G. Petts

Stat2 loss leads to cytokine-independent, cell-mediated lethality in LPS-induced sepsis

Deregulated Toll-like receptor (TLR)-triggered inflammatory responses that depend on NF-κB are detrimental to the host via excessive production of proinflammatory cytokines, including TNF-α. Stat2 is a critical component of type I IFN signaling, but it is not thought to participate in TLR signaling. Our study shows that LPS-induced lethality in Stat2−/− mice is accelerated as a result of increased cellular transmigration. Blocking intercellular adhesion molecule-1 prevents cellular egress and confers survival of Stat2−/− mice. The main determinant of cellular egress in Stat2−/− mice is the genotype of the host and not the circulating leukocyte. Surprisingly, lethality and cellular egress observed on Stat2−/− mice are not associated with excessive increases in classical sepsis cytokines or chemokines. Indeed, in the absence of Stat2, cytokine production in response to multiple TLR agonists is reduced. We find that Stat2 loss leads to reduced expression of NF-κB target genes by affecting nuclear translocation of NF-κB. Thus, our data reveal the existence of a different mechanism of LPS-induced lethality that is independent of NF-κB triggered cytokine storm but dependent on cellular egress.

Defective monocyte oxidative burst predicts infection in alcoholic hepatitis and is associated with reduced expression of NADPH oxidase

Objective In order to explain the increased susceptibility to serious infection in alcoholic hepatitis, we evaluated monocyte phagocytosis, aberrations of associated signalling pathways and their reversibility, and whether phagocytic defects could predict subsequent infection. Design Monocytes were identified from blood samples of 42 patients with severe alcoholic hepatitis using monoclonal antibody to CD14. Phagocytosis and monocyte oxidative burst (MOB) were measured ex vivo using flow cytometry, luminometry and bacterial killing assays. Defects were related to the subsequent development of infection. Intracellular signalling pathways were investigated using western blotting and PCR. Interferon-γ (IFN-γ) was evaluated for its therapeutic potential in reversing phagocytic defects. Paired longitudinal samples were used to evaluate the effect of in vivo prednisolone therapy. Results MOB, production of superoxide and bacterial killing in response to Escherichia coli were markedly impaired in patients with alcoholic hepatitis. Pretreatment MOB predicted development of infection within two weeks with sensitivity and specificity that were superior to available clinical markers. Accordingly, defective MOB was associated with death at 28 and 90 days. Expression of the gp91phox subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was reduced in patients with alcoholic hepatitis demonstrating defective MOB. Monocytes were refractory to IFN-γ stimulation and showed high levels of a negative regulator of cytokine signalling, suppressor of cytokine signalling-1. MOB was unaffected by 7 days in vivo prednisolone therapy. Conclusions Monocyte oxidative burst and bacterial killing is impaired in alcoholic hepatitis while bacterial uptake by phagocytosis is preserved. Defective MOB is associated with reduced expression of NADPH oxidase in these patients and predicts the development of infection and death.

MerTK expressing hepatic macrophages promote the resolution of inflammation in acute liver failure

Objective Acute liver failure (ALF) is characterised by overwhelming hepatocyte death and liver inflammation with massive infiltration of myeloid cells in necrotic areas. The mechanisms underlying resolution of acute hepatic inflammation are largely unknown. Here, we aimed to investigate the impact of Mer tyrosine kinase (MerTK) during ALF and also examine how the microenvironmental mediator, secretory leucocyte protease inhibitor (SLPI), governs this response. Design Flow cytometry, immunohistochemistry, confocal imaging and gene expression analyses determined the phenotype, functional/transcriptomic profile and tissue topography of MerTK+ monocytes/macrophages in ALF, healthy and disease controls. The temporal evolution of macrophage MerTK expression and its impact on resolution was examined in APAP-induced acute liver injury using wild-type (WT) and Mer-deficient (Mer−/−) mice. SLPI effects on hepatic myeloid cells were determined in vitro and in vivo using APAP-treated WT mice. Results We demonstrate a significant expansion of resolution-like MerTK+HLA-DRhigh cells in circulatory and tissue compartments of patients with ALF. Compared with WT mice which show an increase of MerTK+MHCIIhigh macrophages during the resolution phase in ALF, APAP-treated Mer−/− mice exhibit persistent liver injury and inflammation, characterised by a decreased proportion of resident Kupffer cells and increased number of neutrophils. Both in vitro and in APAP-treated mice, SLPI reprogrammes myeloid cells towards resolution responses through induction of a MerTK+HLA-DRhigh phenotype which promotes neutrophil apoptosis and their subsequent clearance. Conclusions We identify a hepatoprotective, MerTK+, macrophage phenotype that evolves during the resolution phase following ALF and represents a novel immunotherapeutic target to promote resolution responses following acute liver injury.

