GM-CSF (granulocyte-macrophage colony-stimulating factor) is a myeloid growth factor that has been implicated in alveolar epithelial type 2 cell (AECII) repair after lung damage. However, the distinct GM-CSF-mediated signaling pathways driving epithelial (stem) cell repair during viral infections remain elusive. Therefore, this study aimed to identify the molecular mechanisms involved in the epithelial regenerative effects of GM-CSF following influenza A virus (IAV)-induced injury.

During IAV infection, GM-CSF is expressed by BASC and AECII which significantly induces their proliferation. In our in vitro bronchioalveolar lung organoid (BALO) model the GM-CSF/GM-CSF receptor axis was necessary for proper alveolarization. Bulk RNA sequencing revealed an upregulation of catabolic pathways in BASC of infected csf2-/- compared to infected WT mice. The treatment of infected AEC from csf2-/- mice with recombinant GM-CSF caused downregulation of AMPK, a master regulator of metabolic pathways, and activation of mTORC1. Accordingly, cell cycle genes, Ccnd1 and cMyc, were upregulated in infected WT but not in csf2-/- AECII. Treatment of lung organoids with AMPK inhibitor or mTORC1 activator supported organoid forming efficiency and alveolarization. Lastly, IAV-infected csf2-/- mice treated with AMPK inhibitor, Compound C, displayed an increase in BASC and AECII proliferation and promoted epithelial barrier repair.

In summary, these data indicate that the protective effects of GM-CSF in BASC and AECII after viral lung injury are mediated by downregulation of AMPK signaling inducing mTORC1 activation, therefore, supporting epithelial stem cells' proliferation and restoration of the alveolar barrier.