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Mechanisms of Itch in Stasis Dermatitis: Significant Role of IL-31 from Macrophages

  • Author Footnotes
    3 These authors contributed equally to this work
    Takashi Hashimoto
    Correspondence
    Takashi Hashimoto, Department of Dermatology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.
    Footnotes
    3 These authors contributed equally to this work
    Affiliations
    Miami Itch Center, Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, Miller School of Medicine, University of Miami, Miami, Florida, USA

    Department of Dermatology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
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  • Christina Dorothy Kursewicz
    Affiliations
    Miami Itch Center, Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, Miller School of Medicine, University of Miami, Miami, Florida, USA
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  • Rachel Alison Fayne
    Affiliations
    Miami Itch Center, Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, Miller School of Medicine, University of Miami, Miami, Florida, USA
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  • Sonali Nanda
    Affiliations
    Miami Itch Center, Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, Miller School of Medicine, University of Miami, Miami, Florida, USA
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  • Serena Maya Shah
    Affiliations
    Miami Itch Center, Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, Miller School of Medicine, University of Miami, Miami, Florida, USA
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  • Leigh Nattkemper
    Affiliations
    Miami Itch Center, Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, Miller School of Medicine, University of Miami, Miami, Florida, USA
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  • Hiroo Yokozeki
    Affiliations
    Department of Dermatology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
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  • Author Footnotes
    3 These authors contributed equally to this work
    Gil Yosipovitch
    Correspondence
    Correspondence: Gil Yosipovitch, Miami Itch Center, Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, Miller School of Medicine, University of Miami, 1600 NW 10th Ave, RMSB 2067B, Miami, Florida 33136.
    Footnotes
    3 These authors contributed equally to this work
    Affiliations
    Miami Itch Center, Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, Miller School of Medicine, University of Miami, Miami, Florida, USA
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  • Author Footnotes
    3 These authors contributed equally to this work
Open ArchivePublished:October 15, 2019DOI:https://doi.org/10.1016/j.jid.2019.09.012
      Stasis dermatitis (SD) is a common disease in the elderly population, with pruritus being one of the troublesome symptoms. However, there are few therapeutic modalities available for SD-associated itch because little is known about its pathophysiological mechanism. Therefore, we sought to investigate the mediators of itch in SD using an immunofluorescence study on patient lesions focusing on IL-31. Ex vivo stimulation studies using murine peritoneal macrophages were also used to elucidate the pathological mechanisms of the generation of IL-31. In SD lesions, dermal infiltrating IL-31(+) cells were increased in number compared with the healthy controls, and the majority of IL-31(+) cells were CD68(+) macrophages. The presence of itch in SD was significantly associated with the amount of CD68(+)/IL-31(+) macrophages and CD68(+)/CD163(+) M2 macrophages. The number of CD68(+)/IL-31(+) macrophages was correlated with the number of dermal C-C chemokine receptor type 4(+) T helper type 2 cells, IL-17(+) cells, basophils, substance P(+) cells, and dermal deposition of periostin and hemosiderin. Furthermore, murine peritoneal macrophages expressed an M2 marker arginase-1 and generated IL-31 when stimulated with a combination of substance P, periostin, and red blood cell lysate (representing hemosiderin). IL-31 from macrophages may play a role in itch in SD.

      Graphical abstract

      Abbreviations:

      Ab (antibody), IENF (intraepidermal nerve fiber), NK1R (neurokinin-1 receptor), OSMRβ (oncostatin M receptor β), pM (peritoneal macrophages), RBC (red blood cell), SD (stasis dermatitis), SP (Substance P), Th (T helper type)

