TRPV1 and TRPA1 Channels Are Both Involved Downstream of Histamine-Induced Itch 

by Jenny Wilzopolski ^1,2,3,*, Manfred Kietzmann ¹, Santosh K. Mishra ², Holger Stark ⁴, Wolfgang Bäumer ^2,3 and Kristine Rossbach¹

¹Department of Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine Hannover, Foundation, 30559 Hannover, Germany

²Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607, USA

³Department of Veterinary Medicine, Institute of Pharmacology and Toxicology, Freie Universität Berlin, 14195 Berlin, Germany

⁴Institute of Pharmaceutical and Medical Chemistry, Heinrich Heine University Düsseldorf, 40225 Duesseldorf, Germany

^*Author to whom correspondence should be addressed. Academic Editors: Emanuela Masini, Laura Lucarini and Alessandro AlaimoBiomolecules2021, 11(8), 1166; https://doi.org/10.3390/biom11081166Received: 13 July 2021 / Revised: 31 July 2021 / Accepted: 4 August 2021 / Published: 6 August 2021(This article belongs to the Special Issue New Developments in Histamine Research)

Abstract: Two histamine receptor subtypes (HR), namely H1R and H4R, are involved in the trans- mission of histamine-induced itch as key components. Although exact downstream signaling mechanisms are still elusive, transient receptor potential (TRP) ion channels play important roles in the sensation of histaminergic and non-histaminergic itch. The aim of this study was to investigate the involvement of TRPV1 and TRPA1 channels in the transmission of histaminergic itch. The potential of TRPV1 and TRPA1 inhibitors to modulate H1R- and H4R-induced signal transmission was tested in a scratching assay in mice in vivo as well as via Ca2+ imaging of murine sensory dorsal root ganglia (DRG) neurons in vitro. TRPV1 inhibition led to a reduction of H1R- and H4R- induced itch, whereas TRPA1 inhibition reduced H4R- but not H1R-induced itch. TRPV1 and TRPA1 inhibition resulted in a reduced Ca2+ influx into sensory neurons in vitro. In conclusion, these results indicate that both channels, TRPV1 and TRPA1, are involved in the transmission of histamine-induced pruritus.

Keywords: histamine; histamine H1 receptor; histamine H4 receptor; itch; signal transduction; TRPV1; TRPA1; dorsal root ganglion neurons (DRG); Ca2+-imaging

Analysis of TRPV channel activation by stimulation of FCεRI and MRGPR receptors in mouse peritoneal mast cells

A. Sol ́ıs-Lo ́ pez1, U. Kriebs1, A. Marx1, S. Mannebach2, W. B. Liedtke3, M. J. Caterina4, M. Freichel1, V. V. Tsvilovskyy1*

1 PharmakologischesInstitut,Ruprecht-Karls-Universita ̈tHeidelberg,Heidelberg,Baden-Wu ̈rttemberg, Germany, 2 Experimentelle und Klinische Pharmakologie und Toxikologie, Universita ̈t des Saarlandes, Homburg, Saarland, Germany, 3 Department of Neurology, School of Medicine Duke University, Durham, North Carolina, United States of America, 4 Departments of Neurosurgery, Biological Chemistry, and Neuroscience, Neurosurgery Pain Research Institute, The Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America

Abstract

The activation of mast cells (MC) is part of the innate and adaptive immune responses and depends on Ca2+ entry across the plasma membrane, leading to the release of preformed inflammatory mediators by degranulation or by de novo synthesis. The calcium conducting channels of the TRPV family, known by their thermo and osmotic sensitivity, have been pro- posed to be involved in the MC activation in murine, rat, and human mast cell models. So far, immortalized mast cell lines and nonspecific TRPV blockers have been employed to charac- terize the role of TRPV channels in MC. The aim of this work was to elucidate the physiological role of TRPV channels by using primary peritoneal mast cells (PMCs), a model of connective tissue type mast cells. Our RT-PCR and NanoString analysis identified the expression of TRPV1, TRPV2, and TRPV4 channels in PMCs. For determination of the functional role of the expressed TRPV channels we performed measurements of intracellular free Ca2+ concentra- tions and beta-hexosaminidase release in PMCs obtained from wild type and mice deficient for corresponding TRPV1, TRPV2 and TRPV4 in response to various receptor-mediated and physical stimuli. Furthermore, substances known as activators of corresponding TRPV-chan- nels were also tested using these assays. Our results demonstrate that TRPV1, TRPV2, and TRPV4 do not participate in activation pathways triggered by activation of the high-affinity receptors for IgE (FcεRI), Mrgprb2 receptor, or Endothelin-1 receptor nor by heat or osmotic stimulation in mouse PMCs.

