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Ursolic acid (UA), a pentacyclic triterpene, exhibits diverse pharmacological effects, including potential treatment for allergic diseases. It downregulates thymic stromal lymphopoietin (TSLP) and disrupts mast cell signaling pathways. However, the exact molecular mechanism by which UA interferes with mast cell action remains unclear. Therefore, the current study aimed to uncover molecular entities underlying the effect of UA on mast cells and its potential antipruritic effect, specifically investigating its modulation of key molecules such as TRPV4, PAR2, and MRGPRX2, which are involved in TSLP regulation and sensation. Calcium imaging experiments revealed that UA pretreatment significantly suppressed MRGPRX2 activation (and its mouse orthologue MrgprB2), a G protein-coupled receptor predominantly expressed in mast cells. Molecular docking predictions suggested potential interactions between UA and MRGPRX2/MrgprB2. UA pretreatment also reduced mast cell degranulation through MRGPRX2 and MrgprB2-dependent mechanisms. In a dry skin mouse model, UA administration decreased tryptase and TSLP production in the skin, and diminished TSLP response in the sensory neurons. While PAR2 and TRPV4 activation enhances TSLP production, UA did not inhibit their activity. Notably, UA attenuated compound 48/80-induced scratching behaviors in mice and suppressed spontaneous scratching in a dry skin model. The present study confirms the effective inhibition of UA on MRGPRX2/MrgprB2, leading to reduced mast cell degranulation and suppressed scratching behaviors. These findings highlight the potential of UA as an antipruritic agent for managing various allergy- or itch-related conditions., Competing Interests: Declaration of competing interest The authors state no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Skin mast cells (MCs) mediate acute allergic reactions in the cutaneous environment and contribute to chronic dermatoses, including urticaria, and atopic or contact dermatitis. The cAMP response element binding protein (CREB), an evolutionarily well conserved transcription factor (TF) with over 4,000 binding sites in the genome, was recently found to form a feedforward loop with KIT, maintaining MC survival. The most selective MC function is degranulation with its acute release of prestored mediators. Herein, we asked whether CREB contributes to the expression and function of the degranulation-competent receptors FcεRI and MRGPRX2. Interference with CREB by pharmacological inhibition (CREBi, 666-15) or RNA interference only slightly affected the expression of these receptors, while KIT was strongly attenuated. Interestingly, MRGPRX2 surface expression moderately increased following CREB-knockdown, whereas MRGPRX2-dependent exocytosis simultaneously decreased. FcεRI expression and function were regulated consistently, although the effect was stronger at the functional level. Preformed MC mediators (tryptase, histamine, β -hexosaminidase) remained comparable following CREB attenuation, suggesting that granule synthesis did not rely on CREB function. Collectively, in contrast to KIT, FcεRI and MRGPRX2 moderately depend on unperturbed CREB function. Nevertheless, CREB is required to maintain MC releasability irrespective of stimulus, insinuating that CREB may operate by safeguarding the degranulation machinery. To our knowledge, CREB is the first factor identified to regulate MRGPRX2 expression and function in opposite direction. Overall, the ancient TF is an indispensable component of skin MCs, orchestrating not only survival and proliferation but also their secretory competence.

Background: Phytosphingosine and its derivative are known for their skin-protective properties. While mYG-II-6, a phytosphingosine derivative, has shown anti-inflammatory and antipsoriatic effects, its potential antipruritic qualities have yet to be explored. This study aimed to investigate mYG-II-6's antipruritic properties., Methods: The calcium imaging technique was employed to investigate the activity of ion channels and receptors. Mast cell degranulation was confirmed through the β-hexosaminidase assay. Additionally, in silico molecular docking and an in vivo mouse scratching behavior test were utilized., Results: Using HEK293T cells transfected with H1R and TRPV1, we examined the impact of mYG-II-6 on histamine-induced intracellular calcium rise, a key signal in itch-mediating sensory neurons. Pretreatment with mYG-II-6 significantly reduced histamine-induced calcium levels and inhibited TRPV1 activity, suggesting its role in blocking the calcium influx channel. Additionally, mYG-II-6 suppressed histamine-induced calcium increase in primary cultures of mouse dorsal root ganglia, indicating its potential antipruritic effect mediated by histamine. Interestingly, mYG-II-6 exhibited inhibitory effects on human MRGPRX2, a G protein-coupled receptor involved in IgE-independent mast cell degranulation. However, it did not inhibit mouse MrgprB2, the ortholog of human MRGPRX2. Molecular docking analysis revealed that mYG-II-6 selectively interacts with the binding pocket of MRGPRX2. Importantly, mYG-II-6 suppressed histamine-induced scratching behaviors in mice., Conclusions: Our findings show that mYG-II-6 can alleviate histamine-induced itch sensation through dual mechanisms. This underscores its potential as a versatile treatment for various pruritic conditions., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterised by itching, erythema, and epidermal barrier dysfunction. The pathogenesis of AD is complex and multifactorial; however,mast cell (MC) activation has been reported to be one of the crucial mechanisms in the pathogenesis of AD. The MC receptor Mas related G protein-coupled receptor-X2 (MRGPRX2) has been identified as a prominent alternative receptor to the IgE receptor in causing MC activation and the subsequent release of inflammatory mediators. The current study aimed to evaluate the therapeutic effect of a novel small molecule MRGPRX2 antagonist GE1111 in AD using in vitro and in vivo approaches., Methods: We developed an in vitro cell culture disease model by using LAD-2 MC, HaCaT keratinocytes and RAW 264.7 macrophage cell lines. We challenged keratinocytes and macrophage cells with CST-14 treated MC supernatant in the presence and absence of GE1111 and measured the expression of tight junction protein claudin 1, inflammatory cytokines and macrophage phagocytosis activity through immunohistochemistry, western blotting, RT-qPCR and fluorescence imaging techniques. In addition to this, we developed a DFNB-induced AD model in mice and evaluated the protective effect and underlying mechanism of GE1111., Results and Discussion: Our in vitro findings demonstrated a potential therapeutic effect of GE1111, which inhibits the expression of TSLP, IL-13, MCP-1, TNF-a, and IL-1ß in MC and keratinocytes. In addition to this, GE1111 was able to preserve the expression of claudin 1 in keratinocytes and the phagocytotic activity of macrophage cells. The in vivo results demonstrated that GE1111 treatment significantly reduced phenotypic changes associated with AD (skin thickening, scaling, erythema and epidermal thickness). Furthermore, immunohistochemical analysis demonstrated that GE1111 treatment preserved the expression of the tight junction protein Involucrin and reduced the expression of the inflammatory mediator periostin in the mouse model of AD. These findings were supported by gene and protein expression analysis, where GE1111 treatment reduced the expression of TSLP, IL-13, and IL-1ß, as well as downstream signalling pathways of MRGPRX2 in AD skin lesions. In conclusion, our findings provide compelling in vitro and in vivo evidence supporting the contribution of MRGPRX2-MC interaction with keratinocytes and macrophages in the pathogenesis of AD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Wong, Choi, Li, Chow and Kumar.)

Initiation of the bradykinin generation cascade is responsible for the occurrence of attacks in some types of angioedema without wheals. Hereditary angioedema due to C1 inhibitor deficiency (HAE-C1-INH) is one such clinical entity. In this paper, we explore the existing evidence that mast cells (MCs) degranulation may contribute to the activation of the kallikrein-kinin system cascade, followed by bradykinin formation and angioedema. We present the multidirectional effects of MC-derived heparin and other polyanions on the major components of the kinin-kallikrein system, particularly on the factor XII activation. Although, bradykinin- and histamine-mediated symptoms are distinct clinical phenomena, they share some common features, such as some similar triggers and a predilection to occur at sites where mast cells reside, namely the skin and mucous membranes. In addition, recent observations indicate a high incidence of hypersensitivity reactions associated with MC degranulation in the HAE-C1-INH patient population. However, not all of these can be explained by IgE-dependent mechanisms. Mast cell-related G protein-coupled receptor-X2 (MRGPRX2), which has recently attracted scientific interest, may be involved in the activation of MCs through a different pathway. Therefore, we reviewed MRGPRX2 ligands that HAE-C1-INH patients may be exposed to in their daily lives and that may affect MCs degranulation. We also discussed the known inter- and intra-individual variability in the course of HAE-C1-INH in relation to factors responsible for possible variability in the strength of the response to MRGPRX2 receptor stimulation. The above issues raise several questions for future research. It is not known to what extent a prophylactic or therapeutic intervention targeting the pathways of one mechanism (mast cell degranulation) may affect the other (bradykinin production), or whether the number of mast cells at a specific body site and their reactivity to triggers such as pressure, allergens or MRGPRX2 agonists may influence the occurrence of HAE-C1-INH attacks at that site., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor MR declared a past co-authorship with the author GP., (Copyright © 2024 Porebski, Dziadowiec, Rybka, Kitel and Kwitniewski.)

