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.

多发性硬化（Multiple sclerosis, MS）是一种中枢神经系统慢性自身免疫性疾病，其与肠-脑轴功能障碍的关联日益受到学界关注。实验性自身免疫性脑脊髓炎（Experimental Autoimmune Encephalomyelitis, EAE）模型可重现多发性硬化样神经炎症，但该模型的构建需要使用完全弗氏佐剂（Complete Freund's Adjuvant, CFA）——一种具有全身效应的强效免疫激活剂。目前，单纯CFA对肠道及菌群改变的影响尚未得到充分探究，这一现状使得EAE研究中肠道变化的解读变得更为复杂。本研究解析了单纯CFA与EAE各自对肠道结构、干细胞功能及菌群组成的独立影响：研究人员对C57BL/6小鼠分别进行单纯CFA处理，或通过在CFA中乳化MOG₃₅–₅₅进行免疫以诱导EAE模型；通过组织学与分子实验分析肠道组织，体外构建肠道类器官，并对粪便样本进行16S rRNA测序。结果显示，单纯CFA可诱导隐窝增生、黏蛋白蓄积以及SOX9定位异常，提示肠干细胞（Intestinal Stem Cell, ISC）龛发生重塑；同时，单纯CFA可引发菌群失调，表现为毛螺菌科（Lachnospiraceae）富集、乳杆菌科（Lactobacillaceae）减少。与之相反，EAE模型小鼠则表现为肠绒毛缩短、黏蛋白缺失以及肌层变薄，其菌群特征与炎症性肠病相似，以嗜黏蛋白阿克曼菌科（Akkermansiaceae）扩增、产丁酸菌减少为典型特征。值得注意的是，源自EAE模型小鼠的肠道类器官呈现增殖亢进但分化延迟的表型，而源自单纯CFA处理小鼠的类器官则表现出结构紊乱。本研究结果揭示了CFA与EAE各自诱导的差异化肠道表型，强调需区分佐剂相关与疾病特异性的肠道改变，以明确肠道在多发性硬化发病机制中的作用。