Identifying Inflammatory Signals Facilitating Ectopic Krt5+ Cell Migration and their Function in the Alveoli

Abstract Severe respiratory viral infections pose a significant risk and can result in debilitating lung diseases such as acute respiratory distress syndrome and subsequent pulmonary fibrosis, which are prominent contributors to global mortality. During severe respiratory viral injury, the lung initiates a distinct form of regeneration that is independent of the functional regeneration facilitated by distal epithelial progenitors. This secondary "dysplastic" mode of regeneration involves the activation of p63+ basal progenitor cells in the upper airways, followed by the upregulation of Krt5, expansion, and migration into the alveoli, ultimately resulting in ectopic bronchiolization of the alveolar epithelium. This dysplastic response is postulated to provide short-term benefits by restoring the epithelial barrier; however, it ultimately results in a reduction of the total functional gas-exchanging surface area of the lung. Ectopic Krt5+ cells are consistently observed in cases of both severe viral lung injury and pulmonary fibrosis but have specifically been found to be strongly associated with fibrotic regions of the lung in cases of pulmonary fibrosis. However, their specific involvement in fibrosis following viral injury and the underlying mechanisms facilitating their ectopic migration from the proximal to distal airways remains poorly understood. In my preliminary data, I observed a significant upregulation of canonical NF-κB transcription factors RelA, Nfkb1, and c-Rel in ectopic Krt5+ cells. The NF-κB pathway is a well-known pro-inflammatory signaling pathway, and its activation has been shown to promote epithelial-mesenchymal transition, migration, and proliferation. Aim 1 of this proposal will assess whether canonical NF-κB signaling is necessary for ectopic Krt5+ cell expansion and migration by utilizing a conditional deletion of IKKβ in p63+ progenitor cells. In vivo experiments will determine the impact of IKKβ deletion on the total quantity of dysplastic Krt5+ epithelium following influenza injury through immunofluorescent imaging and qPCR. Accompanying in vitro experiments will assess the specific migratory and proliferative phenotypes associated with the loss of NF-κB signaling through in vitro gap closure and EdU incorporation assays. Additional preliminary data revealed that ectopic Krt5+ cells highly express CTGF, a known secreted pro-fibrotic growth factor, and are surrounded by more Collagen 1 compared to less-injured regions following H1N1-induced lung injury. Aim 2 of this study will utilize a conditional deletion of CTGF and various metrics, including immunofluorescence and hydroxyproline content, to determine whether CTGF is necessary for the pro-fibrotic function of ectopic Krt5+ cells in vivo. Subsequently, we will elucidate the direct paracrine effect of ectopic Krt5+ cells on fibroblast extracellular matrix deposition in vitro by culturing fibroblasts in conditioned media from Krt5+ cells +/- CTGF and quantifying changes in matrix deposition. These experiments will investigate the role of NF-κB signaling in dysplastic remodeling and the pro-fibrotic function of ectopic Krt5+ cells, thereby providing insight into the underlying mechanisms governing their migration and fibrotic pathology.