Novel Insights on MRGPRX2-Mediated Hypersensitivity to Neuromuscular Blocking Agents And Fluoroquinolones

Neuromuscular blocking agents (NMBAs) like atracurium and rocuronium as well as fluoroquinolones (FQs) cause mast cell-mediated anaphylaxis by activating Mas-related G protein-coupled receptor X2 (MRGPRX2), but many questions remain unanswered. Here, we address three of them, namely whether primary human mast cells show similar activation by these drugs as murine mast cells and mast cell lines, how sugammadex protects from atracurium-induced MRGPRX2-mediated mast cell activation, and why some but not all patients treated with rocuronium develop anaphylaxis. We used peripheral blood-derived cultured mast cells from healthy donors and patients, assessed mast cell activation and degranulation by quantifying intracellular calcium and CD63 expression, respectively, and made use of MRGPRX2-silencing, via electroporation with Dicer-substrate small interfering RNAs, and single cell flow cytometric analyses. Atracurium, ciprofloxacin, and levofloxacin activated and degranulated primary human mast cells, but only MRGPRX2-positive and not MRGPRX2-negative or -silenced mast cells. Sugammadex attenuated the atracurium-induced and MRGPRX2-mediated activation and degranulation of human mast cells by reducing free atracurium levels. The mast cells of patients with IgE-independent anaphylaxis to rocuronium were similar, in their MRGPRX2 expression and function, to those of patients with IgE-mediated anaphylaxis. These findings further improve our understanding of the role and relevance of MRGPRX2-driven mast cell activation in anaphylactic reactions to NMBAs and FQs and may help to improve their prediction, prevention, and treatment.


INTRODUCTION
The activation of Mas-related G protein-coupled receptor X2 (MRGPRX2) on mast cells (MCs) is held to be a major pathway in the pathogenesis of IgE-independent immediate drug hypersensitivity reactions (IDHRs) that occur in response to neuromuscular blocking agents (NMBAs) such as atracurium and rocuronium as well as fluoroquinolones (FQs) such as moxifloxacin and ciprofloxacin. NMBAs and FQs can cause perioperative anaphylaxis, and adverse reactions to these drugs administered during general anesthesia may be very severe and life-threatening, with a mortality rate of up to 9% (1)(2)(3)(4)(5)(6)(7).
As of today, many questions on the role of MRGPRX2 in NMBA-and FQ-induced anaphylaxis remain unanswered and need to be addressed. These include, but are not limited to, 1) whether findings on MRGPRX2 activation by NMBAs and FQs from mouse models or genetically modified cell lines can be extrapolated to humans, 2) what the precise role and mechanisms of action of sugammadex, a NMBA reversal agent, are in atracurium-induced and MRGPRX2-induced adverse reactions (8), and 3) why some but not all patients treated with NMBAs experience MRGPRX2-related IDHRs.
At present, most of the information on the effects of NMBAs and FQs on MRGPRX2 are derived from studies performed with MRGPRX2-expressing neoplastic cells, like the LAD-2 MC line, or with the mouse orthologue, MRGPRB2. It is clear that there are differences between these cellular models and primary human MCs. LAD-2 cells, for example, are more responsive to MRGPRX2-agonists than human peripheral blood-derived cultured MCs (PBCMCs) (9,10), and some NMBAs are less potent activators of MRGPRX2 than MRGPRB2 (11). This makes it difficult, at present, to assess NMBAs and FQs for their MRGPRX2-activating capacity and potency in the human system and to predict the risk that comes with their use. What is needed are results from studies with primary human cells. Such studies, until very recently, were not possible due to the lack of suitable tools, models and assays.
Sugammadex is a modified g-cyclodextrin designed as a selective relaxant-binding agent that acts by encapsulating rocuronium as an inclusion complex and removing it from the neuromuscular junction (12). Although sugammadex does not reverse atracurium-induced neuromuscular block (13), it has recently been shown to attenuate atracurium-induced MRGPRX2dependent MC activation in LAD-2 cells, when used at molar excess (14). Currently, the underlying mechanisms of this effect are unclear. The prevention of atracurium-induced MC activation may be caused by atracurium encapsulation, i.e. the elimination of free atracurium, by a direct inhibition via sugammadex, or by inhibitory effects of sugammadex-atracurium complexes. Addressing this gap of knowledge may guide the development of more effective approaches for the use of sugammadex for the prevention of NMBA-induced IDHRs.
Although MCs in all humans are held to express MRGPRX2, very few patients develop IDHRs when treated with NMBAs such as rocuronium. The reasons for this are unknown, but may include genetic polymorphisms and mutations resulting in an augmented responsiveness of MRGPRX2, distinct receptor binding sites, differences in MRGPRX2 signalosome, epigenetic modifications, post-transcriptional modifications resulting in synthesis of MRGPRX2 variants, temporarily or constitutively varying surface expressions and the influence of co-factors (15) (10). As recently suggested by Chompunud et al. (10), one way to address the role of MRGPRX2 mutations is to compare MCs for MRGPRX2 expression and function between patients who experienced IDHRs and had a positive skin test rocuronium, with and without rocuronium-specific IgE and/or positive BAT rocuronium (16).
Recently, we and others developed tools, models and assays that allow for the investigation of the three questions at hand. These include a primary human MC model in which silencing of MRGPRX2 via DsiRNA electroporation is coupled to flow cytometric analysis (17). In this model, the introduction of MRGPRX2-targeting DsiRNA (MRGPRX2-DsiRNA) almost completely mitigates intracellular calcium elevations and/or degranulation in response to MRGPRX2 ligands such as the opiate morphine, the FQ moxifloxacin, and the NMBA rocuronium (17). Here, we made use of these novel instruments to address important and unanswered questions on the role of MRPRX2 in NMBA-and FQ-induced IDHRs.

