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

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

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

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  1. CIRCULATING MONOCYTE ENDOTOXIN TOLERANCE IN ACUTE LIVER FAILURE: ROLE OF HEPATICALLY DERIVED IL-10 CG. 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.

  2. 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

  3. Functional monocyte deactivation in ALF • ↓ monocyte HLA-DR, ↑circulating IL-10 • Strong correlation with severity of liver injury, organ failure & • outcome r= -0.8; p<0.001 IL-10 pg/ml Antoniades et al, Hepatology ‘06; Berry & Antoniades et al Liv Int ‘10

  4. NORMAL ENDOTOXIN TOLERANCE ↑ HLA-DR ↓ HLA-DR LPS LTA Peptidoglycan IL-10 , PGs, Corticosteriods LPS challenge LPS challenge TNF-α IL-10 TNF-α IL-10

  5. 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 ?

  6. Methods-monocyte phenotype analysis • Patients: • ALF (n=20; all acetaminophen) • Healthy controls (n=15) • Surface expression monocyte HLA-DQ,HLA-DR & CD86: fresh blood analysis Double 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)

  7. Monocyte phenotypic profile in ALF * Abeles RD, Antoniades CG et al , EASL 2011 Poster # 909

  8. 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]

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

  10. Effects of LPS on monocyte TNF-α & IL-10 secretion ALF Normal Controls Basal ALF IL-10 ALF- LPS -induced IL-10 Basal ALF TNF-α ALF- LPS induced TNF-α

  11. 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)

  12. NF-kBp65, MAPKp38 & AKT-1 expression in CD14+ monocytes Phosphoflow: Fresh blood gating strategy

  13. NF-kBp65 expression: ALF vs normal controls LPS [TLR-4] stimulation Zymosan [TLR-2] stimulation

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

  15. STAT 1 & 3 signalling in ALF

  16. 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)

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

  18. 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

  19. Acknowledgments Professor M Thursz Professor D Vergani Dr L Taams Dr Y Ma Ms V Zingarelli

  20. Additional data

  21. Passage of monocytes through hepatic sinusoids Culture of normal monocytes in ALF liver supernatant vs pathological control (fatty liver) ALF liver Pre LPS → LPS stimulation Fatty liver Pre LPS → LPS stimulation

  22. Molecular mechanisms of endotoxin tolerance -

  23. Endotoxin tolerance and infection/inflammation

  24. IL-10 signalling pathways • Induced by both TLR and non-TLR signalling STAT3 MyD88 dependent (ERK, NF-kB, p38 MyD88independent pathways (TRIF) • +ve regulators IL-10 signalling: MAPKp38 (important for phago) ERK STAT3 • -ve regulators of IL-10 signalling: IFN-g DUSP-1 GSK3

  25. IL-10 signalling pathways Sfeir et alCrit Care Med 2001; 29:129 –133

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