Multimodal single-cell analyses reveal mechanisms of perianal fistula in diverse patients with Crohn's disease.

BACKGROUND: Crohn's disease complicated by perianal fistulae is more prevalent and severe in patients of African ancestry. METHODS: We profiled single cells from diverse patients with Crohn's disease with perianal fistula from colorectal mucosa and fistulous tracts. Immunofluorescence was performed to validate predicted cell-cell interactions. Unstimulated monocytes were chronically cultured in diverse cohorts. A subset was analyzed by single-nucleus RNA + ATAC sequencing. FINDINGS: Fistulous tract cells from complete proctectomies demonstrated enrichment of myeloid cells compared to paired rectal tissues. Ligand-receptor analysis highlights myeloid-stromal cross-talk and cellular senescence, with cellular co-localization validated by immunofluorescence. Chitinase-3 like-protein-1 (CHI3L1) is a top upregulated gene in stromal cells from fistulae expressing both destructive and fibrotic gene signatures. Monocyte cultures from patients of African ancestry and controls demonstrated differences in CHI3L1 and oncostatin M (OSM) expression upon differentiation compared to individuals of European ancestry. Activating protein-1 footprints are present in ATAC-seq peaks in stress response genes, including CHI3L1 and OSM; genome-wide chromatin accessibility including JUN footprints was observed, consistent with reported mechanisms of inflammatory memory. Regulon analyses confirm known cell-specific transcription factor regulation and implicate novel ones in fibroblast subsets. All pseudo-bulked clusters demonstrate enrichment of genetic loci, establishing multicellular contributions. In the most significant African American Crohn's genetic locus, upstream of prostaglandin E receptor 4, lymphoid-predominant ATAC-seq peaks were observed, with predicted RORC footprints. CONCLUSIONS: Population differences in myeloid-stromal cross-talk implicate fibrotic and destructive fibroblasts, senescence, epigenetic memory, and cell-specific enhancers in perianal fistula pathogenesis. The transcriptomic and epigenetic data provided here may guide optimization of promising mesenchymal stem cell therapies for perianal fistula. FUNDING: This work was supported by grants U01DK062422, U24DK062429, and R01DK123758.

Clinical predictors of early and late endoscopic recurrence following ileocolonic resection in Crohn's disease.

BACKGROUND AND AIM: Multiple factors are suggested to place Crohn's disease patients at risk of recurrence after ileocolic resection with conflicting associations. We aimed to identify clinical predictors of recurrence at first (early) and further (late) postoperative colonoscopy. METHODS: Crohn's disease patients undergoing ileocolic resection were prospectively recruited at six North American centers. Clinical data was collected and endoscopic recurrence was defined as Rutgeerts score ≥i2. A multivariable model was fitted to analyze variables independently associated with recurrence. RESULTS: A total of 365 patients undergoing 674 postoperative colonoscopies were included with a median age of 32 years, 189 (51.8%) were male and 37 (10.1%) non-Whites. Postoperatively, 133 (36.4%) used anti-TNF and 30 (8.2%) were smokers. At first colonoscopy, 109 (29.9%) had recurrence. Male gender (OR = 1.95, 95% CI 1.12 - 3.40), non-White ethnicity (OR = 2.48, 95% CI 1.09 - 5.63), longer interval between surgery and colonoscopy (OR = 1.09, 95% CI 1.002 - 1.18), and postoperative smoking (OR = 2.78, 95% CI 1.16 - 6.67) were associated with recurrence, while prophylactic anti-TNF reduced the risk (OR = 0.28, 95% CI 0.14 - 0.55). Postoperative anti-TNF prophylaxis had a protective effect on anti-TNF experienced patients but not on anti-TNF naïve patients. Among patients without recurrence at first colonoscopy, Rutgeerts score i1 was associated with subsequent recurrence (OR = 4.43, 95% CI 1.73 - 11.35). CONCLUSIONS: We identified independent clinical predictors of early and late Crohn's disease postoperative endoscopic recurrence. Clinical factors traditionally used for risk stratification failed to predict recurrence and need to be revised.

