Spinal Inhibitory Ptf1a-Derived Neurons Prevent Self-Generated Itch

Augusto Escalante , Rüdiger Klein

Abstract

Chronic itch represents an incapacitating burden on patients suffering from a spectrum of diseases. Despite recent advances in our understanding of the cells and circuits implicated in the processing of itch information, chronic itch often presents itself without an apparent cause. Here, we identify a spinal subpopulation of inhibitory neurons defined by the expression of Ptf1a, involved in gating mechanosensory information self-generated during movement. These neurons receive tactile and motor input and establish presynaptic inhibitory contacts on mechanosensory afferents. Loss of Ptf1a neurons leads to increased hairy skin sensitivity and chronic itch, partially mediated by the classic itch pathway involving gastrin-releasing peptide receptor (GRPR) spinal neurons. Conversely, chemogenetic activation of GRPR neurons elicits itch, which is suppressed by concomitant activation of Ptf1a neurons. These findings shed light on the circuit mechanisms implicated in chronic itch and open novel targets for therapy developments.

Keywords: DREADD receptors; GRPR; Ptf1a; dorsal horn interneurons; intersectional genetics; itch; spinal cord.

A Group of Cationic Amphiphilic Drugs Activates MRGPRX2 and Induces Scratching Behavior in Mice

Katharina Wolf, PhD#*a , Helen Kühn, PhD#a , Felicitas Boehm, MSca , Lisa Gebhardt, MSca , Markus Glaudo, MSc , Konstantin Agelopoulos, PhDb , Sonja Ständer, MDb , Philipp Ectors, PhDc , Dirk Zahn, PhDc , Yvonne K. Riedel, MScd , Dominik Thimm, PhDd , Christa E. Müller, PhDd , Sascha Kretschmann, PhDe , Anita N. Kremer, MD, PhDe , Daphne Chienf, BSc, Nathachit Limjunyawongf , PhD, Qi Pengf , Xinzhong Dong, PhDf , Pavel Kolkhir, MDg,h , Jörg Scheffel, PhDh , Mia Lykke Søgaard, MSca , Benno Weigmann, PhDa , Markus F. Neurath, MDa,i, Tomasz Hawro, MD, PhDh , Martin Metz, MDh , Michael J.M. Fischer, MDj , Andreas E. Kremer, MD, PhD*a

ABSTRACT

Background: Mas gene-related G protein-coupled receptors (MRGPRs) are a GPCR family responsive to various exogenous and endogenous agonists, playing a fundamental role in pain and itch sensation. The primate-specific family member MRGPRX2 and its murine orthologue MRGPRB2 are expressed by mast cells, mediating IgE-independent signaling and pseudo-allergic drug reactions. Objectives: Therefore, knowledge about the function and regulation of MRGPRX2/MRGPRB2 is of major importance in prevention of drug hypersensitivity reactions and drug-induced pruritus.

Methods: To identify novel MRGPR (ant)agonists, we screened a library of pharmacologically active compounds utilizing a high-throughput calcium mobilization assay. Identified hit compounds were analyzed for their pseudo-allergic and pruritogenic effects in mice and human.

Results: We found a class of commonly used drugs activating MRGPRX2 which consists to a large extent of antidepressants, antiallergic drugs, and antipsychotics. Three-dimensional pharmacophore modeling revealed structural similarities of the identified agonists, classifying them as cationic amphiphilic drugs. Mast cell activation was investigated using the three representatively selected antidepressants clomipramine, paroxetine, and desipramine. Indeed, we could show a concentration-dependent activation and MRGPRX2-dependent degranulation of the human mast cell line LAD2. Furthermore, clomipramine, paroxetine, and desipramine were able to induce degranulation of human skin and murine peritoneal mast cells. These substances elicited dose-dependent scratching behavior upon intradermal injection in C57BL/6 mice but less in MRGPRB2-mutant mice as well as wheal-and-flare reactions upon intradermal injections in humans.

Conclusion: Our results contribute to the characterization of structure-activity relationships and functionality of MRGPRX2 ligands and facilitate prediction of adverse reactions like drug induced pruritus to prevent severe drug hypersensitivity reactions.