Morphine derivatives are clinically important anesthetic and sedative drugs, which often show anaphylactic side effects. Mas-related G-protein coupled receptor member X2 (MRGPRX2) triggers mast cell degranulation, which is important process in anaphylactic reactions. MRGPRX2-HEK293 and LAD2 cell membrane chromatographic (CMC) models were used to screen morphine derivatives binding to MRGPRX2. Furthermore, most morphine derivatives significantly enhanced Ca 2+ mobilization. More importantly, thebaine was found to effectively promote histamine release. Thebaine induced the increased release of β-hexosaminidase and high secretion level of cytokines, confirming that thebaine could further trigger anaphylactic reactions and promote subsequent inflammatory reactions. Moreover, the ability of thebaine inducing degranulation and the release of allergenic mediators in mast cells was significantly decreased after MRGPRX2 knockdown, which proved that MRGPRX2 is the key media for thebaine-induced anaphylactic reactions. Significant hind paw swelling and hypothermia in mice after injecting thebaine suggested that thebaine could trigger anaphylactic reactions in vivo., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Pseudo-allergic reactions frequently occur following clinical drug use and sometimes even cause mortal danger. Mas-related G-protein-coupled receptor member X2 (MRGPRX2) is a novel receptor that mediates pseudo-allergy and is an important target in the treatment of allergies. However, to date, there are no synthetic small-molecule inhibitors that prevent anaphylactoid reactions through this pathway. Our preliminary research suggested that B10-S and mubritinib effectively inhibited LAD2 cells. Therefore, two novel derivatives were synthesized by integrating the active substructures of B10-S and mubritinib, according to the molecular docking results. The antiallergic inhibitory effects of the two compounds were preliminarily evaluated in vitro using β-hexosaminidase release, histamine release, and intracellular Ca 2+ mobilization assays, and their binding sites on MRGPRX2 were analyzed by molecular docking. Both substances inhibited β-hexosaminidase and histamine release in LAD2 cells and decreased intracellular Ca 2+ by inhibiting MRGPRX2 in MRGPRX2-HEK293 cells treated with C48/80 in a dose-dependent manner. The docking results suggested that the molecules could competitively bind to the active site on MRGPRX2 and Glu141, which were combined by C48/80. Our study indicated that the two compounds have potential anti-allergic properties, which may provide evidence that will facilitate the development of synthetic molecules with anti-pseudo-allergic activity for clinical use in the future., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Mas-related G protein-coupled receptor X2 (MRGPRX2) mediates the itch response in neurons and is involved in atopic dermatitis (AD)-associated inflammation and itch. Potent and MRGPRX2-selective ligands are essential to an understanding of the detailed function of the receptor and to develop new therapeutic agents for its related diseases. (+)-TAN-67 (1), the enantiomer of the δ-opioid receptor (DOR) selective ligand (-)-TAN-67 (1), has been reported to activate MRGPRX2, although (+)-1 also interacts with DOR, which prevents investigators from interrogating the function of MRGPRX2. Here, we have succeeded in developing a novel unnatural morphinan compound (+)-2a by a transformation based on the structure of (+)-1, which removes the DOR binding affinity. (+)-2a activated both human MRGPRX2 and the mouse orthologue Mrgprb2 in in vitro experiments and induced itch-like behaviors in mice to the same extent as (+)-1. The (+)-2a-induced itch response in mice was suppressed by administration of the tripeptide QWF, an MRGPRX2/Mrgprb2 antagonist, or the antipruritic drug nalfurafine. Together, (+)-2a serves as a useful tool to elucidate the itch-related function/action of MRGPRX2 and its mouse orthologue Mrgprb2., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The G protein-coupled receptor MrgprX2 in mast cells is known to be a crucial receptor for pseudo-allergic reactions. MrgprX2 activation leads to elevated intracellular calcium levels and mast cell degranulation, but the underlying mechanism remains to be elucidated. Herein, we investigated the role of the phosphatidylinositol 3 kinase (PI3K)/serum-threonine kinase (AKT) signaling pathway and phospholipase C gamma (PLCγ) in mast cell degranulation mediated by MrgprX2 in LAD2 human-derived mast cells. The results showed that phosphorylated AKT (p-AKT) and PLCγ up-regulation were accompanied by an increase in intracellular calcium following activation of MrgprX2 by Compound 48/80, an inducer of mast cell degranulation. In contrast, p-AKT and PLCγ were down-regulated and intracellular calcium levels decreased after MrgprX2 knockdown. Mast cell degranulation was clearly suppressed; however, inhibiting PI3K and PLCγ phosphorylation did not influence MrgprX2 expression. The increase in calcium concentration was suppressed and mast cell degranulation was weakened. Furthermore, by inhibiting PI3K and PLCγ phosphorylation in animals, the allergic symptoms caused by C48/80 were obviously reduced. We deduced that during the mast cell degranulation observed in pseudoallergic reactions, MrgprX2 regulated intracellular calcium levels via the PI3K/AKT and PLCγ pathways., (Copyright © 2021. Published by Elsevier B.V.)

Background: P17, a peptide isolated from Tetramorium bicarinatum ant venom, is known to induce an alternative phenotype of human monocyte-derived macrophages via activation of an unknown G protein-coupled receptor (GPCR)., Objective: We sought to investigate the mechanism of action and the immunomodulatory effects of P17 mediated through MRGPRX2 (Mas-related G protein-coupled receptor X2)., Methods: To identify the GPCR for P17, we screened 314 GPCRs. Upon identification of MRGPRX2, a battery of in silico, in vitro, ex vivo, and in vivo assays along with the receptor mutation studies were performed. In particular, to investigate the immunomodulatory actions, we used β-hexosaminidase release assay, cytokine releases, quantification of mRNA expression, cell migration and differentiation assays, immunohistochemical labeling, hematoxylin and eosin, and immunofluorescence staining., Results: P17 activated MRGPRX2 in a dose-dependent manner in β-arrestin recruitment assay. In LAD2 cells, P17 induced calcium and β-hexosaminidase release. Quercetin- and short hairpin RNA-mediated knockdown of MRGPRX2 reduced P17-evoked β-hexosaminidase release. In silico and in vitro mutagenesis studies showed that residue Lys 8 of P17 formed a cation-π interaction with the Phe 172 of MRGPRX2 and [Ala 8 ]P17 lost its activity partially. P17 activated LAD2 cells to recruit THP-1 and human monocytes in Transwell migration assay, whereas MRGPRX2-impaired LAD2 cells cannot. In addition, P17-treated LAD2 cells stimulated differentiation of THP-1 and human monocytes, as indicated by the enhanced expression of macrophage markers cluster of differentiation 11b and TNF-α by quantitative RT-PCR. Immunohistochemical and immunofluorescent staining suggested monocyte recruitment in mice ears injected with P17., Conclusions: Our data provide novel structural information regarding the interaction of P17 with MRGPRX2 and intracellular pathways for its immunomodulatory action., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

