Perturbation of the gut microbiome by *Prevotella* spp. enhances host susceptibility to mucosal inflammation

Intestinal homeostasis is a delicate balance between the gut microbiota and the host immune system. Microbiota-derived metabolites, such as short-chain fatty acids (SCFAs), are crucial signaling molecules influencing both mucosal immunity and epithelial barrier function. Dysbiosis, or disruption of this balance, has been implicated in numerous inflammatory diseases, including inflammatory bowel disease (IBD) and rheumatoid arthritis (RA). While several bacterial species have been linked to IBD (e.g., *E. coli*, *Klebsiella pneumoniae*, *Akkermansia muciniphila*), the precise roles of many gut microbes, including those of the *Prevotella* genus, remain unclear. Conflicting evidence exists regarding the role of *Prevotella*: some studies associate it with beneficial effects (e.g., improved glucose metabolism), while others link it to autoimmune diseases and gut inflammation. This ambiguity stems partly from the diversity of *Prevotella* strains and the difficulty in establishing direct causal links between specific bacteria and complex diseases outside of animal models. This study aimed to investigate the immunomodulatory properties of a novel *Prevotella* species, *P. intestinalis*, isolated from colitogenic *Nlrp6*⁻⁻ mice, and elucidate its mechanism of action in modulating host susceptibility to intestinal inflammation.

The existing literature presents a complex picture of the *Prevotella* genus's impact on the host. While its presence is linked to a plant-rich diet and improved glucose metabolism in some populations, other studies highlight associations with autoimmune diseases like rheumatoid arthritis and gut inflammation. The lack of consistent findings may be attributable to the high diversity of *Prevotella* strains and potential indirect effects mediated by changes in the overall microbial community. Previous work has identified various bacterial species that exacerbate intestinal inflammation in mouse models, but the precise mechanisms often remain unclear. This study aimed to directly assess the causal relationship between *P. intestinalis* and intestinal inflammation.

The researchers isolated a novel *Prevotella* species, *P. intestinalis*, from the colons of *Nlrp6*⁻⁻ mice known for their susceptibility to colitis. They then colonized specific pathogen-free (SPF) wild-type mice, lacking *Prevotella*, with *P. intestinalis* and analyzed the effects on the gut microbiome and host immune responses. 16S rRNA gene sequencing was used to characterize the microbial community composition, while metabolomics assessed SCFA levels. To induce inflammation, the researchers administered dextran sodium sulfate (DSS) to the colonized mice. Colonoscopy, histological analysis, and cytokine/chemokine quantification were used to assess the severity of colitis. Flow cytometry was employed to characterize immune cell populations in the colon. The role of IL-18 was investigated by supplementing recombinant IL-18 to *Prevotella*-colonized mice undergoing DSS-induced colitis. Finally, the researchers replicated some key experiments using a different SPF mouse line and colonized mice lacking adaptive immunity (*Rag2*⁻⁻ mice) to further investigate the mechanism of *P. intestinalis*-induced inflammation. In vitro cultures of *P. intestinalis* were also used to investigate its metabolic capabilities.

Colonization of SPF mice with *P. intestinalis* resulted in high relative abundance (42.5% ± 2.9) of the bacteria, significantly reshaping the gut microbiota, characterized by a decreased Firmicutes-to-Bacteroidetes ratio and reduced microbial diversity. *P. intestinalis* colonization led to a significant reduction in colonic IL-18 levels during homeostasis and an exacerbation of DSS-induced colitis, as evidenced by increased body weight loss, colon shortening, and histological scores. This effect was not limited to a specific SPF mouse line, as similar results were observed in a commercially obtained SPF mouse line (SPF2) colonized with *P. intestinalis*. The enhanced inflammation in *P. intestinalis*-colonized mice was characterized by increased levels of pro-inflammatory cytokines (IL-6, TNF-α) and chemokines (LIX, MCP-1, MIP-1α, MIP-1β) and an increased infiltration of neutrophils. Importantly, the *P. intestinalis*-induced exacerbation of DSS colitis was independent of adaptive immunity, as observed in *Rag2*⁻⁻ mice. The decrease in IL-18 levels was associated with a significant reduction in SCFA concentrations, particularly acetate, in the cecum, colon, and serum. Supplementation with recombinant IL-18 attenuated the colitis severity in *Prevotella*-colonized mice. In vitro studies revealed that *P. intestinalis* does not directly utilize acetate for butyrate production but exhibits a distinct metabolic profile compared to other gut bacteria, particularly *P. goldsteinii*, producing more succinate and less acetate. Similar effects on acetate levels and IL-18 production were observed with another *Prevotella* species and a *Muribaculaceae* species, suggesting a more general link between reduced acetate production and increased inflammation after intestinal injury.

This study demonstrates a causal link between *P. intestinalis* colonization, alteration of the gut microbiome, reduced IL-18 production, and increased susceptibility to intestinal inflammation. The findings highlight the importance of microbiota-derived metabolites, specifically acetate, in maintaining intestinal homeostasis and immune regulation. The reduction in IL-18, likely due to the altered SCFA profile induced by *P. intestinalis*, appears to be a key factor in the enhanced susceptibility to inflammation after intestinal injury. The study's use of SPF mice and a comparison with *Rag2*⁻⁻ mice effectively rules out confounding factors from other commensals and adaptive immunity. The observation that other bacteria caused similar effects on acetate levels further underscores the importance of this metabolite. The study has implications for understanding the complex interactions between the gut microbiome and the host immune system in inflammatory diseases.

This research establishes a causal role for *P. intestinalis* in exacerbating intestinal inflammation, mediated by changes in the gut microbiome and reduced IL-18 production. The findings highlight the importance of SCFA, particularly acetate, in maintaining intestinal health and modulating immune responses. This work provides insights into the mechanisms by which gut dysbiosis can contribute to inflammatory diseases and suggests that targeting IL-18 or acetate levels might offer therapeutic strategies. Future research could focus on identifying specific bacterial interactions within the *Prevotella*-dominated microbiome and exploring the precise mechanisms by which *Prevotella* alters SCFA production.

The study was conducted in mice, and the findings may not directly translate to humans. The DSS-induced colitis model, while widely used, may not fully recapitulate the complexity of human IBD. The study focused primarily on acetate and IL-18; future investigations could explore other metabolites and immune pathways involved in *Prevotella*-mediated inflammation.