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

Low-Threshold Mechanosensitive VGLUT3-Lineage Sensory Neurons Mediate Spinal Inhibition of Itch by Touch

Kent Sakai, Kristen M. Sanders, Shing-Hong Lin, Darya Pavlenko, Hideki Funahashi, Taisa Lozada, Shuanglin Hao, Chih-Cheng Chen and Tasuku Akiyama

Abstract

Innocuous mechanical stimuli, such as rubbing or stroking the skin, relieve itch through the activation of low-threshold mechanoreceptors. However, the mechanisms behind this inhibition remain unknown. We presently investigated whether stroking the skin reduces the responses of superficial dorsal horn neurons to pruritogens in male C57BL/6J mice. Single-unit recordings revealed that neuronal responses to chloroquine were enhanced during skin stroking, and this was followed by suppression of firing below baseline levels after the termination of stroking. Most of these neurons additionally responded to capsaicin. Stroking did not suppress neuronal responses to capsaicin, indicating state-dependent inhibition. Vesicular glutamate transporter 3 (VGLUT3)-lineage sensory nerves compose a subset of low-threshold mechanoreceptors. Stroking-related inhibition of neuronal responses to chloroquine was diminished by optogenetic inhibition of VGLUT3-lineage sensory nerves in male and female Vglut3-cre/NpHR-EYFPmice. Conversely, in male and female Vglut3-cre/ChR2-EYFP mice, optogenetic stimulation of VGLUT3-lineage sensory nerves inhibited firing responses of spinal neurons to pruritogens after the termination of stimulation. This inhibition was nearly abolished by spinal delivery of the κ-opioid receptor antagonist nor-binaltorphimine dihydrochloride, but not the neuropeptide Y receptor Y1 antagonist BMS193885. Optogenetic stimulation of VGLUT3-lineage sensory nerves inhibited pruritogen-evoked scratching without affecting mechanical and thermal pain behaviors. Therefore, VGLUT3-lineage sensory nerves appear to mediate inhibition of itch by tactile stimuli.

Significance Statement 

Rubbing or stroking the skin is known to relieve itch. We investigated the mechanisms behind touch-evoked inhibition of itch in mice. Stroking the skin reduced the activity of itch-responsive spinal neurons. Optogenetic inhibition of VGLUT3-lineage sensory nerves diminished stroking-evoked inhibition, and optogenetic stimulation of VGLUT3-lineage nerves inhibited pruritogen-evoked firing. Together, our results provide a mechanistic understanding of touch-evoked inhibition of itch.

Abstract

Itch is an evolutionarily conserved sensation that facilitates expulsion of pathogens and noxious stimuli from the skin. However, in organ failure, cancer, and chronic inflammatory disorders such as atopic dermatitis (AD), itch becomes chronic, intractable, and debilitating. In addition to chronic itch, patients often experience intense acute itch exacerbations. Recent discoveries have unearthed the neuroimmune circuitry of itch, leading to the development of anti-itch treatments. However, mechanisms underlying acute itch exacerbations remain overlooked. Herein, we identify that a large proportion of patients with AD harbor allergen-specific immunoglobulin E (IgE) and exhibit a propensity for acute itch flares. In mice, while allergen-provoked acute itch is mediated by the mast cell-histamine axis in steady state, AD-associated inflammation renders this pathway dispensable. Instead, a previously unrecognized basophil-leukotriene (LT) axis emerges as critical for acute itch flares. By probing fundamental itch mechanisms, our study highlights a basophil-neuronal circuit that may underlie a variety of neuroimmune processes.

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

Central opioid receptors mediate morphine-induced itch and chronic itch via disinhibition

Zilong Wang, Changyu Jiang, Hongyu Yao, Ouyang Chen, Sreya Rahman, Yun Gu, Junli Zhao, Yul Huh, Ru-Rong Ji 

Abstract

Opioids such as morphine are mainstay treatments for clinical pain conditions. Itch is a common side effect of opioids, particularly as a result of epidural or intrathecal administration. Recent progress has advanced our understanding of itch circuits in the spinal cord. However, the mechanisms underlying opioid-induced itch are not fully understood, although an interaction between µ-opioid receptor (MOR) and gastrin-releasing peptide receptor (GRPR) in spinal GRPR-expressing neurons has been implicated. In this study we investigated the cellular mechanisms of intrathecal opioid-induced itch by conditional deletion of MOR-encoding Oprm1 in distinct populations of interneurons and sensory neurons. We found that intrathecal injection of the MOR agonists morphine or DAMGO elicited dose-dependent scratching as well as licking and biting, but this pruritus was totally abolished in mice with a specific Oprm1 deletion in Vgat+ neurons [Oprm1-Vgat (Slc32a1)]. Loss of MOR in somatostatin+ interneurons and TRPV1+ sensory neurons did not affect morphine-induced itch but impaired morphine-induced antinociception. In situ hybridization revealed Oprm1 expression in 30% of inhibitory and 20% of excitatory interneurons in the spinal dorsal horn. Whole-cell recordings from spinal cord slices showed that DAMGO induced outward currents in 9 of 19 Vgat+ interneurons examined. Morphine also inhibited action potentials in Vgat+ interneurons. Furthermore, morphine suppressed evoked inhibitory postsynaptic currents in postsynaptic Vgat- excitatory neurons, suggesting a mechanism of disinhibition by MOR agonists. Notably, morphine-elicited itch was suppressed by intrathecal administration of NPY and abolished by spinal ablation of GRPR+ neurons with intrathecal injection of bombesin-saporin, whereas intrathecal GRP-induced itch response remained intact in mice lacking Oprm1-Vgat. Intrathecal bombesin-saporin treatment reduced the number of GRPR+ neurons by 97% in the lumber spinal cord and 91% in the cervical spinal cord, without changing the number of Oprm1+ neurons. Additionally, chronic itch from DNFB-induced allergic contact dermatitis was decreased by Oprm1-Vgat deletion. Finally, naloxone, but not peripherally restricted naloxone methiodide, inhibited chronic itch in the DNFB model and the CTCL model, indicating a contribution of central MOR signalling to chronic itch. Our findings demonstrate that intrathecal morphine elicits itch via acting on MOR on spinal inhibitory interneurons, leading to disinhibition of the spinal itch circuit. Our data have also provided mechanistic insights into the current treatment of chronic itch with opioid receptor antagonist such as naloxone.

