Uncovering the role of TL1A/LTα1β2 signalling in perianal fistulising Crohn’s disease

Abstract Background and aims Over 20% of Crohn’s disease (CD) patients will develop perianal fistulising disease (PFD), for which no specific treatments currently exist. Mounting evidence suggests that extracellular matrix (ECM) remodelling is required for fistula formation, and we have recently observed that lymphocyte activation by the tumour necrosis factor-like ligand 1A (TL1A) can promote the remodelling of the rectum mesenchyme in CD patients with PFD through the induction of lymphotoxin beta (LTα1β2). This project seeks to characterise the response to TL1A in a tissue- and disease-specific manner, and to functionally understand PFD-associated stromal rewiring in the context of fistula formation. Methods A cohort of IBD patients with no risk (ulcerative colitis) and high risk (CD) of developing PFD, as well as patients with established PFD (CD+PFD), will be included to measure the response to TL1A at baseline and after TL1A stimulation in T cells isolated from the rectal mucosa. Moreover, the crosstalk between T cells and intestinal fibroblasts upon TL1A stimulation will be assessed by split-pool combinational barcoding single-cell RNA sequencing in a co-culture system. Finally, we will address whether PFD-associated fibroblasts undergo metabolic reprogramming to promote ECM degradation and softening of the matrix. To this end, the mitochondrial activity of primary intestinal fibroblasts stimulated with relevant PFD cytokines (LTα1β2, TNF and TGFβ) will be measured using the Seahorse assay, while the ability of those fibroblasts to alter the stiffness of the ECM in culture will be evaluated with atomic force microscopy (AFM). Anticipated impact By exploring the use of metabolic assays and AFM to mechanistically interrogate the function of fibroblasts in vitro, data derived from this project will shed some light on the cellular mechanisms underlying PFD. Moreover, by thoroughly assessing the T cell-specific response to TL1A, we will evaluate the therapeutic value of TL1A inhibitors for CD+PFD patients.