The current pandemic highlights a growing imperative to understand how the distal lung epithelium repairs
after injury. While 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 (Watanabe 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.