Mast Cell G-protein Coupled Receptors in Immune Modulation of colitis

Summary:
Ulcerative colitis (UC) is a subtype of inflammatory bowel disease with symptoms of abdominal pain
and bloody diarrhea secondary to colonic inflammation. Human colonic mast cells (MCs) express a novel G
protein-coupled receptor known as Mas-related GPCR-X2 (MRGPRX2, mouse ortholog, MrgprB2), and
RNAseq analysis of inflamed UC samples demonstrated enhanced expression of MRGPRX2 agonists and MC
proteases when compared to non-inflamed samples. It is well documented that GPCRs undergo
desensitization following their phosphorylation at Ser/Thr residues. Interestingly, analyses of human GPCR
protein-altering single nucleotide polymorphism (SNP) from exome chip data of inflammatory bowel disease
showed that N62S (rs10833049) mutation of MRGPRX2, which results in the creation of a phosphorylation site,
is associated with protective phenotype in UC. By contrast, we found that another SNP, in which a Ser is
replaced with Leu (S325L) results in reduced desensitization and greater degranulation than the WT receptor.
Based on these findings, we hypothesize that MRGPRX2 expressed in colonic MCs contributes to UC and that
its phosphorylation and desensitization modulates disease outcome. Given that MrgprB2 is the mouse
counterpart of human MRGPRX2, it is expected that MrgprB2−/− mice would display a reduced disease
phenotype in experimental models of UC when compared to WT mice. Surprisingly, however, recent studies
showed that MrgprB2 displays a protective effect in experimental UC in mice. This difference could reflect the
fact that MRGPRX2 and MrgprB2 display low sequence homology (~53%) and suggests that mice expressing
MrgprB2 may not adequately reflect the situation in humans. To overcome this limitation, we utilized
CRISPR/Cas9-mediated gene editing approach to replace MrgprB2 with human MRGPRX2 (MRGPRX2-KI
mice). We found that primary MCs from MRGPRX2-KI mice respond to agonists that are implicated in UC for
substantially greater degranulation at lower concentrations than primary MCs from WT mice expressing
MrgprB2. In aim 1, we will utilize MRGPRX2-KI, WT and MrgprB2-/- mice to test the hypothesis that, unlike
MrgprB2, MRGPRX2 expressed in colonic MCs contributes to the pathogenesis of UC. In aim 2, retrovirus will
be used to express MRGPRX2 and its phosphorylation variants N62S and S325L in MrgprB2−/− mouse bone-
derived mast cells, which will then be engrafted into MC-deficient mice. This strategy will be used to determine
how SNPs on MRGPRX2 modulate experimental UC. Successful completion of this study will lead to the
characterization of a new preclinical model of UC and may provide novel insights on how individual variations
in MRGPRX2 alters disease phenotype.

Grant Number: 1R21AI190597-01A1
NIH Institute/Center: NIH
Principal Investigator: Hydar Ali