Yu, Qigui

Abstract Alcoholic hepatitis (AH) develops in a small percentage of heavy drinkers. The adaptive immune system of AH patients is hyper-activated, yet dysregulated and ineffective against infectious diseases. Mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes that are found in liver, intestinal mucosa, and peripheral blood and contribute to protection against infectious diseases. To determine whether MAIT cells were dysregulated in heavy drinkers and the effect of alcohol abstinence on MAIT cell recovery, we compared peripheral MAIT cells between AH patients and heavy drinkers without liver disease (HDC) at baseline and 6- and 12-month followup. At baseline, AH patients had severely reduced frequency of MAIT cells than HDC and healthy donor controls (HC), whereas MAIT cell frequency was not significantly different between HDC and HC. In addition, MAIT cells in AH patients expressed higher levels of the activation markers (CD69, CD38, and HLA-DR) as well as the immune checkpoint inhibitor PD-1. Multiplex immunoassays showed that plasma levels of several cytokines related to MAIT cell activation were elevated in AH patients (IFN-s, IL-7, IL-15, IL-17, IL-18, and IL-23, and TNF-a). In AH patients, the frequency MAIT cells and CD38-expressing MAIT cells correlated with levels of several cytokines and AST. At followup, AH patients had increased MAIT cell frequency and decreased MAIT cell activation with cessation of alcohol. However, abstinent AH patients still had higher levels of CD38-expressing MAIT cells than HDC at 6-month followup. Conclusion Blood MAIT cells were severely reduced and were dysregulated in AH patients and alcohol abstinence partially reversed abnormalities in MAIT cell frequency and phenotype.

BackgroundHeavy alcohol consumption disrupts gut epithelial integrity, leading to increased permeability of the gastrointestinal tract and subsequent translocation of microbes. Regenerating islet‐derived protein 3α (REG3α) and Trefoil factor 3 (TFF3) are mainly secreted to the gut lumen by Paneth and Goblet cells, respectively, and are functionally linked to gut barrier integrity. Circulating levels of REG3α and TFF3 have been identified as biomarkers for gut damage in several human diseases. We examined whether plasma levels of REG3α and TFF3 were dysregulated and correlated with conventional markers of microbial translocation (MT) and pro‐inflammatory mediators in heavy drinkers with and without alcoholic hepatitis (AH).MethodsCross‐sectional and longitudinal studies were performed to monitor plasma levels of REG3α and TFF3 in 79 AH patients, 66 heavy drinkers without liver disease (HDC), and 46 healthy controls (HC) at enrollment and at 6‐ and 12‐month follow‐ups. Spearman correlation was used to measure the relationships of REG3α and TFF3 levels with MT, disease severity, inflammation, and effects of abstinence from alcohol.ResultsAt enrollment, AH patients had significantly higher levels of REG3α and TFF3 than HDC and HC. The elevated REG3α levels were positively correlated with the 30‐day fatality rate. Plasma levels of REG3α and TFF3 in AH patients differentially correlated with conventional MT markers (sCD14, sCD163, and LBP) and several highly up‐regulated inflammatory cytokines/chemokines/growth factors. At follow‐ups, although REG3α and TFF3 levels were decreased in AH patients with alcohol abstinence, they did not fully return to baseline levels.ConclusionsCirculating levels of REG3α and TFF3 were highly elevated in AH patients and differentially correlated with AH disease severity, MT, and inflammation, thereby serving as potential biomarkers of MT and gut epithelial damage in AH patients.

