Bile acid-induced IRF3 phosphorylation mediates cell death, inflammatory responses, and fibrosis in cholestasis-induced liver and kidney injury via regulation of ZBP1.

BACKGROUND AND AIMS: Cell death and inflammation play critical roles in chronic tissue damage caused by cholestatic liver injury leading to fibrosis and cirrhosis. Liver cirrhosis is often associated with kidney damage, which is a severe complication with poor prognosis. Interferon regulatory factor 3 (IRF3) is known to regulate apoptosis and inflammation, but its role in cholestasis remains obscure. In this study. APPROACH AND RESULTS: We discovered increased IRF3 phosphorylation in the liver of patients with primary biliary cholangitis and primary sclerosing cholangitis. In the bile duct ligation model of obstructive cholestasis in mice, we found that tissue damage was associated with increased phosphorylated IRF3 (p-IRF3) in the liver and kidney. IRF3 knockout ( Irf3-/- ) mice showed significantly attenuated liver and kidney damage and fibrosis compared to wide-type mice after bile duct ligation. Cell-death pathways, including apoptosis, necroptosis, and pyroptosis, inflammasome activation, and inflammatory responses were significantly attenuated in Irf3-/- mice. Mechanistically, we show that bile acids induced p-IRF3 in vitro in hepatocytes. In vivo , activated IRF3 positively correlated with increased expression of its target gene, Z-DNA-Binding Protein-1 (ZBP1), in the liver and kidney. Importantly, we also found increased ZBP1 in the liver of patients with primary biliary cholangitis and primary sclerosing cholangitis. We discovered that ZBP1 interacted with receptor interacting protein 1 (RIP1), RIP3, and NLRP3, thereby revealing its potential role in the regulation of cell-death and inflammation pathways. In conclusion. CONCLUSIONS: Our data indicate that bile acid-induced p-IRF3 and the IRF3-ZBP1 axis play a central role in the pathogenesis of cholestatic liver and kidney injury.

Acute kidney injury in severe alcohol-associated hepatitis treated with anakinra plus zinc or prednisone.

BACKGROUND AIMS: In a recent trial, patients with severe-alcohol-associated-hepatitis (sAH) treated with anakinra-plus-zinc (A+Z) had lower survival and higher acute-kidney-injury (AKI) rates versus prednisone (PRED). We characterize the clinical factors and potential mechanisms associated with AKI development in that trial. APPROACH RESULTS: Data from 147-participants in a multicenter randomized clinical trial (74 A+Z, 73 PRED) were analyzed. AKI, AKI-phenotypes, and kidney-injury biomarkers were compared between participants who did/did not develop AKI in the two treatment-arms. Multivariable competing-risk analyses were performed to identify baseline risk-factors for incident AKI, with death treated as a competing event. Risk-factors considered were age, sex, mean arterial pressure, white blood cell count, albumin, MELD, ascites, hepatic encephalopathy, and treatment arm. At baseline, no participants had AKI; 33% (n=49) developed AKI during follow-up. AKI incidence was higher in A+Z than PRED [45% (n=33) versus 22% (n=16), p=0.001]. AKI-phenotypes were similar between the two treatment-arms (p=0.361) but peak-AKI severity was greater in A+Z than PRED [stage-3 n=21 (63.6%) versus n=8 (50.0%), p=0.035]. At baseline, urine-neutrophil-gelatinase-associated-lipocalin (uNGAL) levels were similar between participants who developed AKI in both treatment-arms (p=0.319). However, day 7 and 14 uNGAL levels were significantly elevated in A+Z-treated participants who developed AKI versus PRED-treated participants who developed AKI (p=0.002 and p=0.032, respectively). On multivariable competing-risk analysis, only A+Z was independently associated with incident AKI (sHR 2.35, p=0.005). CONCLUSIONS: AKI occurred more frequently and was more severe in A+Z-treated participants. A+Z-treated participants with AKI had higher uNGAL, suggesting that A+Z maybe nephrotoxic in sAH patients.

