Complement protein signatures in patients with alcohol-associated hepatitis.

Diagnostic challenges continue to impede development of effective therapies for successful management of alcohol-associated hepatitis (AH), thus creating an unmet need to identify and develop non-invasive biomarkers for AH. In murine models of ethanol-induced liver injury, complement activation contributes to hepatic inflammation and injury. Therefore, we hypothesized that complement proteins could be rational diagnostic/prognostic biomarkers in AH. Here, we performed a comparative analysis of data derived from the human hepatic and serum proteome to identify and characterize complement protein signatures in severe AH (sAH). The quantity of multiple complement proteins was perturbed in liver and serum proteome of patients with sAH. Multiple complement proteins differentiated patients with sAH from those with alcohol cirrhosis (AC), alcohol use disorder (AUD) and healthy controls (HCs). Notably, serum collectin 11 and C1q binding protein were strongly associated with sAH and exhibited good discriminatory performance amongst patients with sAH, AC, AUD, and HCs. Furthermore, complement component receptor 1-like protein (CR1L) was negatively associated with pro-inflammatory cytokines. Additionally, lower serum mannose-binding lectin associated serine protease 1 and coagulation factor II were associated with and independently predicted 90-day mortality. In summary, meta-analysis of proteomic profiles from liver and circulation revealed complement protein signatures of sAH, highlighting a complex perturbation of complement and identifying potential diagnostic and prognostic biomarkers for patients with sAH.

IRF3 regulates neuroinflammatory responses and the expression of genes associated with Alzheimer's disease.

The pathological role of interferon signaling is emerging in neuroinflammatory disorders, yet, the specific role of Interferon Regulatory Factor 3 (IRF3) in neuroinflammation remains poorly understood. Here, we show that global IRF3 deficiency delays TLR4-mediated signaling in microglia and attenuates the hallmark features of LPS-induced inflammation such as cytokine release, microglial reactivity, astrocyte activation, myeloid cell infiltration, and inflammasome activation. Moreover, expression of a constitutively active IRF3 (S388D/S390D:IRF3-2D) in microglia induces a transcriptional program reminiscent of the Activated Response Microglia and the expression of genes associated with Alzheimer's Disease, notably apolipoprotein-e . Lastly, using bulk-RNAseq of IRF3-2D brain myeloid cells, we identified Z-DNA binding protein-1 as a target of IRF3 that is relevant across various neuroinflammatory disorders. Together, our results identify IRF3 as an important regulator of LPS-mediated neuroinflammatory responses and highlight IRF3 as a central regulator of disease-specific gene activation in different neuroinflammatory diseases.

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.

G-CSF increases calprotectin expression, liver damage and neuroinflammation in a murine model of alcohol-induced ACLF.

Background and aims: Granulocyte colony-stimulating factor (G-CSF) has been proposed as a therapeutic option for patients with ACLF, however clinical outcomes are controversial. We aimed at dissecting the role of G-CSF in an alcohol-induced murine model of ACLF. Methods: ACLF was triggered by a single alcohol binge (5 g/kg) in a bile duct ligation (BDL) liver fibrosis model. A subgroup of mice received two G-CSF (200 µg/kg) or vehicle injections prior to acute decompensation with alcohol. Liver, blood and brain tissues were assessed. Results: Alcohol binge administered to BDL-fibrotic mice resulted in features of ACLF indicated by a significant increase in liver damage and systemic inflammation compared to BDL alone. G-CSF treatment in ACLF mice induced an increase in liver regeneration and neutrophil infiltration in the liver compared to vehicle-treated ACLF mice. Moreover, liver-infiltrating neutrophils in G-CSF-treated mice exhibited an activated phenotype indicated by increased expression of CXC motif chemokine receptor 2, leukotriene B4 receptor 1, and calprotectin. In the liver, G-CSF triggered increased oxidative stress, type I interferon response, extracellular matrix remodeling and inflammasome activation. Circulating IL-1ß was also increased after G-CSF treatment. In the cerebellum, G-CSF increased neutrophil infiltration and S100a8/9 expression, induced microglia proliferation and reactive astrocytes, which was accompanied by oxidative stress, and inflammasome activation compared to vehicle-treated ACLF mice. Conclusion: In our novel ACLF model triggered by alcohol binge that mimics ACLF pathophysiology, neutrophil infiltration and S100a8/9 expression in the liver and brain indicate increased tissue damage, accompanied by oxidative stress and inflammasome activation after G-CSF treatment.