MrgprB4 in trigeminal neurons expressing TRPA1 modulates unpleasant sensations

Shota Tobori a, 1, Haruka Hiyama a, 1, Takahito Miyake a, b, Yuichi Yano a, Kazuki Nagayasu a, Hisashi Shirakawa a, *, Takayuki Nakagawa c, Yasuo Mori d, Shuji Kaneko a

a Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
b Department of Systems Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto 606- 8501, Japan

c Department of Clinical Pharmacology and Therapeutics, Kyoto University Hospital, 54 Shogoin -Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
d Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Building A4, Katsura Campus, Nishikyo-ku, Kyoto 615-8510, Japan

ABSTRACT

Gentle touch such as stroking of the skin produces a pleasant feeling, which is detected by a rare subset of sensory neurons that express Mas-related G protein-coupled receptor B4 (MrgprB4) in mice. We examined small populations of MrgprB4-positive neurons in the trigeminal ganglion and the dorsal root ganglion, and most of these were sensitive to transient receptor potential ankyrin 1 (TRPA1) agonist but not TRPV1, TRPM8, or TRPV4 agonists. Deficiency of MrgprB4 did not affect noxious pain or itch be- haviors in the hairless plantar and hairy cheek. Although behavior related to acetone-induced cold sensing in the hind paw was not changed, unpleasant sensory behaviors in response to acetone appli- cation or sucrose splash to the cheek were significantly enhanced in Mrgprb4-knockout mice as well as in TRPA1-knockout mice. These results suggest that MrgprB4 in the trigeminal neurons produces pleasant sensations in cooperation with TRPA1, rather than noxious or cold sensations. Pleasant sensa- tions may modulate unpleasant sensations on the cheek via MrgprB4.

© 2021 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Keywords:

Sensory behavior Hairy skin
MrgprB4
TRPA1
Trigeminal ganglion

MrgprC11+ sensory neurons mediate glabrous skin itch

Proc Natl Acad Sci U S A. 2021 Apr 13;118(15):e2022874118. doi: 10.1073/pnas.2022874118.

Haley R. Steele, Yanyan Xinga, Yuyan Zhu, Henry B. Hilley, Katy Lawson, Yeseul Nho, Taylor Niehoff, and Liang Hana

School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA 30332
Edited by Jeremy Nathans, Johns Hopkins University School of Medicine, Baltimore, MD, and approved March 2, 2021 (received for review November 2, 2020)

Itch arising from glabrous skin (palms and soles) has attracted limited attention within the field due to the lack of methodology. This is despite glabrous itch arising from many medical conditions such as plantar and palmar psoriasis, dyshidrosis, and cholestasis. Therefore, we developed a mouse glabrous skin behavioral assay to investigate the contribution of three previously identified pruriceptive neurons in glabrous skin itch. Our results show that MrgprA3+ and MrgprD+ neurons, although key mediators for hairy skin itch, do not play important roles in glabrous skin itch, dem- onstrating a mechanistic difference in itch sensation between hairy and glabrous skin. We found that MrgprC11+ neurons are the major mediators for glabrous skin itch. Activation of MrgprC11+ neurons induced glabrous skin itch, while ablation of MrgprC11+ neurons reduced both acute and chronic glabrous skin itch. Our study provides insights into the mechanisms of itch and opens up new avenues for future glabrous skin itch research.