PWE-113 The liver biopsy in alcoholic hepatitis: data from the steroids or pentoxifylline in alcoholic hepatitis (stopah) clinical trial

Introduction The European Association for the Study of the Liver guidelines recommend the use of liver biopsy to confirm alcoholic steatohepatitis (ASH) in patients suspected to be suffering from alcoholic hepatitis and who are classified as high risk after prognostic assessment. Despite this it was only in 2014 that Altamirano et alpublished the first prognostic liver biopsy based scoring system for the evaluation of ASH (the Alcoholic Hepatitis Histological Score or AHHS). This work sought to validate their scoring system and further explore the utility of the liver biopsy in ASH. Method Two independent histopathologists, blinded to treatment and outcome, centrally reviewed liver biopsies of patients with clinically high-risk alcoholic hepatitis who had been recruited to the STOPAH trial. Results 93 (47%) of the 208 biopsies received were both adequate in quality and taken between admission and day 5 of trial treatment. 88% (82/93) had histological features diagnostic of ASH. 89% (32/36) of cases obtained as routine clinical practice were diagnostic of ASH compared to 79% (15/19) biopsied for diagnostic uncertainty. Clinically more severe liver disease was associated a higher rate of ASH diagnosis (82% of GAHS ≤8 vs. 97% of GAHS >8). 65% (53/82) of biopsy proven cases of ASH were classified as severe by AHHS. This group had a significantly higher 28 day mortality rate than those classified as mild/moderate (18% vs. 0%, Fisher’s exact p = 0.02). AHHS severity positively correlated with baseline Maddrey’s Discriminant Function and Glasgow Alcoholic Hepatitis Score (r = 0.2, p = 0.045 and r = 0.3, p = 0.01 respectively). Clinical markers of severe disease positively correlated with biopsy features of severe disease including: - serum bilirubin with bilirubinostasis (r = 0.5, p= <0.0001) - serum white cell and neutrophil count with lobular inflammation (r = 0.4, p = <0.001) Elevation of serum alkaline phosphatase (ALP) and bilirubin are usually associated with more severe liver disease, however in this study they were seen to negatively correlate with certain biopsy features of more severe disease: - serum ALP negatively correlated with ductular changes (r = -0.2, p = 0.04) - serum bilirubin negatively correlated with Laennec fibrosis grading (r = -0.3, p = 0.01) Conclusion This work goes some way towards validating the AHHS classification. The work also highlights the parallels between clinical and histological parameters and documents negative correlations seen in other liver diseases but not previously noted in ASH. Disclosure of interest None Declared.

PTH-102 Stat2 is a key inflammatory molecule in human non-alcoholic fatty liver disease and murine liver injury

Introduction In patients with non-alcoholic fatty liver disease (NAFLD), liver inflammation is a key histological feature that distinguishes benign simple steatosis from progressive non-alcoholic steatohepatitis (NASH). The mechanisms that underlie this process are poorly understood. We recently showed that Stat2plays a pivotal role in acute inflammation, independent of its role as an interferon mediator. The aim of the current study is to test the hypothesis that STAT2 is associated with hepatic inflammation in patients with NASH and in a murine model of liver injury. Method Expression of (phosphorylated, p) STAT2 and STAT1 was assessed by immunohistochemistry in anonymised sections of archived liver tissue taken from consenting patients with NASH at The Royal London and St Mary’s Hospitals (London, UK). STAT2-/-and WT mice were injected with incremental doses of carbon tetrachloride (CCl4) for 4 or 6 weeks. Serum and liver tissue were harvested 24 or 72hrs after the last injection. Immunohistochemistry for activated stellate cells (anti-alpha smooth muscle actin, aSMA) and macrophage/Kupfer cells (anti-F4/80) was performed. Results Tissue from 62 patients was examined. Hepatocyte nuclear expression in NASH liver tissue was significantly greater for STAT1 (74% vs 10%, p < 0.0001) and STAT2 (98% vs 35%, p < 0.0001) compared to normal liver. There was a significant correlation between STAT1 expression (r = 0.6, p = 0.02), but not STAT2 and the NAFLD activity score (NAS). Animal models confirm that Stat 2 plays a role in liver inflammation – Stat2-/- mice were protected from CCl4-mediated liver injury with reduced serum alanine aminotransferase (ALT, 2165 vs 1090 iU/ml, p = 0.03), inflammation and necrosis histologically and fibrosis (Picrosirius red staining). There were no significant differences in the number of aSMA or F4/80-positive cells between WT and Stat2-/- mice. Conclusion These data identify a novel role for STAT2 in the development of liver disease, with expression of STAT1 and STAT2 relating to disease grade. The protection afforded by Stat2 loss in murine models is unrelated to macrophage and stellate cell activation, and we hypothesise that the mechanism of protection is therefore reduced production of Stat2-mediated cytokine production. Disclosure of interest None Declared.

