Dual function of MrgprB2 receptor-dependent neural immune axis in chronic pain

Yucui Jiang ^a1 , Fan Ye ^d1 , Jian Zhang ^b1 , Yun Huang ^b , Yingxin Zong ^b , Feiyan Chen ^a , Yan Yang ^b , Chan Zhu ^b , Tao Yang ^c, Guang Yu ^b , Zongxiang Tang ^b

^aSchool of Chinese Medicine, Nanjing University of Chinese Medicine, 138 Xianlin Road, Nanjing 210023, China
^bSchool of Medicine, Nanjing University of Chinese Medicine, 138 Xianlin Road, Nanjing 210023, China
^cState Key Laboratory of Organic Electronics and Information Displays & Institute of Advanced Materials (IAM), Nanjing University of Posts & Telecommunications, 9 Wenyuan Road, Nanjing 210023, China
^dCollege of Pharmacy, Jishou University, Jishou 416000, China

Received 26 March 2024, Revised 26 November 2024, Accepted 26 February 2025, Available online 28 February 2025.

Abstract

Introduction: Neuro-immune interactions have been recognized to be involved in the development of neuropathic pain induced by chemotherapeutic drugs (CINP). However, its role in pain resolution remains largely unknown, particularly concerning mast cells.

Objectives: To investigate the bidirectional modulation of mast cell Mas-related G protein-coupled receptor B2 (MrgprB2)-mediated neuro-immune interactions in CINP.

Methods: CINP model was established in wild-type mice, Mas-related G protein-coupled receptor D knockout (MrgprD^-/-) mice, mast cell-deficient mice, MrgprB2 knockout (MrgprB2^-/-) mice, and MrgprB2-Cre tdTomato mice. The role of MrgprB2 receptor in CINP was investigated by calcium imaging, cytokine antibody arrays, mining of single-cell sequencing databases, immunofluorescence, western blotting, co-immunoprecipitation (Co-IP), among other methodologies.

Results: We observed that cisplatin-induced allodynia was significantly inhibited in MrgprB2^-/- mice, which was attributed to the blockade of tryptase release and the suppression of upregulation of protease-activated receptor 2 (PAR2) expression in dorsal root ganglion (DRG). Thus, the activation of MrgprB2/Tryptase/PAR2 axis contributed to the development of cisplatin-induced pain. In addition, we also found that there was co-expression of PAR2 and MrgprD in DRG neurons. And activation of PAR2 can negatively regulate the expression of MrgprD, whether in a physiological state or in a chronic pain condition. Consequently, MrgprD expression was down-regulated by the activation of the MrgprB2/Tryptase/PAR2 axis during the later stages of CINP, which was associated with pain relief. Therefore, the activation of MrgprB2/Tryptase/PAR2 axis also contributed to the alleviation of cisplatin-induced pain. This finding was in line with the phenomenon that persistent stimulation by cisplatin did not cause a continuous increase in pain.

Conclusions: Our research elucidated the bidirectional modulation of MrgprB2-dependent neural immune axis in CINP. This study emphasized that MrgprB2 is a critical target for early intervention in CINP, and highlighted the necessity of considering the mechanism differences at different stages in pain management.

Keywords: CINP; MrgprB2 receptor; MrgprD receptor; PAR2 receptor.

Scratching promotes allergic inflammation and host defense via neurogenic mast cell activation

Andrew W. Liu^1,2, Youran R. Zhang^1,2, Chien-Sin Chen^1,2, Tara N. Edwards^1,2, Sumeyye Ozyaman^1,2†,
Torben Ramcke^1,2, Lindsay M. McKendrick^1,2, Eric S. Weiss^1,2, Jacob E. Gillis^1,2, Colin R. Laughlin²‡,
Simran K. Randhawa², Catherine M. Phelps², Kazuo Kurihara^1,2, Hannah M. Kang^1,2,
Sydney-Lam N. Nguyen^1,2, Jiwon Kim3, Tayler D. Sheahan3§, Sarah E. Ross3,4, Marlies Meisel^2,5,
Tina L. Sumpter^1,2, Daniel H. Kaplan^1,2*

