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TRPA1 Acts in a Protective Manner in Imiquimod-Induced Psoriasiform Dermatitis in Mice

  • Author Footnotes
    10 These authors contributed equally to this work (joint first authors).
    Ágnes Kemény
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    10 These authors contributed equally to this work (joint first authors).
    Affiliations
    Department of Pharmacology and Pharmacotherapy, University of Pécs Medical School, Pécs, Hungary

    Department of Medical Biology, University of Pécs Medical School, Pécs, Hungary

    János Szentágothai Research Center, University of Pécs, Pécs, Hungary
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    10 These authors contributed equally to this work (joint first authors).
    Xenia Kodji
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    10 These authors contributed equally to this work (joint first authors).
    Affiliations
    Vascular Biology and Inflammation Section, BHF Centre of Cardiovascular Excellence, King’s College London, London, UK
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    10 These authors contributed equally to this work (joint first authors).
    Szabina Horváth
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    10 These authors contributed equally to this work (joint first authors).
    Affiliations
    Department of Dermatology, Venereology and Oncodermatology, University of Pécs, Pécs, Hungary
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  • Rita Komlódi
    Affiliations
    Department of Pharmacology and Pharmacotherapy, University of Pécs Medical School, Pécs, Hungary
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  • Éva Szőke
    Affiliations
    Department of Pharmacology and Pharmacotherapy, University of Pécs Medical School, Pécs, Hungary

    János Szentágothai Research Center, University of Pécs, Pécs, Hungary

    MTA-PTE NAP B Chronic Pain Research Group, University of Pécs, Pécs, Hungary
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  • Zoltán Sándor
    Affiliations
    Department of Pharmacology and Pharmacotherapy, University of Pécs Medical School, Pécs, Hungary

    János Szentágothai Research Center, University of Pécs, Pécs, Hungary
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  • Anikó Perkecz
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    Department of Pharmacology and Pharmacotherapy, University of Pécs Medical School, Pécs, Hungary
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  • Csaba Gyömörei
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    Department of Pathology, University of Pécs Medical School, Pécs, Hungary
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  • György Sétáló
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    Department of Medical Biology, University of Pécs Medical School, Pécs, Hungary
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  • Balázs Kelemen
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    Department of Physiology, University of Debrecen, Debrecen, Hungary
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  • Tamás Bíró
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    Department of Immunology, University of Debrecen, Debrecen, Hungary
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  • Balázs István Tóth
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    Department of Physiology, University of Debrecen, Debrecen, Hungary
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  • Author Footnotes
    11 These authors contributed equally to this work (joint corresponding authors).
    Susan D. Brain
    Footnotes
    11 These authors contributed equally to this work (joint corresponding authors).
    Affiliations
    Vascular Biology and Inflammation Section, BHF Centre of Cardiovascular Excellence, King’s College London, London, UK
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  • Author Footnotes
    11 These authors contributed equally to this work (joint corresponding authors).
    Erika Pintér
    Footnotes
    11 These authors contributed equally to this work (joint corresponding authors).
    Affiliations
    Department of Pharmacology and Pharmacotherapy, University of Pécs Medical School, Pécs, Hungary

    János Szentágothai Research Center, University of Pécs, Pécs, Hungary
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  • Author Footnotes
    11 These authors contributed equally to this work (joint corresponding authors).
    Rolland Gyulai
    Correspondence
    Correspondence: Rolland Gyulai, Department of Dermatology, Venereology and Oncodermatology, University of Pécs, Akác street 1, 7632-Pécs, Hungary.
    Footnotes
    11 These authors contributed equally to this work (joint corresponding authors).
    Affiliations
    Department of Dermatology, Venereology and Oncodermatology, University of Pécs, Pécs, Hungary
    Search for articles by this author
  • Author Footnotes
    10 These authors contributed equally to this work (joint first authors).
    11 These authors contributed equally to this work (joint corresponding authors).
Open ArchivePublished:March 14, 2018DOI:https://doi.org/10.1016/j.jid.2018.02.040
      This study revealed the modulatory role of transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) cation channels in the Aldara-induced (5% imiquimod) murine psoriasis model using selective antagonists and genetically altered animals. We have also developed a refined localized model to enable internal controls and reduce systemic effects. Skin pathology was quantified by measuring skin thickness, scaling, blood flow, and analyzing dermal cellular infiltrate, whereas nocifensive behaviors were also observed. Cytokine gene expression profiles were measured ex vivo. Psoriasiform dermatitis was significantly enhanced in TRPA1 knockout mice and with TRPA1 antagonist (A967079) treatment. By comparison, symptoms were decreased when TRPV1 function was inhibited. Imiquimod induced Ca2+ influx in TRPA1-, but not in TRPV1-expressing cell lines. Immunohistochemical studies revealed that CD4+ T helper cells express TRPA1 but not TRPV1 ion channels in mice skin. Compared with the TRPV1 knockout animals, additional elimination of the TRPA1 channels in the TRPV1/TRPA1 double knockout mice did not modify the outcome of the imiquimod-induced reaction, further supporting the dominant role of TRPV1 in the process. Our results suggest that the protective effects in psoriasiform dermatitis can be mediated by the activation of neuronal and nonneuronal TRPA1 receptors.

