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Disinhibition of Touch-Evoked Itch in a Mouse Model of Psoriasis

Open ArchivePublished:December 17, 2018DOI:https://doi.org/10.1016/j.jid.2018.12.001

      Abbreviations:

      IMQ (imiquimod), LTMR (low-threshold mechanoreceptors), NFH (neurofilament H), QX-314 (N-ethyl-lidocaine), TLR5 (toll-like receptor 5), VH (vehicle)
      To the Editor
      Alloknesis, itch due to light mechanical stimulation, is frequently associated with dry skin and inflammatory skin disorders (e.g., atopic dermatitis, psoriasis). Recent studies have shown that mechanical itch is regulated by neuropeptide Y-positive spinal inhibitory interneurons that are innervated by low-threshold mechanoreceptors (LTMRs) (
      • Bourane S.
      • Duan B.
      • Koch S.C.
      • Dalet A.
      • Britz O.
      • Garcia-Campmany L.
      • et al.
      Gate control of mechanical itch by a subpopulation of spinal cord interneurons.
      ). LTMRs are divided into subtypes based on their action potential conduction velocities and their rates of adaptation to sustained mechanical stimulus (
      • Woo S.H.
      • Lumpkin E.A.
      • Patapoutian A.
      Merkel cells and neurons keep in touch.
      ,
      • Zimmerman A.
      • Bai L.
      • Ginty D.D.
      The gentle touch receptors of mammalian skin.
      ). In hairy skin, Aβ-slowly adapting type Ι LTMRs and Merkel cells compose touch domes to detect skin identification. In dry skin and aged skin, a reduction of the sustained firing of Aβ-slowly adapting type Ι LTMRs due to a loss of Merkel cells relates to alloknesis (
      • Feng J.
      • Luo J.
      • Yang P.
      • Du J.
      • Kim B.S.
      • Hu H.
      Piezo2 channel-Merkel cell signaling modulates the conversion of touch to itch.
      ). We investigated whether the number of Merkel cells or innervation of Merkel cells by Aβ-fibers is decreased in the imiquimod (IMQ)-induced psoriatic mouse model. In addition, we tested whether the targeted silencing of Aβ-fibers is sufficient to produce alloknesis.
      To develop the mouse model for psoriasis, IMQ cream was applied on the rostral back skin for 7 consecutive days. For further information on materials and methods, see the Supplementary Materials online. All experimental procedures were approved by the University of Miami Institutional Animal Care and Use Committee. In naïve mice, myelinated A-fibers labeled with neurofilament H (NFH) contacted keratin 8-positive Merkel cells in the epidermis (Figure 1a). In IMQ-treated mice, the alloknesis score was markedly increased compared with vehicle (VH)-treated mice (control) (Figure 1b). Although the loss of Merkel cells in aged skin and dry skin was reported (
      • Feng J.
      • Luo J.
      • Yang P.
      • Du J.
      • Kim B.S.
      • Hu H.
      Piezo2 channel-Merkel cell signaling modulates the conversion of touch to itch.
      ), the number of keratin 8-positive Merkel cells was comparable between the IMQ and VH groups (Figure 1c and d). NFH-positive epidermal nerve fiber density was decreased in the IMQ group compared with the VH group (VH, 19.6 ± 0.9 nerve fiber counts/mm; IMQ, 9.7 ± 2.1 counts/mm) (Figure 1e and f).
      Figure thumbnail gr1
