Society: DDW

Background: Perianal fistulae are highly morbid complications of Crohn’s disease (CD) with a cumulative incidence of 20-25%; despite demonstrated efficacy of infliximab treatment, estimates of complete resolution are only 20-30%. Ultimately, 20% of patients will require proctectomy. Across adult and pediatric populations, black CD patients are more likely to have colon-only CD, perianal fistula (OR 1.7-2.47, RR: 2.63) and progression to complex disease.
Methods: We performed single cell RNA sequencing (scRNAseq) of paired rectum and colon biopsies taken from equal African and European ancestry patients (n=14, 130,961 cells) with biopsies taken from two patients undergoing complete proctectomy, allowing for direct fistulous tract biopsies; single-nucleus RNAseq + ATAC (10X Multiome) was executed on a subset of n=6 patients (99,085 nuclei). A parallel cohort of healthy donors (n=8) and CD patients (n=11) with equal ancestry representation was recruited for peripheral blood mononuclear cell studies. We isolated CD14+ monocytes and differentiated sans microbial stimuli for 14 days; image analysis, quantitative Reverse Transcription-PCR, and Luminex immunoassays were performed.
Results: Myeloid and stromal cells were subset and re-clustered with the same cell types from the entire cohort of perianal fistula patients; n=20,704 cells were distributed among n=14 granular clusters. CHI3L1 was a top differentially expressed gene (DEG) (fold change=3.51, p-adj<1E-300) between stromal cells of fistula vs. colonic and rectal tissue origin. CHI3L1hi fibroblasts from fistulous tracts disproportionately expressed a destructive gene module (e.g. matrix metalloproteinases) and markers of macrophage activation. After two weeks’ differentiation in culture, isolated CD14+ monocytes differentially assumed a spindle-like morphology (p<0.001), expressed CHI3L1 (p=0.0145), and secreted CHI3L1 (p<0.001) in CD patients of African ancestry compared to European ancestry. Single cell gene regulatory network analyses focused on IBD-associated transcription factors confirmed cell-specific transcription factor enrichment (e.g. GATA2 in mast cells) and a broad array of predicted transcription factors in fibroblasts, including AP-1 (FOS, JUN). Multiome ATAC analysis of the CHI3L1 promoter revealed n=33 footprints bound in peaks from fibroblasts, over 60% of which were transcription factors belonging to the AP-1 family.
Conclusions: We present the first scRNAseq analysis of direct ex-vivo fistula tracts and identify CHI3L1 expressing fibroblasts that expressed common myeloid and known fibroblast destructive gene modules. Epigenetic control of inflammatory memory by AP-1 (JUN, FOS; PMID: 34320411) in a chronic inflammation context represents a promising mechanism of sustained injury in fistula by aberrantly differentiated innate immune cells.

<b>Figure 1.</b> Experimental design and sample sizes of parallel single cell direct <i>ex-vivo</i> (a) and <i>in vitro</i> (b) studies.

Figure 1. Experimental design and sample sizes of parallel single cell direct ex-vivo (a) and in vitro (b) studies.

<b>Figure 2.</b> Multimodal analyses implicate <i>CHI3L1</i> and AP-1 epigenetic control in fibroblasts.<br /> (a) Hematoxylin and eosin stain of a cross section of a fistula tract sampled for this study. (b) Uniform manifold approximation and projection (UMAP) of subclustered myeloid and stromal cells from n=14 patients. (c) Enrichment of functional fibroblast gene modules across myeloid and stromal cell types. (d) Expression of <i>CHI3L1</i> relative to housekeeping after 14 days’ culture in African and European ancestry (AA, EA) CD patients and controls. (e) CHI3L1 protein measured in day 14 CD14+ cell culture supernatants from healthy controls, CD patients, and isolated fistula single cells cultured under the same conditions. (f) Bias corrected peaks in the promoter of <i>CHI3L1</i> with top predicted transcription factor motifs bound. (g) Aggregate footprint of genome wide <i>FOS</i> occupancy across cell categories (top: stromal, myeloid, fibroblast).

Figure 2. Multimodal analyses implicate CHI3L1 and AP-1 epigenetic control in fibroblasts.
(a) Hematoxylin and eosin stain of a cross section of a fistula tract sampled for this study. (b) Uniform manifold approximation and projection (UMAP) of subclustered myeloid and stromal cells from n=14 patients. (c) Enrichment of functional fibroblast gene modules across myeloid and stromal cell types. (d) Expression of CHI3L1 relative to housekeeping after 14 days’ culture in African and European ancestry (AA, EA) CD patients and controls. (e) CHI3L1 protein measured in day 14 CD14+ cell culture supernatants from healthy controls, CD patients, and isolated fistula single cells cultured under the same conditions. (f) Bias corrected peaks in the promoter of CHI3L1 with top predicted transcription factor motifs bound. (g) Aggregate footprint of genome wide FOS occupancy across cell categories (top: stromal, myeloid, fibroblast).

Neha Maskey

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