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A4355 - Primary Cilia Are a Critical Component of the Fibroblast-AEC2 Self-Renewal Axis and Contribute to Protection from Pulmonary Fibrosis
Author Block: C. Trempus1, C. Barkauskas2, S. Garantziotis3; 1IIDL, NIH/NIEHS, Research Triangle Park, NC, United States, 2Pulmonary, Allergy, and Critical Care Medicine, Duke University, Durham, NC, United States, 3NIEHS, Natl Inst of Environmental Hlth Sciences, Research Triangle Park, NC, United States.
Introduction: Idiopathic Pulmonary Fibrosis (IPF) is a fatal lung disease caused by dysregulated and progressive deposition of collagen in the lung, ultimately resulting in intractable scarring of the lung parenchyma, leading to death 3-10 years from diagnosis. While dysregulated collagen accumulation is facilitated in part by myofibroblasts, the underlying mechanism of disease pathogenesis is thought to arise to defects in the regenerative capacity of the alveolar epithelium. Alveolar self-renewal is driven by alveolar type 2 epithelial cells (AEC2), and it is known that PDGFRa+ lipofibroblasts play a crucial role in maintaining the alveolar stem cell niche and stimulating stem cell proliferation. We have evidence that the PDGFRa+ lipofibroblasts present in the alveolar septa express primary cilia (PC) – sensory organelles that regulate key signaling pathways, including the Sonic Hedgehog, WNT and PDGF pathways. The objective of this study was to determine a potential role for PC in alveolar stem cell niche regeneration and response to injury. Methods: In order to investigate the role of lipofibroblasts PC in regeneration of the alveolar stem cell niche, a mouse strain was developed in which fibroblasts are deficient in PC (Ift88 fx/fx;SMa22.Cre). PDGFRa+ (EpCam-, CD45-, CD31-) fibroblasts were isolated from naive FxFxCre and WtWtCre mouse lungs using FACS and combined with AEC2 cells obtained from Sftpc-CreER, Rosa26R-tdTm mice in 3D organoid culture for 14 days. Colony size and numbers were measured using ImageJ software. To test the effect of low dose bleomycin on fibrosis development in mice with PC-deficient fibroblasts, FxFxCre and WtWtCre mice were exposed to a single dose of bleomycin (1 unit/kg) via oropharyngeal instillation and lungs collected 21 days post-exposure. Collagen content was measured using the Sircol assay. Results: PDGFRa+ fibroblasts isolated from FxFxCre and control mice were LipidTox+, identifying them as lipofibroblasts. While all lipofibroblast populations supported growth of AEC2s and development of alveolospheres in 3D culture, colony size and number were significantly reduced with AEC2s were co-cultured with FxFxCre (PC-deficient) FBs. Low dose bleomycin resulted in increased fibrosis in PC-deficient FxFxCre mice compared to controls, as evidenced by significantly higher collagen content in whole lung lysates. Conclusions: In PDGFRa+ lipofibroblasts, PC promote a role in alveolar stem cell niche regeneration and response to injury, with implications in the pathogenesis of pulmonary fibrosis.