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

摘要 纤维化肺重塑代表了各种间质性肺损伤的最终共同呼吸衰竭途径。 儿童和成人的肺部疾病（ILD）。基于最近的范式转变， 上皮细胞功能障碍和异常伤口愈合被假定为肺纤维化的“驱动因素”， ILD治疗发现的机会正在出现。表面活性蛋白C（SP-C）突变 在散发性和家族性ILD中发现了一种肺泡2型细胞（AT 2）限制性蛋白基因[SFTPC 为了解上皮细胞功能障碍在ILD发病中的作用提供重要线索。 我们实验室先前的体外研究表明，在患有糖尿病的成人和儿童中描述的SFTPC突变， ILD导致产生采用非天然构象的异常SP-C前蛋白同种型， 至少2种结局：ER应激和细胞内聚集（BRICHOS）或误作用于非天然 细胞器和大自噬抑制（非BRICHOS）。本申请建议利用两个 新的自发性肺纤维化的基因敲入小鼠模型已经在手，其表达非BRICHOS或 BRICHOS临床SFTPC突变体在AT 2细胞中的等位基因和诱导方式。我们的出版物和 初步数据显示，非BRICHOS突变体（SP-CI 73 T）或BRICHOS突变体（SP-CI 73 T）的表达均与BRICHOS突变体（SP-CI 73 T）的表达相关。 CC 121 G）在体内对肺具有极强的毒性，每种都导致时间依赖性自发性肺纤维化 由3个阶段标记：ii）早期AT 2细胞因子产生和单核细胞募集;（ii）多细胞肺泡炎 和肺损伤;（iii）生理限制性外周纤维化重塑。这些模型还阐述了 在人ILD中报告的与疾病相关的生物标志物。为了定义共识和分歧 将这两种AT 2细胞表型与下游肺损伤和肺纤维化联系起来的分子机制 重塑，我们的实验方法将是利用这些遗传模型的独特功能相结合 我们的项目提供了工具试剂和专业知识。在三个具体目标中，我们建议全面 表征由非BRICHOS表达诱发的转录组和功能性AT 2细胞表型 （SP-CI 73 T）和BRICHOS（SP-CC 121 G）Sftpc突变体[特异性目的1]，以确定关键的 在发育过程中通过AT 2突变体Sftpc表达募集的促炎/促纤维化单核细胞群 纤维化[具体目标2]，并检查上皮-间质串扰的变化， 生态位稳态和促进纤维化重塑[具体目标3]。由于上皮功能障碍尚未得到 在纤维化肺疾病的相关临床前模型的背景下进行了广泛的体内研究， 提供了一个独特的机会，为突变型SFTPC在家族性疾病中的因果作用提供了概念证明。 ILD以及AT 2功能障碍作为炎症细胞和成纤维细胞活性的关键上游驱动因素的作用 促进实质重塑重要的是，采用重点突出的方法确定的机制， 这些Sftpc模型可以交叉使用以更好地理解散发形式ILD的发病机理。

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.