BACKGROUND AND AIMS:Alcohol-associated liver disease (ALD) represents a global health burden with limited therapeutic strategies. While the hepatocyte transcription factor CCAAT/enhancer-binding protein α (CEBPA) regulates hepatic gene expression and liver fibrosis, its role in ALD pathogenesis remains undefined. Here, hepatocyte CEBPA was examined for its modulation of alcohol-associated hepatic steatosis and ALD. APPROACH AND RESULTS:Hepatocyte-specific CEBPA knockout mice, adeno-associated virus serotype transduction studies, and reporter gene assays were carried out using acute and chronic mouse ALD models. Western blotting was performed on liver tissues from human patients with ALD. Hepatic CEBPA expression decreased during ALD progression in human patient cohorts. Hepatocyte-specific Cebpa -knockout mice exhibited exacerbated alcohol-associated steatosis in both the acute and chronic ALD models. Inducible ablation of CEBPA in hepatocytes during late-stage ALD, accelerated disease progression, demonstrating the persistent protective function of CEBPA. Global transcriptomics identified Orm1 encoding orosomucoid 1 (ORM1) as the top CEBPA-upregulated gene in hepatocytes, while reporter assays and chromatin immunoprecipitation (ChIP) revealed that CEBPA directly activated Orm1 transcription by binding CEBPA response elements upstream of the Orm1 promoter. Loss of hepatocyte ORM1 potentiated the severity of ALD in mice. Conversely, restoring CEBPA or ORM1 via i.v. adeno-associated virus serotype 8 delivery or i.p. administeration of recombinant ORM1 protein rescued hepatic lipid accumulation and reduced disease progression. Consistently, serum ORM1, a hepatocyte-secreted hepatokine, inversely correlated with ALD severity in patients. CONCLUSIONS:These findings identify the hepatocyte CEBPA-ORM1 axis as a critical suppressor of ALD, offering therapeutic targets and nominating serum ORM1 as a potential biomarker for staging ALD severity.

Objectives:Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by persistent synovial inflammation, bone erosion, cartilage destruction, and debilitating pain. Current therapies often fall short in preventing structural damage and restoring immune balance. To address these limitations, we evaluated MRG-001 - a fixed-dose combination of plerixafor (AMD3100) and low-dose tacrolimus (FK506) with reported immunoregulatory and regenerative potential - in preclinical models of RA. Methods:The therapeutic efficacy of MRG-001 was assessed in collagen-induced arthritis and collagen antibody-induced arthritis mouse models. Mice received subcutaneous MRG-001, and clinical, histological, molecular, and behavioral endpoints were evaluated to examine inflammation, immune modulation, bone integrity, angiogenesis, and pain. Results:MRG-001 treatment significantly reduced joint swelling, arthritis scores, and pro-inflammatory cytokine expression (TNF-α, IL-6, IL-17a) in both models. It was associated with mobilization of CD45-Sca1+CD29+ mesenchymal stem cells, increased CD4+CD25+Foxp3+ regulatory T cells, and reduced CD4+RORγt+ Th17 cells in peripheral blood. MRG-001 preserved cartilage integrity was associated with reduced osteoclast-mediated bone erosion, and improved trabecular bone structure. It was associated with reduced fibroblast-like synoviocyte activation, lower TGF-β signaling activity, and decreased RANKL expression in joint tissues. Additionally, MRG-001 was associated with reduced pathological angiogenesis (CD31+EMCN+ vessels) and sensory nerve innervation (PGP9.5+, CGRP+ fibers), corresponding with improved pain-like behaviors and physical function. Conclusions:MRG-001 exerts broad therapeutic effects in RA by modulating immune responses, preserving joint architecture, and alleviating pain. These findings support its potential as a novel, multi-modal disease-modifying therapy for RA and warrant further clinical investigation.