MRGPRX2 on mast cells (MCs) is the target that directly mediates MC activation through the activity of small molecular substances. Previous work has attempted to prove that substance P (SP) and PAMP(9-20) induce an MRGPRX2-mediated MC degranulation reaction. However, SP activates MRGPRX2-induced histamine release, which may lead to allergic airway inflammation, while PAMP(9-20)-induced MrgprB2 activation releases more tryptase and fewer monoamines. Due to the lack of direct available comparisons, the different types of sensitizing mediators released by the action of SP and PAMP(9-20) inducing pseudo-allergic reactions via MRGPRX2 are unclear. To investigate whether the action sites of excited MRGPRX2 are different for SP and PAMP(9-20), leading to different effects, the release of inflammatory mediators was measured using MC degranulation reactions and RNA-seq assay in vitro. Mice were treated to observe local inflammation and MC degranulation in vivo. Moreover, site-directed mutagenesis was used to verify the excited sites of SP and PAMP(9-20). SP and PAMP(9-20) both activated MRGPRX2 and led MCs to release inflammatory mediators. Significantly different levels of histamine, tryptase, TNF-α, MCP-1, and other cytokines were released in vivo and in vitro. G165E, D184N, W243R, and H259Y were necessary for SP to activate MRGPRX2, while only D184N and W243R were important for PAMP(9-20). The downstream signaling pathways activated by SP and PAMP(9-20) also differed in the phosphorylation level of PKC. There were differences in the sites via which SP and PAMP(9-20) activate MRGPRX2 and also in the activated downstream signaling pathways, which led to the differences the activation of the pathways and effects of SP- and PAMP(9-20)-induced MRGPRX2 activation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Peptide based therapeutics are desirable owing to their high biological specificity. However, a number of these fail in clinical testing due to an adverse inflammatory response. Mast cells play a key role in directing the host response to drugs and related products. Although the role of FcεRI receptor is well known, Mas-related G-protein coupled receptor X2 (MRGPRX2) binding of endogenous peptides, and drugs will activate mast cells independent of FcεRI. Identifying peptides that activate mast cells through MRGPRX2, and their respective activation potency, can be used to reduce the failure rate of peptide therapeutics at clinical trial. Moreover, it will allow for peptide design where mast cell activation is actually desired. It was found that FRKKW and WNKWAL are two motifs that activate human LAD2 cells similar to PAMP-12 controls. Peptide activators of MRGPRX2 could be reduced to X a -(Y) (n ≥ 3) -X b where: X a is an aromatic residue; X b is a hydrophobic residue; and Y is a minimum 3 residue long sequence, containing a minimum of one positively charged residue with the remainder being uncharged residues. Artificial peptides WKKKW and FKKKF were constructed to test this structural functionality and were similar to PAMP-12 controls. Peptides with different activation potentials were found where FRKKW = WKKKW = FKKKF > PAMP-12 = WNKWAL > YKKKY > FRKKANKWALSR = FRKKWNKAALSR > KWKWK > FRKK = WNKWA > KYKYK > NKWALSR = YKKY = WNK. These sequences should be considered when designing peptide-based therapeutics. STATEMENT OF SIGNIFICANCE: Mast cells release immune regulating molecules upon activation that direct host's immune response. MRGPRX2 receptor provides an alternate pathway for mast cell activation that is independent of FcεRI receptor. It is thought that mast cell activation through MRGPRX2 plays a critical role in high failure rates of drugs in clinical trials. Identifying peptide sequences that activate mast cells through MRGPRX2 can serve two important purposes, namely, sequences to avoid when designing peptide therapeutics, and artificial peptides with different activation potentials for mast cells. Herein, we have identified a general amino acid sequence that induces mast cell activation through MRGPRX2. Furthermore, by modulating the identified sequence, artificial peptides have been designed which activate mast cells by varying degrees for therapeutic applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Acute anaphylaxis to small molecule drugs is largely considered to be antibody-mediated with immunogloblin E (IgE) and mast cell activation being key. More recently, a role for drug-reactive immunoglobulin G (IgG) with neutrophil activation has also been suggested, at least in reactions to neuromuscular blocking agents (NMBAs). However, the mast cell receptor MRGPRX2 has also been highlighted as a possible triggering mechanism in acute anaphylaxis to many clinically used drugs. Significantly, MRGPRX2 activation is not dependent upon the presence of drug-recognising antibody. Given the reasonable assumption that MRGPRX2 is expressed in all individuals, the corollary of this is that in theory, anybody could respond detrimentally to triggering drugs (recently suggested to be around 20% of a drug-like compound library). But this clearly is not the case, as the incidence of acute drug-induced anaphylaxis is very low. In this mini-review we consider antibody-dependent and -independent mechanisms of mast cell activation by small molecule drugs with a focus on the MRGPRX2 pathway. Moreover, as a juxtaposition to these adverse drug actions, we consider how increased understanding of the role of MRGPRX2 in anaphylaxis is important for future drug development and can complement exploration of this receptor as a drug target in broader clinical settings., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mackay, Fernandopulle, Ding, McComish and Soeding.)

Mas-related G-protein-coupled receptor X2 (MRGPRX2) has recently been reported to be associated with anaphylaxis. Detection of MRGPRX2 levels in human peripheral blood might serve as a powerful tool for predicting the predisposition of patients to anaphylactic reactions. For rapid measurement of MRGPRX2, we established a paper-based double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antibody and horseradish peroxidase (HRP)-labelled rabbit polyclonal antibody as capture antibody and detection antibody, respectively. We avoided chemical functionalization of the cellulose paper by introducing bovine serum albumin (BSA) to provide COOH and NH 2 groups for covalent immobilization of the capture antibody. Through amide condensation, a two-layer immobilization strategy was applied with BSA-BSA and BSA-capture antibody networks as the first and second layers, respectively. This strategy improved the quantity, activity and stability of the immobilized antibody. We then established a paper-based ELISA to detect MRGPRX2 in human peripheral blood. Our method is less laborious, easier to implement, and more cost-effective than conventional ELISA, while offering similar sensitivity, specificity, and accuracy. Therefore, it could serve as an innovative clinical point-of-care diagnostic tool, especially in areas that lack advanced clinical equipment., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Mast cells (MCs) act as primary effectors in inflammatory and allergic reactions by releasing intracellularly-stored inflammatory mediators in diseases. The two major pathways for MC activation are known to be immunoglobulin E (IgE)-dependent and -independent. Although IgE-dependent signaling is the main pathway to MC activation, IgE-independent pathways have also been found to serve pivotal roles in the pathophysiology of various inflammatory conditions. Recent studies have shown that human and mouse MCs express several regulatory receptors such as toll-like receptors (TLRs), CD48, C300a, and GPCRs, including mas-related GPCR-X2 (MRGPRX2). MRGPRX2 has been reported as a novel GPCR that is expressed in MCs activated by basic secretagogues, neurokinin peptides, host defense antimicrobial peptides, and small molecule compounds (e.g., neuromuscular blocking agents) and leads to MC degranulation and eicosanoids release under in vitro experimental condition. Functional analyses of MRGPRX2 and Mrgprb2 (mouse ortholog) indicate that MRGPRX2 is involved in MC hypersensitivity reactions causing neuroinflammation such as postoperative pain, type 2 inflammation, non-histaminergic itch, and drug-induced anaphylactic-like reactions. In this review, we discuss the roles in innate immunity through functional studies on MRGPRX2-mediated IgE-independent MC activation and also the therapeutic potential of MRGPRX2 inhibitors on allergic and inflammatory diseases.

Cutaneous mast cells (MCs) express Mas-related G protein-coupled receptor-X2 (MRGPRX2; mouse ortholog MrgprB2), which is activated by an ever-increasing number of cationic ligands. Antimicrobial host defense peptides (HDPs) generated by keratinocytes contribute to host defense likely by 2 mechanisms, one involving direct killing of microbes and the other via MC activation through MRGPRX2. However, its inappropriate activation may cause pseudoallergy and likely contribute to the pathogenesis of rosacea, atopic dermatitis, allergic contact dermatitis, urticaria, and mastocytosis. Gain- and loss-of-function missense single nucleotide polymorphisms in MRGPRX2 have been identified. The ability of certain ligands to serve as balanced or G protein-biased agonists has been defined. Small-molecule HDP mimetics that display both direct antimicrobial activity and activate MCs via MRGPRX2 have been developed. In addition, antibodies and reagents that modulate MRGPRX2 expression and signaling have been generated. In this article, we provide a comprehensive update on MrgprB2 and MRGPRX2 biology. We propose that harnessing MRGPRX2's host defense function by small-molecule HDP mimetics may provide a novel approach for the treatment of antibiotic-resistant cutaneous infections. In contrast, MRGPRX2-specific antibodies and inhibitors could be used for the modulation of allergic and inflammatory diseases that are mediated via this receptor., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: MM has received honoraria (advisory board, speaker) and/or institutional grant/research support from Allakos, Amgen, Astra-Zeneca, Bayer, Dr. Pfleger, FAES, Genentech, GSK, Innate Pharma, Kyowa Kirin, Lilly, Merckle Recordati, Moxie, Novartis, Regeneron, Roche, Sanofi, MSD, UCB, and Uriach. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Elst, Maurer, Sabato, Faber, Bridts, Mertens, Van Houdt, Van Gasse, van der Poorten, De Puysseleyr, Hagendorens, Van Tendeloo, Lion, Campillo-Davo and Ebo.)

