Idiopathic pulmonary fibrosis (IPF) is a progressive scarring interstitial lung disease (ILD) that affects mainly older adults. Recently, a paradigm shift has occurred wherein the concepts of epithelial cell dysfunction and abnormal wound healing have been placed at center stage as mechanisms driving fibrotic lung remodeling offering new opportunities for therapeutic discovery for IPF. Surfactant protein C (SP-C), an alveolar type 2 (AT2) cell-specific hydrophobic protein that enhances the biophysical activity of surfactant phospholipid, has provided an important clue for understanding epithelial cell dysfunction in IPF pathogenesis as the heterozygous expression of over 50 mutations in the SFTPC gene in humans is associated with chronic ILD. During the current funding period we have shown that sequence alterations in the SP-C primary translation product (proSP-C) associated with clinical ILD phenotypes result in either of 2 distinct aberrant cellular expression patterns, each capable of triggering a series of aberrant cellular responses. ILD-associated, aggregation-prone SP- C isoforms resulting from mutations within the distal COOH domain of the SP-C proprotein (termed ?BRICHOS?) produce vigorous induction of an unfolded protein response (UPR), ER stress, and apoptosis. We have also made the seminal observation that SFTPC mutations found in the more proximal proSP-C COOH linker domain (?Non-BRICHOS?) are mistrafficked to the plasma membrane with a secondary disruption of endosomal / lysosomal function. The induced cellular phenotype includes a late block in macroautophagy, impaired mitophagy, and alterations in general proteostasis repertoires. Building on this, the overall goal of this Merit Review renewal is to now use SFTPC mutants as substrates in vivo to identify and translate molecular mechanisms underlying the disrupted cellular quality control and epithelial dysfunction to the pathophysiology of IPF/ILDs. This proposal will leverage a novel mouse model also generated in the current cycle which expresses the disease-causing clinical non-BRICHOS SFTPC mutant, SP-CI73T, exclusively in AT2 cells. Our Preliminary Data reveals that SP-CI73T mice exhibit alterations in normal proSP-C biosynthetic routing, acquire disruptions in AT2 cell autophagy, and develop diffuse parenchymal lung remodeling. Our experimental approach will be to exploit the unique features of this genetic model combined with tools and reagents available in our program designed to interogate cell quality control and integrated stress responses to first define the ontogeny of and cellular mechanisms mediating the aberrant lung injury, repair, and remodeling responses induced by non-BRICHOS SP-C mutations in vivo [Specific Aim 1]. This will be combined with reductionist studies using primary AT2 cell culures isolated from SP-CI73T mice at key time points in the development of the lung phenotype to characterize the biosynthesis and specific AT2 cellular responses to mutant SP-CI73T in vitro [Specific Aim 2]. Finally, to obtain a more complete model of IPF pathogenesis, the effect of exogenous ?second hits? such as oxidative-nitrative stress and infection on the SP-CI73T mouse lung phenotype will be investigated [Specific Aim 3]. As epithelial dysfunction and susceptibility to injury have not been studied extensively in vivo in the context of fibrotic lung diseases, this approach offers the unique opportunity to comprehensively identify mechanisms mediating responses to the mutant SFTPC substrate by AT2 cells, and to assess the pathways promoting crosstalk between AT2 cells, inflammatory cells and fibroblasts that drive parenchymal remodeling. By understanding the path to epithelial injury from mutant SP-C, the mechanisms identified using these systems can be cross-purposed to better understand the pathogenesis of both sporadic and familial IPF.

Public Health Relevance

Pulmonary fibrosis is a devastating interstitial lung disease (ILD) marked by unrelenting respiratory failure and death. It is increasingly recognized that lung epithelial cell dysfunction plays an important role in the pathogenesis of ILD. Over 50 different mutations in the Surfactant Protein C (SP-C) gene in humans are associated with lung fibrosis. Since SP-C is made and secreted by alveolar type 2 epithelial cells, SP-C mutations can be used as model substrates to probe the role of epithelial cell dysfunction in ILD pathogenesis. Using novel cell systems and mouse models expressing these mutations we will characterize molecular mechanisms regulating cellular quality control responses (e.g. the unfolded protein response, proteasomal degradation, autophagy) used by epithelial cells for maintenance of homeostasis in health and how their failure contributes to ILD development. Results funded by this project will permit rapid assessment of new therapeutic targets for ILD treatment.

National Institute of Health (NIH)
Veterans Affairs (VA)
Non-HHS Research Projects (I01)
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Philadelphia VA Medical Center
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