BackgroundAlcoholic hepatitis (AH) is a severe inflammatory liver disease that develops in some heavy drinkers. AH patients have intense hepatic infiltration of leukocytes. Up‐regulation of cell adhesion molecules (CAMs) upon endothelial cell (EC) activation plays an important role in leukocyte transendothelial migration. CAMs can shed from EC surface and accumulate in the blood, serving as soluble markers for EC activation. In this study, we examined the impact of heavy drinking on expression of soluble forms of EC activation markers (CD146, ICAM‐1, VCAM‐1, and VEGF‐A) and the effect of alcohol abstinence on the reversal of these abnormalities in heavy drinkers with and without AH.MethodsELISA and multiplex immunoassays were used to measure soluble EC activation markers in plasma samples from 79 AH patients, 66 heavy drinkers without overt liver disease (HDC), and 44 healthy controls (HC) at baseline, 31 AH patients and 30 HDC at 6‐month follow‐up, and 18 AH patients and 25 HDC at 12‐month follow‐up.ResultsAt baseline, the 4 soluble markers were significantly up‐regulated in AH patients compared with HDC and HC, whereas only sVCAM‐1 was elevated in HDC relative to HC. At follow‐ups, plasma levels of CD146, VCAM‐1, and VEGF‐A remained higher in AH patients, even for those who stopped drinking. These dysregulated markers correlated with AH disease severity, clinical parameters, and several soluble inflammatory factors.ConclusionsThe levels of soluble CD146, ICAM‐1, VCAM‐1, and VEGF‐A were highly elevated in AH patients, and alcohol abstinence did not completely reverse these abnormalities.

Alcoholic hepatitis (AH) is a severe inflammatory liver disease that develops in some heavy drinkers. The immune system in patients with AH is hyperactive and yet dysfunctional. Here, we investigated whether this immune‐dysregulated state is related to the alcoholic impact on immune checkpoints (ICPs). We used multiplex immunoassays and enzyme‐linked immunosorbent assay to quantify plasma levels of 18 soluble ICPs (sICPs) from 81 patients with AH, 65 heavy drinkers without liver diseases (HDCs), and 39 healthy controls (HCs) at baseline, 33 patients with AH and 32 HDCs at 6‐month follow‐up, and 18 patients with AH and 29 HDCs at 12‐month follow‐up. We demonstrated that baseline levels of 6 sICPs (soluble T‐cell immunoglobulin and mucin domain 3 [sTIM‐3], soluble cluster of differentiation [sCD]27, sCD40, soluble Toll‐like receptor‐2 [sTLR‐2], soluble herpesvirus entry mediator [sHVEM], and soluble lymphotoxin‐like inducible protein that competes with glycoprotein D for herpes virus entry on T cells [sLIGHT]) were up‐regulated, while 11 sICPs (soluble B‐ and T‐lymphocyte attenuator [sBTLA], sCD160, soluble cytotoxic T‐lymphocyte‐associated protein 4 [sCTLA‐4], soluble lymphocyte‐activation gene 3 [sLAG‐3], soluble programmed death 1 [sPD‐1], sPD ligand 1 [sPD‐L1], sCD28, soluble glucocorticoid‐induced tumor necrosis factor receptor‐related protein [sGITR], sGITR ligand [sGITRL], sCD80, and inducible T‐cell costimulator [sICOS]) were down‐regulated in patients with AH compared to HDCs. The up‐regulated sICPs except sLIGHT and down‐regulated sCD80, sCD160, sCTLA‐4, and sLAG‐3 correlated positively or negatively with AH disease severity, bacterial translocation, and inflammatory factors. At follow‐up, abstinent patients with AH still had higher levels of several sICPs compared to HDCs. We also compared expression of 10 membrane‐bound ICPs (mICPs) on peripheral blood mononuclear cells (PBMCs) from patients with AH and HCs by flow cytometry and found that several mICPs were dysregulated on blood cells from patients with AH. The function and regulation of sICPs and mICPs were studied using PBMCs from patients with AH and HCs. Recombinant sHVEM affected tumor necrosis factor (TNF)‐α and interferon‐γ production by T cells from patients with AH and HCs. Conclusion: Both sICPs and mICPs were dysregulated in patients with AH, and alcohol abstinence did not fully reverse these abnormalities. The HVEM axis plays a role in regulating T‐cell function in patients with AH.

