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.