A mouse model of the regression of alcoholic hepatitis: Monitoring the regression of hepatic steatosis, inflammation, oxidative stress, and NAD+ metabolism upon alcohol withdrawal.

This study aimed to develop a well-characterized mouse model of alcoholic hepatitis (AH) regression. Male C57BL/6J mice were fed a Lieber-DeCarli (LD) control diet or LD containing 5% ethanol for ten days followed by one binge, which is the chronic-binge model of AH developed by the National Institute on Alcohol Abuse and Alcoholism. To determine AH regression, mice previously exposed to ethanol were put on LD control diet and metabolic and inflammatory features were monitored weekly for three weeks. Serum alcohol, total cholesterol, and alanine transaminase levels were increased in ethanol-fed mice, which declined to those of no ethanol controls within one and three weeks after ethanol withdrawal, respectively. Serum malondialdehyde was increased with ethanol feeding, but it was restored to no ethanol control levels within one week. Ethanol-induced changes in the hepatic expression of genes involved in lipogenesis, fatty acid oxidation, ethanol metabolism, and antioxidant response were restored to those of no ethanol controls after 3 weeks of ethanol withdrawal. Also, ethanol-induced hepatic inflammation was gradually decreased during the 3 weeks of ethanol withdrawal. Hepatic nicotinamide adenine dinucleotide (NAD+) levels and the expression of enzymes involved in the NAD+ salvage pathway were decreased by ethanol feeding, which was mitigated after ethanol withdrawal. Ethanol significantly lowered hepatic sirtuin 1 expression, but its levels were restored with ethanol cessation. This study established a mouse model of AH regression, which can be used as a preclinical model to study the potential of dietary bioactives or therapeutic agents on AH regression.

Disseminated Human Parvovirus B19 Infection Induced Multiple Organ Dysfunction Syndrome in an Adult Patient With Alcoholic Hepatitis Complicated by Hemolytic Anemia: A Case Report and Literature Review.

Background: Human parvovirus B19 (B19) can cause acute hepatitis and is attributed to the high mortality of alcoholic hepatitis (AH). B19 infection is generally self-healing in previously healthy people, but it can cause fatal effects in some high-risk groups and increase its virulence and infectivity. Disseminated B19 infection-induced multiple organ dysfunction syndrome (MODS) in patients with AH has not been reported yet. Here, we described B19 viremia in an adult patient with AH accompanied by hemolytic anemia (HA), leading to disseminated infection and secondary MODS, as well as self-limiting B19 infections in seven nurses caring for him. Meanwhile, we reviewed the literature on AH and B19 infection. Case Presentation: A 43-year-old male patient with AH accompanied by HA was transferred to the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China, on March 31, 2021. After supportive treatment, his transaminase and bilirubin levels were reduced, but his anemia worsened. He received a red blood cell (RBC) infusion on April 9 for hemoglobin (Hb) lower than 6 g/dl. On April 13, he suddenly had a high fever. Under empirical anti-infection, his high fever dropped and maintained at a low fever level; however, his anemia worsened. On April 25, he was transferred to the medical intensive care unit (MICU) due to severe pneumonia, acute respiratory distress syndrome (ARDS), acute aplastic crisis (AAC), and hemophagocytic syndrome (HPS), which were subsequently confirmed to be related to B19 infection. After methylprednisolone, intravenous immunoglobulin (IVIG), empirical anti-infection, and supportive treatment, the lung infection improved, but hematopoietic and liver abnormalities aggravated, and systemic B19 infection occurred. Finally, the patient developed a refractory arrhythmia, heart failure, and shock and was referred to a local hospital by his family on May 8, 2021. Unfortunately, he died the next day. Fourteen days after he was transferred to MICU, seven nurses caring for him in his first two days in the MICU developed self-limiting erythema infectiosum (EI). Conclusions: B19 infection is self-limiting in healthy people, with low virulence and infectivity; however, in AH patients with HA, it can lead to fatal consequences and high contagion.

Risk Prediction of Nosocomial and Posthospital Discharge Infections in Alcohol-Associated Hepatitis.