Pseudomonas aeruginosa is a frequent cause of hospital-acquired wound infection and is difficult to treat because it forms biofilms and displays antibiotic resistance. Previous studies in mice demonstrated that mast cells (MCs) not only contribute to P. aeruginosa eradication but also promote wound healing via an unknown mechanism. We recently reported that host defense peptides (HDPs) induce human MC degranulation via Mas-related G protein-coupled receptor-X2 (MRGPRX2). Small molecule HDP mimetics have distinct advantages over HDPs because they are inexpensive to synthesize and display high stability, bioavailability, and low toxicity. Murepavadin is a lipidated HDP mimetic, (also known as POL7080), which displays antibacterial activity against a broad panel of multi-drug-resistant P. aeruginosa . We found that murepavadin induces Ca 2+ mobilization, degranulation, chemokine IL-8 and CCL3 production in a human MC line (LAD2 cells) endogenously expressing MRGPRX2. Murepavadin also caused degranulation in RBL-2H3 cells expressing MRGPRX2 but this response was significantly reduced in cells expressing missense variants within the receptor's ligand binding (G165E) or G protein coupling (V282M) domains. Compound 48/80 induced β-arrestin recruitment and promoted receptor internalization, which resulted in substantial decrease in the subsequent responsiveness to the MRGPRX2 agonist. By contrast, murepavadin did not cause β-arrestin-mediated MRGPRX2 regulation. Murepavadin induced degranulation in mouse peritoneal MCs via MrgprB2 (ortholog of human MRGPRX2) and caused increased vascular permeability in wild-type mice but not in MrgprB2 -/- mice. The data presented herein demonstrate that murepavadin activates human MCs via MRGPRX2 and murine MCs via MrgprB2 and that MRGPRX2 is resistant to β-arrestin-mediated receptor regulation. Thus, besides its direct activity against P. aeruginosa , murepavadin may contribute to bacterial clearance and promote wound healing by harnessing MC's immunomodulatory property via the activation of MRGPRX2., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Amponnawarat, Chompunud Na Ayudhya and Ali.)

The neuropeptide substance P (SP) mediates neurogenic inflammation and pain and contributes to atopic dermatitis in mice through the activation of mast cells (MCs) via Mas-related G protein-coupled receptor (GPCR)-B2 (MrgprB2, human ortholog MRGPRX2). In addition to G proteins, certain MRGPRX2 agonists activate an additional signaling pathway that involves the recruitment of β-arrestins, which contributes to receptor internalization and desensitization (balanced agonists). We found that SP caused β-arrestin recruitment, MRGPRX2 internalization, and desensitization. These responses were independent of G proteins, indicating that SP serves as a balanced agonist for MRGPRX2. A tyrosine residue in the highly conserved NPxxY motif contributes to the activation and internalization of many GPCRs. We have previously shown that Tyr 279 of MRGPRX2 is essential for G protein-mediated signaling and degranulation. To assess its role in β-arrestin-mediated MRGPRX2 regulation, we replaced Tyr 279 in the NPxxY motif of MRGPRX2 with Ala (Y279A). Surprisingly, we found that, unlike the wild-type receptor, Y279A mutant of MRGPRX2 was resistant to SP-induced β-arrestin recruitment and internalization. This study reveals the novel findings that activation of MRGPRX2 by SP is regulated by β-arrestins and that a highly conserved tyrosine residue within MRGPRX2's NPxxY motif contributes to both G protein- and β-arrestin-mediated responses.

Background: Iohexol is a typical iodinated radiocontrast medium and widely used in clinical angiography. Hypersensitivity reactions induced by iohexol are common side effects known to increase the risk for patients. Iodine is the main functional group of iohexol, and it can induce delayed anaphylaxis. However, iohexol also induces immediate-type allergies, but the underlying mechanism is still not clear. MRGPRX2 is a key receptor present on mast cells, which mediates pseudo-allergic reactions induced by various drugs., Methods: We aimed to verify the relationship between iohexol-induced anaphylactic reactions and MRGPRX2. MRGPRX2-mediated pseudo-allergic reactions induced by iohexol were investigated in vivo and in vitro using a mouse model of local and systemic anaphylaxis and mast cell degranulation assays, respectively., Results: Iohexol caused pseudo-allergic reactions in wild-type (WT) mice by activating mast cells to release histamine and cytokines. However, it did not induce a similar phenomenon in Kit W-sh/W-sh (MUT) mice. Iohexol stimulated intracellular calcium ion (Ca 2+ ) influx in MRGPRX2-HEK293, MrgprB2-HEK293, and LAD2 cells but not in NC-HEK293 cells. After knockdown of MRGPRX2 expression in LAD2 cells, the degree of iohexol-induced degranulation was reduced. In addition, after structural modification of iohexol by removal of iodine, a reduction in iohexol-induced effects, such as local and systemic anaphylaxis in mice and degranulation of LAD2 cells, could be observed. Iohexol was shown to induce immediate-type pseudo-allergic reactions via MRGPRX2, which was dependent on the presence of iodine., Conclusions: Conclusively, inhibition of MRGPRX2-mediated mast cell degranulation and cytokine release is important to prevent iohexol-induced immediate-type pseudo-allergic reactions., (Copyright © 2021 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Background: Neuromuscular-blocking agents (NMBAs) can cause both IgE-dependent and IgE-independent anaphylactic reactions, with activation of the mast cell receptor MRGPRX2 being important to the latter. Sugammadex, a reversal agent for certain aminosteroid NMBAs, has been proposed as an antidote for these anaphylactic events with conflicting outcomes., Objective: We further characterize the involvement of MRGPRX2 in NMBA-induced mast cell activation and determine how this is influenced by sugammadex. We then apply these in vitro results to infer the possible utility of sugammadex in the acute management of non-IgE-dependent anaphylaxis., Methods: The LAD2 human mast cell line and a MRGPRX2 knock-down derivative were used to validate the involvement of MRGPRX2 and to test the effect of sugammadex on mast cell activation by NMBAs and other MRGPRX2 agonists., Results: All MRGPRX2 agonists tested were shown to induce MRGPRX2-dependent LAD2 mast cell calcium mobilization and cytokine release and all, apart from rocuronium, induced degranulation. Co-treatment of mast cells with sugammadex and some MRGPRX2 agonists significantly reduced cell activation, but if sugammadex was administered a few minutes following stimulation, degranulation was not attenuated. However, addition of sugammadex up to 180 min following LAD2 MRGPRX2 stimulation, significantly reduced CCL2 mRNA and protein induction., Conclusions and Clinical Relevance: We show that sugammadex, known to reverse muscle blockade by certain NMBAs, is also able to reduce MRGPRX2 activation by NMBAs and other, but not all, MRGPRX2 agonists. As sugammadex was ineffective in attenuating mast cell degranulation when added rapidly post MRGPRX2 activation, this suggests against the agent having efficacy in controlling acute symptoms of anaphylaxis to NMBAs caused by MRGPRX2 activation. Interestingly, however, sugammadex did impair MRGPRX2-induced CCL2 release, suggesting that it may have some benefit in perhaps dampening less well-defined adverse effects of MRGPRX2-dependent anaphylaxis associated with the more slowly elaborated mast cell mediators., (© 2020 John Wiley & Sons Ltd.)

Symptoms that resemble allergic reactions, such as pruritus, flushing, and hypotension, are common side effects of therapeutic drugs. In a true allergic reaction, Immunoglobulin E (IgE) antibodies recognize the drug and trigger mediator release from mast cells through cross-linking of IgE receptors. However, many drugs can bypass this pathway and can activate mast cells directly through MRGPRX2, a G protein-coupled receptor that responds to a wide range of small molecules, peptides, and proteins that have little in common except for a net positive charge. This review will provide an overview of MRGPRX2, including its expression pattern, studies of its pharmacology, and its orthologs. It also will review evidence for MRGPRX2 activation by many drugs closely associated with these reactions., (Copyright © 2021. Published by Elsevier B.V.)

Mast cells (MCs) are crucial effectors in inflammation and allergic reactions. The Mas-related G-protein-coupled receptor X2 (MRGPRX2) was the MC-specific receptor and play a key role in IgE-independent allergic reactions. The activation of the Nuclear factor erythroid derived 2-related factor 2 (Nrf2) is involved in IgE-mediated MC degranulation. Resveratrol (Res) is a polyphenolic compound in red wine and has been reported to exert a variety of pharmacological effects. In the current study, we investigated the effect of Res in regulating MRGPRX2-mediated MC activation and its underlyingmechanism. We demonstrated that Res reduced compound 48/80 (C48/80)-induced calcium flux in MCs and inhibited MCs degranulation in vitro. Res also suppressed C48/80-induced hind paw extravasation, active systemic anaphylaxis, and MCs degranulation in mouse models of pseudo-allergy in vivo. Furthermore, PCR and immunohistochemistry assay suggest that Res up-regulates Nrf2 expression and Nrf2 inhibitor attenuates the protective effects of Res. In conclusion, Res exerts an inhibitory effect on MRGPRX2-mediated MCs activation by targeting Nrf2 pathway and may present a promising new therapeutic agent for the treatment of MRGPRX2-dependent anaphylactoid reactions., (Copyright © 2021. Published by Elsevier B.V.)