¹Department of Dermatology, University of Pittsburgh, Pittsburgh, PA, USA. ²Department of Immunology, University of Pittsburgh, Pittsburgh, PA, USA. ³Department of Anesthesiology, University of Pittsburgh, Pittsburgh, PA, USA. ⁴Pittsburgh Center for Pain Research, Pittsburgh, PA, USA. ⁵Cancer Immunology and Immunotherapy Program, UPMC Hillman Cancer Center, Pittsburgh, PA, USA.
*Corresponding author. Email: dankaplan@pitt.edu
†Present address: Department of Histology and Embryology, School of Medicine, Istanbul Medipol University, Istanbul, Turkey.
‡Present address: Department of Immunobiology, Yale University, New Haven, CT, USA.
§Present address: Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA.

Editor’s summary

Itch, the sensation that stimulates scratching behavior, is often triggered by skin irritants and inflammation. Liu et al. found that ablating itch-sensing neurons or physically preventing scratching decreased the inflammation associated with antigen-dependent mast cell responses in response to chemicals that induce allergic immune responses (see the Perspective by Ver Heul). Scratching promoted pain-sensing neurons to release a neuropeptide that stimulated mast cells, and this peptide hormone synergized with antigen-dependent activation to increase the mast cell’s degranulation and ability to produce inflammatory mediators. In a model of skin infection associated with antigen-specific mast cell responses, scratching contributed to decreasing the bacterial load. —Sarah H. Ross

MrgprX2 regulates mast cell degranulation through PI3K/AKT and PLCγ signaling in pseudo-allergic reactions

https://doi.org/10.1016/j.intimp.2021.108389

Fan Zhang ^ab, Fang Hong ^ab, Lu Wang ^ac, Renjie Fu ^a, Jin Qi ^a, Boyang Yu^ab

^aJiangsu Key Laboratory for TCM Evaluation and Translational Research, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 21198, China
^bResearch Center for Traceability and Standardization of TCMs, China Pharmaceutical University, Nanjing 211198, China
^cNanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine, China

Received 10 August 2021, Revised 27 October 2021, Accepted 9 November 2021, Available online 15 December 2021, Version of Record 15 December 2021.

Highlights

• •MrgprX2 is a key receptor for pseudo-allergic reactions.
• •PI3K/AKT signaling pathway and PLCγ are important downstream of MrgprX2.
• •Inhibiting PI3K/AKT signaling pathway and PLCγ remarkable weakened pseudo-allergic reactions both in vivo and in vitro.

Abstract

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 PI3Kand 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.

Keywords

Pseudo-allergic reactions, Mast cell, MRGPRX2, PI3K/AKT, PLCγ

Clarithromycin-treated chronic spontaneous urticaria with the negative regulation of FcεRΙ and MRGPRX2 activation via CD300f

Delu Che ¹, Tao Zhang ², Tianxiao Zhang ³, Yi Zheng ⁴, Yajing Hou ², Songmei Geng ⁵, Langchong He ⁶

Affiliations Expand

• PMID: 35853276
• DOI: 10.1016/j.intimp.2022.109063

Abstract

Mast cells (MCs) are main effector cells in chronic spontaneous urticaria (CSU). Both Fc epsilon RI (FcεRΙ)- and MAS-related G coupled receptor-X2 (MRGPRX2)-mediated MC activations affect CSU course. Leukocyte mono-immunoglobulin-like receptor 3 (CD300f) has been shown to regulate FcεRΙ activation. However, no study has verified CD300f is a target to cure CSU. Therefore this study aimed to verify whether clarithromycin (CLA) regulates FcεRΙ- and MRGPRX2-mediated MC activations via CD300f and shows therapeutic effect on CSU. The target of CLA was verification. CLA inhibited FcεRΙ- and MRGPRX2-mediated MC activations were shown in vivo and in vitro. A single-center, self-comparison study was performed, and CLA-treated CSU was investigated in 28 patients who were not sensitive to the third-generation antihistamines. Serum inflammatory mediators in patients before and after CLA administration were analyzed. CLA effectively inhibited type Ι anaphylactic reactions and pseudo-allergic reactions in mice. Moreover, CLA inhibited FcεRΙ- and MRGPRX2-mediated MC signaling pathway activation. Regulatory effects of CLA were decreased significantly after CD300f knockdown. CLA effectively alleviated the symptoms of wheal and itch and reduced serum cytokine levels in patients. CLA negatively regulated FcεRΙ- and MRGPRX2-mediated MC activation via CD300f and showed significant therapeutic effect on CSU.

