Journal club 2016-04-01

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Transient receptor potential vanilloid 4 ion channel functions as a pruriceptor in epidermal keratinocytes to evoke histaminergic itch

  1. Yong Chen,
  2. Quan Fang,
  3. Zilong Wang,
  4. Jennifer Y. Zhang,
  5. Amanda S. MacLeod,
  6. Russell P. Hall and
  7. Wolfgang B. Liedtke*

+ Author Affiliations


  1. Duke University, United States

Abstract

TRPV4 ion channels function in epidermal keratinocytes and in innervating sensory neurons, however, the contribution of the channel in either cell to neurosensory function remains to be elucidated. We recently reported TRPV4 as a critical component of the keratinocyte machinery that responds to UVB, and functions critically to convert the keratinocyte into a pain-generator cell after excess UVB exposure. One key mechanism in keratinocytes was increased expression and secretion of endothelin-1, which is also a known pruritogen. Here we address the question whether TRPV4 in skin keratinocytes functions in itch, as a particular form of forefront signaling in non-neural cells. Our results support this novel concept, based on attenuated scratching behavior in response to histaminergic (histamine, compound 48/80, endothelin-1), not non-histaminergic (chloroquine) pruritogens in Trpv4 keratinocyte-specific and inducible knockout mice. We demonstrate that keratinocytes rely on TRPV4 for calcium influx in response to histaminergic pruritogens. TRPV4 activation in keratinocytes evokes phosphorylation of MAP-kinase, ERK, for histaminergic pruritogens. This finding is relevant because we observed robust anti-pruritic effects with topical applications of selective inhibitors for TRPV4 and also for MEK, the kinase upstream of ERK, suggesting that calcium influx via TRPV4 in keratinocytes leads to ERK-phosphorylation, which in-turn rapidly converts the keratinocyte into an organismal itch-generator cell. In support of this concept we found that scratching behavior, evoked by direct intradermal activation of TRPV4, was critically dependent on TRPV4-expression in keratinocytes. Thus, TRPV4 functions as a pruriceptor-TRP in skin keratinocytes in histaminergic itch, a novel basic concept with translational-medical relevance.

TRPV4 in histaminergic itch

Journal club 2016-02-19

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Voltage-Gated Potassium Channels Involved in Regulation of Physiological Function in MrgprA3-Specific Itch Neurons.

Tang M1, Wu G2, Wang Z3, Yang N3, Shi H3, He Q4, Zhu C3, Yang Y3, Yu G3, Wang C3, Yuan X3, Liu Q5, Guan Y6, Dong X7, Tang Z8.

Abstract

Itch is described as an unpleasant or irritating skin sensation that elicits the desire or reflex to scratch. MrgprA3, one of members of the Mrgprs family, is specifically expressed in a subpopulation of dorsal root ganglion (DRG) in the peripheral nervous system (PNS). These MrgprA3-expressing DRG neurons have been identified as itch-specific neurons. They can be activated by the compound, chloroquine, which is used as a drug to treat malaria. In the present study, we labeled these itch-specific neurons using the method of molecular genetic markers, and then studied their electrophysiological properties. We also recorded the cutaneous MrgprA3– neurons retrogradely labeled by Dil dye (MrgprA3–-Dil). We first found that MrgprA3+ neurons have a lower excitability than MrgprA3– neurons (MrgprA3–-non-Dil and MrgprA3–-Dil). The number of action potential (AP) was reduced more obviously in MrgprA3+ neurons than that of in MrgprA3– neurons. In most cases, MrgprA3+ neurons only generated single AP; however, in MrgprA3– neurons, the same stimulation could induce multiple AP firing due to the greater voltage-gated potassium (Kv) current existence in MrgprA3+ than in MrgprA3– neurons. Thus, Kv current plays an important role in the regulation of excitability in itch-specific neurons.

Copyright © 2016. Published by Elsevier B.V.