MRGPRX2 Silencing by DsiRNA Electroporation
PBCMCs at a concentration of 1x10 6 cells/mL were washed twice in cold serum-free Opti-MEM I medium (Gibco Invitrogen) and resuspended in 200 µL of the same medium. Cells were transferred to a 4.0-mm electroporation cuvette (Cell Projects), and 1 µM pool of two DsiRNA against MRGPRX2 at a 1:1 ratio or a non-targeting control DsiRNA (Integrated DNA Technologies, Catalog #:51-01-14-03) were added to the cuvette (Duplex sequences: DsiRNA 1: 5'-GGCAUUCAGUGGUUCCUAAUAU UAT-3' and 3'-AACCGUAAGUCACCAAGGA UUAUAAUA-5', DsiRNA 2: 5'GUUACGUGUUCCA CAGAAUAAAATA-3' and 3'-UUCA AUGCACAAGGUGUCUUAUUUUAU-5'). A square wave protocol (500 V, 5 ms, 0 gap, 1 pulse) was used to electroporate the cells (Gene Pulser Xcell ™ device, Bio-Rad Laboratories). Immediately after electroporation, cells were transferred to 5 mL of IMDM medium (Thermofischer Scientific) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich) (pre-heated at 37°C) and incubated for 20 min at 37°C and 5% CO 2 . Thereafter, cells were centrifuged and transferred to IMDM medium with SCF and IL-6. Five days after electroporation, a repeated analysis of MRGPRX2 expression and functionality of the PBCMCs was performed as described above. Based on the known affinity of sugammadex for atracurium, we estimated the remaining free atracurium concentrations of S-A-Cx 1, 2, and 3 to be 263 µM, 407 µM, and 612 µM, respectively. Therefore, outcomes from the S-A-Cx analyses were compared with results obtained in cells exposed to the corresponding free atracurium concentration. The activation and degranulation were studied using both intracellular calcium staining and CD63-upregulation, according to the protocol described above.

