Molecular signature of pruriceptive MrgprA3+ neurons

Yanyan Xing, Junyu Chen, Henry Hilley, Haley Steele, Jingjing Yang, Liang Han

Abstract

Itch, initiated by the activation of sensory neurons, is frequently associated with dermatological diseases. MrgprA3+ sensory neurons have been identified as one of the major itch-sensing neuronal populations. Mounting evidence has demonstrated that peripheral pathological conditions induce physiological regulations of sensory neurons, which is critical for the maintenance of chronic itch sensation. However, the underlying molecular mechanisms are not clear. Here we performed RNA sequencing of genetically labeled MrgprA3+ neurons under both naïve and allergic contact dermatitis conditions. Our results revealed the unique molecular signature of itch-sensing neurons and the distinct transcriptional profile changes that result in response to dermatitis. We found enrichment of nine Mrgpr family members and two histamine receptors in MrgprA3+ neurons, suggesting that MrgprA3+ neurons are a direct neuronal target for histamine and Mrgprs agonists. In addition, Ptpn6 and Pcdh12 were identified as highly selective markers of MrgprA3+ neurons. We also discovered that MrgprA3+ neurons respond to skin dermatitis in a way that is unique from other sensory neurons by regulating a combination of transcriptional factors, ion channels, and key molecules involved in synaptic transmission. These results significantly increase our knowledge of itch transmission and uncover potential targets for combating itch.

Activation of Different Heterodimers of TLR2 Distinctly Mediates Pain and Itch

Ting-Ting Wang, a,c,dy Xian-Yun Xu, a,b,cy Wei Lin, e Dan-Dan Hu, a,b,c Wu Shi, a,b,c Xin Jia, a,b,c Hui Wang, a Ning-Jing Song, d Yu-Qiu Zhang e and Ling Zhang a,b,c*

a The First Rehabilitation Hospital of Shanghai, Tongji University School of Medicine, Shanghai 200090, China
b Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopaedic Department of Tongji

Hospital, School of Medicine, Tongji University, Shanghai, 200065, China

c Department of Anatomy and Histology, Tongji University School of Medicine, Shanghai 200092, China

d Department of Dermatology, Tongren Hospital Shanghai Jiao Tong University School of Medicine Shanghai 200336, China

e State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Department of Translational Neuroscience, Institutes of Brain Science, Fudan University, Shanghai 200032, China

Abstract—Toll-like receptors (TLRs) have been implicated in pain and itch regulation. TLR2, a TLR family member that detects microbial membrane components, has been implicated in pathologic pain. However, the role of TLR2 in pruritic and nociceptive responses has not been thoroughly investigated. In this study, we found that TLR2 was expressed in mouse dorsal root ganglia (DRG) and trigeminal ganglia (TG) neurons. Itch and pain behaviors, including histamine-dependent and histamine-independent acute itching, acetone/diethyl ether/water and 2,4-dini trofluorobenzene-induced chronic itching and inflammatory pain, were largely attenuated in TLR2 knockout (KO) mice. The TLR2 agonist Pam3CSK4, which targets TLR2/1 heterodimers, evoked pain and itch behavior, whereas lipoteichoic acid (LTA) and zymosan, which recognize TLR2/6 heterodimers, produced only pain response. The TLR2 agonist-induced nociceptive and pruritic behaviors were largely diminished in transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) KO mice. Finally, Pam3Csk4 and zymosan increased the [Ca2+]i in DRG neurons from wild-type mice. However, the enhancement of [Ca2+]i was largely inhibited in the DRG neurons from TRPV1 and TRPA1 KO mice. Our results demonstrate that TLR2 is involved in different itch and pain behaviors through activating TLR1/TLR2 or TLR6/TLR2 heterodimers via TRPV1 and TRPA1 channels. ! 2020 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: TLR2, pain, itch, TRPV1, TRPA1.

Complementary roles of murine Na_V1.7, Na_V1.8 and Na_V1.9 in acute itch signalling

Helen Kühn*, Leonie Kappes, Katharina Wolf, Lisa Gebhardt, Markus F. Neurath, Peter Reeh, Michael J. M. Fischer & Andreas E. Kremer* 