Neutrophil extracellular traps activate hepatic stellate cells and monocytes via NLRP3 sensing in alcohol-induced acceleration of MASH fibrosis.

OBJECTIVE: Alcohol use in metabolic dysfunction-associated steatohepatitis (MASH) is associated with an increased risk of fibrosis and liver-related death. Here, we aimed to identify a mechanism through which repeated alcohol binges exacerbate liver injury in a high fat-cholesterol-sugar diet (MASH diet)-induced model of MASH. DESIGN: C57BL/6 mice received either chow or the MASH diet for 3 months with or without weekly alcohol binges. Neutrophil infiltration, neutrophil extracellular traps (NETs) and fibrosis were evaluated. RESULTS: We found that alcohol binges in MASH increase liver injury and fibrosis. Liver transcriptomic profiling revealed differential expression of genes involved in extracellular matrix reorganisation, neutrophil activation and inflammation compared with alcohol or the MASH diet alone. Alcohol binges specifically increased NET formation in MASH livers in mice, and NETs were also increased in human livers with MASH plus alcohol use. We discovered that cell-free NETs are sensed via Nod-like receptor protein 3 (NLRP3). Furthermore, we show that cell-free NETs in vitro induce a profibrotic phenotype in hepatic stellate cells (HSCs) and proinflammatory monocytes. In vivo, neutrophil depletion using anti-Ly6G antibody or NET disruption with deoxyribonuclease treatment abrogated monocyte and HSC activation and ameliorated liver damage and fibrosis. In vivo, inhibition of NLRP3 using MCC950 or NLRP3 deficiency attenuated NET formation, liver injury and fibrosis in MASH plus alcohol diet-fed mice (graphical abstract). CONCLUSION: Alcohol binges promote liver fibrosis via NET-induced activation of HSCs and monocytes in MASH. Our study highlights the potential of inhibition of NETs and/or NLRP3, as novel therapeutic strategies to combat the profibrotic effects of alcohol in MASH.

Inflammasomes in chronic liver disease: hepatic injury, fibrosis progression and systemic inflammation.

Chronic liver disease (CLD) leads to hepatocellular injury that triggers a pro-inflammatory state in several parenchymal and non-parenchymal hepatic cell types ultimately resulting in liver fibrosis, cirrhosis, portal hypertension (PH) and liver failure. Thus, an improved understanding of the inflammasomes - as key molecular drivers of liver injury - supports the development of novel diagnostic or prognostic biomarkers and effective therapeutics. In liver disease, innate immune cells respond to hepatic noxes by activating cell-intrinsic inflammasomes via toll-like receptors (TLRs) and nuclear factor kappa-B (NF-κB) and release of pro-inflammatory cytokines (such as IL-1ß, IL-18, TNF-α and IL-6). Subsequently, cells of the adaptive immune system are recruited to fuel hepatic inflammation, and liver parenchymal cells may undergo programmed cell-death mediated by gasdermin D, termed pyroptosis. With liver disease progression, there is a shift towards a type 2 inflammatory response, which promotes tissue repair but also fibrogenesis. Inflammasome activation may also occur at extrahepatic sites, such as the white adipose tissue in metabolic dysfunction-associated steatohepatitis (MASH). In end-stage liver disease, flares of inflammation (e.g., in severe alcohol-related hepatitis) that spark on a dysfunctional immune system, contribute to inflammasome-mediated liver injury and potentially result in organ dysfunctions/failures, as seen in acute-on-chronic liver failure (ACLF). This review provides an overview on current concepts regarding inflammasome activation in liver disease progression and related biomarkers and therapeutic approaches that are being developed for patients with liver disease.

Randomized trial of anakinra plus zinc vs. prednisone for severe alcohol-associated hepatitis.

