D1.430 - Extracellular vesicles isolated after cholinergic urticaria challenge carry MRGPRX2 and induce pro-inflammatory mast cell responses

Mast cell activation is a recognized driver of disease in cholinergic urticaria (CholU). Clinical response in patients suggests that non-IgE-mediated triggers are likely to be involved, but the exact underlying mechanisms are still partly undiscovered. Extracellular vesicles (EVs) are known mediators of cell to cell communication, capable of interacting with MCs. Likewise, the MRGPRX2 receptor is an alternate inducer of MC degranulation. In this study, we sought to evaluate how EVs isolated from patients with cholinergic urticaria (before and after performing exercise) stimulated MCs, and to investigate a potential role of the MRGPRX2 receptor in this interaction. 

Patients clinically diagnosed with cholinergic urticaria were recruited at our center in Pamplona, Spain. Healthy atopic and non-atopic individuals were included as controls. All participants were tested with an exercise challenge, and serum samples were extracted before and after symptoms appeared in cases, and at paired time points in controls. EVs were isolated from the samples using standardized guidelines and evaluated for the presence of MRGPRX2 using flow cytometry. Skin-derived MCs were incubated overnight with EVs and then stimulated over the next 24 hours with substance P (SP). Supernatant contents were tested for cytokine release using RT-qPCR. Experiments were replicated in a similar fashion using the LAD2 cell line.

Six CholU cases and six controls were included in the study. MRGPRX2 was detected only in EVs obtained from CholU cases after exercise, and not in those from controls and CholU cases before exercise. Stimulation of skin MCs with CholU EVs induced greater expression of TPSAB1, HDC, IL-1beta, IL-8, and PTSG2 than stimulation with EVs from controls, when substance P was present.  This effect was especially pronounced when using CholU EVs obtained after exercise, rather than before exercise. CCL2, TGFB1, and IL-10 release was higher when cells were incubated with control EVs obtained after exercise. Cytokine patterns released by LAD2 MCs under similar incubation conditions were comparable. 

EVs obtained from patients with cholinergic urticaria after exercise, which were confirmed to contain the MRGPRX2 receptor, triggered pro-inflammatory marker release in MCs, in the presence of an MRPGRX2 agonist. Our study’s findings support targeting MRGPRX2, and potentially, EV-mediated pathways, as potential novel targets for the treatment of refractory CholU.