      Abbreviations:

      IMQ (imiquimod), KO (knockout), RTX (resiniferatoxin), TNF-α (tumor necrosis factor-α), TLR (toll-like receptor), TRPA1 (transient receptor potential ankyrin 1), TRPV1 (transient receptor potential vanilloid 1), WT (wild type)

      Introduction

      Psoriasis is a chronic, recurrent immune-mediated inflammatory skin disease, affecting 2–3% of the population. The most common type of the disease is psoriasis vulgaris that occurs in approximately 90% of the patients. Clinically, the disease is characterized by sharply demarcated, scaly, erythematous skin lesions, and pruritus (
      • Nestle F.O.
      • Kaplan D.H.
      • Barker J.
      Psoriasis.
      ). It is widely accepted that T lymphocytes play a key role in psoriasis, as they chronically colonize the skin and promote the proliferation of keratinocytes. T-cell activation is initiated and driven by dendritic cells that, on onset or exacerbation of psoriasis, produce various proinflammatory cytokines, including tumor necrosis factor-α (TNF-α) and IL-23. The critical roles for immune cells and cytokines in psoriasis pathogenesis are supported by the observation that treatments targeting the immune system, such as antibodies against TNF-α, IL-12/23, or IL-17, are highly effective in improving the disease (
      • Nestle F.O.
      • Kaplan D.H.
      • Barker J.
      Psoriasis.
      ).
      Imiquimod (IMQ)-induced psoriasiform skin inflammation in mice is the most frequently used animal model to study the pathomechanism of psoriasis (
      • Swindell W.R.
      • Michaels K.A.
      • Sutter A.J.
      • Diaconu D.
      • Fritz Y.
      • Xing X.
      • et al.
      Imiquimod has strain-dependent effects in mice and does not uniquely model human psoriasis.
      ,
      • van der Fits L.
      • Mourits S.
      • Voerman J.S.A.
      • Kant M.
      • Boon L.
      • Laman J.D.
      • et al.
      Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
      ). The IMQ model recapitulates many of the phenotypic changes of psoriasis and mimics its IL-17/IL-23-driven pathology. IMQ acts primarily via the ligation of toll-like receptor (TLR) 7 (in mice and human) or TLR8 (only in human) (
      • Hemmi H.
      • Kaisho T.
      • Takeuchi O.
      • Sato S.
      • Sanjo H.
      • Hoshino K.
      • et al.
      Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway.
      ,
      • Jurk M.
      • Heil F.
      • Vollmer J.
      • Schetter C.
      • Krieg A.M.
      • Wagner H.
      • et al.
      Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848.
      ), although a TLR7-independent mechanism has also been proposed (
      • Kanneganti T.-D.
      • Ozören N.
      • Body-Malapel M.
      • Amer A.
      • Park J.-H.
      • Franchi L.
      • et al.
      Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3.
      ,
      • Schön M.P.
      • Schön M.
      • Klotz K.-N.
      The small antitumoral immune response modifier imiquimod interacts with adenosine receptor signaling in a TLR7- and TLR8-independent fashion.
      ). Despite some controversy, the roles for cytokines in this model, especially TNF-α and the IL-17/IL-23 axes, have been shown (
      • Van Belle A.B.
      • de Heusch M.
      • Lemaire M.M.
      • Hendrickx E.
      • Warnier G.
      • Dunussi-Joannopoulos K.
      • et al.
      IL-22 is required for imiquimod-induced psoriasiform skin inflammation in mice.
      ,
      • Vinter H.
      • Kragballe K.
      • Steiniche T.
      • Gaestel M.
      • Iversen L.
      • Johansen C.
      Tumour necrosis factor-alpha plays a significant role in the Aldara-induced skin inflammation in mice.
      ).
      Recently,
      • Riol-Blanco L.
      • Ordovas-Montanes J.
      • Perro M.
      • Naval E.
      • Thiriot A.
      • Alvarez D.
      • et al.
      Nociceptive sensory neurons drive interleukin-23-mediated psoriasiform skin inflammation.
      provided evidence that the desensitization of NaV1.8+/TRPV1+ nociceptive sensory neurons by resiniferatoxin (RTX) pretreatment significantly decreased the inflammatory reaction in IMQ-induced psoriasiform dermatitis in mice by blocking of IL-23 production in dermal dendritic cells. Because transient receptor potential ankyrin 1 (TRPA1) and transient receptor potential vanilloid 1 (TRPV1) nonselective cation channels are expressed on the sensory nerve endings, RTX pretreatment causes selective elimination of the functions of these neuronal receptors. Nevertheless, chemical denervation by RTX does not influence mRNA and protein expression of nonneuronal TRPV1 and TRPA1 (
      • Kun J.
      • Helyes Z.
      • Perkecz A.
      • Bán Á.
      • Polgár B.
      • Szolcsányi J.
      • et al.
      Effect of surgical and chemical sensory denervation on non-neural expression of the transient receptor potential vanilloid 1 (TRPV1) receptors in the rat.
      ). In addition, these TRP channels have previously been shown to play important roles in pruritus (
      • Fernandes E.S.
      • Vong C.T.
      • Quek S.
      • Cheong J.
      • Awal S.
      • Gentry C.
      • et al.
      Superoxide generation and leukocyte accumulation: key elements in the mediation of leukotriene B4-induced itch by transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1.
      ,
      • Wilson S.R.R.
      • Thé L.
      • Batia L.M.M.
      • Beattie K.
      • Katibah G.E.E.
      • McClain S.P.P.
      • et al.
      The epithelial cell-derived atopic dermatitis cytokine TSLP activates neurons to induce itch.
      ) and contact dermatitis (
      • Liu B.
      • Escalera J.
      • Balakrishna S.
      • Fan L.
      • Caceres A.I.
      • Robinson E.
      • et al.
      TRPA1 controls inflammation and pruritogen responses in allergic contact dermatitis.
      ). Hence, this study aimed to investigate the role for TRPA1 and TRPV1 in the IMQ model of psoriasis.