In Vivo and Ex Vivo Testing for Responses to Rocuronium
We assessed eight patients with previous IDHRs and rocuronium hypersensitivity was documented by skin testing, as previously described (19). Their PBCMCs were investigated for MRGPRX2 levels and function. Skin tests were done with rocuronium (Esmeron ® ; Merck Sharp and Dohme, Brussels, Belgium), saline buffer as a negative control and histamine as a positive control (10 mg/mL; HAL Allergy Benelux NV, Haarlem, The Netherlands), first as skin prick tests (SPTs) and, if negative, by intradermal tests (IDTs). The maximal non-irritating rocuronium concentration was 10 mg/mL for SPTs and 0.05 mg/mL for IDTs. Rocuronium was diluted immediately before use. SPTs with a wheal ≥ 3 mm with surrounding erythema after 15 minutes were considered positive. For IDTs, injection of 0.02 mL was performed, and reactions were read after 20-30 minutes. IDT responses with a wheal surrounded by an erythema ≥ 8 mm (or doubling compared to injection bleb) were considered positive. The direct basophil activation test (BAT) for rocuronium using the patients' basophils was performed as described (20). Results were expressed as the net percentages of CD63 + basophils, and the threshold of positivity was set at 4% (20). Total IgE and specific IgE (sIgE) to morphine were quantified by the FEIA ImmunoCAP system (Phadia Thermo Fisher, Uppsala, Sweden) according to the manufacturer's instructions (21). As shown in Table 1, patients were stratified by the results of their rocuronium basophil test and/or levels of specific IgE as positive (i.e. IgE-dependent, patients 1-4) and negative (i.e. non IgEdependent and probably MRGPRX2-mediated, patients 5-8). MRGPRX2 expression and functionality of their PBCMCs were compared between the two groups (16).

Flow Cytometric Analyses
Flow cytometric analyses were performed on a calibrated FACSCalibur (BD Immunocytometry systems) with argon-ion lasers (488nm and 633nm), for the intracellular calcium measurements, or on a calibrated FACSCanto II flow cytometer (BD Immunocytometry Systems, San Jose, CA) equipped with three lasers (405 nm, 488 nm and 633 nm), for the CD63 measurement. Correct compensation settings were performed using BD CompBeads (BD Biosciences). Flow cytometric data were analyzed using Kaluza Analysis 2.1 software (Beckman Coulter) and FCS6 Express 6 flow research edition (de novo software, Glendale, California). A fluorescence minus one (FMO) sample was used to set a marker between positive and negative cells according to the 99 th percentile. The results of the calcium measurements were expressed as fold increase against the basal intensity. Results of CD63 measurements were expressed as the net value of percentages of positive cells, i.e. the percentage of CD63 + cells in stimulated cells minus the percentage of CD63 + cells in resting cells. Al least 500 PBCMCs were counted per sample.

Statistical Analysis
GraphPad Prism version 7 software was used for data analysis and paired Student's t-tests were performed. Results are expressed as mean ± SEM. A P-value of < 0.05 was considered significant. The "n" in the figures denotes the total number of different donors used.

Ethical Considerations
Our study was approved by the Ethical Committee of the Antwerp University Hospital (Belgium B300201837509). All participants gave written informed consent.

RESULTS
Ciprofloxacin, Levofloxacin, and Atracurium, but Not Succinylcholine, Induce Activation and Degranulation of PBCMCs via MRGPRX2 The FQs ciprofloxacin and levofloxacin potently activated PBCMCs, with a rapid increase in intracellular calcium ( Figure 1A and Supplementary Figure 3). They also induced degranulation, with upregulated surface expression of the lysosomal degranulation marker CD63 ( Figure 1B). Of the two NMBAs tested, atracurium, but not succinylcholine, induced significant activation of PBCMCs as demonstrated by increase in intracellular calcium ( Figure 1A and Supplementary Figure 3), and degranulation, i.e. upregulation of CD63 ( Figure 1B). EC50 values are displayed in Table 2.
Only PBCMCs that expressed MRGPRX2, but not MRGPRX2-negative PBCMCs, showed significant activation by ciprofloxacin, levofloxacin, and atracurium, as assessed by calcium flux (Figure 2A and Supplementary Figure 4) as well as degranulation ( Figure 2B). Moreover, degranulation of MRGPRX2-positive PBCMCs was most pronounced in cells with the highest surface expression of MRGPRX2 ( Figure 2B). Silencing of MRGPRX2 in PBCMCs, by treatment with MRGPRX2-DsiRNA, reduced MRGPRX2 expression by more than 70% and prevented PBCMCs activation and degranulation by ciprofloxacin, levofloxacin, and atracurium ( Figure 3 and Supplementary Figures 5-7).

