Impact of a <scp>GABA</scp> ‐Producing <scp> <i>Lactococcus lactis</i> </scp> on Microbiota and Mycobiota During <scp>CNS</scp> Inflammatory Demyelination

Gut microbes are key regulators of immune homeostasis. Their composition fluctuates over time and between individuals and is also influenced by disease. We and others have reported changes in gut bacterial composition following induction of experimental autoimmune encephalomyelitis (EAE), a well-established model for multiple sclerosis (MS). Specifically, we observed reductions in the abundance of bacteria capable of producing gamma-aminobutyric acid (GABA). Because GABA regulates immune cell function, we genetically engineered a <i>Lactococcus lactis</i> strain to overproduce GABA (P8s-GAD <i>L. lactis</i>) and hypothesized that this strain would have protective activity in EAE. To test this hypothesis, a suspension of P8s-GAD <i>L. lactis</i> was administered by gavage to C57BL/6 Envigo (Env) and Jackson Laboratories (Jax) mice at the time of EAE induction. Controls included mice treated with unmodified <i>L. lactis (P-L. lactis)</i> and mice treated with sterile bacterial medium. P8s-GAD <i>L. lactis</i> was clinically protective in Env mice but not in Jax mice. To understand the lack of protection in Jax mice, we examined the effects of treatments on intestinal micro- and mycobiota using 16S rRNA and IST sequencing, and samples were collected at disease induction, 14 days after, and at the end of the experiment (day 28). We also examined the impact of treatments on the brain, using whole-brain proteomics (day 28). Despite the lack of disease protection, P8s-GAD <i>L. lactis</i> significantly modified the gut microbiome by affecting broad taxonomic composition, as quantified by beta-diversity changes over time, and the CNS protein profile, including an increase in Gabra6 expression, the alpha-6 subunit of the GABA type A (GABA_RA) receptor. These changes, combined with reduced EAE severity observed in Env mice, suggest that GABA-producing bacteria could be considered for the treatment of neuroinflammatory conditions. The study also highlights the importance of controlling the mouse source in probiotic and microbiota research within experimental models of immune-mediated diseases.