Epithelial Immunomodulation by Aerosolized Toll-like Agonists Prevents Allergic Lung Inflammation by Reducing Th2 Polarization

Abstract In recent decades, the number of people with chronic allergic lung diseases has steadily increased. Reversing the trend of these allergic diseases requires the development of new prophylactic-based strategies. We previously demonstrated that therapeutic immunomodulation following inhalation of the synergistic agents ODN M362, a TLR9 ligand, and Pam2CSK4, a TLR2/6 ligand (collectively, “Pam2ODN”) protects mice against a variety of lung allergy disease models, including allergic inflammation brought on by mouse house dust mite (HDM) extract. By preventing sensitization, Pam2ODN has been shown to reduce HDM-induced eosinophilic and lymphocytic inflammation. How Pam2ODN pretreatment affects lung epithelial cells, dendritic cells, and T cell polarization to prevent lung eosinophilic inflammation has not been investigated. We used flow cytometry, RNAseq, and scRNAseq analysis to examine the HDM model of lung allergic disease to better comprehend the mechanisms underlying this protection. Here, we show that giving BALB/c mice a single dose of Pam2ODN seven days prior to HDM sensitization reduces the polarization of lung Th2 CD4+ T cells following repeated HDM challenges without affecting the immune responses of Th1 and Treg cells. Analysis of mice immune responses at the time of sensitization reveals that Pam2ODN pre-treatment inhibits the recruitment of lung monocyte-derived dendritic cells (moDCs) and conventional type2 migratory dendritic cells (cDC2s) on the one hand and prevents the HDM-induced decrease of resident alveolar macrophages (AMs) and conventional type1 migratory dendritic cells (cDC1s) on the other hand. Bulk RNAseq of the lung homogenate revealed that Pam2ODN pre-treatment restricts the expression of HDM sensitization-induced proinflammatory transcripts including Il1β, Il33, Il6, Tnf, Ccl2, Ccl20, Nlrp3, Myd88, etc. This tolerogenic effect of Pam2ODN pre-treatment against HDM sensitization was also reflected at the single-cell level in alveolar type II (AT2) lung epithelial cells, where many proinflammatory genes and pathways were inhibited. These results suggest that Pam2ODN-induced immunomodulation reduces HDM-induced allergic lung inflammation by preventing the release of lung proallergic DCs chemoattractant and proinflammatory cytokines by lung epithelial cells while maintaining the populations of protective AMs and cDC1s, which prevents lung injury and Th2 polarization. Together these data suggest that the development of Th2-induced chronic allergic lung diseases may be mitigated by epithelium-targeted immunomodulatory strategies.