      Results

      Loss of TRPA1 function enhances IMQ-induced psoriasiform skin inflammation

      Clinical signs of psoriasis such as skin thickening and scaling were consistently observed in IMQ- but not vaseline-treated skin using the two different disease induction techniques in both male and female mice (Figure 1a and b , Supplementary Figure S1 online). Dorsal skin erythema, one of the main clinical signs of psoriasis, was assessed using the laser Doppler blood flowmetry technique. The measurement of dorsal skin blood flow quantitatively in this model, to our knowledge, has been previously unreported. A significant increase in dorsal skin blood flow was observed using both IMQ treatment techniques, reaching maximal responses after the last treatment (Figure 1g and h), highlighting a useful application for the laser Doppler technique to assess the dorsal skin erythema in a quantitative manner. Both the “whole back model” and the “localized model,” where IMQ application in the Finn chamber technique was used, resulted in similar trends of skin thickening and scaling (Figure 1d, e, j, and k). Thus, the localized model, to our knowledge, is a previously unreported way of inducing skin inflammation with less systemic side effects, as indicated by the development of reduced splenomegaly (Supplementary Figure S2 online).
      Figure 1
      Figure 1Effect of topical Aldara treatment on skin thickness, blood perfusion, and skin scaling response in TRPA1 WT and KO mice and A967079 treatment. (a, c) Aldara (75 mg) was applied to the back skin of TRPA1 WT animals. A967079 or vehicle (0.5% methylcellulose in water) was administered orally at 30 minutes and 5 hours after topical skin treatment. (b) 25 mg vaseline (V) or Aldara (A) was applied to the shaved back skin of TRPA1 WT and KO animals using Finn chambers. (d, e, f) Percent change in skin thickness after vaseline or Aldara treatment in TRPA1 WT or KO male mice (d) and TRPA1 WT or KO female animals using the Finn chamber (e) or A967079 pretreated TRPA1 WT male mice (f). (g, h, i) Percent change in dorsal skin blood flow after vaseline or Aldara treatment in TRPA1 WT or KO (g) and TRPA1 WT or KO animals using the Finn chamber (h) or after A967079 pretreatment in TRPA1 WT mice (i). (j, k, l) Skin scaling PASI scores after vaseline or Aldara treatment in TRPA1 WT or KO (j) and TRPA1 WT or KO animals using Finn chambers (k) or in A967079 pretreated TRPA1 WT mice (l). Data are mean ± standard error of the mean for n = 6–12/group. *P < 0.05; **P < 0.01; ***P < 0.001 vaseline versus Aldara-treated sites, #P < 0.05; ##P < 0.01; ###P < 0.001 Aldara-treated WT versus Aldara-treated KO or Aldara-treated WT/Veh versus Aldara-treated knockout/A967079-treated group, based on repeated measures two-way analysis of variance followed by Bonferroni’s post hoc test. KO, knockout; PASI, psoriasis area and severity index; TRPA1, transient receptor potential ankyrin 1; WT, wild type.
      Interestingly, TRPA1 knockout (KO) mice showed exacerbated skin pathology in both disease induction models, with increased skin thickness and enhanced erythema from day 2 onward (Figure 1). This effect was independent of gender differences, and systemic inflammatory effects, highlighting a specific, localized protective role for TRPA1 in this model (Supplementary Figures S1 and S2). To eliminate the possibility of compensatory mechanisms in the KO animals, experiments involving pretreatment with the selective TRPA1 antagonist A967079 were also carried out. Twice daily application of A967079 over 4 days substantially enhanced IMQ-mediated skin thickening and scaling in wild-type (WT) mice similar to the observed profile in TRPA1 KO mice (Figure 1c, f, and l). In contrast to the observation in TRPA1 KO mice, A967079 treatment did not markedly influence blood perfusion response in the IMQ-treated TRPA1 WT groups (Figure 1i).

      Histologic analysis confirms the protective role for TRPA1 in IMQ-mediated psoriasiform dermatitis

      The typical histologic hallmarks of human psoriasis (keratinocyte hyperproliferation, hyperkeratosis, parakeratosis, Munro’s microabscesses) were observed in IMQ-treated TRPA1 WT and KO mice. Infiltration of inflammatory cells (e.g., lymphocytes, granulocytes, macrophages) could be observed in the epidermal and dermal layers of the treated skin (Figure 2a, Supplementary Figure S3 online). Accumulation of neutrophils in the epidermis resulted in Munro’s microabscesses after IMQ treatment (Figure 2a). Histopathologic scoring assessing characteristic parameters in psoriasis (Munro’s microabscesses, epidermal thickness, and cell layers) was significantly enhanced in IMQ-treated skin samples of TRPA1 KO or A967079-treated mice compared with WT samples (Figure 2b and c).
      Figure 2
      Figure 2Representative histologic view of TRPA1 WT and TRPA1 KO mouse dorsal skin after Aldara treatment. (a) Upper panel: vaseline-treated control skin of TRPA1 WT and KO animal at ×100 magnification (scale bar = 200 μm); middle panel: Aldara-treated dorsal skin tissue of TRPA1 WT and KO mouse at ×200 magnification (scale bar = 200 μm); bottom panel: Aldara-treated dorsal skin tissue of TRPA1 WT and KO mouse (×400, scale bar = 100 μm). (b) Arbitrary score of vaseline- or Aldara-treated dorsal skin samples of TRPA1 WT and KO animals. (c) Arbitrary score of vaseline- or Aldara-treated dorsal skin samples of TRPA1 WT animals pretreated with A967079 or its vehicle. Data are mean ± standard error of the mean for n = 4–7/group. **P < 0.01; ***P < 0.001 vaseline versus Aldara-treated sites, #P < 0.05 Aldara-treated WT versus Aldara-treated KO group or vehicle+Aldara-treated versus A967079+Aldara-treated group, based on two-way analysis of variance followed by Bonferroni’s post hoc test. HK, hyperkeratosis; KO, knockout; MM, Munro’s microabscesses; PK, parakeratosis; TRPA1, transient receptor potential ankyrin 1; WT, wild type.

