O39 IL22 producing iNKT and CD4+ T-cells drive dysregulation of the extracellular matrix in perianal fistulas

Abstract

<h3>Introduction</h3> Perianal fistulas are a common complication of Crohn’s disease (CD) affecting approximately 25% of patients, often predicting a more complicated disease course. Dysregulated immune responses and epithelial-to-mesenchymal transition (EMT) have been implicated in fistulizing disease; however, they have been poorly studied. In this study, we investigated the immune phenotype of patients with perianal fistulizing disease and its relationship with tissue remodelling. <h3>Methods</h3> Immune cells were isolated from fistula curettage samples (n=31) and paired peripheral blood from patients with perianal Crohn’s (pCD) or idiopathic fistulizing disease. Multiparameter flow cytometry was performed to evaluate lymphocyte populations including invariant natural killer T-cells (iNKTs), gamma-delta (γδ) T-cells, CD161⁺ mucosal-associated invariant T-cells (MAIT), CD4⁺ T-cells and CD8⁺ T-cells. Gene expression profiling of fistula (pCD n=11, idiopathic n=11)) and rectal biopsies (pCD n=9, idiopathic n=9)) was performed by RNA-sequencing. Cytokine treated intestinal epithelial organoids were used to probe the impact of selective cytokines on disease relevant pathways. <h3>Results</h3> iNKTs and CD4⁺ T-cells were expanded in perianal fistulas compared to peripheral blood. Deeper analysis of the phenotype of these populations revealed enrichment of CD8^- CD4^- CD161⁺ iNKTs producing IL22, and CD4⁺ CD161⁺ T-cells producing IL13 and IL22. Interestingly, pCD and idiopathic fistulas displayed similar immunophenotypes. Fistulas exhibited distinct transcriptional profiles to rectal tissue, although the phenotype of pCD and idiopathic fistulas appeared similar. Pathways related to the extracellular matrix (ECM) and EMT were more activated in fistulas compared to the rectal mucosa. IL13, IL22 and TNFα responsive transcripts were enriched in fistula tissue, and in the case of IL22, was shown to regulate key matrisome components. <h3>Conclusions</h3> Perianal fistulas are characterised by increased infiltration of CD161⁺ iNKT cells and CD4⁺ T-cells, producing IL22 and IL13. These pro-inflammatory cytokines are likely important drivers of ECM dysregulation and tissue remodelling in perianal fistulizing disease and targeting of this axis may represent a novel target in the treatment of fistulizing disease.