      Introduction

      Stasis dermatitis (SD) is a common disease that predominantly affects the lower legs of elderly patients, with a prevalence of 6.2% among those older than 65 years (
      • Yalçin B.
      • Tamer E.
      • Toy G.G.
      • Öztaş P.
      • Hayran M.
      • Alli N.
      The prevalence of skin diseases in the elderly: analysis of 4099 geriatric patients.
      ). The main causes of SD are chronic venous insufficiency and venous hypertension. Venous hypertension promotes the cellular accumulation of inflammatory cells (e.g., T cells and macrophages) and extravasation of red blood cells (RBCs) in the affected skin (
      • Saharay M.
      • Shields D.A.
      • Porter J.B.
      • Scurr J.H.
      • Coleridge Smith P.D.
      Leukocyte activity in the microcirculation of the leg in patients with chronic venous disease.
      ,
      • Thomas P.R.
      • Nash G.B.
      • Dormandy J.A.
      White cell accumulation in dependent legs of patients with venous hypertension: a possible mechanism for trophic changes in the skin.
      ). Extravasation and disruption of the RBCs are followed by decomposition of hemoglobin, which results in excessive tissue iron stored as hemosiderin (
      • Caggiati A.
      • Rosi C.
      • Casini A.
      • Cirenza M.
      • Petrozza V.
      • Acconcia M.C.
      • et al.
      Skin iron deposition characterises lipodermatosclerosis and leg ulcer.
      ). Hemoglobin and hemosiderin can further induce monocyte/macrophage-recruitment through hemoglobin scavenger receptor CD163 (
      • Rubio-Navarro A.
      • Amaro Villalobos J.M.
      • Lindholt J.S.
      • Buendía I.
      • Egido J.
      • Blanco-Colio L.M.
      • et al.
      Hemoglobin induces monocyte recruitment and CD163-macrophage polarization in abdominal aortic aneurysm.
      ). Accumulating macrophages and other cells promote inflammation and induce the abnormal histological features of SD (e.g., epidermal spongiotic changes, papillary structure alternation, and capillary proliferation) partially through the secretion of proteolytic enzymes (
      • Sundaresan S.
      • Migden M.R.
      • Silapunt S.
      Stasis dermatitis: pathophysiology, evaluation, and management.
      ,
      • Wenk J.
      • Foitzik A.
      • Achterberg V.
      • Sabiwalsky A.
      • Dissemond J.
      • Meewes C.
      • et al.
      Selective pick-up of increased iron by deferoxamine-coupled cellulose abrogates the iron-driven induction of matrix-degrading metalloproteinase 1 and lipid peroxidation in human dermal fibroblasts in vitro: a new dressing concept.
      ).
      One of the troublesome symptoms reported in SD is pruritus. In an elderly population, the dermatosis that was most commonly reported to cause the complaint of itch was SD (
      • Valdes-Rodriguez R.
      • Mollanazar N.K.
      • González-Muro J.
      • Nattkemper L.
      • Torres-Alvarez B.
      • López-Esqueda F.J.
      • et al.
      Itch prevalence and characteristics in a Hispanic Geriatric population: A comprehensive study using a standardized itch questionnaire.
      ). Itch in SD not only impairs the patients’ quality of life but also induces scratching, which aggravates wounds and increases the risk of skin infection. Unfortunately, there are few therapeutic modalities available for itch because little is known about the pathophysiological mechanisms of SD-associated itch.
      The phenomenon of itch is divided into two subgroups: histaminergic and non-histaminergic (
      • Yosipovitch G.
      • Rosen J.D.
      • Hashimoto T.
      Itch: From mechanism to (novel) therapeutic approaches.
      ). Itch in SD appears to be non-histaminergic, as antihistamines do not necessarily improve SD-associated itch. Non-histaminergic itch involves various itch mediators, including cytokines/chemokines (e.g., interleukin [IL]-31), amines, proteases and their associated receptors (e.g., protease-activated receptor-2), neuropeptides and receptors (e.g., substance P [SP] and its receptor neurokinin-1 receptor [NK1R]), ion channels (e.g., transient receptor potential ankyin-1 and vanilloid-1), and immune cells (e.g., T cells, mast cells, eosinophils, and basophils).
      IL-31, a T helper type (Th)2-related pruritogenic cytokine, has recently gained attention as a potential therapeutic target for inflammatory skin conditions with itch (
      • Furue M.
      • Yamamura K.
      • Kido-Nakahara M.
      • Nakahara T.
      • Fukui Y.
      Emerging role of interleukin-31 and interleukin-31 receptor in pruritus in atopic dermatitis.
      ). IL-31 is involved in itch in various diseases, such as atopic dermatitis (
      • Nattkemper L.A.
      • Tey H.L.
      • Valdes-Rodriguez R.
      • Lee H.
      • Mollanazar N.K.
      • Albornoz C.
      • et al.
      The genetics of chronic itch: gene expression in the skin of patients with atopic dermatitis and psoriasis with severe itch.
      ,
      • Sonkoly E.
      • Muller A.
      • Lauerma A.I.
      • Pivarcsi A.
      • Soto H.
      • Kemeny L.
      • et al.
      IL-31: A new link between T cells and pruritus in atopic skin inflammation.
      ), prurigo nodularis (
      • Sonkoly E.
      • Muller A.
      • Lauerma A.I.
      • Pivarcsi A.
      • Soto H.
      • Kemeny L.
      • et al.
      IL-31: A new link between T cells and pruritus in atopic skin inflammation.
      ), psoriasis (
      • Nattkemper L.A.
      • Tey H.L.
      • Valdes-Rodriguez R.
      • Lee H.
      • Mollanazar N.K.
      • Albornoz C.
      • et al.
      The genetics of chronic itch: gene expression in the skin of patients with atopic dermatitis and psoriasis with severe itch.
      ), and cutaneous T-cell lymphoma (
      • Nattkemper L.A.
      • Martinez-Escala M.E.
      • Gelman A.B.
      • Singer E.M.
      • Rook A.H.
      • Guitart J.
      • et al.
      Cutaneous T-cell lymphoma and pruritus: the expression of IL-31 and its receptors in the skin.
      ). IL-31 exerts its function through its receptor complex, comprising IL-31RA and oncostatin M receptor β (OSMRβ) (
      • Sonkoly E.
      • Muller A.
      • Lauerma A.I.
      • Pivarcsi A.
      • Soto H.
      • Kemeny L.
      • et al.
      IL-31: A new link between T cells and pruritus in atopic skin inflammation.
      ). Blocking IL-31RA has been shown to improve itch in atopic dermatitis (
      • Kabashima K.
      • Furue M.
      • Hanifin J.M.
      • Pulka G.
      • Wollenberg A.
      • Galus R.
      • et al.
      Nemolizumab in patients with moderate-to-severe atopic dermatitis: randomized, phase II, long-term extension study.
      ). However, the involvement of IL-31 and its receptor complex in SD-associated itch is unknown.
      Thus, we sought to investigate the pathophysiological mechanisms of SD-associated itch focusing on IL-31 and other major itch mediators through an immunofluorescence study of patient lesions. In addition, we sought to elucidate the pathological mechanisms of IL-31 generation through ex vivo stimulation studies using murine peritoneal macrophages.

