Pharmacological evidence of involvement of nitric oxide pathway in anti-pruritic effects of sumatriptan in chloroquine-induced scratching in mice

Pharmacological evidence of involvement of nitric oxide pathway in anti-pruritic effects of sumatriptan in chloroquine-induced scratching in mice

Nazgol-Sadat Haddadia,b, Sattar Ostadhadia,b,c, Saeed Shakibaa,b, Khashayar Afsharia,b, Nastaran Rahimia,b, Arash Foroutana,b, Ahmad-Reza Dehpoura,b* aExperimental Medicine Research Center, Tehran University of Medical Sciences, Poorsina St., Enghelab Ave., Tehran, Iran bDepartment of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Poorsina St., Enghelab Ave., Tehran, Iran cBrain and Spinal Cord Injury Research Center, Neuroscience Institute, Tehran University of Medical Sciences, Chamran highway, Bagherkhan St., Imam Khomeini Hospital, Tehran, Iran

Chloroquine (CQ) induces histamine-independent itch in human and mice. We recently reported the role of intradermal nitric oxide (NO)/cyclic guanosine monophosphate pathway in CQ-evoked scratching in mice. Chloroquine stimulates neuronal nitric oxide synthase (nNOS) activity to over-producing NO in the skin. Sumatriptan, a 5-hydroxytryptamine 1b/1d receptors (5-HTR1b/1d) agonist, is involved in pain and used to treat migraine and cluster headaches. According to previous studies, sumatriptan inhibits NOS activity. Thus, we aimed to investigate the effect of sumatriptan on CQ-induced scratching. We used the rostral back model of itch. Chloroquine was injected intradermally into the rostral back of NMRI mice, and the scratching behavior was evaluated by measuring the number of bouts over 30 min. We evaluated the effect of sumatriptan and combination of sumatriptan and a non-selective NO synthase inhibitor, L-N-nitro arginine methyl ester (L-NAME), on the scratching behavior. Additionally, the changes of skin, hip- pocampus, and cortical nitrite level after different treatments were studied. Intraperitoneal and intradermal sumatriptan attenuates CQ-induced itch which reversed by GR-127935, the selective 5-HTR1b and 5-HTR1d antagonist. Co- administration of subeffective doses of sumatriptan and L-NAME significantly decreases the scratching behavior. Intradermal injection of CQ significantly increases the intradermal nitrite levels while it does not have any significant effects on hippocampal or cortical nitrite concentrations. Likewise, the effective doses of intraperitoneal and intradermal sumatriptan significantly reduce intradermal nitrite levels. We concluded that sumatriptan suppresses CQ-induced itch most likely by activating 5-HT1b/1d receptors. This effect probably mediates through NO pathway.

Transient receptor potential vanilloid 4–expressing macrophages and keratinocytes contribute differentially to allergic and nonallergic chronic itch

TRP4-expressing macrophages and keratinocytes contribute differentially to allergic and nonallergic chronic itch

Jialie Luo, PhD,a* Jing Feng, PhD,a* Guang Yu, PhD,a,b Pu Yang, PhD,a Madison R. Mack, BA,a Junhui Du, PhD,c Weihua Yu, PhD,d Aihua Qian, PhD,e Yujin Zhang, PhD,f Shenbin Liu, PhD,a Shijin Yin, PhD,g Amy Xu, BS,a Jizhong Cheng, PhD,h Qingyun Liu, PhD,i Roger G. O’Neil, PhD,j Yang Xia, PhD,f Liang Ma, PhD,k
Susan M. Carlton, PhD,c Brian S. Kim, MD,a,k Kenneth Renner, PhD,l Qin Liu, PhD,a and Hongzhen Hu, PhDa
St Louis, Mo; Nanjing, Chongqing, Shanghai, and Wuhan, China; Galveston and Houston, Tex; and Vermillion, SD

