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

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.

Keywords: G-Protein Coupled Receptors; Mast Cells; Single Cell Sequencing; Ulcerative Colitis.

Figure 1.. Mast cell activation in inflamed and uninflamed UC tissues and genetic associations implicate MRGPRX2 and altered arrestin recruitment.

(A-B) Analysis of 401 UC bulk mRNASeq samples from the Mount Sinai Crohn’s and Colitis Registry (A) Bulk mRNA analysis shows upregulation of mast cell-specific tryptases, histamine, and carboxypeptidase in non-inflamed and inflamed sigmoid and rectum, respectively. (B) The carboxypeptidase enzyme-substrate, ADM, is more highly expressed in inflamed compared to uninflamed UC. The UC non-inflamed sigmoid and inflamed rectum comparisons reflect paired samples from the same patients. (C) Carboxypeptidase secretion from immune cells isolated from three, paired inflamed and uninflamed UC tissues. MRGPRX2 specific ligand (R)-ZINC3573 stimulation was performed for 90 minutes, and supernatants were analyzed with CPA3 ELISA. One, two, and three asterisks correspond to p-values less than 0.05, 0.01, and 0.001, respectively. (D) Quantile-quantile association plot of protein-altering GPCR alleles from Ashkenazi Jewish UC patients from an exome chip case-control cohort^,. (E) Model for ADM-CPA3-PAMP12-MRGPRX2 positive feedback inflammatory loop in inflamed colon. (F) Sequence motifs predicted to alter arrestin recruitment, with the 62Ser allele predicted to result in a gain of arrestin recruitment.

Figure 2.. Dose-response stimulation with synthetic and natural MRGPRX2 ligands shows that the serine allele enhances beta-arrestin recruitment compared to the asparagine allele

(A) Ligands and downstream signaling pathways of MRGPRX2. (B-C) pEC50 (B) and Emax (C) of MRGPRX2 ligands between Asn62 (red) and 62Ser (green) (D) Electroporation of Asn62 (red), 62Ser (green) MRGPRX2 or empty (blue) expression vectors with a β-Gal ProLink Peptide Tag was performed into CHO cells containing PathHunter β-Arrestin containing inactive beta-galactosidase. Beta-galactosidase luminescence reflects the recruitment of β-arrestin to MRGPRX2. (E) Difference in luminescence between serine and asparagine alleles. One, two, three and four asterisks correspond to p-values less than 0.05, 0.01, 0.001, and 0.0001.

Figure 3.. Dose-response stimulation shows enhancement of MRGPRX2 activation as measured by IP1 accumulation for the asparagine allele

(A) Stable transfectants of CHO cells were sorted and normalized for equivalent expression vector expression for Asn62 (red), 62Ser (green) or empty vector (blue). Stimulation of (R)-ZINC3573, PAMP-12, CSP-1, S-Zinc3573, and LL-37 was performed for 120 minutes and IP1 concentration was measured through a FRET based assay read on an HTRF plate reader. (B) Net change in IP1 between alleles. One, two, three and four asterisks correspond to p-values less than 0.05, 0.01, 0.001 and 0.0001.

Figure 4.. Genotype-differences in pERK expression in mast cells

(A-B) Representative blot of stable transfectants of human mast cells (HMCs) was performed for Asn62 and 62Ser. Stimulation of PAMP-12 was performed at 10 μM and 20 μM. Cell lysates were collected and pERK expression was quantified through western blot analysis. Immunoblotting of pERK (A), ERK (B) and loading control α/β tubulin (Figure S3 B-C). (C) Quantification of pERK of Asn62 (red) and 62Ser (green). All pERK expression levels were normalized to total ERK and α/β Tubulin. n = 3 independent replicates. *, p < 0.05 (Figure S3 D).

Figure 5.. Comparative gene expression in uninflamed and inflamed UC by immunohistochemistry and scRNAseq

(A) Co-localization of MRGPRX2 (purple) with tryptase (brown) in inflamed and uninflamed UC colon. Bar = 100 and 20 microns. (B) scRNASeq TSNE plot from four UC patients having active disease and a clear demarcation separating inflamed and uninflamed tissues. (C) Violin plots of selected transcripts, including mast cell proteases (CPA3, TPSAB1) and MRGPRX2 mRNA precursors (ADM, CAMP) of reported endogenous ligands (PAMP-12, LL-37, respectively).

Figure 6.. Mast cell genes demonstrate primarily induction of gene expression in inflamed UC tissues, with upstream regulators expressed primarily by T cell clusters.

(A) Heatmap of differentially expressed genes in the mast cell cluster of scRNAseq. (B) Circos plot of genes differentially expressed by mast cells between inflamed and uninflamed UC tissues. Shown are the mast cell genes differentially expressed by scRNASeq and regulated by the most significant predicted upstream regulators using Ingenuity Pathway Analysis package, namely TNF and IFNG. Red and green transcripts designate up- and down-regulated transcripts in inflamed and uninflamed tissues, respectively. (C) Heat-map of TNF and IFNG ligands in the composite UC scRNASeq dataset. (D) Violin plots of IRF1, PIM1, and PSMB8 from scRNASeq mast cell cluster of scRNAseq comparing inflamed and uninflamed UC. The expression level is shown as log2 normailized expression. (E) RT-PCR of IRF1, PIM1 and PSMB8 from LAD2 mast cells treated with TNF, IFNG or IL-13 treatment for 4 hours. One, two, three and four asterisks correspond to p-values less than 0.05, 0.01, 0.001 and 0.0001.

Figure 7.. Model for MRGPRX2 mediated, genotype-dependent differences in mast cell activation, contributing to UC inflammation.

T cell derived IFNG modulates mast cell gene expression. Model for ADM production from inflamed epithelial cells and activated fibroblasts from inflamed regions being proteolyzed by CPA3, resulting in a feedback loop of continued mast cell activation.