OBJECTIVES: Alcoholic hepatitis (AH) develops in approximately 30% of chronic heavy drinkers. The immune system of patients with AH is hyperactivated, yet ineffective against infectious diseases. Mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes that are highly enriched in liver, mucosa, and peripheral blood and contribute to antimicrobial immunity. We aimed to determine whether MAIT cells were dysregulated in heavy drinkers with and without AH and the effects of alcohol abstinence on MAIT cell recovery. METHODS: MR1 tetramers loaded with a potent MAIT cell ligand 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil were used in multiparameter flow cytometry to analyze peripheral blood MAIT cells in 59 healthy controls (HC), 56 patients with AH, and 45 heavy drinkers without overt liver disease (HDC) at baseline and 6- and 12-month follow-ups. Multiplex immunoassays were used to quantify plasma levels of cytokines related to MAIT cell activation. Kinetic Turbidimetric Limulus Amebocyte Lysate Assay and ELISA were performed to measure circulating levels of 2 surrogate markers for bacterial translocation (lipopolysaccharide and CD14), respectively. RESULTS: At baseline, patients with AH had a significantly lower frequency of MAIT cells than HDC and HC. HDC also had less MAIT cells than HC (median 0.16% in AH, 0.56% in HDC, and 1.25% in HC). Further, the residual MAIT cells in patients with AH expressed higher levels of activation markers (CD69, CD38, and human leukocyte antigen [HLA]-DR), the effector molecule granzyme B, and the immune exhaustion molecule PD-1. Plasma levels of lipopolysaccharide and CD14 and several cytokines related to MAIT cell activation were elevated in patients with AH (interferon [IFN]-α, interleukin [IL]-7, IL-15, IL-17, IL-18, IL-23, IFN-γ, and tumor necrosis factor α). Decreased MAIT cell frequency and upregulated CD38, CD69, and HLA-DR correlated negatively and positively, respectively, with aspartate aminotransferase level. MAIT cell frequency negatively correlated with IL-18. HLA-DR and CD38 levels correlated with several cytokines. At follow-ups, abstinent patients with AH had increased MAIT cell frequency and decreased MAIT cell activation. However, MAIT cell frequency was not fully normalized in patients with AH (median 0.31%). DISCUSSION: We showed that HDC had a reduction of blood MAIT cells despite showing little evidence of immune activation, whereas patients with AH had a severe depletion of blood MAIT cells and the residual cells were highly activated. Alcohol abstinence partially reversed those abnormalities.

Intestinal dysbiosis is implicated in alcoholic hepatitis (AH). However, changes in the circulating microbiome, its association with the presence and severity of AH, and its functional relevance in AH is unknown. Qualitative and quantitative assessment of changes in the circulating microbiome were performed by sequencing bacterial DNA in subjects with moderate AH (MAH) (n = 18) or severe AH (SAH) (n = 19). These data were compared with heavy drinking controls (HDCs) without obvious liver disease (n = 19) and non–alcohol‐consuming controls (NACs, n = 20). The data were related to endotoxin levels and markers of monocyte activation. Linear discriminant analysis effect size (LEfSe) analysis, inferred metagenomics, and predictive functional analysis using PICRUSt were performed. There was a significant increase in 16S copies/ng DNA both in MAH ( P < 0.01) and SAH ( P < 0.001) subjects. Compared with NACs, the relative abundance of phylum Bacteroidetes was significantly decreased in HDCs, MAH, and SAH ( P < 0.001). In contrast, all alcohol‐consuming groups had enrichment with Fusobacteria ; this was greatest for HDCs and decreased progressively in MAH and SAH. Subjects with SAH had significantly higher endotoxemia ( P = 0.01). Compared with alcohol‐consuming groups, predictive functional metagenomics indicated an enrichment of bacteria with genes related to methanogenesis and denitrification. Furthermore, both HDCs and SAH showed activation of a type III secretion system that has been linked to gram‐negative bacterial virulence. Metagenomics in SAH versus NACs predicted increased isoprenoid synthesis via mevalonate and anthranilate degradation, known modulators of gram‐positive bacterial growth and biofilm production, respectively. Conclusion: Heavy alcohol consumption appears to be the primary driver of changes in the circulating microbiome associated with a shift in its inferred metabolic functions. (H epatology 2018;67:1284‐1302).

