Hyperglycemic environments directly compromise intestinal epithelial barrier function in an organoid model and hyaluronan (∼35 kDa) protects via a layilin dependent mechanism.

BACKGROUND: Metabolic syndrome and diabetes in obese individuals are strong risk factors for development of inflammatory bowel disease (IBD) and colorectal cancer. The pathogenic mechanisms of low-grade metabolic inflammation, including chronic hyperglycemic stress, in disrupting gut homeostasis are poorly understood. In this study, we sought to understand the impact of a hyperglycemic environment on intestinal barrier integrity and the protective effects of small molecular weight (35 kDa) hyaluronan on epithelial barrier function. METHODS: Intestinal organoids derived from mouse colon were grown in normal glucose media (5 mM) or high glucose media (25 mM) to study the impact of hyperglycemic stress on the intestinal barrier. Additionally, organoids were pretreated with 35 kDa hyaluronan (HA35) to investigate the effect of hyaluronan on epithelial barrier under high glucose stress. Immunoblotting as well as confocal imaging was used to understand changes in barrier proteins, quantitative as well as spatial distribution, respectively. Alterations in barrier function were measured using trans-epithelial electrical resistance and fluorescein isothiocyanate flux assays. Untargeted proteomics analysis was performed to elucidate mechanisms by which HA35 exerts a protective effect on the barrier. Intestinal organoids derived from receptor knockout mice specific to various HA receptors were utilized to understand the role of HA receptors in barrier protection under high glucose conditions. RESULTS: We found that high glucose stress decreased the protein expression as well as spatial distribution of two key barrier proteins, zona occludens-1 (ZO-1) and occludin. HA35 prevented the degradation or loss of ZO-1 and maintained the spatial distribution of both ZO-1 and occludin under hyperglycemic stress. Functionally, we also observed a protective effect of HA35 on the epithelial barrier under high glucose conditions. We found that HA receptor, layilin, was involved in preventing barrier protein loss (ZO-1) as well as maintaining spatial distribution of ZO-1 and occludin. Additionally, proteomics analysis showed that cell death and survival was the primary pathway upregulated in organoids treated with HA35 under high glucose stress. We found that XIAP associated factor 1 (Xaf1) was modulated by HA35 thereby regulating apoptotic cell death in the intestinal organoid system. Finally, we observed that spatial organization of both focal adhesion kinase (FAK) as well as F-actin was mediated by HA35 via layilin. CONCLUSION: Our results highlight the impact of hyperglycemic stress on the intestinal barrier function. This is of clinical relevance, as impaired barrier function has been observed in individuals with metabolic syndrome. Additionally, we demonstrate barrier protective effects of HA35 through its receptor layilin and modulation of cellular apoptosis under high glucose stress.

Mincle-GSDMD-mediated release of IL-1ß small extracellular vesicles from hepatic macrophages in ethanol-induced liver injury.

BACKGROUND: Macrophage-inducible C-type lectin (Mincle) is expressed on hepatic macrophages and senses ethanol (EtOH)-induced danger signals released from dying hepatocytes and promotes IL-1ß production. However, it remains unclear what and how EtOH-induced Mincle ligands activate downstream signaling events to mediate IL-1ß release and contribute to alcohol-associated liver disease (ALD). In this study, we investigated the association of circulating ß-glucosylceramide (ß-GluCer), an endogenous Mincle ligand, with severity of ALD and examined the mechanism by which ß-GluCer engages Mincle on hepatic macrophages to release IL-1ß in the absence of cell death and exacerbates ALD. METHOD AND RESULTS: Concentrations of ß-GluCer were increased in serum of patients with severe AH and correlated with disease severity. Challenge of hepatic macrophages with lipopolysaccharide and ß-GluCer induced formation of a Mincle and Gsdmd-dependent secretory complex containing chaperoned full-length gasdermin D (Hsp90-CDC37-NEDD4) with polyubiquitinated pro-IL-1ß and components of the Caspase 8-NLRP3 inflammasome loaded as cargo in small extracellular vesicles (sEVs). Gao-binge EtOH exposure to wild-type, but not Mincle-/- and Gsdmd-/-, mice increased release of IL-1ß-containing sEVs from liver explant cultures. Myeloid-specific deletion of Gsdmd similarly decreased the formation of sEVs by liver explant cultures and protected mice from EtOH-induced liver injury. sEVs collected from EtOH-fed wild-type, but not Gsdmd-/-, mice promoted injury of cultured hepatocytes and, when injected into wild-type mice, aggravated Gao-binge EtOH-induced liver injury. CONCLUSION: ß-GluCer functions as a danger-associated molecular pattern activating Mincle-dependent gasdermin D-mediated formation and release of IL-1ß-containing sEVs, which in turn exacerbate hepatocyte cell death and contribute to the pathogenesis of ALD.