      TRPA1 KO and TRPA1 antagonist-treated mice exhibit significantly increased nocifensive behavior

      The effect of IMQ on nocifensive and itch behavior was determined using the “whole back model.” The trends for hind paw scratching were inconsistent and highly variable in both TRPA1 WT and KO mice (Figure 3b). However, IMQ resulted in significantly increased biting/licking and flinching responses (Figure 3c and d). Consistent with worsening skin pathology, deletion or long-term inhibition of TRPA1 function showed trends of increased biting/licking and flinching, reaching significance on day 4 (Figure 3). A similar profile was observed in female mice (Supplementary Figure S4 online).
      Figure 3
      Figure 3Repeated TRPA1 inhibition resulted in increased spontaneous nocifensive behaviors. Aldara (75 mg) was applied to the back skin of male TRPA1 WT or KO animals. A967079 or vehicle (0.5% methylcellulose in water) was administered orally at 30 minutes and 5 hours after topical skin treatment. Behavioral observation was performed daily at 4 hours after topical skin treatment, for 30 minutes. Mice were acclimatized to the setup for 2 days (days 3 and 2). (a) Day 4 spontaneous behaviors, (b) hind paw scratching, (c) biting/licking of treated dorsal skin, (d) flinching of the dorsal region in TRPA1 WT or KO animals. (e) Day 4 spontaneous behaviors, (f) hind paw scratching, (g) biting/licking of treated dorsal skin, (h) flinching of the dorsal region in vehicle or A967079 pretreated animals. Data are mean ± standard error of the mean for n = 5–7/group. *P < 0.05; **P < 0.01; ***P < 0.001 vaseline versus Aldara-treated sites, #P < 0.05; ##P < 0.01; ###P < 0.001 Aldara-treated Veh versus Aldara-treated A967079 groups, based on repeated measures two-way analysis of variance followed by Bonferroni’s post hoc test. KO, knockout; TRPA1, transient receptor potential ankyrin 1; WT, wild type.

      Temporal inflammatory cytokine mRNA expression in TRPA1 WT and KO mice

      IMQ induced a marked increase in the mRNA expression of IL-1β, TNF-α, IL-23, IL-17, and IL-22 in WT mice, in agreement with previous publications (
      • Flutter B.
      • Nestle F.O.
      TLRs to cytokines: mechanistic insights from the imiquimod mouse model of psoriasis.
      ,
      • Riol-Blanco L.
      • Ordovas-Montanes J.
      • Perro M.
      • Naval E.
      • Thiriot A.
      • Alvarez D.
      • et al.
      Nociceptive sensory neurons drive interleukin-23-mediated psoriasiform skin inflammation.
      ,
      • van der Fits L.
      • Mourits S.
      • Voerman J.S.A.
      • Kant M.
      • Boon L.
      • Laman J.D.
      • et al.
      Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
      ) (Figure 4). TNF-α and IL-23 mRNA levels peaked at 6 hours (Figure 4b and c), whereas for IL-1β, IL-17, and IL-22, the highest mRNA expression was observed at 48 hours (Figure 4a, d, and e). After 96 hours, all of the cytokine mRNA levels started to return to baseline (data not shown). A significantly increased expression of IL-1β, TNF-α, and IL-22 mRNA was detected in the TRPA1 KO compared with WT mice, reaching maximal differences at 48 hours. The trends of cytokines expression reflect the worsening skin pathology in TRPA1 KO mice, which is initiated as early as 6 hours after the first IMQ application.
      Figure 4
      Figure 4Imiquimod-induced relative expression of distinct inflammatory cytokines in TRPA1 WT and KO mice at different time points. (a) IL-1β relative quantity, (b) TNF-α relative quantity, (c) IL-23 relative quantity, (d) IL-17 relative quantity, (e) IL-22 relative quantity measured with quantitative real-time reverse transcriptase-PCR. Data are mean ± standard error of the mean for n = 5/group. *P < 0.05; ***P < 0.001 vaseline versus Aldara-treated sites, ##P < 0.01; ###P < 0.001 Aldara-treated wild-type versus Aldara-treated knockout group, based on Student’s t-test. KO, knockout; TNF-α, tumor necrosis factor-α; TRPA1, transient receptor potential ankyrin 1; WT, wild type.