Inflamed Ulcerative Colitis Regions Associated With MRGPRX2-Mediated Mast Cell Degranulation and Cell Activation Modules, Defining a New Therapeutic Target.

BACKGROUND & AIMS: Recent literature has implicated a key role for mast cells in murine models of colonic inflammation, but their role in human ulcerative colitis (UC) is not well established. A major advance has been the identification of mrgprb2 (human orthologue, MRGPX2) as mediating IgE-independent mast cell activation. We sought to define mechanisms of mast cell activation and MRGPRX2 in human UC. METHODS: Colon tissues were collected from patients with UC for bulk RNA sequencing and lamina propria cells were isolated for MRGPRX2 activation studies and single-cell RNA sequencing. Genetic association of all protein-altering G-protein coupled receptor single-nucleotide polymorphism was performed in an Ashkenazi Jewish UC case-control cohort. Variants of MRGPRX2 were transfected into Chinese hamster ovary (CHO) and human mast cell (HMC) 1.1 cells to detect genotype-dependent effects on ß-arrestin recruitment, IP-1 accumulation, and phosphorylated extracellular signal-regulated kinase. RESULTS: Mast cell-specific mediators and adrenomedullin (proteolytic precursor of PAMP-12, an MRGPRX2 agonist) are up-regulated in inflamed compared to uninflamed UC. MRGPRX2 stimulation induces carboxypeptidase secretion from inflamed UC. Of all protein-altering GPCR alleles, a unique variant of MRGPRX2, Asn62Ser, was most associated with and was bioinformatically predicted to alter arrestin recruitment. We validated that the UC protective serine allele enhances ß-arrestin recruitment, decreases IP-1, and increases phosphorylated extracellular signal-regulated kinase with MRGPRX2 agonists. Single-cell RNA sequencing defines that adrenomedullin is expressed by activated fibroblasts and epithelial cells and that interferon gamma is a key upstream regulator of mast cell gene expression. CONCLUSION: Inflamed UC regions are distinguished by MRGPRX2-mediated activation of mast cells, with decreased activation observed with a UC-protective genetic variant. These results define cell modules of UC activation and a new therapeutic target.

Common and Rare Variant Prediction and Penetrance of IBD in a Large, Multi-ethnic, Health System-based Biobank Cohort.

BACKGROUND AND AIMS: Polygenic risk scores (PRS) may soon be used to predict inflammatory bowel disease (IBD) risk in prevention efforts. We leveraged exome-sequence and single nucleotide polymorphism (SNP) array data from 29,358 individuals in the multiethnic, randomly ascertained health system-based BioMe biobank to define effects of common and rare IBD variants on disease prediction and pathophysiology. METHODS: PRS were calculated from European, African American, and Ashkenazi Jewish (AJ) reference case-control studies, and a meta-GWAS run using all three association datasets. PRS were then combined using regression to assess which combination of scores best predicted IBD status in European, AJ, Hispanic, and African American cohorts in BioMe. Additionally, rare variants were assessed in genes associated with very early-onset IBD (VEO-IBD), by estimating genetic penetrance in each BioMe population. RESULTS: Combining risk scores based on association data from distinct ancestral populations improved IBD prediction for every population in BioMe and significantly improved prediction among European ancestry UK Biobank individuals. Lower predictive power for non-Europeans was observed, reflecting in part substantially lower African IBD case-control reference sizes. We replicated associations for two VEO-IBD genes, ADAM17 and LRBA, with high dominant model penetrance in BioMe. Autosomal recessive LRBA risk alleles are associated with severe, early-onset autoimmunity; we show that heterozygous carriage of an African-predominant LRBA protein-altering allele is associated with significantly decreased LRBA and CTLA-4 expression with T-cell activation. CONCLUSIONS: Greater genetic diversity in African populations improves prediction across populations, and generalizes some VEO-IBD genes. Increasing African American IBD case-collections should be prioritized to reduce health disparities and enhance pathophysiological insight.