Journal Club-2021.07.30

P17 induces chemotaxis and differentiation of monocytes via MRGPRX2-mediated mast cell–line activation

Journal of allergy and clinical immunology, ~14.11 (IF)

Karthi Duraisamy, Kailash Singh, Mukesh Kumar, Benjamin Lefranc, Elsa Bonnafe, Michel Treilhou, Jerome Leprince and Billy K. C. Chow, (Hong Kong, China; and Rouen and Albi, France).

Abstract

Background: P17, a peptide isolated from Tetramorium bicarinatum ant venom, is known to induce an alternative phenotype of human monocyte–derived macrophages via activation of an unknown G protein–coupled receptor (GPCR).

Objective: We sought to investigate the mechanism of action and the immunomodulatory effects of P17 mediated through MRGPRX2 (Mas related G protein–coupled receptor X2).

Methods: To identify the GPCR for P17, we screened 314 GPCRs. Upon identification of MRGPRX2, a battery of in silico, in vitro, ex vivo, and in vivo assays along with the receptor mutation studies were performed. In
particular, to investigate the immunomodulatory actions, we used b-hexosaminidase release assay, cytokine releases, quantification of mRNA expression, cell migration and differentiation assays,
immunohistochemical labeling, hematoxylin and eosin, and immunofluorescence staining.

Results: P17 activated MRGPRX2 in a dose-dependent manner in b-arrestin recruitment assay. In LAD2 cells, P17 induced calcium and b-hexosaminidase release. Quercetin- and short hairpin RNA–mediated knockdown of MRGPRX2 reduced P17- evoked b-hexosaminidase release. In silico and in vitro mutagenesis studies showed that residue Lys8 of P17 formed a cation-p interaction with the Phe172 of MRGPRX2 and [Ala8
] P17 lost its activity partially. P17 activated LAD2 cells to recruit THP-1 and human monocytes in Transwell migration assay, whereas MRGPRX2-impaired LAD2 cells cannot. In addition, P17-treated LAD2 cells stimulated differentiation of THP-1 and human monocytes, as indicated by the enhanced expression of macrophage markers cluster of differentiation 11b and TNF-α by quantitative RT-PCR. Immunohistochemical and immunofluorescent staining suggested monocyte recruitment in mice ears injected with P17.

Conclusion: Our data provide novel structural information regarding the interaction of P17 with MRGPRX2 and intracellular pathways for its immunomodulatory action.

Supplementary datas:-