The role of TAM receptors in immuneparesis in tissue compartments in liver failure syndromes

Background: Decompensated liver cirrhosis and acute-on-chronic liver failure (ACLF) – liver failure syndromes - are associated with increased incidence of bacterial infection, often spontaneous bacterial peritonitis (SBP), and a subsequent higher mortality. Within a model of pathological bacterial translocation, immuneparesis increases the susceptibility to infection. TAM receptor MerTK, expressed on monocytes and macrophages, mediates apoptotic cell clearance and attenuates responses to microbial challenge. Whilst MerTK+ circulating monocytes are expanded in ACLF, its role in conjunction with other TAM receptors, Axl and Tyro3, is not known in tissue compartments, such as the peritoneum and liver. In this thesis, I describe the tissue compartmental macrophage phenotype and function in cirrhosis and ACLF, focusing on TAM receptors, and the relationship to microenvironmental mediators. Methods: Combinations of flow cytometry, gene expression analyses and live cell imaging determined the phenotypic, functional and transcriptomic profile of MerTK+ monocytes/macrophages in cirrhosis and ACLF, in the circulation, peritoneum and liver. The composition of the ascitic microenviroment was determined by enzyme-linked immunosorbent assay (ELISA) and 1H-NMR metabonomics. Results: Circulating monocyte MerTK expression in ACLF was markedly elevated whilst Axl was low-level and Tyro3 variable. The peritoneum in cirrhosis was immunotolerant, characterized by MerTK[high] Axl[low] macrophages primed for apoptotic cell clearance, with impaired antimicrobial cytokine and oxidative burst responses but preserved phagocytosis. In ACLF, ascites contained pro-inflammatory cytokines, metabolites from anaerobic and lipid metabolism and increased apoptotic/necrotic cell death markers in conjunction with higher TAM receptor expression. Hepatic macrophages displayed intermediate MerTK expression. Conclusions: The work presented in this thesis has determined the TAM receptor expression across tissue compartments, the ascitic microenvironment in cirrhosis and ACLF and identified MerTK as a promising immunotherapeutic target to restore innate immune function in the evolution of ACLF.Open Acces

Increased Expression of Cytotoxic T-Lymphocyte-Associated Protein 4 by T Cells, Induced by B7 in Sera, Reduces Adaptive Immunity in Patients With Acute Liver Failure.

BACKGROUND & AIMS: Patients with acute liver failure (ALF) have defects in innate immune responses to microbes (immune paresis) and are susceptible to sepsis. Cytotoxic T-lymphocyte-associated protein 4 (CTLA4), which interacts with the membrane receptor B7 (also called CD80 and CD86), is a negative regulator of T-cell activation. We collected T cells from patients with ALF and investigated whether inhibitory signals down-regulate adaptive immune responses in patients with ALF. METHODS: We collected peripheral blood mononuclear cells from patients with ALF and controls from September 2013 through September 2015 (45 patients with ALF, 20 patients with acute-on-chronic liver failure, 15 patients with cirrhosis with no evidence of acute decompensation, 20 patients with septic shock but no cirrhosis or liver disease, and 20 healthy individuals). Circulating CD4+ T cells were isolated and analyzed by flow cytometry. CD4+ T cells were incubated with antigen, or agonist to CD3 and dendritic cells, with or without antibody against CTLA4; T-cell proliferation and protein expression were quantified. We measured levels of soluble B7 molecules in supernatants of isolated primary hepatocytes, hepatic sinusoidal endothelial cells, and biliary epithelial cells from healthy or diseased liver tissues. We also measured levels of soluble B7 serum samples from patients and controls, and mice with acetaminophen-induced liver injury using enzyme-linked immunosorbent assays. RESULTS: Peripheral blood samples from patients with ALF had a higher proportion of CD4+ CTLA4+ T cells than controls; patients with infections had the highest proportions. CD4+ T cells from patients with ALF had a reduced proliferative response to antigen or CD3 stimulation compared to cells from controls; incubation of CD4+ T cells from patients with ALF with an antibody against CTLA4 increased their proliferative response to antigen and to CD3 stimulation, to the same levels as cells from controls. CD4+ T cells from controls up-regulated expression of CTLA4 after 24-48 hours culture with sera from patients with ALF; these sera were found to have increased concentrations of soluble B7 compared to sera from controls. Necrotic human primary hepatocytes exposed to acetaminophen, but not hepatic sinusoidal endothelial cells and biliary epithelial cells from patients with ALF, secreted high levels of soluble B7. Sera from mice with acetaminophen-induced liver injury contained high levels of soluble B7 compared to sera from mice without liver injury. Plasma exchange reduced circulating levels of soluble B7 in patients with ALF and expression of CTLA4 on T cells. CONCLUSIONS: Peripheral CD4+ T cells from patients with ALF have increased expression of CTLA4 compared to individuals without ALF; these cells have a reduced response to antigen and CD3 stimulation. We found sera of patients with ALF and from mice with liver injury to have high concentrations of soluble B7, which up-regulates CTLA4 expression by T cells and reduces their response to antigen. Plasma exchange reduces levels of B7 in sera from patients with ALF and might be used to restore antimicrobial responses to patients