Background: Chronic itch is a highly debilitating symptom that underlies many medical disorders with no universally effective treatments. Although unique neuronal signaling cascades in the sensory ganglia and spinal cord have been shown to critically promote the pathogenesis of chronic itch, the role of skin- associated cells remains poorly understood.
Objective: We sought to examine the cutaneous mechanisms underlying transient receptor potential vanilloid 4 (TRPV4)–mediated allergic and nonallergic chronic itch. Methods: Expression of TRPV4 in chronic itch and healthy control skin preparations was examined by using real-time RT-PCR. Trpv4eGFP mice were used to study the expression and function of TRPV4 in the skin by means of immunofluorescence staining, flow cytometry, calcium imaging, and patch-clamp recordings. Genetic and pharmacologic approaches were used to examine the role and underlying mechanisms of TRPV4 in mouse models of dry skin–associated chronic itch and spontaneous scratching associated with squaric acid dibutylester–induced allergic contact dermatitis.
Results: TRPV4 is selectively expressed by dermal macrophages and epidermal keratinocytes in mice. Lineage-specific deletion of TRPV4 in macrophages and keratinocytes reduces allergic and nonallergic chronic itch in mice, respectively. Importantly, TRPV4 expression is significantly increased in skin biopsy specimens from patients with chronic idiopathic pruritus in comparison with skin from healthy control subjects. Moreover, TRPV4-dependent chronic itch requires 5-hydroxytryptamine (5-HT) signaling secondary to activation of distinct 5-HT receptors in both patients with allergic and those with nonallergic chronic itch conditions.
Conclusion: Our study reveals previously unrecognized mechanisms by which TRPV4-expressing epithelial and immune cells in the skin critically and dynamically mediate chronic itch and unravels novel targets for therapeutics in the setting of chronic itch. (J Allergy Clin Immunol 2017;nnn:nnn-nnn.)
Key words: Transient receptor potential vanilloid 4, chronic itch, macrophage, keratinocyte

TRPC3 Is Dispensable for β-Alanine Triggered Acute Itch
Peter Dong1, Changxiong Guo2, Shengxiang Huang2,3, Minghong Ma1, Qin Liu2 & Wenqin Luo1

TRPC3 Is Dispensable for β-Alanine Triggered Acute Itch

The detection of pruritic (itchy) stimuli is mediated by a variety of receptors and channels expressed
by primary sensory neurons. The G protein-coupled receptor (GPCR) MRGPRD is selectively expressed by a subset of mouse non-peptidergic nociceptors and functions as the molecular receptor for the itch-inducing chemical β-alanine. However, the channels responsible for generating electrical signals downstream of MRGPRD remain unclear. Here, we found that a member of the canonical TRP channel family, TRPC3, is highly expressed in MRGPRD+ non-peptidergic nociceptors, raising the possibility of whether TRPC3 functions as a downstream channel in the MRGPRD signaling pathway. We tested TrpC3 null mice for β-alanine induced itch, and found that these mice exhibit normal responses to β-alanine. At the cellular level, calcium in ux triggered by β-alanine is also unchanged in cultured DRG neurons from TrpC3 null mice compared to wild type. Together, our results demonstrate that mouse TrpC3 is dispensable for β-alanine-induced acute itch.

Sensory Neurons Co-opt Classical Immune Signaling Pathways to Mediate Chronic Itch

Sensory Neurons Co-opt Classical Immune Signaling Pathways to Mediate Chronic Itch

Landon K. Oetjen,1,2 Madison R. Mack,1,2 Jing Feng,1,3 Timothy M. Whelan,1,2 Haixia Niu,1,2 Changxiong J. Guo,1,3
Sisi Chen,4 Anna M. Trier,1,2 Amy Z. Xu,1,2 Shivani V. Tripathi,1,2 Jialie Luo,1,3 Xiaofei Gao,1,3 Lihua Yang,5
Samantha L. Hamilton,5 Peter L. Wang,6 Jonathan R. Brestoff,6 M. Laurin Council,2 Richard Brasington,7
Andra ́ s Schaffer,2,6 Frank Brombacher,8 Chyi-Song Hsieh,6,7 Robert W. Gereau IV,3 Mark J. Miller,5 Zhou-Feng Chen,1,3 Hongzhen Hu,1,3 Steve Davidson,4 Qin Liu,1,3 and Brian S. Kim1,2,3,6,9,*
1Center for the Study of Itch, Washington University School of Medicine, St. Louis, MO 63110, USA
2Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA
3Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA
4Department of Anesthesiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA
5Division of Infectious Diseases, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA 6Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA
7Division of Rheumatology, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA 8International Centre for Genetic Engineering and Biotechnology and Institute of Infectious Disease and Molecular Medicine, Division of Immunology, University of Cape Town, Cape Town 7700, South Africa
9Lead Contact
*Correspondence: briankim@wustl.edu
http://dx.doi.org/10.1016/j.cell.2017.08.006

