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Sinomenine hydrochloride (SH) is commonly used in the treatment of rheumatoid arthritis. It activates mast cells and induces anaphylaxis in the clinical setting. Adverse drug reactions can be caused by activation of MAS-associated G proteincoupled receptor X2 (MRGPRX2) on mast cells. Because the ligand binding site of MRGPRX2 is easily contacted in dilute solvents, it can be activated by many opioid drug structures. N-Demethylsinomenine (M-3) has a similar chemical structure to that of the opioid scaffold and is a major metabolite of SH. We sought to clarify whether M-3 induces anaphylaxis synergistically with its prototype in a mouse model. Molecular docking computer simulations suggested a similar binding effect between M-3 and SH. M-3 was chemically synthesized and analyzed by surface plasmon resonance to reveal its affinity for MRGPRX2. Temperature monitoring, in vivo hindlimb swelling and exudation test, and in vitro mast cell degranulation test were used to explore the mechanism of MRGPrx2 mediated allergic reaction triggered by M-3. Reduced M-3-induced inflammation was evident in MrgprB2 (the ortholog of MRGPRX2) conditional (Cpa3-Cre/MrgprB2flox) knockout (MrgprB2-CKO) mice. Additionally, LAD2 human mast cells with MRGPRX2 knockdown showed reduced degranulation. M-3 activated LAD2 cells synergistically with SH as regulated by GRK2 signaling and IP3R/PLC/PKC/P38 molecular signaling pathways. The results indicate that the M-3 metabolite can activate mast cells synergistically with its prototype SH via MRGPRX2 and aggravate anaphylaxis. These findings provide important insights into drug safety. [ABSTRACT FROM AUTHOR]

Chenrui Zhao,1,2,* Yuanyuan Ding,2,* Yihan Huang,2 Chao Wang,2 Bin Guo,1 Tao Zhang2 1Department of Anesthesiology, Xi’an Honghui Hospital, Xi’an Jiaotong University, Xi’an, 710054, People’s Republic of China; 2College of Pharmacy, Xi’an Jiaotong University, Xi’an, 710061, People’s Republic of China*These authors contributed equally to this workCorrespondence: Bin Guo, Department of Anesthesiology, Xi’an Honghui Hospital, Xi’an Jiaotong University, Xi’an, 710054, People’s Republic of China, Email xuan_310755@sina.comBackground: CMRF35-like molecule-1 (CLM-1) is a receptor of the CD300 family that inhibits MRGPRX2-mediated mast cell degranulation. Understanding the role and mechanism of CLM-1 agonist has significant implications for the treatment of allergic disease. Quercetin is a natural small molecule compound derived from plants and vegetables that has been shown to prevent histamine release by immune cells.Objective: This study aims to examine the inhibitory effects of quercetin on MRGPRX2-mediated mast cell degranulation via CLM-1.Results: We found that C48/80 stimulation resulted in significantly increased release of β-hexosaminidase, histamine and Ca2+ in CLM-1-knockdown LAD2 cells than in NC-LAD2 cells. Surface plasmon resonance (SPR) and molecular docking analyses revealed high-affinity binding between quercetin and CLM-1 (KD = 2.962× 10− 5 mol/L) mediated by the formation of hydrogen bonds. In addition, quercetin can selectively bind to CLM-1 on mast cells, leading to SHP-1 phosphorylation and subsequent inhibition of downstream MyD88/IKK/NF-κB signaling. Furthermore, activation of CLM-1 modulated the surface expression of MRGPRX2 by inhibiting F-actin, leading to internalization of the MRGPRX2 receptor via the PI3K/AKT/ Rac1/Cdc42 pathway.Conclusion: Quercetin is a promising treatment for allergic diseases by acting as a CLM-1 agonist that inhibits MRGPRX2-mediated mast cell degranulation. Keywords: quercetin, CLM-1, inhibition, MRGPRX2, mast cell degranulation

Mast cells (MCs) are tissue-resident immune cells of a hematopoietic origin that play vital roles in innate and adaptive immunity. Human MCs can be isolated and differentiated from various tissue sources, including cord blood, when supplemented with cytokines such as stem cell factor, interleukin 3, and interleukin 6. Our current research study has shown significant differences in the marker expressions of human cord blood-derived mast cells (hCBMCs) based on donor dependency and the type of medium used for culturing and differentiation. These findings are particularly relevant given the challenges of obtaining specialty media influencing MC phenotypic marker expressions. We found that hCBMCs cultured in StemSpanTM-XF medium had a moderate expression of mast/stem cell growth factor receptor Kit (c-KIT) (mRNA and protein), low expressions of FcεRI (mRNA) and TLR2 (mRNA and protein) but had high levels of MRGPRX2 (mRNA and protein) expressions. In contrast, hCBMCs cultured in Stem Line II medium expressed FcεRI and TLR2 (mRNA and protein) with higher c-KIT but had lower MRGPRX2 expressions compared to the hCBMCs cultured in the StemSpanTM-XF medium. These results suggest that it is crucial to consider both donor dependency and the medium when investigating MC functions and that further research is needed to fully understand the impact of these factors on the hCBMC marker expressions.

Background: Mast cells are critically involved in IgE-mediated diseases, e.g., allergies and asthma. Human mast cells are heterogeneous, and mast cells from different anatomical sites have been shown to respond differently to certain stimuli and drugs. The origin of the mast cells is therefore of importance when setting up a model system, and human lung mast cells are highly relevant cells to study in the context of asthma. We therefore set out to optimize a protocol of IgE-mediated activation of human lung mast cells. Methods: Human lung mast cells were extracted from lung tissue obtained from patients undergoing pulmonary resection by enzyme digestion and mechanical disruption followed by CD117 magnetic-activated cell sorting (MACS) enrichment. Different culturing media and conditions for the IgE-mediated degranulation were tested to obtain an optimized method. Results: IgE crosslinking of human lung mast cells cultured in serum-free media gave a stronger response compared to cells cultured with 10% serum. The addition of stem cell factor (SCF) did not enhance the degranulation. However, when the cells were put in fresh serum-free media 30 minutes prior to the addition of anti-IgE antibodies, the cells responded more vigorously. Maximum degranulation was reached 10 minutes after the addition of anti-IgE. Both CD63 and CD164 were identified as stable markers for the detection of degranulated mast cells over time, while the staining with anti-CD107a and avidin started to decline 10 minutes after activation. The levels of CD203c and CD13 did not change in activated cells and therefore cannot be used as degranulation markers of human lung mast cells. Conclusions: For an optimal degranulation response, human lung mast cells should be cultured and activated in serum-free media. With this method, a very strong and consistent degranulation response with a low donor-to-donor variation is obtained. Therefore, this model is useful for further investigations of IgE-mediated mast cell activation and exploring drugs that target human lung mast cells, for instance, in the context of asthma. [ABSTRACT FROM AUTHOR]

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-1G 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-1&, 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