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

Immunotherapy in the treatment and prevention of infection in acute-on-chronic liver failure

Chronic liver disease, depicted by gradual destruction and fibrosis of the liver, is a condition with high and probably increasing prevalence worldwide. Its deterioration, acute-on-chronic liver failure (ACLF), is characterized by an in-hospital mortality of up to 65%. Infectious complications are the main precipitants eliciting ACLF and concurrently the main cause of death from ACLF. Patients have a marked susceptibility to bacterial infections, which is thought to arise a consequence of an inadequate immune response to microbial challenge, termed immuneparesis. The pathophysiologic mechanisms remain poorly understood. Treatments aimed at restoring the patients’ immune function may prevent onset of ACLF and death from secondary infections. A number of drugs approved for patients with liver disease bear immunomodulatory potential such as albumin, glucocorticoids, N-acetylcysteine. Specific targets have been defined that may lead to development of new immunotherapeutic agents. Here, we summarize the pathophysiology of immuneparesis in ACLF and drug candidates to restore immune function and improve survival in the future.</jats:p

Balanced haemostasis with both hypo- and hyper-coagulable features in critically ill patients with acute-on-chronic-liver failure

Background: Cirrhotic patients have complex haemostatic abnormalities. Current evidence suggests stable cirrhotic (SC) patients have a "re-balanced" haemostatic state. However, limited data exists in acute decompensated (AD) or acute on chronic liver failure (ACLF) patients. Methods: We utilised thrombin generation analysis, fibrinolysis assessment, and evaluation of haemostatic parameters to assess haemostasis in liver disease of progressive severity. Results: The study cohorts were comprised of: SC, n = 8; AD n = 44; ACLF, n = 17; and Healthy Control (HC), n = 35. There was a progressive increase across the cohorts in INR (p = 0.0001), Factor VIII (p = 0.0001) and VWF levels (p = 0.0001) and a correspondingly decrease in anti-thrombin (p = 0.0001), ADAMTS-13 (p = 0.01) and fibrinogen levels (p = 0.0001). In the presence of thrombomodulin, thrombin generation was equivalent or significantly higher in all the cohorts compared to HC (p = 0.0001). Compared to AD, ACLF had a lower ETP (p = 0.002) and thrombin peak (p = 0.0001). There was no significant difference across the cohorts in clot lysis time (p = 0.07), although compared to HC, AD had a significantly shorter lysis time (p = 0.001). Conclusions: Our cohorts, despite significant differences in haemostatic parameters, displayed intact thrombin generation but progressive hypo-functional clot stability and potentially but not universal hyper-functional haemostasis