Sugammadex Attenuates Atracurium-Induced Activation and Degranulation of PBCMCs
Atracurium dose-dependently activated and degranulated PBCMCs, via MRGPRX2, whereas sugammadex did not cause PBCMC activation or degranulation (Figure 4 and Supplementary  Figure 4). Complexing of atracurium with sugammadex, at a 1:1 molar ratio, markedly reduced, but did not completely eliminate the activation and degranulation of PBCMCs (Figure 4). Degranulation by free atracurium, induced at the concentrations achieved by complexing with sugammadex, was comparable to that induced by the sugammadex-atracurium complexes ( Figure 5).

Rocuronium Causes Similar Activation and Degranulation of PBCMCs From Rocuronium-Hypersensitive Patients Who Have Specific IgE and Those Who Do Not
PBCMCs obtained from patients with rocuronium hypersensitivity, i.e. a history of rocuronium-induced IDHR, showed similar levels of activation and degranulation in patients who had specific IgE and/ or a positive BAT to rocuronium as compared to those who did not (Figures 6 and 7). Rocuronium-hypersensitive patients with and without specific IgE and/or a positive BAT to rocuronium had similar rates of MRGPRX2-positive cells, 65 ± 11% and 63 ± 4%, respectively (Supplementary Figure 8).

DISCUSSION
Here, we answer three important questions on the role and relevance of the MRGPRX2 pathway in MC-dependent immediate hypersensitivity reactions to NMBAs and FQs (11,15,24,25). We show 1) that atracurium, ciprofloxacin and levofloxacin degranulate human MCs via MRGPRX2, 2) that sugammadex attenuates atracurium-induced and MRGPRX2mediated MC degranulation by reducing free atracurium, and 3) that rocuronium-hypersensitive patients with and without sensitization to this NMBA show similar mast cell MRGPRX2 expression and function. These findings help our understanding  and may improve the prevention of anaphylactic reactions to NMBAs and FQs. Our results of studies with primary human MCs show that degranulation in response to atracurium, ciprofloxacin and levofloxacin is restricted to the MRGPRX2 + subpopulation and that these responses can be effectively mitigated by silencing of MRGPRX2. In contrast, succinylcholine did not degranulate human MCs. These observations are in line with the results of previous studies with murine cells and human MC lines (11,14,25,26). Our results also confirm that selective MRGPRX2 silencing in primary human MCs via electroporation of DsiRNA against MRGPRX2 enables studying the effects of receptor activation on both calcium mobilization and degranulation (17). As shown previously, this silencing approach has a high transfection efficiency and results in markedly downregulated cell surface levels of MRGPRX2 (17). Combined with the novel flow cytometric technique we used in this study, this approach enables analyses at the single cell level, which allows for the identification and characterization of cellular subsets in non-homogeneous populations, such as PBCMCs with MRGPRX2 positive and negative subsets. This also explains why traditional mediator release tests, which only provide averaged results across all cells, How does sugammadex, a NMBA reversal agent, prevent atracurium-induced and MRGPRX2-induced adverse reactions? Earlier work with leukemic LAD-2 cells demonstrated that coincubation of atracurium with sugammadex, in molar excess, can inhibit atracurium-induced MRGPRX2-mediated calcium mobilization and degranulation (14). Our findings from studies with primary human PBCMCs confirm that this is indeed the case.
More importantly, our study shows that sugammadex reduces MRGPRX2 responses by sequestration of free atracurium. Sugammadex-atracurium complexes, by themselves, are not inhibitory. While this is interesting, mechanistically, it may be of limited clinical relevance, as recent reports suggest that sugammadex does not stop atracurium-induced basophil and MC degranulation, once initiated (14,19). This argues against the use of sugammadex in patients with ongoing atracurium-mediated perioperative anaphylaxis (27).
