Integrated multi-omics analysis identifies microbial and metabolic signatures and drivers of CNS autoimmunity

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) driven by genetic and environmental determinants. The gut microbiome of people with MS (pwMS) is distinct and influences disease through immunomodulatory metabolite production. Circulating metabolites are altered in pwMS, but identifying microbial-metabolic drivers remains challenging. We previously showed that colonization by the gut commensal <i>Limosilactobacillus reuteri</i> (<i>L. reuteri</i>) exacerbates disease in the experimental autoimmune encephalomyelitis (EAE) model of MS, in a tryptophan-dependent manner. Here, we integrated microbiomic and metabolomic datasets from a longitudinal EAE study utilizing high and low tryptophan diets in mice colonized or not with <i>L. reuteri</i>. Gut microbiome dynamics under short- and long-term alterations in tryptophan bioavailability, were affected by diet, microbiome context, or disease. During short-term dietary intervention, <i>L. reuteri</i> colonization exerted a greater impact on microbiome composition than tryptophan bioavailability. With longer dietary exposure and EAE progression, high dietary tryptophan and <i>L. reuteri</i> colonization synergized to elicit profound microbiota changes, including alterations in Lachnospiraceae, <i>Blautia</i>, and <i>Akkermansia</i>. Integration of metabolomic and microbiomic datasets using joint Robust Aitchison PCA revealed clusters of associated metabolites and microbiota enriched for functional pathways, including bile acid and tryptophan metabolism. Metabolites outperformed microbiota in predicting EAE severity, identifying p-cresols and indoles as top disease-associated metabolites. Treatment with p-cresol or 3-indoleglyoxylic acid exacerbated EAE, enhanced proinflammatory T cell responses, and increased cerebellar pathology. These data demonstrate that dietary responses are shaped by gut microbiome composition and that integrated microbiomic-metabolomic analyses can identify drivers of disease worsening in MS.