Differential MRGPRX2-dependent activation of human mast cells by polymyxins and octapeptins

The emergence of multi-drug resistant Gram-negative bacteria has led to renewed interest in the antimicrobial activity of polymyxins and novel polymyxin analogues (e.g. nonapeptides and octapeptin). In some individuals, clinically used polymyxins can cause acute hypersensitivity reactions through mast cell activation, with a recent study attributing this effect to activation of the MAS-related G protein-coupled receptor X2 (MRGPRX2). In the present study, HEK293 cells expressing human MRGPRX2 and the human mast cell line LAD2 were used to characterize the activity of the broader family of polymyxins. Octapeptin C4, polymyxin B and colistin produced concentration-dependent calcium mobilization, degranulation, and CCL-2 (MCP-1) release in LAD2 mast cells, with the former being highly potent. CRISPR-Cas9 knockdown of MRGPRX2 in LAD2 cells and a MRGPRX2 inverse agonist caused a significant reduction in calcium mobilization, degranulation, and CCL-2 release, demonstrating dependency on MRGPRX2 expression. In contrast, polymyxin nonapeptides were far less potent calcium mobilisers and failed to induce functional degranulation in LAD2 cells. Our results confirm that activation of mast cells induced by polymyxin-related antibiotics is MRGPRX2-dependent and reveal that octapeptin C4 might be more liable, whilst nonapeptides are less liable, to trigger immediate hypersensitivity reactions clinically. The mechanism underpinning the difference in MRGPRX2 activation between polymyxin-related antibiotics is important to better understand as it may help design new, safer polymyxins and guide the optimal clinical use of existing polymyxin drugs.