      Influence of TRPV1 on the protective role of TRPA1 in IMQ response

      To determine potential interactions between TRPV1 and TRPA1 receptors in this model, we induced skin inflammation in TRPV1/TRPA1 double KO animals (Figure 5e and f ). Double TRPA1/V1 KO led to an inhibition of skin fold thickness and reduced dorsal skin perfusion (Figure 5e and f). A similar tendency was also observed when TRPA1 KO mice were treated with a TRPV1 antagonist (SB366791). The enhanced skin pathology and dorsal skin erythema phenotype were inhibited with SB366791 treatment (Figure 5c and d). TRPV1 KO mice also showed improved skin pathology and showed trends of reduced erythema in comparison to WT mice (Figure 5a and b). Hence, these results suggest an opposing interaction between TRPA1 and TRPV1 in the IMQ model.
      Figure 5
      Figure 5Effect of TRPA1 and TRPV1 loss of function on Aldara-mediated skin inflammation (a, b, c, d, e, f). Imiquimod-induced Ca2+ influx in TRPA1-, TRPV1-expressing CHO cells or HaCaT cell lines (g, h). (a, b) Vaseline (25 mg) or Aldara was applied to the shaved back skin of TRPV1 WT and KO animals in Finn chambers. Percent change of skin thickness (a) and blood perfusion (b) were compared with the vaseline-treated groups. (c, d) Aldara (75 mg) was applied to the back skin of TRPA1 WT or KO animals. SB366791 TRPV1 receptor antagonist (3 mg/kg, intraperitoneally) or its vehicle (Veh, 10% DMSO in saline) was administered 30 minutes before Aldara-treatment. Percent change of skin thickness (c) and dorsal skin blood flow (d) was monitored as described in the Methods section. (e, f) Vaseline (25 mg) or Aldara was applied to the shaved back skin of TRPV1-, TRPA1, or TRPA1/V1 WT and KO animals in Finn chambers. Percent change of skin thickness (e) and blood perfusion (f) were compared with the Aldara-treated WT groups of each mice strains. Data are mean ± standard error of the mean for n = 6–12/group. *P < 0.05; **P < 0.01; ***P < 0.001 vaseline-treated versus Aldara-treated groups or Aldara-treated TRPA1 WT versus Aldara-treated TRPA1 KO+vehiculum (veh) groups, #P < 0.05; ##P < 0.01; ###P < 0.001 Aldara-treated TRPV1 WT versus Aldara-treated TRPV1 KO groups (a, b) or Aldara-treated TRPA1 KO+vehiculum (Veh) versus Aldara-treated TRPA1 KO+SB366791 groups (c, d) or TRPA1 KO versus TRPV1- and double KO animals (e, f), based on repeated measures two-way analysis of variance followed by Bonferroni’s post hoc test. (g) Dose-response curve induced by IMQ in TRPA1 or TRPV1 expressing CHO cells or in HaCaT cell line monitored by radioactive 45Ca2+ uptake. (h) IMQ-induced Fluo-4 signal ratio in TRPA1+ CHO cells after mustard oil (MO), imiquimod (IMQ), or different concentrations of A967079 (A96) treatments, ***P < 0.001 A96+50 μM IMQ vs. 50 μM IMQ-treated groups based on the unpaired t test. KO, knockout; TRPA1, transient receptor potential ankyrin 1; TRPV1, transient receptor potential vanilloid 1; WT, wild type.

      Effect of IMQ in TRPA1 or TRPV1 expressing CHO, HaCaT, and HEK293T cell lines

      To investigate if IMQ could directly activate TRPA1 or TRPV1 receptors or keratinocytes, we used radioactive 45Ca2+ uptake studies with TRPA1 or TRPV1 expressing CHO cell lines and HaCaT cells, a human cell line derived from keratinocytes. IMQ induced Ca2+ influx dose-dependently (with an EC50 of 4.03 μM) in TRPA1 expressing but not in TRPV1 expressing CHO cells. Only a low level of Ca2+ influx was detected in HaCaT cells even at 100 μM IMQ (Figure 5g). To ensure that the observed effect was TRPA1 dependent, in subsequent experiments, varying doses of TRPA1 antagonist (A967079) were coadministered with the IMQ treatment. A967079 dose-dependently inhibited Ca2+ influx in TRPA1 expressing cells in response to IMQ, highlighting that IMQ specifically activates TRPA1 (Figure 5h). IMQ induced a mainly outwardly rectifying transmembrane current on HEK293T cells overexpressing recombinant human TRPA1, which was abolished in the presence of HC030031, a potent and selective inhibitor of TRPA1 measured with the patch clamp technique (Supplementary Figure S5 online).

      Expression of TRPA1, but not TRPV1 receptors on CD4+ T cells

      Using confocal microscopy of IMQ-treated skin, we observed TRPA1+ staining, overlapping with CD4+ expression on dermal CD4+ T cells. TRPA1 was mainly expressed on the plasma membrane of CD4+ T cells (Figure 6, left panel). Intriguingly, there were no TRPV1 and CD4 colocalization in mouse dorsal skin sections (Figure 6, right panel).
      Figure 6
      Figure 6Immunohistologic detection of TRPA1+/CD4+ T helper cells or TRPV1+/CD4+ T helper cells in imiquimod-treated mouse dorsal skin samples. Single optical sections have been taken using a ×40 fluorescent objective with phase-contrast capacity (scale bar = 100 μm). TRPA1, transient receptor potential ankyrin 1; TRPV1, transient receptor potential vanilloid 1.