Mammals have evolved neurophysiologic reflexes, such as coughing and scratching, to expel invading pathogens and noxious environmental stimuli. It is well established that these responses are also asso- ciated with chronic inflammatory diseases, including asthma and atopic dermatitis. However, the mecha- nisms by which inflammatory pathways promote sensations such as itch remain poorly understood. Here, we show that type 2 cytokines directly activate sensory neurons in both mice and humans. Further, we demonstrate that chronic itch is dependent on neuronal IL-4Ra and JAK1 signaling. We also observe that patients with recalcitrant chronic itch that failed other immunosuppressive therapies mark- edly improve when treated with JAK inhibitors. Thus, signaling mechanisms previously ascribed to the immune system may represent novel therapeutic targets within the nervous system. Collectively, this study reveals an evolutionarily conserved para- digm in which the sensory nervous system employs classical immune signaling pathways to influence mammalian behavior.

Interleukin 31 receptor (IL-31RA) is a novel Type I cytokine
receptor that pairs with oncostatinMreceptor to mediate IL-31
signaling. Binding of IL-31 to its receptor results in the phosphorylation
and activation of STATs, MAPK, and JNK signaling
pathways. IL-31 plays a pathogenic role in tissue inflammation,
particularly in allergic diseases. Recent studies demonstrate
IL-31RA expression and signaling in non-hematopoietic cells,
but this receptor is poorly studied in immune cells. Macrophages
are key immune-effector cells that play a critical role in
Th2-cytokine-mediated allergic diseases. Here, we demonstrate
that Th2 cytokines IL-4 and IL-13 are capable of up-regulating
IL-31RA expression on both peritoneal and bone marrowderived
macrophages from mice. Our data also demonstrate
that IL-4R-driven IL-31RA expression is STAT6 dependent
in macrophages. Notably, the inflammation-associated genes
Fizz1 and serum amyloid A (SAA) are significantly up-regulated
inM2macrophages stimulated with IL-31, but not in IL-4 receptor-
deficient macrophages. Furthermore, the absence of Type II
IL-4 receptor signaling is sufficient to attenuate the expression
of IL-31RA in vivo during allergic asthma induced by soluble egg
antigen, which may suggest a role for IL-31 signaling in Th2
cytokine-driven inflammation and allergic responses. Our study
reveals an important counter-regulatory role between Th2 cytokine
and IL-31 signaling involved in allergic diseases.Th2 Cytokines Augment IL-31IL-31RA Interactions via

Intraperitoneal administration of AAV9-shRNA inhibits target gene expression in the dorsal root ganglia of neonatal mice
Akira Machida1, Hiroya Kuwahara1,4, Azat Mayra1, Takayuki Kubodera1, Takashi Hirai2, Fumiko Sunaga1, Mio Tajiri1, Yukihiko Hirai3, Takashi Shimada3, Hidehiro Mizusawa1 and Takanori Yokota1,4,5*

Intraperitoneal administration of AAV9-shRNA inhibits target gene expression in the dorsal root ganglia of neonatal mice

Abstract
Background: There is considerable interest in inducing RNA interference (RNAi) in neurons to study gene function and identify new targets for disease intervention. Although short interfering RNAs (siRNAs) have been used to silence genes in neurons, in vivo delivery of RNAi remains a major challenge, especially by systemic administration. We have developed a highly efficient method for in vivo gene silencing in dorsal root ganglia (DRG) by using short hairpin RNA–expressing single-stranded adeno-associated virus 9 (ssAAV9-shRNA).
Results: Intraperitoneal administration of ssAAV9-shRNA to neonatal mice resulted in highly effective and specific silencing of a target gene in DRG. We observed an approximately 80% reduction in target mRNA in the DRG, and 74.7% suppression of the protein was confirmed by Western blot analysis. There were no major side effects, and the suppression effect lasted for more than three months after the injection of ssAAV9-shRNA.
Conclusions: Although we previously showed substantial inhibition of target gene expression in DRG via intrathecal ssAAV9-shRNA administration, here we succeeded in inhibiting target gene expression in DRG neurons via intraperitoneal injection of ssAAV9-shRNA. AAV9-mediated delivery of shRNA will pave the way for creating animal models for investigating the molecular biology of the mechanisms of pain and sensory ganglionopathies.
Keywords: RNA interference, Adeno-associated virus 9, Dorsal root ganglia, Blood–nerve barrier