The IL-1 family cytokine IL-33 activates and re-shapes mast cells (MCs), but whether and by what mechanisms it elicits cytokines in MCs from human skin remains poorly understood. The current study found that IL-33 activates CCL1, CCL2, IL-5, IL-8, IL-13, and TNF-α, while IL-1β, IL-6, IL-31, and VEGFA remain unaffected in cutaneous MCs, highlighting that each MC subset responds to IL-33 with a unique cytokine profile. Mechanistically, IL-33 induced the rapid (1-2 min) and durable (2 h) phosphorylation of p38, whereas the phosphorylation of JNK was weaker and more transient. Moreover, the NF-κB pathway was potently activated, as revealed by IκB degradation, increased nuclear abundance of p50/p65, and vigorous phosphorylation of p65. The activation of NF-κB occurred independently of p38 or JNK. The induced transcription of the cytokines selected for further study (CCL1, CCL2, IL-8, TNF-α) was abolished by interference with NF-κB, while p38/JNK had only some cytokine-selective effects. Surprisingly, at the level of the secreted protein products, p38 was nearly as effective as NF-κB for all entities, suggesting post-transcriptional involvement. IL-33 did not only instruct skin MCs to produce selected cytokines, but it also efficiently co-operated with the allergic and pseudo-allergic/neurogenic activation networks in the production of IL-8, TNF-α, CCL1, and CCL2. Synergism was more pronounced at the protein than at the mRNA level and appeared stronger for MRGPRX2 ligands than for FcεRI. Our results underscore the pro-inflammatory nature of an acute IL-33 stimulus and imply that especially in combination with allergens or MRGPRX2 agonists, IL-33 will efficiently amplify skin inflammation and thereby aggravate inflammatory dermatoses.

The Mas-related G-protein-coupled receptor X2 (MRGPRX2) is prominently expressed by mast cells and induces degranulation upon binding by different ligands. Its activation has been linked to various mast cell-related diseases, such as chronic spontaneous urticaria, atopic dermatitis and asthma. Therefore, inhibition of MRGPRX2 activity represents a therapeutic target for these conditions. However, the exact pathophysiology of this receptor is still unknown. In vitro research with mast cells is often hampered by the technical limitations of available cell lines. The human mast cell types LAD2 and HuMC (human mast cells cultured from CD34+ progenitor cells) most closely resemble mature human mast cells, yet have a very slow growth rate. A fast proliferating alternative is the human mast cell line HMC1, but they are considered unsuitable for degranulation assays due to their immature phenotype. Moreover, the expression and functionality of MRGPRX2 on HMC1 is controversial. Here, we describe the MRGPRX2 expression and functionality in HMC1 cells, and compare these with LAD2 and HuMC. We also propose a model to render HMC1 suitable for degranulation assays by pre-incubating them with latrunculin-B (Lat-B). Expression of MRGPRX2 by HMC1 was proven by RQ-PCR and flowcytometry, although at lower levels compared with LAD2 and HuMC. Pre-incubation of HMC1 cells with Lat-B significantly increased the overall degranulation capacity, without significantly changing their MRGPRX2 expression, phenotype or morphology. The MRGPRX2 specific compound 48/80 (C48/80) effectively induced degranulation of HMC1 as measured by CD63 membrane expression and β-hexosaminidase release, albeit in lower levels than for LAD2 or HuMC. HMC1, LAD2 and HuMC each had different degranulation kinetics upon stimulation with C48/80. Incubation with the MRGPRX2 specific inhibitor QWF inhibited C48/80-induced degranulation, confirming the functionality of MRGPRX2 on HMC1. In conclusion, HMC1 cells have lower levels of MRGPRX2 expression than LAD2 or HuMC, but are attractive for in vitro research because of their high growth rate and stable phenotype. HMC1 can be used to study MRGPRX2-mediated degranulation after pre-incubation with Lat-B, which provides the opportunity to explore MPRGRX2 biology in mast cells in a feasible way., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hermans, van Stigt, van de Meerendonk, Schrijver, van Daele, van Hagen, van Splunter and Dik.)

The identification of the Mas-related G-protein-coupled receptors (Mrgpr) as targets of diverse stimuli of mast cells (MCs), including neuropeptides and pseudo-allergy causing drugs, has placed these receptors at a prime position in MC research. However, the species-dependent diversity of these receptors raises the need for an adequate model for investigating the human MRGPRX2 receptor. RBL-2H3 cells, stably transfected with MRGPRX2 (RBL-MRGPRX2), are increasingly used for this purpose. Therefore, we investigated whether ectopically expressed MRGPRX2, in rat MCs, recapitulates its authentic signaling. To this purpose, we performed a broad comparative study of the responses of human LAD-2 MCs that express MRGPRX2 endogenously, and RBL-MRGPRX2 cells to compound 48/80, substance P and vancomycin, three proto-type ligands of MRGPRX2. We demonstrate that both models share similar dose-response relationships, kinetics and sensitivities to a wide range of signaling targeting drugs. Therefore, our results indicate that ectopically expressed MRGPRX2 preserves the signaling pathways employed to evoke human MC degranulation, which we show to rely on ERK1/2 MAP kinases, phospholipase C (PLC) and autophagy-related signaling. Importantly, we also show that the underlying mechanisms of MRGPRX2-triggered MC degranulation in either LAD-2 or RBL-MRGPRX2 cells are different from those elicited by its rodent orthologs.

Background: Allergic asthma is a common inflammatory lung disease associated with complex pathogenesis. Mast cell (MC) is one of the key drivers of allergic asthma, Mas-related G protein-coupled receptor X2 (MRGPRX2) on the MC could mediate MC activation and trigger a pseudo-allergic reaction. Imperatorin (IMP), the main active compound of Radix Angelicae Dahuricae, has been reported to exert various pharmacological effects. In this study, we focused on the therapeutical mechanism of IMP on MRGPRX2-induced pseudo-allergy and allergic asthma., Methods: We examined the effect of IMP on MRGPRX2 related mast cell activation in mouse peritoneal MC (MPMC), Human Laboratory of Allergic Disease 2 MCs (LAD2 cells) and Mrgprx2-expressing HEK293 cells. Molecular docking and Surface plasmon resonance (SPR) were taken to reveal the binding character between IMP and MRPGRX2. MRGPRX2 downstream proteins were also detected by western blotting. IgE-independent responses was evaluated by using passive cutaneous anaphylaxis (PCA) and active systemic anaphylaxis (ASA) models. The therapeutic effect of IMP on asthma was evaluated by a lung inflammation mouse model which was induced by ovalbumin (OVA)., Results: IMP was found to reduce substance P (SP) induced calcium flux and suppressed degranulation of MC. SP can promote the phosphorylation of ERK and CamKII, which regulates the synthesis of inflammatory factors such as MIP-2 and TNF-α in MC. In vivo assays revealed that IMP can mitigate SP-induced mouse PCA and ASA. IMP could also mitigate lung inflammation in an OVA induced mice model by inhibiting MC activation in the lung tissue. Furthermore, IMP binds well to MRGPRX2 protein. The binding constant (K D ) is 4.48 ± 0.49 × 10 -7 M. The data suggeste that IMP is a novel inhibitor of MRGPRX2 to treat allergic asthma., (Copyright © 2020. Published by Elsevier Inc.)