PTH-101 The role of tissue factor pathway inhibitor (tfpi) in liver injury

Introduction Studies have demonstrated that pro-coagulant states are associated with rapidly advancing liver fibrosis and that anticoagulation limits this. In acute liver injury studies have suggested that decreased coagulation cascade activation results in reduced acute liver injury. TFPI is a serine protease inhibitor that acts as a homeostatic brake in the coagulation cascade. TFPI is a potential target for the inhibition of the coagulation cascade to modify liver disease. To further investigate this, transgenic mice expressing human TFPI were used in models of liver injury. The transgenic mice carry a genetic modification that allows cells expressing alpha smooth muscle actin (αSMA) to simultaneously express TFPI. This results in cells, such as activated hepatic stellate cells, expressing TFPI in a manner not seen in wild type mice. Method Transgenic and wild type mice were used in a chronic liver injury model (carbon tetrachloride, CCl4) or an acute liver injury model (paracetamol) and culled at set time points after dosing. Results Chronic liver injury: At 24 h after the last dose of CCl4there was a significant decrease in αSMA expression in the transgenic mice compared to wild type (Mann-Whitney test, p = 0.003). At 24 h there was also a decrease in tissue inhibitor of metalloproteinase (TIMP) -1 gene expression but normal matrix metalloproteinase (MMP) -2 and -9 gene expression. This suggested a microenvironment that would promote fibrosis resolution through decreased hepatic stellate activation and loss of inhibition of MMPs. However after 24 h this difference was lost. At 24 h after the last dose of CCl4and at all subsequent time points there was no significant difference between fibrosis in transgenic and wild type mice as demonstrated by Sirius red staining, hydroxyproline assay and collagen 1α1 gene expression. Acute liver injury: In early paracetamol induced liver injury (2–12 h post-dose) there was no significant difference in parenchymal necrosis in transgenic compared to wild type mice. However, at 24 h and 48 h after dosing there was a significant decrease in parenchymal necrosis in transgenic compared to wild type mice (24 h: mean percentage necrosis 6% vs. 30% respectively, Mann-Whitney test p = 0.008. 48 h: percentage necrosis 2% vs. 20% respectively, Mann-Whitney test p = 0.036). Conclusion These results suggest that TFPI is not a potential therapeutic target in chronic liver injury. However in acute paracetamol induced liver injury TFPI appears to reduce liver injury in a sustained manner from 24 h after the initial insult. This suggests a role for TFPI in managing acute liver injury and warrants further investigation. Disclosure of interest None Declared.

PTH-096 Monocyte oxidative burst defect in alcoholic hepatitis is resistant to interferon gamma