Keywords: Chronic spontaneous urticarial; Clarithromycin; Leukocyte mono-immunoglobulin-like receptor 3; Mast cell; Negatively regulation.

Copyright © 2022 Elsevier B.V. All rights reserved.

Mast cell activation induced by tamoxifen citrate via MRGPRX2 plays a potential adverse role in breast cancer treatment

Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm

Jiapan Gao a,b,1, Xinyue Su a,b,1, Yuxiu Zhang a,b, Xiaoyu Ma a,b, Bingxi Ren a,b, Panpan Lei a,b, Jiming Jin c, Weina Ma a,b,*

a School of Pharmacy, Xi’an Jiaotong University, Xi’an 710061, PR China
b State Key Laboratory of Shaanxi for Natural Medicines Research and Engineering, Xi’an 710061, PR China
c First School of Clinical Medicine, Shaanxi University of Chinese Medicine, Xi’an 712046, PR China

ABSTRACT
Breast cancer is the most common malignant tumor endangering women’s life and health. Tamoxifen citrate (TAM) is the first-line drug of adjuvant endocrine therapy for estrogen receptor-positive (ER+) breast cancer patients. Some sporadic cases have described rare adverse reactions of TAM with potentially life-threatening dermatological manifestations, which were associated with skin allergy. Mas related G protein-coupled recep- tor X2 (MRGPRX2) on human mast cells is the key target for skin allergy. We aimed to investigate the mechanism of TAM-induced allergic reactions and their potential effects on TAM treatment for breast cancer. In our study, TAM can specifically bind with MRGPRX2, which was mainly driven by hydrophobic force. TAM formed hydrogen bonds with TRP243, TRP248, and GLU164 residues in MRGPRX2. TAM induced calcium mobilization and degranulation of mast cells via MRGPRX2. Besides, TAM induced passive cutaneous anaphylaxis and active systemic anaphylaxis in C57BL/6 mice. The release of β-hexosaminidase, histamine, tumor necrosis factor-α, monocyte chemoattractant protein 1, and interleukin-8 were increased by TAM in vitro and in vivo. Furthermore, we found that MCF-7 and T-47D breast cancer cells can recruit mast cells to adjacent cancerous tissues. Besides, mast cell activation induced by TAM via MRGPRX2 significantly promoted the proliferation and migration of MCF-7 and T-47D cells, which can be effectively reversed by mast cell membrane stabilizer clarithromycin and MRGPRX2 silencing. This study proposed an anti-allergic therapeutic strategy for breast cancer treatment with TAM, while also the potential of MRGPRX2 as an adjunctive target.

S1P/S1PRs-TRPV4 axis is a novel therapeutic target for persistent pain and itch in chronic dermatitis

inyu Zhang1,2 | Yuan Zhou2 | ChangmingWang2 | JiahuiRen2 | YinWang2 |Pei Liu1 | WeimengFeng1 | XueLi2 | MingxinQi2 | YanYang2 |Chan Zhu2 | FangWang3 | YuxiangMa4 | ZongxiangTang2 | GuangYu1,2

1Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing
University of Chinese Medicine, Nanjing, China
2Key Laboratory for Chinese Medicine of Prevention and Treatment in Neurological Diseases, School of Medicine, Nanjing University of Chinese Medicine, Nanjing, China
3Department of Dermatology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
4School of Life Science, China Pharmaceutical University, Nanjing, China

Abstract
Background and Purpose: While pain and itch are both commonly associated with chronic dermatitis (CD), the molecular mechanisms underlying these debilitating symptoms is not well understood. This study aims to identify novel, endogenous compounds that mediate CD-associated pain and itch.

