P061 The molecular landscape of perianal fistula in Crohn’s disease: opportunities for new therapeutic approaches

Abstract

Abstract Background Perianal Crohn’s disease (CD) is a debilitating condition, often refractory to medical therapy and requiring repetitive surgical interventions. Nonetheless, its pathophysiology is very poorly understood. Hence, we molecularly characterised the fistula tract and compared it to the molecular landscape of its inner rectal orifice. Methods We collected paired surgical biopsies from the fistula tract and the inner rectal fistula orifice in 29 CD patients with draining perianal fistula, requiring surgical examination under anaesthesia. RNA was extracted and single-end RNA sequencing performed using Illumina HiSeq4000. Sequencing data were analysed through differential gene expression (DESeq2) and corrected for the presence of proctitis. A false discovery rate of 0.001 was considered significant. In addition, cellular deconvolution methods (CIBERSORT) were applied to study the cellular composition of the fistula tract. Results Differential gene expression revealed 2701 transcripts being differentially expressed (1727 up, 974 down in fistula) between the fistula tract and the paired rectal mucosa. The top upregulated gene, LBP (fold change [FC]=2858.8, p = 3.6E−13), highlights the potential contribution of the microbiome. LBP has a central role in the innate immune system by binding to bacterial lipopolysaccharides (LPS) and facilitating the affinity between LPS and CD14, with the subsequent release of various cytokines. Several extracellular matrix proteins could be identified within the top 25 of upregulated genes, including MMP13 (FC = 358.8, p = 1.3E−11), ADAM12 (FC = 175.6, p = 1.4E−12), COL1A1 (FC = 77.1, p = 2.7E−10) and COL5A3 (FC = 32.1, p = 1.7E−12), emphasising the intense tissue remodelling going on in the fistula tract. Despite correcting for the confounding effect of proctitis, the fistula tract expressed higher levels of IL6 (FC = 133.1, p = 3.7E−9), TNF(FC = 14.2, p = 4.8E−5), OSM (FC = 24.3, p = 8.9E−5), IL12p40 (FC = 10.0, p = 8.1E−3), integrin α 4 (FC = 4.5, p = 9.3E−3), integrin β 7(FC = 3.8, p = 4.1E−3) but not IL23p19 (FC = 1.2, p = 0.9). Top downregulated genes were linked to the intestinal epithelium, including KRT19 (FC = −489.1, p = 5.5E−17), KRT8 (FC = −324.0, p = 1.1E−16), CEACAM6 (FC=−515.1, p = 4.5E−16) and MUC2 (FC=−795.4, p = 3.0E−15). Cellular deconvolution identified CD4 memory resting T cells (18.5%), M0 macrophages (17%), M2 macrophages (15.2%), neutrophils (9.2%) and plasma cells (7.5%) as the most abundant cells within the fistula tract. Conclusion We molecularly characterised the fistula tract in perianal CD and identified clear biological differences in comparison to the luminal tract, highlighting the potential of new therapeutic targets and cell types driving this debilitating condition.