Intranasal probiotic administration attenuates allergic airway inflammation and is associated with immune–stromal changes in mice

BACKGROUND: Asthma is a chronic inflammatory airway disease characterized by Th2- dominant immune responses, airway hyperresponsiveness, and structural remodeling. Although inhaled corticosteroids and biologics are effective for many patients, a substantial proportion remains poorly controlled and experiences treatment-related adverse effects. Probiotics have emerged as immunomodulatory agents in asthma, but existing studies predominantly focus on oral administration and gut-lung axis regulation. Whether direct respiratory administration of probiotics can modulate the pulmonary immune microenvironment and alleviate asthma remains largely unexplored. METHODS: An ovalbumin (OVA)-induced asthma model was established in C57BL/6 J mice. Clostridium butyricum, Lactobacillus casei, or Bifidobacterium infantis were administered intranasally during the challenge phase. Inflammation and remodeling were evaluated using bronchoalveolar lavage fluid (BALF) cell counts, serum IgE measurement, and histological staining (H&E, PAS, Masson). Lung immune and stromal cell heterogeneity and intercellular interactions were analyzed by single-cell RNA sequencing, while airway microbiota composition was characterized by 16S rDNA sequencing. RESULTS: Intranasal probiotic administration attenuated OVA-induced airway hyperresponsiveness, eosinophilic inflammation, and was associated with reduced mucus hypersecretion and collagen-associated histological changes. Probiotics suppressed Th2-biased immune responses, evidenced by reduced Th2 cell proportions, downregulation of Gata3, and decreased expression of Il4, Il5, and Il13. These effects were associated with inhibited dendritic cell activation and weakened DC-T cell interactions, particularly via the Cxcl16-Cxcr6 axis. Probiotic treatment was associated with an increased proportion of M2 macrophages, reduced pro-inflammatory signaling, reduced predicted macrophage-fibroblast communication through the Osm-Osmr pathway, and a lower proportion of fibrotic fibroblasts. Additionally, intranasal probiotic treatment was associated with changes in airway microbial composition, including a reduced relative abundance of Neisseria, which was positively correlated with Th2 cytokine expression. CONCLUSION: Intranasal probiotic administration was associated with attenuation of airway inflammation and early remodeling-associated changes, accompanied by coordinated changes in Th2 immunity, dendritic cell activation, macrophage polarization, fibroblast subtypes, and the airway microbiota. These effects appeared to be strain-specific, with Lactobacillus casei, Bifidobacterium infantis, and Clostridium butyricum being predominantly associated with suppression of Th2 immunity, modulation of predicted DC-T cell communication, and macrophage polarization, respectively. These findings provide preclinical evidence supporting further investigation of intranasal probiotics as a potential strategy for asthma management.