unclear

Rolando et al, Hepatology ‘00

Functional monocyte deactivation in ALF

0

200

400

600

800

1000

1200

0 20 40 60 80

hladr

Observed

Logarithmic

IL-1

0 pg

/ml r= -0.8; p<0.001

• ↓ monocyte HLA-DR, ↑circulating IL-10

• Strong correlation with severity of liver injury, organ failure & outcome

Antoniades et al, Hepatology ‘06; Berry & Antoniades et al Liv Int ‘10

LPS

LPS challengeLPS challenge

TNF-α

IL-10TNF-α

IL-10TNF-α

IL-10

TNF-αIL-10

↑ HLA-DR↑ HLA-DR ↓ HLA-DR↓ HLA-DR

IL-10 , PGs, Corticosteriods

LTA

Peptidoglycan

NORMAL ENDOTOXIN TOLERANCE

Hypothesis and Aims

• Do circulating monocytes exhibit phenotypic and functional features of endotoxin tolerance in ALF?

• What are the potential causes of endotoxin tolerance in ALF?– liver derived circulating inflammatory mediators?– gut-derived microbial products? – passage of monocytes through hepatic sinusoids ?

Methods-monocyte phenotype analysis

• Patients:– ALF (n=20; all acetaminophen) – Healthy controls (n=15)

• Surface expression monocyte HLA-DQ,HLA-DR & CD86:fresh blood analysisDouble colour flow cytometry antibodies for HLA-DR/DQ

and CD86 & monocyte specific marker-CD14 Results expressed as %

• Serum IL-10 measured using ELISA (pg/ml)

Monocyte phenotypic profile in ALF

* Abeles RD, Antoniades CG et al , EASL 2011 Poster # 909

Methods-monocyte cytokineresponses to LPS

• Patients recruited:ALF (n=12; all acetaminophen induced ALF)Chronic liver disease (n=10)Normal controls (n=10)

• PBMCs labelled CD14 & CD3 monolonal antibodies and incubated following permeabilisation with anti-IL-10 or anti-TNF-α monoclonal antibodies

• Percentage CD14+ monocytes expressing intracellular IL-10 and TNF-α was estimated by flow cytometry

• TNF-α and IL-10 secretion was evaluated using ELISPOT in PBMC– Baseline & following 6 hour stimulation with 100ng/ml LPS

• Results are expressed as the median of specific spot forming cells/106 PBMC [spSFC/106 PBMC]

Ex-vivo monocyte TNF-α & IL-10 production

Effects of LPS on monocyte TNF-α & IL-10 secretion

Basal ALF IL-10 ALF- LPS -induced IL-10

Basal ALF TNF-α ALF- LPS induced TNF-α

Normal Controls

ALF

Methods – TLR and STAT signalling pathways

• Phosphoflow: identify changes in regulators of TLR signalling (NF-kBp65, MAPK p38, AKT-1) and IL-10 signalling (STAT 3 vs STAT 1) – ex-vivo CD14+ monocytes ALF (n=10) & normal controls (n=8)– ex-vivo CD33+ monocytes ALF (n=5) vs normal controls (n=5)

• Experimental conditions: Unstimulated (RPMI)LPS [TLR-4] (100ng/ml)Zymosan [TLR-2] (50µg/ml)IL-10 (50ng/ml)IFN-γ (10ng/ml)

• FACS Canto analysis (MFI)

• Results expressed as MFI & ratio of activation (MFI post stimulation/MFI unstimulated)

NF-kBp65, MAPKp38 & AKT-1 expression in CD14+ monocytesPhosphoflow:

Fresh blood gating strategy

NF-kBp65 expression: ALF vs normal controls

LPS [TLR-4] stimulation Zymosan [TLR-2] stimulation

LPS [TLR-4] effects on MAPKp38, AKT-1 expression in ALF vs normal controls

MAPKp38 AKT-1

STAT 1 & 3 signalling in ALF

STAT3 expression in healthy control (HC) and ALF patient

Baseline IL-100

500

1000

1500

2000HCALF

STAT

3 MFI

STAT1 expression in healthy control (HC) and ALF patient

Baseline IFN-g0

500

1000

1500

2000HCALF

STAT

1 MFI

Systemic and regional cytokine measurement

• TNF-α & IL-10 levels [pg/ml]

• Hepatic:– Protein array - liver

homogenates – 10 ALF & 8 pathological controls

• Porto-hepatic gradient:– 5 ALF patients

• Circulation:ALF (n=35)Normal controls (n=15)

Liver

Hepatic necrosis and apoptosisMetabolic dysfunctionCytokine and inflammatory mediators releaseCoagulopathyChanges in blood flow

oxygen gradientzone 1 to 3relative hypoxia

“Spill-over hypothesis”:Liver derived anti-inflammatory mediators

Conclusions• Endotoxin tolerant monocytes in ALF

– Circulating immunosuppressive monocyte phenotype ↓HLA-DR, CD86, ↑HLA-DQ

– Expansion of IL-10 producing monocytes

– Anti-inflammatory cytokine secretion profile following LPS stimulation (IL-10>TNF-α)

– Down-regulation of positive regulators TLR signalling and monocyte survival pathways

• Hepatic derived IL-10 – promote induction of endotoxin tolerance in ALF

• Anti-inflammatory monocyte responses to microbial challenge - increase susceptibility to sepsis

Acknowledgments

Professor M ThurszProfessor D VerganiDr L TaamsDr Y MaMs V Zingarelli

Additional data

Passage of monocytes through hepatic sinusoids Culture of normal monocytes in ALF liver supernatant vs pathological control (fatty liver)

Fatty liverPre LPS → LPS stimulation

ALF liverPre LPS → LPS stimulation

Molecular mechanisms of endotoxin tolerance

-

Endotoxin tolerance and infection/inflammation

IL-10 signalling pathways• Induced by both TLR and non-TLR

signalling STAT3MyD88 dependent (ERK, NF-kB, p38 MyD88independent pathways (TRIF)

• +ve regulators IL-10 signalling:MAPKp38 (important for

phago)ERKSTAT3

• -ve regulators of IL-10 signalling:IFN-gDUSP-1GSK3

IL-10 signalling pathways

Sfeir et alCrit Care Med 2001; 29:129 –133