Alcohol-associated hepatitis (AAH) is a severe form of liver injury with mortality as high as 30%-40% at 90 days. As a result of altered immune function in AAH, bacterial infections are common and are associated with poor outcomes. However, determining the risk and subsequent development of infection in patients with AAH remain challenging. We performed a retrospective study of consecutive patients admitted with a diagnosis of AAH at two independent tertiary centers from 1998 to 2018 (test cohort, n = 286) who developed infections following hospitalization. The diagnosis of AAH was confirmed by manual chart review according to the recent National Institute on Alcohol Abuse and Alcoholism definition. Infections were categorized by location and time of diagnosis as hospital-acquired infection (48 hours after admission until discharge) and posthospital infections (up to 6 months following discharge). The cohort was 66% men, and the median age was 48 (21-83) years. Corticosteroids were used in 32% of all patients with AAH. The overall infection rate was 24%. Of those with infections, 46% were hospital acquired and 54% were acquired after hospitalization. Variables found to be significant risk factors for bacterial infection included the presence of ascites on admission (hazard ratio [HR], 2.06), corticosteroid administration (HR, 1.70), Model for End-Stage Liver Disease (MELD) >23 (HR, 2.61), and white blood cell (WBC) count on admission per point (HR, 1.02). Conclusion: In this multicenter cohort study of patients hospitalized with AAH, MELD score, ascites, WBC count, and use of corticosteroids were identified as significant predictors of the development of bacterial infection. We created a novel predictive equation that may be used to aid in the identification of patients with AAH at high risk of infection.

Early loss of T lymphocyte 4-1BB receptor expression is associated with higher short-term mortality in alcoholic hepatitis.

OBJECTIVES: In alcoholic hepatitis (AH), dysfunctional T lymphocytes may contribute to the high mortality from infections. T lymphocyte activation is governed by the expression of co-stimulatory receptors such as 4-1BB balanced by inhibitory receptors such as Programmed Death receptor 1 (PD-1). 4-1BB expression is unaccounted for in AH, while PD-1 is elevated. We characterized expression of 4-1BB and PD-1 and the associated T lymphocyte functional status in AH and investigated whether these were associated with short-term mortality. METHODS: Thirty-five patients with AH (at diagnosis and days 7 and 90) were compared with healthy controls (HC). Spontaneous and in vitro stimulated receptor expression were quantified by flow cytometry, and plasma proteins by ELISA. RESULTS: At diagnosis, the patients showed increased stimulated 4-1BB responses of CD4+ T lymphocytes. Also, the frequencies of PD-1+ T lymphocytes both with and without co-expressed 4-1BB were increased. Further, interferon-gamma was predominantly produced in T lymphocytes co-expressing 4-1BB. A decrease in the frequency of spontaneous 4-1BB+ T lymphocytes and an increase in soluble 4-1BB during the first week after diagnosis were associated with higher mortality at day 90 in AH. PD-1 expression showed no systematic dynamics related to mortality. CONCLUSIONS: We found an increased stimulated 4-1BB response of T lymphocytes in AH and early loss of these lymphocytes was associated with a higher short-term mortality. This suggests a role of T lymphocyte 4-1BB expression in the progression of AH.

Clinical Course and Risk Factors for Infection in Severe Forms of Alcohol-Associated Liver Disease.

BACKGROUND AND AIMS: Infection is a major driver of mortality in patients with advanced alcohol-associated liver disease (ALD). The epidemiology and clinical course of patients infected with life-threatening forms of ALD, including severe alcohol-associated hepatitis (sAH) and decompensated alcohol-associated cirrhosis (DAC), and specific risk factors for infection remain mostly unknown. APPROACH AND RESULTS: In this observational study, we assessed all infectious episodes occurring within a 90-day period from diagnosis in all consecutive patients with biopsy-proven sAH (modified Maddrey's discriminant function ≥ 32, Model for End-Stage Liver Disease [MELD] ≥ 18) and DAC (MELD ≥ 18) without alcohol-associated hepatitis in our tertiary hospital between 2003 and 2016. A total of 207 patients were included: 139 with sAH and 68 with DAC. One hundred seventeen (84%) patients with sAH and 41 (60%) patients with DAC experienced at least one infection episode at 90 days (P < 0.001). In multivariable analysis, factors associated with the development of infection were the presence of sAH and baseline MELD score. Bacterial infections represented the most common infection in the two groups, and only the MELD score was independently associated with the occurrence of bacterial infection. In both groups, pneumonia was the most prevalent bacterial infection, and gram-negative bacilli were the main pathogens. Invasive fungal infections (IFI) occurred in 20 (14.5%) patients with sAH and 3 (4.5%) with patients with DAC (P < 0.05). Multivariable regression showed that younger age, higher MELD, and corticosteroid therapy were independently associated with IFI. The 90-day cumulative incidence of death in patients infected with sAH and patients infected with DAC was 46% and 41.5%, respectively (P = 0.43). CONCLUSIONS: Patients with sAH are more susceptible to develop infection than those with DAC. In life-threatening forms of ALD, patients who were infected share a similar mortality rate. Corticosteroid treatment, not sAH, seems to be the main risk factor triggering IFI.

