Biology subjects

Nagy, L. E.

Publications and source records attributed to Nagy, L. E..

8 recordsLinked to original sources

bioRxiv2026

Integrated multi-omics reveals adaptive anti-oxidant remodeling in early alcohol-associated liver disease

Alcohol-associated liver disease (ALD) is a leading cause of liver-related morbidity and mortality. Although various omics approaches have revealed early metabolic alterations, individual datasets provide limited mechanistic insight. Here, we integrated RNA sequencing with mass spectrometry-based analyses to quantify gene expression, protein abundance, proteome and acetylome dynamics, and metabolic fluxes in livers of alcohol-fed mice. This multi-layered approach revealed extensive metabolic rewiring characterized by suppressed mitochondrial energy metabolism and compensatory upregulation of glutathione (GSH) production, utilization, and recycling, establishing a high-flux antioxidant network. These changes were coupled to epigenetic histone H3 remodeling, marked by increased permissive acetylation and decreased suppressive methylation, linking alcohol-induced metabolic and redox alterations to chromatin reprogramming. ChEA-based in silico upstream transcription factor analysis, identified hepatocyte nuclear factor 4 (HNF4) and nuclear factor erythroid 2-related factor 2 (NRF2) as key regulatory nodes. Alcohol exposure was associated with a modest HNF4 suppression alongside increased expression of NRF2, indicating a shift from HNF4-driven metabolic programs toward NRF2-mediated antioxidant responses. Despite acetylation-associated impairment of mitochondrial proteins, GSH-related enzymes were preserved, supporting a protective, high-turnover antioxidant response that limits early oxidative stress and defines an adaptive state maintaining redox homeostasis while potentially predisposing to ALD progression.

biochemistry↗

bioRxiv2026

Urine proteomic profiling at admission reveals complement biomarkers linked to alcohol-associated liver disease.

Background and aimsCirculating complement is associated with occurrence of alcohol-associated hepatitis (AH) and is a potential biomarker to distinguish AH from alcohol cirrhosis (AC). Complement contributes to kidney injury, a condition often occurring in patients with alcohol-associated liver disease (ALD). However, little is known regarding complement in cross talk between liver and kidney in ALD. Here we tested the hypothesis that urinary complement would provide potential biomarkers for ALD and insights into mechanisms of liver-kidney crosstalk in the pathogenesis of ALD. MethodsPlasma and urine were collected at admission from patients with sAH, healthy controls (HC), and heavy drinkers without liver disease (HD) (from the multicenter Alcohol Hepatitis Network) and with AC (from the Northern Ohio Alcohol Center). Urine was subjected to unbiased proteomics analysis and plasma complement assessed by multiplex/ELISA assays. 30- and 90-day mortality was tracked in patients with sAH. ResultsAll three complement activation pathways were perturbed in plasma and urine of patients with sAH and AC compared to HC and HD. Components of the lectin and classical pathways in urine were associated with 30- and 90-day mortality in patients with sAH. When 4 complement proteins were combined, they distinguished sAH from AC (AUC 0.78), equivalent to that of MELD (AUC 0.65). There was no correlation between complement in plasma and urine, suggesting an independent impact of sAH on complement in kidney and liver. ConclusionThe urinary proteome revealed complement protein signatures associated with sAH and AC, providing valuable insights into the potential for complement biomarkers and the mechanisms of liver-kidney crosstalk in ALD.

immunology↗

bioRxiv2025

RIP3 and MLKL regulate Hepatic ER stress in alcohol-associated liver disease and pharmacological ER stress models: insights beyond necroptosis

Background and AimEndoplasmic reticulum (ER) stress is an important contributor to liver disease progression, including alcohol-associated liver disease (ALD). While receptor-interacting protein kinase-3 (RIP3) and mixed lineage kinase domain-like pseudokinase (MLKL) are known for their roles in necroptosis, emerging evidence highlights their non-canonical functions in metabolic regulation and cellular stress responses. However, their specific role in regulating hepatic ER stress remains unclear. This study investigates how RIP3, its kinase activity, and MLKL regulate ER stress pathways during chronic ethanol exposure and pharmacological ER stress induction. MethodsRip3-/-, Rip3K51A/K51A and Mlkl-/- mice alongside WT controls and pharmacological necroptosis inhibitors were used to study the role of RIP3 and MLKL in modulating ER stress. Chronic ethanol feeding and pharmacological agents (tunicamycin, thapsigargin) were utilized to induce ER stress in vivo and in isolated primary hepatocytes. ER stress markers were assessed by qPCR and western blot, ER expansion was evaluated by confocal microscopy, and hepatocyte viability was measured using MTS assay. ResultsChronic ethanol increased expression of ER stress markers in WT mice; this response was attenuated in Rip3-/- mice. Tunicamycin exposure increased hepatic ER stress markers in WT mice; this response was diminished in Rip3-/-, Rip3K51A/K51A and Mlkl-/-mice. In primary hepatocytes, genetic and pharmacological inhibition of RIP3 and MLKL also reduced thapsigargin-induced ER stress responses. Hepatocytes isolated from Rip3-/-, Rip3K51A/K51Aand Mlkl-/- mice exhibited enhanced cell viability under ER stress conditions compared to hepatocytes from WT mice, which was associated with ER expansion as a potential mechanism for mitigating ER stress. ConclusionThis study highlights a novel function of RIP3 and MLKL in regulating hepatic ER stress responses, expanding their known roles beyond programmed necrosis. Impact and ImplicationsThis study provides new mechanistic insight into how RIP3 and MLKL regulate hepatic ER stress responses, extending their roles beyond necroptosis. By demonstrating that genetic or pharmacological inhibition of Rip3, RIP3 kinase activity and Mlkl attenuates ER stress signaling, reduces cell death, and promotes adaptive ER remodeling, our findings identify these proteins as key modulators of hepatocyte survival under stress. These results are important for researchers and clinicians focused on alcohol-associated liver disease and other ER stress-driven liver disorders, as they highlight novel therapeutic targets. In practical terms, modulation of the RIP3- MLKL axis could inform the development of interventions aimed at enhancing ER stress resilience, with potential applications in drug development for ER stress-associated liver injury.

