Soluble TIM3 and Its Ligands Galectin-9 and CEACAM1 Are in Disequilibrium During Alcohol-Related Liver Disease and Promote Impairment of Anti-bacterial Immunity.

BACKGROUND AND AIMS: Immunoregulatory checkpoint receptors (CR) contribute to the profound immunoparesis observed in alcohol-related liver disease (ALD) and in vitro neutralization of inhibitory-CRs TIM3/PD1 on anti-bacterial T-cells can rescue innate and adaptive anti-bacterial immunity. Recently described soluble-CR forms can modulate immunity in inflammatory conditions, but the contributions of soluble-TIM3 and soluble-PD1 and other soluble-CRs to immune derangements in ALD remain unclear. METHODS: In Alcoholic Hepatitis (AH; n = 19), alcohol-related cirrhosis (ARC; n = 53) and healthy control (HC; n = 27) subjects, we measured by Luminex technology (i) plasma levels of 16 soluble-CRs, 12 pro/anti-inflammatory cytokines and markers of gut bacterial translocation; (ii) pre-hepatic, post-hepatic and non-hepatic soluble-CR plasma levels in ARC patients undergoing TIPS; (iii) soluble-CRs production from ethanol-treated immunocompetent precision cut human liver slices (PCLS); (iv) whole-blood soluble-CR expression upon bacterial challenge. By FACS, we assessed the relationship between soluble-TIM3 and membrane-TIM3 and rescue of immunity in bacterial-challenged PBMCs. RESULTS: Soluble-TIM3 was the dominant plasma soluble-CR in ALD vs. HC (p = 0.00002) and multivariate analysis identified it as the main driver of differences between groups. Soluble-CRs were strongly correlated with pro-inflammatory cytokines, gut bacterial translocation markers and clinical indices of disease severity. Ethanol exposure or bacterial challenge did not induce soluble-TIM3 production from PCLS nor from whole-blood. Bacterial challenge prompted membrane-TIM3 hyperexpression on PBMCs from ALD patient's vs. HC (p < 0.002) and was inversely correlated with plasma soluble-TIM3 levels in matched patients. TIM3 ligands soluble-Galectin-9 and soluble-CEACAM1 were elevated in ALD plasma (AH > ARC; p < 0.002). In vitro neutralization of Galectin-9 and soluble-CEACAM1 improved the defective anti-bacterial and anti-inflammatory cytokine production from E. coli-challenged PBMCs in ALD patients. CONCLUSIONS: Alcohol-related liver disease patients exhibit supra-physiological plasma levels of soluble-TIM3, particularly those with greater disease severity. This is also associated with increased levels of soluble TIM3-ligands and membrane-TIM3 expression on immune cells. Soluble-TIM3 can block the TIM3-ligand synapse and improve anti-bacterial immunity; however, the increased levels of soluble TIM3-binding ligands in patients with ALD negate any potential immunostimulatory effects. We believe that anti-TIM3 neutralizing antibodies currently in Phase I clinical trials or soluble-TIM3 should be investigated further for their ability to enhance anti-bacterial immunity. These agents could potentially represent an innovative immune-based supportive approach to rescue anti-bacterial defenses in ALD patients.

Mucosa-associated invariant T cells link intestinal immunity with antibacterial immune defects in alcoholic liver disease.

BACKGROUND/AIMS: Intestinal permeability with systemic distribution of bacterial products are central in the immunopathogenesis of alcoholic liver disease (ALD), yet links with intestinal immunity remain elusive. Mucosa-associated invariant T cells (MAIT) are found in liver, blood and intestinal mucosa and are a key component of antibacterial host defences. Their role in ALD is unknown. METHODS/DESIGN: We analysed frequency, phenotype, transcriptional regulation and function of blood MAIT cells in severe alcoholic hepatitis (SAH), alcohol-related cirrhosis (ARC) and healthy controls (HC). We also examined direct impact of ethanol, bacterial products from faecal extracts and antigenic hyperstimulation on MAIT cell functionality. Presence of MAIT cells in colon and liver was assessed by quantitative PCR and immunohistochemistry/gene expression respectively. RESULTS: In ARC and SAH, blood MAIT cells were dramatically depleted, hyperactivated and displayed defective antibacterial cytokine/cytotoxic responses. These correlated with suppression of lineage-specific transcription factors and hyperexpression of homing receptors in the liver with intrahepatic preservation of MAIT cells in ALD. These alterations were stronger in SAH, where surrogate markers of bacterial infection and microbial translocation were higher than ARC. Ethanol exposure in vitro, in vivo alcohol withdrawal and treatment with Escherichia coli had no effect on MAIT cell frequencies, whereas exposure to faecal bacteria/antigens induced functional impairments comparable with blood MAIT cells from ALD and significant MAIT cell depletion, which was not observed in other T cell compartments. CONCLUSIONS: In ALD, the antibacterial potency of MAIT cells is compromised as a consequence of contact with microbial products and microbiota, suggesting that the 'leaky' gut observed in ALD drives MAIT cell dysfunction and susceptibility to infection in these patients.