BACKGROUND & AIMS: Severe alcohol-associated hepatitis (SAH) is associated with high 90-day mortality. Glucocorticoid therapy for 28 days improves 30- but not 90-day survival. We assessed the efficacy and safety of a combination of anakinra, an IL-1 antagonist, plus zinc (A+Z) compared to prednisone using the Day-7 Lille score as a stopping rule in patients with SAH. METHODS: In this phase IIb double-blind randomized trial in adults with SAH and MELD scores of 20-35, participants were randomized to receive either daily anakinra 100 mg subcutaneously for 14 days plus daily zinc sulfate 220 mg orally for 90 days, or daily prednisone 40 mg orally for 30 days. Prednisone or prednisone placebo was stopped if Day-7 Lille score was >0.45. All study drugs were stopped for uncontrolled infection or ≥5 point increase in MELD score. The primary endpoint was overall survival at 90 days. RESULTS: Seventy-three participants were randomized to prednisone and 74 to A+Z. The trial was stopped early after a prespecified interim analysis showed prednisone was associated with higher 90-day overall survival (90% vs. 70%; hazard ratio for death = 0.34, 95% CI 0.14-0.83, p = 0.018) and transplant-free survival (88% vs. 64%; hazard ratio for transplant or death = 0.30, 95% CI 0.13-0.69, p = 0.004) than A+Z. Acute kidney injury was more frequent with A+Z (45%) than prednisone (22%) (p = 0.001), but rates of infection were similar (31% in A+Z vs. 27% in prednisone, p = 0.389). CONCLUSIONS: Participants with SAH treated with prednisone using the Day-7 Lille score as a stopping rule had significantly higher overall and transplant-free 90-day survival and lower incidence of acute kidney injury than those treated with A+Z. IMPACT AND IMPLICATIONS: There is no approved treatment for severe alcohol-associated hepatitis (SAH). In this double-blind randomized trial, patients with SAH treated with prednisone using the Lille stopping rule on Day 7 had higher 90-day overall and transplant-free survival and lower rates of acute kidney injury compared to patients treated with a combination of anakinra and zinc. The data support continued use of glucocorticoids for patients with SAH, with treatment discontinuation for those with a Lille score >0.45 on Day 7. TRIAL REGISTRATION: NCT04072822.

Safety, Pharmacokinetics, and Efficacy Signals of Larsucosterol (DUR-928) in Alcohol-Associated Hepatitis.

INTRODUCTION: This study is to evaluate the safety and pharmacokinetics (PK) of larsucosterol (DUR-928 or 25HC3S) in subjects with alcohol-associated hepatitis (AH), a devastating acute illness without US Food and Drug Administration-approved therapies. METHODS: This phase 2a, multicenter, open-label, dose escalation study evaluated the safety, PK, and efficacy signals of larsucosterol in 19 clinically diagnosed subjects with AH. Based on the model for end-stage liver disease (MELD) score, 7 subjects were considered to have moderate AH and 12 to have severe AH. All subjects received 1 or 2 intravenous infusions (72 hours apart) of larsucosterol at a dose of 30, 90, or 150 mg and were followed up for 28 days. Efficacy signals from a subgroup of subjects with severe AH were compared with those from 2 matched arms of those with severe AH treated with standard of care (SOC), including corticosteroids, from a contemporaneous study. RESULTS: All 19 larsucosterol-treated subjects survived the 28-day study. Fourteen (74%) of all subjects including 8 (67%) of the subjects with severe AH were discharged ≤72 hours after receiving a single infusion. There were no drug-related serious adverse events nor early terminations due to the treatment. PK profiles were not affected by disease severity. Biochemical parameters improved in most subjects. Serum bilirubin levels declined notably from baseline to day 7 and day 28, and MELD scores were reduced at day 28. The efficacy signals compared favorably with those from 2 matched groups treated with SOC. Lille scores at day 7 were <0.45 in 16 of the 18 (89%) subjects with day 7 samples. Lille scores from 8 subjects with severe AH who received 30 or 90 mg larsucosterol (doses used in phase 2b trial) were statistically significantly lower ( P < 0.01) than those from subjects with severe AH treated with SOC from the contemporaneous study. DISCUSSION: Larsucosterol was well tolerated at all 3 doses in subjects with AH without safety concerns. Data from this pilot study showed promising efficacy signals in subjects with AH. Larsucosterol is being evaluated in a phase 2b multicenter, randomized, double-blinded, placebo-controlled (AHFIRM) trial.

