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

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.

Automated summary

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.