Idiopathic pulmonary fibrosis (IPF) is a genetically mediated, progressive, lung disorder. Evidence points to dysfunctional epithelial telomeres as a core disease-initiating abnormality in IPF. There is limited knowledge of the relationship between alveolar type 2 (AT2) cell telomere dysfunction and the cellular and molecular elements of the remodeled fibrotic lung. Attempts to elucidate this connection in mouse models of telomere dysfunction have been limited by the inherently long telomeres of mice and the use additional fibrogenic stimuli such as low dose bleomycin, which may obscure the distinct input of dysfunctional telomeres to the pathobiology. This proposal seeks to overcome these limitations by applying a novel model of progressive lung fibrosis driven solely by telomere dysfunction isolated to AT2 cells (TRF1SC mice) to study mechanisms of lung fibrosis. The hypothesis is that events developing in TRF1SC mice mimic early epithelial-specific events in the pathogenesis of IPF and that this model will provide new insights into the origins of epithelial cell remodeling observed in IPF. To test this hypothesis, the following aims will be explored: Aim 1 proposes to use TRF1SC/p53-/- mice to establish whether p53 is a molecular mediator of progressive epithelial transdifferentiation and remodeling driven by AT2 cell telomere dysfunction in TRF1SC mice. Single cell RNAseq (scRNAseq) will be used to define the role of p53 on reprogramming of TRF1SC AT2 cells after telomere uncapping, and the appearance of other profibrotic epithelial cell subtypes. Aim 2 proposes to study TRF1SC/TZAP-/- mice to test the hypothesis that the latency of onset of fibrosis in TRF1SC mice requires telomere trimming. TRF1SC/TZAP-/- mice will be treated with tamoxifen for 9 months and measures of telomere attrition, epithelial remodeling, and fibrosis compared to control TRF1SC mice. scRNAseq will be used to define the contribution of telomere trimming to AT2 cell reprogramming in control, TRF1SC, and TRF1SC/TZAP-/- mice. Telomere trimming in normal AT2 cells will be investigated by studying changes in telomere lengths in aged TZAP-/- mice. Aim 3 proposes to culture IPF and control AT2 cells in the presence and absence of hTERT overexpression in an organoid system to establish whether augmenting telomere length restores epithelial cell fate and function of IPF lung epithelial cells. Comparative endpoints include: telomere length measures, DNA damage detected by co-staining for γH2AX and TRF2, senescence reprogramming by β-galactosidase and p16 staining. AT2 cell transdifferentiation into AT1 cells and by qPCR and immunostaining. The influence of hTERT overexpression on other IPF epithelial cell subtypes will be studied by comparing gene expression by scRNAseq of mixed populations of IPF epithelial cells cultured in the presence or absence of hTERT. If successful, these studies will provide an understanding into cell-cell interactions, mediated by telomere dysfunction,...