IntroductionBecause MRGPRX2 is now recognized as the mast cell receptor for basic secretagogues, there is currently a tremendous interest in whether MRGRPX2 could play an important role in various pruritic dermatoses such as chronic spontaneous urticaria. Therefore, we sought to identify new potent and selective antagonists to pharmacologically characterize the biological role of MRGPRX2.MethodsVarious relevant in vitro, ex vivo, and in vivo model systems were used to investigate the role of MRGPRX2. This included the study of freshly isolated human skin mast cells and human basophils as well as an ex vivo human skin microdialysis preparation. The additivity of MRGPRX2 and FcεR1-mediated degranulation was also investigated. Human MRGPRX2 knock-in mice were generated to interrogate pharmacokinetic/pharmacodynamic relationships because both antagonists studied were shown to be human specific.ResultsTwo novel and structurally distinct MRGPRX2 antagonists were identified with one, Compound B, being orally active and demonstrating high potency in blocking Substance P–mediated degranulation using freshly isolated human skin mast cells with half maximal inhibitory concentration (IC50) at 0.42 nM. Compound B also potently blocked Substance P–stimulated histamine release from resident mast cells in a human skin explant setup as well as blocking itch in an established behavioral scratching model using MRGPRX2 knock-in mice. Unlike human mast cells, Substance P failed to elicit a functional response in human basophils.ConclusionThese data fully support the investigation of MRGPRX2 receptor antagonists in mast cell–driven allergic skin disorders such as chronic spontaneous urticaria.

Mast cells (MCs) are immune cells that reside in tissues; particularly in the skin, and in the gastrointestinal and respiratory tracts. In recent years, there has been considerable interest in the Mas-Related G Protein-Coupled Receptor X2 (MRGPRX2), which is present on the surface of MCs and can be targeted by multiple exogenous and endogenous ligands. It is potentially implicated in non-IgE-mediated pseudoallergic reactions and inflammatory conditions such as asthma or atopic dermatitis. In this paper, we review natural products and herbal medicines that may potentially interact with MRGPRX2. They mainly belong to the classes of polyphenols, flavonoids, coumarins, and alkaloids. Representative compounds include rosmarinic acid, liquiritin from licorice extract, osthole, and sinomenine, respectively. While evidence-based medicine studies are still required, these compounds have shown diverse effects, such as antioxidant, analgesic, anti-inflammatory, or neuroprotective. However, despite potential beneficial effects, their use is also burdened with risks of fatal reactions such as anaphylaxis. The role of MRGPRX2 in these reactions is a subject of debate. This review explores the literature on xenobiotic compounds from herbal medicines that have been shown to act as MRGPRX2 ligands, and their potential clinical significance.

Mas-related G-protein-coupled receptor X2 (MRGPRX2), expressed on mast cells, is associated with drug-induced pseudo-allergic reactions. Although it is well known that there are differences of sensitivity between species in the pseudo-allergic reactions, no platform for evaluating a human risk of the pseudo-allergic reactions observed in nonclinical studies has been established. Valemetostat tosylate, developed as an anti-cancer drug, induced histamine release in a nonclinical study with dogs. The purpose of the current study was to identify the mechanism and assess the human risk of valemetostat- tosylate-induced histamine release using dog and human MRGPRX2-expressing cells. In an experiment with human or dog MRGPRX2-expressing cells, valemetostat tosylate caused activation of human and dog MRGPRX2. Importantly, the EC50 for dog MRGPRX2 was consistent with the Cmax value at which histamine release was observed in dogs. Furthermore, the EC50 for human MRGPRX2 was ca. 27-fold higher than that for dog MRGPRX2, indicating a species difference in histamine-releasing activity. In a clinical trial, histamine release was not observed in patients receiving valemetostat tosylate. In conclusion, an in vitro assay using human and animal MRGPRX2-expressing cells would be an effective platform to investigate the mechanism and predict the human risk of histamine release observed in nonclinical studies. [ABSTRACT FROM AUTHOR]

Mast cells (MCs) are immune cells that reside in tissues; particularly in the skin, and in the gastrointestinal and respiratory tracts. In recent years, there has been considerable interest in the Mas-Related G Protein-Coupled Receptor X2 (MRGPRX2), which is present on the surface of MCs and can be targeted by multiple exogenous and endogenous ligands. It is potentially implicated in non-IgE-mediated pseudoallergic reactions and inflammatory conditions such as asthma or atopic dermatitis. In this paper, we review natural products and herbal medicines that may potentially interact with MRGPRX2. They mainly belong to the classes of polyphenols, flavonoids, coumarins, and alkaloids. Representative compounds include rosmarinic acid, liquiritin from licorice extract, osthole, and sinomenine, respectively. While evidence-based medicine studies are still required, these compounds have shown diverse effects, such as antioxidant, analgesic, anti-inflammatory, or neuroprotective. However, despite potential beneficial effects, their use is also burdened with risks of fatal reactions such as anaphylaxis. The role of MRGPRX2 in these reactions is a subject of debate. This review explores the literature on xenobiotic compounds from herbal medicines that have been shown to act as MRGPRX2 ligands, and their potential clinical significance. [ABSTRACT FROM AUTHOR]

Mast cells (MCs) are an important part of the immune system, responding both to pathogens and toxins, but they also play an important role in allergic diseases, where recent data show that non-IgE-mediated activation is also of relevance, especially in chronic urticaria (CU) and atopic dermatitis (AD). Skin MCs express Mas-related G-protein-coupled receptor X2 (MRGPRX2), a key protein in non-IgE-dependent MC degranulation, and its overactivity is one of the triggering factors for the above-mentioned diseases, making MRGPRX2 a potential therapeutic target. Reviewing the latest literature revealed our need to focus on the discovery of MRGPRX2 activators as well as the ongoing vast research towards finding specific MRGPRX2 inhibitors for potential therapeutic approaches. Most of these studies are in their preliminary stages, with one drug currently being investigated in a clinical trial. Future studies and improved model systems are needed to verify whether any of these inhibitors may have the potential to be the next therapeutic treatment for CU, AD, and other pseudo-allergic reactions. [ABSTRACT FROM AUTHOR]

IntroductionAtopic 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. MethodsWe 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 DiscussionOur 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.

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.

The mast cell receptor Mrgprb2, a mouse orthologue of human Mrgprx2, is known as an inflammatory receptor and its elevated expression is associated with various diseases such as ulcerative colitis. We aimed to elucidate the role of Mrgprb2/x2 and the effect of its ligands on a chemically induced murine colitis model. We showed that in Mrgprb2-/- mice, there is a differential regulation of cytokine releases in the blood plasma and severe colonic damages after DSS treatment. Unexpectedly, we demonstrated that known Mrgprb2/x2 agonists (peptide P17, P17 analogues and CST-14) and antagonist (GE1111) similarly increased the survival rate of WT mice subjected to 4% DSS-induced colitis, ameliorated the colonic damages of 2.5% DSS-induced colitis, restored major protein mRNA expression involved in colon integrity, reduced CD68+ and F4/80+ immune cell infiltration and restored cytokine levels. Collectively, our findings highlight the eminent role of Mrpgrb2/x2 in conferring a beneficial effect in the colitis model, and this significance is demonstrated by the heightened severity of colitis with altered cytokine releases and inflammatory immune cell infiltration observed in the Mrgprb2 knockout mice. Elevated expression of Mrgprb2 in WT colitis murine models may represent the organism’s adaptive protective mechanism since Mrgprb2 knockout results in severe colitis. On the other hand, both agonist and antagonist of Mrgprb2 analogously mitigated the severity of colitis in DSS-induced colitis model by altering Mrgprb2 expression, immune cell infiltration and inflammatory cytokine releases.