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.

A novel score of IL-13 and age predicts 90-day mortality in severe alcohol-associated hepatitis: A multicenter plasma biomarker analysis.

BACKGROUND: Severe alcoholic hepatitis (AH) has a high short-term mortality rate. The MELD assesses disease severity and mortality; however, it is not specific for AH. We screened plasma samples from patients with severe AH for biomarkers of multiple pathological processes and identified predictors of short-term mortality. METHODS: Plasma was collected at baseline from 85 patients with severe AH (MELD≥20, Maddrey's discriminant function≥32) enrolled in the Defeat Alcoholic Steatohepatitis clinical trial (investigating IL-1 receptor antagonist+pentoxifylline+zinc vs. methylprednisolone+placebo). Samples were analyzed for 43 biomarkers and the markers' association with 28- and 90-day mortalities was assessed. RESULTS: Thirty-one (36.5%) patients died during the 90-day follow-up with similar ratios in the treatment groups. Eight biomarkers showed an association with mortality. IL-6, IL-22, interferon-α2, soluble TNF receptor 1, lipocalin-2, and α-fetoprotein levels were associated with 28-day mortality, while IL-6, IL-13, and endotoxin levels with 90-day mortality. In multivariable Cox regression, encephalopathy, lipocalin-2, and α-fetoprotein levels were independent predictors of 28-day mortality, and IL-6, IL-13, international normalized ratio levels, and age were independent predictors of 90-day mortality. The combination of IL-13 and age had superior performance in predicting 90-day mortality compared with MELD in the total cohort and the individual treatment groups. CONCLUSIONS: We identified predictors of short-term mortality in a cohort exclusively involving patients with severe AH. We created a composite score of IL-13 and age that predicts 90-day mortality regardless of the treatment type with a performance superior to MELD in severe AH.

Dysregulated meta-organismal metabolism of aromatic amino acids in alcohol-associated liver disease.

BACKGROUND: Chronic alcohol consumption impairs gut barrier function and perturbs the gut microbiome. Although shifts in bacterial communities in patients with alcohol-associated liver disease (ALD) have been characterized, less is known about the interactions between host metabolism and circulating microbe-derived metabolites during the progression of ALD. METHODS: A large panel of gut microbiome-derived metabolites of aromatic amino acids was quantified by stable isotope dilution liquid chromatography with online tandem mass spectrometry in plasma from healthy controls (n = 29), heavy drinkers (n = 10), patients with moderate (n = 16) or severe alcohol-associated hepatitis (n = 40), and alcohol-associated cirrhosis (n = 10). RESULTS: The tryptophan metabolites, serotonin and indole-3-propionic acid, and tyrosine metabolites, p-cresol sulfate, and p-cresol glucuronide, were decreased in patients with ALD. Patients with severe alcohol-associated hepatitis and alcohol-associated cirrhosis had the largest decrease in concentrations of tryptophan and tyrosine-derived metabolites compared to healthy control. Western blot analysis and interrogation of bulk RNA sequencing data from patients with various liver pathologies revealed perturbations in hepatic expression of phase II metabolism enzymes involved in sulfonation and glucuronidation in patients with severe forms of ALD. CONCLUSIONS: We identified several metabolites decreased in ALD and disruptions of hepatic phase II metabolism. These results indicate that patients with more advanced stages of ALD, including severe alcohol-associated hepatitis and alcohol-associated cirrhosis, had complex perturbations in metabolite concentrations that likely reflect both changes in the composition of the gut microbiome community and the ability of the host to enzymatically modify the gut-derived metabolites.

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.

Therapeutic inhibition of miR-155 attenuates liver fibrosis via STAT3 signaling.