Myeloid-cell-specific role of Gasdermin D in promoting lung cancer progression in mice.

The activities of the NLRP3 and AIM2 inflammasomes and Gasdermin D (GsdmD) are implicated in lung cancer pathophysiology but it's not clear if their contributions promote or retard lung cancer progression. Using a metastatic Lewis lung carcinoma (LLC) cell model, we show that GsdmD knockout (GsdmD-/-) mice form significantly fewer cancer foci in lungs, exhibit markedly decreased lung cancer metastasis, and show a significant ∼50% increase in median survival rate. The cleaved forms of GsdmD and IL-1ß were detected in lung tumor tissue, indicating inflammasome activity in lung tumor microenvironment (TME). Increased migration and growth of LLC cells was observed upon exposure to the conditioned media derived from inflammasome-induced wild type, but not the GsdmD-/-, macrophages. Using bone marrow transplantations, we show a myeloid-specific contribution of GsdmD in lung cancer metastasis. Taken together, our data show that GsdmD plays a myeloid-specific role in lung cancer progression.

Macrophage-derived MLKL in alcohol-associated liver disease: Regulation of phagocytosis.

BACKGROUND AND AIMS: Mixed lineage kinase domain-like pseudokinase (MLKL), a key terminal effector of necroptosis, also plays a role in intracellular vesicle trafficking that is critical for regulating liver inflammation and injury in alcohol-associated liver disease (ALD). Although receptor interacting protein kinase 3 (Rip3)-/- mice are completely protected from ethanol-induced liver injury, Mlkl-/- mice are only partially protected. Therefore, we hypothesized that cell-specific functions of MLKL may contribute to ethanol-induced injury. APPROACH AND RESULTS: Bone marrow transplants between Mlkl-/- mice and littermates were conducted to distinguish the role of myeloid versus nonmyeloid Mlkl in the Gao-binge model of ALD. Ethanol-induced hepatic injury, steatosis, and inflammation were exacerbated in Mlkl-/- →wild-type (WT) mice, whereas Mlkl deficiency in nonmyeloid cells (WT→ Mlkl-/- ) had no effect on Gao-binge ethanol-induced injury. Importantly, Mlkl deficiency in myeloid cells exacerbated ethanol-mediated bacterial burden and accumulation of immune cells in livers. Mechanistically, challenging macrophages with lipopolysaccharide (LPS) induced signal transducer and activator of transcription 1-mediated expression and phosphorylation of MLKL, as well as translocation and oligomerization of MLKL to intracellular compartments, including phagosomes and lysosomes but not plasma membrane. Importantly, pharmacological or genetic inhibition of MLKL suppressed the phagocytic capability of primary mouse Kupffer cells (KCs) at baseline and in response to LPS with/without ethanol as well as peripheral monocytes isolated from both healthy controls and patients with alcohol-associated hepatitis. Further, in vivo studies revealed that KCs of Mlkl-/- mice phagocytosed fewer bioparticles than KCs of WT mice. CONCLUSION: Together, these data indicate that myeloid MLKL restricts ethanol-induced liver inflammation and injury by regulating hepatic immune cell homeostasis and macrophage phagocytosis.

A gut microbial metabolite of dietary polyphenols reverses obesity-driven hepatic steatosis.