MRGPRX2, the human member of the MAS-related G-protein-coupled receptors (GPCRs), mediates the immunoglobulin E (IgE)-independent responses of a subset of mast cells (MCs) that are associated with itch, pain, neurogenic inflammation, and pseudoallergy to drugs. The mechanisms underlying the responses of MRGPRX2 to its multiple and diverse ligands are still not completely understood. Given the close association between GPCR location and function, and the key role played by Rab GTPases in controlling discrete steps along vesicular trafficking, we aimed to reveal the vesicular pathways that directly impact MRGPRX2-mediated exocytosis by identifying the Rabs that influence this process. For this purpose, we screened 43 Rabs for their functional and phenotypic impacts on MC degranulation in response to the synthetic MRGPRX2 ligand compound 48/80 (c48/80), which is often used as the gold standard of MRGPRX2 ligands, or to substance P (SP), an important trigger of neuroinflammatory MC responses. Results of this study highlight the important roles played by macropinocytosis and autophagy in controlling MRGPRX2-mediated exocytosis, demonstrating a close feedback control between the internalization and post-endocytic trafficking of MRGPRX2 and its triggered exocytosis. [ABSTRACT FROM AUTHOR]

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Simple Summary: Anaphylaxis to paclitaxel requires desensitization protocols for safe drug re-administration. Hypersensitivity severity and skin tests (STs) guide desensitization protocol choices. The only study reporting on the basophil activation test (BAT) in diagnosing hypersensitivity to paclitaxel showed unsatisfactory diagnostic performance. Here, we present a modified BAT that shows unprecedented sensitivity (90.91%) and specificity (90.91%) in diagnosing hypersensitivity to paclitaxel and demonstrate that most patients with hypersensitivity to paclitaxel who score ST-negative score BAT-positive. We propose that the present BAT should be used to personalize patient management. Moreover, we predict the interactions of paclitaxel with the degranulation-competent MRGPRX2 receptor using an in silico molecular docking study. Immediate hypersensitivity reactions (iHSRs) to taxanes are observed in 6% and 4% of gynecologic and breast cancer patients, respectively. Drug desensitization is the only option, as no comparable alternative therapy is available. Surfactants in the taxane formulation have been implicated in the immunopathogenesis of iHSRs, although sporadic skin test (ST) positivity and iHSRs to nab-paclitaxel have suggested the involvement of the taxane moiety and/or IgE-mediated pathomechanisms. In vitro diagnostic tests might offer insights into mechanisms underlying iHSRs to taxanes. The aim of the present study was to address this unmet need by developing a novel basophil activation test (BAT). The study included patients (n = 31) undergoing paclitaxel/carboplatin therapy. Seventeen patients presented with iHSRs to paclitaxel (iHSR-Taxpos), and eleven were tolerant (iHSR-Taxneg). Fourteen patients presented with iHSRs to carboplatin (iHSR-Plpos), and fourteen were tolerant (iHSR-Plneg). The BAT median stimulation index (SI) values were 1.563 (range, 0.02–4.11; n = 11) and −0.28 (range −4.88–0.07, n = 11) in iHSR-Taxpos and iHSR-Taxneg, respectively. The BAT median SI values were 4.45 (range, 0.1–26.7; n = 14) and 0 (range, −0.51–1.65; n = 12) in iHSR-Plpos and iHSR-Plneg, respectively. SI levels were not associated with iHSR severity grading. Comparing BAT results in iHSR-Taxpos and iHSR-Taxneg showed the area under the receiver operator characteristic (ROC) curve to be 0.9752 (p = 0.0002). The cutoff calculated by the maximized likelihood ratio identified 90.91% of iHSR-Taxpos patients and 90.91% of iHSR-Taxneg patients. Comparing BAT results for iHSR-Plpos and iHSR-Plneg showed the area under the ROC curve to be 0.9286 (p = 0.0002). The cutoff calculated by the maximized likelihood ratio identified 78.57% of iHSR-Plpos patients and 91.67% of iHSR-Plneg patients. Most iHSR-Taxpos patients for which ST was available (10/11) scored ST-negative and BAT-positive, whereas most iHSR-Plpos patients for which ST was available (14/14) scored both BAT- and ST-positive. This suggested the intervention of non-IgE-mediated mechanisms in iHSR-Taxpos patients. Consistent with this view, an in silico molecular docking analysis predicted the high affinity of paclitaxel to the degranulation-competent MRGPRX2 receptor. This hypothesis warrants further in vitro investigations. In conclusion, the present study provides preliminary proof-of-concept evidence that this novel BAT has potential utility in understanding mechanisms underlying iHSRs to taxanes. [ABSTRACT FROM AUTHOR]

Skin mast cells (MCs) express high levels of MRGPRX2, FcεRI, and ST2, and vigorously respond to their ligands when triggered individually. IL-33/ST2 also potently synergizes with other receptors, but the molecular underpinnings are poorly understood. Human skin-derived MCs were stimulated via different receptors individually or jointly in the presence/absence of selective inhibitors. TNF was quantified by ELISA. Signaling cascades were studied by immunoblot. TNF was stimulated by FcεRI ≈ ST2 > MRGPRX2. Surprisingly, neither FcεRI nor MRGPRX2 stimulation elicited NF-κB activation (IκB degradation, p65 phosphorylation) in stark contrast to IL-33. Accordingly, TNF production did not depend on NF-κB in FcεRI- or MRGPRX2-stimulated MCs, but did well so downstream of ST2. Conversely, ERK1/2 and PI3K were the crucial modules upon FcεRI/MRGPRX2 stimulation, while p38 was key to the IL-33-elicited route. The different signaling prerequisites were mirrored by their activation patterns with potent pERK/pAKT after FcεRI/MRGPRX2, but preferential induction of pp38/NF-κB downstream of ST2. FcεRI/MRGPRX2 strongly synergized with IL-33, and some synergy was still observed upon inhibition of each module (ERK1/2, JNK, p38, PI3K, NF-κB). IL-33's contribution to synergism was owed to p38 > JNK > NF-κB, while the partner receptor contributed through ERK > PI3K ≈ JNK. Concurrent IL-33 led to slightly prolonged pERK (downstream of MRGPRX2) or pAKT (activated by FcεRI), while the IL-33-elicited modules (pp38/NF-κB) remained unaffected by co-stimulation of FcεRI/MRGPRX2. Collectively, the strong synergistic activity of IL-33 primarily results from the complementation of highly distinct modules following co-activation of the partner receptor rather than by altered signal strength of the same modules. [ABSTRACT FROM AUTHOR]