Abstract

Acute pruritus occurs in various disorders. Despite severe repercussions on quality of life treatment options remain limited. Voltage-gated sodium channels (Na_V) are indispensable for transformation and propagation of sensory signals implicating them as drug targets. Here, Na_V1.7, 1.8 and 1.9 were compared for their contribution to itch by analysing Na_V-specific knockout mice. Acute pruritus was induced by a comprehensive panel of pruritogens (C48/80, endothelin, 5-HT, chloroquine, histamine, lysophosphatidic acid, trypsin, SLIGRL, β-alanine, BAM8-22), and scratching was assessed using a magnet-based recording technology. We report an unexpected stimulus-dependent diversity in Na_V channel-mediated itch signalling. Na_V1.7^−/− showed substantial scratch reduction mainly towards strong pruritogens. Na_V1.8^−/− impaired histamine and 5-HT-induced scratching while Na_V1.9 was involved in itch signalling towards 5-HT, C48/80 and SLIGRL. Furthermore, similar microfluorimetric calcium responses of sensory neurons and expression of itch-related TRP channels suggest no change in sensory transduction but in action potential transformation and conduction. The cumulative sum of scratching over all pruritogens confirmed a leading role of Na_V1.7 and indicated an overall contribution of Na_V1.9. Beside the proposed general role of Na_V1.7 and 1.9 in itch signalling, scrutiny of time courses suggested Na_V1.8 to sustain prolonged itching. Therefore, Na_V1.7 and 1.9 may represent targets in pruritus therapy.

Neuronal branching of sensory neurons is associated with BDNF-positive eosinophils in atopic dermatitis

Guseva D, Rüdrich U, Kotnik N, Gehring M, Patsinakidis N, Agelopoulos K, Ständer S, Homey B, Kapp A, Gibbs BF, Ponimaskin E, Raap U.

Abstract

BACKGROUND: Pruritus is a major symptom of atopic dermatitis (AD) and is transmitted by a subpopulation of non-myelinated C-type free nerve endings in the epidermis and upper dermis. Stimulation of these nerve terminals is affected by histamine, neurotrophins and physical factors. Eosinophils of patients with AD are a source of neurotrophins, including brain-derived neurotrophic factor (BDNF), levels of which correlate with disease severity.

OBJECTIVE: The purpose of this study was to determine the anatomical localization of eosinophils in the skin of patients with AD with regard to peripheral nerves and to investigate whether eosinophils induce sprouting and neurite outgrowth in murine sensory neurons.

RESULTS: Dermal axon density and the proximity of eosinophils to nerve fibres were significantly higher in AD patients vs NA. Both neuronal projections and eosinophils expressed BDNF. Furthermore, activated eosinophil supernatants induced BDNF-dependent mouse DRG neuron branching.

CONCLUSIONS AND CLINICAL RELEVANCE: Our results indicate that BDNF-positive eosinophils are also localized in close proximity with nerve fibres in AD, suggesting a functional relationship between BDNF-expressing eosinophils and neuronal projections. These observations suggest that eosinophils may have considerable impact on pruritus by supporting sensory nerve branching.

Abstract

While imiquimod (IMQ) has been widely used in dermatology, its side effect manifested as dermatitis couldn’t be ignored. However, the underlying mechanism has not been fully understood. Considering the clinical features of IMQ-related dermatitis similar to pseudo-allergic reaction and the presence of large numbers of mast cell in tissues treated with IMQ, the possibility that IMQ-related dermatitis mediated by mast cell-specific Mas-related G protein-coupled receptor X2 (MRGPRX2) should be addressed. To investigate the role of MRGPRX2 in vivo, MrgprB2, the mice homology of human MRGPRX2, was detected in IMQ-induced dermatitis mouse model. Histopathological changes including mast cell degranulation and footpad swelling were assayed in wild-type and MrgprB2^−/− mice. The results showed that IMQ application induced dermatitis and footpad swelling with inflammatory cells infiltration plus mast cell activation in the skin of wild-type mice but reduced significantly in MrgprB2^−/− mice. Further, compared to wild-type mice, serum histamine and inflammatory cytokine levels were compromised in MrgprB2^−/− mice treated with IMQ, while the serum IgE level didn’t change significantly. In vitro studies, levels of mediators released from murine peritoneal mast cells (MPMCs) after IMQ treatment were increased in a dose-dependent manner, which was much mild in MPMCs from MrgprB2^−/− mice. Intracellular Ca²⁺ concentration was increased in a dose-dependent manner after IMQ treatment both in MrgprB2-HEK293 and MRGPRX2-HEK293 cells. Moreover, β-hexosaminidase released after IMQ treatment was blocked by siRNA directed at the MRGPRX2 receptor in LAD2 cells. In summary, MrgprB2 /MRGPRX2 mediate mast cell activation and participate in IMQ-related dermatitis.