The molecular mechanisms by which dietary fruits and vegetables confer cardiometabolic benefits remain poorly understood. Historically, these beneficial properties have been attributed to the antioxidant activity of flavonoids. Here, we reveal that the host metabolic benefits associated with flavonoid consumption hinge, in part, on gut microbial metabolism. Specifically, we show that a single gut microbial flavonoid catabolite, 4-hydroxyphenylacetic acid (4-HPAA), is sufficient to reduce diet-induced cardiometabolic disease (CMD) burden in mice. The addition of flavonoids to a high fat diet heightened the levels of 4-HPAA within the portal plasma and attenuated obesity, and continuous delivery of 4-HPAA was sufficient to reverse hepatic steatosis. The antisteatotic effect was shown to be associated with the activation of AMP-activated protein kinase α (AMPKα). In a large survey of healthy human gut metagenomes, just over one percent contained homologs of all four characterized bacterial genes required to catabolize flavonols into 4-HPAA. Our results demonstrate the gut microbial contribution to the metabolic benefits associated with flavonoid consumption and underscore the rarity of this process in human gut microbial communities.

Phosphoproteomics identifies pathways underlying the role of receptor-interaction protein kinase 3 in alcohol-associated liver disease and uncovers apoptosis signal-regulating kinase 1 as a target.

Receptor-interaction protein kinase 3 (RIP3), a critical determinant of the necroptotic pathway of programmed cell death, contributes to injury in murine models of alcohol-associated liver disease (ALD); however, the underlying mechanisms are unknown. We investigated the effect of chronic ethanol feeding on the hepatic phosphoproteome in C57BL/6 and RIP3-deficient (Rip3-/- ) mice, focusing on death receptor (DR) signaling pathways. C57BL/6 and Rip3-/- mice were fed an ethanol-containing liquid diet or pair-fed control diet. A label-free mass spectrometry-based approach identified differentially phosphorylated proteins that were mapped to pathways affected by ethanol and Rip3 genotype. Identified targets were validated in both the murine model of ALD and in liver tissue from patients with alcohol-associated hepatitis (AH) and healthy controls. Chronic ethanol dysregulated hepatic tumor necrosis factor-induced DR signaling pathways. Of particular importance, chronic ethanol feeding to C57BL/6 mice decreased the phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) at serine (S)1036/S1040 (S1029/S1033 human), sites linked with the inhibition of ASK1 death-promoting activity. This decrease in phosphorylation of inhibitory sites was muted in Rip3-/- mice. Decreased phosphorylation at S1033 was also lower in liver of patients with severe AH compared to healthy controls, and phosphorylation at the ASK1 activation site (threonine [Thr]-838) was increased in patients with AH. The net impact of these changes in phosphorylation of ASK1 was associated with increased phosphorylation of p38, a downstream target of ASK1, in patients with AH and C57BL/6 but not Rip3-/- mice. Similarly, chronic ethanol feeding affected the c-Jun N-terminal kinase pathway in C57BL/6 but not Rip3-/- mice. Taken together, our data indicate that changes in inhibitory phosphorylation of ASK1 are an important target in ALD and suggest the involvement of noncanonical functions of Rip3 in ALD.

SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure.

Alcohol abuse, reported by 1/8th critically ill patients, is an independent risk factor for death in sepsis. Sepsis kills over 270,000 patients/year in the US. We reported that the ethanol-exposure suppresses innate-immune response, pathogen clearance, and decreases survival in sepsis-mice via sirtuin 2 (SIRT2). SIRT2 is an NAD+-dependent histone-deacetylase with anti-inflammatory properties. We hypothesized that in ethanol-exposed macrophages, SIRT2 suppresses phagocytosis and pathogen clearance by regulating glycolysis. Immune cells use glycolysis to fuel increased metabolic and energy demand of phagocytosis. Using ethanol-exposed mouse bone marrow- and human blood monocyte-derived macrophages, we found that SIRT2 mutes glycolysis via deacetylating key glycolysis regulating enzyme phosphofructokinase-platelet isoform (PFKP), at mouse lysine 394 (mK394, human: hK395). Acetylation of PFKP at mK394 (hK395) is crucial for PFKP function as a glycolysis regulating enzyme. The PFKP also facilitates phosphorylation and activation of autophagy related protein 4B (Atg4B). Atg4B activates microtubule associated protein 1 light chain-3B (LC3). LC3 is a driver of a subset of phagocytosis, the LC3-associated phagocytosis (LAP), which is crucial for segregation and enhanced clearance of pathogens, in sepsis. We found that in ethanol-exposed cells, the SIRT2-PFKP interaction leads to decreased Atg4B-phosphorylation, decreased LC3 activation, repressed phagocytosis and LAP. Genetic deficiency or pharmacological inhibition of SIRT2 reverse PFKP-deacetylation, suppressed LC3-activation and phagocytosis including LAP, in ethanol-exposed macrophages to improve bacterial clearance and survival in ethanol with sepsis mice.