Role of MIF in coordinated expression of hepatic chemokines in patients with alcohol-associated hepatitis.

The chemokine system of ligands and receptors is implicated in the progression of alcohol-associated hepatitis (AH). Finding upstream regulators could lead to novel therapies. This study involved coordinated expression of chemokines in livers of healthy controls (HC) and patients with AH in 2 distinct cohorts of patients with various chronic liver diseases. Studies in cultured hepatocytes and in tissue-specific KO were used for mechanistic insight into a potential upstream regulator of chemokine expression in AH. Selected C-X-C chemokine members of the IL-8 chemokine family and C-C chemokine CCL20 were highly associated with AH compared with HC but not in patients with liver diseases of other etiologies (nonalcoholic fatty liver disease [NAFLD] and hepatitis C virus [HCV]). Our previous studies implicate macrophage migration inhibitory factor (MIF) as a pleiotropic cytokine/chemokine with the potential to coordinately regulate chemokine expression in AH. LPS-stimulated expression of multiple chemokines in cultured hepatocytes was dependent on MIF. Gao-binge ethanol feeding to mice induced a similar coordinated chemokine expression in livers of WT mice; this was prevented in hepatocyte-specific Mif-KO (MifΔHep) mice. This study demonstrates that patients with AH exhibit a specific, coordinately expressed chemokine signature and that hepatocyte-derived MIF might drive this inflammatory response.

Gene Deconvolution Reveals Aberrant Liver Regeneration and Immune Cell Infiltration in Alcohol-Associated Hepatitis.

BACKGROUND AND AIMS: Acute liver damage causes hepatocyte stress and death, but in chronic liver disease impaired hepatocyte regeneration and immune cell infiltration prevents recovery. While the roles of both impaired liver regeneration and immune infiltration have been studied extensively in chronic liver diseases, the differential contribution of these factors is difficult to assess. APPROACH AND RESULTS: We combined single-cell RNA-sequencing (RNA-seq) data from healthy livers and peripheral immune cells to measure cell proportions in chronic liver diseases. Using bulk RNA-seq data from patients with early alcohol-associated hepatitis, severe AH (sAH), HCV, HCV with cirrhosis, and NAFLD, we performed gene deconvolution to predict the contribution of different cell types in each disease. Patients with sAH had the greatest change in cell composition, with increases in both periportal hepatocytes and cholangiocyte populations. Interestingly, while central vein hepatocytes were decreased, central vein endothelial cells were expanded. Endothelial cells are thought to regulate liver regeneration through WNT signaling. WNT2, important in central vein hepatocyte development, was down in sAH, while multiple other WNTs and WNT receptors were up-regulated. Immunohistochemistry revealed up-regulation of FZD6, a noncanonical WNT receptor, in hepatocytes in sAH. Immune cell populations also differed in disease. In sAH, a specific group of inflammatory macrophages was increased and distinct from the macrophage population in patients with HCV. Network and correlation analyses revealed that changes in the cell types in the liver were highly correlated with clinical liver function tests. CONCLUSIONS: These results identify distinct changes in the liver cell populations in chronic liver disease and illustrate the power of using single-cell RNA-seq data from a limited number of samples in understanding multiple different diseases.

Interleukin-20 exacerbates acute hepatitis and bacterial infection by downregulating IκBζ target genes in hepatocytes.