A novel TRPM8 agonist relieves dry eye discomfort

A novel TRPM8 agonist relieves dry eye discomfort S

A novel TRPM8 agonist relieves dry eye discomfort

Jee Myung Yang1,2†, Fengxian Li3,4†, Qin Liu4, Marco Rüedi5, Edward Tak Wei6, Michael Lentsman7, Hyo Seok Lee2, Won Choi2, Seong Jin Kim8*† and Kyung Chul Yoon2*†

Abstract
Background: Physical cooling of the eye surface relieves ocular discomfort, but translating this event to drug treatment of dry eye discomfort not been studied. Here, we synthesized a water-soluble TRPM8 receptor agonist called cryosim-3 (C3, 1-diisopropylphosphorylnonane) which selectively activates TRPM8 (linked to cooling) but not TRPV1 or TRPA1 (linked to nociception) and tested C3 in subjects with mild forms of dry eye disease.
Methods: A set of 1-dialkylphosphoryalkanes were tested for activation of TRPM8, TRPV1 and TRPA1 receptors in transfected cells. The bioactivity profiles were compared by perioral, topical, and intravenous delivery to anesthetized rats. The selected lead candidate C3 or vehicle (water) was applied with a cotton gauze pad to upper eyelids of patients with dry eye disease (n = 30). Cooling sensation, tear film break-up time (TBUT), basal tear secretion, and corneal staining were evaluated. C3 was then applied four times daily for 2 weeks to patients using a pre-loaded single unit applicator containing 2 mg/mL of C3 in water (n = 20) or water only. TBUT, basal tear secretion, and corneal staining, and three questionnaires surveys of ocular discomfort (VAS scale, OSDI, and CVS symptoms) were analyzed before and at 1 and 2 weeks thereafter.
Results: C3 was a selective and potent TRPM8 agonist without TRPV1 or TRPA1 activity. In test animals, the absence of shaking behavior after C3 perioral administration made it the first choice for further study. C3 increased tear secretion in an animal model of dry eye disease and did not irritate when wiped on eyes of volunteers. C3 singly applied (2 mg/ml) produced significant cooling in <5 min, an effecting lasting 46 min with an increase in tear secretion for 60 min. C3 applied for 2 weeks also significantly increased basal tear secretion with questionnaire surveys of ocular discomfort indices clearly showing improvement of symptoms at 1 and 2 weeks. No complaints of irritation or pain were reported by any subject.
Conclusions: C3 is a promising candidate for study of TRPM8 function on the eye surface and for relief of dry eye discomfort.
Trial registration: ISRCTN24802609 and ISRCTN13359367. Registered 23 March 2015 and 2 September 2015. Keywords: Dry eye, Eyelid, Ocular discomfort, TRPM8

Involvement of TRPV1 and TDAG8 in Pruriception Associated with Noxious Acidosis

Shing-Hong Lin1, Martin Steinhoff2,3,4, Akihiko Ikoma4, Yen-Ching Chang5, Yuan-Ren Cheng1,6, Ravi Chandra Kopparaju1,7, Satoshi Ishii8, Wei-Hsin Sun5 and Chih-Cheng Chen1,6,7,9

Acid-Sensing Ion Channel and Pruritus

Itch and pain are closely related but are distinct sensations. Intradermal injection of acid generates pain in both rodents and humans; however, few studies have addressed the intriguing question of whether acid (protons) can evoke itch like other algogens by spatial contrast activation of single nociceptors. Here, we report that (i) citric acid (0.2 mol/L) pH-dependently induced a scratching response in mice when applied intradermally to nape or cheek skin, (ii) acidified buffer elevated intracellular calcium levels in dorsal root ganglion pruri- ceptors, and (iii) injection of intradermal citric acid (pH 3.0) into the nape induced a pruritogen-like but not algogen-like c-Fos immunoreactivity pattern in the cervical spinal cord. Using pharmacological and genetic approaches, we identified potential acid-sensing channels/receptors involved in acidic citrate-evoked itch. Results indicate that TRPV1, but neither ASIC3 nor TRPA1, is involved in the acidic citrate-induced scratching response. Furthermore, one of the proton-sensing G-proteinecoupled receptors, TDAG8, was highly (w71%) expressed in Nppbþ dorsal root ganglion pruriceptors. Itch induced by acidic citrate, but not a-methyl-5- hydroxytryptamine, chloroquine, compound 48/80, or bile acid, was markedly decreased in TDAG8e/e mice. In a heterologous expression system, TDAG8 potentiated the acid-induced calcium response by regulating TRPV1. Thus, protons could evoke pruriception by acting on TDAG8 to regulate TRPV1 activation with its mechanism of future therapeutic relevance.