Alcohol-induced extracellular ASC specks perpetuate liver inflammation and damage in alcohol-associated hepatitis even after alcohol cessation.

BACKGROUND AIMS: Prolonged systemic inflammation contributes to poor clinical outcomes in severe alcohol-associated hepatitis (AH) even after the cessation of alcohol use. However, mechanisms leading to this persistent inflammation remain to be understood. APPROACH RESULTS: We show that while chronic alcohol induces nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation in the liver, alcohol binge results not only in NLRP3 inflammasome activation but also in increased circulating extracellular apoptosis-associated speck-like protein containing a caspase recruitment domain (ex-ASC) specks and hepatic ASC aggregates both in patients with AH and in mouse models of AH. These ex-ASC specks persist in circulation even after the cessation of alcohol use. Administration of alcohol-induced-ex-ASC specks in vivo in alcohol-naive mice results in sustained inflammation in the liver and circulation and causes liver damage. Consistent with the key role of ex-ASC specks in mediating liver injury and inflammation, alcohol binge failed to induce liver damage or IL-1ß release in ASC-deficient mice. Our data show that alcohol induces ex-ASC specks in liver macrophages and hepatocytes, and these ex-ASC specks can trigger IL-1ß release in alcohol-naive monocytes, a process that can be prevented by the NLRP3 inhibitor, MCC950. In vivo administration of MCC950 reduced hepatic and ex-ASC specks, caspase-1 activation, IL-1ß production, and steatohepatitis in a murine model of AH. CONCLUSIONS: Our study demonstrates the central role of NLRP3 and ASC in alcohol-induced liver inflammation and unravels the critical role of ex-ASC specks in the propagation of systemic and liver inflammation in AH. Our data also identify NLRP3 as a potential therapeutic target in AH.

Neutrophil extracellular traps contribute to liver damage and increase defective low-density neutrophils in alcohol-associated hepatitis.

BACKGROUND & AIMS: In alcohol-associated hepatitis (AH), inflammation and neutrophil counts correlate with poor clinical outcomes. Here, we investigated how neutrophils contribute to liver damage in AH. METHODS: We isolated blood neutrophils from individuals with AH to examine neutrophil extracellular traps (NETs) and performed RNA sequencing to explore their unique characteristics. RESULTS: We observed a significant increase in NET production in AH. We also observed a unique low-density neutrophil (LDN) population in individuals with AH and alcohol-fed mice that was not present in healthy controls. Transcriptome analysis of peripheral LDNs and high-density neutrophils (HDNs) from individuals with AH revealed that LDNs exhibit a functionally exhausted phenotype, while HDNs are activated. Indeed, AH HDNs exhibited increased resting reactive oxygen species (ROS) production and produced more ROS upon lipopolysaccharide stimulation than control HDNs, whereas AH LDNs failed to respond to lipopolysaccharide. We show that LDNs are generated from HDNs after alcohol-induced NET release in vitro, and this LDN subset has decreased functionality, including reduced phagocytic capacity. Moreover, LDNs showed reduced homing capacity and clearance by macrophage efferocytosis; therefore, dysfunctional neutrophils could remain in the circulation and liver. Depletion of both HDNs and LDNs in vivo prevented alcohol-induced NET production and liver damage in mice. Granulocyte-colony stimulating factor treatment also ameliorated alcohol-induced liver injury in mice. CONCLUSION: Neutrophils contribute to liver damage through increased NET formation which increases defective LDNs in AH. Alcohol induces phenotypic changes in neutrophils; HDNs are activated whereas LDNs are defective. Our findings provide mechanistic insights that could guide the development of therapeutic interventions for AH. IMPACT AND IMPLICATIONS: In this study we discovered heterogeneity of neutrophils in alcohol-associated hepatitis, including high-density and low-density neutrophils that show hyper-activated or exhausted transcriptomic profiles, respectively. We found that alcohol induces neutrophil extracellular trap (NET) formation, which contributes to liver damage. NET release by high-density neutrophils resulted in low-density neutrophils that reside in the liver and escape clean-up by macrophages. Our findings help to understand the opposing neutrophil phenotypes observed in individuals with alcohol-associated hepatitis and provide mechanistic insights that could guide therapeutic strategies targeting neutrophils.