Astrocytic STAT3 activation and chronic itch require IP3R1/TRPC-dependent Ca21 signals in mice

Miho Shiratori-Hayashi, PhD,a Chiharu Yamaguchi, MPharm,a Kazushi Eguchi, MPharm,a Yuto Shiraishi, BPharm,a Keita Kohno, BPharm,a Katsuhiko Mikoshiba, MD, PhD,b,c,d Kazuhide Inoue, PhD,e Motohiro Nishida, PhD,f,g and Makoto Tsuda, PhDa 

Background: Chronic itch is a debilitating symptom of inflammatory skin diseases, but the underlying mechanism is poorly understood. We have recently demonstrated that astrocytes in the spinal dorsal horn become reactive in models of atopic and contact dermatitis via activation of the transcription factor signal transducer and activator of transcription 3 (STAT3) and critically contribute to chronic itch. In general, STAT3 is transiently activated; however, STAT3 activation in reactive astrocytes of chronic itch model mice persistently occurs via an unknown mechanism.
Objective: We aimed to determine the mechanisms of persistent activation of astrocytic STAT3 in chronic itch conditions. Methods: To determine the factors that are required for persistent activation of astrocytic STAT3, Western blotting and calcium imaging with cultured astrocytes or spinal cord slices were performed. Thereafter, chronic itch model mice were used for genetic and behavioral experiments to confirm the role of the factors determined to mediate persistent STAT3 activation from in vitro and ex vivo experiments in chronic itch.

Results: IP3 receptor type 1 (IP3R1) knockdown in astrocytes suppressed IL-6–induced persistent STAT3 activation and expression of lipocalin-2 (LCN2), an astrocytic STAT3- dependent inflammatory factor that is required for chronic itch. IP3R1-dependent astrocytic Ca21 responses involved Ca21 influx through the cation channel transient receptor potential canonical (TRPC), which was required for persistent STAT3 activation evoked by IL-6. IL-6 expression was upregulated in dorsal root ganglion neurons in a mouse model of chronic itch. Dorsal root ganglion neuron–specific IL-6 knockdown, spinal astrocyte–specific IP3R1 knockdown, and pharmacologic spinal TRPC inhibition attenuated LCN2 expression and chronic itch.

Conclusion: Our findings suggest that IP3R1/TRPC channel– mediated Ca21 signals elicited by IL-6 in astrocytes are necessary for persistent STAT3 activation, LCN2 expression, and chronic itch, and they may also provide new targets for therapeutic intervention. (J Allergy Clin Immunol 2021;147:1341-53.)

Key words: Contact dermatitis, chronic itch, astrocytes, STAT3, lip- ocalin-2, Ca21 signal, IP3R1, TRPC, IL-6, primary afferent sensory neuron

Transient receptor potential ankyrin 1 (TRPA1) positively regulates imiquimod‐induced, psoriasiform dermal inflammation in mice

YanZhou1,2 | DanHan1,2 BoWang4 | ZhenruiShi2 Samuel T. Hwang2 TaylorFollansbee3 | XuesongWu2 | SebastianYu2 | | DanT.Domocos3 | MirelaCarstens3 | EarlCarstens3 |

1Department of Dermatology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi, China

2Department of Dermatology, University of California, Davis, California

3Department of Neurobiology, Physiology and Behavior, University of California, Davis, California

4Center for Translational Medicine, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi, China

Abstract

Transient receptor potential ankyrin 1 (TRPA1), a membrane protein ion channel, is known to mediate itch and pain in skin. The function of TRPA1, however, in psori‐ asiform dermatitis (PsD) is uncertain. Herein, we found that expression of TRPA1 is highly up‐regulated in human psoriatic lesional skin. To study the role of TRPA1 in PsD, we assessed Psoriasis Severity Index (PSI) scores, transepidermal water loss (TEWL), skin thickness and pathology, and examined dermal inflammatory infiltrates, Th17‐related genes and itch‐related genes in c57BL/6 as wild‐type (WT) and TRPA1 gene knockout (KO) mice following daily application of topical IMQ cream for 5 days. Compared with WT mice, clinical scores, skin thickness change and TEWL scores were similar on day 3, but were significantly decreased on day 5 in IMQ‐treated TRPA1 KO mice (vs WT mice), suggesting reduced inflammation and skin barrier de‐ fects. Additionally, the relative area of epidermal Munro’s microabscesses and mRNA levels of neutrophil inducible chemokines (S100A8, S100A9 and CXCL1) were de‐ creased in the treated skin of TRPA1 KO mice, suggesting that neutrophil recruitment was impaired in the KO mice. Furthermore, mast cells, CD31+ blood vascular cells, CD45+ leukocytes and CD3+ T cells were all reduced in the treated skin of TRPA1 KO mice. Lastly, mRNA expression levels of IL‐1β, IL‐6, IL‐23, IL‐17A, IL‐17F and IL‐22 were decreased in TRPA1 KO mice. In summary, these results suggest a key role for TRPA1 in psoriasiform inflammation and raising its potential as a target for therapeu‐ tic intervention.