      Figure 1Decrease in epidermal innervation density of myelinated nerve endings in imiquimod-induced psoriatic mouse model. The skin was dissected from mice (a) untreated or (c–f) after 7 days of vehicle or imiquimod treatment. (a) Skin sections were immunostained with an antibody for NFH (red) and K8 (green) to visualize myelinated nerve fibers and Merkel cells, respectively. The scale bar indicates 10 μm. (b) Alloknesis score was measured in mice treated with VH or IMQ for 7 days. Error bars show standard error of the mean (n = 7). (c) Typical examples of K8 expression in the skin treated with VH (left) or IMQ (right) for 7 days. Scale bar = 10 μm. (d) The number of K8-positive Merkel cells in the skin treated with VH or IMQ for 7 days. Error bars show standard error of the mean (n = 5). (e) Typical examples of NFH expression in the skin treated with VH (left) or IMQ (right) for 7 days. Scale bar = 20 μm. (f) NFH-positive epidermal nerve fiber density was quantified in the skin treated with VH (open bar) or IMQ (blue bar) for 7 days. Error bars show standard error of the mean (n = 4–5). P < 0.05, significant difference from VH, unpaired t test. CNT, control; ENFD, epidermal nerve fiber density; IMQ, imiquimod; K8, keratin 8; NFH, neurofilament H; VH, vehicle.
      Toll-like receptor 5 (TLR5) was recently proposed as a marker for Aβ-fibers (
      • Xu Z.Z.
      • Kim Y.H.
      • Bang S.
      • Zhang Y.
      • Berta T.
      • Wang F.
      • et al.
      Inhibition of mechanical allodynia in neuropathic pain by TLR5-mediated A-fiber blockade.
      ). We observed TLR5 expression in the majority of NFH-positive fibers in the rostral back skin (Figure 2a). Moreover, TLR5 is expressed by NFH-positive fibers innervating the touch dome (see Supplementary Figure S1 online). TLR5-positive epidermal nerve fiber density was decreased in the IMQ group compared with the VH group (VH, 16.8 ± 0.8 nerve fiber counts/mm; IMQ, 8.4 ± 1.2 counts/mm) (Figure 2b). To directly test the contribution of myelinated Aβ-fibers in regulating alloknesis, we used a strategy of the activity-dependent silencing of Aβ-fibers by targeted delivery of a membrane-impermeable lidocaine derivative, N-ethyl-lidocaine (QX-314) (
      • Binshtok A.M.
      • Bean B.P.
      • Woolf C.J.
      Inhibition of nociceptors by TRPV1-mediated entry of impermeant sodium channel blockers.
      ,
      • Roberson D.P.
      • Gudes S.
      • Sprague J.M.
      • Patoski H.A.
      • Robson V.K.
      • Blasl F.
      • et al.
      Activity-dependent silencing reveals functionally distinct itch-generating sensory neurons.
      ,
      • Xu Z.Z.
      • Kim Y.H.
      • Bang S.
      • Zhang Y.
      • Berta T.
      • Wang F.
      • et al.
      Inhibition of mechanical allodynia in neuropathic pain by TLR5-mediated A-fiber blockade.
      ). Activation of TLR5 with its ligand flagellin leads to selective QX-314 entry into Aβ-fibers and subsequent blockade of sodium currents in these fibers (
      • Xu Z.Z.
      • Kim Y.H.
      • Bang S.
      • Zhang Y.
      • Berta T.
      • Wang F.
      • et al.
      Inhibition of mechanical allodynia in neuropathic pain by TLR5-mediated A-fiber blockade.
      ). In line with this finding, co-injection of QX-314 and flagellin abolished responses of low-threshold mechanosensitive fibers to brush stimuli without affecting responses of high-threshold mechanosensitive fibers to pinch stimuli (see Supplementary Figure S2 online). Given the decrease in TLR5-positive epidermal nerve fiber density in the mouse model of IMQ-induced psoriasis, we hypothesized that inhibition of TLR5-positive nerves induces a mechanical itch phenotype resembling the alloknesis seen in the psoriasis mice. Intradermal co-application of QX-314 and flagellin produced touch-evoked scratching (Figure 2c). The alloknesis score became increased at 15 minutes, peaked at 0.5–1 hours, was maintained at 3 hours, and recovered at 24 hours. Intradermal injection of either flagellin or QX-314 alone did not induce mechanical itch phenotype in naïve mice (Figure 2c). Touch-evoked scratching was observed only when the mechanical stimulus was delivered to skin adjacent to a site of injection but not skin in the center of the injection site. Consistently, it was previously reported that touch-evoked scratching was elicited only by stimulation at the skin adjacent to a site of acute itch or the border of the dry skin-treated area on the rostral back (