PAR2 mediates itch via TRPV3 signaling in keratinocytes

Jiahui Zhao, Admire Munanairi, Xian-Yu Liu, Jie Zhang, Linghan Hu, Meiqin Hu, Dingfang Bu, Lingling Liu, Zhiqiang Xie, Brian S. Kim, Yong Yang, Zhou-Feng Chen

The Journal of Investigative Dermatology

20 August 2019
10 December 2019 12 January 2020

Please cite this article as: Zhao J, Munanairi A, Liu X-Y, Zhang J, Hu L, Hu M, Bu D, Liu L, Xie Z,
Kim BS, Yang Y, Chen Z-F, PAR2 mediates itch via TRPV3 signaling in keratinocytes, The Journal of Investigative Dermatology (2020), doi: https://doi.org/10.1016/j.jid.2020.01.012.

Abstract

Animal studies have suggested that transient receptor potential (TRP) ion channels and G protein-coupled receptors (GPCRs) play important roles in itch transmission. TRPV3 gain-of-function mutations have been identified in patients with Olmsted syndrome which is associated with severe pruritus. However, the mechanisms causing itch remain poorly understood. Here, we show that keratinocytes lacking TRPV3 impair the function of protease activated receptor 2 (PAR2), resulting in reduced neuronal activation and scratching behavior in response to PAR2 agonists. Moreover, we show that TRPV3 and PAR2 were upregulated in skin biopsies from patients and mice with atopic dermatitis (AD), whereas their inhibition attenuated scratching and inflammatory responses in mouse AD models. Taken together, these results reveal a previously unrecognized link between TRPV3 and PAR2 in keratinocytes to convey itch information and suggest that a blockade of PAR2 or TRPV3 individually or both may serve as a potential approach for antipruritic therapy in AD.

Key Words

TRPV3PAR2itchkeratinocytescalciumatopic dermatitis

Abbreviations

ADatopic dermatitisBAM 8-22bovine adrenal medulla peptide 8-22DRGdorsal root gangliaGPCRG protein coupled receptorMrgprMas-related G-protein coupled receptorPAR2protease-activated receptor 2TRPV3transient receptor potential cation channel V3TSLPthymic stromal lymphopoietin

Quantitative Characterization of the Neuropeptide Level Changes in Dorsal Horn and Dorsal Root Ganglia Regions of the Murine Itch Models

Emily G Tillmaand, Krishna D B Anapindi, Eduardo De La Toba, Changxiong J Guo, Jessica Krebs, Ashley E Lenhart, Qin Liu, Jonathan V Sweedler

Abstract

Chronic itch can be extremely devastating and, in many cases, difficult to treat. One challenge in treating itch disorders is the limited understanding of the multitude of chemical players involved in the communication of itch sensation from the peripheral to central nervous system. Neuropeptides are intercellular signaling molecules that are known to be involved in the transmission of itch signals from primary afferent neurons, which detect itch in the skin, to higher-order circuits in the spinal cord and brain. To investigate the role neuropeptides play in transmitting itch signals, we generated two mouse models of chronic itch-Acetone-Ether-Water (AEW, dry skin) and calcipotriol (MC903, atopic dermatitis). For peptide identification and quantitation, we analyzed the peptide content of dorsal root ganglia (DRG) and dorsal horn (DH) tissues from chronically itchy mice using liquid chromatography coupled to tandem mass spectrometry. De novo-assisted database searching facilitated the identification and quantitation of 335 peptides for DH MC903, 318 for DH AEW, 266 for DRG MC903, and 271 for DRG AEW. Of these quantifiable peptides, we detected 30 that were differentially regulated in the tested models, after accounting for multiple testing correction (q<0.1). These include several peptide candidates derived from neuropeptide precursors, such as proSAAS, protachykinin-1, proenkephalin and calcitonin gene-related peptide, some of them previously linked to itch. The peptides identified in this study may help elucidate our understanding about these debilitating disorders. Data are available via ProteomeXchange with identifier PXD015949.