Background and aims Fibrosis is considered the gold standard for diagnosing chronic pancreatitis (CP) but has not been systematically studied across a large cohort of patients with and without pancreatitis undergoing different pancreatic surgeries. Methods Demographic, clinical and radiologic data for patients undergoing total pancreatectomy with islet autotransplantation (TPIAT) for indeterminate CP, recurrent acute pancreatitis (RAP) and definite CP; Whipple/Frey for indeterminate and definite CP; and distal pancreatectomy for serous cystadenoma (SCA) between 2004-2023 were reviewed. Definite and indeterminate CP were defined by M-ANNHEIM criteria; and RAP defined as ≥2 episodes of imaging-documented acute pancreatitis. The Ammann Fibrosis Score (FS) was tabulated for all surgical pathology specimens. Patients with SCA were included as controls. Data were summarized as median[Q1,Q3] or n(%). Results There were 206 patients including 24 controls, 31 with indeterminate CP, 36 with RAP, and 115 with definite CP. The median FS was 0.33[0.2,0.5], 2.8[1,6], 6.5[3.7,8], 8.5[5.9,11] in controls, indeterminate CP, RAP, and definite CP, respectively. Post hoc Dunn tests with Holm adjustment showed significant differences in total Fibrosis Score across all diagnostic groups, with most component scores also differing significantly between groups. There was no correlation between FS and number of AP episodes; however, genetic/idiopathic RAP patients with ≥7 episodes of AP had similar FS to genetic/idiopathic CP(p=0.08). Conclusion Fibrosis quantification may provide complementary information to existing clinical, imaging, and functional assessments when evaluating patients across the pancreatitis continuum. Current diagnostic criteria for indeterminate CP are leading to an overdiagnosis of CP if fibrosis remains the criteria standard. Among genetic/idiopathic etiologies, RAP patients with ≥7 episodes of AP have similar quantities of fibrosis to CP.

Kidney transplantation is the gold standard treatment strategy for end-stage renal disease. Deceased donor kidneys usually undergo cold storage until kidney transplantation, leading to cold ischemia injury that may contribute to poor graft outcomes. However, the molecular characterization of potential mechanisms of cold ischemia injury remains incomplete. To bridge this knowledge gap, we leveraged spatial transcriptomics technology to perform full transcriptome characterization of cold ischemia injury (0-48 hours) using a murine model. We developed a computational workflow to identify spatiotemporal transcriptomic changes that accompany the injury pathophysiology in a compartment-specific manner. We identified potential metabolic reprogramming preferentially within the kidney inner medulla displaying strong oxidative phosphorylation signature in an ischemic environment. We found commonalities between the spatiotemporal transcriptomic presentation of cold ischemia and warm ischemia‒reperfusion injury, including an induction of an anti-viral like immune response throughout the renal tissue. Altogether, these systems-level biological insights enabled by our full transcriptome temporal characterization unveil a molecular basis for how cold ischemia injury may negatively affect kidney outcomes. Moreover, our spatial analyses highlight pathological developments deep within the renal tissue, suggesting potential opportunities for new insights beyond biopsy-focused superficial tissue examinations. We also developed an interactive online browser at https://jef.works/vitessce-cold-ischemia/ to facilitate exploration of our results by the broader scientific and clinical community.

Alcohol-associated liver disease (ALD) is a growing global health concern, with alcohol-associated hepatitis (AH) leading to the highest morbidity and mortality. Available therapies are limited and often inadequate. Platelets contribute in a variety of ways to liver disease pathogenesis, but their role in AH remains largely unexplored. In this study, we addressed the hypothesis that platelets contribute to pathological inflammation in AH. Using patient samples and a multiomics approach, we found that platelets undergo proinflammatory transcriptomic and proteomic changes in AH, with 2 alarmins, S100A8 and S100A9, being among the top upregulated genes/proteins. Additionally, the abundance of platelet-derived microparticles containing S100A8 and S100A9 in AH patient plasma was increased and correlated with disease severity (assessed by model for end-stage liver disease sodium [MELD-Na]) and endotheliopathy (assessed by ICAM1, CXCL8, and vWF). We mechanistically linked S100A9 with endotheliopathy via crosstalk between primary human liver sinusoidal endothelial cells and primary human monocytes. We also demonstrated that IL-6 upregulates S100A9 in megakaryocytic cells in a JAK/STAT-dependent manner, modeling changes occurring in the bone marrow in patients with AH. Our studies establish proinflammatory platelets as important contributors to AH pathology. Moreover, antiplatelet agents — or, more specifically, S100A9 targeted drugs — are potential therapeutic strategies in AH.

