PAD2 Deficiency Reveals Sex-Divergent Colonic Homeostasis, β-Catenin Remodeling, and Antibiotic-Sensitive Phenotypes

Peptidylarginine deiminase 2 (PAD2) catalyzes protein citrullination and is implicated in inflammatory bowel disease and colorectal cancer. However, its role in regulating the intestinal epithelium and the influence of biological sex and the microbiome remain poorly defined. This study investigated the relationships among PAD2 deficiency, sex-divergent epithelial states, β-catenin regulation, and the intestinal microbiome. Global PAD2 knockout and wild-type littermate mice of both sexes were characterized under basal conditions and following dextran sodium sulfate-induced colitis. Intestinal epithelial phenotypes were assessed by histology, immunofluorescence, immunoblotting, quantitative RT-PCR, and immunoprecipitation. Gut microbial communities were evaluated by shotgun metagenomics, while broad-spectrum antibiotic treatment, fecal protein immunoblotting, and recombinant human PAD2 exposure in vitro were used to examine relationships among antibiotic-sensitive factors, β-catenin regulation, and epithelial phenotypes. PAD2 deficiency was associated with distinct epithelial states in male and female colons before injury. Male KO mice displayed increased β-catenin phosphorylation and increased susceptibility to DSS colitis, whereas female KO mice demonstrated increased total β-catenin abundance and preservation of structural epithelial features following injury. Female KO microbiota exhibited genotype-associated differences in microbial community composition by exploratory Bray-Curtis PERMANOVA. Broad-spectrum antibiotic treatment altered the female epithelial phenotype, reduced active β-catenin, and progressively depleted luminal PAD2-immunoreactive proteins. Exogenous recombinant human PAD2 increased epithelial protein citrullination and altered active β-catenin abundance in HT29 cells. Collectively, these findings demonstrate both sex-specific and sex-divergent colonic phenotypes while supporting a framework connecting PAD2 biology, epithelial architecture, and the microbial environment during intestinal maintenance and injury.