Backgrounds: Perianal fistulas occur in ~30-40% of Crohn’s disease (CD) patients associated with high morbidity and an impaired quality of life. The management of perianal fistulizing CD is a major clinical challenge, where its underlying etiology remains poorly understood.
Methods: We recruited patients with 1) CD with perianal fistulas (PCD; n = 12); 2) CD without perianal involvement (NPCD; n = 10); 3) idiopathic perianal fistulas (IPF; n = 21). Biopsies were taken from the fistula tracts, external opening of the fistulas, and rectal mucosa during examination under anesthesia or colonoscopy. Mucosal immune cells were analyzed using mass cytometry (or CyTOF), while all immune and stromal cells from PCD and IPF fistula tracts (n = 5 or 6) were characterized using single-cell RNA-sequencing (scRNA-seq).
Results: CyTOF revealed a skewed mucosal immune landscape in PCD. For example, PCD fistula tracts harbored elevated CD45RO+ T cells including Tregs with increased expression of TIGIT and CD226 compared to IPF. PCD also expanded Th17 cells in the fistula tracts and IL-17-producing CD8 T cells (Tc17) in the rectum. Altered exhaustion markers CD39 and CD127 were present in both CD4 and CD8 T cells from PCD fistula tracts, external fistula openings, and rectum compared to IPF and NPCD. Regulatory B cells (Bregs), with immunomodulatory function in the gut, were dramatically diminished in PCD compared to IPF. In addition, PCD fistula tracts, fistula opening, and rectum all exhibited substantially higher CD172+TREM1+ macrophages, which were previously found to promote luminal CD (Figure 1; only fistula tract data are shown). Using scRNA-seq, we unraveled immune and stromal cell compartments in PCD and IPF fistula tracts (Figure 2). We also identified a small mast cell population, which was diminished in PCD. Mast cells are involved in tissue repair and remodeling in other diseases, although their presence in perianal fistulas was previously unknown. Three fibroblast populations (cluster 4, 6, 7 in figure 2D), with altered activities in interferon signaling, IL-6-JAK-STAT3 pathway, TNF signaling, as well as TGF-B and epithelial-mesenchymal transition, were dramatically increased in PCD compared to IPF. Ongoing studies are focusing on spatial transcriptomics and functional analysis of fistula-associated immune cells and fibroblasts.
Conclusions: Using single cell analysis of the fistula tracts and nearby tissues from an extensive patient population, we revealed previously unknown immune and stromal cell landscapes of PCD. Our findings highlight the pathogenic roles of IL-17 signaling, T cell exhaustion, pathogenic macrophages, and pro-inflammatory/remodeling fibroblasts in the pathogenesis of PCD. This study may open new avenues for disease-specific therapies for this challenging condition.

CyTOF of perianal CD

scRNA-seq of perianal CD

Khai Nguyen

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