Keywords: inhibitory interneurons; itch; lymphoma; opioid; spinal cord.

Abstract

Background: Tick bites severely threaten human health because they allow the transmission of many deadly pathogens, including viruses, bacteria, protozoa and helminths. Pruritus is a leading symptom of tick bites, but its molecular and neural bases remain elusive.

Objective: To discover potent drugs and targets for the specific prevention and treatment of tick bite-induced pruritus and arthropod-related itch. Methods We used live-cell calcium imaging, patch-clamp recordings, and genetic ablation and evaluated mouse behavior to investigate the molecular and neural bases of tick bite-induced pruritus.

Results: We found that two tick salivary peptides, IPDef1 and IRDef2, induced itch in mice. IPDef1 was further revealed to have a stronger pruritogenic potential than IRDef2 and to induce pruritus in a histamine-independent manner. IPDef1 evoked itch by activating mouse MrgprC11 and human MrgprX1 on dorsal root ganglion (DRG) neurons. IPDef1-activated MrgprC11/X1 signaling sensitized downstream ion channel TRPV1 on DRG neurons. Moreover, IPDef1 also activated mouse MrgprB2 and its ortholog human MrgprX2 selectively expressed on mast cells, inducing the release of inflammatory cytokines and driving acute inflammation in mice, although mast cell activation did not contribute to IP-O-induced itch. Conclusion Our study identifies tick salivary peptides as a new class of pruritogens that initiate itch through MrgprC11/X1-TRPV1 signaling in pruritoceptors. Our work will provide potential drug targets for the prevention and treatment of pruritus induced by the bites or stings of tick and maybe other arthropods.

Key words: Tick; Peptide; Itch; Mrgprs; TRP channel

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

TACAN Is an Ion Channel Involved in Sensing Mechanical Pain

Lou Beaulieu-Laroche ¹, Marine Christin ¹, Annmarie Donoghue ², Francina Agosti ³, Noosha Yousefpour ⁴, Hugues Petitjean ¹, Albena Davidova ¹, Craig Stanton ¹, Uzair Khan ⁵, Connor Dietz ⁵, Elise Faure ⁶, Tarheen Fatima ¹, Amanda MacPherson ⁵, Stephanie Mouchbahani-Constance ¹, Daniel G Bisson ⁷, Lisbet Haglund ⁷, Jean A Ouellet ⁸, Laura S Stone ⁹, Jonathan Samson ¹⁰, Mary-Jo Smith ¹¹, Kjetil Ask ¹², Alfredo Ribeiro-da-Silva ⁴, Rikard Blunck ¹³, Kate Poole ¹⁴, Emmanuel Bourinet ³, Reza Sharif-Naeini ¹⁵

Abstract

Mechanotransduction, the conversion of mechanical stimuli into electrical signals, is a fundamental process underlying essential physiological functions such as touch and pain sensing, hearing, and proprioception. Although the mechanisms for some of these functions have been identified, the molecules essential to the sense of pain have remained elusive. Here we report identification of TACAN (Tmem120A), an ion channel involved in sensing mechanical pain. TACAN is expressed in a subset of nociceptors, and its heterologous expression increases mechanically evoked currents in cell lines. Purification and reconstitution of TACAN in synthetic lipids generates a functional ion channel. Finally, a nociceptor-specific inducible knockout of TACAN decreases the mechanosensitivity of nociceptors and reduces behavioral responses to painful mechanical stimuli but not to thermal or touch stimuli. We propose that TACAN is an ion channel that contributes to sensing mechanical pain.

Keywords: TACAN; bilayer; ion channel; mechanosensitive; mechanotransduction; nociceptor; pain; patch clamp; pillar.