TL1A-activated Tcells as upstream regulators of perianal fistulizing disease-associated changes in the rectum of Crohn's Disease patients [scRNA-seq]

Single-cell RNA sequencing revealed the increased abundance of some cell types, such as intestinal fibroblasts, in the CD-PFD rectal mucosa relative to CD. Within the stromal subset, the lamina propria S1 fibroblasts showed the highest number of differentially expressed genes (DEGs) between CD and CD-PFD patients, including an upregulation of TFPI2 and a downregulation of the TGFβ-responding gene CTGF. These changes were validated by bulk RNA analysis, where we also observed overexpression of the previously described PFD target genes MMP1 and MMP3. In vitro studies with intestinal fibroblasts confirmed the negative regulation by TGFβ of the PFD-signature, thus ruling out TGFβ as a driver of the PFD-associated fibroblast signature. Next, we investigated DEGs in the lymphoid compartment and identified lymphotoxin beta (LTB) as being upregulated in CD-PFD intestinal CD4+ T cells. LTB interacts with its receptor LTBR, which is expressed in stromal, epithelial and myeloid cells. Interestingly, LTB-LTBR interactions were increased in the CD-PFD group. In vitro studies in primary fibroblasts showed that LTB upregulates TFPI2, while it downregulates CTGF, thus reproducing the PFD-associated signature. Finally, we submitted the top 50 genes overexpressed by CD-PFD CD4+ T cells to the immune dictionary1, which identified TL1A as its most likely driver. In line with this, the TL1A receptor TNFRSF25 was overexpressed in CD-PFD CD4+ T cells. As a link between TL1A and LTB is not described in the literature, we stimulated anti-CD3-activated peripheral T cells with TL1A and observed the significant upregulation of LTa1b2, the functional form of LTB. Intriguingly, we also observed that while TNF can drive the PFD-associated signature in fibroblasts, it cannot induce LTa1b2 in T cells, suggesting that TNF and TL1A/LTB signalling are two independent but potentially complementary pathways in PFD. In summary, we propose a previously unrecognized TL1A-LTB axis as being responsible for inducing PFD-associated alterations in the rectal mucosa, and we hypothesise that inhibition of TL1A might be worth exploring in the context of perianal disease.