A genetic risk score and diabetes predict development of alcohol-related cirrhosis in drinkers.

BACKGROUND & AIMS: Only a minority of excess alcohol drinkers develop cirrhosis. We developed and evaluated risk stratification scores to identify those at highest risk. METHODS: Three cohorts (GenomALC-1: n = 1,690, GenomALC-2: n = 3,037, UK Biobank: relevant n = 6,898) with a history of heavy alcohol consumption (≥80 g/day (men), ≥50 g/day (women), for ≥10 years) were included. Cases were participants with alcohol-related cirrhosis. Controls had a history of similar alcohol consumption but no evidence of liver disease. Risk scores were computed from up to 8 genetic loci identified previously as associated with alcohol-related cirrhosis and 3 clinical risk factors. Score performance for the stratification of alcohol-related cirrhosis risk was assessed and compared across the alcohol-related liver disease spectrum, including hepatocellular carcinoma (HCC). RESULTS: A combination of 3 single nucleotide polymorphisms (SNPs) (PNPLA3:rs738409, SUGP1-TM6SF2:rs10401969, HSD17B13:rs6834314) and diabetes status best discriminated cirrhosis risk. The odds ratios (ORs) and (95% CIs) between the lowest (Q1) and highest (Q5) score quintiles of the 3-SNP score, based on independent allelic effect size estimates, were 5.99 (4.18-8.60) (GenomALC-1), 2.81 (2.03-3.89) (GenomALC-2), and 3.10 (2.32-4.14) (UK Biobank). Patients with diabetes and high risk scores had ORs of 14.7 (7.69-28.1) (GenomALC-1) and 17.1 (11.3-25.7) (UK Biobank) compared to those without diabetes and with low risk scores. Patients with cirrhosis and HCC had significantly higher mean risk scores than patients with cirrhosis alone (0.76 ± 0.06 vs. 0.61 ± 0.02, p = 0.007). Score performance was not significantly enhanced by information on additional genetic risk variants, body mass index or coffee consumption. CONCLUSIONS: A risk score based on 3 genetic risk variants and diabetes status enables the stratification of heavy drinkers based on their risk of cirrhosis, allowing for the provision of earlier preventative interventions. LAY SUMMARY: Excessive chronic drinking leads to cirrhosis in some people, but so far there is no way to identify those at high risk of developing this debilitating disease. We developed a genetic risk score that can identify patients at high risk. The risk of cirrhosis is increased >10-fold with just two risk factors - diabetes and a high genetic risk score. Risk assessment using this test could enable the early and personalised management of this disease in high-risk patients.

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.

Non-coding RNA crosstalk with nuclear receptors in liver disease.

The dysregulation of nuclear receptors (NRs) underlies the pathogenesis of a variety of liver disorders. Non-coding RNAs (ncRNAs) are defined as RNA molecules transcribed from DNA but not translated into proteins. MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are two types of ncRNAs that have been extensively studied for regulating gene expression during diverse cellular processes. NRs as therapeutic targets in liver disease have been exemplified by the successful application of their pharmacological ligands in clinics. MiRNA-based reagents or drugs are emerging as flagship products in clinical trials. Advancing our understanding of the crosstalk between NRs and ncRNAs is critical to the development of diagnostic and therapeutic strategies. This review summarizes recent findings on the reciprocal regulation between NRs and ncRNAs (mainly on miRNAs and lncRNAs) and their implication in liver pathophysiology, which might be informative to the translational medicine of targeting NRs and ncRNAs in liver disease.

