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CIRCULATING MONOCYTE ENDOTOXIN TOLERANCE IN ACUTE LIVER FAILURE: ROLE OF HEPATICALLY DERIVED IL-10. - PowerPoint PPT Presentation
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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