KEYWORDS:

DRG; Itch; Kv current; MrgprA3

 

voltage gated K+ channel and Mrgpra3 specific itchneuron

journal club 2016-01-08

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3-iodothyroacetic acid, a metabolite of thyroid hormone, induces itch and reduces threshold to noxious and to painful heat stimuli in mice.

Laurino A1, De Siena G, Resta F, Masi A, Musilli C, Zucchi R, Raimondi L.

Abstract

BACKGROUND AND PURPOSE:

Itch is associated with increased sensitization to nociceptive stimuli. We investigated whether 3-iodothyroacetic acid (TA1), by releasing histamine, induces itch and increases sensitization to noxious and painful heat stimuli.

EXPERIMENTAL APPROACH:

Itch was evaluated after s.c. administration of TA1 (0.4, 1.32 and 4 μg·kg(-1) ). Mice threshold to noxious (NHT) and to painful heat stimuli were evaluated by the increasing-temperature hot plate (from 45.5 to 49.5°C) or by the hot plate (51.5°C) test, respectively, 15 min after i.p. injection of TA1 (0.4, 1.32 and 4 μg·kg(-1) ). Itch, NHT and pain threshold evaluation were repeated in mice pretreated with pyrilamine. Itch and NHT were also measured in HDC(+/+) and HDC(-/-) following injection of saline or TA1 (1.32, 4 and 11 μg·kg(-1) ; s.c. and i.p.). pERK1/2 levels were determined by Western blot in dorsal root ganglia (DRG) isolated from CD1 mice 15 min after they received (i.p.): saline, saline and noxious heat stimulus (46.5°C), TA1 (0.1, 0.4, 1.32, 4 μg·kg(-1) ) or TA1 1.32 μg·kg(-1) and noxious heat stimulus.

KEY RESULTS:

TA1 0.4 and 1.32 μg·kg(-1) induced itch and reduced NHT; pyrilamine pretreatment prevented both of these effects. TA1 4 μg·kg(-1) (i.p.) reduced pain threshold without inducing itch or modifying NHT. In HDC(-/-) mice, TA1 failed to induce itch and to reduce NHT. In DRG, pERK1/2 levels were significantly increased by noxious heat stimuli and by TA1 0.1, 0.4 and 1.32 μg·kg(-1) ; i.p.

CONCLUSIONS AND IMPLICATIONS:

Increased TA1 levels induce itch and an enhanced sensitivity to noxious heat stimuli suggesting that TA1 might represent a potential cause of itch in thyroid diseases.

Journal club 2015-12-04

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Incoherent feed-forward regulatory loops control segregation of C-mechanoreceptors, nociceptors, and pruriceptors.

Lou S1, Pan X2, Huang T1, Duan B1, Yang FC1, Yang J2, Xiong M3, Liu Y4, Ma Q5.

Abstract

Mammalian skin is innervated by diverse, unmyelinated C fibers that are associated with senses of pain, itch, temperature, or touch. A key developmental question is how this neuronal cell diversity is generated during development. We reported previously that the runt domain transcription factor Runx1 is required to coordinate the development of these unmyelinated cutaneous sensory neurons, including VGLUT3(+) low-threshold c-mechanoreceptors (CLTMs), MrgprD(+) polymodal nociceptors, MrgprA3(+) pruriceptors, MrgprB4(+) c-mechanoreceptors, and others. However, how these Runx1-dependent cutaneous sensory neurons are further segregated is poorly illustrated. Here, we find that the Runx1-dependent transcription factor gene Zfp521 is expressed in, and required for establishing molecular features that define, VGLUT3(+) CLTMs. Furthermore, Runx1 and Zfp521 form a classic incoherent feedforward loop (I-FFL) in controlling molecular identities that normally belong to MrgprD(+) neurons, with Runx1 and Zfp51 playing activator and repressor roles, respectively (in genetic terms). A knock-out of Zfp521 allows prospective VGLUT3 lineage neurons to acquire MrgprD(+) neuron identities. Furthermore, Runx1 might form other I-FFLs to regulate the expression of MrgprA3 and MrgprB4, a mechanism preventing these genes from being expressed in Runx1-persistent VGLUT3(+) and MrgprD(+) neurons. The evolvement of these I-FFLs provides an explanation for how modality-selective sensory subtypes are formed during development and may also have intriguing implications for sensory neuron evolution and sensory coding

