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

Lead Research Organisation: King's College London


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 phenotypes
We 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 healing
We 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.

Technical Summary

More than half of Crohn's disease (CD) patients develop life-threatening fibrotic gut strictures; however, gold-standard treatments that alleviate inflammation do not resolve fibrosis nor alter the progression of stricturing disease. This suggests that intestinal fibrosis is auto-propagative and likely driven by pathological changes to the composition/stiffness of the tissue, but there are currently no treatments for CD that target the intestinal matrix. Our work combining hiPSC-derived intestinal organoids (HIO), synthetic hydrogels, and immune cells established a novel model to study gut pathological matrix remodelling in vitro. Here, we hypothesise that by encapsulating HIO within hydrogels that mimic the dysregulated matrix in stricturing CD, which is marked by its aberrant composition and stiffness, we can unpick its contribution to CD-like disease signatures in the epithelium, and that healing can be promoted by tuning the mesenchyme's stiffness and matrisome.

Using strictured and normal human intestinal tissue, we will perform mass spectrometry to profile the matrisome and atomic force microscopy (AFM) to measure stiffness. We will then encapsulate HIO within synthetic hydrogels that match the stiffness of strictured tissue and that incorporate CD-enriched matrisome cues and monitor their impact on HIO epithelia. Promising findings will be pursued using RNAseq, knockout/overexpression, pSILAC, and AFM measurements of local matrix stiffening. Together, this will allow us to identify potential signaling pathways impacted by physical/matrisome cues, and whether they prompt further aberrant matrix synthesis. Our approaches will also identify whether HIO response is mediated through specific matrisome components or cell-mediated changes in local stiffness. Finally, we will use an IFNg-induced model of HIO damage to ask how physical/matrisome cues impact epithelial healing, and screen small molecules/antibodies that target the matrix to promote regeneration.


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