Surfactant Protein C Mutations and Interstitial Lung Disease

摘要 特发性肺纤维化（IPF）是一种进行性瘢痕性间质性肺病（ILD），主要影响 老年人仍然存在显著的未满足的治疗需求。虽然病理生理 IPF的基础仍然不完全清楚，这是更好地发展的另一个关键障碍。 IPF的治疗结果缺乏诊断相关的临床前模型。根据最近的 范式转变，其中对功能失调的、脆弱的肺泡上皮细胞的重复性损伤的概念 随着异常伤口愈合反应被假定为疾病“驱动因素”，新的机会正在出现， 用于IPF的治疗发现。肺泡2型细胞（AT 2）限制性表面活性蛋白中超过60个突变 C [SP-C]基因[SFTPC]在散发性和家族性IPF中被发现，并提供了重要的线索， 了解IPF发病机制。为了解决IPF退伍军人未满足的需求，本提案基于 在我们之前工作的坚实基础上，由该优点审查计划资助，描述了细胞生物学特征 SP-C生物合成的最终结果是产生两种新的自发肺的基因敲入小鼠模型 其在AT 2细胞中以等位基因和诱导方式表达临床SP-C突变体。我们 已发表的数据表明，临床IPF相关的SFTPC突变产生异常SP-C 前蛋白同种型在功能上分离成2种AT 2细胞应激表型： 细胞内SP-C错误折叠（“BRICHOS”）或继发于proSP-C的受损自噬/线粒体自噬 误作用于非天然细胞器（“非BRICHOS”）。当在体内肺上皮中表达时， 非BRICHOS突变体（SftpcI 73 T）和BRICHOS突变体（SftpcC 121 G）对肺具有极强的毒性， 每一种都足以引起时间依赖性的、生理限制性的外周纤维化肺表型， 阐述了人类IPF中报告的预防相关生物标志物。在此基础上， 现在将利用这些Sftpc突变小鼠来绘制IPF中的远端肺细胞群， 翻译分子机制，连接中断的细胞质量控制，上皮功能障碍， IPF/ILD的病理生理学。在3个具体目标中，我们的实验方法将利用 这些遗传模型结合我们的程序中可用的工具和试剂，旨在interrogate细胞 质量控制和综合应激反应，以首先确定关键的肺泡小生境细胞群， 体内SP-C突变诱导的启动、损伤放大和纤维化阶段[特定目的1]。然后 有了这个功能图，我们将Sftpc小鼠与简化模型如AT 2类器官配对， 确定内源性内质网（ER）应激在AT 2功能障碍中的作用， IPF中发现的损伤/修复途径[特定目标2]。最后，我们将评估 破坏了上皮细胞对AT 2代谢重编程和线粒体动力学的质量控制， 将其对AT 2表型和纤维化重塑的影响置于背景中[具体目标3]。肺泡上皮细胞 在纤维化肺疾病的情况下，尚未在体内广泛研究功能障碍，这种方法提供了 这是一个独特的机会，可以全面确定介导对突变体反应的独特机制， 通过AT 2细胞的SFTPC底物，并评估促进AT 2细胞之间的串扰的途径，炎性 细胞和成纤维细胞驱动实质重塑。通过了解上皮损伤的途径， 突变SP-C，使用这些模型确定的机制可以交叉使用，以更好地了解IPF 一般的发病机制，促进识别新的靶向途径，并测试新型IPF治疗。

ABSTRACT Idiopathic pulmonary fibrosis (IPF) is a progressive scarring interstitial lung disease (ILD) that affects mainly older adults for which there remains a significant unmet therapeutic need. While the pathophysiologic underpinnings of IPF remain incompletely understood, an additional critical barrier to developing better therapeutic outcomes for IPF has been a dearth of translationally relevant preclinical models. Based on a recent paradigm shift wherein the concepts of repetitive injury to a dysfunctional, vulnerable, alveolar epithelium coupled with an abnormal wound healing response are postulated as disease “drivers”, new opportunities are emerging for therapeutic discovery in IPF. Over 60 mutations in the alveolar type 2 cell (AT2) restricted, Surfactant Protein C [SP-C] gene [SFTPC], have been found in sporadic and familial IPF and provide important clues for understanding IPF pathogenesis. To address the unmet need for veterans with IPF, this proposal builds upon on a strong foundation of our prior work funded by this Merit Review program characterizing the cell biology of SP-C biosynthesis that culminated in generation of two novel knock-in mouse models of spontaneous lung fibrosis already in hand which express clinical SP-C mutants in AT2 cells in an allelic and inducible fashion. Our Published Data has demonstrated that clinical, IPF-associated SFTPC mutations produce aberrant SP-C proprotein isoforms that functionally segregate into 2 AT2 cell stress phenotypes: ER stress induced by intracellular SP-C misfolding (“BRICHOS”) or impaired autophagy/mitophagy secondary to proSP-C mistrafficking to non-native organelles (“Non-BRICHOS”). When expressed in the lung epithelium in vivo, both the non-BRICHOS mutant (SftpcI73T) and the BRICHOS mutant (SftpcC121G) are extremely toxic to the lung and each is sufficient to evoke a time-dependent, physiologically restrictive peripheral fibrotic lung phenotype that elaborates translationally relevant biomarkers reported in human IPF. Building on this, our Merit Review renewal will now leverage these Sftpc mutant mice to map distal lung cell populations in IPF while also identifying and translating molecular mechanisms linking the disrupted cellular quality control, epithelial dysfunction, and pathophysiology of IPF/ILDs. In 3 specific aims, our experimental approach will be to exploit the unique features of these genetic models combined with tools and reagents available in our program designed to interogate cell quality control and integrated stress responses to first define key alveolar niche cell populations emerging during initiation, injury amplification, and fibrosis stages induced by SP-C mutations in vivo [Specific Aim 1]. Then armed with this functional map we will couple Sftpc mice with reductionist models such as AT2 organoids to define the role of endogenous endoplasmic reticulum (ER) stress in AT2 dysfunction and the aberrant injury/repair pathways found in IPF [Specific Aim 2]. Finally, we will assess the downstream consequences of disrupted epithelial cell quality control for AT2 metabolic reprogramming and mitochondrial dynamics and then contextualize their impact on AT2 phenotypes and fibrotic remodeling [Specific Aim 3]. As alveolar epithelial dysfunction has not been studied extensively in vivo in the setting of fibrotic lung diseases, this approach offers the unique opportunity to comprehensively identify unique 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 models can be cross-purposed to better understand IPF pathogenesis in general, promote identification of new target pathways, and test novel IPF therapies.