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.

GRPR/Extracellular SignaleRegulated Kinase and NPRA/Extracellular SignaleRegulated Kinase Signaling Pathways Play a Critical Role in Spinal Transmission of Chronic Itch

Abstract

Intractable or recurrent chronic itch greatly reduces the patients’ QOL and impairs their daily activities. In this study, we investigated whether there are certain key signaling molecules downstream of the recently identified peptides mediating itch in the spinal cord. RNA sequencing analysis of mouse spinal cord in chronic itch models induced by squaric acid dibutylester and imiquimod showed that extracellular signaleregulated kinase (ERK) 1/2 cascade is the most significantly upregulated gene cluster in both models. In four different mouse models of chronic itch, sustained ERK phosphorylation was detected mainly in spinal neurons, and MAPK/ERK kinase inhibitors significantly inhibited chronic itch in these models. Phosphorylated ERK was observed in the interneurons expressing the receptors of different neuropeptides for itch, including gastrin-releasing peptide receptor, natriuretic peptide receptor A, neuromedin B receptor, and sst2A. Blocking gastrin-releasing peptide receptor and natriuretic peptide receptor A by genetic approaches or toxins in mice significantly attenuated or ablated spinal phosphorylated ERK. When human embryonic kidney 293T cells transfected with these receptors were exposed to their respective agonists, ERK was the most significantly activated intracellular signaling molecule. Together, our work showed that phosphorylated ERK is a unique marker for itch signal transmission in the spinal cord and an attractive target for the treatment of chronic itch.

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.

Allantoin induces pruritus by activating MrgprD in chronic kidney disease 

Yan Yang, Yulin Sun, Donglang Guan, Dan Chen, Dijun Wang, Tongtong Liu, Meixiao Sheng, Tao Jing, Shi Jun, Chan Zhu, Guang Yu, Xinzhong Dong, Zongxiang Tang

Abstract

Chronic kidney disease is a disease with decreased, irreversible renal function. Pruritus is the most common skin symptom in patients with chronic kidney disease, especially in end-stage renal disease (AKA chronic kidney disease-associated pruritus [CKD-aP]); however, the underlying molecular and neural mechanism of the CKD-aP in patients remains obscure. Our data show that the level of allantoin increases in the serum of CKD-aP and CKD model mice. Allantoin could induce scratching behavior in mice and active DRG neurons; the calcium influx and the action potential were significantly reduced in DRG neurons of MrgprD KO or TRPV1 KO mice. U73122, an antagonist of PLC, could also block calcium influx in DRG neurons induced by allantoin. Thus, our results concluded that allantoin plays an important role in CKD-aP, mediated by MrgprD and TrpV1, in CKD patients.

FGF13 is required for histamine-induced itch sensation by interaction with NaV1.7

Abstract

Itch can be induced by activation of small-diameter dorsal root ganglion (DRG) neurons which express abundant intracellular fibroblast growth factor 13 (FGF13). Although FGF13 is revealed to be essential for heat nociception, its role in mediating itch remains to be investigated. Here, we reported that loss of FGF13 in mouse DRG neurons impaired the histamine-induced scratching behavior. Calcium imaging showed that the percentage of histamine-responsive DRG neurons was largely decreased in FGF13-deficient mice, and consistently, electrophysiological recording exhibited that histamine failed to evoke action potential firing in most DRG neurons from these mice. Given that the reduced histamine-evoked neuronal response was caused by knockdown of FGF13 but not by FGF13A deficiency, FGF13B was supposed to mediate this process. Furthermore, overexpression of histamine type 1 receptor H1R, but not H2R, H3R nor H4R, increased the percentage of histamine-responsive DRG neurons, and the scratching behavior in FGF13-deficient mice was highly reduced by selective activation of H1R, suggesting that H1R is mainly required for FGF13-mediated neuronal response and scratching behavior induced by histamine. However, overexpression of H1R failed to rescue the histamine-evoked neuronal response in FGF13-deficient mice. Histamine enhanced the FGF13 interaction with NaV1.7. Disruption of this interaction by a membrane-permeable competitive peptide, GST-Flag-NaV1.7CT-TAT, reduced the percentage of histamine-responsive DRG neurons, and impaired the histamine-induced scratching, indicating that the FGF13/NaV1.7 interaction is a key molecular determinant in the histamine-induced itch sensation. Therefore, our study reveals a novel role of FGF13 in mediating itch sensation via the interaction of NaV1.7 in peripheral nervous system.

Substance P activates Mas-related G protein-coupled receptors to induce itch.

Azimi E1, Reddy VB1, Pereira PJ2, Talbot S3, Woolf CJ3, Lerner EA4.

Abstract

BACKGROUND: Substance P (SP) is linked to itch and inflammation through activation of receptors on mast cells and sensory neurons. There is increasing evidence that SP functions through Mas-related G protein-coupled receptors (Mrgprs) in addition to its conventional receptor, neurokinin-1.

OBJECTIVE: Because Mrgprs mediate some aspects of inflammation that had been considered mediated by neurokinin-1 receptor (NK-1R), we sought to determine whether itch induced by SP can also be mediated by Mrgprs.

METHODS: Genetic and pharmacologic approaches were used to evaluate the contribution of Mrgprs to SP-induced scratching behavior and activation of cultured dorsal root ganglion neurons from mice.

RESULTS: SP-induced scratching behavior and activation of cultured dorsal root ganglion neurons was dependent on Mrgprs rather than NK-1R.

CONCLUSION: We deduce that SP activates MrgprA1 on sensory neurons rather than NK-1R to induce itch.

KEYWORDS: Mas-related G protein–coupled receptors; Substance P; calcium imaging; dorsal root ganglion neurons; knockout mice; receptor antagonist