KEYWORDS:

Runx1; Zfp521; low-threshold c-mechanoreceptors; nociceptors; pruriceptors; sensory subtype specification

 

5317.full

journal club 2015-05-01

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Mutual upregulation of endothelin-1 and IL-25 in atopic dermatitis.

Aktar MK1, Kido-Nakahara M, Furue M, Nakahara T

Abstract

BACKGROUND:

Endothelin 1 (ET-1) has been reported to evoke histamine-independent pruritus in　mammals. However, its association with pruritus or inflammation of atopic dermatitis (AD) has not been clarified. We sought to investigate the role of ET-1 in the skin inflammation of AD.

METHODS:

To examine the role of ET-1 in AD, we investigated the expression of ET-1 and IL-25 in the skin of an AD mouse model and AD patients, and examined the mutual regulatory relationship between ET-1 and IL-25, one of the important cytokines in AD, using the human HaCaT keratinocyte cell line.

RESULTS:

We immunohistochemically confirmed the upregulation of ET-1 and IL-25 expression in the epidermis of both the AD mouse model and AD patients. In vitro, IL-25 upregulated ET-1 mRNA and protein expression in a concentration- and time-dependent fashion in HaCaT cells. This IL-25-induced ET-1 expression was inhibited by ERK1/2 or JNK inhibitor. In a reciprocal manner, ET-1 also induced IL-25 upregulation. The enhancing effect of ET-1 on IL-25 was inhibited by an endothelin A receptor antagonist, ERK1/2 inhibitor or p38 inhibitor, but not by an endothelin B receptor antagonist or JNK inhibitor.

CONCLUSION:

These findings suggest that mutual upregulation of ET-1 and IL-25 takes place in the epidermis in AD, which may be a future target for anti-pruritic agents. This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

KEYWORDS:

MAPK ; IL-25; Keratinocyte; atopic dermatitis; endothelin-1

 

http://onlinelibrary.wiley.com/doi/10.1111/all.12633/pdf

journal clb 27-3-2015

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Gamisasangja-tang suppresses pruritus and atopic skin inflammation in the NC/Nga murine model of atopic dermatitis

Abstract

Ethnopharmacological relevance

Gamisasangja-tang (GST) is a traditional herbal formula prescribed for patients with intractable pruritus in association with various inflammatory skin diseases. To evaluate the effects of GST on pruritic skin inflammation and investigate its cellular and molecular mechanisms.

Materials and methods

We orally administered GST to NC/Nga (NC) mice, an animal model of atopic dermatitis. Scratching frequency and the dermatitis index were evaluated, and histological examination was performed using hematoxylin and eosin and toluidine blue staining. The levels of interleukin (IL)-31 and T-helper cell type 2 (TH2) cytokines were determined in both the dorsal skin and cultured splenocytes by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The serum levels of chemokines and immunoglobulin E (IgE) were determined by ELISA. Changes in the inflammatory cell population were analyzed by a hemocytometer.

Results

GST significantly lowered scratching frequency and inhibited increases in dermatitis index, thickness of epidermis/dermis and infiltration of chemokine (C-C motif) receptor 3 (CCR3)+ and cluster of differentiation (CD)117+/FcεRIα (Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide)+ cells in atopic skin. Both IL-31 mRNA expression and production were significantly reduced by GST, which was accomrease in the levels of IL-4, IL-5, and IL-13. Further, GST treatment suppressed the secretion of eotaxin, TARC (thymus and activation-regulated chemokine), IgE, and increases in the number of basophils and eosinophils in the blood.