That NMBAs induced MRGPRX2-related IDHRs occur only in some patients and not most or all has been suggested to be due to higher levels of MRGPRX2 expression or susceptibility to activation in the former (10,15). Our findings suggest that this is probably not the case. The mast cells of rocuronium-hypersensitive patients without specific IgE to rocuronium and a negative BAT, i.e. patients with IgE-independent IDHRs to rocuronium, had the same levels of MRGPRX2 expression and the same susceptibility to MRGPRX2 activation as rocuronium-sensitized patients, i.e. patients with IgE-dependent IDHRs to rocuronium (16). Further Incubation with substance P (74 µM), the natural agonist of MRGPRX2, anti-FcϵRI (2.5 µg/mL), succinylcholine (5536 µM), atracurium (2152 µM), ciprofloxacin (755 µM) or levofloxacin (2767 µM). In all experiments, n=5. Area under the curves were compared using a paired student t-test, p < 0.05*, p < 0.01**, p < 0.001***, p < 0.0001****. Note that the effect of MRGPRX2 silencing align with the differences observed between MRGPRX2 + and MRGPRX2cells as shown in Figure 1B and Supplementary Figure 4. As shown previously, MRGPRX2 silencing has no significant effect on anti-FcϵRI-dependent intracellular calcium signalling and degranulation (17). s, seconds. studies aimed at identifying those at risk of rocuronium IDHRs should focus on alternative mechanisms (15). Our results come with two caveats. First, we analyzed patient PBCMCs rather than skin mast cells (there are some differences) (10), and second, our conclusions are based on the assumption that positive sIgE and BAT results are indicative for an IgE-dependent reaction and that the absence of sIgE and a negative BAT point to a MRGPRX2dependent reaction. There is, currently, no validated assay to test patients for MRGPRX2-dependent anaphylaxis. Exclusion of other mechanisms, i.e. IgE-mediated activation, by sIgE and BAT is the best approach to identifying patients with probably MRGPRX2dependent reactions (16,28). Unlike MCs, resting circulation basophils barely express MRGPRX2 (29,30) and have repeatedly been shown not to respond in a non-specific manner to potent MRGPRX2 agonists such as opiates (e.g. morphine, codeine and pholcodine) (31,32), FQs (e.g. moxifloxacin, ciprofloxacin, levofloxacin) (6, 33) and certain NMBAs (e.g. atracurium, mivacurium) (3,34,35). Positive CD63-BAT results together with sIgE and wortmannin inhibition experiments point to patients with specific sensitization and IgE-dependent reactions (32,33,36).
Our study has several strengths and limitations. Its major strength is that we assessed MRGPRX2, across all three questions we addressed, with novel and powerful models and techniques including primary human MCs generated from peripheral blood, silencing of MRGPRX2 via DsiRNA electroporation, and single cell flow cytometric analyses. Its limitations include the relatively low number of patients analyzed, which did not allow for meaningful comparisons of subpopulations of patients, owed largely to the rare occurrence of NMBA-mediated IDHRs. Another limitation of our approach, as in many in vitro and ex vivo experiments, lies in the necessity of supra-therapeutic (sometimes near toxic) stimulation concentrations. In all experiments, n=5. Area under the curves were compared using a paired student t-test, p < 0.05*, p < 0.01**, p < 0.001***, p < 0.0001***. s, seconds.   Taken together, this is the first study to show, in primary human MCs, that the NMBA atracurium as well as the FQs ciprofloxacin and levofloxacin induce degranulation via MRGPRX2, that sugammadex attenuates atracurium-induced MRGPRX2activation and downstream MC degranulation by reducing free atracurium, and that MCs of rocuronium-hypersensitive patients with IgE-independent IDHRs to this NMBA are not different in their MRGPRX2 expression and function as compared to patients with IgE-dependent IDHRs. We also conclude that the use of PBCMCs together with MRGPRX2-targeted silencing can contribute to answer further questions on the role and relevance of MRGPRX2 in IDHRs and beyond (37). Our results should encourage such studies, as a better understanding of the MRGPRX2 pathway is needed to improve the prevention and treatment of anaphylactic reactions to NMBAs and FQs.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusion of this article are available on request from the corresponding author.

ETHICS STATEMENT
Our study was approved by the Ethical Committee of the Antwerp University Hospital (Belgium B300201837509). All participants gave written informed consent. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.