BACKGROUND & AIMS: Interleukin (IL)-20 and IL-22 belong to the IL-10 family. IL-10 is a well-documented anti-inflammatory cytokine while IL-22 is well known for epithelial protection and its antibacterial function, showing great therapeutic potential for organ damage; however, the function of IL-20 remains largely unknown. METHODS: Il20 knockout (Il20-/-) mice and wild-type littermates were generated and injected with Concanavalin A (ConA) and Klebsiella pneumoniae (K.P.) to induce acute hepatitis and bacterial infection, respectively. RESULTS: Il20-/- mice were resistant to acute hepatitis and exhibited selectively elevated levels of the hepatoprotective cytokine IL-6. Such selective inhibition of IL-6 by IL-20 was due to IL-20 targeting hepatocytes that produce high levels of IL-6 but a limited number of other cytokines. Mechanistically, IL-20 upregulated NAD(P)H: quinone oxidoreductase 1 (NQO1) expression and subsequently promoted the protein degradation of transcription factor IκBζ, resulting in selective downregulation of the IκBζ-dependent gene Il6 as well several other IκBζ-dependent genes including lipocalin-2 (Lcn2). Given the important role of IL-6 and LCN2 in limiting bacterial infection, we examined the effect of IL-20 on bacterial infection and found Il20-/- mice were resistant to K.P. infection and exhibited elevated levels of hepatic IκBζ-dependent antibacterial genes. Moreover, IL-20 upregulated hepatic NQO1 by binding to IL-22R1/IL-20R2 and activating ERK/p38MAPK/NRF2 signaling pathways. Finally, the levels of hepatic IL1B, IL20, and IκBζ target genes were elevated, and correlated with each other, in patients with severe alcoholic hepatitis. CONCLUSIONS: IL-20 selectively inhibits hepatic IL-6 production rather than exerting IL-10-like broad anti-inflammatory properties. Unlike IL-22, IL-20 aggravates acute hepatitis and bacterial infection. Thus, anti-IL-20 therapy could be a promising option to control acute hepatitis and bacterial infection. LAY SUMMARY: Several interleukin (IL)-20 family cytokines have been shown to play important roles in controllimg inflammatory responses, infection and tissue damage, but the role of IL-20 remains unclear. Herein, we elucidated the role of IL-20 in liver disease and bacterial infection. We show that IL-20 can aggravate hepatitis and bacterial infection; thus, targeting IL-20 holds promise for the treatment of patients with liver disease.

Diagnostic and Prognostic Significance of Complement in Patients With Alcohol-Associated Hepatitis.

BACKGROUND AND AIMS: Given the lack of effective therapies and high mortality in acute alcohol-associated hepatitis (AH), it is important to develop rationally designed biomarkers for effective disease management. Complement, a critical component of the innate immune system, contributes to uncontrolled inflammatory responses leading to liver injury, but is also involved in hepatic regeneration. Here, we investigated whether a panel of complement proteins and activation products would provide useful biomarkers for severity of AH and aid in predicting 90-day mortality. APPROACH AND RESULTS: Plasma samples collected at time of diagnosis from 254 patients with moderate and severe AH recruited from four medical centers and 31 healthy persons were used to quantify complement proteins by enzyme-linked immunosorbent assay and Luminex arrays. Components of the classical and lectin pathways, including complement factors C2, C4b, and C4d, as well as complement factor I (CFI) and C5, were reduced in AH patients compared to healthy persons. In contrast, components of the alternative pathway, including complement factor Ba (CFBa) and factor D (CFD), were increased. Markers of complement activation were also differentially evident, with C5a increased and the soluble terminal complement complex (sC5b9) decreased in AH. Mannose-binding lectin, C4b, CFI, C5, and sC5b9 were negatively correlated with Model for End-Stage Liver Disease score, whereas CFBa and CFD were positively associated with disease severity. Lower CFI and sC5b9 were associated with increased 90-day mortality in AH. CONCLUSIONS: Taken together, these data indicate that AH is associated with a profound disruption of complement. Inclusion of complement, especially CFI and sC5b9, along with other laboratory indicators, could improve diagnostic and prognostic indications of disease severity and risk of mortality for AH patients.