The Beneficial Effects of Lactobacillus GG Therapy on Liver and Drinking Assessments in Patients with Moderate Alcohol-Associated Hepatitis.

INTRODUCTION: We investigated the effect of daily oral Lactobacillus rhamnosus GG (LGG) in reducing liver injury/severity and drinking in patients with alcohol use disorder and moderately severe alcohol-associated hepatitis. METHODS: Forty-six male and female individuals with alcohol use disorder and moderate alcohol-associated hepatitis (12 ≤ model for end-stage liver disease score < 20, aged 21-67 years) received either LGG (n = 24) or placebo (n = 22). Data were collected/assessed at baseline and at 1, 3, and 6 months. RESULTS: LGG treatment was associated with a significant reduction in liver injury after 1 month. Six months of LGG treatment reduced heavy drinking levels to social or abstinence levels. DISCUSSION: LGG treatment was associated with an improvement in both liver injury and drinking.

IL-1 receptor antagonist plus pentoxifylline and zinc for severe alcohol-associated hepatitis.

BACKGROUND AND AIMS: Patients with severe alcohol-associated hepatitis (AH) have high mortality. Corticosteroids improve survival only for 30 days. We targeted inflammation, cellular injury, and gut leakiness in a randomized clinical trial comparing combination therapy to corticosteroids on 180-day survival. APPROACH AND RESULTS: Subjects with a clinical diagnosis of severe AH (Model for End-Stage Liver Disease [MELD] >20, Maddrey discriminant function [MDF] >32) were randomized to receive methylprednisolone (PRED; 28 days) or a combination of anakinra (14 days) plus pentoxifylline (28 days) plus zinc (COMB; 180 days). The primary endpoint was survival at 180 days. The study was designed in 2013, initiated in October 2014, and completed in March 2018. Five hundred patients were screened to randomize 104 subjects with a clinical diagnosis of AH with a MELD score >20. Fifty-three patients were randomized into the COMB and 50 to the PRED treatment; 1 dropped out of the study before randomization. Mean age was 45.3 ± 10.4 years; 60.6% were males, 92.3% White, and mean MELD 25.7 ± 3.9. Kaplan-Meier survival estimate at 180 days was 67.9% in COMB and 56% in PRED (HR = 0.69; p = 0.3001). Survival curves separated by 90 days (COMB, 69.8%; PRED, 58.0%; HR = 0.69; p = 0.28). Survival at 28 days was similar between the COMB (83.4%) and PRED groups (81.2%; HR = 0.91; p = 0.85). There were no unexpected serious adverse events, and incidence of infection was comparable between groups. MELD 20-25 and MELD >26 strata showed nonsignificant treatment effects in favor of COMB. CONCLUSIONS: A combination of anakinra, pentoxifylline plus zinc provides similar survival benefits compared to corticosteroid therapy in severe AH.

Research methodologies to address clinical unmet needs and challenges in alcohol-associated liver disease.