Background: Mycosis fungoides (MF) is an indolent T-cell lymphoma that mainly affects the skin and presents with itch in more than half of the patients. Recently, the expression of Mas-related G protein-coupled receptor X2 (MRGPRX2), a receptor of mast cell (MC) responsible for the IgE-independent non-histaminergic itch, has been shown in lesional skin of patients with pruritic skin diseases, including chronic urticaria, prurigo, and mastocytosis. As of yet, limited knowledge exists regarding the MRGPRX2 expression in the skin of patients with MF. Objectives: To investigate the number of MRGPRX2-expressing (MRGPRX2+) cells in the skin of patients with MF and its correlation with clinical and laboratory characteristics of the disease. Methods: MRGPRX2 was analyzed in lesional and non-lesional skin of MF patients and healthy skin tissues by immunohistochemistry. Co-localization of MRGPRX2 with the MC marker tryptase was assessed by immunofluorescence. Public single-cell RNAseq data was reanalyzed to identify the MRGPRX2 expression on the distinct cell types. Results: In lesional skin of MF patients, MRGPRX2+ cell number was higher than in non-lesional skin and healthy control skin (mean:15.12 vs. 6.84 vs. 5.51 cells/mm², p=0.04), and correlated with MC numbers (r=0.73, p=0.02). MC was the primary cell type expressing MRGPRX2 in MF patients. The ratio of MRGPRX2+ MCs to MRGPRX2+ cells in lesional and non-lesional skin correlated with the severity of disease (r=0.71, p=0.02 and r=0.67, p=0.03, respectively). Conclusions: Our findings point to the role of MRGPRX2 and MC in the pathogenesis of MF that should be investigated in further studies. [ABSTRACT FROM AUTHOR]

MRGPRX2, a novel Gaq‐coupled human mast cell receptor, mediates non‐immune adverse reactions without the involvement of antibody priming. Constitutively expressed by human skin mast cells, MRGPRX2 modulates cell degranulation producing pseudoallergies manifesting as itch, inflammation and pain. The term pseudoallergy is defined in relation to adverse drug reactions in general and immune/non‐immune‐mediated reactions in particular. A list of drugs with MRGPRX2 activity is presented, including a detailed examination of three important and widely used approved therapies: neuromuscular blockers, quinolones and opioids. For the clinician, the significance of MRGPRX2 is considered as an aid in distinguishing and ultimately identifying specific immune and non‐immune inflammatory reactions. Anaphylactoid/anaphylactic reactions, neurogenic inflammation and inflammatory diseases with a clear or strongly suspected association with MRGPRX2 activation are examined. Inflammatory diseases include chronic urticaria, rosacea, atopic dermatitis, allergic contact dermatitis, mastocytosis, allergic asthma, ulcerative colitis and rheumatoid arthritis. MRGPRX2‐ and allergic IgE/FcεRI‐mediated reactions may be clinically similar. Importantly, the usual testing procedures do not distinguish the two mechanisms. Currently, identification of MRGPRX2 activation and diagnosis of pseudoallergic reactions is generally viewed as a process of exclusion once other non‐immune and immune processes, particularly IgE/FcεRI‐mediated degranulation of mast cells, are ruled out. This does not take into account that MRGPRX2 signals via β‐arrestin, which can be utilized to detect MRGPRX2 activation by employing MRGPRX2 transfected cells to assess MRGPRX2 activation via two pathways, the G‐protein‐independent β‐arrestin pathway and the G‐protein‐dependent Ca2+ pathway. Testing procedures, interpretations for distinguishing mechanisms, patient diagnosis, agonist identification and drug safety evaluations are addressed. [ABSTRACT FROM AUTHOR]

Mast cells are activated through a variety of different receptors to release preformed granules and mediators synthesized de novo. However, the physiology and function of mast cells are not fully understood. Traditional studies of mast cell activation in humans have utilized cultures of tissue-derived mast cells including CD34+ progenitor cells or well-characterized commercially available cell lines. One limitation of these methods is that mast cells are no longer in a natural state. Therefore, their applicability to human skin disorders may be limited. Human skin explant models have been utilized to investigate the short-term effects of cell mediators, drugs, and irritants on skin while avoiding the ethical concerns surrounding in vivo stimulation studies with non-approved agents. Nonetheless, few studies have utilized intact human tissue to study mast cell degranulation. This “Methods” paper describes the development and application of an intact skin explant model to study human mast cell activation. In this manuscript, we share our protocol for setting up ex vivo human skin explants and describe the results of stimulation experiments and techniques to minimize trauma-induced histamine release. Skin explants were generated using de-identified, full-thickness, non-diseased skin specimens from plastic and reconstructive surgeries. Results were reproducible and demonstrated FcɛRI- and MRGPRX2-induced mediator release which was inhibited with the use of a BTK inhibitor and QWF, respectively. Thus, this explant model provides a quick and accessible method of assessing human skin mast cell activation and inhibition.

The signs and symptoms of chronic urticaria (CU) are caused by the activation and degranulation of skin mast cells (MCs). Recent studies have added to our understanding of how and why skin MCs are involved and different in CU. Also, novel and relevant mechanisms of MC activation in CU have been identified and characterized. Finally, the use of MC-targeted and MC mediator-specific treatments has helped to better define the role of the skin environment, the contribution of specific MC mediators, and the relevance of MC crosstalk with other cells in the pathogenesis of CU. Here, we review these recent findings and their impact on our understanding of CU, with a focus on chronic spontaneous urticaria (CSU). Also, we highlight open questions, issues of controversy, and unmet needs, and we suggest what studies should be performed moving forward.

Objectives: The activation of mast cell (MC) plays an important part in the pathogenesis of chronic urticaria (CU), and the expression of MRGPRX2 (Mas-related G-protein coupled receptor X2) and the circulating levels of SP (substance P) in skin MC of CU patients increased. Fisetin is a natural flavonoid with anti-inflammatory and antiallergic pharmacological effects. This study aimed to investigate the inhibitory effect of fisetin on CU via MRGPRX2 and its possible molecular mechanisms. Methods: OVA/SP co-stimulated and SP-stimulated CU like murine models were used to evaluate the effect of fisetin on CU. MRGPRX2/HEK293 cells and LAD2 cells were used to perform the antagonism effect of fisetin on MC via MRGPRX2. Key findings: The results indicated that fisetin prevented urticaria-like symptoms in murine CU models, and inhibited MCs activation by suppressing calcium mobilization and degranulation of cytokines and chemokines via binding to MRGPRX2. The bioinformatics analysis showed that fisetin might have an interaction relationship with Akt in CU. The western blotting experiments showed that fisetin downregulated the phosphorylation levels of Akt, P38, NF-κB, and PLCγ in C48/80 activated LAD2 cells. Conclusions: Fisetin alleviates CU progression by inhibiting mast cell activation via MRGPRX2, which may be a novel therapeutic candidate for CU. [ABSTRACT FROM AUTHOR]