Journal of Investigative Dermatology (2017) 137, 170e178; doi:10.1016/j.jid.2016.07.037

Abstract

Persistent itch often accompanies allergic contact dermatitis (ACD), but the underlying mechanisms remain largely unexplored. We previously demonstrated that CXCL10/ CXCR3 signaling activated a subpopulation of cutaneous primary sensory neurons and mediated itch response after contact hypersensitivity (CHS), a murine model of ACD, induced by squaric acid dibutylester. The purpose of this study was to determine the ionic mechanisms underlying CXCL10-induced neuronal activation and allergic itch. In whole-cell recordings, CXCL10 triggered a current in dorsal root ganglion (DRG) neurons innervating the area of CHS. This current was modulated by intracellular Cl- and blocked by the general Cl- channel inhibitors. Moreover, increasing Ca2+ buffering capacity reduced this current. In addition, blockade of Cl- channels significantly suppressed CXCL10-induced Ca2+ response. In behavioral tests, injection of CXCL10 into CHS site exacerbated itch-related scratching behaviors. Moreover, the potentiating behavioral effects of CXCL10 were attenuated by either of two Cl- channel blockers. Thus, we suggest that the Cl- channel acts as a downstream target mediating the excitatory and pruritic behavioral effects of CXCL10. Cl- channels may provide a promising therapeutic target for the treatment of allergic itch in which CXCL10/CXCR3 signaling may participate.
jn.00187.2017.fulljn.00187.2017.full

TNF-a/TNFR1 Signaling is Required for the Full Expression of Acute and Chronic Itch in Mice via Peripheral and Central Mechanisms

art10.1007s12264-017-0124-3

Xiuhua Miao1 • Ya Huang2 • Teng-Teng Liu2 • Ran Guo2 • Bing Wang2 • Xue-Long Wang3 • Li-Hua Chen4 • Yan Zhou2 • Ru-Rong Ji5,6 • Tong Liu1,2
Received: 11 November 2016 / Accepted: 9 February 2017
Ó Shanghai Institutes for Biological Sciences, CAS and Springer Science+Business Media Singapore 2017

Abstract Increasing evidence suggests that cytokines and chemokines play crucial roles in chronic itch. In the present study, we evaluated the roles of tumor necrosis factor-alpha (TNF-a) and its receptors TNF receptor subtype-1 (TNFR1) and TNFR2 in acute and chronic itch in mice. Compared to wild-type (WT) mice, TNFR1-knockout (TNFR1-KO) and TNFR1/R2 double-KO (DKO), but not TNFR2-KO mice, exhibited reduced acute itch induced by compound 48/80 and chloroquine (CQ). Application of the TNF-synthesis inhibitor thalidomide and the TNF-a antagonist etanercept dose-dependently suppressed acute itch. Intradermal injection of TNF-a was not sufficient to evoke scratching, but potentiated itch induced by com- pound 48/80, but not CQ. In addition, compound 48/80 induced TNF-a mRNA expression in the skin, while CQ induced its expression in the dorsal root ganglia (DRG) and spinal cord. Furthermore, chronic itch induced by dry skin was reduced by administration of thalidomide and etaner- cept and in TNFR1/R2 DKO mice. Dry skin induced TNF- a expression in the skin, DRG, and spinal cord and TNFR1 expression only in the spinal cord. Thus, our findings suggest that TNF-a/TNFR1 signaling is required for the full expression of acute and chronic itch via peripheral and central mechanisms, and targeting TNFR1 may be benefi- cial for chronic itch treatment.
Keywords Itch Tumor necrosis factor Tumor necrosis factor receptor Spinal cord Central sensitization