      Discussion

      IMQ-induced skin inflammation is widely accepted as a murine model for psoriasis (
      • Flutter B.
      • Nestle F.O.
      TLRs to cytokines: mechanistic insights from the imiquimod mouse model of psoriasis.
      ). Indeed, we observed similar degrees of psoriasiform dermatitis, in vivo and histopathologically, using two different techniques of IMQ application. We have characterized a “localized model” involving the Finn chamber application technique, in which both IMQ and vaseline treatment were applied on the dorsal skin of the same mouse, thus allowing lesional and nonlesional samples to be obtained from the same mouse. In this model, involving lower total IMQ levels, mice showed reduced splenomegaly, indicating a less systemic inflammatory component. The overall effect of IMQ activation results in the production of various proinflammatory cytokines, such as TNF-α (
      • Perera G.K.
      • Di Meglio P.
      • Nestle F.O.
      Psoriasis.
      ,
      • Quivy V.
      • Van Lint C.
      Regulation at multiple levels of NF-kappaB-mediated transactivation by protein acetylation.
      ) and IL-1β (
      • Kanneganti T.-D.
      • Ozören N.
      • Body-Malapel M.
      • Amer A.
      • Park J.-H.
      • Franchi L.
      • et al.
      Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3.
      ), whereas the activation of T cells leads to the production of IL-17/IL-22/IL-23 (
      • Van Belle A.B.
      • de Heusch M.
      • Lemaire M.M.
      • Hendrickx E.
      • Warnier G.
      • Dunussi-Joannopoulos K.
      • et al.
      IL-22 is required for imiquimod-induced psoriasiform skin inflammation in mice.
      ,
      • van der Fits L.
      • Mourits S.
      • Voerman J.S.A.
      • Kant M.
      • Boon L.
      • Laman J.D.
      • et al.
      Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
      ). Our current observations confirmed that the upregulation of these cytokines within 48 hours in our “localized model” is similar to previous publications (
      • van der Fits L.
      • Mourits S.
      • Voerman J.S.A.
      • Kant M.
      • Boon L.
      • Laman J.D.
      • et al.
      Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
      ). Hence, this is a suitable model for studying IMQ-induced psoriasis.
      Recently, Riol-Blanco et al. used RTX pretreatment as an established technique to selectively deplete peripheral sensory nerve endings. Because TRPA1 and TRPV1 are co-expressed on these neurons (
      • Kobayashi K.
      • Fukuoka T.
      • Obata K.
      • Yamanaka H.
      • Dai Y.
      • Tokunaga A.
      • et al.
      Distinct expression of TRPM8, TRPA1, and TRPV1 mRNAs in rat primary afferent neurons with Aδ/C-fibers and colocalization with Trk receptors.
      ), this technique eliminates both receptors from neuronal elements, but has no effect on nonneuronal receptor structures (
      • Kun J.
      • Helyes Z.
      • Perkecz A.
      • Bán Á.
      • Polgár B.
      • Szolcsányi J.
      • et al.
      Effect of surgical and chemical sensory denervation on non-neural expression of the transient receptor potential vanilloid 1 (TRPV1) receptors in the rat.
      ). This leads to the following assumptions. (i) Activation of TRP or TLR-7 receptors by IMQ on nonneuronal cells is not capable of triggering psoriasiform dermatitis. (ii) The peripheral nociceptors/sensory nerve endings are inevitable for the development of the IMQ-induced inflammation via the activation of dermal DCs, which then release IL-23.
      Here, we investigated the role of TRP channels in this process by using various TRP KO mice. Interestingly, TRPV1 KO mice showed similar improvement as the RTX-treated animals, suggesting that the presence of TRPV1 receptors is essential on the peripheral nociceptors for the IMQ-induced psoriasis. However, we have shown that this could not be a direct effect of IMQ via the TRPV1 receptor as IMQ does not activate TRPV1 expressing CHO cells. Hence, we propose that the TRPV1 receptor is activated by an as yet unknown mediator released by other cells (e.g., keratinocytes) in response to IMQ. In contrast, additional elimination of the TRPA1 channels in the TRPV1/TRPA1 double KO mice did not modify the outcome of the IMQ-induced pathology, further supporting the dominant role of TRPV1 in the process. Surprisingly, when TRPV1 is intact, genetic deletion or pharmacologic blockade of TRPA1 led to a significantly enhanced IMQ-induced pathology, suggesting that under normal circumstances TRPA1 downregulates the inflammatory process. Here we have provided evidence that the expression of Iba-1 mRNA, a marker of the activation of macrophages in the dorsal root ganglia, is significantly elevated after IMQ treatment in TRPA1 KO mice compared with WT animals (see Supplementary Figure S6 online). It is possible that TRPV1 and TRPA1 channels can form a heterodimer in sensory neurons during basal conditions, and both are able to cross regulate each other’s activity (cross-sensitization/desensitization) during inflammation (
      • Gouin O.
      • L’Herondelle K.
      • Lebonvallet N.
      • Le Gall-Ianotto C.
      • Sakka M.
      • Buhé V.
      • et al.
      TRPV1 and TRPA1 in cutaneous neurogenic and chronic inflammation: pro-inflammatory response induced by their activation and their sensitization.
      ). This suggests that TRPA1 likely acts by modulating neuronal excitability, possibly via the regulation of TRPV1 activity.
      In addition, spontaneous nocifensive behaviors were also significantly enhanced with TRPA1 deletion or inhibition. This is in contrast to the previous observation for the role of TRPA1 in pruritus (
      • Liu B.
      • Escalera J.
      • Balakrishna S.
      • Fan L.
      • Caceres A.I.
      • Robinson E.
      • et al.
      TRPA1 controls inflammation and pruritogen responses in allergic contact dermatitis.
      ,
      • Wilson S.R.
      • Nelson A.M.
      • Batia L.
      • Morita T.
      • Estandian D.
      • Owens D.M.
      • et al.
      The ion channel TRPA1 is required for chronic itch.
      ) as well as in nociception (
      • Chen J.
      • Joshi S.K.
      • DiDomenico S.
      • Perner R.J.
      • Mikusa J.P.
      • Gauvin D.M.
      • et al.
      Selective blockade of TRPA1 channel attenuates pathological pain without altering noxious cold sensation or body temperature regulation.
      ,
      • Fernandes E.S.
      • Vong C.T.
      • Quek S.
      • Cheong J.
      • Awal S.
      • Gentry C.
      • et al.
      Superoxide generation and leukocyte accumulation: key elements in the mediation of leukotriene B4-induced itch by transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1.
      ). However, this is unsurprising in the present model, as the enhanced nocifensive behaviors correlate with the increase in skin pathology.
      Although the proinflammatory effects of TRPA1 activation are well established, there is emerging evidence for its protective effects. Recently, capsazepine, originally classified as a TRPV1 antagonist (
