A stage specific NETs-related signature in alcoholic steatohepatitis: from molecular subtyping to therapeutic vulnerabilities.

BACKGROUND: Alcohol-associated steatohepatitis (ASH) is a globally prevalent liver disease, with robust evidence implicating neutrophil extracellular traps (NETs) as a central pathological phenomenon driving inflammation and progression. However, the core genomic signatures that govern NETs and underlying molecular mechanisms within the ASH microenvironment remain poorly defined. METHODS: Building on the prominent NETs formation phenomenon in ASH, we established a core pool of NETs-related hub genes through intersection of ASH-derived differentially expressed genes (DEGs), key WGCNA modules, and a curated NETs gene set. From this NET-focused pool, a consensus of three machine learning algorithms (LASSO, SVM, RF) distilled a final diagnostic signature, which was rigorously validated in training and external cohorts via ROC analysis and neural networks. Patient heterogeneity was then investigated using consensus clustering with this signature, followed by immune profiling and functional validation in human and mouse ASH models. Therapeutic potential was explored through drug database enrichment and molecular docking. RESULTS: A NETs-focused three-gene signature (FOS, MMP7, CXCL6) achieved exceptional diagnostic accuracy for ASH (AUC = 1.00 in training; 0.983 in validation). It stratified ASH into a Metabolic-dominant (C1) subtype and a Pro-inflammatory (C2) subtype, the latter exhibiting higher MMP7/CXCL6, lower FOS, and enriched cytotoxic infiltration. In vivo, FOS rose in acute injury but declined in chronic models and human ASH, whereas MMP7/CXCL6 remained elevated, suggesting a temporal shift from acute FOS-dominant response to sustained MMP7/CXCL6-mediated inflammation. Finally, drug-gene interaction analysis identified several potential therapeutic modulators, including N-acetylcysteine (NAC), with predicted high binding affinities to FOS and MMP7. CONCLUSION: FOS, MMP7, and CXCL6 constitute a clinically actionable signature capturing the stage-specific dynamics of NETs-driven inflammation in ASH. Beyond its diagnostic and stratifying utility, this signature highlights potential therapeutic avenues for clinical intervention.