Low Interleukin-22 Binding Protein Is Associated With High Mortality in Alcoholic Hepatitis and Modulates Interleukin-22 Receptor Expression.

INTRODUCTION: In alcoholic hepatitis (AH), high interleukin (IL)-22 production is associated with disease improvement, purportedly through enhanced infection resistance and liver regeneration. IL-22 binding protein (BP) binds and antagonizes IL-22 bioactivity, but data on IL-22BP in liver disease suggest a complex interplay. Despite the scarcity of human data, IL-22 is in clinical trial as treatment of AH. We, therefore, in patients with AH, described the IL-22 system focusing on IL-22BP and associations with disease course, and mechanistically pursued the human associations in vitro. METHODS: We prospectively studied 41 consecutive patients with AH at diagnosis, days 7 and 90, and followed them for up to 1 year. We measured IL-22 pathway proteins in liver biopsies and blood and investigated IL-22BP effects on IL-22 in hepatocyte cultures. RESULTS: IL-22BP was produced in the gut and was identifiable in the patients with AH' livers. Plasma IL-22BP was only 50% of controls and the IL-22/IL-22BP ratio thus elevated. Consistently, IL-22-inducible genes were upregulated in AH livers at diagnosis. Low plasma IL-22BP was closely associated with high 1-year mortality. In vitro, IL-22 stimulation reduced IL-22 receptor (R) expression, but coincubation with IL-22BP sustained IL-22R expression. In the AH livers, IL-22R mRNA expression was similar to healthy livers, although IL-22R liver protein was higher at diagnosis. DISCUSSION: Plasma IL-22BP was associated with an adverse disease course, possibly because its low level reduces IL-22R expression so that IL-22 bioactivity was reduced. This suggests the IL-BP interplay to be central in AH pathogenesis, and in future treatment trials (see Visual abstract, Supplementary Digital Content 5, http://links.lww.com/CTG/A338).

Interleukin-22 in alcoholic hepatitis and beyond.

Alcoholic hepatitis (AH) is a clinical syndrome characterized by jaundice and progressive inflammatory liver injury in patients with a history of prolonged periods of excess alcohol consumption and recent heavy alcohol abuse. Severe AH is a life-threatening form of alcohol-associated liver disease with a high short-term mortality rate around 30-50% at one month from the initial presentation. A large number of pro-inflammatory mediators, metabolic pathways, transcriptional factors and epigenetic factors have been suggested to be associated with the development and progression of AH. Several factors may contribute to liver failure and mortality in patients with severe AH including hepatocyte death, inflammation, and impaired liver regeneration. Although the pathogeneses of AH have been extensively investigated and many therapeutic targets have been identified over the last five decades, no new drugs for AH have been successfully developed. In this review, we discuss interleukin-22 (IL-22) biology and its roles of anti-apoptosis, anti-fibrosis, anti-oxidation, anti-bacterial infection and regenerative stimulation in protecting against liver injury in many preclinical models including several recently developed models such as chronic-plus-binge ethanol feeding, acute-on-chronic liver failure, C-X-C motif chemokine ligand 1 plus high-fat diet-induced nonalcoholic steatohepatitis. Finally, clinical trials of IL-22 for the treatment of AH are also discussed, which showed some promising benefits for AH patients.

Differential Activation of Unconventional T Cells, Including iNKT Cells, in Alcohol-Related Liver Disease.