Most chronic liver diseases progress to liver fibrosis, which, when left untreated, can lead to cirrhosis and hepatocellular carcinoma. MicroRNA (miRNA)-targeted therapeutics have become attractive approaches to treat diseases. In this study, we investigated the therapeutic effect of miR-155 inhibition in the bile duct ligation (BDL) mouse model of liver fibrosis and evaluated the role of miR-155 in chronic liver fibrosis using miR-155-deficient (miR-155 knockout [KO]) mice. We found increased hepatic miR-155 expression in patients with cirrhosis and in the BDL- and CCl4-induced mouse models of liver fibrosis. Liver fibrosis was significantly reduced in miR-155 KO mice after CCl4 administration or BDL. To assess the therapeutic potential of miR-155 inhibition, we administered an rAAV8-anti-miR-155 tough decoy in vivo that significantly reduced liver damage and fibrosis in BDL. BDL-induced protein levels of transforming growth factor ß (TGF-ß), p-SMAD2/3, and p-STAT3 were attenuated in anti-miR-155-treated compared with control mice. Hepatic stellate cells from miR-155 KO mice showed attenuation in activation and mesenchymal marker expression. In vitro, miR-155 gain- and loss-of-function studies revealed that miR-155 regulates activation of stellate cells partly via STAT3 signaling. Our study suggests that miR-155 is the key regulator of liver fibrosis and might be a potential therapeutic target to attenuate fibrosis progression.

Prolonged hypoxia alleviates prolyl hydroxylation-mediated suppression of RIPK1 to promote necroptosis and inflammation.

The prolyl hydroxylation of hypoxia-inducible factor 1α (HIF-1α) mediated by the EGLN-pVHL pathway represents a classic signalling mechanism that mediates cellular adaptation under hypoxia. Here we identify RIPK1, a known regulator of cell death mediated by tumour necrosis factor receptor 1 (TNFR1), as a target of EGLN1-pVHL. Prolyl hydroxylation of RIPK1 mediated by EGLN1 promotes the binding of RIPK1 with pVHL to suppress its activation under normoxic conditions. Prolonged hypoxia promotes the activation of RIPK1 kinase by modulating its proline hydroxylation, independent of the TNFα-TNFR1 pathway. As such, inhibiting proline hydroxylation of RIPK1 promotes RIPK1 activation to trigger cell death and inflammation. Hepatocyte-specific Vhl deficiency promoted RIPK1-dependent apoptosis to mediate liver pathology. Our findings illustrate a key role of the EGLN-pVHL pathway in suppressing RIPK1 activation under normoxic conditions to promote cell survival and a model by which hypoxia promotes RIPK1 activation through modulating its proline hydroxylation to mediate cell death and inflammation in human diseases, independent of TNFR1.

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.

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.

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.

Design of a multicenter randomized clinical trial for treatment of Alcohol-Associated Hepatitis.

Background: Mortality is high for severe alcohol-associated hepatitis (AH). Corticosteroids are the standard of care for patients without contraindications. Recent data showed that interleukin-1ß receptor antagonist anakinra attenuated inflammation and liver damage. We designed a multicenter, double-blind, randomized controlled trial to assess the safety and efficacy of anakinra compared to prednisone. Methods: Patients meeting the clinical and biochemical criteria for severe AH with MELD scores between 20 and 35 were recruited at eight clinical sites. Eligible patients enrolled in the study were randomized to anakinra, 100 mg subcutaneous injection for 14 days, plus zinc sulfate 220 mg for 90 days, vs. prednisone 40 mg PO daily for 30 days. Matching placebos for anakinra, zinc, and prednisone were provided to mask the treatment. Participants were followed for 180 days. The primary outcome was overall survival at 90 days. An unadjusted log-rank test was used to compare the survival of the two treatments in the first 90 days. Between July 10, 2020, and March 4, 2022, we screened 1082 patients with severe AH, and 147 eligible patients were enrolled and randomized. The average baseline MELD score was 25 [range 20-35], Maddrey discriminant function (MDF) was 59.4 [range 20.2-197.5]. The mean aspartate transaminase (AST)-to-alanine transaminase (ALT) ratio was 3.5. The baseline characteristics were not statistically different between the two treatment groups. Conclusions: The study provided a direct comparison of the survival benefits and safety profiles of anakinra plus zinc vs. prednisone in patients with severe AH.

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.

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.