Conclusion

GST may have potential as an effective treatment for pruritic skin disease such as atopic dermatitis

gamisasangja tang

journal club 2015-02-06

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Enhanced non-peptidergic intraepidermal fiber density and an expanded subset of chloroquine-responsive trigeminal neurons in a mouse model of dry skin itch

Manouela V. Valtcheva

,

Vijay K. Samineni

,

Judith P. Golden

,

Robert W. Gereau

,

Steve Davidson

Abstract

Chronic pruritic conditions are often associated with dry skin and loss of epidermal barrier integrity. In this study, repeated application of acetone and ether, followed by water (AEW) to the cheek skin of mice produced persistent scratching behavior with no increase in pain-related forelimb wiping, indicating the generation of itch without pain. Cheek skin immunohistochemistry showed a 64.5% increase in total epidermal innervation in AEW-treated mice compared to water-treated controls. This increase was independent of scratching, because mice prevented from scratching by Elizabethan collars showed similar hyperinnervation. To determine the effects of dry skin treatment on specific subsets of peripheral fibers, we examined Ret-positive, CGRP-positive, and GFRα3-positive intraepidermal fiber density. AEW treatment increased Ret-positive fibers, but not CGRP-positive or GFRα3-positive fibers, suggesting that a specific subset of non-peptidergic fibers could contribute to dry skin itch. To test whether trigeminal ganglion neurons innervating the cheek exhibited altered excitability after AEW treatment, primary cultures of retrogradely labeled neurons were examined using whole-cell patch clamp electrophysiology. AEW treatment produced no differences in measures of excitability compared to water-treated controls. In contrast, a significantly higher proportion of trigeminal ganglion neurons were responsive to the non-histaminergic pruritogen chloroquine after AEW treatment. We conclude that non-peptidergic, Ret-positive fibers and chloroquine-sensitive neurons may contribute to dry skin pruritus.

February 2015

Journal club 2015-01-05

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LPS sensitizes TRPV1 via activation of TLR4 in trigeminal sensory neurons.

Diogenes A1, Ferraz CC, Akopian AN, Henry MA, Hargreaves KM.

Abstract

Recent studies have demonstrated that the lipopolysaccharide (LPS) receptor (TLR4) is expressed in TRPV1 containing trigeminal sensory neurons. In this study, we evaluated whether LPS activates trigeminal neurons, and sensitizes TRPV1 responses via TLR4. To test this novel hypothesis, we first demonstrated that LPS binds to receptors in trigeminal neurons using competitive binding. Second, we demonstrated that LPS evoked a concentration-dependent increase in intracellular calcium accumulation (Ca(2+))(i) and inward currents. Third, LPS significantly sensitized TRPV1 to capsaicin measured by (Ca(2+))(i), release of calcitonin gene-related peptide, and inward currents. Importantly, a selective TLR4 antagonist blocked these effects. Analysis of these data, collectively, demonstrates that LPS is capable of directly activating trigeminal neurons, and sensitizing TRPV1 via a TLR4-mediated mechanism. These findings are consistent with the hypothesis that trigeminal neurons are capable of detecting pathogenic bacterial components leading to sensitization of TRPV1, possibly contributing to the inflammatory pain often observed in bacterial infections.

LPS

journal club 2014.12.01

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Antipruritic mechanisms of topical E6005, a phosphodiesterase 4 inhibitor: Inhibition of responses to proteinase-activated receptor 2 stimulation mediated by increase in intracellular cyclic AMP

Abstract

Background

Phosphodiesterase 4 (PDE4), which catalyses the conversion of cyclic adenosine 3′,5′-monophosphate (cAMP) to 5′-AMP, plays a critical role in the pathogenesis of inflammatory disorders. Pruritus is the main symptom of dermatitides, such as atopic dermatitis, and is very difficult to control. Recent studies have shown that the activation of proteinase-activated receptor 2 (PAR2) is involved in pruritus in dermatoses in humans and rodents.