KEYWORDS

imiquimod, inflammation, itch, mice, psoriasis, TRPA1

Genetic priming of sensory neurons in mice that overexpress PAR2 enhances allergen responsiveness

Joao M. Braza,1, Todd Demboa,1, Alexandra Charruyerb, Ruby Ghadiallyb,c, Marlys S. Fassettc,d, and Allan I. Basbauma,2

aDepartment of Anatomy, University of California, San Francisco, CA 94158; bDivision of Dermatology, San Francisco Veteran’s Administration Medical Center, San Francisco, CA 94121; cDepartment of Dermatology, University of California, San Francisco, CA 94158; and dDepartment of Microbiology and Immunology, University of California, San Francisco, CA 94158

Contributed by Allan I. Basbaum, January 13, 2021 (sent for review October 14, 2020; reviewed by Diana M. Bautista and Earl Carstens)

Pruritus is a common symptom of inflammatory skin conditions, including atopic dermatitis (AD). Although primary sensory neu- rons that transmit pruritic signals are well-cataloged, little is known about the neuronal alterations that occur as a result of skin disruption in AD. To address this question, we examined the mo- lecular and behavioral consequences of challenging Grhl3PAR2/+ mice, which overexpress PAR2 in suprabasal keratinocytes, with serial topical application of the environmental allergen house dust mite (HDM). We monitored behavior and used RNA sequencing, qPCR, and in situ hybridization to evaluate gene expression in tri- geminal ganglia (TG), before and after HDM. We found that nei- ther Grhl3PAR2/+ nor wild-type (WT) mice exhibited spontaneous scratching, and pruritogen-induced acute scratching did not differ. In contrast, HDM exacerbated scratching in Grhl3PAR2/+ mice. Despite the absence of scratching in untreated Grhl3PAR2/+ mice, several TG genes in these mice were up-regulated compared to WT. HDM treat- ment of the Grhl3PAR2/+ mice enhanced up-regulation of this set of genes and induced additional genes, many within the subset of TG neurons that express TRPV1. The same set of genes was up- regulated in HDM-treated Grhl3PAR2/+ mice that did not scratch, but at lesser magnitude. Finally, we recorded comparable transcrip- tional changes in IL31Tg mice, demonstrating that a common ge- netic program is induced in two AD models. Taken together, we conclude that transcriptional changes that occur in primary sensory neurons in dermatitis-susceptible animals underlie a genetic priming that not only sensitizes the animal to chronic allergens but also contributes to pruritus in atopic skin disease.

itch | dermatitis | trigeminal neurons | PAR2 | RNA sequencing

Thymic Stromal Lymphopoietin Promotes MRGPRX2-Triggered Degranulation of Skin Mast Cells in a STAT5-Dependent Manner with Further Support from JNK

Magda Babina *, Zhao Wang, Kristin Franke and Torsten Zuberbier

Mast Cell Biology Unit, Department of Dermatology and Allergy, Charité–Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 10117 Berlin, Germany; zhao.wang@charite.de (Z.W.); kristin.franke@charite.de (K.F.); torsten.zuberbier@charite.de (T.Z.)
* Correspondence: magda.babina@charite.de; Tel.: +49-30-1751649539; Fax: +49-30-450518900

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

Keywords: mast cell; MRGPRX2; pseudo-allergy; Substance P; TSLP; skin

Resolvin D3 controls mouse and human TRPV1-positive neurons and preclinical progression of psoriasis

Sang Hoon Lee1, Raquel Tonello1, Sang-Taek Im2, Hawon Jeon3, Jeongsu Park3, Zachary Ford1, Steve Davidson1, Yong Ho Kim2,3, Chul-Kyu Park2,3 and Temugin Berta1

1. Pain Research Center, Department of Anesthesiology, University of Cincinnati Medical Center, Cincinnati, OH, USA.
2. Gachon Pain Center and Department of Physiology, College of Medicine, Gachon University, Incheon 21999, Republic of Korea.
3. Department of Health Sciences and Technology, GAIHST, Gachon University, Incheon 21999, Korea, Republic of Korea.

