bREATH-EASy: UNDERSTANDING THE ROLE OF INFECTION & EXTRACELLULAR MATRIX IN IDIOPATHIC PULMONARY FIBROSIS USING STEM CELL DERIVED ALVEOLAR CELLS

在英国，1 / 4的人死于呼吸系统疾病。难以获得肺样本，无法培养肺细胞以及缺乏复制人类肺部疾病的动物模型，使我们对如何诊断和治疗肺部疾病的理解不足。特发性肺纤维化（IPF）就是这些问题的例证，这种疾病的快速瘢痕形成会降低肺功能，导致平均3年内死亡。IPF的病因不明，预后难以预测。需要更多地了解肺部疾病，包括IPF，以便改进诊断、预后和治疗。人类多能干细胞为建模和理解疾病提供了新的途径。然而，直到最近，还没有肺病的模型。我们最近发表的工作已经解决了这一需求，我们现在可以生产大量的肺泡细胞，这是受IPF影响的细胞类型之一。我们现在可以制作3D肺类器官，允许对复杂的生物功能和相互作用进行建模，并且可以利用这些技术来理解IPF。为此，我们组建了一个国际团队来解决3个相互关联的目标：1)生成IPF的hIPSC模型，以了解SFTPC突变如何影响AE2细胞的细胞表型和基因表达。目前还没有已知的突变导致IPF，然而表面活性剂蛋白C （SFTPC）的突变可导致遗传性IPF，也称为家族性IPF，但其机制尚不清楚。我们将通过对SFTPC突变患者进行皮肤活检，然后使用我们既定的方案将其皮肤细胞转化为肺细胞，从而生成家族性IPF的干细胞模型。接下来，我们将利用这些细胞研究该突变如何导致肺细胞损伤、纤维化，并寻找治疗ipf的新靶点。2)体外模拟呼吸道感染，了解感染对携带SFTPC突变的AE2细胞的影响。IPF患者有更多的呼吸道感染，每次感染的死亡率约为90%，细菌感染如何加重IPF尚不清楚。使用在Obj. 1中创建的SFTPC突变细胞，我们将使用我们的3D培养平台来生长肺类器官，然后用细菌或病毒感染它们。我们将创建一个响应感染的基因表达变化的概况，并将其与未感染的细胞进行比较。这将有助于了解呼吸道感染如何影响正常肺细胞功能，并重要地揭示感染加剧ipf的机制。3)ECM成分的模型变化，以了解ECM如何影响细胞表型。随着IPF的发展，将肺部连接在一起的支架细胞外基质（ECM）发生了巨大的变化，导致肺部变得僵硬，缺乏弹性，同时减少了氧气的吸收。ECM的变化如何影响肺细胞功能，以及肺细胞是否积极促进进一步的ECM变化尚未得到很好的表征。我们将通过在简单的3D凝胶中生长在Obj. 1中创建的细胞来发展我们对这一过程的理解，这将使我们能够模拟在IPF的早期和后期阶段ECM的变化。然后我们将评估这些变化如何影响细胞功能和基因表达。这将深入了解ECM如何影响肺细胞功能，并提供潜在的治疗靶点，旨在预防或逆转IPF中ECM的变化。这些目标将解决难以获得人类IPF疾病模型的问题，并为IPF中肺纤维化和感染的发病机制提供见解，为开发令人兴奋的新药物靶点以及疾病的诊断和预后标志物提供机会。重要的是，这个平台可以被修改来模拟其他纤维化肺部疾病，以及其他具有纤维化成分的疾病。

In the UK 1 in 4 people die from respiratory disease. Poor access to lung samples, an inability to culture lung cells and a lack of animal models that reproduce human lung disease has left us with an inadequate understanding of how to diagnose and treat lung disease. These issues are exemplified by idiopathic pulmonary fibrosis (IPF), a disease where rapid scarring reduces lung function leading to death within an average of 3 years. The cause of IPF is unknown and prognosis is difficult to predict. A greater knowledge of lung disease including IPF is needed so diagnosis, prognosis and treatment can be improved.Human pluripotent stem cells are providing new ways to model and understand disease. Until recently however, models for lung diseases were not available. Our recently-published work has addressed this need and we can now produce large numbers of lung alveoli cells, one of the cell types affected by IPF. We can now make 3D lung organoids allowing modelling of complex biological functions and interactions, and can utilise these technologies to understand IPF.To this end we have assembled an international team to address 3 interlinked objectives:1) Generate a hIPSC model of IPF to understand how SFTPC mutations affect cellular phenotype and gene expression in AE2 cells. There are no known mutations that cause of IPF, however mutations in surfactant protein C (SFTPC) can cause inherited IPF also known as familial IPF, the mechanism however remains elusive. We will generate a stem cell model of familial IPF by taking a skin biopsy from patients with a SFTPC mutation and then turning their skin cells into lung cells using our established protocol. We will then use the cells to study how the mutation causes lung cell damage, fibrosis and to identify new therapeutic targets to treat IPF.2) Model respiratory infection in-vitro to understand the effect of infection on AE2 cells carrying SFTPC mutation. IPF patients have more frequent respiratory infections and >90% mortality rate per infection, how bacterial infection exacerbates IPF is not well understood. Using the SFTPC mutant cells created in Obj. 1 we will use our 3D culture platform to grow lung organoids and then infect them with bacteria or viruses. We will create a profile of changes in gene expression in response to infection and compare this to uninfected cells. This will provide insight into how respiratory infection affects normal lung cell function and importantly deliver insight into the mechanism by which infection exacerbates IPF.3) Model change in ECM composition to understand how ECM impacts cell phenotype. As IPF progresses, the scaffold that holds the lungs together, the extracellular matrix (ECM), dramatically changes, causing the lungs to become stiff and less elastic as well as reducing oxygen absorption. How the changes in ECM affect lung cell function, and whether lung cells actively contribute to further ECM changes has not been well characterised. We will develop our understanding of this process by growing cells created in Obj. 1 in simple 3D gels that will allow us to model how the ECM changes during early and late stages of IPF. We will then assess how these changes affect cell functionality and gene expression. This will provide insight into how ECM influences lung cell function and importantly deliver potential therapeutic targets that aim to prevent or reverse changes to ECM in IPF. These objectives will address poor access to human IPF disease models, and provide insight to the pathogenesis of lung fibrosis and infection in IPF, offering the opportunity to develop exciting new drug targets as well as diagnostic and prognostic markers for the disease. Importantly, this platform could be modified to model other fibrotic lung diseases, and other diseases that have a fibrotic component.