BACKGROUND: Liver is enriched in several innate-like unconventional T cells, but their role in alcohol-related liver disease (ALD) is not fully understood. Studies in several acute alcohol feeding models but not in chronic alcoholic steatohepatitis (ASH) model have shown that invariant natural killer T (iNKT) cells play a pathogenic role in ALD. Here, we investigated the activation of iNKT cells in an intragastric (iG) infusion model of chronic ASH as well as the frequency and cytokine phenotype of 3 different unconventional T cells: iNKT, mucosal-associated invariant T (MAIT), and CD8+ CD161hi Vα7.2- cells in peripheral blood of ALD patients. METHODS: Hepatic iNKT cells were investigated using the iG model of chronic ASH that combines feeding of high-cholesterol/high-fat diet (HCFD) with intragastric feeding of ethanol diet (HCFD + iG Alc). Human iNKT, MAIT, and CD8+ CD161hi Vα7.2- cells were examined by flow cytometry in peripheral blood of patients with severe alcoholic hepatitis (SAH) and chronic alcoholics (ChA) and compared with healthy controls. RESULTS: In the iG model of chronic ASH, IFNγ+ iNKT cells accumulate in their livers compared with pair-fed control mice and activated hepatic iNKT cells show high expression of Fas and FasL. Notably, IFNγ+ iNKT cells are also significantly increased in peripheral blood of ChA patients compared with SAH patients. MAIT cells are significantly reduced in all ALD patients, but CD8+ CD161hi Vα7.2- cells are increased in SAH patients. Although MAIT and CD8+ CD161hi Vα7.2- cells displayed a similar cytokine production profile, the production of IFNγ and TNFα is significantly increased in SAH patients, while significant IL-17A production is found in ChA patients. CONCLUSIONS: We found that the 3 unconventional T cells are activated in ALD patients showing interesting differences in their frequency and cytokine production profile between SAH and ChA patients. In the iG murine model of chronic ASH, iNKT cells are also activated secreting proinflammatory cytokines suggesting their involvement in liver disease.

Epigenetic basis for monocyte dysfunction in patients with severe alcoholic hepatitis.

BACKGROUND & AIMS: Severe forms of alcohol-related liver disease are associated with increased susceptibility to infections which are associated with poor prognosis. The cellular and molecular mechanisms responsible for this altered host defense are incompletely understood. METHODS: We performed whole blood phenotypic analysis and ex vivo stimulation with various pathogen-associated molecular patterns (PAMPs). We included 34 patients with alcohol-related cirrhosis (18 of whom had biopsy-proven severe alcoholic hepatitis [sAH]), 12 healthy controls and 11 patients with chronic alcohol consumption without significant liver disease. We also evaluated the transcriptomic (RNA-seq) and chromatin accessibility (ATAC-seq) profiles of CD14+ monocytes from a subset of patients. RESULTS: Circulating monocytes and conventional dendritic cells (DCs) from patients with sAH displayed complex alterations characterized by increased expression of both activating and inhibitory surface markers and an impaired pro-inflammatory response upon stimulation with PAMPs representative of gram-negative bacteria (lipopolysaccharide, Pam3CSK4) or fungal pathogens (Zymosan). Their decreased ability to produce more than 1 cytokine (polyfunctionality) upon PAMP stimulation correlated with the risk of developing infection at 28 days or mortality at 90 days. The presence of acute-on-chronic liver failure in patients with sAH did not significantly modify the immune profile of monocytes and DCs. Moreover, CD14+ monocytes of patients with sAH displayed altered transcriptional and epigenomic profiles characterized by downregulation of key innate immune and metabolic pathways and upregulation of important immunomodulatory factors. CONCLUSIONS: In patients with sAH, the altered transcriptional program and functional properties of monocytes that contribute to patients' susceptibility to infection have strong epigenetic determinants. LAY SUMMARY: Patients with severe alcoholic hepatitis are at increased risk of infections, which contribute to the poor prognosis associated with the disease. Herein, we show that epigenetic determinants underly the immune cell dysfunction and inappropriate responses to pathogens that are associated with severe alcoholic hepatitis.

IL-33/ST2 pathway regulates neutrophil migration and predicts outcome in patients with severe alcoholic hepatitis.