Ethanol Exposure Attenuates Immune Response in Sepsis via Sirtuin 2 Expression.

BACKGROUND: Sepsis and septic shock kill over 270,000 patients per year in the United States. Sepsis transitions from a hyper-inflammatory to a hypo-inflammatory phase. Alcohol dependence is a risk factor for mortality from sepsis. Ethanol (EtOH) exposure impairs pathogen clearance through mechanisms that are not fully understood. Sirtuin 2 (SIRT2) interferes with pathogen clearance in immune cells but its role in the effects of EtOH on sepsis is unknown. We studied the effect of EtOH exposure on hyper- and hypo-inflammation and the role of SIRT2 in mice. METHODS: We exposed C57Bl/6 (WT) mice to EtOH via drinking water and used intraperitoneal cecal slurry (CS)-induced sepsis to study: (i) 7-day survival, (ii) leukocyte adhesion (LA) in the mesenteric microcirculation during hyper- and hypo-inflammation, (iii) peritoneal cavity bacterial clearance, and (iv) SIRT2 expression in peritoneal macrophages. Using EtOH-exposed and lipopolysaccharide (LPS)-stimulated RAW 264.7 (RAW) cell macrophages for 4 hours or 24 hours, we studied: (i) tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), and SIRT2 expression, and (ii) the effect of the SIRT2 inhibitor AK-7 on inflammatory response at 24 hours. Lastly, we studied the effect of EtOH on sepsis in whole body Sirt2 knockout (SIRT2KO) mice during hyper- and hypo-inflammation, bacterial clearance, and 7-day survival. RESULTS: WT EtOH-sepsis mice showed: (i) Decreased survival, (ii) Muted LA in the microcirculation, (iii) Lower plasma TNF-α and IL-6 expression, (iv) Decreased bacterial clearance, and (v) Increased SIRT2 expression in peritoneal macrophages versus vehicle-sepsis. EtOH-exposed LPS-stimulated RAW cells showed: (i) Muted TNF-α, IL-6, and increased IL-10 expression at 4 hours, (ii) endotoxin tolerance at 24 hours, and (iii) reversal of endotoxin tolerance with the SIRT2 inhibitor AK-7. EtOH-exposed SIRT2KO-sepsis mice showed greater 7-day survival, LA, and bacterial clearance than WT EtOH-sepsis mice. CONCLUSION: EtOH exposure decreases survival and reduces the inflammatory response to sepsis via increased SIRT2 expression. SIRT2 is a potential therapeutic target in EtOH with sepsis.

Activated Protein Phosphatase 2A Disrupts Nutrient Sensing Balance Between Mechanistic Target of Rapamycin Complex 1 and Adenosine Monophosphate-Activated Protein Kinase, Causing Sarcopenia in Alcohol-Associated Liver Disease.

BACKGROUND AND AIMS: Despite the high clinical significance of sarcopenia in alcohol-associated cirrhosis, there are currently no effective therapies because the underlying mechanisms are poorly understood. We determined the mechanisms of ethanol-induced impaired phosphorylation of mechanistic target of rapamycin complex 1 (mTORC1) and adenosine monophosphate-activated protein kinase (AMPK) with consequent dysregulated skeletal muscle protein homeostasis (balance between protein synthesis and breakdown). APPROACH AND RESULTS: Differentiated murine myotubes, gastrocnemius muscle from mice with loss and gain of function of regulatory genes following ethanol treatment, and skeletal muscle from patients with alcohol-associated cirrhosis were used. Ethanol increases skeletal muscle autophagy by dephosphorylating mTORC1, circumventing the classical kinase regulation by protein kinase B (Akt). Concurrently and paradoxically, ethanol exposure results in dephosphorylation and inhibition of AMPK, an activator of autophagy and inhibitor of mTORC1 signaling. However, AMPK remains inactive with ethanol exposure despite lower cellular and tissue adenosine triphosphate, indicating a "pseudofed" state. We identified protein phosphatase (PP) 2A as a key mediator of ethanol-induced signaling and functional perturbations using loss and gain of function studies. Ethanol impairs binding of endogenous inhibitor of PP2A to PP2A, resulting in methylation and targeting of PP2A to cause dephosphorylation of mTORC1 and AMPK. Activity of phosphoinositide 3-kinase-γ (PI3Kγ), a negative regulator of PP2A, was decreased in response to ethanol. Ethanol-induced molecular and phenotypic perturbations in wild-type mice were observed in PI3Kγ-/- mice even at baseline. Importantly, overexpressing kinase-active PI3Kγ but not the kinase-dead mutant reversed ethanol-induced molecular perturbations. CONCLUSIONS: Our study describes the mechanistic underpinnings for ethanol-mediated dysregulation of protein homeostasis by PP2A that leads to sarcopenia with a potential for therapeutic approaches by targeting the PI3Kγ-PP2A axis.

