Alveolar epithelial stress-induced polyploidization in lung injury and repair

The current pandemic highlights a growing imperative to understand how the distal lung epithelium repairs after injury. 1/\/hile surfactant producing-alveolar type 2 pneumocytes (AT2 cells) also give rise to gas-exchange promoting alveolar type 1 pneumocytes (AT1 cells), recent studies reinforce the vulnerability of this morphogenetic step. Mouse injury studies reveal that AT2 cells convert to an AT1 fate through an intermediate transition-state manifesting activation of integrated stress response (ISR) pathways, a transcriptional state also found in alveolar epithelial cells from patients with fibrosis or injury secondary to viral pneumonia. We have discovered that an Inhibitor of the Integrated Stress Response (ISRIB) can facilitate the AT2-to-AT1 transition step, ultimately attenuating fibrosis in murine models (1/\/atanabe et al., PNAS, 2021). While these data suggest persistent activation of the ISR in AT2 cells can thwart alveolar epithelial repair, conceptual gaps remain: What are the upstream morphogenetic events driving this stress response after injury? And how does modulating the ISR improve the AT2-to-AT1 morphogenetic transition? We have discovered that injury leads to AT2 hypertrophy and polyploidization-notably binucleated AT2s---during the acute injury response. Ex vivo analysis suggests the route to polyploidy is via failed cytokinesis during the AT2-to-AT1 flattening process. Attenuating the ISR inhibits the abundance of hypertrophic, polyploid AT2 cells. Based on our published and preliminary data, we hypothesize lung injury persistently activates the ISR in AT2 cells as they enlarge and flatten to repair the alveolar epithelium, increasing the susceptibility to mitotic slippage into polyploid AT2 cells. Accordingly, Aim 1 will determine whether activation of the ISR underpins AT2 polyploidization during the development of lung fibrosis in mice and humans. Aim 2 will determine whether AT2 polyploidy is necessary or sufficient to worsen fibrosis in response to subsequent injury. Aim 3 will determine whether AT2 polyploidization results from failed cytokinesis downstream of injury-induced signals that collapse the actin cytoskeleton. We use treatment with ISRIB throughout to understand molecular processes that guide AT2 cells through the morpho-genetically stressful AT2-to-AT1 transition required for repair. Overall, we propose AT2 cell divisions are intrinsically vulnerable to injury-induced signals that target F-actin organization, leading to mitotic failures that promote the polyploid state. A key consequence of AT2 polyploidization is loss of the AT2 stem cell daughter, compromising future regenerative potential.