Surfactant Protein C Mutations and Interstitial Lung Disease

特发性肺纤维化（IPF）是一种进行性瘢痕性间质性肺病（ILD）， 主要是老年人。最近，发生了范式转变，其中上皮细胞的概念 功能障碍和异常伤口愈合已被置于中心阶段， 纤维化肺重塑为IPF的治疗发现提供了新的机会。表面活性 蛋白C（SP-C），肺泡2型（AT 2）细胞特异性疏水蛋白，其增强肺泡2型（AT 2）细胞的免疫应答。 表面活性剂磷脂的生物物理活性，提供了一个重要的线索，了解 IPF发病机制中的上皮细胞功能障碍，表现为超过50种突变的杂合表达 人类SFTPC基因的突变与慢性ILD相关。在目前的融资期内，我们 已经表明SP-C初级翻译产物（proSP-C）中的序列改变与 临床ILD表型导致2种不同的异常细胞表达模式， 能够引发一系列异常的细胞反应ILD相关、易聚集SP- 由SP-C前蛋白的远端COOH结构域内的突变产生的C同种型（称为 “BRICHOS”）产生未折叠蛋白应答（UPR）、ER应激和 凋亡我们还进行了开创性的观察，即SFTPC突变在更多的人中发现， 近端proSP-CCOOH接头结构域（“非BRICHOS”）错误地与血浆结合 膜，伴有内体/溶酶体功能的继发性破坏。所诱导的细胞 表型包括大细胞自噬的晚期阻滞、线粒体自噬受损和一般的改变。 蛋白质稳定库在此基础上，本次Merit Review更新的总体目标是现在使用 SFTPC突变体作为体内底物，以鉴定和翻译SFTPC突变体的分子机制。 破坏了IPF/ILD病理生理学的细胞质量控制和上皮功能障碍。这 该提案将利用一种新的小鼠模型，该模型也是在当前周期中产生的， 致病性临床非BRICHOS SFTPC突变体SP-CI 73 T，仅在AT 2细胞中存在。我们 初步数据显示SP-CI 73 T小鼠在正常proSP-C生物合成中表现出改变， 路由，获得AT 2细胞自噬的破坏，并发展为弥漫性实质性肺 重塑我们的实验方法将是利用这种遗传模型的独特功能 结合我们计划中的工具和试剂，旨在对细胞质量控制进行交互 和整合的应激反应，以首先确定个体发育和细胞机制介导 非BRICHOS SP-C诱导的异常肺损伤、修复和重塑反应 体内突变[具体目标1]。这将与还原主义者的研究相结合， 在肺发育的关键时间点从SP-CI 73 T小鼠分离的AT 2细胞培养物 表型，以表征生物合成和特异性AT 2细胞对突变体SP-CI 73 T的反应， 体外[特定目标2]。最后，为了获得更完整的IPF发病机制模型， 外源性“二次打击”，如氧化-硝化应激和感染对SP-CI 73 T小鼠肺的影响 将研究表型[具体目标3]。由于上皮功能障碍和对损伤的敏感性 尚未在纤维化肺疾病的背景下进行广泛的体内研究， 提供了一个独特的机会，以全面确定调解机制， 突变SFTPC底物的AT 2细胞，并评估促进串扰之间的途径 AT 2细胞、炎性细胞和成纤维细胞驱动实质重塑。通过了解 突变SP-C导致上皮损伤的途径，使用这些系统鉴定的机制可以 交叉目的是更好地了解散发性和家族性IPF的发病机制。

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.