      • Bevan S.
      • Hothi S.
      • Hughes G.
      • James I.F.
      • Rang H.P.
      • Shah K.
      • et al.
      Capsazepine: a competitive antagonist of the sensory neurone excitant capsaicin.
      ), has been shown to protect the development of experimental colitis via TRPA1 agonism (
      • Kistner K.
      • Siklosi N.
      • Babes A.
      • Khalil M.
      • Selescu T.
      • Zimmermann K.
      • et al.
      Systemic desensitization through TRPA1 channels by capsazepine and mustard oil—a novel strategy against inflammation and pain.
      ), whereas TRPA1 deletion was shown to enhance inflammatory responses in various colitis animal models (
      • Bertin S.
      • Aoki-Nonaka Y.
      • Lee J.
      • de Jong P.R.
      • Kim P.
      • Han T.
      • et al.
      The TRPA1 ion channel is expressed in CD4+ T cells and restrains T-cell-mediated colitis through inhibition of TRPV1.
      ,
      • Kun J.
      • Szitter I.
      • Kemény A.
      • Perkecz A.
      • Kereskai L.
      • Pohóczky K.
      • et al.
      Upregulation of the transient receptor potential ankyrin 1 ion channel in the inflamed human and mouse colon and its protective roles.
      ). These results imply that the nonneuronal TRPA1 activation has a major anti-inflammatory activity in contrast to the proinflammatory contribution of sensory nerves.
      Although the main mechanism of action for IMQ is considered to be via TLR7 on cutaneous macrophages and dendritic cells in mice (
      • Hemmi H.
      • Kaisho T.
      • Takeuchi O.
      • Sato S.
      • Sanjo H.
      • Hoshino K.
      • et al.
      Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway.
      ), various TLR7-independent pathways have also been proposed (
      • Kanneganti T.-D.
      • Ozören N.
      • Body-Malapel M.
      • Amer A.
      • Park J.-H.
      • Franchi L.
      • et al.
      Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3.
      ). To our knowledge, here we present the first evidence that IMQ has TRPA1 agonist activity. It induced dose-dependent Ca2+ influx in TRPA1-transfected CHO cells and a mainly outwardly rectifying transmembrane current on HEK293T cells overexpressing recombinant human TRPA1, and this response was selectively inhibited by the TRPA1 antagonists A967079 and HC030031. By comparison, IMQ did not induce Ca2+ influx in CHO cells transfected with recombinant TRPV1, indicating that it is unlikely that IMQ would directly influence the functions of cutaneous nociceptors via TRPV1. Thus, IMQ, as a TRPA1 agonist, may potentially activate neural and nonneural cells by their TRPA1 channels, and exert anti-inflammatory activity in the skin.
      As TRPA1 KO mice showed a more prominent increase in T helper 1-associated cytokines, it was proposed that TRPA1 may have an important role in regulating CD4+ T-cell signaling. Indeed,
      • Bertin S.
      • Aoki-Nonaka Y.
      • Lee J.
      • de Jong P.R.
      • Kim P.
      • Han T.
      • et al.
      The TRPA1 ion channel is expressed in CD4+ T cells and restrains T-cell-mediated colitis through inhibition of TRPV1.
      previously showed an interaction between TRPA1 and TRPV1 in CD4+ T-cell activation toward the T helper 1 phenotype. We found that TRPA1 is also colocalized in CD4+ T cells in IMQ-induced skin inflammation, in support for Bertin et al., but, in contrast to the CD4+ T cells in the gut, we did not detect TRPV1 expression on skin infiltrating CD4+ cells. This indicates that this mechanism is unlikely to have major involvement here. Furthermore, we did not detect any difference in the number of dermal CD4+ T cells between TRPA1 WT and KO animals.
      Inflammatory mediators are known to regulate TRPV1 activity via a range of pathways, including specific G-protein coupled receptors (
      • Gouin O.
      • L’Herondelle K.
      • Lebonvallet N.
      • Le Gall-Ianotto C.
      • Sakka M.
      • Buhé V.
      • et al.
      TRPV1 and TRPA1 in cutaneous neurogenic and chronic inflammation: pro-inflammatory response induced by their activation and their sensitization.
      ). Of note, cutaneous denervation in the KC-Tie2 psoriasisform mouse model also results in improvements in acanthosis, decreases in CD4+ T cells, and an elimination of CD11c+ cells concomitant with decreased IL-23 protein expression, and this effect was suggested to be mediated by nerve-derived substance P and calcitonin gene-related peptide (
      • Ostrowski S.M.
      • Belkadi A.
      • Loyd C.M.
      • Diaconu D.
      • Ward N.L.
      Cutaneous denervation of psoriasiform mouse skin improves acanthosis and inflammation in a sensory neuropeptide-dependent manner.
      ). Potentially, IMQ, similar to capsazepine and isopetasin, may directly activate TRPA1 on sensory neurons, leading to the partial desensitization of these nociceptors to TRP-mediated (and perhaps other) stimuli in psoriasis. This, in turn, may result in reduced release of neuropeptides from nerve endings, leading to attenuated dendritic cell activation, decreased IL-12 or IL-23 release, and diminished subsequent generation of T helper 1 or T helper 17 T cells, and cutaneous inflammation. Potentially supporting this is our observation that the nocifensive behaviors associated with the early IMQ treatments at day 1 in the mice were not observed in TRPA1 KO or antagonist-treated mice (see Figure 3a and e), indicating that the TRPA1 channel is indispensable for the early IMQ-evoked pruritus. This phenomenon was very similar in the colitis model, where the first capsazepine administration caused pain in the WT mice, whereas TRPA1 KOs did not show pain-related behavior (
      • Kistner K.
      • Siklosi N.
      • Babes A.
      • Khalil M.
      • Selescu T.
      • Zimmermann K.
      • et al.
      Systemic desensitization through TRPA1 channels by capsazepine and mustard oil—a novel strategy against inflammation and pain.
      ). The more intensive nociceptive behavior at the later time points in the TRPA1 KO mice in the IMQ-induced dermatitis model provides further evidence for a protective phenotype of TRPA1.
      In conclusion, to our knowledge, these results are the first to show that TRPA1 has a protective role in IMQ-induced psoriasiform dermatitis. We provide evidence that IMQ can directly activate cells through TRPA1 (but not TRPV1). Potentially, the use of TRPA1 agonists may lead to further inhibition of psoriasis-like inflammation. Mustard oil (marketed as a natural anti-psoriasis remedy) and mustard seed, which contains the TRPA1 agonist allyl-isothiocyanate, were recently shown to reduce IMQ inflammation (
      • Yang R.
      • Zhou Q.
      • Wen C.
      • Hu J.
      • Li H.
      • Zhao M.
      • et al.
      Mustard seed (Sinapis Alba Linn) attenuates imiquimod-induced psoriasiform inflammation of BALB/c mice.
      ). Thus, there is some evidence to support this strategy. Further studies are required, however, to fully characterize and exploit the pro- and anti-inflammatory potential of the TRPA1 receptor in cutaneous inflammatory reactions.

