Understanding how alveolar epithelial cell stress drives aberrant repair in interstitial lung disease

项目概要 间质性肺疾病（ILD）是一类病理学上以间质纤维化为特征的肺部疾病 和炎症。由于我们对发病机制上游启动子的了解有限，ILD 的研究进展缓慢。 预后和有限的治疗方法。 2 型肺泡上皮细胞 (AEC2) 限制性表面活性剂的突变 ILD 患者子集中的蛋白 C (SP-C) 基因 (SFTPC) 支持越来越多的假设，即 AEC2 功能障碍是疾病的驱动因素。当我们在体外模拟这些与疾病相关的 SFTPC 突变时，它们 根据突变的 SP-C 同工型如何对细胞通路施加压力，将其分为两大类 管理异常蛋白质：抑制巨自噬的突变和导致内质的突变 网状（ER）压力。然而，这些 AEC2 如何影响 ILD 中的表型？ 没有 SFTPC 突变的患者与 ILD 发展的关系仍知之甚少。 为了理解这种关系，我们生成了两种独特的 Sftpc 突变敲入小鼠模型，一种是 表达诱导 AEC2 巨自噬功能障碍 (SP-CI73T) 的 SP-C 亚型，以及其他诱导 内质网应激 (SP-CC121G)。这些突变中的每一个在成年小鼠肺中表达时都会导致自发的 肺泡炎和肺损伤，随后异常修复导致纤维化 ILD。这些模型因此提供了证据 AEC2 应激能够驱动自发性肺部病理学并且是稳健的临床前概念 平台。这些模型还支持 ILD 发病机制的第二种新兴理论：ILD AEC2 理论，该理论 必须通过增殖和分化来修复肺损伤后充当关键的兼性祖细胞 上皮细胞受损，祖细胞能力出现功能障碍。我们发现虽然相似的肺 我们的每个 Sftpc 模型中都有不同的病理学发展，因此存在不同的 AEC2 增殖表型 肺损伤：SP-CI73T AEC2 过度增殖，SP-CC121G AEC2 凋亡并 增殖不良。因此，我们的模型支持以下假设：异常的 AEC2 祖细胞功能在 在 ILD 发展中发挥核心作用，并创建一个平台来发展对如何进行机械理解 离散的 AEC2 应激特征导致祖细胞容量明显缺陷。 该提案具有三个相互关联的目标，旨在了解分子和细胞机制 将 AEC2 细胞应激、功能失调的祖细胞容量和 ILD 联系起来。目标 1 使用生物信息学和体内 谱系追踪来表征每个参与的 AEC2 应激信号和祖细胞功能途径 模型。目标 2 通过以下方式提供了细胞应激信号传导与祖细胞功能障碍之间的机制联系： 离体类器官培养和体内建模。在目标 3 中，我们将生成第一个 ILD 患者衍生的 iPSC ER应激相关SFTPC突变的培养模型作为研究途径的人源化平台 目标 1 和 2 中确定的。该提案还将为受训者提供多样化和全面的知识 开发关于上皮在 ILD 中的作用的多模式独立研究计划所需的培训。

PROJECT SUMMARY Interstitial lung diseases (ILDs) are a class of pulmonary diseases pathologically defined by interstitial fibrosis and inflammation. Owing to our limited understanding of the upstream initiators of pathogenesis, ILDs have poor prognoses and limited therapeutics. Mutations in the Alveolar Epithelial Type 2 Cell (AEC2) restricted Surfactant Protein C (SP-C) gene (SFTPC) in a subset of ILD patients supports a growing hypothesis that AEC2 dysfunction is a driver of disease. When we model these disease-related SFTPC mutations in vitro they segregate into two major classes based upon how the mutated SP-C isoform stresses cellular pathways that manage abnormal proteins: mutations that inhibit macroautophagy and mutations that cause endoplasmic reticulum (ER) stress. However, how these AEC2 stress phenotypes, which have also been identified in ILD patients without SFTPC mutations, relate to ILD development remains poorly understood. To understand this relationship, we have generated two unique Sftpc mutation knock-in mouse models, one expressing an SP-C isoform that induces AEC2 macroautophagy dysfunction (SP-CI73T) and the other inducing ER stress (SP-CC121G). Each of these mutations when expressed in the adult mouse lung results in spontaneous alveolitis and lung injury followed by aberrant repair with resultant fibrotic ILD. These models thus provide proof of concept that AEC2 stress is capable of driving spontaneous lung pathology and are robust preclinical platforms. These models also support a second emerging theory of ILD pathogenesis: that in ILD AEC2s, which must act as critical facultative progenitor cells after lung injury by both proliferating and differentiating to repair damaged epithelium, develop dysfunction in their progenitor cell capacity. We discovered that while similar lung pathology develops in each of our Sftpc models, there are divergent AEC2 proliferation phenotypes following lung injury: SP-CI73T AEC2s become hyperpoliferative and SP-CC121G AEC2s become apoptotic and hypoproliferative. Thus, our models support the hypothesis that abnormal AEC2 progenitor cell function plays a central role in ILD development, and also create a platform to develop a mechanistic understanding of how discrete AEC2 stress signatures result in distinct defects in progenitor cell capacity. This proposal has three interrelated aims that seek to understand the molecular and cellular mechanisms that relate AEC2 cell stress, dysfunctional progenitor cell capacity, and ILD. Aim 1 uses bioinformatics and in vivo linage-tracing to characterize the AEC2 stress signaling and progenitor function pathways involved in each model. Aim 2 provides a mechanistic link between cell stress signaling and progenitor cell dysfunction through ex vivo organoid culture and in vivo modeling. In Aim 3 we will generate the first ILD patient-derived iPSC culture model of an ER stress associated SFTPC mutation as a humanized platform to study the pathways identified in Aims 1 and 2. This proposal will also provided the trainee with the diverse and comprehensive training necessary to develop a multimodal independent research program on the role of the epithelium in ILD.