BACKGROUND & AIMS: Severe alcoholic hepatitis (SAH) is associated with a high risk of infection. The IL-33/ST2 pathway is involved in sepsis control but data regarding its role in alcohol-related liver disease (ALD) are lacking. We aimed to characterize the role of IL-33/ST2 in the polymorphonuclear neutrophils (PMNs) of patients with ALD and SAH. METHODS: Serum and circulating neutrophils were collected from patients with SAH, alcoholic cirrhosis and healthy controls. We quantified IL-33/ST2 pathway activity and CXCR2 at baseline and after exposure to IL-33. We also determined the migration capacity of PMNs. RESULTS: The decoy receptor of IL-33 (soluble ST2 [sST2]) was increased in SAH vs. cirrhosis and controls, demonstrating the defect in this pathway during ALD. The sST2 level was associated with response to treatment, 2-month survival, infection-free survival and probability of infection in SAH. Endotoxemia was weakly correlated with sST2. GRK2, a negative regulator of CXCR2, was overexpressed in PMNs of patients with SAH and cirrhosis and was decreased by IL-33. CXCR2 levels on PMNs were lower in SAH vs. cirrhosis and controls. Treatment with IL-33 partially restored CXCR2 expression in SAH and cirrhosis. PMN migration upon IL-8 was lower in patients with SAH and cirrhosis vs. controls. Treatment with IL-33 partially restored migration in those with SAH and cirrhosis. Interestingly, the migration capacity of PMNs and the response to IL-33 were enhanced in responders to corticosteroids (Lille <0.45) compared to non-responders. CONCLUSION: The IL33/ST2 pathway is defective in SAH and predicts outcome. This defect is associated with decreased CXCR2 expression on the surface of PMNs and lower migration capacity, which can be corrected by IL-33, especially in patients responding to steroids. These results suggest that IL-33 has therapeutic potential for SAH and its infectious complications. LAY SUMMARY: The neutrophils of patients with severe alcoholic hepatitis are associated with a defect in the IL-33/ST2 pathway. This defect is associated with lower migration capacities in neutrophils and a higher probability of getting infected. Administration of IL-33 to the neutrophils at least partly restores this defect and may be effective at reducing the risk of infection in patients with severe alcoholic hepatitis.

Intestinal Fungal Dysbiosis and Systemic Immune Response to Fungi in Patients With Alcoholic Hepatitis.

Chronic alcohol consumption causes increased intestinal permeability and changes in the intestinal microbiota composition, which contribute to the development and progression of alcohol-related liver disease. In this setting, little is known about commensal fungi in the gut. We studied the intestinal mycobiota in a cohort of patients with alcoholic hepatitis, patients with alcohol use disorder, and nonalcoholic controls using fungal-specific internal transcribed spacer amplicon sequencing of fecal samples. We further measured serum anti-Saccharomyces cerevisiae antibodies (ASCA) as a systemic immune response to fungal products or fungi. Candida was the most abundant genus in the fecal mycobiota of the two alcohol groups, whereas genus Penicillium dominated the mycobiome of nonalcoholic controls. We observed a lower diversity in the alcohol groups compared with controls. Antibiotic or steroid treatment was not associated with a lower diversity. Patients with alcoholic hepatitis had significantly higher ASCA levels compared to patients with alcohol use disorder and to nonalcoholic controls. Within the alcoholic hepatitis cohort, patients with levels of at least 34 IU/mL had a significantly lower 90-day survival (59%) compared with those with ASCA levels less than 34 IU/mL (80%) with an adjusted hazard ratio of 3.13 (95% CI, 1.11-8.82; P = 0.031). Conclusion: Patients with alcohol-associated liver disease have a lower fungal diversity with an overgrowth of Candida compared with controls. Higher serum ASCA was associated with increased mortality in patients with alcoholic hepatitis. Intestinal fungi may serve as a therapeutic target to improve survival, and ASCA may be useful to predict the outcome in patients with alcoholic hepatitis.

The Major Histocompatibility Complex Class II-CD4 Immunologic Synapse in Alcoholic Hepatitis and Autoimmune Liver Pathology: The Role of Aberrant Major Histocompatibility Complex Class II in Hepatocytes.

The major histocompatibility complex class II (MHC II)-CD4 immunologic synapse is classically described between the T-cell receptor of CD4-positive lymphocytes and MHC II on antigen-presenting cells. This interaction and others between surrounding costimulatory and checkpoint molecules promote differentiation of naïve CD4 T lymphocytes into helper T cells subtypes, including types 1, 2, and 17 helper T cells, that have more tailored immunologic responses. Although MHC II is mainly produced by professional antigen-presenting cells, it can be aberrantly produced by other cell types, including hepatocytes in various liver pathologies, such as autoimmune hepatitis and alcoholic hepatitis. This can lead to direct targeting of hepatocytes by CD4-positive lymphocytes, which form an immunologic synapse with the hepatocyte. The lymphocytes internalize the MHC II-CD4 complexes in a phagocytosis-like mechanism and in the process eat the hepatocyte piece by piece. We review the evidence for this mechanism and the role of these autoimmune responses in various liver diseases, including alcoholic hepatitis, autoimmune hepatitis, and primary biliary cirrhosis. The role of aberrant MHC II in malignancy, including hepatocellular carcinoma, is also reviewed. Further understanding of this mechanism can lead to better understanding of the immune mechanisms involved in these liver pathologies, with potential diagnostic and therapeutic applications.