      Materials and Methods

      Full methods are in the Supplementary Material online.

      Animals

      Animal experiments were performed in Hungary and the UK, according to the Animal Research: Reporting In Vivo Experiments guidelines, in accordance with the Hungarian and UK law and ethics approval. Female and male TRPV1 WT, TRPA1 WT (+/+), TRPV1 receptor gene KO (–/–), TRPA1 KO, and TRPA1/V1 KO mice (8–10 weeks, 20–25 g) were used with respective background strains.

      Induction of psoriasiform skin inflammation

      Psoriasiform dermatitis was induced by Aldara cream (5% IMQ, Meda Pharma, Hungary) and vaseline, for vehicle control, using two techniques administered daily for 4 days. Dorsal skin was shaved and depilated before application. The first technique involved the application of 75 mg of Aldara on a 4 cm2 area, known as “the whole back model” as previously characterized (
      • Roller A.
      • Perino A.
      • Dapavo P.
      • Soro E.
      • Okkenhaug K.
      • Hirsch E.
      • et al.
      Blockade of phosphatidylinositol 3-kinase (PI3K)δ or PI3Kγ reduces IL-17 and ameliorates imiquimod-induced psoriasis-like dermatitis.
      ) and was used for nociceptive behavior experiments. The second model involved a modification of the technique using Finn chambers to enable a more localized application and a lower dose of IMQ in the “localized model.” This technique reduced the systemic effects associated with the IMQ model and allowed each mouse to be its own internal control, with lesional and nonlesional skin. For pharmacologic studies, the TRPA1 antagonist (A967079; 60 mg/kg, orally) or vehicle (0.5% methylcellulose in water) was administered twice daily. TRPV1 antagonist (SB366791; 3 mg/kg, intraperitoneally) or vehicle (10% DMSO in saline) was administered once daily, 30 minutes before topical skin treatment.

      Measurement of dorsal skin thickness

      Double-fold dorsal skin thickness at the treated areas was measured with micrometers (Moore and Wright, Sheffield, England) with 0.1 mm accuracy.

      Measurement of blood flow perfusion changes

      Measurement of dorsal skin blood flow was assessed in the dorsal skin by laser Doppler flowmetry.

      Skin scaling score

      The skin scaling score was evaluated and graded 0 to 4 (0: absent to 4: severe).

      Radioactive 45Ca2+ uptake and Ca2+ influx experiments

      IMQ-induced radioactive 45Ca2+ uptake or Ca2+ influx was determined on HaCaT (human immortalized keratinocytes) and TRPA1 or TRPV1 receptor-expressing CHO cell lines by scintillation counter or flow cytometry.

      Patch clamp recording

      HEK293T cells were seeded on coverslips and whole cell patch clamp measurements were carried out using the Axopatch 1.D amplifier and Clampex 10.2 software (Molecular Devices, San Jose, CA) to record TRPA1-mediated currents.

      Nocifensive behavioral studies

      After acclimatization, daily observation was carried out at 4 hours after topical skin treatment for 30 minutes. Studies showed three typical spontaneous, nocifensive behaviors in IMQ-treated mice: (i) hind paw scratching, (ii) biting/licking of the treated area, and (ii) flinching, defined as rotational flipping of the dorsal area (
      • Wheeler-Aceto H.
      • Porreca F.
      • Cowan A.
      The rat paw formalin test: comparison of noxious agents.
      ).

      Histology and immunochemistry

      Skin tissue samples were formalin-fixed (6%) and embedded in paraffin for hematoxylin-eosin or for chloroacetate esterase staining. Scoring parameters and values were determined to evaluate inflammatory alterations. Formalin-fixed paraffin-embedded tissue sections (5 μm) were prepared for anti-mouse CD4, TRPV1, and TRPA1 immunohistochemistry.

      Quantitative real-time reverse transcriptase-PCR

      Dorsal skin samples or dorsal root ganglia were collected at various time points and were prepared for real-time reverse transcriptase-PCR mRNA profiling of inflammatory cytokine or Iba-1 gene expression as previously described (
      • Sághy É.
      • Sipos É.
      • Ács P.
      • Bölcskei K.
      • Pohóczky K.
      • Kemény Á.
      • et al.
      TRPA1 deficiency is protective in cuprizone-induced demyelination—a new target against oligodendrocyte apoptosis.
      ).

      Statistical analysis

      Results are expressed as mean ± standard error of the mean and analyzed statistically using an appropriate test. P < 0.05 accepted as significant.

      ORCID

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      This work was performed with the financial support of KTIA_NAP_13-1-2013-001; OTKA-NN-114458, GINOP-2.3.2-15-2016-00050 (SH, AK, EP, RG) and British Pharmacological Society’s AJ Clark Studentship (XK). RG was supported by the PTE ÁOK-KA-2015/11 grant. EP was supported by A2-SZJÖ-TOK-13-0149. TB and BIT were supported by ÚNKP-17-4 New National Excellence Program of the Ministry of Human Capacities and by Hungarian research grants NKFI K_16 120187. We thank Mária Reiszné Horváth for the professional technical assistance and Tamás Palkovics, who helped the PCR measurements. This work is dedicated to the 650th anniversary of University of Pécs.

      Supplementary Material

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