Clostridium leptum attenuates allergic airway inflammation via tDC‒Treg axis activation in a murine model of gut dysbiosis-associated asthma

Allergic pulmonary inflammation is driven by allergen-specific Th2/Th17 responses counterregulated by tolerogenic dendritic cells (tDCs) and regulatory T cells (Tregs). While early-life colonization with commensal Clostridia induces the generation of colonic Tregs, the therapeutic potential of Clostridium leptum (CL) monotherapy to treat asthma patients with comorbid gut dysbiosis remains unexplored. Therefore, this study aimed to investigate whether 14 days of oral CL administration restores gut-lung immune homeostasis and suppresses allergic inflammation via tDC/Treg induction in a dysbiosis-asthma comorbidity model. To this end, adult BALB/c mice were subjected to ovalbumin (OVA) sensitization/challenge and antibiotic-induced gut dysbiosis. Interventions included 14 days of oral CL (5 × 10⁹ CFUs/day) and/or 5 days of budesonide (BUD) nebulization. The gut microbiota (16 S rRNA sequencing), metabolome (untargeted LC‒MS), and immune parameters were assessed via histopathology, flow cytometry (tDCs: CD11c⁺CD80^-CD86^-; Tregs: CD4⁺CD25⁺FoxP3⁺), cytokine profiling (ELISA), and analyses of the TLR/NF-κB pathway (IHC/qPCR/WB). As a result, CL monotherapy restored gut microbial α diversity and enriched tryptophan-derived indole derivatives. These microbial metabolites suppressed TLR2/4-NF-κB activation in pulmonary tDCs, driving Treg differentiation while inhibiting Th2 and Th17 responses. Similar to budesonide (BUD), CL significantly reduced airway hyperresponsiveness and eosinophilic infiltration, with a synergistic effect observed for the combination therapy. In conclusion, oral CL alleviates allergic asthma by the microbiota-mediated production of immunoregulatory indoles that activate the TLR2/4-tDC-Treg axis, suggesting a microbiota-targeted strategy for treating steroid-resistant asthma.