      Results

       CD68(+)/CD163(+) macrophages express IL-31 and correlate with the presence of itch in SD

      In SD lesions, the number of dermal infiltrating IL-31(+) cells was significantly increased than in healthy subjects (t-test, P = 0.001), while epidermal expression was not (t-test, P = 0.80). In addition, SD with severe itch, which was defined as a level 4 itch using the Likert scale from zero (no itch) to 4 (severe itch), showed greater infiltration of IL-31(+) cells in the dermis compared to SD without severe itch, but no statistical significance was found (t-test, P = 0.16) (Figure 1a).
      Figure thumbnail gr1
      Figure 1CD68(+) macrophages express IL-31 and correlate with itch in stasis dermatitis. Representative images of SD lesions and healthy skin with quantification of staining. (a) Epidermal expression of IL-31 was not enhanced in SD lesions. The number of IL-31(+) cells and CD68(+) macrophages was increased in SD lesions. (b) The majority of IL-31(+) cells expressed CD68. The number of CD68(+)/IL-31(+) cells was increased in SD lesions and correlated with itch. (c) In SD with severe itch, CD68(+) macrophages expressed an M2 macrophage marker CD163. The number of CD68(+)/CD163(+) cells was increased in SD lesions and correlated with itch. *P < 0.05, unpaired t-test. Dotted lines indicate the dermo-epidermal junction. Vertical bars indicate the standard deviation. AU, arbitrary unit; NS, not significant; SD, statis dermatitis; w/, with; w/o, without. Bar = 100 μm.
      Next, we investigated the cellular sources of IL-31. We detected a massive dermal infiltrate comprising various types of immune cells (Figures 1a). Most common among them were CD68(+) macrophages, with the number of these cells significantly increased in SD lesions than healthy controls (t-test, P = 0.0016) (Figure 1a). Therefore, we then examined IL-31 expression by macrophages. As expected, the majority of IL-31(+) cells were CD68(+) cells. In addition, the number of IL-31(+)/CD68(+) cells was significantly associated with the presence of severe itch, whereas the number of CD68(+) cells was approximately the same in patients with and without severe itch (average ratio of IL-31(+)/CD68(+) cells to CD68(+) cells was 88.2% in patients with severe itch vs 46.5% in patients without severe itch; t-test, P = 0.0096) (Figure 1b).
      Macrophages are generally divided into two groups: M1 and M2 (
      • Wang N.
      • Liang H.
      • Zen K.
      Molecular mechanisms that influence the macrophage m1-m2 polarization balance.
      ). Our group previously reported that M2 macrophages are capable of generating IL-31 (
      • Hashimoto T.
      • Satoh T.
      • Yokozeki H.
      Pruritus in ordinary scabies: il-31 from macrophages induced by overexpression of thymic stromal lymphopoietin and periostin.
      ). Thus, we examined the expression of an M2 marker, CD163, by CD68(+) cells. The number of CD163(+)/CD68(+) cells was increased in SD with severe itch compared with SD without severe itch (t-test, P = 0.004) (Figure 1c).

       Lesional expression of IL-31RA and OSMRβ

      Both the epidermal and dermal expression of IL-31RA was increased in SD lesions compared with healthy controls (t-test, P < 0.0005 and P = 0.005, respectively) but were not associated with the presence of severe itch (t-test, P = 0.74 and 0.58, respectively). OSMRβ expression in the epidermis and the dermis was not enhanced in SD lesions compared with healthy skin (t-test, P = 0.95 and 0.086, respectively) and was not associated with the presence of severe itch (t-test, P = 0.18 and 0.78, respectively) (Figure 2).
      Figure thumbnail gr2
      Figure 2Expression of IL-31 receptor complex components, IL-31RA and OSMRβ. Representative images of SD lesions and healthy skin with quantification of staining. Both epidermal and dermal expression of IL-31RA was enhanced in the SD lesions, but did not correlate with itch. The expression of OSMRβ in the epidermis and dermis was not increased in the SD lesions and did not correlate with itch. *P < 0.05, unpaired t-test. Dotted lines indicate the dermo-epidermal junction. Vertical bars indicate the standard deviation. AU, arbitrary unit; NS, not significant; OSMRβ, oncostatin M receptor; w/, with; w/o, without. Bar = 100 μm.