Hepatobiliary malignancies have distinct peripheral myeloid-derived suppressor cell signatures and tumor myeloid cell profiles.

Myeloid-derived suppressor cells (MDSCs) are immunosuppressive cells that are increased in patients with numerous malignancies including viral-derived hepatocellular carcinoma (HCC). Here, we report an elevation of MDSCs in the peripheral blood of patients with other hepatobiliary malignancies including non-viral HCC, neuroendocrine tumors (NET), and colorectal carcinoma with liver metastases (CRLM), but not cholangiocarcinoma (CCA). The investigation of myeloid cell infiltration in HCC, NET and intrahepatic CCA tumors further established that the frequency of antigen-presenting cells was limited compared to benign lesions, suggesting that primary and metastatic hepatobiliary cancers have distinct peripheral and tumoral myeloid signatures. Bioinformatics analysis of The Cancer Genome Atlas dataset demonstrated that a high MDSC score in HCC patients is associated with poor disease outcome. Given our observation that MDSCs are increased in non-CCA malignant liver cancers, these cells may represent suitable targets for effective immunotherapy approaches.

Design and rationale of a multicenter defeat alcoholic steatohepatitis trial: (DASH) randomized clinical trial to treat alcohol-associated hepatitis.

BACKGROUND/AIMS: Despite high mortality of alcohol-associated hepatitis, there has been limited advancement in treatment strategies. Defeat Alcoholic Steatohepatitis (DASH) is a multicenter, randomized, double-blind controlled trial whose primary objective was to evaluate the safety and efficacy of a novel combination of 3 drugs targeting different perturbations in AH. METHODS: Severe AH was diagnosed by liver biopsy or clinical and biochemical criteria and model for end stage liver disease (MELD) score ≥ 20 stratified by MELD scores (20-25 and ≥ 26) and randomized to a combination of an interleukin receptor 1 antagonist, Anakinra(100 mg daily for 14 days) to suppress acute inflammation, pentoxifylline (400 mg three times a day for 28 days) to prevent hepatorenal syndrome, and zinc sulfate (220 mg orally once daily for 6 months) or the standard of care therapy including methylprednisolone 32 mg orally once daily for 28 days. The primary efficacy outcome was the unadjusted log-rank test of the Kaplan-Meier survival estimates for the two treatment groups at 180 days. RESULTS: Between July 2012 to March 2018, 500 subjects with severe AH were screened of which 104 subjects were enrolled with MELD score of 25.6 ± 3.2 (20.0-35.0) in the investigational arm and 25.8 ± 4.5 (20.0-40.0) in the standard of care arm. Causes of screen failures included not meeting eligibility criteria (n = 347), declining to participate (n = 39), and other reasons (n = 10). CONCLUSIONS: Data from the DASH consortium studies will determine if a combination of drugs targeting multiple mechanisms of injury in the severe AH will improve clinical outcomes.

Alcoholic-Hepatitis, Links to Brain and Microbiome: Mechanisms, Clinical and Experimental Research.

