Journal
JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY
Volume 149, Issue 1, Pages 275-291Publisher
MOSBY-ELSEVIER
DOI: 10.1016/j.jaci.2021.04.040
Keywords
Mast cell activation; innate immunity; immune modulation; MRGPRX2; mast cells; monocytes; human-monocyte-derived macrophages (h-MDMs); monocyte recruitment; chemotaxis; cytokine release
Categories
Funding
- HK government Research Grants Council (RGC) grant [GRF 1711320, 17111421]
- National Natural Science Foundation of China/RGC
- University of Hong Kong seed fund for basic research [201910159222]
- Institut National de la Sante et de la Recherche Medicale (Inserm)
- Normandy University (Rouen)
- Region Normandy
- European Union (PHEDERCPG project)
- European Regional Development Fund
- European Union (3R project)
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This study investigates the mechanism of action and immunomodulatory effects of P17 mediated through MRGPRX2. The results show that P17 activates MRGPRX2 in a dose-dependent manner and induces various immune responses, including cell migration and differentiation. These findings provide important insights into the molecular mechanisms of immune modulation.
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 beta-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 beta-arrestin recruitment assay. In LAD2 cells, P17 induced calcium and beta-hexosaminidase release. Quercetin- and short hairpin RNA-mediated knockdown of MRGPRX2 reduced P17-evoked beta-hexosaminidase release. In silico and in vitro mutagenesis studies showed that residue Lys(8) of P17 formed a cation-pi interaction with the Phe(172) of MRGPRX2 and [Ala(8)] 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-alpha by quantitative RT-PCR. Immunohistochemical and immunofluorescent staining suggested monocyte recruitment in mice ears injected with P17. Conclusions: Our data provide novel structural information regarding the interaction of P17 with MRGPRX2 and intracellular pathways for its immunomodulatory action.
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