       Th2 and Th17 immune responses are predominant in SD lesions and correlated with IL-31 expression by macrophages

      M2-macrophage skewing is closely related with Th2 immunity (
      • Wang N.
      • Liang H.
      • Zen K.
      Molecular mechanisms that influence the macrophage m1-m2 polarization balance.
      ). The number of dermal cells expressing C-C chemokine receptor type 4, which is preferentially expressed by Th2 cells, increased in SD lesions compared with healthy controls (t-test, P = 0.0026) and correlated with the number of CD68(+)/IL-31(+) cells (Spearman’s correlation, r = 0.453, P = 0.020). The number of IL-17(+) cells was also increased in the SD lesions compared with healthy skin (t-test, P = 0.0045) and correlated with the number of CD68(+)/IL-31(+) cells (Spearman’s correlation, r = 0.405, P = 0.040). In contrast, the number of cells that expressed C-X-C chemokine receptor type 3, a preferential marker for Th1 cells, did not change between SD and the healthy controls (t-test, P = 0.698) and was not correlated with the number of CD68(+)/IL-31(+) cells (Spearman’s correlation, r = 0.326, P = 0.104), even though there was a difference in the number of C-X-C chemokine receptor type 3(+) cells between the SD lesions with severe itch and those without severe itch (t-test, P = 0.036).
      Basophils and eosinophils are involved in Th2 immunity (
      • Hashimoto T.
      • Satoh T.
      Immunological Perspectives: Th2 Cells/Mast Cells/Basophils/Eosinophils.
      ). The number of dermal basophils was increased in SD lesions compared with healthy controls (t-test, P <0.0005) and correlated with the number of CD68(+)/IL-31(+) cells (Spearman’s correlation, r = 0.603, P = 0.001). The number of dermal eosinophils was neither increased in SD compared with healthy controls (t-test, P = 0.106) nor correlated with the number of CD68(+)/IL-31(+) cells (Spearman’s correlation, r = 0.145, P = 0.481) (Figure 3).
      Figure thumbnail gr3
      Figure 3Infiltration of immune cells in stasis dermatitis. Representative images of SD lesions and healthy skin with quantification of staining. The number of C-X-C chemokine receptor type 3 (CXCR3)(+) cells was not increased in the SD lesions and did not correlate with the number of CD68(+)/IL-31(+) cells. In contrast, the number of C-C chemokine receptor type 4 (CCR4)(+) cells and IL-17(+) cells was increased in the SD lesions and correlated with the number of CD68(+)/IL-31(+) cells. The number of dermal basophils, but not dermal eosinophils, was increased in the SD lesions and correlated with the number of CD68(+)/IL-31(+) cells. *P < 0.05, unpaired t-test. Vertical bars indicate the standard deviation. CXCR3, C-X-C chemokine receptor type 3; NS, not significant; r, Spearman’s rank correlation coefficient; SD, statis dermatitis; w/, with; w/o, without. Bar = 100 μm.

       Periostin, substance P, and hemosiderin, but not thymic stromal lymphopoietin, are correlated with IL-31 expression by macrophages

