[scATAC-seq]: Inhaled PRR Agonists Reprogram Lung Epithelial Cells to Prevent Type 2 Allergic Inflammation

The increasing prevalence of chronic allergic lung diseases demands development of new preventative strategies. We previously demonstrated that therapeutic inhalation of the synergistic agents ODN M362, a TLR9 ligand, and Pam2CSK4, a TLR2/6 ligand (collectively, “Pam2ODN”) protects mice against both infectious challenges and allergic lung disease models, including allergic inflammation caused by mouse house dust mite (HDM) extract. By preventing sensitization, Pam2ODN reduces HDM-induced eosinophilic and lymphocytic inflammation. How Pam2ODN affects the interactions of lung epithelial cells, dendritic cells, and T cells to prevent lung eosinophilic inflammation has not been established. In the present study, we show that a single inhaled dose of Pam2ODN prior to HDM sensitization reduces Th2 polarization of airway CD4+ T cells without affecting the immune responses of Th1 or Treg cells. Furthermore, Pam2ODN pre-treatment inhibits recruitment of lung monocyte-derived dendritic cells (moDCs) and conventional type 2 migratory dendritic cells (DC2s) while preventing HDM-induced decrease of conventional type 1 migratory dendritic cells (DC1s). Bulk RNA-seq of whole lung homogenates reveals that Pam2ODN pretreatment restricts the expression of HDM sensitization-induced proinflammatory transcripts. This tolerogenic effect is also reflected at the single-cell level in lung epithelial cells where many proinflammatory transcripts, pathways and chromatin accessibility are inhibited. These results indicate that Pam2ODN reprograms lung epithelial cells to attenuate allergen-induced lung proallergic DC cytokines while maintaining the population of protective DC1s, which prevents lung injury and Th2 polarization. Collectively, our findings suggest that the development of Th2-induced chronic allergic lung diseases may be mitigated by lung epithelium-targeted immunomodulatory strategies. Overall design: A single aerosol treatment of PBS or Pam2ODN was administered 7-days before intratracheal sensitization of naïve Balb/c mice with 100 µg HDM. Whole lungs were harvested 12 hours after the HDM administration. Lungs were enzymatically digested, and single cell suspensions prepared for flow cytometry sorting of lung immune cells (CD45+) and epithelial cell populations, followed by subsequent single-cell ATAC sequencing analysis.