Club cell RhoA activation amplifies allergic airway inflammation by regulating epithelial integrity and C1qα⁺...

Ras homolog family member A (RhoA) activation in alveolar type 2 cells has been implicated in regulating allergen-induced allergic airway inflammation; however, its role in large airway, particularly Club cells, remains unclear.

We sought to determine the function of RhoA in Club cells during allergic airway inflammation.

A Club cell-specific RhoA knockout mouse model (RhoA^CKO) was generated, and allergic airway inflammation was assessed. Air-liquid interface cultures were used to evaluate epithelial barrier integrity. Multidimensional flow cytometry, bulk RNA sequencing, and single-cell RNA sequencing were used to characterize immune responses and delineate downstream pathways.

RhoA deletion in Club cells significantly attenuated allergic airway inflammation. Air-liquid interface cultures derived from RhoA^CKO epithelium exhibited increased transepithelial electrical resistance, decreased permeability, and reduced cytokine and chemokine (CCL24, TSLP, and IL-33) production. Multidimensional flow cytometry revealed that interstitial macrophages (IMs) were the most profoundly affected immune population. Bulk RNA-sequencing pinpointed IL-13 and Ccl24 as key mediators downstream of Club cell RhoA signaling. Single-cell RNA sequencing confirmed allergen-induced expansion of IMs with high expression of the C1q gene family, which was markedly reduced in RhoA^CKO lungs. Integrated bulk and single-cell RNA-sequencing data revealed Ccl24 as one of the most significantly downregulated genes in IMs from RhoA^CKO lungs. Immunofluorescence colocalization further demonstrated reduced C1qa⁺ IMs and Ccl24⁺C1qa⁺ IM subsets. Functionally, recombinant CCL24 disrupted epithelial barrier integrity, whereas CCL24 neutralization significantly ameliorated airway inflammation.

These findings suggest that RhoA activation in Club cells plays a pivotal role in orchestrating allergic airway inflammation by impairing epithelial barrier function and altering macrophage dynamics.