Alveolar Epithelial Cell Dysfunction in Pulmonary Fibrosis: Leveraging SFTPC Mutations for Discovery of Molecular and Cellular Targets

摘要 纤维化肺重塑是各种间质呼吸衰竭的最终共同途径 儿童和成人肺部疾病(ILD)。基于最近的范式转变，其中的概念 上皮细胞功能障碍和创面愈合异常被认为是肺纤维化的“驱动因素”，新发现 ILD的治疗发现机会正在出现。表面活性蛋白C的突变 [SFTPC]基因是一种肺泡2型细胞(AT2)限制性蛋白，已在散发性和家族性ILD中发现 为了解上皮细胞功能障碍在ILD发病机制中的作用提供重要线索。 我们实验室先前的体外研究表明，SFTPC突变在成人和儿童中都有描述 ILD导致产生采用非天然构象的异常SP-C前蛋白亚型，从而导致 至少两种结果：内质网应激和细胞内聚集(Brichos)或误选为非本地人 细胞器和抑制大型自噬(非Brichos)。此应用程序建议利用两个 已有的新型自发性肺纤维化小鼠模型已有表达非Brichos或 Brichos在AT2细胞中以等位基因和可诱导的方式发生临床SFTPC突变。我们出版的和 初步数据显示，非Brichos突变体(SP-CI73T)或Brichos突变体(SP-CI73T)的表达 CC121G)对体内的肺有极强的毒性，每种药物都会导致时间依赖性的自发性肺纤维化 以3个阶段为特点：ii)早期AT2细胞因子分泌和单核细胞募集；(Ii)多细胞性肺泡炎 和肺损伤；(Iii)生理性限制性外周纤维化重塑。这些模型还详细阐述了 人类ILD中报道的翻译相关生物标记物。为了给共识和分歧下定义 这两种AT2细胞表型与下游肺损伤和纤维化肺的分子机制 重塑，我们的实验方法将是利用这些遗传模型组合的独特功能 利用我们计划中提供的工具、试剂和专业知识。在三个具体目标上，我们全面提出 非Brichos基因表达对AT2细胞转录和功能表型的影响 (SP-CI73T)和Brichos(SP-CC121G)Sftpc突变体[特定目标1]，以定义密钥的作用 AT2突变型Sftpc在发育过程中招募的促炎/促纤维化单核细胞群 纤维化[特定目标2]，并检查扰乱肺泡的上皮-间充质串扰的变化 利基平衡和促进纤维化重塑[特定目标3]。因为上皮性功能障碍还没有 在纤维化肺疾病的相关临床前模型的背景下，在体内进行了广泛的研究，这种方法 提供了独特的机会，为突变型SFTPC在家族性心脏病中的因果关系提供概念证明 ILD和AT2功能障碍作为炎症细胞和成纤维细胞活动的关键上游驱动因素的作用 促进实质重塑的物质。重要的是，使用集中方法确定的机制 这些Sftpc模型可以交叉使用，以更好地理解散发性ILD的发病机制。

ABSTRACT Fibrotic lung remodeling represents the final common pathway to respiratory failure for a variety of Interstitial Lung Diseases (ILD) in children and adults. Based on a recent paradigm shift wherein the concepts of epithelial cell dysfunction and abnormal wound healing are postulated as “drivers” of pulmonary fibrosis, new opportunities are emerging for therapeutic discovery for ILD. Mutations in the Surfactant Protein C [SP-C] gene [SFTPC], an alveolar type 2 cell (AT2) restricted protein, have been found in sporadic and familial ILD and can provide important clues for understanding the role of epithelial cell dysfunction in ILD pathogenesis. Prior in vitro studies from our lab have shown that SFTPC mutations described in both adults and children with ILD result in production of aberrant SP-C proprotein isoforms that adopt non-native conformations resulting in at least 2 outcomes: ER stress and intracellular aggregation (BRICHOS) or mistrafficking to non-native organelles and inhibition of macroautophagy (Non-BRICHOS). This application proposes to leverage two novel knock-in mouse models of spontaneous lung fibrosis already in hand which express Non-BRICHOS or BRICHOS clinical SFTPC mutants in AT2 cells in an allelic and inducible fashion. Our Published and Preliminary Data reveal that expression of either a non-BRICHOS mutant (SP-CI73T) or BRICHOS mutant (SP- CC121G) is extremely toxic to the lung in vivo with each resulting in time-dependent spontaneous lung fibrosis marked by 3 phases: ii) early AT2 cytokine elaboration and monocyte recruitment,; (ii) a polycellular alveolitis and lung injury; (iii) physiologically restrictive peripheral fibrotic remodeling. These models also elaborate translationally relevant biomarkers reported in human ILD. In order to define both consensus and divergent molecular mechanisms linking these two AT2 cell phenotypes with the downstream lung injury and fibrotic lung remodeling, our experimental approach will be to exploit the unique features of these genetic models combined with tools, reagents, and expertise available in our program. In 3 specific aims, we propose to comprehensively characterize the transcriptomic and functional AT2 cell phenotypes evoked by expression of non-BRICHOS (SP-CI73T) and BRICHOS (SP-CC121G) Sftpc mutants [Specific Aim 1], to define the role of a key proinflammatory/profibrotic monocyte population recruited by AT2 mutant Sftpc expression in the development of fibrosis [Specific Aim 2], and to examine changes in epithelial-mesenchymal crosstalk that disrupt alveolar niche homeostasis and promote fibrotic remodeling [Specific Aim 3]. As epithelial dysfunction has not been studied extensively in vivo in the context of relevant preclinical models of fibrotic lung disease, this approach offers the unique opportunity to provide proof of concept both for the causal effect of mutant SFTPC in familial ILD and for the role of AT2 dysfunction as a key upstream driver of inflammatory cell and fibroblast activities that promote parenchymal remodeling. Importantly, mechanisms identified using a focused approach with these Sftpc models can be cross-purposed to better understand the pathogenesis of sporadic forms of ILD.