GM-CSF (granulocyte-macrophage colony-stimulating factor) is a key myeloid growth factor involved in alveolar epithelial repair after lung damage. However, the exact mechanisms by which GM-CSF promotes epithelial stem cell repair during viral infections remain unclear. This study aimed to investigate the signaling pathways that mediate GM-CSF's regenerative effects following influenza A virus (IAV) injury.

During IAV infection, GM-CSF is predominantly expressed by bronchioalveolar stem cells (BASC) and alveolar epithelial cells type II (AECII), significantly inducing their proliferation. In our ex vivo bronchioalveolar lung organoid (BALO) model, the GM-CSF/GM-CSF receptor axis was essential for proper alveolarization. Bulk RNA sequencing revealed an upregulation of catabolic pathways in BASC from infected Csf2-/- (GM-CSF knock-out) compared to infected WT mice. Treatment of infected AEC from Csf2-/- mice with recombinant GM-CSF caused downregulation of AMPK, a key metabolic regulator, and activation of mTORC1. Accordingly, cell cycle genes, Ccnd1 and cMyc, were upregulated in infected WT but not in Csf2-/- AECII. Notably, treatment of lung organoids with GM-CSF or AMPK inhibitor supported organoid forming efficiency and alveolarization. IAV-infected Csf2-/- mice treated with AMPK inhibitor displayed a significant increase in BASC and AECII proliferation, promoting epithelial barrier repair.

In summary, these data suggest that GM-CSF exerts protective effects on BASC and AECII following viral lung injury by downregulating AMPK signaling, which activates mTORC1. This activation supports the proliferation of epithelial stem cells and aids in the restoration of alveolar barrier function.