3 - MULTI-OMICS CHARACTERIZATION OF PERIANAL FISTULIZING CROHN'S DISEASE IDENTIFY PATHOGENESIS AND NOVEL THERAPEUTIC TARGETS

Introduction:  The management of perianal fistulizing CD (PCD) is a major clinical challenge, where its underlying etiology remains elusive.

Materials &

Methods: We recruited patients with 1) PCD (n = 24); 2) CD without perianal disease (NPCD; n = 10); 3) idiopathic or cryptoglandular perianal fistulas (IPF; n = 29). Biopsies were taken from the fistula tracts, openings of the fistulas, and rectal mucosa during examination under anesthesia or colonoscopy. CyTOF, single cell RNA-sequencing (scRNA-seq), spatial transcriptomics, immunohistochemistry (IHC), ex vivo cell stimulation, and integrated analysis of published scRNA-seq datasets were performed.

Results: CyTOF revealed a skewed mucosal immune landscape in PCD compared to NPCD and IPF. PCD expanded Th17 cells in the fistula tracts and IL17-producing CD8 T cells (Tc17) in the rectum. Altered exhaustion markers CD39 and CD127 were present in CD4 and CD8 T cells from PCD fistula tracts, fistula openings, and rectum. Regulatory B cells, with immunomodulatory function in the gut, were dramatically diminished in PCD compared to IPF. In addition, PCD fistula tracts, fistula openings, and rectum all exhibited substantially higher CD172+TREM1+ macrophages, which are associated with anti-TNF resistance in luminal CD. ScRNA-seq unraveled immune and non-immune cell compartments in PCD and IPF fistula tracts. PCD fistulas showed hyperactivated pathogenic pathways including interferon (IFN)G response, TNF signaling, and IL6-JAK-STAT3 in myeloid cells. Similarly, stromal cells from PCD fistulas exhibited higher activities in IFNG and TNF response, TNF signaling, and epithelial-mesenchymal transition (EMT). In addition to fistula tracts, luminal (rectal and ileal) cells from PCD patients also expressed greater levels of IFNG-responsive and EMT genes compared to those without perianal disease (Figure 1). Moreover, we showed that both fistula tracts and ileal mucosa from PCD patients harbored expanded IFNG+ Th17 cells with elevated expression of inflammatory mediators (Figure 2). Further analysis revealed cellular modules associated with anti-TNF therapy in PCD patients. The findings were independently validated using spatial transcriptomics, IHC, and ex vivo stimulation of PCD cells.

Conclusion: Using multi-omics analysis of perianal fistula tracts and luminal tissues from extensive patient cohorts, we revealed previously unknown immune, stromal, and epithelial cell landscapes of PCD. In addition to IL17 signaling, T cell exhaustion, pathogenic macrophages, and pro-inflammatory/remodeling fibroblasts, our data highlight the pathogenic role of hyperactivated IFNG signaling in both fistula tracts and luminal mucosa of patients with PCD. This study identified IFNG as a potential therapeutic target for PCD.