      Thymic stromal lymphopoietin and periostin promote M2 skewing (
      • Furudate S.
      • Fujimura T.
      • Kakizaki A.
      • Kambayashi Y.
      • Asano M.
      • Watabe A.
      • et al.
      The possible interaction between periostin expressed by cancer stroma and tumor-associated macrophages in developing mycosis fungoides.
      ,
      • Han H.
      • Headley M.B.
      • Xu W.
      • Comeau M.R.
      • Zhou B.
      • Ziegler S.F.
      Thymic stromal lymphopoietin amplifies the differentiation of alternatively activated macrophages.
      ) and IL-31 generation from M2 macrophages (
      • Hashimoto T.
      • Satoh T.
      • Yokozeki H.
      Pruritus in ordinary scabies: il-31 from macrophages induced by overexpression of thymic stromal lymphopoietin and periostin.
      ). Epidermal expression of thymic stromal lymphopoietin was not enhanced in the SD lesions compared with healthy controls (t-test, P = 0.182). It was also not associated with the presence of severe itch (t-test, P = 0.355) or with the number of CD68(+)/IL-31(+) cells (Spearman’s correlation, r = 0.213, P = 0.295). In contrast, the dermal deposition of perisotin was increased in SD lesions compared with healthy controls (t-test, P <0.001) and correlated with the number of CD68(+)/IL-31(+) cells (Spearman’s correlation, r = 0.552, P = 0.003) but was not directly related to the presence of severe itch (t-test, P = 0.803).
      SP and hemosiderin are also capable of promoting M2 skewing (
      • Leal E.C.
      • Carvalho E.
      • Tellechea A.
      • Kafanas A.
      • Tecilazich F.
      • Kearney C.
      • et al.
      Substance P promotes wound healing in diabetes by modulating inflammation and macrophage phenotype.
      ,
      • Lim J.E.
      • Chung E.
      • Son Y.
      A neuropeptide, substance-P, directly induces tissue-repairing M2 like macrophages by activating the PI3K/Akt/mTOR pathway even in the presence of IFNγ.
      ,
      • Rubio-Navarro A.
      • Amaro Villalobos J.M.
      • Lindholt J.S.
      • Buendía I.
      • Egido J.
      • Blanco-Colio L.M.
      • et al.
      Hemoglobin induces monocyte recruitment and CD163-macrophage polarization in abdominal aortic aneurysm.
      ). The number of dermal SP(+) cells and dermal deposition of hemosiderin were increased in the SD lesions compared with healthy controls (t-test, P <0.001 and P = 0.005, respectively) and correlated with the number of CD68(+)/IL-31(+) cells (Spearman’s correlation, r = 0.463, P = 0.017 and r = 0.506, P = 0.008, respectively) but was not related to the presence of severe itch (t-test, P = 0.802 and 0.859, respectively) (Figure 4).
      Figure thumbnail gr4
      Figure 4Periostin, hemosiderin, and substance P, but not TSLP, are correlated with the presence of IL-31(+) macrophages. Representative images of SD lesion and healthy skin with the quantification of staining. The epidermal expression of TSLP was not enhanced in the SD lesions and did not correlate with the number of CD68(+)/IL-31(+) cells. The dermal deposition of both periostin and hemosiderin, as well as the number of substance P(+) cells, was increased in the SD lesions and correlated with CD68(+)/IL-31(+) cells. All the aforementioned factors were not significantly higher in SD with severe itch compared to SD without severe itch. Bar = 100 μm for TSLP and substance P. Bar = 1 mm for Periostin, and bar = 200 μm for hemosiderin. *P < 0.05, unpaired t-test. Dotted lines indicate the dermo-epidermal junction. Vertical bars indicate standard deviation. AU, arbitrary unit; NS, not significant; SD, statis dermatitis; TSLP, thymic stromal lymphopoietin; w/, with; w/o, without.

       CD68(+) macrophages express NK1R

      SP exerts its function mainly, but not exclusively, through its receptor NK1R (
      • Azimi E.
      • Reddy V.B.
      • Pereira P.J.S.
      • Talbot S.
      • Woolf C.J.
      • Lerner E.A.
      Substance P activates Mas-related G protein–coupled receptors to induce itch.
      ,
      • Ständer S.
      • Yosipovitch G.
      Substance P and neurokinin 1 receptor are new targets for the treatment of chronic pruritus.
      ). SP stimulates NK1R expressing cells, resulting in the release of additional itch mediators and evoking itch (
      • Yosipovitch G.
      • Rosen J.D.
      • Hashimoto T.
      Itch: From mechanism to (novel) therapeutic approaches.
      ). Epidermal and dermal expression of NK1R was enhanced in SD compared with healthy controls (t-test, P = 0.001 and P < 0.001, respectively) but was not associated with the presence of severe itch (t-test, P = 0.483 and 0.559, respectively) (Figure 5a). Of note, almost all the CD68(+) macrophages expressed NK1R (Figure 5b).
      Figure thumbnail gr5
      Figure 5CD68(+) macrophages express neurokinin-1 receptor. Representative images of SD lesions and healthy skin with quantification of staining. (a) Both epidermal expression of neurokinin-1 receptor (NK-1R) and the number of dermal NK-1R(+) cells were increased in SD lesions but did not correlate with severe itch. (b) CD68(+) cells expressed NK-1R. *P < 0.05, unpaired t-test. Dotted lines indicate the dermo-epidermal junction. Vertical bars indicate standard deviation. AU, arbitrary unit; NK-1R, neurokinin-1 receptor; NS, not significant; SD, statis dermatitis; w/, with; w/o, without. Bar = 100 μm.