      • Akiyama T.
      • Carstens M.I.
      • Ikoma A.
      • Cevikbas F.
      • Steinhoff M.
      • Carstens E.
      Mouse model of touch-evoked itch (alloknesis).
      ,
      • Iwanaga T.
      • Tominaga M.
      • Hirata Y.
      • Matsuda H.
      • Shimanuki T.
      • Ogawa H.
      • et al.
      Effects of film dressings on itch hypersensitivity using murine dry skin models.
      ). Therefore, it appears that, although Aβ-fibers innervating the chemical-injected site or the skin within the treatment area drive a circuit for antimechanical itch, a subset of LTMRs innervating the adjacent skin mediates touch-evoked itch.
      Figure thumbnail gr2
      Figure 2Targeted silencing of Aβ-fibers produces alloknesis. (a) Skin sections were immunostained with an antibody for NFH (green) and TLR5 (red). Scale bar = 10 μm. (b) TLR5-positive epidermal nerve fiber density was quantified in the skin treated with vehicle (open bar) or imiquimod (blue bar) for 7 days. Error bars show standard error of the mean (n = 4–5). P < 0.05, significant difference from VH, unpaired t test. (c) Time-dependent changes in alloknesis score after intradermal injection of either QX-314 (white square), flagellin (black triangles), or QX + FL (blue dots). Error bars show standard error of the mean (n = 6). P < 0.05, F(5,30) = 26.6, significant difference from time 0, one-way analysis of variance followed by Bonferroni test. ENFD, epidermal nerve fiber density; FL, flagellin; h, hour; IMQ, imiquimod; NFH, neurofilament H; QX, QX-314; TLR5, toll-like receptor 5; VH, vehicle.
      In summary, we show that alloknesis in an IMQ-induced psoriatic mouse model is associated with a loss of the epidermal nerve endings of Aβ-fibers but not Merkel cells. By contrast, alloknesis in dry skin and aged skin is associated with a loss of Merkel cells (
      • Feng J.
      • Luo J.
      • Yang P.
      • Du J.
      • Kim B.S.
      • Hu H.
      Piezo2 channel-Merkel cell signaling modulates the conversion of touch to itch.
      ). Considering that the Merkel cells are innervated by Aβ-fibers, the loss of sustained discharge of Aβ-fibers from Merkel cells is essential for alloknesis. Aβ-fibers in hairy skin likely innervate neuropeptide Y-positive inhibitory interneurons in the spinal cord to gate mechanical itch in normal conditions (
      • Bourane S.
      • Duan B.
      • Koch S.C.
      • Dalet A.
      • Britz O.
      • Garcia-Campmany L.
      • et al.
      Gate control of mechanical itch by a subpopulation of spinal cord interneurons.
      ).
      Our findings suggest that reactivation of Aβ-fibers can reduce alloknesis under chronic itch condition. High-frequency transcutaneous electrical nerve stimulation activates Aβ-fibers and temporarily relives generalized itch (
      • Wallengren J.
      Cutaneous nerve stiumulation in treatment of localized itch.
      ). Additionally, massage therapy works for certain types of itch (
      • Gürol A.P.
      • Polat S.
      • Akçay M.N.
      Itching, pain, and anxiety levels are reduced with massage therapy in burned adolescents.
      ,
      • Schachner L.
      • Field T.
      • Hernandez-Reif M.
      • Duarte A.M.
      • Krasnegor J.
      Atopic dermatitis symptoms decreased in children following massage therapy.
      ). Although antipruritic effects of those therapies are expected to be temporary, the approach to regain Aβ-fibers could be considered as a novel strategy for treating chronic itch.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      This work was supported by a National Institutes of Health grant ( AR063228 to TA).

      Supplementary Material

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