The following review article presents clinical and experimental features of alcohol-induced liver disease (ALD). Basic aspects of alcohol metabolism leading to the development of liver hepatotoxicity are discussed. ALD includes fatty liver, acute alcoholic hepatitis with or without liver failure, alcoholic steatohepatitis (ASH) leading to fibrosis and cirrhosis, and hepatocellular cancer (HCC). ALD is fully attributable to alcohol consumption. However, only 10-20% of heavy drinkers (persons consuming more than 40 g of ethanol/day) develop clinical ALD. Moreover, there is a link between behaviour and environmental factors that determine the amount of alcohol misuse and their liver disease. The range of clinical presentation varies from reversible alcoholic hepatic steatosis to cirrhosis, hepatic failure, and hepatocellular carcinoma. We aimed to (1) describe the clinico-pathology of ALD, (2) examine the role of immune responses in the development of alcoholic hepatitis (ASH), (3) propose diagnostic markers of ASH, (4) analyze the experimental models of ALD, (5) study the role of alcohol in changing the microbiota, and (6) articulate how findings in the liver and/or intestine influence the brain (and/or vice versa) on ASH; (7) identify pathways in alcohol-induced organ damage and (8) to target new innovative experimental concepts modeling the experimental approaches. The present review includes evidence recognizing the key toxic role of alcohol in ALD severity. Cytochrome p450 CYP2E1 activation may change the severity of ASH. The microbiota is a key element in immune responses, being an inducer of proinflammatory T helper 17 cells and regulatory T cells in the intestine. Alcohol consumption changes the intestinal microbiota and influences liver steatosis and liver inflammation. Knowing how to exploit the microbiome to modulate the immune system might lead to a new form of personalized medicine in ALF and ASH.

MLKL-dependent signaling regulates autophagic flux in a murine model of non-alcohol-associated fatty liver and steatohepatitis.

BACKGROUND & AIMS: Autophagy maintains cellular homeostasis and plays a critical role in the development of non-alcoholic fatty liver and steatohepatitis. The pseudokinase mixed lineage kinase domain-like (MLKL) is a key downstream effector of receptor interacting protein kinase 3 (RIP3) in the necroptotic pathway of programmed cell death. However, recent data reveal that MLKL also regulates autophagy. Herein, we tested the hypothesis that MLKL contributes to the progression of Western diet-induced liver injury in mice by regulating autophagy. METHODS: Rip3+/+, Rip3-/-, Mlkl+/+ and Mlkl-/- mice were fed a Western diet (FFC diet, high in fat, fructose and cholesterol) or chow for 12 weeks. AML12 and primary mouse hepatocytes were exposed to palmitic acid (PA). RESULTS: The FFC diet increased expression, phosphorylation and oligomerization of MLKL in the liver. Mlkl, but not Rip3, deficiency protected mice from FFC diet-induced liver injury. The FFC diet also induced accumulation of p62 and LC3-II, as well as markers of endoplasmic reticulum stress, in Mlkl+/+ but not Mlkl-/- mice. Mlkl deficiency in mice also prevented the inhibition of autophagy by a protease inhibitor, leupeptin. Using an mRFP-GFP-LC3 reporter in cultured hepatocytes revealed that PA blocked the fusion of autophagosomes with lysosomes. PA triggered MLKL expression and translocation, first to autophagosomes and then to the plasma membrane, independently of Rip3. Mlkl, but not Rip3, deficiency prevented inhibition of autophagy in PA-treated hepatocytes. Overexpression of Mlkl blocked autophagy independently of PA. Additionally, pharmacologic inhibition of autophagy induced MLKL expression and translocation to the plasma membrane in hepatocytes. CONCLUSIONS: Taken together, these data indicate that MLKL-dependent, but RIP3-independent, signaling contributes to FFC diet-induced liver injury by inhibiting autophagy. LAY SUMMARY: Autophagy is a regulated process that maintains cellular homeostasis. Impaired autophagy contributes to cell injury and death, thus playing a critical role in the pathogenesis of a number of diseases, including non-alcohol-associated fatty liver and steatohepatitis. Herein, we show that Mlkl-dependent, but Rip3-independent, signaling contributed to diet-induced liver injury and inflammatory responses by inhibiting autophagy. These data identify a novel co-regulatory mechanism between necroptotic and autophagic signaling pathways in non-alcoholic fatty liver disease.

Specifically Sized Hyaluronan (35 kDa) Prevents Ethanol-Induced Disruption of Epithelial Tight Junctions Through a layilin-Dependent Mechanism in Caco-2 Cells.

