Lung fibrosis mediated by telomere dysfunction in alveolar type II cells

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Idiopathic pulmonary fibrosis (IPF) is a progressive lung disorder whose prevalence increases with age. IPF alveolar type II cells (AECII cell) have shortened telomeres and express markers of senescence such as senescence associated beta-galactosidase (SA-βgal). Although short telomeres and a senescent phenotype are always found in IPF AECII cells, there is little data linking these abnormalities, to molecular mediators facilitating the lung remodeling found in IPF patients. This lack of insight is due to the absence of a model of lung remodeling mediated by telomere dysfunction. This proposal seeks to overcome these knowledge gaps by using a model of lung fibrosis mediated by telomere dysfunction isolated to alveolar type II cells (TRF1SC mice) to examine whether telomere dysfunction in AECII cells is a molecular switch that drives pathologic abnormalities found in IPF. Aim 1. To establish the contribution of macrophages to lung remodeling in TRF1SC mice. This aim uses pharmacologic (clodronate) and genetic approaches to deplete alveolar macrophages in TRF1SC mice and define the contribution of macrophage expansion to lung fibrosis. It explores whether AECII cell expression of the chemokine CCL2 mediates macrophage expansion by crossing CCL2F/F mice to TRF1SC mice. At key time-points lungs will be harvested from clodronate treated or CCL2F/F/TRF1SC mice and measures of remodeling and fibrosis quantified and compared to relevant controls. Aim 2. To establish the profibrotic molecular mediators generated by AECII cells in TRF1SC mice. The AECII cell-specific, TGFβ activating, integrin αvβ6, will be quantified at key time-points and correlated with active TGFβ levels. Capacity for senescent AECII cells to activate TGFβ will be quantified in the MLE TGFβ activation assay +/- β6 blocking Ab. Contribution of β6–mediated of TGFβ activation to lung fibrosis will be tested by crossing β6 deficient mice to TRF1SC mice and measures of lung remodeling quantified. RNAseq will identify pro-fibrotic elements of the senescence associated secretory proteome in TRF1SC mice, and establish whether they change over time. Aim 3. To establish that senescence programming can be leveraged to identify and target profibrotic AECII cells. SA-βgal staining will be used to isolate senescent AECII cells from IPF lungs. Measures of the capacity for senescent IPF AECIIs to activate TGFβ will be compared between SA-βgal+ to SA-βgal- control AECII cells in the presence and absence of β6 blocking Ab. Profibrotic pathways will identified in senescent IPF AECIIs by comparing mRNA levels between the senescent SA-βgal+ IPF AECII cells and normal controls. AECII cells isolated from IPF or TRF1SC mice will be treated with a senolytic drug (ABT263) and expression of senolytic markers and profibrotic mediators compared between treated and untreated cells. To test effect of senolysis on lung remodeling in vivo, TRF1SC mice will be treated with ABT263 and at various time-points after treatment, measures of senescence and remodeling compared with vehicle treated controls.