cell biology↗

bioRxiv2025

Preventing Microglial Reactivity Protects from Acute and Progressive Neuronal Dysfunction, Motor Impairments and Sedation following Alcohol Abuse

Alcohol abuse is the primary risk factor for alcohol use disorder (AUD), a leading cause of preventable morbidity and mortality, characterized by systemic inflammation, multi-organ damage, and neurological impairments. While direct effects of alcohol on brain function are well-established, the role of microglia in acute and chronic neurological dysfunction in AUD remains unclear. Using longitudinal in vivo imaging in mice during acute and repeated alcohol abuse, we found that microglia exhibit dynamic morphological responses that precede but parallel ethanol-induced sedation. Ethanol also induced microglia-dependent synapse elimination and reduced neuronal activity and density. Genetic disruption of microglial MyD88 reversed these ethanol-associated changes in microglial reactivity, neuronal structure, and function, while protecting against alcohol-induced intoxication and motor impairments. These findings identify microglia as cellular drivers of acute and chronic brain dysfunction following alcohol abuse, and highlight MyD88 as a critical therapeutic target for the detrimental neurological consequences of AUD.

neuroscience↗

bioRxiv2025

Mitochondria-derived dsRNA-induced stress granules promote IRF3-mediated fibrotic responses

Nucleic acid-induced activation of the anti-viral transcription factor interferon regulatory factor 3 (IRF3) promotes transforming growth factor-beta (TGF{beta})-induced fibrotic responses of hepatic stellate cells in vitro. Herein, we identified molecular mechanisms underlying TGF{beta}-induced IRF3 activation. Gene silencing of IRF3 with short interfering RNA (siRNA) in LX2 human hepatic stellate cells decreased TGF{beta}-induced fibrotic responses. An unbiased proteomic analysis of homeostatic IRF3-interacting proteins revealed striking enrichment in cytoplasmic stress granule components, including G3BP stress granule assembly factor 1 (G3BP1), G3BP2, and cell cycle associated protein 1 (CAPRIN1). TGF{beta} induced cytoplasmic accumulation of mitochondria-derived double-stranded RNA (mt-dsRNA) and assembly of IRF3-containing stress granules. Finally, blockade of G3BP1 activity or depletion of mt-dsRNA decreased TGF{beta}-induced fibrotic responses. Collectively, these findings identify a novel TGF{beta}-mt-dsRNA-stress granule axis in hepatic stellate cells that promotes IRF3-mediated fibrotic responses and potentially implicate cytoplasmic stress granules as a global regulator of fibrosis.

cell biology↗

bioRxiv2023

Membrane Bound O-Acyltransferase 7 (MBOAT7) Shapes Lysosomal Lipid Homeostasis and Function to Control Alcohol-Associated Liver Injury