Anaphylaxis is a life-threatening or even fatal systemic hypersensitivity reaction. The incidence of anaphylaxis has risen at an alarming rate in the past decades in the majority of countries. Generally, the most common causes of severe or fatal anaphylaxis are medication, foods and Hymenoptera venoms. Anaphylactic reactions are characterized by the activation of mast cells and basophils and the release of mediators. These cells express a variety of receptors that enable them to respond to a wide range of stimulants. Most studies of anaphylaxis focus on IgE-dependent reactions. The mast cell has long been regarded as the main effector cell involved in IgE-mediated anaphylaxis. This paper reviews IgE-independent anaphylaxis, with special emphasis on mast cells, basophils, anaphylactic mediators, risk factors, triggers, and management. [ABSTRACT FROM AUTHOR]

BackgroundMycosis fungoides (MF) is an indolent T-cell lymphoma that mainly affects the skin and presents with itch in more than half of the patients. Recently, the expression of Mas-related G protein-coupled receptor X2 (MRGPRX2), a receptor of mast cell (MC) responsible for the IgE-independent non-histaminergic itch, has been shown in lesional skin of patients with pruritic skin diseases, including chronic urticaria, prurigo, and mastocytosis. As of yet, limited knowledge exists regarding the MRGPRX2 expression in the skin of patients with MF.ObjectivesTo investigate the number of MRGPRX2-expressing (MRGPRX2+) cells in the skin of patients with MF and its correlation with clinical and laboratory characteristics of the disease.MethodsMRGPRX2 was analyzed in lesional and non-lesional skin of MF patients and healthy skin tissues by immunohistochemistry. Co-localization of MRGPRX2 with the MC marker tryptase was assessed by immunofluorescence. Public single-cell RNAseq data was reanalyzed to identify the MRGPRX2 expression on the distinct cell types.ResultsIn lesional skin of MF patients, MRGPRX2+ cell number was higher than in non-lesional skin and healthy control skin (mean:15.12 vs. 6.84 vs. 5.51 cells/mm2, p=0.04), and correlated with MC numbers (r=0.73, p=0.02). MC was the primary cell type expressing MRGPRX2 in MF patients. The ratio of MRGPRX2+ MCs to MRGPRX2+ cells in lesional and non-lesional skin correlated with the severity of disease (r=0.71, p=0.02 and r=0.67, p=0.03, respectively).ConclusionsOur findings point to the role of MRGPRX2 and MC in the pathogenesis of MF that should be investigated in further studies.

Mast cell (MC) activation is implicated in the pathogenesis of multiple immunodysregulatory skin disorders. Activation of an IgE-independent pseudo-allergic route has been recently found to be mainly mediated via Mas-Related G protein-coupled receptor X2 (MRGPRX2). Ryanodine receptor (RYR) regulates intracellular calcium liberation. Calcium mobilization is critical in the regulation of MC functional programs. However, the role of RYR in MRGPRX2-mediated pseudo-allergic skin reaction has not been fully addressed. To study the role of RYR in vivo, we established a murine skin pseudo-allergic reaction model. RYR inhibitor attenuated MRGPRX2 ligand substance P (SP)-induced vascular permeability and neutrophil recruitment. Then, we confirmed the role of RYR in an MC line (LAD2 cells) and primary human skin-derived MCs. In LAD2 cells, RYR inhibitor pretreatment dampened MC degranulation (detected by bhexosaminidase retlease), calcium mobilization, IL-13, TNF-α, CCL-1, CCL-2 mRNA, and protein expression activated by MRGPRX2 ligands, namely, compound 48/80 (c48/80) and SP. Moreover, the inhibition effect of c48/80 by RYR inhibitor was verified in skin MCs. After the confirmation of RYR2 and RYR3 expression, the isoforms were silenced by siRNA-mediated knockdown. MRGPRX2-induced LAD2 cell exocytosis and cytokine generation were substantially inhibited by RYR3 knockdown, while RYR2 had less contribution. Collectively, our finding suggests that RYR activation contributes to MRGPRX2-triggered pseudo-allergic dermatitis, and provides a potential approach for MRGPRX2-mediated disorders. [ABSTRACT FROM AUTHOR]

The signs and symptoms of chronic urticaria (CU) are caused by the activation and degranulation of skin mast cells (MCs). Recent studies have added to our understanding of how and why skin MCs are involved and different in CU. Also, novel and relevant mechanisms of MC activation in CU have been identified and characterized. Finally, the use of MC-targeted and MC mediator-specific treatments has helped to better define the role of the skin environment, the contribution of specific MC mediators, and the relevance of MC crosstalk with other cells in the pathogenesis of CU. Here, we review these recent findings and their impact on our understanding of CU, with a focus on chronic spontaneous urticaria (CSU). Also, we highlight open questions, issues of controversy, and unmet needs, and we suggest what studies should be performed moving forward. [ABSTRACT FROM AUTHOR]

MRGPRX2, a G-protein-coupled-seven transmembrane domain receptor, is mainly expressed in mast cells and neurons and is involved in skin immunity and pain. It is implicated in the pathophysiology of non-IgE-mediated immediate hypersensitivity and has been related to adverse drug reactions. Moreover, a role has been proposed in asthma, atopic dermatitis, contact dermatitis, and chronic spontaneous urticaria. Although it has a prominent role in disease, its signaling transduction is poorly understood. This study shows that MRGPRX2 activation with substance P increased Lysyl t-RNA synthetase (LysRS) translocation to the nucleus. LysRS is a moonlighting protein with a dual role in protein translation and IgE signaling in mast cells. Upon allergen- IgE-FcεRI crosslinking, LysRS is translocated to the nucleus and activates microphthalmia-associated transcription factor (MITF) activity. In this study, we found that MRGPRX2 triggering led to MITF phosphorylation and increased MITF activity. Therefore, overexpression of LysRS increased MITF activity after MRGPRX2 activation. MITF silencing reduced MRGPRX2-dependent calcium influx and mast cell degranulation. Furthermore, a MITF pathway inhibitor, ML329, impaired MITF expression, calcium influx, and mast cell degranulation. Moreover, drugs such as atracurium, vancomycin, and morphine, reported to induce MRGPRX2-dependent degranulation, increased MITF activity. Altogether, our data show that MRGPRX2 signaling enhances MITF activity, and its abrogation by silencing or inhibition resulted in defective MRGPRX2 degranulation. We conclude that MRGPRX2 signaling involves the LysRS and MITF pathway. Thus, MITF and MITF-dependent targets may be considered therapeutic approaches to treat pathologies where MRGPRX2 is implicated. [ABSTRACT FROM AUTHOR]