Alcohol-associated liver disease (ALD) is emerging worldwide as the leading cause of liver-related morbidity, mortality, and indication for liver transplantation. The ALD Special Interest Group and the Clinical Research Committee at the digital American Association for the Study of Liver Diseases meeting in November 2020 held the scientific sessions to identify clinical unmet needs in ALD, and addressing these needs using clinical research methodologies. Of several research methodologies, the sessions were focused on (a) studying disease burden of ALD using large administrative databases, (b) developing biomarkers for noninvasive diagnosis of alcohol-associated hepatitis (AH) and estimation of disease prognosis, (c) identifying therapeutic targets for ALD and AH, (d) deriving accurate models to predict prognosis or posttransplant alcohol relapse as a basis for developing treatment algorithm and a uniform protocol on patient-selection criteria for liver transplantation, and (e) examining qualitative research methodologies in studying the barriers to implementation of multidisciplinary integrated care model by hepatology and addiction teams for the management of dual pathology of liver disease and of alcohol use disorder. Prospective multicenter studies are required to address many of these clinical unmet needs. Further, multidisciplinary care models are needed to improve long-term outcomes in patients with ALD.

Lipoprotein Z, a hepatotoxic lipoprotein, predicts outcome in alcohol-associated hepatitis.

BACKGROUND AND AIMS: Lipoprotein Z (LP-Z) is an abnormal free cholesterol (FC)-enriched LDL-like particle discovered from patients with cholestatic liver disease. This study aims to define the diagnostic value of LP-Z in alcohol-associated hepatitis (AH) and interrogate the biology behind its formation. APPROACH AND RESULTS: We measured serum levels of LP-Z using nuclear magnetic resonance spectroscopy, a well-established clinical assay. Serum levels of LP-Z were significantly elevated in four AH cohorts compared with control groups, including heavy drinkers and patients with cirrhosis. We defined a Z-index, calculated by the ratio of LP-Z to total apolipoprotein B-containing lipoproteins, representing the degree of deviation from normal VLDL metabolism. A high Z-index was associated with 90-day mortality independent from the Model for End-Stage Liver Disease (MELD) and provided added prognosticative value. Both a Z-index ≤ 0.6 and a decline of Z-index by ≥0.1 in 2 weeks predicted 90-day survival. RNA-sequencing analyses of liver tissues demonstrated an inverse association in the expression of enzymes responsible for the extrahepatic conversion of VLDL to LDL and AH disease severity, which was further confirmed by the measurement of serum enzyme activity. To evaluate whether the FC in LP-Z could contribute to the pathogenesis of AH, we found significantly altered FC levels in liver explant of patients with AH. Furthermore, FC in reconstituted LP-Z particles caused direct toxicity to human hepatocytes in a concentration-dependent manner, supporting a pathogenic role of FC in LP-Z. CONCLUSIONS: Impaired lipoprotein metabolism in AH leads to the accumulation of LP-Z in the circulation, which is hepatotoxic from excessive FC. A Z-index ≤ 0.6 predicts 90-day survival independent from conventional biomarkers for disease prognostication.

Altered ethanol metabolism and increased oxidative stress enhance alcohol-associated liver injury in farnesoid X receptor-deficient mice.

BACKGROUND & AIMS: Pharmacological activation of farnesoid X receptor (FXR) ameliorates liver injury, steatosis and inflammation in mouse models of alcoholic liver disease (ALD), but the underlying mechanisms of the protective effect of FXR against ALD remain unclear. METHODS: To investigate the role of FXR in ALD, we used the NIAAA model of chronic plus binge ethanol feeding in FXR-deficient knockout (FXR KO) mice. RESULTS: Ethanol-mediated liver injury and steatosis were increased in FXR KO mice, while both WT and FXR KO mice consumed the same amount of alcohol. Ethanol feeding induced liver inflammation and neutrophil infiltration that were further increased in FXR KO mice. In addition, collagen accumulation and expression of profibrotic genes were markedly elevated in the liver of alcohol-fed FXR KO compared to wild-type mice, suggesting that ethanol-induced liver fibrosis is enhanced in the absence of FXR. Surprisingly, FXR KO mice showed reduced blood alcohol levels post-binge, while CYP2E1 and ALDH1A1 were upregulated compared to WT mice, suggesting that alcohol metabolism is altered in FXR KO mice. Notably, exacerbated liver injury in FXR KO mice was associated with increased oxidative stress. ALDH1A1 activity was upregulated in FXR-deficient mouse primary hepatocytes, contributing to reactive oxygen species (ROS) generation, in vitro. Finally, using an ALDH1A1 inhibitor, we showed that ALDH1A1 activity is a key contributor to alcohol-induced ROS generation in FXR-deficient hepatocytes, in vitro. CONCLUSION: ALD pathogenesis in FXR KO mice correlates with altered ethanol metabolism and increased oxidative stress, providing new insights into the protective function of FXR in ALD.

