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

肺纤维化中的肺泡上皮细胞功能障碍：利用 SFTPC 突变发现分子和细胞靶点

• 批准号: 10165806
• 负责人: MICHAEL FRANCIS BEERS
• 金额: $ 55.14万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10165806
• 关键词: Adopted; Adult; Affect; Alleles; Alveolar; Alveolitis; Appearance; Architecture; Biological Markers; CCL2 gene; Cell Communication; Cells; Cessation of life; Child; Cicatrix; Clinical; Codon Nucleotides; Complex; Complication; Consensus; Data; Development; Distal; Epithelial; Epithelial Cells; Event; Evolution; Fibroblasts; Fibrosis; Functional disorder; Funding; Gases; Gene Expression; Genes; Genetic; Genetic Models; Genetic Transcription; Growth; Hand; Homeostasis; Human; In Vitro; Inflammatory; Injury; Interstitial Lung Diseases; Kinetics; Knock-in Mouse; Ligands; Link; Lung; Lung diseases; Mediator of activation protein; Medical; Mesenchymal; Modeling; Molecular; Molecular Conformation; Molecular Target; Mus; Mutation; Organelles; Organoids; Outcome; Paracrine Communication; Pathogenesis; Pathogenicity; Pathology; Pathway interactions; Peripheral; Phase; Phenotype; Physiological; Population; Pre-Clinical Model; Process; Production; Protein Isoforms; Proteins; Publishing; Pulmonary Fibrosis; Pulmonary Surfactant-Associated Protein C; Reagent; Reporting; Respiratory Failure; Role; Signal Pathway; Signal Transduction; Testing; Therapeutic; Time; Tissues; alveolar epithelium; base; cellular targeting; cysteinylglycine; cytokine; endoplasmic reticulum stress; genetic variant; in vitro Model; in vivo; insight; lung injury; monocyte; mouse model; multimodality; mutant; non-Native; novel; older patient; progenitor; programs; receptor; recruit; response; stem cell function; stem cells; tool; transcriptomics; wound healing; young adult

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.

抽象的 纤维化肺重塑代表了多种间质性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，因此出现了以下概念： 上皮细胞功能障碍和伤口愈合异常被认为是肺纤维化的“驱动因素”，新研究 表面活性蛋白 C [SP-C] 突变的治疗机会正在出现。 基因 [SFTPC] 是一种肺泡 2 型细胞 (AT2) 限制蛋白，已在散发性和家族性 ILD 中发现 可为理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。 我们实验室之前的体外研究表明，成人和儿童中都存在 SFTPC 突变。 ILD 导致产生异常的 SP-C 蛋白原亚型，采用非天然构象，从而导致 至少 2 个结果：内质网应激和细胞内聚集 (BRICHOS) 或误流入非本地 细胞器和巨自噬的抑制（非 BRICHOS） 该应用建议利用两个。 新型自发性肺纤维化敲入小鼠模型已在手，其表达非 BRICHOS 或 AT2 细胞中的 BRICHOS 临床 SFTPC 突变体以等位基因和诱导方式出现。 初步数据表明，非 BRICHOS 突变体 (SP-CI73T) 或 BRICHOS 突变体 (SP- CC121G）在体内对肺具有极大的毒性，每种都会导致时间依赖性自发性肺纤维化 分为 3 个阶段：ii) 早期 AT2 细胞因子形成和单核细胞募集；(ii) 多细胞肺泡炎 和肺损伤；（iii）生理限制性外周纤维化重塑。 人类 ILD 中报道的翻译相关生物标志物，以定义共识和分歧。 将这两种 AT2 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组合的独特特征 借助我们计划中可用的工具、试剂和专业知识，我们建议全面实现 3 个具体目标。 表征非 BRICHOS 表达引起的转录组和功能 AT2 细胞表型 (SP-CI73T) 和 BRICHOS (SP-CC121G) Sftpc 突变体 [具体目标 1]，定义密钥的作用 发育过程中 AT2 突变体 Sftpc 表达招募的促炎/促纤维化单核细胞群 纤维化 [具体目标 2]，并检查破坏肺泡的上皮-间质串扰的变化 生态位稳态并促进纤维化重塑[具体目标 3]。 主要在纤维化肺病的相关临床前模型的背景下进行体内研究，这种方法 提供了独特的机会来证明突变 SFTPC 在家族中的因果效应 ILD 以及 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 重要的是，使用有针对性的方法确定了促进实质重塑的机制。 这些 Sftpc 模型可以交叉使用，以更好地了解散发性 ILD 的发病机制。