Experimental Approach: Lesional skin of CD model mice was examined using unbiased metabolomic analysis to identify candidate pain or itch inducing compounds in CD. Sphingosine-1-phosphate (S1P) concentration in CD model skin was analysed using UPLC/MS/MS. Behaviour, calcium imaging and immunofluorescence staining were used to determine the pain and itch effects and mechanisms of the identified CD-related compounds.

Key Results: In the lesional skin of CD model mice, 136 compounds were significantly changed. These compounds are predominately associated with the sphingolipids metabolism pathway. S1P is significantly increased in the lesional skin . The TRPV4 channel was critical for S1P induced itch and pain. Sphingosine kinase 2 (SPHK2), the key enzyme controlling S1P synthesis, was significantly increased in lesional skin. ABC294640, a SPHK2 inhibitor, significantly decreased S1P concentration in lesional CD model skin, as well as in model associated epidermal hyperplasia and chronic pain and itch. In CD patients, SPHK2 expression and S1P concentration were significantly elevated compared to healthy control skin.

Conclusion and Implications: Our results indicate that, in CD, increased S1P induces chronic pain and itch partly through TRPV4. Inhibition of S1P synthesis or the S1P/S1P receptor-TRPV4 pathway are promising treatment strategies for CD associated pain and itch.

KEYWORDS
chronic dermatitis, itch, pain, S1P, TRPV4

Neuronal substance P-driven MRGPRX2-dependent mast cell degranulation products differentially promote vascular permeability

 https://doi.org/10.3389/fimmu.2024.1477072

iPSC-derived human sensory neurons reveal a subset of TRPV1 antagonists as anti-pruritic compounds

Shermaine Huiping Tay, Jeremy Kah Sheng Pang, Winanto Ng, Chong Yi Ng, Zi Jian Khong, Zheng-Shan Chong, Boon Seng Soh & Shi-Yan Ng 

Scientific Reports volume 14, Article number: 31182 (2024) 

Cite this article

Abstract

Signaling interplay between the histamine 1 receptor (H1R) and transient receptor potential cation channel subfamily V member 1 (TRPV1) in mediating histaminergic itch has been well-established in mammalian models, but whether this is conserved in humans remains to be confirmed due to the difficulties in obtaining human sensory neurons (SNs) for experimentation. Additionally, previously reported species-specific differences in TRPV1 function indicate that use of human SNs is vital for drug candidate screening to have a higher chance of identifying clinically effective TRPV1 antagonists. In this study, we built a histamine-dependent itch model using peripheral SNs derived from human induced pluripotent stem cells (hiPSC-SNs), which provides an accessible source of human SNs for pre-clinical drug screening. We validated channel functionality using immunostaining, calcium imaging, and multielectrode array (MEA) recordings, and confirmed the interdependence of H1R and TRPV1 signalling in human SNs. We further tested the amenability of our model for pre-clinical studies by screening multiple TRPV1 antagonists in parallel, identifying SB366791 as a potent inhibitor of H1R activation and potential candidate for alleviating histaminergic itch. Notably, some of the results using our model corroborated with efficacy and side effect findings from human clinical trials, underscoring the importance of this species-specific platform. Taken together, our results present a robust in vitro human model for histaminergic itch, which can be used to further interrogate the molecular basis of human SN function as well as screen for TRPV1 activity-modifying compounds for a number of clinical indications.

Phytother Res. 2023 Aug;37(8):3572-3582. doi: 10.1002/ptr.7835. Epub 2023 Apr 28.

