The role of GM-CSF/GM-CSFRβ signaling in modulating alveolar epithelial cell states in bronchioalveolar lung organoids and in vivo

GM-CSF is a key myeloid growth factor aiding alveolar repair post-influenza A virus infection by promoting epithelial stem cell proliferation via AMPK (adenosine monophosphate kinase) inhibition and mTORC1 (mechanistic target of rapamycin complex 1) activation. However, its role in alveolar epithelial cell (AEC) transitions remains unclear. Thus, this study investigated GM-CSF-induced transcriptional changes in the (bronchio)alveolar epithelium at the single-cell level. Bronchioalveolar lung organoids (BALO) generated from Csf2rb-/- (GM-CSF receptor β) mice exhibited reduced alveolar complexity compared to WT BALOs. To investigate the underlying reasons, single-cell RNA sequencing was performed on WT and Csf2rb-/- BALOs. The analysis identified an aberrant CD14⁺ AEC subpopulation in the absence of GM-CSF signaling. This finding was validated in AECs from Csf2rb-/- compared to WT mice using FACS analysis as well as immunofluorescence staining of lung sections and BALOs. Trajectory analysis of the single-cell data further suggested stalled differentiation of CD14⁺ immature AECs into alveolar epithelial cells type I (AECI), essential for proper alveolarization. Additionally, differential gene expression analysis revealed an inflammatory phenotype in CD14⁺ immature AECs upon lacking GM-CSF signaling. These findings suggest that absent GM-CSF signaling results in immature CD14⁺ AECs with limited differentiation into AECIs. The elevated expression of inflammation-associated genes indicates that the functional unit between alveolar macrophages and the alveolar epithelial compartment might be implicated in the emergence of CD14⁺ AECs, guiding future research.