       Skin innervation and other itch mediators

      Intraepidermal nerve fibers (IENF) are involved in itch (
      • Pereira M.P.
      • Mühl S.
      • Pogatzki-Zahn E.M.
      • Agelopoulos K.
      • Ständer S.
      Intraepidermal nerve fiber density: diagnostic and therapeutic relevance in the management of chronic pruritus: a review.
      ). We found reduced IENF density in the SD lesions compared with healthy controls (t-test, P = 0.010) but no association between IENF density and the presence of severe itch (t-test, P = 0.28).
      Mast cells can release various itch mediators, including histamine, prostaglandins, and proteases (
      • Steinhoff M.
      • Buddenkotte J.
      • Lerner E.A.
      Role of mast cells and basophils in pruritus.
      ). The number of dermal mast cells did not change between SD and healthy controls, or between SD with severe itch and SD without severe itch.
      Protease-activated receptor 2 is a representative receptor for non-histaminergic itch (
      • Steinhoff M.
      • Neisius U.
      • Ikoma A.
      • Fartasch M.
      • Heyer G.
      • Skov P.S.
      • et al.
      Proteinase-activated receptor-2 mediates itch: a novel pathway for pruritus in human skin.
      ). The epidermal expression of protease-activated receptor 2 was not enhanced in SD compared with healthy controls (t-test, P = 0.734) and was reduced in SD without severe itch compared to SD with severe itch (t-test, P = 0.022).
      Transient receptor potential ankyin-1 and transient receptor potential vanilloid-1 are ion channels that are involved in itch (
      • Kittaka H.
      • Tominaga M.
      The molecular and cellular mechanisms of itch and the involvement of TRP channels in the peripheral sensory nervous system and skin.
      ). The epidermal expression of transient receptor potential ankyin-1 and transient receptor potential vanilloid-1 was neither enhanced in SD compared with healthy controls (t-test, P = 0.071 and 0.283, respectively) nor related to the presence of severe itch (t-test, P = 0.375 and 0.139, respectively) (Supplementary Figure S1).

       Murine peritoneal macrophages generate IL-31 in response to the combination of substance P, periostin, and red blood cell lysate

      To elucidate the mechanisms of IL-31-generation from macrophages, we employed an ex vivo stimulation test with murine peritoneal macrophages (pM) (
      • Hashimoto T.
      • Satoh T.
      • Yokozeki H.
      Pruritus in ordinary scabies: il-31 from macrophages induced by overexpression of thymic stromal lymphopoietin and periostin.
      ). pM were stimulated with substance P, periostin, and/or RBC lysate, which represents hemosiderin. Stimulation with SP alone did not significantly increase IL-31 mRNA expression in pM (Supplementary Figure S2). In contrast, stimulation with periostin alone or periostin and SP significantly increased IL-31 mRNA expression. Of note, stimulation with the combination of SP, periostin, and RBC lysate dramatically enhanced IL-31 mRNA expression (Figure 6a). The protein expression of IL-31 was confirmed with flow cytometric analysis. Flow cytometric analysis also revealed that MOMA-2(+)/IL-31(+) pM express an M2 marker arginase-1, indicating that M2 macrophages generate IL-31 in response to a combination of SP, periostin and hemosiderin (Figure 6b, c).
      Figure thumbnail gr6
      Figure 6Murine peritoneal macrophages generate IL-31 in response to a combination of substance P, periostin, and RBC lysate ex vivo. Murine peritoneal macrophages were stimulated with substance P, periostin, and/or RBC lysate ex vivo for 24 hours. (a) IL-31 mRNA expression was significantly increased in response to periostin, substance P plus periostin, and a combination of substance P + periostin + RBC lysate. (b, c) Flow cytometric analysis of intracellular IL-31 and arginase-1 in MOMA-2 gated murine peritoneal macrophages without stimulation (medium-treated, non-stimulated macrophages) or in response to substance P plus periostin plus RBC lysate (stimulated macrophages). Representative results of at least two independent experiments are shown. Values represent the mean + SD. *P < 0.05, unpaired t-test, compared with medium-treated, non-stimulated macrophages. Arg-1, arginase-1; MFI, mean fluorescence intensity, MOMA, monocyte/macrophage; RBC, red blood cell.