Mast cells play an important role in disease pathogenesis by secreting immunomodulatory molecules. Mast cells are primarily activated by the crosslinking of their high affinity IgE receptors (FceRI) by antigen bound immunoglobulin (Ig)E antibody complexes. However, mast cells can also be activated by the mas related G protein-coupled receptor X2 (MRGPRX2), in response to a range of cationic secretagogues, such as substance P (SP), which is associated with pseudo-allergic reactions. We have previously reported that the in vitro activation of mouse mast cells by basic secretagogues is mediated by the mouse orthologue of the human MRGPRX2, MRGPRB2. To further elucidate the mechanism of MRGPRX2 activation, we studied the time-dependent internalization of MRGPRX2 by human mast cells (LAD2) upon stimulation with the neuropeptide SP. In addition, we performed computational studies to identify the intermolecular forces that facilitate ligand-MRGPRX2 interaction using SP. The computational predictions were tested experimentally by activating LAD2 with SP analogs, which were missing key amino acid residues. Our data suggest that mast cell activation by SP causes internalization of MRGPRX2 within 1 min of stimulation. Hydrogen bonds (h-bonds) and salt bridges govern the biding of SP to MRGPRX2. Arg1 and Lys3 in SP are key residues that are involved in both h-bonding and salt bridge formations with Glu164 and Asp184 of MRGPRX2, respectively. In accordance, SP analogs devoid of key residues (SP1 and SP2) failed to activate MRGPRX2 degranulation. However, both SP1 and SP2 caused a comparable release of chemokine CCL2. Further, SP analogs SP1, SP2 and SP4 did not activate tumor necrosis factor (TNF) production. We further show that SP1 and SP2 limit the activity of SP on mast cells. The results provide important mechanistic insight into the events that result in mast cell activation through MRGPRX2 and highlight the important physiochemical characteristics of a peptide ligand that facilitates ligand-MRGPRX2 interactions. The results are important in understanding activation through MRGPRX2, and the intermolecular forces that govern ligand-MRGPRX2 interaction. The elucidation of important physiochemical properties within a ligand that are needed for receptor interaction will aid in designing novel therapeutics and antagonists for MRGPRX2. [ABSTRACT FROM AUTHOR]

Hydroquinone (HQ) is one of the most effective drugs to treat hyperpigmentary disorders, but often causes skin irritation in clinic. Mast cell plays an important role in contact dermatitis and triggering pseudo‐allergic reactions via MRGPRX2. Whether HQ‐induced skin irritant reaction through activating mast cells via MRGPRX2 remains unknown. To investigate the role of mast cells in HQ‐induced skin irritant reaction and verify whether MRGPRX2 participated in the HQ effect on mast cells which contributed to the pathogenesis of skin irritant reaction, a mouse model of HQ‐induced skin irritation was established to observe the local and systemic inflammation associated with mast cell receptor MrgprB2. Human mast cell LAD2 was used to verify the effect of HQ on mast cells via MRGPRX2 by knocking down with siRNA. As a result, mast cells were involved in the development of HQ‐induced irritant reaction, and local inflammation is closely related to mast cell receptor MrgprB2. HQ could activate mast cells via MRGPRX2, causing changes in calcium concentration, degranulation and release of inflammatory cytokines which lead to skin irritant reaction. In conclusion, HQ‐induced skin irritant reaction could be skin pseudo‐allergic reactions achieved by activating mast cells via MRGPRX2. [ABSTRACT FROM AUTHOR]

Mast cells play a pivotal role in initiating and directing host's immune response. They reside in tissues that primarily interface with the external environment. Activated mast cells respond to environmental cues throughout acute and chronic inflammation through releasing immune mediators via rapid degranulation, or long-term de novo expression. Mast cell activation results in the rapid release of a variety of unique enzymes and reactive oxygen species. Furthermore, the increased density of mast cell unique receptors like mas related G protein-coupled receptor X2 also characterizes the inflamed tissues. The presence of these molecules (either released mediators or surface receptors) are particular to the sites of active inflammation, and are a result of mast cell activation. Herein, the molecular design principles for capitalizing on these novel mast cell properties is discussed with the goal of manipulating localized inflammation. Mast cells are immune regulating cells that play a crucial role in both innate and adaptive immune responses. The activation of mast cells causes the release of multiple unique profiles of biomolecules, which are specific to both tissue and disease. These unique characteristics are tightly regulated and afford a localized stimulus for targeting inflammatory diseases. Herein, these important mast cell attributes are discussed in the frame of highlighting strategies for the design of bioresponsive functional materials to target regions of inflammations. [Display omitted] [ABSTRACT FROM AUTHOR]

Insights into the pathophysiology of many non-immune-mediated drug reactions referred to as toxicities, sensitivities, intolerances, or pseudoallergies have resulted from research identifying the mastocyte-related G-protein-coupled receptor (GPCR) member X2 (MRGPRX2), a human mast cell receptor mediating adverse reactions without the involvement of antibody priming. Opioid-induced degranulation of mast cells, particularly morphine, provoking release of histamine and other preformed mediators and causing hemodynamic and cutaneous changes seen as flushing, headache and wheal and flare reactions in the skin, is an example of results of MRGPRX2 activation. Opioids including morphine, codeine, dextromethorphan and metazocine as well as endogenous prodynorphin opioid peptides activate MRGPRX2 at concentrations causing mast cell degranulation. Unlike the canonical opioid receptors, MRGPRX2 shows stereochemical recognition preference for dextro rather than levo opioid enantiomers. Opioid analgesic drugs (OADs) display a range of histamine-releasing potencies from the strong releaser morphine to doubtful releasers like hydromorphone and the non-releaser fentanyl. Whether there is a correlation between histamine release by individual OADs, MRGPRX2 activation, and presence or absence of adverse cutaneous effects is not known. To investigate the question, ongoing research with recently pursued methodologies and strategies employing basophil and mast cell tests resulting from MRGPRX2 insights should help to elucidate whether or not an opioid's histamine-releasing potency, and its property of provoking an adverse reaction, are each a reflection of its activation of MRGPRX2. [ABSTRACT FROM AUTHOR]

Mast cells play an important role in disease pathogenesis by secreting immunomodulatory molecules. Mast cells are primarily activated by the crosslinking of their high affinity IgE receptors (FcεRI) by antigen bound immunoglobulin (Ig)E antibody complexes. However, mast cells can also be activated by the mas related G protein-coupled receptor X2 (MRGPRX2), in response to a range of cationic secretagogues, such as substance P (SP), which is associated with pseudo-allergic reactions. We have previously reported that the in vitro activation of mouse mast cells by basic secretagogues is mediated by the mouse orthologue of the human MRGPRX2, MRGPRB2. To further elucidate the mechanism of MRGPRX2 activation, we studied the time-dependent internalization of MRGPRX2 by human mast cells (LAD2) upon stimulation with the neuropeptide SP. In addition, we performed computational studies to identify the intermolecular forces that facilitate ligand-MRGPRX2 interaction using SP. The computational predictions were tested experimentally by activating LAD2 with SP analogs, which were missing key amino acid residues. Our data suggest that mast cell activation by SP causes internalization of MRGPRX2 within 1 min of stimulation. Hydrogen bonds (h-bonds) and salt bridges govern the biding of SP to MRGPRX2. Arg1 and Lys3 in SP are key residues that are involved in both h-bonding and salt bridge formations with Glu164 and Asp184 of MRGPRX2, respectively. In accordance, SP analogs devoid of key residues (SP1 and SP2) failed to activate MRGPRX2 degranulation. However, both SP1 and SP2 caused a comparable release of chemokine CCL2. Further, SP analogs SP1, SP2 and SP4 did not activate tumor necrosis factor (TNF) production. We further show that SP1 and SP2 limit the activity of SP on mast cells. The results provide important mechanistic insight into the events that result in mast cell activation through MRGPRX2 and highlight the important physiochemical characteristics of a peptide ligand that facilitates ligand-MRGPRX2 interactions. The results are important in understanding activation through MRGPRX2, and the intermolecular forces that govern ligand-MRGPRX2 interaction. The elucidation of important physiochemical properties within a ligand that are needed for receptor interaction will aid in designing novel therapeutics and antagonists for MRGPRX2.