Protective role of cGAS in NASH is related to the maintenance of intestinal homeostasis.

BACKGROUND & AIMS: Various intracellular pathways regulate inflammation in NASH. Cyclic GMP-AMP synthase (cGAS) is a DNA sensor that activates STING and plays a role in inflammatory diseases. Here, we explored the role of cGAS in hepatic damage, steatosis, inflammation, and liver fibrosis in mouse models of NASH. METHODS: cGAS deficient (cGAS-KO) and STING deficient (STING-KO) mice received high fat-high cholesterol-high sugar diet (HF-HC-HSD) or relevant control diets. Livers were evaluated after 16 or 30 weeks. RESULTS: HF-HC-HSD diet, both at 16 and 30 weeks, resulted in increased cGAS protein expression as well as in increased ALT, IL-1ß, TNF-α and MCP-1 in wild-type (WT) mice compared to controls. Surprisingly, liver injury, triglyceride accumulation, and inflammasome activation were greater in HF-HC-HSD cGAS-KO compared to WT mice at 16 and to a lesser extent at 30 weeks. STING, a downstream target of cGAS was significantly increased in WT mice after HF-HC-HSD. In STING-KO mice after HF-HC-HSD feeding, we found increased ALT and attenuated MCP1 and IL-1ß expression compared to WT mice. Markers of liver fibrosis were increased in cGAS- and STING-KO mice compared to WT on HF-HC-HSD. We discovered that cGAS-KO mice had a significant increase in circulating endotoxin levels on HF-HC-HSD that correlated with changes in intestinal morphology which was exacerbated by HF-HC-HSD compared to WT mice. CONCLUSION: Our findings indicate that cGAS or STING deficiency exacerbate liver damage, steatosis, and inflammation in HF-HC-HSD diet-induced NASH, which might be linked to the disruption of the gut barrier.

Therapeutic advances in alcohol-associated hepatitis.

In recent years, there have been important advances in our understanding of alcohol-associated hepatitis (AH), which have occurred in parallel with a surge in clinical trial activity. Meanwhile, the broader medical field has seen a transformation in care paradigms based on emerging digital technologies. This review focuses on breakthroughs in our understanding of AH and how these breakthroughs are leading to new paradigms for biomarker discovery, clinical trial activity, and care models for patients. It portends a future in which multimodal data from genetic, radiomic, histologic, and environmental sources can be integrated and synthesised to generate personalised biomarkers and therapies for patients with AH.

Two Faces of Neutrophils in Liver Disease Development and Progression.

Neutrophils, the most abundant type of leukocyte in human blood, play a major role in host defense against invading pathogens and in sterile injury. Neutrophil infiltration is characteristic of inflammation because of its antimicrobial and cytotoxic activities. Neutrophils also actively participate in the resolution of inflammation and subsequent tissue repair by acting as a critical mediator between the inflammation and resolution phases of tissue damage. However, neutrophils that are consistently exposed to inflammatory conditions lose their self-resolving capabilities and maintain an inflammatory phenotype, further exacerbating tissue damage. The current review describes how neutrophils interact with tissue microenvironments and acquire disease-specific phenotypes under chronic inflammatory conditions. Here, we aim to provide a better understanding of neutrophil-mediated pathogenesis of various liver diseases.

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.