BACKGROUND & AIMS:Disruption of the intestinal barrier facilitates microbial translocation to the liver and contributes to chronic liver disease. We aimed to study the role of the fecal proteome in disease progression in patients with alcohol-associated hepatitis. METHODS:We used fecal proteomics data from a multicenter cohort of patients with alcohol-associated hepatitis (n = 80), alcohol use disorder (n = 20), and controls (n = 19) (InTeam), and a cathepsin B activity assay in an independent multicenter cohort of patients with alcohol-associated hepatitis (n = 80), alcohol use disorder (n = 20), and controls (n = 18) (AlcHepNet). Mice lacking cathepsin B in myeloid cells and transgenic mice overexpressing occludin in intestinal epithelial cells, were subjected to the chronic-plus-binge ethanol feeding model (NIAAA). RESULTS:Fecal proteomics and activity analysis revealed that the protease cathepsin B progressively increased with alcohol use disorder and alcohol-associated hepatitis compared to controls, and is associated with higher short-term mortality in patients with alcohol-associated hepatitis. Cathepsin B is predominantly expressed in intestinal macrophages and is upregulated by ethanol. Cathepsin B deficiency in myeloid cells or oral treatment with the gut-restricted cathepsin B inhibitor CA074 stabilized the gut barrier by preserving the tight junction protein occludin, lowered serum lipopolysaccharide levels, and attenuated ethanol-induced steatohepatitis. Transgenic overexpression of occludin in intestinal epithelial cells sufficed to reduce steatohepatitis and blunted the effects of CA074 in ethanol-fed mice. Cathepsin B proteolytically cleaves occludin in enzymatic assays, and its inhibition prevented occludin degradation and barrier disruption in intestinal organoids and epithelial monolayers. Molecular modeling and peptide profiling reveal specific cathepsin B-induced cleavage sites in the extracellular region of occludin. CONCLUSIONS:Intestinal cathepsin B is an essential mediator of gut barrier dysfunction and a potential therapeutic target in alcohol-associated liver disease. IMPACT AND IMPLICATIONS:Intestinal barrier disruption facilitates microbial translocation to the liver, contributing to the progression of alcohol-associated liver disease; however, the molecular mechanisms driving barrier dysfunction remain incompletely understood. Our study identifies the protease cathepsin B as a key contributor to alcohol-associated liver disease progression by degrading the extracellular region of the tight junction protein occludin in the intestine, thereby leading to barrier disruption. This work advances the field by establishing causality, uncovering the molecular target, and proposing cathepsin B as a promising therapeutic target in alcohol-associated hepatitis - a condition for which liver transplantation remains the only effective treatment in a limited subset of patients.

Background:Alcohol-associated hepatitis (AH) is a severe inflammatory liver condition driven by dysregulated immune responses. CD8 T cells accumulate in the liver during AH, yet their functional role remains poorly defined.Methods:A murine NIAAA chronic-binge model with lipopolysaccharide (LPS) challenge was used. Single-cell RNA sequencing of liver and blood immune cells defined CD8 T cell subsets, followed by pathway analysis and reanalysis of human AH datasets. Spatial relationships were validated by immunohistochemistry of human AH liver tissue with CD8 TRM and liver sinusoidal endothelial cell (LSEC) co-staining. LSEC-CD8 T cell interactions were examined using in vitro co-culture, and signaling pathways were assessed by bulk RNA sequencing and phospho-protein analyses. IL15 function was evaluated by in vivo neutralization.Results:Single-cell RNA sequencing in the murine AH model revealed a distinct population of CD8 tissue-resident memory T cells (TRM) with heightened activation and proinflammatory cytokine production. Analysis of human AH liver single-cell RNA sequencing data, along with immunohistochemistry validation, supported CD8 TRM enrichment in diseased tissue. IL15 emerged as a prominent pathway linked to TRM activation, and IL15 blockade reduced TRM abundance and attenuated EtOH/ lipopolysaccharide-induced liver injury. Mechanistically, LSECs not only provided a structural niche for TRM retention but also amplified IL15 signalling. In vitro, coculture experiments demonstrated that LSECs intensified activation in pre-stimulated CD8 T cells in a stimulus-dependent manner: under IL15 stimulation, LSECs boosted effector function without inducing cell death, whereas under T-cell receptor stimulation, LSECs drove hyperactivation and activation-induced cell death. Bulk RNA-seq and phospho-protein analysis identified the PI3K-AKT pathway as a shared pathway enhanced by LSEC coculture in activated CD8 T cells. These findings define a context-dependent mechanism in which LSECs promote IL15-driven signaling through AKT pathway amplification, promoting TRM persistence and inflammatory activity in AH.Conclusions:IL15-associated signaling within the hepatic microenvironment, shaped by LSEC-CD8 T-cell interactions, promotes activation and persistence of CD8 TRM cells in AH. Targeting the IL15-LSEC-AKT axis may disrupt pathogenic TRM niches and represent a promising therapeutic strategy for severe AH.