Corresponding authors: Temugin Berta, Pain Research Center, Department of Anesthesiology, University of Cincinnati Medical Center, Cincinnati, Ohio 45267, USA. E-mail: temugin.berta@uc.edu & Chul-Kyu Park, Gachon Pain Center and Department of Physiology, College of Medicine, Gachon University, Incheon 21999, Republic of Korea. Email: pck0708@gachon.ac.kr

© The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.

Received: 2020.08.18; Accepted: 2020.10.12; Published: 2020.10.26

Abstract

Rationale: Psoriasis is a chronic inflammatory disease caused by a complex interplay between the immune and nervous systems with recurrent scaly skin plaques, thickened stratum corneum, infiltration and activation of inflammatory cells, and itch. Despite an increasing availability of immune therapies, they often have adverse effects, high costs, and dissociated effects on inflammation and itch. Activation of sensory neurons innervating the skin and TRPV1 (transient receptor potential vanilloid 1) are emerging as critical components in the pathogenesis of psoriasis, but little is known about their endogenous inhibitors. Recent studies have demonstrated that resolvins, endogenous lipid mediators derived from omega-3 fatty acids, are potent inhibitors of TRP channels and may offer new therapies for psoriasis without known adverse effects.

Methods: We used behavioral, electrophysiological and biochemical approaches to investigate the therapeutic effects of resolvin D3 (RvD3), a novel family member of resolvins, in a preclinical model of psoriasis consisting of repeated topical applications of imiquimod (IMQ) to murine skin, which provokes inflammatory lesions that resemble human psoriasis.

Results: We report that RvD3 specifically reduced TRPV1-dependent acute pain and itch in mice. Mechanistically, RvD3 inhibited capsaicin-induced TRPV1 currents in dissociated dorsal root ganglion (DRG) neurons via the N-formyl peptide receptor 2 (i.e. ALX/FPR2), a G-protein coupled receptor. Single systemic administration of RvD3 (2.8 mg/kg) reversed itch after IMQ, and repeated administration largely prevented the development of both psoriasiform itch and skin inflammation with concomitant decreased in calcitonin gene-related peptide (CGRP) expression in DRG neurons. Accordingly, specific knockdown of CGRP in DRG was sufficient to prevent both psoriasiform itch and skin inflammation similar to the effects following RvD3 administration. Finally, we elevated the translational potential of this study by showing that RvD3 significantly inhibited capsaicin-induced TRPV1 activity and CGRP release in human DRG neurons.

Conclusions: Our findings demonstrate a novel role for RvD3 in regulating TRPV1/CGRP in mouse and human DRG neurons and identify RvD3 and its neuronal pathways as novel therapeutic targets to treat psoriasis.

Key words: psoriasis, skin inflammation, sensory neurons, pruritus, resolvin D3, TRPV1, CGRP

Behavioral characterization of a CRISPR-generated TRPA1 knockout rat in models of pain, itch, and asthma

Rebecca M. Reese1,4, Michelle Dourado1,4, Keith Anderson 3, Søren Warming 3, Kimberly L. Stark1, Alessia Balestrini2, eric Suto2, Wyne Lee2, Lorena Riol-Blanco2, Shannon D. Shields1 & David H. Hackos1*

The transient receptor potential (TRP) superfamily of ion channels has garnered significant attention by the pharmaceutical industry. In particular, TRP channels showing high levels of expression in sensory neurons such as TRPV1, TRPA1, and TRPM8, have been considered as targets for indications where sensory neurons play a fundamental role, such as pain, itch, and asthma. Modeling these indications in rodents is challenging, especially in mice. The rat is the preferred species for pharmacological studies
in pain, itch, and asthma, but until recently, genetic manipulation of the rat has been technically challenging. Here, using CRISPR technology, we have generated a TRPA1 KO rat to enable more sophisticated modeling of pain, itch, and asthma. We present a detailed phenotyping of the TRPA1
KO rat in models of pain, itch, and asthma that have previously only been investigated in the mouse. With the exception of nociception induced by direct TRPA1 activation, we have found that the TRPA1 KO rat shows apparently normal behavioral responses in multiple models of pain and itch. Immune cell infiltration into the lung in the rat OVA model of asthma, on the other hand, appears to be dependent on TRPA1, similar to was has been observed in TRPA1 KO mice. Our hope is that the TRPA1 KO rat will become a useful tool in further studies of TRPA1 as a drug target.