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.
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
Bacterial and fungal
7%
chest sepsis
13%
No infection
42%
fungal
5%
Bacterial
33%
Rolando et al, Hepatology ‘00
Functional monocyte deactivation in ALF
• ↓ monocyte HLA-DR, ↑circulating IL-10
• Strong correlation with severity of liver injury, organ failure &
outcome
Observed
1200
Logarithmic
1000
IL-10 pg/ml
800
r= -0.8; p<0.001
600
400
200
0
0
20
40
hladr
Antoniades et al, Hepatology ‘06; Berry & Antoniades et al Liv Int ‘10
60
80
NORMAL
ENDOTOXIN TOLERANCE
↑ HLA-DR
↓ HLA-DR
LPS
LTA
Peptidoglycan
IL-10 , PGs,
Corticosteriods
LPS challenge
TNF-α
IL-10
LPS challenge
TNF-α
IL-10
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 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)
Monocyte phenotypic profile in ALF
* Abeles RD, Antoniades CG et al , EASL 2011 Poster # 909
Methods-monocyte cytokine
responses 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
ALF
Basal ALF IL-10
ALF- LPS -induced IL-10
Normal
Controls
Basal ALF TNF-α
ALF- LPS induced TNF-α
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+ monocytes
Phosphoflow:
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
2000
HC
ALF
1500
STAT1 MFI
STAT3 MFI
2000
STAT1 expression in healthy control (HC) and ALF patient
1000
1500
1000
500
500
0
0
Baseline
IL-10
HC
ALF
Baseline
IFN-g
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 apoptosis
Metabolic dysfunction
Cytokine and inflammatory
mediators release
Coagulopathy
Changes in blood flow
oxygen gradient
zone 1 to 3
relative 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 Thursz
Professor D Vergani
Dr L Taams
Dr Y Ma
Ms V Zingarelli
Additional data
Passage of monocytes through hepatic sinusoids
Culture of normal monocytes in ALF liver supernatant vs pathological control (fatty liver)
Fatty liver
Pre LPS
→ LPS stimulation
ALF liver
Pre 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
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
•
IL-10 signalling pathways
Sfeir et alCrit Care Med 2001; 29:129 –133