BACKGROUND. In chronic alcohol consumers, immune cells may drive the progression from mild liver injury to more severe alcohol-associated liver disease (ALD), including alcohol-associated hepatitis (AAH) and cancer. Liver macrophages, both resident and infiltrating, express allograft inflammatory factor 1 (AIF1), which is upregulated during inflammation and enhances immune activation. METHODS. Using serum and urine samples from 868 individuals classified as having alcohol use disorder or not, based on DSM-IV/V criteria, along with serum and liver biopsy tissue from a second cohort of 27 patients diagnosed with AAH, we evaluated the impact of the AIF1 promoter single-nucleotide polymorphism (SNP) (rs3132451; C/C, C/G, G/G) on liver function markers and immune cell profiles. RESULTS. AIF1 transcript levels were genotype dependent: C/C homozygotes expressed 5.2% of the levels observed in G/G individuals, while C/G heterozygotes expressed 46%. Unlike most SNPs associated with harmful effects, the G/G genotype is highly prevalent, present in about 70% of patients. Among chronic alcohol users, G/G individuals exhibited elevated markers of liver injury and a more than 3-fold increase in hepatic immune cells, including infiltrating AIF1+ macrophages and neutrophils. Despite similar durations of alcohol misuse, G/G individuals had higher Model for End-Stage Liver Disease scores compared with C/G individuals, indicating a significantly greater 90-day mortality risk. Notably, some immune abnormalities, such as elevated neutrophils, persisted in G/G males even after alcohol abstinence. CONCLUSION. These findings suggest that functional genetic variation in AIF1 may contribute to the severity and persistence of ALD. TRIAL REGISTRATION. ClinicalTrials.gov NCT02231840. FUNDING. Research support was provided from the National Institute on Alcohol Abuse and Alcoholism of the NIH under grants 1ZIAAA000440-02 and R24AA025017.

Xenotransplantation using genetically engineered pig organs offers a promising solution to the shortage of donor organs for life-saving transplantations. However, human-preformed antibodies against unknown pig xenoantigens remain a significant barrier to successful xenotransplantation. Current methods for characterizing these antibodies or xenoantigens are limited to cellular-level cross-match assays. In this study, we developed a novel approach to identify pig xenoantigens, including peptide and glycopeptide epitopes, that react with human-preformed antibodies. First, human-preformed antibodies against xenoantigens were enriched from plasma using immobilized pig kidney proteins. The enriched antibodies were then immobilized and used to isolate pig kidney proteins, peptides, and intact glycopeptides, followed by liquid chromatography-tandem mass spectrometry analysis. This dual-level approach identified 221 peptides corresponding to 153 proteins, with a significant enrichment of plasma membrane and extracellular proteins. Notably, 11 peptides were unique to pig sequences, suggesting their potential role in driving xenogeneic immune responses. Glycoproteomic analysis identified 122 intact glycopeptides, predominantly complex/hybrid glycoforms, and Neu5Gc-containing glycans. Our method effectively identifies peptides and intact glycopeptides reactive to human-preformed antibodies, providing critical insights for discovering xenoantigens. These findings could guide genetic engineering strategies and enhance recipient candidate screening for xenotransplantation, ultimately increasing the feasibility and success of xenogeneic organ transplantation.

Patients with alcohol-associated cirrhosis (AC) may develop severe alcohol-associated hepatitis (sAH), a disease with high short-term mortality. Our previous studies demonstrated that sAH, but not AC livers, are infiltrated with a high number of self-sustaining IL-8+ neutrophils that likely drive the transition from AC to sAH. Monocyte-derived macrophages (MoMFs) also infiltrate the liver in sAH, but their roles remain largely obscure. In the present study, we characterized liver macrophages in human liver explants from sAH and AC patients. Our data revealed a marked reduction in Kupffer cells, whereas MoMFs were increased in sAH and AC. Single-cell RNA-Seq analyses revealed several populations in both AC and sAH, including C1Q+, S100A8+, APOE+, TNF+ and VSIG4+ macrophages, with sAH containing unique C1Q+ macrophages potentially playing a role in removing apoptotic neutrophils in sAH. C1Q+ macrophages also express many genes involved in phagocytosis and proinflammatory and anti-inflammatory functions, suggesting that C1Q+ macrophages have diverse functions in sAH. The roles of C1Q, S100A8, and APOE were further examined in experimental models of alcohol-induced liver injury. Our data revealed that C1q KO mice and macrophage-specific S100a8 KO mice presented similar alcohol-induced liver injury and hepatic neutrophil infiltration, while Apoe KO mice developed much more severe liver injury than did WT mice following chronic-plus-binge ethanol challenge. Taken together, sAH and AC are infiltrated with multiple populations of macrophages that perform diverse functions to drive chronic disease progression. Unique C1Q+ macrophages in sAH play a compensatory role in removing dead cells but may also promote inflammation in sAH.