Calcium imaging of primary canine sensory neurons: Smalldiameter neurons responsive to pruritogens and algogens

Joy Rachel C. Ganchingco| Tomoki Fukuyama | Jeffrey A. Yoder | Wolfgang Bäumer

Abstract

Calcium imaging of primary canine sensory neurons: Smalldiameter neurons responsive to pruritogens and algogensAbstract Introduction: Rodent primary sensory neurons are commonly used for studying itch and pain neurophysiology, but translation from rodents to larger mammals and humans is not direct and requires further validation to make correlations. Methods: This study developed a primary canine sensory neuron culture from dorsal root ganglia (DRG) excised from cadaver dogs. Additionally, the canine DRG cell cultures developed were used for single‐cell ratiometric calcium imaging, with the activation of neurons to the following pruritogenic and algogenic substances: histamine, chloroquine, canine protease‐activated receptor 2 (PAR2) activating peptide (SLIGKT), compound 48/80, 5‐hydroxytryptamine receptor agonist (5‐HT), bovine adrenal medulla peptide (BAM8‐22), substance P, allyl isothiocyanate (AITC), and capsaicin. Results: This study demonstrates a simple dissection and rapid processing of DRG collected from canine cadavers used to create viable primary sensory neuron cultures to measure responses to pruritogens and algogens. Conclusion: Ratiometric calcium imaging demonstrated that small‐diameter canine sensory neurons can be activated by multiple stimuli, and a single neuron can react to both a pruritogenic stimulation and an algogenic stimulation.

K E Y W O R D S dorsal laminectomy dissection, dorsal root ganglia cell culture, fura‐2AM, ratiometric calcium imagingCalcium imaging of primary canine sensory neurons: Smalldiameter neurons responsive to pruritogens and algogens

Activation of mas-related G-protein-coupled receptors by the house dust mite cysteine protease Der p1 provides a new mechanism linking allergy and inflammation.

Reddy VB¹, Lerner EA².

Author information

Abstract

Cysteine and serine proteases function via protease-activated and mas-related G-protein-coupled receptors (Mrgprs) to contribute to allergy and inflammation. Der p1 is a cysteine protease and major allergen from the house dust mite and is associated with allergic rhinitis and allergic asthma. Der p1 activates protease-activated receptor 2 and induces the release of the pro-inflammatory cytokine IL-6 from cells. However, the possibility that Der p1 acts on Mrgprs has not been considered. We report here that ratiometric calcium imaging reveals that Der p1 activates the human receptor MRGPRX1 and the mouse homolog MrgprC11, implicated previously in itch. Der p1 cleavage of N-terminal receptor peptides followed by site-directed mutagenesis of the cleavage sites links receptor activation to specific amino acid residues. Der p1 also induced the release of IL-6 from heterologous cells expressing MRGPRX1. In summary, activation of Mrgprs by the allergen Der p1 may contribute to inflammation.

Polymyxin B and polymyxin E induce anaphylactoid response through T mediation of Mas-related G protein–coupled receptor X2

Yingzhuan Zhan, Nan Ma, Rui Liu, Nan Wang, Tao Zhang, Langchong He* School of Pharmacy, Health Science Center, Xi’an Jiaotong University, Xi’an, Shaanxi Province, 710061, PR China

Keywords: Polymyxin B, Polymixin E, Anaphylactoid reaction, MRGPRX2

ABSTRACT

Polymyxin B (PMB) and polymyxin E (PME) are cyclic, peptide antibiotics which derived from various species of Paenibacillus (Bacillus) polymyxa. They are decapeptide antibiotics with an antimicrobial spectrum that includes Gram-negative bacteria, and reused as therapeutic agents due to the emergence of multidrug-resistant (MDR) Gram-positive bacteria. PMB or PME-induced anaphylactoid reactions in the clinic have been documented. However, the mechanism underlying anaphylactoid reaction induced by polymyxin has not yet been reported. Here, we report that human Mas-related G protein-coupled receptor X2 (MRGPRX2) and its mouse homologue Mas-related G protein-coupled receptor B2 (MrgprB2) are the receptors mediating the anaphylactoid response provoked by PMB and PME. We firstly investigated the anaphylactoid reactions induced by PMB and PME in LAD2 cells in vitro and in vivo, and found that treatment with PMB and PME led to significant release of mast cell granules such as histamine and β-hexosaminidase, secretion of pro-inflammatory cytokines, such as TNF-α and PGD2, and provocation of calcium flux in LAD2 cells. Furthermore, treatment with PMB and PME led to reduced release of β-hexosaminidase in MRGPRX2 knockdown-LAD2 cells, and obvious increased calcium release in MRGPRX2 overexpressing HEK293 cells, which suggested that MRGPRX2 are involved in mast cell activation provoked by PMB or PME. In vivo, MRGPRB2 knockout mice exhibited lower pseudo-allergic reactions than wild type mice. Activation of MrgprB2 also triggers increased capillary permeability and paw swelling. Our results elucidated the role of MRGPRX2 in PMB and PME-induced anaphylactoid response and suggested that MRGPRX2 as a potential therapeutic target to control the anaphylactoid reactions which triggered by PMB or PME.