Several recent genome-wide association studies (GWAS) have identified single nucleotide polymorphism (SNPs) near the gene encoding membrane-bound O-acyltransferase 7 (MBOAT7) that is associated with advanced liver diseases. In fact, a common MBOAT7 variant (rs641738), which is associated with reduced MBOAT7 expression, confers increased susceptibility to non-alcoholic fatty liver disease (NAFLD), alcohol-associated liver disease (ALD), and liver fibrosis in those chronically infected with hepatitis viruses B and C. The MBOAT7 gene encodes a lysophosphatidylinositol (LPI) acyltransferase enzyme that produces the most abundant form of phosphatidylinositol 38:4 (PI 18:0/20:4). Although these recent genetic studies clearly implicate MBOAT7 function in liver disease progression, the mechanism(s) by which MBOAT7-driven LPI acylation regulates liver disease is currently unknown. Previously we showed that antisense oligonucleotide (ASO)-mediated knockdown of Mboat7 promoted non-alcoholic fatty liver disease (NAFLD) in mice (Helsley et al., 2019). Here, we provide mechanistic insights into how MBOAT7 loss of function promotes alcohol-associated liver disease (ALD). In agreement with GWAS studies, we find that circulating levels of metabolic product of MBOAT7 (PI 38:4) are significantly reduced in heavy drinkers compared to age-matched healthy controls. Hepatocyte specific genetic deletion (Mboat7HSKO), but not myeloid-specific deletion (Mboat7MSKO), of Mboat7 in mice results in enhanced ethanol-induced hepatic steatosis and high concentrations of plasma alanine aminotransferase (ALT). Given MBOAT7 is a lipid metabolic enzyme, we performed comprehensive lipidomic profiling of the liver and identified a striking reorganization of the hepatic lipidome upon ethanol feeding in Mboat7HSKO mice. Specifically, we observed large increases in the levels of endosomal/lysosomal lipids including bis(monoacylglycero)phosphates (BMP) and phosphatidylglycerols (PGs) in ethanol-exposed Mboat7HSKO mice. In parallel, ethanol-fed Mboat7HSKO mice exhibited marked dysregulation of autophagic flux and lysosomal biogenesis when exposed to ethanol. This was associated with impaired transcription factor EB (TFEB)-mediated lysosomal biogenesis and accumulation of autophagosomes. Collectively, this works provides new molecular insights into how genetic variation in MBOAT7 impacts ALD progression in humans and mice. This work is the first to causally link MBOAT7 loss of function in hepatocytes, but not myeloid cells, to ethanol-induced liver injury via dysregulation of lysosomal biogenesis and autophagic flux.

cell biology↗

bioRxiv2022

Gut Microbial Trimethylamine is Elevated in Alcohol-Associated Hepatitis and Contributes to Ethanol-Induced Liver Injury in Mice

BACKGROUNDThere is mounting evidence that microbes resident in the human intestine contribute to diverse alcohol-associated liver diseases (ALD) including the most deadly form known as alcohol-associated hepatitis (AH). However, mechanisms by which gut microbes synergize with excessive alcohol intake to promote liver injury are poorly understood. Furthermore, whether drugs that selectively target gut microbial metabolism can improve ALD has never been tested. METHODSWe used liquid chromatography tandem mass spectrometry to quantify the levels of microbe and host choline co-metabolites in healthy controls and AH patients, finding elevated levels of the microbial metabolite trimethylamine (TMA) in AH. In subsequent studies, we treated mice with non-lethal bacterial choline TMA lyase (CutC/D) inhibitors to blunt gut microbe-dependent production of TMA in the context of chronic ethanol administration. Indices of liver injury were quantified by complementary RNA sequencing, biochemical, and histological approaches. In addition, we examined the impact of ethanol consumption and TMA lyase inhibition on gut microbiome structure via 16S rRNA sequencing. RESULTSWe show the gut microbial choline metabolite trimethylamine (TMA) is elevated in AH patients and correlates with reduced hepatic expression of the TMA oxygenase flavin-containing monooxygenase 3 (FMO3). Provocatively, we find that small molecule inhibition of gut microbial CutC/D activity protects mice from ethanol-induced liver injury. CutC/D inhibitor-driven improvement in ethanol-induced liver injury is associated with distinct reorganization of the gut microbiome and host liver transcriptome. CONCLUSIONSThe microbial metabolite TMA is elevated in patients with AH, and inhibition of TMA production from gut microbes can protect mice from ethanol-induced liver injury.

biochemistry↗

bioRxiv2021

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

The molecular mechanisms by which dietary fruits and vegetables confer cardiometabolic benefits remain poorly understood. Historically, these beneficial properties have been attributed to the antioxidant activity of flavonoids. Here, we reveal that the host metabolic benefits associated with flavonoid consumption actually hinge on gut microbial metabolism. We show that a single gut microbial flavonoid catabolite is sufficient to reduce diet-induced cardiometabolic disease burden in mice. Dietary supplementation with elderberry extract attenuated obesity and continuous delivery of the catabolite 4-hydroxphenylacetic acid was sufficient to reverse hepatic steatosis. Analysis of human gut metagenomes revealed that under one percent contains a flavonol catabolic pathway, underscoring the rarity of this process. Our study will impact the design of dietary and probiotic interventions to complement traditional cardiometabolic treatment strategies. One-Sentence SummarySelect gut microbes can metabolize flavonoids from a fruit and vegetable diet to monophenolic acids, which improve fatty liver disease. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/460661v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1c4f56borg.highwire.dtl.DTLVardef@1cc4be3org.highwire.dtl.DTLVardef@18a4af5org.highwire.dtl.DTLVardef@3f44a5_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