Rna Silencing: A Model To Explore The Mrgprx2-Pathway In Cultured Human Mast Cells.

MRGPRX2 is a G-protein-coupled receptor that is expressed on human mast cells (hMC). Substance P MRGPRX2-pathway is a recent described IgE-independent activation route, such as off-target occupation through drugs. Unfortunately, there are no specific MRGPRX2 inhibitors allowing to deepen our insights in the MRGPRX2-activation pathway(s). Therefore, the aim of this study is to explore whether MRGPRX2-expression can be silenced via siRNA technology. Human MC were cultured out of CD34+ progenitor cells that were obtained from peripheral blood. MCs were electroporated, with the introduction of a 1000 nM mixture of two MRGPRX2-specific siRNA in a 1:1 ratio or with a negative control, using a Square Wave protocol (500V, 5ms 1 pulse). Five days after electroporation, cells were stimulated with the MRGPRX2 antagonist substance P and anti-FcεRI. For staining of intracellular calcium a Fluo-4 technique was applied. Degranulation were flow cytometric analysed by CD63-upregulation. Results are expressed as median decrease with range (n=3). After inserting MRGPRX2-specific siRNA, expression of MRGPRX2 decreases with 83% (76-97). This reduced expression resulted in a significant decrease of 75% (55-77) intracellular calcium staining and CD63 appearance of 95% (89-95) after stimulation with substance P for 20 minutes. In contrast, introduction of MRGPRX2-specific siRNA did not affect intracellular calcium staining (0% (0-11)) nor appearance of CD63 (0% (0-29)) in response to anti-FcεRI. Our siRNA model is an effective way to silence MRGPRX2-expression and functionality in cultured hMC. Therefore, our technique can be used to explore signaling in the MRGPRX2-pathway and to study pathogenic effects of off-target occupation.