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，目前还没有针对肠道纤维化的CD治疗方法。我们之前已经证明，当我们将人类肠道有机物质(盘中迷你肠道，HIO)与一种胶状材料(水凝胶)相结合时，我们可以在实验室中研究肠道纤维化。在这里，我们假设通过调节我们的水凝胶来模拟僵硬的、患病的CD基质，我们可以使用HIO来更好地了解纤维化如何促进肠上皮中的CD，并确定解决或预防患者纤维化的新方法。CD的黄金标准抗炎治疗帮助了三分之二的患者，但它们无法预防或逆转纤维化。我们的目标是通过明确关注纤维化基质来确定治疗CD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中包围肠道的纤维基质的硬度和组成的变化如何导致疾病。通过解开这些相互作用，我们希望通过确定靶向基质以逆转或预防纤维化的方法来拓宽治疗CD的治疗策略。为了实现这一点，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的人类肠道组织的硬度，并将HIO包裹在模拟这些和其他物理线索的水凝胶中。然后，我们将分析封装的HIO，以了解像僵硬这样的物理线索如何影响HIO上皮内的信号通路，以及水凝胶僵硬和可降解性是否单独可以促使HIO在其周围形成纤维样基质。目标2：确定矩阵组线索是否在驱动CD样上皮表型中发挥作用我们将使用质谱学来描述正常和纤维化的人类肠道组织的组成(基质组)，以确定在疾病组织中更丰富的蛋白质。我们将使用生物工程策略将HIO包裹在水凝胶中，以结合或隔离成纤维的基质成分，然后分析HIO以确定病变基质中的特定蛋白质是否影响HIO，或者病变基质的组成是否促使HIO使其局部环境变得僵硬。目的3：确定紊乱的基质是否影响上皮细胞的愈合我们将在HIO中诱导损伤，并使用目标1和2中的类似僵硬和基质的水凝胶来研究CD样僵硬或基质成分是否影响肠道愈合，以及这是否通过特定的信号通路或HIO分泌的蛋白质来调节。我们还将使用我们的模型来测试针对基质的现有药物，以确定它们是否可以促进肠道愈合。潜在的应用和好处：减轻CD患者炎症的黄金标准治疗仅使三分之二的患者受益，无法逆转或预防肠道纤维化。尽管如此，CD的大多数研究都集中在缓解炎症以促进愈合。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，从组织水平上看待CD和肠道愈合。在这个项目中，我们的目标是通过关注纤维化的基质来发现治疗CD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.