Abstract Follicular Th (Tfh) cells are a distinct subset of Th cells that help B cells produce class-switched antibodies. Studies have demonstrated that Tfh cells are highly prone to HIV infection and replication. However, the molecular mechanisms underlying this phenomenon are largely unclear. Here, we show that murine and human Tfh cells have diminished constitutive expression of IFN-stimulated genes (ISGs) inclusive of antiviral resistance factor MX dynamin-like GTPase 2 (MX2) and IFN-induced transmembrane 3 (IFITM3) compared with non-Tfh cells. A lower antiviral resistance in Tfh was consistent with a higher susceptibility to retroviral infections. Mechanistically, we found that BCL6, a master regulator of Tfh cell development, binds to ISG loci and inhibits the expression of MX2 and IFITM3 in Tfh cells. We demonstrate further that inhibition of the BCL6 BR-C, ttk, and bab (BTB) domain function increases the expression of ISGs and suppresses HIV infection and replication in Tfh cells. Our data reveal a regulatory role of BCL6 in inhibiting antiviral resistance factors in Tfh cells, thereby promoting the susceptibility Tfh cells to viral infections. Our results indicate that the modulation of BCL6 function in Tfh cells could be a potential strategy to enhance Tfh cell resistance to retroviral infections and potentially decrease cellular reservoirs of HIV infection.

Alcoholic hepatitis (AH) develops in only a small proportion of heavy drinkers. To better understand the mechanisms underlying this disparity, we conducted a study to define the relationship between AH development and dysregulated immune responses that might be ameliorated by alcohol abstinence. Sixty‐eight AH patients, 65 heavy drinking controls without liver disease (HDC), and 20 healthy controls were enrolled and followed up to 12 months. At baseline, HDC and healthy controls had no significant differences in their plasma levels of 38 inflammatory cytokines/chemokines measured using multiplex immunoassays. However, compared to HDC, AH patients had higher baseline levels of 11 cytokines/chemokines (tumor necrosis factor alpha, interleukin 6 [IL‐6], IL‐8, interferon gamma–induced protein 10, IL‐4, IL‐9, IL‐10, fibroblast growth factor 2, IL‐7, IL‐15, and transforming growth factor alpha) but lower levels of the anti‐inflammatory macrophage‐derived chemokine. AH patients also had more activated yet dysfunctional immune cells as monocytes, T cells, and B cells expressed higher levels of cluster of differentiation 38 (CD38) and CD69 but low levels of human leukocyte antigen DR, CD80, and CD86 at baseline. In addition, CD4 T cells produced less interferon‐gamma in response to T‐cell stimulation. Up‐regulated IL‐6, IL‐8, CD38, and CD69 and down‐regulated macrophage‐derived chemokine, human leukocyte antigen DR, CD86, and CD80 correlated positively and negatively, respectively, with disease severity. Longitudinal analysis indicated that levels of IL‐6, IL‐8, CD38, and CD69 were reduced, whereas levels of macrophage‐derived chemokine, human leukocyte antigen DR, CD80, and CD86 were increased in abstinent AH patients. All of the cellular immune abnormalities were reversed by day 360 in abstinent AH patients; however, plasma levels of tumor necrosis factor alpha, IL‐8, IL‐10, fibroblast growth factor 2, and IL‐7 remained higher. Conclusion: AH patients were in a highly immune‐dysregulated state, whereas HDC showed little evidence of immune activation; alcohol abstinence reversed most, but not all, of the immunological abnormalities. (Hepatology 2017;66:575–590).