Dysbiosis induced by CFA and EAE impairs gut regeneration and ISC differentiation via gut-brain axis remodeling
Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system increasingly linked to gut-brain axis dysfunction. The Experimental Autoimmune Encephalomyelitis (EAE) model recapitulates MS-like neuroinflammation but requires Complete Freund's Adjuvant (CFA), a potent immune activator with systemic effects. The impact of CFA alone on intestinal and microbiota alterations remains underexplored, complicating the interpretation of gut changes in EAE studies. Here, we dissected the independent effects of CFA and EAE on intestinal architecture, stem cell function, and microbiota composition. C57BL/6 mice were treated with CFA alone or immunized with MOG35–55 in CFA to induce EAE. Intestinal tissues were analyzed via histology and molecular assays; organoids were derived ex vivo; and fecal samples underwent 16S rRNA sequencing. CFA induced crypt hyperplasia, mucin accumulation, and SOX9 mislocalization, indicating ISC niche remodeling. CFA also caused dysbiosis, with enrichment of Lachnospiraceae and loss of Lactobacillaceae. In contrast, EAE led to villus shortening, mucin depletion, and muscularis thinning, with microbial profiles resembling inflammatory bowel disease—marked by Akkermansiaceae expansion and depletion of butyrate producers. Notably, EAE-derived organoids showed delayed differentiation with hyperproliferation, while CFA-derived organoids exhibited structural disorganization. These findings highlight distinct intestinal phenotypes driven by CFA and EAE, underscoring the need to disentangle adjuvant- from disease-specific effects to clarify the gut's role in MS.