Steatosis, inflammasome upregulation, and fibrosis are attenuated in miR-155 deficient mice in a high fat-cholesterol-sugar diet-induced model of NASH.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease globally. miRNAs (miRs) regulate various cellular events that lead to NAFLD. In this study we tested the hypothesis that miR-155 is an important regulator of steatohepatitis and fibrosis pathways. Wild type (WT) or miR-155 deficient (KO) mice received a high fat-high cholesterol-high sugar-diet (HF-HC-HS) for 34 weeks and liver tissues were analyzed. In patients with nonalcoholic steatohepatitis and in the mouse model of HF-HC-HS diet we found increased miR-155 levels in the liver compared to normal livers. Upon HF-HC-HS diet feeding, miR-155 KO mice displayed less liver injury, decreased steatosis, and attenuation in fibrosis compared to WT mice. ALT, triglyceride levels, and genes involved in fatty acid metabolic pathway were increased in WT mice whereas miR-155 KO mice showed attenuation in these parameters. HF-HC-HS diet-induced significant increase in the expression of NLRP3 inflammasome components in the livers of WT mice compared to chow fed diet. Compared to WT mice, miR-155 KO showed attenuated induction in the NLRP3, ASC, and caspase1 inflammasome expression on HF-HC-HS diet. Fibrosis markers such as collagen content and deposition, αSMA, Zeb2, and vimentin were all increased in WT mice and miR-155 KO mice showed attenuated fibrosis marker expression. Overall, our findings highlight a role for miR-155 in HF-HC-HS diet-induced steatosis and liver fibrosis.

Pathophysiology of decompensated cirrhosis: Portal hypertension, circulatory dysfunction, inflammation, metabolism and mitochondrial dysfunction.

Patients with acutely decompensated cirrhosis have a dismal prognosis and frequently progress to acute-on-chronic liver failure, which is characterised by hepatic and extrahepatic organ failure(s). The pathomechanisms involved in decompensation and disease progression are still not well understood, and as specific disease-modifying treatments do not exist, research to identify novel therapeutic targets is of the utmost importance. This review amalgamates the latest knowledge on disease mechanisms that lead to tissue injury and extrahepatic organ failure - such as systemic inflammation, mitochondrial dysfunction, oxidative stress and metabolic changes - and marries these with the classical paradigms of acute decompensation to form a single paradigm. With this detailed breakdown of pathomechanisms, we identify areas for future research. Novel disease-modifying strategies that break the vicious cycle are urgently required to improve patient outcomes.

Provider Attitudes and Practices for Alcohol Screening, Treatment, and Education in Patients With Liver Disease: A Survey From the American Association for the Study of Liver Diseases Alcohol-Associated Liver Disease Special Interest Group.

BACKGROUND & AIMS: While abstinence-promoting behavioral and pharmacotherapies are part of the therapeutic foundation for alcohol use disorder (AUD) and alcohol-associated liver disease (ALD), these therapies, along with alcohol screening and education, are often underutilized. Our aim was to examine provider attitudes and practices for alcohol screening, treatment and education in patients with liver disease. METHODS: We conducted a survey of primarily (89%) hepatology and gastroenterology providers within (80%) and outside the United States (20%). Surveys were sent to 921 providers with 408 complete responses (44%), of whom 343 (80%) work in a tertiary liver transplant center. RESULTS: While alcohol screening rates in liver disease patients was nearly universal, less than half of providers reported practicing with integrated addiction providers, using alcohol biomarkers and screening tools. Safe alcohol use by liver disease patients was felt to exist by 40% of providers. While 60% of providers reported referring AUD patients for behavioral therapy, 71% never prescribed AUD pharmacotherapy due to low comfort (84%). Most providers (77%) reported low addiction education and 90% desired more during GI/hepatology fellowship training. Amongst prescribers, baclofen was preferred, but with gaps in pharmacotherapy knowledge. Overall, there was low adherence to the 2019 AASLD practice guidance for ALD, although higher in hepatologists and experienced providers. CONCLUSIONS: While our survey of hepatology and gastroenterology providers demonstrated higher rates of alcohol screening and referrals for behavioral therapy, we found low rates of prescribing AUD pharmacotherapy due to knowledge gaps from insufficient education. Further studies are needed to assess interventions to improve provider alignment with best practices for treating patients with AUD and ALD.