Deconstructing the fibrotic microenvironment in Crohn's disease to promote tissue healing

研究背景：超过一半的克罗恩病（CD）患者在肠道周围形成疤痕（纤维化），这需要改变生活的手术。然而，目前还没有针对肠纤维化的乳糜泻治疗方法。我们之前已经证明，当我们将人类肠道类器官（微型肠道培养皿，HIO）与一种果冻状材料（水凝胶）结合在一起时，我们可以在实验室中研究肠道纤维化。水凝胶可以模拟发生纤维化的肠道周围组织（基质）。在这里，我们假设通过调节我们的水凝胶来模拟僵硬的、病变的CD基质，我们可以使用HIO来更好地了解纤维化如何在肠上皮中促进CD，并确定解决或预防患者纤维化的新方法。黄金标准的抗炎治疗对2/3的乳糜泻患者有帮助，但它们不能预防或逆转纤维化。我们的目标是通过明确关注纤维化基质来确定治疗乳糜泻的新方法。目的和目的：我们的目标是使用HIO和合成水凝胶来了解CD患者肠道周围纤维化基质的硬度和组成的变化是如何导致疾病的。通过解开这些相互作用，我们希望通过确定靶向基质逆转或预防纤维化的方法来拓宽治疗CD的治疗策略。为了实现这一目标，我们将：目标1：确定物理线索是否在驱动cd样上皮表型中发挥作用。我们将使用原子力显微镜测量正常和纤维化人类肠道组织的硬度，并将HIO封装在模拟这些和其他物理提示的水凝胶中。然后，我们将分析封装的HIO，以了解硬度等物理线索如何影响HIO上皮内的信号通路，以及水凝胶硬度和可降解性是否能单独促使HIO在自身周围形成纤维样基质。目的2：确定基质提示是否在驱动cd样上皮表型中发挥作用我们将使用质谱分析正常和纤维化人类肠道组织的组成（基质），以确定病变组织中更丰富的蛋白质。我们将把HIO封装在水凝胶中，水凝胶使用生物工程策略合并或隔离纤维化基质成分，然后分析HIO以确定病变基质中的特定蛋白质是否会影响HIO，或者病变基质的组成是否会促使HIO硬化其局部环境。目的3：确定失调的基质是否影响上皮愈合我们将诱导HIO损伤，并使用目的1和2中的硬度和基质模拟水凝胶来研究cd样硬度或基质成分是否影响肠道愈合，以及这是否由HIO分泌的特定信号通路或蛋白质介导。我们还将使用我们的模型来测试现有的靶向基质的药物，以确定它们是否能促进肠道愈合。潜在的应用和益处：金标准治疗减轻了乳糜泻患者的炎症，仅使2/3的患者受益，并且不能逆转或预防肠纤维化。尽管如此，大多数关于乳糜泻的研究都集中在减轻炎症以促进愈合上。我们的研究方法旨在通过关注上皮和基质之间的相互作用，从组织水平的角度研究乳糜泻和肠道愈合。在这个项目中，我们的目标是通过关注纤维化基质来发现治疗乳糜泻的新方法。

Research context: Over half of Crohn's disease (CD) patients develop scarring (fibrosis) around the intestine which requires life-altering surgery. However, there are currently no treatments for CD that target intestinal fibrosis. We have previously shown that when we combine human intestinal organoids (mini-intestines in-a-dish, HIO) with a jelly-like material (hydrogel) that mimics the tissue surrounding the gut where fibrosis takes place (matrix), we can study gut fibrosis in the lab. Here, we hypothesise that by modulating our hydrogels to mimic the stiff, diseased CD matrix, we can use HIO to better understand how fibrosis contributes to CD in the intestinal epithelium, and identify new ways to resolve or prevent fibrosis in patients. Gold-standard anti-inflammatory treatments for CD help 2/3 of patients, but they do not prevent or reverse fibrosis. Our goal is to identify new ways to treat CD by explicitly focusing on the fibrotic matrix. Aims and objectives: We aim to use HIO and synthetic hydrogels to understand how changes in the stiffness and composition of the fibrotic matrix that surrounds the gut in CD contribute to disease. By untangling these interactions, we hope to broaden therapeutic strategies for treating CD by identifying ways to target the matrix to reverse or prevent fibrosis. To accomplish this, we will:Objective 1: Determine if physical cues play a role in driving CD-like epithelial phenotypes. We will measure the stiffness of normal and fibrotic human intestinal tissue using atomic force microscopy, and encapsulate HIO within hydrogels that mimic these and other physical cues. We will then analyse encapsulated HIO to understand how physical cues like stiffness impact signaling pathways within the HIO epithelium, and whether hydrogel stiffness and degradability alone can prompt HIO to form fibrotic-like matrix around themselves.Objective 2: Determine if matrisome cues play a role driving CD-like epithelial phenotypesWe will use mass spectrometry to profile the composition (matrisome) of normal and fibrotic human intestinal tissue to identify proteins that are more abundant in diseased tissue. We will encapsulate HIO within hydrogels that use bioengineering strategies to incorporate or sequester fibrotic matrix components, and then analyse HIO to determine if specific proteins in the diseased matrix impact HIO or if the composition of the diseased matrix prompts HIO to stiffen their local surroundings. Objective 3: Determine whether the dysregulated matrix impacts epithelial healingWe will induce damage in HIO and use stiffness- and matrix-mimicking hydrogels from Objectives 1&2 to ask if CD-like stiffness or matrix composition impact intestinal healing, and whether this is mediated by specific signalling pathways or proteins secreted by HIO. We will also use our models to test existing drugs that target the matrix to determine if they can promote intestinal healing.Potential applications and benefits: Gold-standard treatments that alleviate inflammation in CD patients only benefit 2/3 of patients and cannot reverse or prevent intestinal fibrosis. Despite this, most research in CD focuses on moderating inflammation to promote healing. Our research approach aims to take a tissue-level perspective on CD and intestinal healing by focussing on reciprocal interactions between the epithelium and the matrix. In this project, we aim to discover new ways to treat CD by focusing on the fibrotic matrix.