Background Alcohol-associated liver disease (ALD) is a global health problem without an effective treatment. Mallory-Denk body (MDB) is a protein aggregate commonly found in alcohol-associated hepatitis (AH). MDB primarily contains ubiquitinated proteins, cytokeratin 8 and sequestosome 1 (SQSTM1)/p62. Stress granule (SG) is a cytosolic, membrane-less aggregate composed of various RNA-binding proteins and untranslated mRNA. However, the role and mechanisms of MDB and SG induced by alcohol and their implications in the pathogenesis of ALD remain largely unknown.Methods SQSTM1/p62 whole body knockout and matched wild-type mice were subjected to the Gao-binge alcohol model or fed a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet alongside Gao-binge alcohol model. ALD mouse liver tissues and human AH liver tissues underwent immunohistochemistry (IHC) staining and western blot analysis for SG and MDB markers.Results We found that the livers of patients with AH had higher levels of SQSTM1/p62 (MDB marker) and Ras-GTPase-activating protein-binding protein 1 (an SG marker) using IHC staining, and these increased protein levels were enriched in detergent-insoluble fractions compared with healthy individuals. We further discovered that Gao-binge alcohol feeding increased insoluble SG markers, such as phosphorylated eukaryotic initiation factor 2 in mouse livers. Mice fed a DDC diet with Gao-binge alcohol had greater hepatic MDB formation and liver injury than those fed either diet alone. Loss of SQSTM1/p62 led to reduced protein aggregation involved in SGs and MDBs but increased liver injury in DDC plus Gao-binge alcohol-fed mice, indicating that SQSTM1/p62 is required for MDB formation and protects against alcohol-induced liver injury.Conclusion Chronic plus binge alcohol exposure increases hepatic MDBs and moderate levels of SGs. p62/SQSTM1 is critical for the formation but is not essential for the clearance of MDBs, a process that may act as an adaptive protective mechanism against ALD.

Individuals with progressive liver failure risk dying without liver transplantation. However, our understanding of why regenerative responses are disrupted in failing livers is limited. Here, we perform multiomic profiling of healthy and diseased human livers using bulk and single-nucleus RNA- and ATAC-seq. We report that in alcohol-associated liver disease, alterations in the hepatic immune milieu prevent hepatocytes from transitioning to proliferative progenitors. We also find differences in RNA binding protein expression, particularly of the ESRP, PTBP, and SR families, leading to misregulation of developmentally controlled RNA splicing. Our data pinpoint ESRP2 as a disease-sensitive splicing factor and support a causal role for its deficiency in the pathogenesis of severe alcoholic hepatitis. Notably, splicing defects in Tcf4 and Slk, two ESPR2 targets, alter their nuclear localization and activities, disrupting WNT and Hippo signaling pathways that are critical for normal liver regeneration. We further demonstrate that changes in stromal cell populations enrich failing livers with TGF-β, which suppresses the ESRP2-driven epithelial splicing program and replaces functional parenchyma with quasi-progenitor-like cells lacking liver-specific functions. Taken together, these findings indicate that misspliced RNAs are effective biomarkers for alcohol-associated liver disease, and targeting them could improve recovery in affected individuals.