      Discussion

      This data showed that dermal IL-31 appeared to play a significant role in itch in SD. Notably, the majority of IL-31-expressing cells were CD68(+) macrophages.
      It is generally established that IL-31 is generated mainly by activated Th2 cells (
      • Dillon S.R.
      • Sprecher C.
      • Hammond A.
      • Bilsborough J.
      • Rosenfeld-Franklin M.
      • Presnell S.R.
      • et al.
      Interleukin 31, a cytokine produced by activated T cells, induces dermatitis in mice.
      ,
      • Sonkoly E.
      • Muller A.
      • Lauerma A.I.
      • Pivarcsi A.
      • Soto H.
      • Kemeny L.
      • et al.
      IL-31: A new link between T cells and pruritus in atopic skin inflammation.
      ). Other cellular types are also capable of generating IL-31, including macrophages (
      • Cornelissen C.
      • Brans R.
      • Czaja K.
      • Skazik C.
      • Marquardt Y.
      • Zwadlo-Klarwasser G.
      • et al.
      Ultraviolet B radiation and reactive oxygen species modulate interleukin-31 expression in T lymphocytes, monocytes and dendritic cells.
      ), eosinophils (
      • Kunsleben N.
      • Rüdrich U.
      • Gehring M.
      • Novak N.
      • Kapp A.
      • Raap U.
      IL-31 induces chemotaxis, calcium mobilization, release of reactive oxygen species, and CCL26 in eosinophils, which are capable to release IL-31.
      ), mast cells (
      • Niyonsaba F.
      • Ushio H.
      • Hara M.
      • Yokoi H.
      • Tominaga M.
      • Takamori K.
      • et al.
      Antimicrobial peptides human beta-Defensins and cathelicidin LL-37 induce the secretion of a pruritogenic cytokine IL-31 by human mast cells.
      ), basophils (
      • Raap U.
      • Gehring M.
      • Kleiner S.
      • Rüdrich U.
      • Eiz-Vesper B.
      • Haas H.
      • et al.
      Human basophils are a source of - and are differentially activated by - IL-31.
      ), and keratinocytes (
      • Nattkemper L.A.
      • Martinez-Escala M.E.
      • Gelman A.B.
      • Singer E.M.
      • Rook A.H.
      • Guitart J.
      • et al.
      Cutaneous T-cell lymphoma and pruritus: the expression of IL-31 and its receptors in the skin.
      ). Among these, macrophages are reported to be the main cellular source in human skin lesions of scabies (
      • Hashimoto T.
      • Satoh T.
      • Yokozeki H.
      Pruritus in ordinary scabies: il-31 from macrophages induced by overexpression of thymic stromal lymphopoietin and periostin.
      ), polymorphic light eruption (
      • Patra V.
      • Strobl J.
      • Gruber-Wackernagel A.
      • Vieyra-Garcia P.
      • Stary G.
      • Wolf P.
      CD11b+ cells markedly express the itch cytokine interleukin 31 in polymorphic light eruption.
      ), and atopic dermatitis (
      • Kato A.
      • Fujii E.
      • Watanabe T.
      • Takashima Y.
      • Matsushita H.
      • Furuhashi T.
      • et al.
      Distribution of IL-31 and its receptor expressing cells in skin of atopic dermatitis.
      ).
      In SD lesions with severe itch, the majority of CD68(+)/IL-31(+) macrophages expressed CD163, indicating they were M2 macrophages. In SD lesions without severe itch, a smaller population of CD68(+) macrophages expressed CD163. These findings are congruent with our previous study in scabies lesions with severe itch, which showed that dermal accumulating M2 macrophages are the main cellular sources of IL-31 (
      • Hashimoto T.
      • Satoh T.
      • Yokozeki H.
      Pruritus in ordinary scabies: il-31 from macrophages induced by overexpression of thymic stromal lymphopoietin and periostin.
      ).
      M2-macrophage polarization is promoted by Th2 immunity (
      • Wang N.
      • Liang H.
      • Zen K.
      Molecular mechanisms that influence the macrophage m1-m2 polarization balance.
      ). We found that a significant number of C-C chemokine receptor type 4(+) Th2 cells and basophils infiltrated the SD lesions. The dermal deposition of periostin, a Th2-related protein (
      • Masuoka M.
      • Shiraishi H.
      • Ohta S.
      • Suzuki S.
      • Arima K.
      • Aoki S.
      • et al.
      Periostin promotes chronic allergic inflammation in response to Th2 cytokines.
      ), was also enhanced. These findings indicate Th2 immunity-predominance in the SD lesions. Even though these factors were not directly associated with the presence of itch, they were significantly correlated with the number of CD68(+)/IL-31(+) cells.
      Interestingly, the number of IL-17(+) cells was increased in the SD lesions, indicating that Th17 immunity is also predominant in SD. The number of IL-17(+) cells was not directly related to the presence of itch, but it was significantly correlated with the number of CD68(+)/IL-31(+) cells. IL-17 is reported to promote M2 macrophage skewing (
      • Nakai K.
      • He Y.Y.
      • Nishiyama F.
      • Naruse F.
      • Haba R.
      • Kushida Y.
      • et al.
      IL-17A induces heterogeneous macrophages, and it does not alter the effects of lipopolysaccharides on macrophage activation in the skin of mice.
      ), and M2 macrophages can promote Th17 cell-expansion (
      • Haribhai D.
      • Ziegelbauer J.
      • Jia S.
      • Upchurch K.
      • Yan K.
      • Schmitt E.G.
      • et al.
      Alternatively activated macrophages boost induced regulatory T and Th17 cell responses during immunotherapy for colitis.