Mast cells are innate immune cells located at many barrier sites in the body and known to protect the host against environmental threats and to be involved in allergic diseases. More recently, new studies have investigated their roles in the regulation of skin inflammation and transmission of pain and itch sensations. Mast cell signalling through the Mas-related G protein-coupled receptor (MRGPR) X2 or its mouse orthologue MRGPRB2 has been reported to be one of the major mechanism by which mast cell can regulate such processes. MRGPRX2 and MRGPRB2 can induce mast cell degranulation upon binding to a broad panel of cationic molecules such as neuropeptides, bacteria-derived quorum sensing molecules, venom peptides, host defense peptides and, unfortunately, various FDA-approved drugs. Upon activation, mast cells release granule-associated proteases, lipids and multiple cytokines that can modulate vascular permeability, immune cells recruitment and activation status of tissue-projecting nociceptive sensory neurons (ie nociceptors). Here, we discuss the modality of MRGPRX2-dependent mast cell activation and its different consequences on the patterns of skin inflammation and associated diseases. We notably emphasize how MRGPRX2-dependent skin mast cell activation might trigger various pathological traits such as pruritus, pain and inflammation and therefore become a potential therapeutic target for inflammatory pain, itch, atopic dermatitis and drugs-induced injection site reactions., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Clozapine is one of the most widely used second-generation antipsychotics in clinic. However, allergy-like symptoms such as rash and angioedema have been reported frequently, and the mechanism is still not clear. Mas-related G protein-coupled receptor X2 (MRGPRX2) expressed on mast cells is a crucial receptor for drug induced pseudo-allergic reactions. Therefore, we explored whether the symptoms induced by clozapine were associated with allergic reaction through MRGPRX2., Methods: The effects of clozapine on pseudo-allergic reactions were evaluated by mast cells degranulation and calcium mobilization assay in vitro , and mice hindpaw swelling, serum histamine detection, avidin and H&E staining assay in vivo . The overexpressed MRGPRX2 cells membrane chromatography (MRGPRX2-HEK293/CMC), MRGPRX2-HEK293 cells calcium mobilization assay and molecular docking were applied to research the correlation between clozapine and MRGPRX2., Results: The study showed that clozapine induced the release of β-hexosaminidase, histamine and monocyte chemoattractant protein-1 (MCP-1), and trigged calcium mobilization in mast cells. In vivo , clozapine induced paw swelling, degranulation and vasodilation. Furthermore, clozapine could activate the calcium mobilization obviously in MRGPRX2-HEK293 cells, not in NC-HEK293 cells. Clozapine also had a good retention characteristic on MRGPRX2-HEK293/CMC column and the K D value is (2.33 ± 0.21)×10 -01 M., Conclusions: Our findings demonstrated that clozapine could induce pseudo-allergic reactions and MRGPRX2 might be the critical receptor for it.

The Mas-related G protein-coupled receptor X2 (MRGPRX2) is a multiligand receptor responding to various exogenous and endogenous stimuli. Being highly expressed on skin mast cells, MRGPRX2 triggers their degranulation and release of proinflammatory mediators, and it promotes multicellular signaling cascades, such as itch induction and transmission in sensory neurons. The expression of MRGPRX2 by skin mast cells and the levels of the MRGPRX2 agonists (eg, substance P, major basic protein, eosinophil peroxidase) are upregulated in the serum and/or skin of patients with inflammatory and pruritic skin diseases, such as chronic spontaneous urticaria or atopic dermatitis. Therefore, MRGPRX2 and its agonists might be potential biomarkers for the progression of cutaneous inflammatory diseases and the response to treatment. In addition, they may represent promising targets for prevention and treatment of signs and symptoms in patients with skin diseases or drug reactions. To assess this possibility, this review explores the role and relevance of MRGPRX2 and its activators in cutaneous inflammatory disorders and chronic pruritus., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Perioperative hypersensitivity (POH) to the neuromuscular blocking drug (NMBD) rocuronium was previously thought to be IgE and mast cell (MC)-mediated. However, the recent seminal observation that rocuronium induces degranulation in murine peritoneal MCs (PMCs) via Mas-related G protein-coupled receptor B2 (MrgprB2) led to the idea that POH to this drug involves the activation of MRGPRX2 (human ortholog of MrgprB2). Furthermore, based on the demonstration that a patient with POH to rocuronium displayed three missense mutations (M196I, L226P and L237P) in MRGPRX2's transmembrane domains, it was proposed that this hypersensitivity reaction resulted from aberrant activation of this receptor. We found that rocuronium at 20 µg/mL caused degranulation in mouse PMCs via MrgprB2 but required at least 500 µg/mL to induce degranulation in human MCs via MRGPRX2. Furthermore, RBL-2H3 cells transiently expressing M196I, L226P and L237P variants did not display enhanced degranulation in response to rocuronium when compared to the wild-type receptor. These findings provide the first demonstration that rocuronium induces degranulation in human MCs via MRGPRX2. Furthermore, the important differences between MrgprB2 and MRGPRX2 and the inability of rocuronium to induce enhanced response in cells expressing MRGPRX2 variants suggest that the mechanism of its POH is more complex than previously thought.

Thymic stromal lymphopoietin (TSLP) is released by epithelial cells following disturbed homeostasis to act as "alarmin" and driver of Th2-immunity. Aberrant TSLP expression is a hallmark of atopic diseases, including atopic dermatitis (AD). Mast cells (MCs) are overabundant in AD lesions and show signs of degranulation, but it remains unknown whether TSLP contributes to granule discharge. Degranulation of skin MCs proceeds via two major routes, i.e., FcεRI-dependent (allergic) and MRGPRX2-mediated (pseudo-allergic/neurogenic). Evidence is accumulating that MRGPRX2 may be crucial in the context of skin diseases, including eczema. The current study reveals TSLP as a novel priming factor of human skin MCs. Interestingly, TSLP selectively cooperates with MRGPRX2 to support granule discharge, while it does not impact spontaneous or FcεRI-driven exocytosis. TSLP-assisted histamine liberation triggered by compound 48/80 or Substance P, two canonical MRGPRX2 agonists, was accompanied by an increase in CD107a+ cells (a MC activation marker). The latter process was less potent, however, and detectable only at the later of two time points, suggesting TSLP may prolong opening of the granules. Mechanistically, TSLP elicited phosphorylation of STAT5 and JNK in skin MCs and the reinforced degranulation critically depended on STAT5 activity, while JNK had a contributory role. Results from pharmacological inhibition were confirmed by RNA-interference, whereby silencing of STAT5 completely abolished the priming effect of TSLP on MRGPRX2-mediated degranulation. Collectively, TSLP is the first factor to favor MRGPRX2- over FcεRI-triggered MC activation. The relevance of TSLP, MCs and MRGPRX2 to pruritis and atopic skin pathology indicates broad repercussions of the identified connection.

Roxithromycin (ROX) is a macrolide antibiotic with a variety of immunological effects. Mast cells (MCs) play a key role in host defense, mediating hypersensitivity and pseudo-allergic reactions. Mas-related G protein-coupled receptor X2 (MrgprX2) is the main receptor related to pseudo-allergy. In this study, we investigated the anti-pseudo-allergy effect of ROX and its underlying mechanism. The effects of ROX on passive cutaneous anaphylaxis (PCA) and active systemic allergy were examined, degranulation, Ca 2+ influx, and cytokine release were studied in vivo and in vitro. Interactions between ROX and MrgprX2 protein were also detected through surface plasmon resonance. The PCA and active systemic allergy induced by compound 48/80 were inhibited by ROX. An intermolecular interaction was detected between the ROX and MrgprX2 protein. In conclusion, ROX could inhibit pseudo-allergic reactions, and this effect involves the Ca 2+ /PLC/IP3 pathway of MrgprX2. This study provides new insight into the anti-pseudo-allergy effects of ROX., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mast cells play key roles in allergy, anaphylaxis/anaphylactoid reactions, and defense against pathogens/toxins. These cells contain cytoplasmic granules with a wide spectrum of pleotropic mediators that are released upon activation. While mast cell degranulation (MCD) occurs upon clustering of the IgE receptor bound to IgE and antigen, MCD is also triggered through non-IgE-mediated mechanisms, one of which is via Mas-related G protein-coupled receptor X2 (MRGPRX2). MRGPRX2 can be activated by many basic biogenic amines and peptides. Consequently, MRGPRX2-mediated MCD is an important potential safety liability for peptide therapeutics. To facilitate peptide screening for this liability in early preclinical drug development, a rapid, high-throughput engineered CHO-K1 cell-based MRGPRX2 activation assay was evaluated and compared to histamine release in CD34 + stem cell-derived mature human mast cells as a reference assay, using 30 positive control and 29 negative control peptides for MCD. Both G protein-dependent (Ca 2+ endpoint) and -independent (β-arrestin endpoint) pathways were assessed in the MRGPRX2 activation assay. The MRGPRX2 activation assay had a sensitivity of 100% for both Ca 2+ and β-arrestin endpoints and a specificity of 93% (β-arrestin endpoint) and 83% (Ca 2+ endpoint) compared to histamine release in CD34 + stem cell-derived mature human mast cells. These findings suggest that assessing MRGPRX2 activation in an engineered cell model can provide value as a rapid, high-throughput, economical mechanism-based screening tool for early MCD hazard identification during preclinical safety evaluation of peptide-based therapeutics.

