A standardized fecal microbiota transplantation protocol enables consistent, microbiota-driven colitis in IL-10-deficient mice.

Gut microbiota dysbiosis is a central feature of inflammatory bowel disease (IBD), yet experimental systems that enable controlled investigation of microbiota-driven inflammation remain limited. In interleukin-10-deficient (Il10-/-) mice, intestinal inflammation is strictly dependent on the presence of commensal microbiota; however, disease onset and severity are highly variable, reflecting differences in microbial composition across environments. To overcome this limitation, pharmacologic approaches such as piroxicam administration have been widely used to synchronize disease, but these methods introduce epithelial injury and non-microbiota-dependent inflammatory pathways that confound mechanistic interpretation. Here, we describe a standardized fecal microbiota transplantation (FMT) protocol that enables controlled microbiota-driven induction of colitis in Il10-/- recipient mice without the use of chemical triggers. In this model, recipient mice aged 8-10 weeks receive fecal microbiota via oral gavage from either colitic Il10-/-; Itgb7-/- double knockout (DKO) donor mice or non-colitic young Il10-/- controls. The DKO donors exhibit impaired mucosal immune regulation and reduced IgA responses, features associated with the emergence of a colitogenic microbial community. Repeated FMT administration over 9 weeks promotes uniform disease induction and reduces variability in disease kinetics across experimental cohorts. Importantly, this approach preserves microbiota-driven disease mechanisms while improving experimental consistency compared with conventional spontaneous Il10-/- models and avoids the confounding effects of pharmacologic synchronization. The protocol is compatible with downstream histological, immunological, and microbiome analyses and provides a practical platform for investigating host-microbiota interactions and microbiome-targeted therapeutic strategies in IBD.