Silibinin attenuated pseudo-allergic reactions and mast cell degranulation via PLCγ and PI3K/Akt signaling pathway

Yuejin Wang ¹, Shiting Hu ¹, Baowen Dang ¹, Yonghui Zhang ¹, Guodong Zheng ¹, Chenrui Zhao ¹, Yihan Huang ¹, Tao Zhang ¹

Affiliations expand

• PMID: 37115717
• DOI: 10.1002/ptr.7835

Abstract

Anaphylaxis is a type of potentially fatal hypersensitivity reaction resulting from the activation of mast cells. Many endogenous or exogenous factors could cause this reaction. Silibinin is the main chemical component of silymarin and has been reported to have pharmacological activities. However, the anti-allergic reaction effect of silibinin has not yet been investigated. This study aimed to evaluate the effect of silibinin to attenuate pseudo-allergic reactions in vivo and to investigate the underlying mechanism in vitro. In this study, calcium imaging was used to assess Ca²⁺ mobilization. The levels of cytokines and chemokines, released by stimulated mast cells, were measured using enzyme immunoassay kits. The activity of silibinin was evaluated in a mouse model of passive cutaneous anaphylaxis (PCA). Western blotting was used to explore the related molecular signaling pathways. In results, silibinin markedly inhibited mast cell degranulation, calcium mobilization, and preventing the release of cytokines and chemokines in a dose-dependent manner via the PLCγ and PI3K/Akt signaling pathway. Silibinin also attenuated PCA in a dose-dependent manner. In summary, silibinin has an anti-pseudo-allergic pharmacological activity, which makes it a potential candidate for the development of a novel agent to arrest pseudo-allergic reactions.

Keywords: PI3K/Akt; mast cell; pseudo-allergy; silibinin.

https://pubmed.ncbi.nlm.nih.gov/37115717/

The role of PAR2 in regulating MIF release in house dust mite-induced atopic dermatitis

Lingxuan Zhou¹Guohong Zhang¹Kai Zhang¹Ziyan Rao²Zhanli Tang³Yang Wang¹Jiahui Zhao^1,4*

• ¹Department of Dermatology, Peking University First Hospital, Beijing Key Laboratory of Molecular Diagnosis on Dermatoses, National Clinical Research Center for Skin and Immune Disease, National Medical Products Administration (NMPA) Key Laboratory for Quality Control and Evaluation of Cosmetics, Beijing, China
• ²Department of Biomedical Informatics, School of Basic Medical Sciences, State Key Laboratory of Vascular Homeostasis and Remodeling, Peking University, Beijing, China
• ³Department of Dermatology, Qilu Hospital of Shandong University, Jinan, China
• ⁴Chinese Institute for Brain Research (CIBR), Beijing, China

Atopic dermatitis (AD) is a chronic disease characterized by relapsed eczema and intractable itch, and is often triggered by house dust mites (HDM). PAR2 is a G-protein coupled receptor on keratinocytes and may be activated by HDM to affect AD processes. We first established a HDM-derived AD mouse model in wild-type (WT) and Par2^-/-mice. Single cell RNA sequencing of the diseased skins found a stronger cellular communication between the ligand macrophage migration inhibitory factor (MIF) from keratinocytes and its receptors on antigen-presenting cells, suggesting the critical role of MIF in AD. HDM-WT mice showed severer skin lesions and pathological changes with stronger immunofluorescence MIF signals in skin sections than HDM-Par2^-/- mice. Primary keratinocytes from WT mice stimulated with HDM or SLIGRL (PAR2 agonist) secreted more MIF in cultured medium and induced stronger immunofluorescence MIF signals than those from Par2^-/- mice. The skin section of HDM-WT mice showed higher immunofluorescence signals of P115 (relating to MIF secretion) and KIF13B (possibly relating to intracellular trafficking of MIF) than that of HDM-Par2^-/- mice. Acetylation of α-tubulin increased after stimulation by SLIGRL in WT keratinocytes but not in Par2^-/- keratinocytes. HDM-WT mice treated with the MIF antagonist ISO-1 displayed improvement of AD-like presentations and lower expressions of IL-4, IL-13, TSLP and Arg1 (a biomarker of M2 macrophage) mRNAs. We conclude that MIF is an important cytokine and is significantly increased in the AD model. PAR2 affects AD changes by regulating the expression, intracellular trafficking, and secretion of MIF in epidermis.