Background: Mast cells are a major actor in the development of abdominal pain, a hallmark symptom of IBS. We recently showed that a local IgE production triggers mast cell activation leading to food-induced abdominal pain in mice. However, little is known about the phenotype of gut mast cells in health and IBS. Aim: Our study aims to characterize mast cells across colon layers in health and in patients with IBS using single cell transcriptomics. Methods: Five healthy volunteers were recruited by public advertisement (2M, 3F; 18-43 years). Nineteen participants with IBS meeting the ROME III criteria were recruited from the outpatient clinic of the Leuven University Hospitals (3M, 16F; 18-51 years) and classified into IBS-D, IBS-C, and IBS-M. Rectal biopsies were taken during proctoscopy. Resection tissue from three patients (2M, 1F; 53-68 years) undergoing hemicolectomy for colon carcinoma were collected and colon layers were dissected. Single cell suspensions were obtained by enzymatic digestion. Live immune cells were FACS-sorted and 10X Genomics libraries were prepared. 280903 cells from rectal biopsies and 62063 cells from colon were integrated at the CD45 level. Results: 4148 mast cells, identified based on TPSAB1 and KIT, were separated into five clusters (MC1-MC5) exhibiting a layer-specific distribution in the healthy colon and distinct cytokine, chemokine, protease, and transcription factor profiles. In the mucosa, MC1 are primed for immune cross-talk: they express HLA class I and II, co-stimulatory genes, and multiple cytokine receptors. MC4 and MC3 are connective tissue mast cells, enriched in the submucosa and muscularis, respectively. Both subsets express CMA1, HPGD, and CTSG as well as NTM, a neural adhesion molecule. MC2 has an intermediate phenotype, expresses quiescence markers, and has low cytokine levels. MC5 expresses APOE and is specific to the rectum. In IBS, mast cells clustered similarly to healthy, apart from IBS-M which had MC2 and MC5 cells but lacked MC1 and MC4. Moreover, we found 77 differentially expressed genes between IBS and healthy mast cells (log fold change>0.25, p<0.05), including targets potentially involved in pain development. We next investigated changes in rectal antibody-producing cells to evaluate a role for IgE-mediated mast cell activation in patients with IBS. Plasma cells and B cells were identified using SDC1 and CD19, respectively. Plasma cells were increased in IBS-D and IBS-C and B cells were increased in IBS-M. Interestingly, IGHE⁺ plasma cells and B cells were enriched in IBS compared to healthy. Conclusions: We have characterized mast cell populations in the human gut and identified five novel subsets with distinct putative functions and transcription factor expression. Moreover, our results confirm IgE-mediated mast cell activation as a mechanism involved in IBS development in patients.

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SINGLE CELL TRANSCRIPTOMICS REVEAL MAST CELL HETEROGENEITY IN THE HUMAN GUT IN HEALTH AND IBS

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