Background:Chronic alcohol consumption leads to lipid accumulation, oxidative stress, cellular damage, and inflammation in the liver, collectively referred to as alcohol-associated liver disease (ALD). FAF2/UBXD8/ETEA (Fas-associated factor 2) is a ubiquitin ligase adaptor protein that plays a crucial role in the ubiquitin-mediated degradation of misfolded proteins in the endoplasmic reticulum. A recent genome-wide association study indicated an association between FAF2 and ALD; however, the exact contribution of FAF2 to ALD pathogenesis remains unclear.Methods:FAF2 was knocked down using AAV-delivered shRNA in C57/BL6 mice. Mice were subjected to a chronic-plus-single binge ethanol feeding (NIAAA) model. Nine hours after gavage, liver, blood, and other organs of interest were collected for gene expression and biochemical analyses.Results:We first observed a significant elevation in hepatic FAF2 protein expression in individuals with ALD and in mice subjected to an ethanol-binge model. Interestingly, knocking down FAF2 in the liver using adeno-associated virus serotype 8-delivered short hairpin RNA conferred a protective effect against alcohol-induced liver steatosis in ethanol-binged mice. Transcriptomic analysis revealed that differentially expressed genes were enriched in multiple lipid metabolism regulation pathways. Further analysis of transcription factors regulating these differentially expressed genes suggested potential regulation by SREBP1. Several SREBP1 target genes, including Fasn, Scd1, Lpin1, and Pcsk9 (proprotein convertase subtilisin/kexin type 9), were dysregulated in the livers of ethanol-fed FAF2 knockdown mice. Additionally, Pcsk9 could be regulated through the FOXO3-SIRT6 pathway in the livers of ethanol-fed FAF2 knockdown mice, leading to increased liver low-density lipoprotein receptor expression and reduced plasma LDL cholesterol levels. Furthermore, FAF2 knockdown in mouse liver enhanced adipose triglyceride lipase lipolytic activity by upregulating the adipose triglyceride lipase activator, comparative gene identification-58, and downregulating the adipose triglyceridelipase transport inhibitor, Elmod2, contributing to the alleviation of liver steatosis.Conclusions:Our study uncovers a novel mechanism involving FAF2 in the pathogenesis of ALD.

Nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), a nicotinamide adenine dinucleotide (NAD+) synthetase in Preiss-Handler and salvage pathways, governs nuclear NAD+ homeostasis. This study investigated the role of NMNAT1 in alcohol-associated liver disease (ALD). Decreased NMNAT1 expression and activity were observed in the liver of patients with alcohol-associated hepatitis and either liver or primary hepatocytes from ALD mice. F-box and WD repeat domain containing 7 (FBXW7)-regulated interferon regulatory factor 1 (IRF1) ubiquitination degradation contributed to the alcohol-inhibited NMNAT1 transcriptional level. Hepatic NMNAT1 knockout aggravated alcohol-induced hepatic NAD+ decline and further hepatic steatosis and liver injury. Metabolomics and transcriptomics interaction revealed that the cysteine sulfinic acid decarboxylase (CSAD)-regulated taurine pathway was involved in NMNAT1-disrupted hepatic lipid metabolism in ALD. Hepatic CSAD overexpression or taurine supply attenuated hepatic NMNAT1 knockout-aggravated ALD. Hepatic NMNAT1 loss inhibited NMN-protected ALD. Replenishing hepatic NMNAT1 reversed liver lipid accumulation in ALD mice. These findings identified NMNAT1 as a promising therapeutic target for ALD.

Chronic alcohol use causes pancytopenia and diminished immune responses against pathogens. However, it remains unclear whether chronic alcohol consumption directly induces inflammation in human hematopoietic stem progenitor cells (HSPCs), and if aging modifies the impact of chronic alcohol consumption in HSPCs. To examine how chronic alcohol use affects HSPCs, we performed single-cell RNA-seq in human and murine HSPCs and single-cell ATAC-seq in aged murine HSPCs following alcohol exposure. In xenotransplanted human HSPCs, chronic alcohol feeding resulted in a significant myeloid bias, heightened inflammation, double-stranded RNA (dsRNA) sensor upregulation, and type 1 interferon responses. In the native murine bone marrow, chronic alcohol exposure primed HSPCs to differentiate into myeloid cells and to exhibit heightened inflammation, DNA damage, and epigenetic reactivation of transposable elements (TEs) in an age-dependent manner. Alcohol-exposed aged long-term hematopoietic stem cells (LT-HSCs) displayed increased chromatin accessibility at TE-containing loci correlated with aberrant TE transcription. This transposon derepression was associated with the accumulation of dsRNAs in aged bone marrow cells, and activation of innate immune pathways, perpetuating HSC inflammaging. Furthermore, old mice showed two epigenomically distinct LT-HSC clusters, LT-HSC1 and LT-HSC2, in which the LT-HSC2 cluster expanded in response to chronic alcohol drinking and resembled inflammatory HSCs. Notably, secondary transplantation revealed unperturbed long-term self-renewal capacity in both human and murine HSCs, suggesting that HSC function may recover following alcohol cessation. Our data illuminate potential interactions between alcohol and aging that can reinforce inflammaging and epigenetic dysregulation in HSPCs. Keypoints:Chronic alcohol consumption triggers age-dependent myeloid bias and inflammation in HSPCs without impairing self-renewalChronic alcohol consumption alters epigenome, driving heightened transposon upregulation in aged HSPCs.