Introduction An important cause of mortality in patients with severe alcoholic hepatitis (SAH) is infection. We and others have found that the oxidative burst response of phagocytic cells to E. coliis impaired in SAH which may contribute to susceptibility to infection. This is analogous to Chronic Granulomatous Disease, an inherited condition caused by a mutation in the enzyme NADPH oxidase which leads to predisposition to recurrent life-threatening infections. Treatment with interferon gamma (IFN-γ) reduces the frequency of severe infections in these patients. We sought to investigate the molecular mechanism underlying the monocyte oxidative burst (mOB) defect in SAH and determine whether IFN-γ can restore this defect in vitro . Method Peripheral blood mononuclear cells (PBMCs) were isolated from 6 patients with SAH and 4 healthy controls (HCs). Monocytes were isolated from PBMCs using magnetic MACS beads and negative selection. Monocytes were incubated with 50 ng/ml IFN-γ supplemented by 10% healthy serum for 24 h and the mOB to E. coliwas quantified by oxidation of fluorogenic rhodamine and flow cytometry as previously described. Western blotting was used to quantify protein using monoclonal antibodies to components of the IFN-γ signalling pathway (pSTAT-1 and negative regulator SOCS-1) and glucose-6-phosphate dehydrogenase (G6PD, which generates NADPH substrate required for NAPDH oxidase function), in the monocytes of HCs and AH patients. G6PD enzyme activity was measured using a colorimetric kit. Quantitative PCR was used to determine expression of STAT-1 in monocytes stimulated for 24 h in 50 ng/ml IFN-γ. Results Surprisingly, SAH mOB defect was resistant to 24 h’ stimulation by 50 ng/mlIFN-γ. The level of unstimulated phosphorylated STAT-1 protein, a transcription factor activated by IFN-γ signalling was significantly reduced in monocytes of SAH patients compared to healthy controls (pSTAT-1: housekeeping gene 0.90 HC vs 0.14 SAH, p < 0.05). q-PCR confirmed STAT-1 expression following stimulation with IFN-γ was significantly reduced in SAH compared to HCs (ΔCT 0.97 HC vs SAH 0.54, p < 0.05). The level of SOCS-1 protein was significantly increased in monocytes of SAH patients compared to HCs (SOCS-1: housekeeping gene 0.98 HC vs 2.24 AH, p < 0.05). There was no difference in G6PD enzyme activity or protein expression between groups. Conclusion Our study suggests that elevated levels of the inhibitory factor SOCS-1 may reduce phosphorylation and activation of STAT-1. Reduced pSTAT-1 may explain the resistance to IFN-γ observed and hence the burst defect in SAH. This work provides a novel insight into the impaired mOB in SAH and furthers our knowledge of the predisposition to infection observed in this patient group. Disclosure of interest None Declared.

390 TARGETED INHIBITION OF TISSUE FACTOR AND THROMBIN ON CD31 EXPRESSING CELLS SUPPRESSES HEPATIC FIBROSIS IN CCL4 TREATED MICE

Introduction Recent evidence suggests a role for the coagulation cascade in promoting liver fibrosis. However, the cellular basis for this relationship is unclear. In order to explore this relationship we employed two unique transgenic mice strains expressing membrane–tethered tissue factor pathway inhibitor (TFPI) or hirudin (anti-thrombin) fusion proteins driven by a CD31 promoter. These strains allow for the selective inhibition of tissue factor (TF) or thrombin on endothelial cells, monocytes and platelets expressing CD31. Aims To evaluate the impact of the targeted inhibition of tissue factor and thrombin on effector cells of liver fibrosis in murine hepatic fibrosis induced by CCl 4 . Methods Liver fibrosis was induced in CD31-TFPI, CD31-Hirudin and wild type control mice with 4 weeks carbon tetrachloride (CCl 4 ) administered by intraperitoneal injection. Animals were culled and livers extracted for histological and biochemical analysis. Fibrosis was scored using a four point semi-quantitative system and quantified by digital image analysis to determine percentage area of fibrosis. Immunohistochemistry to determine α-SMA expression, a marker of hepatic stellate cell activation was performed and the mean number of activated stellate cells was quantified per high power field. Results The percentage area of fibrosis was significantly less in CD31-TFPI (1.89%±0.26, p=0.001) and CD31-Hirudin mice (1.04%±0.16, p=0.00003) in comparison to control mice (3.66%±0.39). Semiquantitative fibrosis scoring showed a significant difference between the CD31-TFPI (median 2/4) and CD31-Hirudin (median 3/4) mice in comparison with control mice (median 3/4, p=0.009) but the difference between the two transgenic strains was not significant. Both transgenic strains demonstrated a significantly reduced mean number of α-SMA stellate cells per high power field in comparison to control mice (CD31-TFPI vs control, 7.4 vs 29.4, p=0.0002; CD31-hirudin vs control, 5.25 vs 29.4, p=0.0002). Conclusion CD31 targeted inhibition of TF and thrombin significantly reduces liver fibrosis and stellate cell activation in a murine CCl 4 model. The data supports the use of this novel murine model as a tool for investigating the cellular biology of the role of coagulation in liver fibrogenesis. It will also provide information for developing new treatments of human liver fibrosis. Competing interests None declared.