Overexpression of MHCII by hepatocytes in alcoholic hepatitis (AH) compared to non-alcoholic steatohepatitis (NASH) and normal controls.

Previously we have shown that in autoimmune hepatitis CD4 positive lymphocytes form an immunologic synapse with hepatocytes, leading to gradual diminishing and elimination of the hepatocyte. We wondered whether a similar mechanism may occur in alcoholic hepatitis (AH) and non-alcoholic steatohepatitis (NASH). We conducted immunofluorescence studies of expression of MHCII, the binding partner of CD4, on patient liver biopsies of AH, NASH, and normal controls. In cases of alcoholic hepatitis, there was prominent sinusoidal expression of MHC II; In NASH biopsies there was comparatively lower expression of MHC II, but still more than control tissue. Immunohistochemical stain for CD4 showed CD4 positive lymphocytes closely associated with hepatocytes in AH biopsies. Furthermore, expression levels of the multifunctional cytokine IL-1α was higher in AH compared to NASH and control biopsies. These results underlie the more severe nature of alcoholic hepatitis and underscore the autoimmune mechanisms involved in the liver damage found in alcoholic hepatitis.

Alcohol Abstinence Does Not Fully Reverse Abnormalities of Mucosal-Associated Invariant T Cells in the Blood of Patients With Alcoholic Hepatitis.

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.

miRNAs Involved in M1/M2 Hyperpolarization Are Clustered and Coordinately Expressed in Alcoholic Hepatitis.

The innate immune system, including monocytes/macrophages, is critical to the progression of alcoholic liver disease (ALD). In response to chronic ethanol, Kupffer cells, the resident macrophage of livers, and peripheral monocytes become sensitized to bacterial lipopolysaccharides (LPS), express more pro-inflammatory cytokines and exhibit macrophage M1/M2 hyperpolarization. Since miRNAs play an important role in the regulation of M1/M2 polarization, we hypothesized that miRNAs regulating macrophage polarization would be dysregulated after chronic ethanol consumption. miRNA sequencing data from Kupffer cells isolated from rats fed an ethanol diet vs. control diet and qPCR data from PBMCs isolated from alcoholic hepatitis (AH) patients and healthy controls were used to assess the role of miRNAs in macrophage hyperpolarization in ALD. Differential expression analyses revealed 40 misregulated miRNAs in Kupffer cells from the chronic ethanol-fed rats compared to pair-fed controls. Nine of these miRNAs are known to be associated with macrophage polarization and consist of a mixture of M1- and M2-associated miRNAs, indicative of hyperpolarization. Twenty-three of the 40 differentially expressed miRNAs were localized to miRNA clusters throughout the genome. Correlation analyses revealed that miRNAs in three of these clusters were co-regulated and located within antisense non-coding RNAs. Similar to Kupffer cells from ethanol-fed rats, M1 and M2 polarization markers, as well as sensitivity to LPS, were elevated in PBMCs from AH patients compared to healthy controls. These increases were associated with an up-regulation of polarization-associated miRNAs, including miR-125a-5p, a miRNA associated with hyperpolarization. miR-125a-5p is clustered in the genome with other miRNAs inside a host gene, Spaca6, which was also upregulated in PBMCs, as well as isolated monocytes, from AH patients. Finally, correlation analyses revealed co-regulation of human polarization-associated miRNA clusters. While expression of polarization-associated miRNAs in clusters was upregulated in AH compared to healthy controls, co-regulation of the miRNAs within a cluster was independent of disease state. Together, these results reveal that global changes in miRNA regulation are associated with polarization phenotypes in Kupffer cells from rat after chronic ethanol as well as in PBMCs from patients with AH. Importantly, polarization-associated miRNAs were localized to coordinately regulated clusters.