OBJECTIVES:Fibrosis is considered the criterion standard for diagnosing chronic pancreatitis (CP) but adequate tissue specimens are difficult to obtain, carry risk and are often obtained at the time of surgery in advanced stages of CP. Noninvasive biomarkers that correlate with fibrosis across the continuum of pancreatitis are needed. Our aim was to determine which clinical variables are associated with fibrosis in patients with recurrent acute pancreatitis (RAP) or CP undergoing total pancreatectomy with islet autotransplantation (TPIAT). METHODS:The demographic, clinical and radiologic data for patients undergoing TPIAT for RAP or CP between 2011 and 2023 were reviewed. Excisional biopsies from the proximal and distal pancreas were each scored from 0 to 6 for both perilobular and intralobular fibrosis, and the score of each biopsy was the sum of perilobular and intralobular fibrosis (0-12). The fibrosis score (FS), ranging from 0 to 12, was the mean FS from the proximal and distal pancreas. RESULTS:There were 88 patients with a mean age 38 ± 14 years and 46 (52.3 %) were female. There were 35 (39.8 %) and 53 (60.2 %) with RAP and CP, respectively. Genetic (52.3 %) and idiopathic (37.5 %) were the most common etiologies. The mean FS was 6.52 ± 3.53. Large duct CP (β = 3, p = 0.001), exocrine pancreatic insufficiency (EPI) (β = 1.5, p = 0.037) and a genetic etiology (β = 1.6, p = 0.03) were significant predictors of fibrosis after adjusting for age, BMI, disease duration and use of oral hypoglycemic drugs and/or insulin. CONCLUSION:Large duct CP, genetic etiology and EPI are all independent predictors of pancreatic fibrosis in a cohort of patients undergoing TPIAT. Computed tomography (CT) imaging and fecal elastase-1 (FE-1) concentration may be sufficient to estimate fibrosis without acquisition of a tissue specimen.

BACKGROUND:Gut-liver crosstalk plays an important role in alcohol-associated liver disease (ALD) pathogenesis; but underlying mechanisms remain obscure. OBJECTIVE:We examined the regulation of intestinal and intrahepatic CD8+ T lymphocytes and their contribution to ALD. DESIGN:ALD patients were recruited for evaluation of intestinal and liver T cells. Single-cell RNA sequencing (scRNA seq) was performed to analyse intrahepatic and peripheral T cells in ALD. Wildtype, CD8-specific Bcl2 transgenic (Cd8 Bcl-2), and Cd8 -/- mice were subjected to chronic-plus-binge ethanol feeding. RESULTS:In ALD patients, duodenal CD8+ T cells were selectively reduced and negatively correlated with liver injury and bacterial translocation markers, while intrahepatic CD8+ T cells were markedly increased. ScRNA seq analysis of ALD patient livers revealed several populations of CD8+ T cells expressing activation and survival genes (eg, Bcl2). Transcriptomics and functional studies revealed a key role of prosurvival BCL2 in this opposite regulation of CD8+ T cells. Mechanistically, chronic-plus-binge ethanol feeding reduced CD8+ T cells specifically in the duodenum where ethanol levels are high. Inducing BCL2 in CD8+ T cells reversed ethanol-induced loss of duodenal CD8+ T cells, improved gut barrier function and ameliorated ALD, while CD8 deficiency was linked to enhanced neutrophil and macrophage infiltration in the liver, exacerbating ALD in mice. CONCLUSIONS:ALD is associated with loss of duodenal CD8+ T cells but elevation of intrahepatic CD8+ T cells, which aggravates and ameliorates ALD, respectively. Restoration of survival and functions of intestinal and intrahepatic CD8+ T cells may represent a novel therapeutic strategy for ALD patients.