BACKGROUND: Specific-sized species of the carbohydrate hyaluronan elicit a variety of cellular responses mediating tissue integrity and repair, as well as regulating inflammatory responses. Orally provided hyaluronan with an average molecular weight of 35 kDa (HA35) protects mice from short-term ethanol (EtOH)-induced liver injury. This protection was associated with maintenance of the colocalization of zonula occludens-1 (ZO-1) and occludin at tight junctions in the proximal colon. However, it is not known whether HA35 also protects other regions of the intestine or whether protection is due to a direct and/or indirect interaction of HA35 with the intestinal epithelium. METHODS: Female C57BL/6J mice were fed an EtOH containing diet or pair-fed control diet (4 days) and treated with or without HA35 via daily gavage during the last 3 days of EtOH feeding. Intestinal morphology and tight junction integrity were assessed. Differentiated Caco-2 cells were transfected or not with scrambled siRNA or siRNA targeting layilin, a hyaluronan receptor. Caco-2 cells were treated with or without HA35 prior to challenge with EtOH. Localization of tight junction proteins, fluorescein isothiocyanate (FITC)-dextran permeability, and transepithelial electrical resistance (TEER) were evaluated. RESULTS: While short-term EtOH did not result in any apparent changes in the gross morphology of the intestine, colocalization of ZO-1 and occludin at tight junctions was decreased in the proximal and distal colon. HA35 prevented these effects of EtOH. In differentiated Caco-2 cells, EtOH decreased the localization of ZO-1 and occludin at tight junctions and increased permeability of FITC-dextran. At higher concentrations, EtOH also decreased TEER. Pretreatment with HA35 prevented these changes. When the hyaluronan receptor layilin was knocked down in Caco-2 cells, HA35 no longer protected cells from EtOH-induced loss of tight junctions. CONCLUSIONS: Taken together, these data indicate that HA35 interacts with layilin on intestinal epithelial cells and maintains intestinal tight junction integrity during short-term EtOH exposure.

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.

Novel Role of Macrophage Migration Inhibitory Factor in Upstream Control of the Unfolded Protein Response After Ethanol Feeding in Mice.

BACKGROUND: Macrophage migration inhibitory factor (MIF), a pluripotent immune regulator, is an emerging mediator in alcohol-related liver disease (ALD). MIF is associated with ALD progression through its chemokine- and cytokine-like activities. METHODS: Mechanistic studies into the role of MIF in ethanol (EtOH)-induced liver injury were performed in Mif-/- mice and in C57BL/6J mice treated with a small-molecule MIF antagonist, MIF098, after Gao-Binge (acute-on-chronic) EtOH feeding, an EtOH feeding protocol associated with hepatic neutrophilia and induction of the unfolded protein response (UPR). RESULTS: The MIF axis, for example, MIF and MIF receptors invariant polypeptide of major histocompatibility complex, class II antigen-associated (CD74), CXCR2, CXCR4, and CXCR7, was enhanced in the livers of alcoholic hepatitis (AH) patients as compared to healthy controls. Mif-/- mice were protected from hepatocellular injury after Gao-Binge feeding, independent of neutrophilia and inflammation, but were associated with the UPR. Interestingly, the UPR signature in AH patients and in mice following Gao-Binge feeding was biased toward cell death with increased expression of pro-cell death CCAAT-enhancer-binding protein homologous protein (CHOP) and decreased prosurvival GRP78. The UPR and liver injury 6 hours after binge were prevented both in Mif-/- mice and in MIF098-treated mice. However, both MIF interventions led to increased liver injury and exacerbated the hepatic UPR 9 hours after binge. Induction of upstream UPR signaling and expression of CHOP protein by thapsigargin in alpha mouse liver 12 hepatocytes were blunted by coexposure to MIF098, directly connecting MIF to UPR in hepatocytes. CONCLUSIONS: The current study revealed that, in addition to its cytokine/chemokine functions, MIF is an upstream regulator of UPR in response to EtOH feeding in mice. Importantly, both MIF and UPR can either protect or contribute to liver injury, dependent upon the stage or severity of EtOH-induced liver injury.