Clara Cell RhoA Activation Aggravates Allergic Airway Inflammation by Disrupting Airway Barrier Integrity via Interstitial Macrophage Interaction
Background: Our previous research demonstrated that Ras homolog family member A (RhoA) activation in alveolar type 2 cells can prevent airway hyperresponsiveness and mitigate allergic airway inflammation. Objective: We aim to investigate the role of RhoA activation in larger airways, particularly Clara cells,  during allergic airway inflammation and explore its underlying mechanisms. Methods: RhoA-deficient mice in Clara cells were generated by crossing CC10-CreERTM mice with RhoAfl/fl mice (CC10-CreERTM; RhoAfl/fl, RhoACKO). Airway epithelial barrier function was assessed by Trans-Epithelial Electrical Resistance (TEER) and permeability across Air-Liquid Interface (ALI) cultures. Multiparameter flow cytometry was applied to characterize various lung immune cells. Both bulk RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq) were also utilized. Results: Deletion of RhoA in Clara cells significantly reduced allergen-induced airway inflammation and remodeling. In vitro analysis demonstrated that airway epithelial cells from RhoACKO mice exhibited decreased TEER and increased permeability. multi-dimensional flow cytometry analysis revealed a significant reduction in interstitial macrophages (IMs) in RhoACKO mice compared to wild-type controls. Notably, scRNA-seq identified IMs as the most up-regulated population following cockroach allergen exposure. RNA-seq analysis revealed down-regulation of several genes, including Ccl24, IL-13, Mcpt1, and Tdo2. Integration of single-cell and bulk RNA-seq identified differentially expressed genes in IMs, such as Tlr1, Ccl24, Tmem26, and Msx3. Further experiments demonstrated that Ccl24 contributes to barrier damage, and blocking CCL24 attenuated allergic airway inflammation. Conclusion: This research underscores the importance of RhoA in Clara cells in airway inflammation and epithelial barrier integrity through regulating interstitial macrophage and its key mediator Ccl24. Overall design: Comparative gene expression profiling analysis of RNA-seq data for PBS or CRE treated Cc10-Cre mice and Cc10-Cre;Rhoafl/fl mice