Ernie Chen1, Ling-Shiang Chuang1, Mamta Giri1, Nicole Villaverde1, Nai-Yun Hsu1, Ksenija Sabic1, Sari Joshowitz1, Kyle Gettler1, Shikha Nayar1, Zhi Chai2, Isaac L Alter2, Colleen C Chasteau1, Ujunwa M Korie1, Siarhei Dzedzik3, Tin Htwe Thin3, Aayushee Jain1, Arden Moscati1, Gerardus Bongers4, Richard H Duerr5, Mark S Silverberg6, Steven R Brant7, John D Rioux8, Inga Peter1, L Philip Schumm9, Talin Haritunians10, Dermot P McGovern10, Yuval Itan1, Judy H Cho11. 1. The Charles Bronfman Institute for Personalized Medicine, Icahn School of Medicine at Mount Sinai, New York; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York. 2. The Charles Bronfman Institute for Personalized Medicine, Icahn School of Medicine at Mount Sinai, New York. 3. Department of Pathology, Icahn School of Medicine at Mount Sinai, New York. 4. Precision Immunology Institute at the Icahn School of Medicine at Mount Sinai, New York. 5. Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh, Pittsburgh, Pennsylvania. 6. Zane Cohen Centre for Digestive Diseases, Division of Gastroenterology, Mount Sinai Hospital, University of Toronto, Ontario, Canada, Toronto, Ontario, Canada. 7. Crohns and Colitis Center of New Jersey, Division of Gastroenterology and Hepatology, Department of Medicine, Rutgers Robert Wood Johnson Medical School, New Brunswick, New Jersey. 8. Research Centre, Montreal Heart Institute, Montréal, Quebec, Canada; Faculty of Medicine, Université de Montréal, Montréal, Quebec, Canada. 9. Department of Health Sciences, University of Chicago, Chicago, Illinois. 10. F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, California. 11. The Charles Bronfman Institute for Personalized Medicine, Icahn School of Medicine at Mount Sinai, New York; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York. Electronic address: Judy.cho@mssm.edu.
Abstract
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 β-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 β-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.
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 β-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 β-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.
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