MRGPRX2, a G-protein-coupled-seven transmembrane domain receptor, is mainly expressed in mast cells and neurons and is involved in skin immunity and pain. It is implicated in the pathophysiology of non-IgE-mediated immediate hypersensitivity and has been related to adverse drug reactions. Moreover, a role has been proposed in asthma, atopic dermatitis, contact dermatitis, and chronic spontaneous urticaria. Although it has a prominent role in disease, its signaling transduction is poorly understood. This study shows that MRGPRX2 activation with substance P increased Lysyl t-RNA synthetase (LysRS) translocation to the nucleus. LysRS is a moonlighting protein with a dual role in protein translation and IgE signaling in mast cells. Upon allergen- IgE-FcεRI crosslinking, LysRS is translocated to the nucleus and activates microphthalmia-associated transcription factor (MITF) activity. In this study, we found that MRGPRX2 triggering led to MITF phosphorylation and increased MITF activity. Therefore, overexpression of LysRS increased MITF activity after MRGPRX2 activation. MITF silencing reduced MRGPRX2-dependent calcium influx and mast cell degranulation. Furthermore, a MITF pathway inhibitor, ML329, impaired MITF expression, calcium influx, and mast cell degranulation. Moreover, drugs such as atracurium, vancomycin, and morphine, reported to induce MRGPRX2-dependent degranulation, increased MITF activity. Altogether, our data show that MRGPRX2 signaling enhances MITF activity, and its abrogation by silencing or inhibition resulted in defective MRGPRX2 degranulation. We conclude that MRGPRX2 signaling involves the LysRS and MITF pathway. Thus, MITF and MITF-dependent targets may be considered therapeutic approaches to treat pathologies where MRGPRX2 is implicated.

MRGPRX2, the human member of the MAS-related G-protein-coupled receptors (GPCRs), mediates the immunoglobulin E (IgE)-independent responses of a subset of mast cells (MCs) that are associated with itch, pain, neurogenic inflammation, and pseudoallergy to drugs. The mechanisms underlying the responses of MRGPRX2 to its multiple and diverse ligands are still not completely understood. Given the close association between GPCR location and function, and the key role played by Rab GTPases in controlling discrete steps along vesicular trafficking, we aimed to reveal the vesicular pathways that directly impact MRGPRX2-mediated exocytosis by identifying the Rabs that influence this process. For this purpose, we screened 43 Rabs for their functional and phenotypic impacts on MC degranulation in response to the synthetic MRGPRX2 ligand compound 48/80 (c48/80), which is often used as the gold standard of MRGPRX2 ligands, or to substance P (SP), an important trigger of neuroinflammatory MC responses. Results of this study highlight the important roles played by macropinocytosis and autophagy in controlling MRGPRX2-mediated exocytosis, demonstrating a close feedback control between the internalization and post-endocytic trafficking of MRGPRX2 and its triggered exocytosis.

Mast cells (MCs) are an important part of the immune system, responding both to pathogens and toxins, but they also play an important role in allergic diseases, where recent data show that non-IgE-mediated activation is also of relevance, especially in chronic urticaria (CU) and atopic dermatitis (AD). Skin MCs express Mas-related G-protein-coupled receptor X2 (MRGPRX2), a key protein in non-IgE-dependent MC degranulation, and its overactivity is one of the triggering factors for the above-mentioned diseases, making MRGPRX2 a potential therapeutic target. Reviewing the latest literature revealed our need to focus on the discovery of MRGPRX2 activators as well as the ongoing vast research towards finding specific MRGPRX2 inhibitors for potential therapeutic approaches. Most of these studies are in their preliminary stages, with one drug currently being investigated in a clinical trial. Future studies and improved model systems are needed to verify whether any of these inhibitors may have the potential to be the next therapeutic treatment for CU, AD, and other pseudo-allergic reactions.

Skin mast cells (MCs) express high levels of MRGPRX2, FcεRI, and ST2, and vigorously respond to their ligands when triggered individually. IL-33/ST2 also potently synergizes with other receptors, but the molecular underpinnings are poorly understood. Human skin-derived MCs were stimulated via different receptors individually or jointly in the presence/absence of selective inhibitors. TNF was quantified by ELISA. Signaling cascades were studied by immunoblot. TNF was stimulated by FcεRI ≈ ST2 > MRGPRX2. Surprisingly, neither FcεRI nor MRGPRX2 stimulation elicited NF-κB activation (IκB degradation, p65 phosphorylation) in stark contrast to IL-33. Accordingly, TNF production did not depend on NF-κB in FcεRI- or MRGPRX2-stimulated MCs, but did well so downstream of ST2. Conversely, ERK1/2 and PI3K were the crucial modules upon FcεRI/MRGPRX2 stimulation, while p38 was key to the IL-33-elicited route. The different signaling prerequisites were mirrored by their activation patterns with potent pERK/pAKT after FcεRI/MRGPRX2, but preferential induction of pp38/NF-κB downstream of ST2. FcεRI/MRGPRX2 strongly synergized with IL-33, and some synergy was still observed upon inhibition of each module (ERK1/2, JNK, p38, PI3K, NF-κB). IL-33’s contribution to synergism was owed to p38 > JNK > NF-κB, while the partner receptor contributed through ERK > PI3K ≈ JNK. Concurrent IL-33 led to slightly prolonged pERK (downstream of MRGPRX2) or pAKT (activated by FcεRI), while the IL-33-elicited modules (pp38/NF-κB) remained unaffected by co-stimulation of FcεRI/MRGPRX2. Collectively, the strong synergistic activity of IL-33 primarily results from the complementation of highly distinct modules following co-activation of the partner receptor rather than by altered signal strength of the same modules.

Perioperative hypersensitivity constitutes an important health issue, with potential dramatic consequences of diagnostic mistakes. However, safe and correct diagnosis is not always straightforward, mainly because of the application of incorrect nomenclature, absence of easy accessible in-vitro/ex-vivo tests and uncertainties associated with the non-irritating skin test concentrations. In this editorial we summarize the time line, seminal findings, and major realizations of 25 years of research on the mechanisms, diagnosis, and management of perioperative hypersensitivity.