Objective

To investigate the inhibitory effect of E6005, a topically effective PDE4 inhibitor, on PAR2-associated itching in mice.

Methods

Mice were given an intradermal injection of SLIGRL-NH2 (100 nmol/site), a PAR2 agonist peptide, into the rostral part of the back. E6005 and 8-bromo-cAMP were applied topically and injected intradermally, respectively, to the same site. Scratching bouts were observed as an itch-related behavior, and firing activity of the cutaneous nerve was electrophysiologically recorded. Keratinocytes were isolated from the skin of neonatal mice and cultured for in vitro experiments. The concentrations of cAMP and leukotriene B4 (LTB4) were measured by enzyme immunoassay. The distribution of PDE4 subtypes in the skin was investigated by immunostaining.

Results

Topical E6005 and intradermal 8-bromo-cAMP significantly inhibited SLIGRL-NH2-induced scratching and cutaneous nerve firing. Topical E6005 increased cutaneous cAMP content. Topical E6005 and intradermal 8-bromo-cAMP inhibited cutaneous LTB4 production induced by SLIGRL-NH2, which has been shown to elicit LTB4-mediated scratching. E6005 and 8-bromo-cAMP inhibited SLIGRL-NH2-induced LTB4 production in the cultured murine keratinocytes also. PDE4 subtypes were mainly expressed in keratinocytes and mast cells in the skin.

Conclusions

The results suggest that topical E6005 treatment inhibits PAR2-associated itching. Inhibition of LTB4 production mediated by an increase in cAMP may be partly involved in the antipruritic action of E6005.

Abbreviations

  • cAMP, cyclic adenosine 3′,5′-monophosphate;
  • EIA, enzyme immunoassay;
  • IgG, immunoglobulin G;
  • LTB4, leukotriene B4;
  • PDE, phosphodiesterase;
  • PAR2, proteinase-activated receptor 2;
  • PBS, phosphate-buffered saline

dec presentation

journal club 2014-11-10

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The Gardenia jasminoides extract and its constituent, geniposide, elicit anti-allergic effects on atopic dermatitis by inhibiting histamine in vitro and in vivo

Abstract

Ethnopharmacological relevance

Gardenia jasminoides Ellis has been used in traditional medicine for treatment of inflammation, edema, and dermaitis. The aim of this study was to investigate the mechanism by which Gardenia jasminoides extract (GJE) elicits anti-allergic effects in mast cells and in mice with atopic dermatitis (AD).

Materials and methods

We investigated the effects of GJE and its fractions on compound 48/80-induced histamine release from MC/9 cells and Dermatophagoides farinae-exposed NC/Nga mice. The effects of its constituents on histamine release from MC/9 cells were also investigated.

Results

GJE and its ethyl acetate fraction (GJE-EA) inhibited compound 48/80-induced histamine release from MC/9 mast cells. The topical application of GJE or GJE-EA to Dermatophagoides farinae-exposed NC/Nga mice reduced the symptoms of AD, inhibited the infiltration of inflammatory cells, and lowered the serum levels of immunoglobulin E and histamine. Both GJE and GJE-EA reduced the expression of cytokines (interleukin [IL]-4, IL-6, and tumor necrosis factor-alpha) and adhesion molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) in ear lesions. In addition, the quantitative analysis of GJE and GJE-EA by high-performance liquid chromatography revealed the presence of crocin and geniposide. Geniposide, but not crocin, inhibited the release of histamine from mast cells, which may contribute to the anti-allergic effect of GJE and GJE-EA.

Conclusions

These results suggest that GJE and GJE-EA can suppress mast cell degranulation-induced histamine release, and geniposide may be potential therapeutic candidates for AD

 

gardenia paper

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