MRGPRX2 and its role in drug-induced anaphylaxis

© 2021 Nithya Asiri FernandopulleDrug-induced anaphylaxis is a growing concern with both the better characterised IgE-dependent mechanism and less well-established IgE-independent mechanisms being implicated. IgE-independent anaphylaxis has recently been suggested to be associated with direct mast cell activation via the newly identified G protein-coupled receptor, MRGPRX2. While the clinical relevance of the receptor is not yet well understood, a range of polybasic agonists, including therapeutic drugs like neuromuscular blocking agents (NMBAs), have been found to be agonists at the receptor. Presumably, mast cells in all humans express MRGPRX2, therefore the reason why only certain individuals are susceptible to sometimes extreme adverse reactions induced by these drugs remains unclear. In this project, we aimed to better characterise the role and activation of MRGPRX2 by: 1) Investigating if MRGPRX2-mediated mast cell activation can be halted with chemical antagonists; 2) Examining if receptor activation is subject to biased agonism so that some compounds more effectively trigger mast cell activation; 3) Determining if MRGPRX2-mediated anaphylaxis can be predicted through the identification of insightful biomarkers. To achieve this, we quantified mast cell activation through calcium mobilization, degranulation, and cytokine CCL2 release, using an in vitro human mast cell model (LAD2). We also examined clinical samples from patients who experienced an anaphylactic reaction during surgery to identify possible predictive biomarkers, whether genomic or serum components. Firstly, we generated a novel CRISPR-Cas9 MRGPRX2 knock down LAD2 model to verify the activity of known and proposed MRGPRX2 agonists at the receptor. Following this, we observed that chemical antagonism of some MRGPRX2 agonists using sugammadex (counter ligand of rocuronium) and heparin (counter ligand of protamine), when administered after stimulation, does not affect the release of mediators contributing to the immediate symptoms of anaphylaxis. However, these agents were shown to inhibit release of the late phase mediator, cytokine CCL2, which may perhaps be associated with the more delayed effects of anaphylaxis. Secondly, we demonstrated evidence in LAD2 cells that some NMBAs known to cause perioperative anaphylaxis (rocuronium and atracurium) are paradoxically biased against degranulation and cytokine release compared to calcium mobilization. Another agonist, substance P, was shown to be a full unbiased agonist, while protamine was identified as a partial yet unbiased agonist. Lastly, we have identified a novel double polymorphic form of MRGPRX2 in a patient who experienced anaphylaxis. We have also shown that sera from two anaphylactic patients had the ability to activate resting LAD2 mast cells. Whilst these findings were not broadly predictive of anaphylaxis across the cohort they may contribute in part to susceptibility to anaphylaxis in those individual patients. The lack of efficacy of post hoc chemical antagonism of MRGPRX2 stimuli on mast cell degranulation is not unexpected given the rapidity of the degranulation process. However, the inhibition of the more slowly released cytokine mediators may have at least some theoretical benefit in patient recovery from anaphylaxis, although further work is needed to establish the role of cytokines in anaphylaxis. In addition, that certain NMBAs were biased against MRGPRX2-induced degranulation suggests against this mechanism underpinning NMBA-induced anaphylaxis. This, however, assumes that the LAD2 mast cell model is broadly representative of human mast cells, but it is possible that patients susceptible to MRGPRX2-dependent anaphylaxis may have ‘overreactive’ mast cells where NMBAs trigger a full degranulatory response. Our ambitious attempts to identify a genetic or serum biomarker that defined patients who experienced anaphylaxis were not however fruitful. Broader genomic, transcriptomic, and proteomic studies on a larger, more clearly characterised patient cohort are necessary to identify useful biomarkers that help firstly establish and perhaps then predict drug-induced, MRGPRX2-dependent anaphylaxis. In summary, the findings presented in this thesis lay important groundwork for the better understanding of MRGPRX2, and can be effectively utilised in future research to define its role in drug-induced anaphylaxis