The discovery of MRGPRX2 marks an important change in MC biology, explaining non-IgE-mediated clinical phenomena relying on MCs. As receptor for multiple drugs, MRGPRX2 is crucial to drug-induced hypersensitivity. However, not only drugs, but also endogenous mediators like neuropeptides and host defense peptides activate MRGPRX2, suggesting its broad impact in cutaneous pathophysiology. Here, we give a brief overview of MRGPRX2 and its regulation by microenvironmental stimuli, which support MCs and can be altered in skin disorders, and briefly touch on the functional programs elicited by MRGPRX2 ligation. Studies in Mrgprb2-deficient mice (the murine ortholog) help illuminate MRGPRX2's function in health and disease. Recent advances in this model support the long-suspected operational unit between MCs and nerves, with MRGPRX2 being a vital component. Based on the limited evidence for a major contribution of FcεRI/IgE-activated MCs to atopic dermatitis (AD), we develop the hypothesis that MRGPRX2 constitutes the missing link connecting MCs and AD, at least in selected endotypes. Support comes from the multifold changes in the MC-neuronal system of AD skin (eg greater density of MCs and closer connections between MCs and nerves, increased PAR-2/Substance P). We theorize that these deregulations suffice to initiate AD, but external triggers, many of which activating MRGPRX2 themselves (eg Staphylococcus aureus) further feed into the loop. Itch, the most burdensome hallmark of AD, is mostly non-histaminergic but tryptase-dependent, and tryptase is preferentially released upon MRGPRX2 activation. Because MRGPRX2 is a very active research field, some of the existing gaps are likely to be closed soon., (© 2020 The Authors. Experimental Dermatology published by John Wiley & Sons Ltd.)

Dextromethorphan (DM) is a cough suppressant available in many prescribed and over-the-counter medications. Adverse reactions induced by DM have been regularly reported, including allergic skin reactions in some cases. However, the underlying mechanisms of local anaphylaxis induced by DM have not been elucidated. In this study, we found that DM could activate mast cells to increase calcium mobilization and release β-hexosaminidase, histamine, tumor necrosis factor-α, MCP-1, and IL-8 in a dose-dependent manner. The allergic reactions were confirmed by hind paw swelling and extravasation assay in vivo. Furthermore, DM was revealed to induce local anaphylaxis via MRGPRX2 by the mast cell-deficient kit W-sh/W-sh mice and MRGPRX2 knockdown mast cells. And the MRGPRX2-HEK293/CMC analysis and frontal analysis also showed that DM has a considerable affinity with MRGPRX2. Together, our findings suggest that close monitoring should be drawn on patients with DM for its potential anaphylaxis via MRGPRX2., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Numerous inflammatory skin disorders display a high prevalence of itch. The Mas-related G protein coupled receptor X2 (MRGPRX2) has been shown to modulate itch by inducing non-IgE-mediated mast cell degranulation and the release of endogenous inducers of pruritus. Various substances collectively known as basic secretagogues, which include inflammatory peptides and certain drugs, can trigger MRGPRX2 and thereby induce pseudo-allergic reactions characterized by histamine and protease release as well as inflammation. Here, we investigated the capacity of an immunomodulatory single-stranded oligonucleotide (ssON) to modulate IgE-independent mast cell degranulation and, more specifically, its ability to inhibit the basic secretagogues compound 48/80 (C48/80)-and LL-37 in vitro and in vivo . We examined the effect of ssON on MRGPRX2 activation in vitro by measuring degranulation in a human mast cell line (LAD2) and calcium influx in MRGPRX2-transfected HEK293 cells. To determine the effect of ssON on itch, we performed behavioral studies in established mouse models and collected skin biopsies for histological analysis. Additionally, with the use of a rosacea mouse model and RT-qPCR, we investigated the effect on ssON on LL-37-induced inflammation. We reveal that both mast cell degranulation and calcium influx in MRGPRX2 transfected HEK293 cells, induced by the antimicrobial peptide LL-37 and the basic secretagogue C48/80, are effectively inhibited by ssON in a dose-dependent manner. Further, ssON demonstrates a capability to inhibit LL-37 and C48/80 activation in vivo in two mouse models. We show that intradermal injection of ssON in mice is able to block itch induced via C48/80 in a dose-dependent manner. Histological staining revealed that ssON inhibits acute mast cell degranulation in murine skin treated with C48/80. Lastly, we show that ssON treatment ameliorates LL-37-induced inflammation in a rosacea mouse model. Since there is a need for new therapeutics targeting non-IgE-mediated activation of mast cells, ssON could be used as a prospective drug candidate to resolve itch and inflammation in certain dermatoses., (Copyright © 2020 Dondalska, Rönnberg, Ma, Pålsson, Magnusdottir, Gao, Adam, Lerner, Nilsson, Lagerström and Spetz.)

Mast cells play an important role in inflammatory and allergic diseases. MAS-related G protein-coupled receptor X2 (MRGPRX2) is a novel G protein-coupled receptor in mast cells that mediates drug-induced anaphylactoid reactions. Piperine has been reported to have anti-inflammatory and anti-allergic pharmacological activities. However, whether the pharmacological effects are regulated by MRGPRX2 has not yet been reported. The purpose of this study was to assess the anti-anaphylactoid effect of Piperine and to explore its potential mechanism. The anti-anaphylactoid effect of Piperine was assessed by an in vivo mouse hindpaw extravasation model. Mast cell intracellular calcium mobilization was measured by a calcium imaging assay. An enzyme immunoassay was used to evaluate the release of pro-inflammatory factors from stimulated mast cells. Activated mast cell related signals were assessed by western blot. A cell membrane chromatography assay was used to determine the binding characteristics of Piperine and MRGPRX2. The results showed that Piperine suppressed mast cell intracellular Ca 2+ mobilization, inhibited cytokines and chemokines release, and down-regulated the phosphorylation level of phospholipase Cγ1, protein kinase C, inositol 1,4,5-triphate receptor, P38, protein kinase B, and ERK. Meanwhile, Piperine can bind to MRGPRX2 as a specific antagonist. Hence, Piperine can be served as a novel therapeutic drug candidate for MRGPRX2-mediated anaphylactoid reactions., (© 2020 John Wiley & Sons, Ltd.)

Objectives: Screen and identify the anti-pseudo-allergic activity components of Perilla frutescens leaves that interacted with MRGPRX2 (a new reported pseudo-allergic reaction-related receptor)., Methods: An overexpressed MRGPRX2 cell membrane chromatography (CMC) coupled with HPLC-ESI-IT-TOF system has been established to screen and identify the effective components from P. frutescens leaves. A frontal analysis method was performed to investigate the binding affinity between ligands and MRGPRX2. Their activity of relieving pseudo-allergic reaction was evaluated in vitro by histamine release assay, β-hexosaminidase release assay and intracellular Ca 2+ mobilization assay., Key Findings: Extract of P. frutescens leaves was proved to be effective in anti-pseudo-allergic reaction by inhibiting MRGPRX2. Apigenin (API) and rosmarinic acid (ROS) were confirmed to be the potential anti-allergy compounds that could bind with MRGPRX2. The binding affinity (K D ) of ROS and API with MRGPRX2 was (8.79 ± 0.13) × 10 -8  m and (6.54 ± 1.69) × 10 -8  m, respectively. The IC 50 of API inhibiting laboratory of allergic disease 2 cells degranulation was also determined to be (51.96 ± 0.18) μm., Conclusions: A MRGPRX2/CMC coupled with HPLC-ESI-IT-TOF system was successfully established and applied to discover the effective components from P. frutescens leaves., (© 2020 Royal Pharmaceutical Society.)

Objective: Mice and rats are important animal models for mast cell (MC) study. However, rat Mas-related-GPCR-B3 receptor (MRGPRB3) has been less studied than its mouse counterpart. Therefore, we aimed to characterize rat MRGPRB3., Methods: Mrgprb3 mRNA expression was assessed in peritoneal cells (RPCs) and peritoneal MCs (RPMCs) of wild-type rats, RPCs of MC-deficient rats, and RBL-2H3 cells by reverse-transcriptase polymerase chain reaction (RT-PCR). RPMCs, MRGPRX2-transfected and non-transfected RBL-2H3 cells were activated by 15-30 min incubation with DNP-BSA, substance-P (SP), or compound-48/80. L732138 or CP96344 was used as a tachykinin/neurokinin-1-receptor antagonist. Histamine release from MCs was measured by HPLC fluorometry., Results: Mrgprb3 mRNA expression was found in all cells, with the highest level in wild-type RPCs. All cells responded to DNP-BSA, but only MRGPRX2-transfected-RBL-2H3 cells and RPMCs responded to all activators. L732138 (0.1-10 μM) and CP96344 (1-100 μM) suppressed SP (10 μM)-induced RPMC activation. L732138 inhibition was dose independent, whereas CP96344 inhibition occurred in a dose-dependent manner. Additionally, only CP96344 suppressed SP (100 μM)- and compound-48/80 (10 μg/mL)-induced RPMC activation., Conclusions: RPMCs expressing functional MRGPRB3 response upon MRGPRX2 ligands to regulated MC-mediated activities. It`s provide novel insights for future pseudo-allergic studies in rodents.