Background: Occupancy of MRGPRX2 heralds a new era in our understandings of immediate drug hypersensitivity reactions (IDHRs), but a constitutive expression of this receptor by basophils is debated. Objective: To explore the expression and functionality of MRGPRX2 in and on basophils. Methods: Basophils from patients with birch pollen allergy, IDHRs to moxifloxacin, and healthy controls were studied in different conditions, that is, in rest, after stimulation with anti-IgE, recombinant major birch pollen allergen (rBet v 1), moxifloxacin, fMLP, substance P (SP), or other potential basophil secretagogues. In a separate set of experiments, basophils were studied after purification and resuspension in different media. Results: Resting whole blood basophils barely express MRGPRX2 on their surface and are unresponsive to SP or moxifloxacin. However, surface MRGPRX2 is quickly upregulated upon incubation with anti-IgE or fMLP. Pre-stimulation with anti-IgE can induce a synergic effect on basophil degranulation in IgE-responsive subjects after incubation with SP or moxifloxacin, provided that basophils have been obtained from patients who experienced an IDHR to moxifloxacin. Cell purification can trigger a “spontaneous” and functional upregulation of MRGPRX2 on basophils, not seen in whole blood cells, and its surface density can be influenced by distinct culture media. Conclusion: Basophils barely express MRGPRX2 in resting conditions. However, the receptor can be quickly upregulated after stimulation with anti-IgE, fMLP, or after purification, making cells responsive to MRGPRX2 occupation. We anticipate that such “conditioned” basophils constitute a model to explore MRGPRX2 agonism or antagonism, including IDHRs originating from the occupation of this receptor. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • UA interferes with the activity of MRGPRX2/MrgprB2, reducing mast cell degranulation. • UA inhibits TSLP-related itch signaling pathway without affecting TRPV4 and PAR2 in keratinocytes. • UA alleviates scratching induced by MRGPRX2/MrgprB2 agonists, suggesting its antipruritic potential. [ABSTRACT FROM AUTHOR]

Depuis la description séminale impliquant l'occupation du récepteur X2 couplé à la protéine G liée à Mas (MRGPRX2) dans la dégranulation des mastocytes (MCs) par les médicaments, de nombreuses études ont été entreprises sur ce nouvel endotype potentiel de réactions d'hypersensibilité immédiate aux médicaments (RHMIs). Cependant, les preuves actuelles de ce mécanisme proviennent principalement de modèles animaux (mutants) ou d'études in vitro. Des preuves cliniques irréfutables chez l'homme font défaut. De plus, la traduction de ces résultats précliniques en pertinence clinique chez l'homme est difficile et doit être interprétée de manière critique. En partant de nos priorités cliniques et de notre expérience des analyses fonctionnelles des basophiles, MCs et lymphocytes T, l'objectif de cette revue est d'identifier certaines de ces difficultés, de souligner les obstacles qui pourraient entraver la transposition des observations précliniques en clinique et de mettre en évidence les différences entre les réactions médiées par les sIgE et par le MRGPRX2. Finalement, nous proposons un algorithme mécanistique théorique qui pourrait faciliter la discrimination entre la dégranulation des MCs due à l'activation de MRGPRX2 et la réticulation des anticorps IgE liés à la membrane et réactifs aux médicaments. [ABSTRACT FROM AUTHOR]

Mast cells (MCs) are tissue-resident immune cells of a hematopoietic origin that play vital roles in innate and adaptive immunity. Human MCs can be isolated and differentiated from various tissue sources, including cord blood, when supplemented with cytokines such as stem cell factor, interleukin 3, and interleukin 6. Our current research study has shown significant differences in the marker expressions of human cord blood-derived mast cells (hCBMCs) based on donor dependency and the type of medium used for culturing and differentiation. These findings are particularly relevant given the challenges of obtaining specialty media influencing MC phenotypic marker expressions. We found that hCBMCs cultured in StemSpanTM-XF medium had a moderate expression of mast/stem cell growth factor receptor Kit (c-KIT) (mRNA and protein), low expressions of FcεRI (mRNA) and TLR2 (mRNA and protein) but had high levels of MRGPRX2 (mRNA and protein) expressions. In contrast, hCBMCs cultured in Stem Line II medium expressed FcεRI and TLR2 (mRNA and protein) with higher c-KIT but had lower MRGPRX2 expressions compared to the hCBMCs cultured in the StemSpanTM-XF medium. These results suggest that it is crucial to consider both donor dependency and the medium when investigating MC functions and that further research is needed to fully understand the impact of these factors on the hCBMC marker expressions.

BackgroundOccupancy of MRGPRX2 heralds a new era in our understandings of immediate drug hypersensitivity reactions (IDHRs), but a constitutive expression of this receptor by basophils is debated.ObjectiveTo explore the expression and functionality of MRGPRX2 in and on basophils.MethodsBasophils from patients with birch pollen allergy, IDHRs to moxifloxacin, and healthy controls were studied in different conditions, that is, in rest, after stimulation with anti-IgE, recombinant major birch pollen allergen (rBet v 1), moxifloxacin, fMLP, substance P (SP), or other potential basophil secretagogues. In a separate set of experiments, basophils were studied after purification and resuspension in different media.ResultsResting whole blood basophils barely express MRGPRX2 on their surface and are unresponsive to SP or moxifloxacin. However, surface MRGPRX2 is quickly upregulated upon incubation with anti-IgE or fMLP. Pre-stimulation with anti-IgE can induce a synergic effect on basophil degranulation in IgE-responsive subjects after incubation with SP or moxifloxacin, provided that basophils have been obtained from patients who experienced an IDHR to moxifloxacin. Cell purification can trigger a “spontaneous” and functional upregulation of MRGPRX2 on basophils, not seen in whole blood cells, and its surface density can be influenced by distinct culture media.ConclusionBasophils barely express MRGPRX2 in resting conditions. However, the receptor can be quickly upregulated after stimulation with anti-IgE, fMLP, or after purification, making cells responsive to MRGPRX2 occupation. We anticipate that such “conditioned” basophils constitute a model to explore MRGPRX2 agonism or antagonism, including IDHRs originating from the occupation of this receptor.

The development of vaccines against SARS-CoV2 brought about several challenges, including the management of hypersensitivity reactions to these formulations. The search for underlying mechanisms involved in these adverse events initially focused on excipients which may trigger mast cell activation responses via non-IgE pathways: polyethylene glycol and trometamol. We sought to determine whether these components, in their pure form, were capable of stimulating mast cells directly. To test this hypothesis, we used an in vitro model for non-IgE-mediated activation that has previously shown degranulation responses induced via MRGPRX2 with known drug agonists of the receptor. Human LAD2 mast cells were incubated with different concentrations (1, 10, 50 mg/ml) of trometamol and of purified polyethylene glycol/Macrogol (molecular weights: 2,000, 3,350, 4,000, and 6,000). Mast cell degranulation was assessed using a beta-hexosaminidase read-out. Interestingly, degranulation responses for all reagents tested showed no significant differences from those obtained from the negative control (basal degranulation). Receptor-silencing assays were therefore not conducted. In summary, purified PEG and trometamol did not induce mast cell degranulation in this in vitro model for the study of non-IgE mechanisms of drug hypersensitivity, previously shown to be useful in the investigation of MRGPRX2 ligands. Studies using complete vaccine formulations, lipid conjugates, and receptor gene variants are needed to further clarify mechanisms of vaccine hypersensitivity.