Background: The gut microbiota plays an essential role in mucosal immunity, with secretory immunoglobulin A (IgA) acting as a key effector in neutralizing pathogens and maintaining host-microbiota homeostasis. IgA production occurs via T cell-dependent (TD) and -independent pathways, with T follicular helper (Tfh) cells driving high-affinity, antigen-specific IgA responses. However, the specific microbial taxa and metabolites that regulate Tfh-mediated IgA responses under steady-state conditions remain poorly understood. This study investigated how gut microbiota-derived signals shape Tfh responses and IgA production, with implications for enhancing mucosal vaccine efficacy.

Results: We demonstrate that Peyer's patches (PP)-derived Tfh cells exhibit superior IgA-inducing capacity compared to splenic Tfh cells. RNA sequencing revealed distinct transcriptional profiles in PP-Tfh cells, including upregulation of the genes associated with Tfh differentiation and activation (Bcl6, Cd40lg, Maf), T-B cell interactions (Il21, Sh2d1a, Fyn), and migration (Ccr6, Cxcr5). Functionally, PP-Tfh cells formed larger T-B cell contact areas and induced significantly higher IgA secretion in co-culture than their splenic counterparts. Microbiota depletion experiments revealed that eliminating neomycin-depleted bacteria reduced fecal IgA levels and diminished PP-Tfh cell frequencies. Fecal microbiota transplantation from neomycin-treated mice restored both IgA production and Tfh responses in germ-free (GF) mice. Bioinformatic analysis (PICRUSt2 and LEfSe) identified butyrate-producing Lachnospiraceae and Ruminococcaceae as key drivers of the Tfh-IgA axis. Butyrate supplementation enhanced Tfh differentiation and IgA⁺ germinal center B cell development in vitro and increased fecal IgA levels in vivo. Mechanistically, butyrate promoted IgA production via GPR43 signaling, as its effect was lost in co-cultures with Gpr43⁻/⁻ Tfh cells. Moreover, treatment with tributyrin, a butyrate prodrug, enhanced vaccine-induced IgA and protected mice against Salmonella Typhimurium infection, reducing bacterial burden and tissue damage. These findings define a functional microbiota-Tfh-IgA axis sustained by neomycin-depleted, butyrate-producing bacteria.

Conclusions: Our study underscores the crucial role of the gut microbiota, particularly neomycin-depleted butyrate producing taxa, in regulating PP-Tfh cell function and IgA production. Butyrate emerges as a metabolite linking microbial metabolism to Tfh differentiation and IgA class switching. Together, these findings establish a microbiota-metabolite-Tfh cell axis essential for mucosal immune homeostasis and suggest novel strategies for enhancing vaccine efficacy and protection against enteric infections. Video Abstract.

Keywords: Salmonella Typhimurium; Butyrate; Gut microbiota; Immunoglobulin A; Mucosal immunity; Peyer’s patches; T follicular helper cell.