Background: Mast cells (MCs) are crucial effectors in allergic disorders by secreting inflammatory mediators. The Mas-related G-protein-coupled receptor X2 (Mrgprx2) was shown to have a key role in IgE-independent allergic reactions. Therefore, potential drug candidates that directly target Mrgprx2 could be used to treat pseudo-allergic diseases. Shikonin, an active ingredient derived from Lithospermum erythrorhizon Sieb. et Zucc has been used for its anti-inflammatory properties since ancient China., Purpose: To investigate the inhibitory effects of Shikonin on IgE-independent allergy both in vitro and in vivo, as well as the mechanism underlying its effects., Methods/study Designs: The anti-anaphylactoid activity of Shikonin was evaluated in PCA and systemic anaphylaxis models, Calcium imaging was used to assess intracellular Ca 2+ mobilization. The release of cytokines and chemokines was measured using enzyme immunoassay kits. Western blot analysis was conducted to investigate the molecules of PLCγ-PKC-IP3 signaling pathway. The analytical method of surface plasmon resonance was employed to study the interaction between Shikonin and potential target protein Mrgprx2., Results: Shikonin can suppress compound 48/80 (C48/80)-induced PCA, active systemic anaphylaxis, and MCs degranulation in mice in a dose-dependent manner. In addition, Shikonin reduced C48/80-induced calcium flux and suppressed LAD2 cell degranulation via PLCγ-PKC-IP3 signaling pathway. Moreover, Shikonin was found to inhibit C48/80-induced Mrgprx2 expression in HEK cells, displaying specific interactions with the Mrgprx2 protein., Conclusion: Shikonin could be a potential antagonist of Mrgprx2, thereby inhibiting pseudo-allergic reactions through Ca 2+ mobilization., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Anaphylactoid shock is a fatal hypersensitivity response caused by non-IgE mediated mast cell activation. These reactions are mediated by a family of G protein-coupled receptors (GPCRs) known as Mas related GPCRX2 (MRGPRX2). Several US FDA approved drugs which are used in day to day life have been reported to cause anaphylactoid shock. Surprisingly, no therapeutic drugs are available which can directly target MRGPRX2 for treatment of anaphylactoid shock. Genistein is a non-steroidal polyphenol known for its diverse physiological and pharmacological activities. In recent studies, Genistein has been reported for its anti-inflammatory activity on mast cells. However, the effects and mechanistic pathways of Genistein on anaphylactoid reaction remain unknown. In the present study, we designed a battery of in-vitro, in-silico and in-vivo experiments to evaluate the anti-anaphylactoid activity of Genistein in order to understand the possible molecular mechanisms of its action. The in-vitro results demonstrated the inhibitory activity of Genistein on MRGPRX2 activation. Further, a mouse model of anaphylactoid shock was used to evaluate the inhibitory activity of Genistein on blood vessel leakage and hind paw edema. Taken together, our findings have demonstrated a therapeutic potential of Genistein as a lead compound in the treatment of anaphylactoid shock via MRGPRX2., Competing Interests: “The authors declare no conflict of interest.”

Mas-related G protein-coupled receptor-X2 (MRGPRX2) expressed on mast cells (MCs) has been shown to be a pivotal target for pseudo-allergic diseases. Therefore, MRGPRX2 might be a therapeutic target for allergic contact dermatitis, atopic dermatitis, and red man syndrome. Paeoniflorin (PF) was reported to have an antiinflammatory effect in neuroinflammation, enteritis, and so forth. In this study, we investigated the anti-pseudo-allergic effect of PF and the underlying molecular mechanisms. Our results showed that PF can suppress compound 48/80 (C48/80)-induced PCA and MCs degranulation in vivo, in a dose-dependent manner. Moreover, PF can reduce C48/80-induced calcium influx and suppress MC degranulation and chemokines release in vitro. PF can downregulate the phosphorylation levels of key kinases in PLCγ-regulated calcium influx and subsequent cytokine synthesis pathways. Our study revealed that PF could inhibit C48/80-induced allergic responses both in vivo and in vitro. As such, it may be regarded as a novel inhibitor for preventing MRGPRX2-mediated allergic diseases., (© 2019 John Wiley & Sons, Ltd.)

Basophils and mast cells (MCs) are important effector cells in the immune system. For a long time, it has been known that these cells can be activated though the cross-linking of IgE antibodies bound to their high-affinity receptor (FcεRI). However, evidence has accumulated suggesting that these cells can also be activated by various IgE-independent mechanisms. Occupation of MAS Related GPR Family Member X2 (MRGPRX2), a G protein-coupled receptor, is described as an alternative IgE-independent activation mechanism. Here we describe a flow cytometric technique to analyze MRGPRX2 expression and its functionality on cultured human MCs and conditioned basophils, that is, basophils with upregulated surface expression of MRGPRX2.

Activation of MrgX2, an orphan G protein-coupled receptor expressed on mast cells, leads to degranulation and histamine release. Human MrgX2 binds promiscuously to structurally diverse peptides and small molecules that tend to have basic properties (basic secretagogues), resulting in acute histamine-like adverse drug reactions of injected therapeutic agents. We set out to identify MrgX2 orthologues from other mammalian species used in nonclinical stages of drug development. Previously, the only known orthologue of human MrgX2 was from mouse, encoded by Mrgprb2 . MrgX2 genes of rat, dog (beagle), minipig, pig, and Rhesus and cynomolgus monkey were identified by bioinformatic approaches and verified by their ability to mediate calcium mobilization in transfected cells in response to the classical MrgX2 agonist, compound 48/80. The peptide GSK3212448 is an inhibitor of the PRC2 epigenetic regulator that caused profound anaphylactoid reactions upon intravenous infusion to rat. We showed GSK3212448 to be a potent MrgX2 agonist particularly at rat MrgX2. We screened sets of drug-like molecules and peptides to confirm the highly promiscuous nature of MrgX2. Approximately 20% of drug-like molecules activated MrgX2 (pEC 50 ranging from 4.5 to 6), with the principle determinant being basicity. All peptides tested of net charge +3 or greater exhibited agonist activity, including the cell penetrating peptides polyarginine (acetyl-Arg 9 -amide) and TAT (49-60), a fragment of HIV-1 TAT protein. Finally, we showed that the glycopeptide antibiotic vancomycin, which is associated with clinical pseudo-allergic reactions known as red man syndrome, is an agonist of MrgX2., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2019 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

The neuropeptide substance P (SP) contributes to neurogenic inflammation through the activation of human mast cells via Mas-related G protein-coupled receptor-X2 (MRGPRX2). Using pertussis toxins and YM-254890, we demonstrated that SP induces Ca 2+ mobilization and degranulation via both the Gαi and Gαq family of G proteins in rat basophilic leukemia (RBL-2H3) cells stably expressing MRGPRX2. To determine the roles of MRGPRX2's transmembrane (TM) and intracellular domains on SP-induced responses, we utilized information obtained from both structural modeling and naturally occurring MRGPRX2 missense variants. We found that highly conserved residues in TM6 (I225) and TM7 (Y279) of MRGPRX2 are essential for SP-induced Ca 2+ mobilization and degranulation in transiently transfected RBL-2H3 cells. Cells expressing missense variants in the receptor's conserved residues (V123F and V282M) as well as intracellular loops (R138C and R141C) failed to respond to SP. By contrast, replacement of all five Ser/Thr residues with Ala and missense variants (S325L and L329Q) in MRGPRX2's carboxyl-terminus resulted in enhanced mast cell activation by SP when compared to the wild-type receptor. These findings suggest that MRGPRX2 utilizes conserved residues in its TM domains and intracellular loops for coupling to G proteins and likely undergoes desensitization via phosphorylation at Ser/Thr residues in its carboxyl-terminus. Furthermore, identification of gain and loss of function MRGPRX2 variants has important clinical implications for SP-mediated neurogenic inflammation and other chronic inflammatory diseases.