      ,
      • Mao H.
      • Pan F.
      • Guo H.
      • Bu F.
      • Xin T.
      • Chen S.
      • et al.
      Feedback mechanisms between M2 macrophages and Th17 cells in colorectal cancer patients.
      ). Thus, Th17 immunity is likely to be linked to M2 macrophage skewing.
      The number of SP(+) cells and amount of dermal deposition of hemosiderin were not directly associated with itch, but were significantly correlated with the number of CD68(+)/IL-31(+) macrophages. Mechanisms of the overexpression of SP and periostin in SD are unclear. SP is expressed by peripheral nerve fibers and various cell types, including macrophages (
      • Marriott I.
      • Bost K.L.
      IL-4 and IFN-gamma up-regulate substance P receptor expression in murine peritoneal macrophages.
      ) and T cells (
      • Lai J.P.
      • Douglas S.D.
      • Ho W.Z.
      Human lymphocytes express substance P and its receptor.
      ). These cells are abundant in SD lesions and can be sources of SP.
      To confirm the ability of macrophages to generate IL-31, we conducted ex vivo stimulation studies with murine pM. We previously demonstrated that canonical Th2 cytokines IL-4 and IL-13 do not stimulate pM to secrete a significant amount of IL-31. Instead, periostin promotes M2 skewing and induces IL-31 generation from pM (
      • Hashimoto T.
      • Satoh T.
      • Yokozeki H.
      Pruritus in ordinary scabies: il-31 from macrophages induced by overexpression of thymic stromal lymphopoietin and periostin.
      ). The current study shows that IL-31 mRNA expression was dramatically increased in response to the combination of periostin, SP, and RBC lysate. In contrast, SP or RBC lysate alone did not increase expression. Of note, IL-31-producing macrophages expressed arginase-1, indicating that they were M2 macrophages. Both human and murine macrophages express the SP receptor NK1R, hemosiderin receptor CD163, and periostin receptor integrin αV (
      • Hashimoto T.
      • Satoh T.
      • Yokozeki H.
      Pruritus in ordinary scabies: il-31 from macrophages induced by overexpression of thymic stromal lymphopoietin and periostin.
      ,
      • Siebenhaar F.
      • Sharov A.A.
      • Peters E.M.J.
      • Sharova T.Y.
      • Syska W.
      • Mardaryev A.N.
      • et al.
      Substance P as an immunomodulatory neuropeptide in a mouse model for autoimmune hair loss (alopecia areata).
      ). Collectively, SP, hemosiderin, and periostin in SD lesions can be essentially involved in IL-31 generation from M2 macrophages. The SP/NK1R pathway can also be involved in the generation of IL-31 by other cell populations such as eosinophils (
      • Hashimoto T.
      • Kursewicz C.D.
      • Fayne R.A.
      • Nanda S.
      • Shah S.M.
      • Nattkemper L.
      • et al.
      Pathophysiological mechanisms of itch in bullous pemphigoid.
      ).
      We also investigated other itch mediators and IENF density in SD. Decreased IENF density is reported to be involved in chronic itch in many skin diseases, such as AD, psoriasis, and prurigo nodularis (
      • Schuhknecht B.
      • Marziniak M.
      • Wissel A.
      • Phan N.Q.
      • Pappai D.
      • Dangelmaier J.
      • et al.
      Reduced intraepidermal nerve fibre density in lesional and nonlesional prurigo nodularis skin as a potential sign of subclinical cutaneous neuropathy.
      ,
      • Tan Y.
      • Ng W.J.
      • Lee S.Z.X.
      • Lee B.T.K.
      • Nattkemper L.A.
      • Yosipovitch G.
      • et al.
      3-Dimensional optical clearing and imaging of pruritic atopic dermatitis and psoriasis skin reveals downregulation of epidermal innervation.
      ). IENF density was also reduced in SD lesions. No significant changes were observed in the number of mast cells and in the expression of protease-activated receptor 2, transient receptor potential ankyin-1, and transient receptor potential vanilloid-1 between SD and healthy controls. Therefore, we conclude that these factors do not significantly contribute to itch in SD.
      IL-31 directly stimulates peripheral nerve fibers through IL-31RA and OSMRβ (
      • Cevikbas F.
      • Wang X.
      • Akiyama T.
      • Kempkes C.
      • Savinko T.
      • Antal A.
      • et al.
      A sensory neuron-expressed IL-31 receptor mediates T helper cell-dependent itch: involvement of TRPV1 and TRPA1.
      ). In addition, IL-31 stimulates IL-31RA expressing cells, including macrophages, basophils, and keratinocytes, to enhance inflammation (
      • Nakashima C.
      • Otsuka A.
      • Kabashima K.
      Interleukin-31 and interleukin-31 receptor: new therapeutic targets for atopic dermatitis.
      ). Our study showed the overexpression of IL-31RA, but not OSMRβ, both in the epidermis and the dermis. Targeting IL-31 itself or IL-31RA may be beneficial in treating itch in SD. Macrophages could also be potential therapeutic targets, in addition to SP and NK1R, periostin, and hemosiderin. However, these targets require further consideration. In wound sites, M2 macrophages contribute to wound healing through the promotion of fibrosis and angiogenesis (
      • Kim S.Y.
      • Nair M.G.
      Macrophages in wound healing: activation and plasticity.
      ,
      • Snyder R.J.
      • Lantis J.
      • Kirsner R.S.
      • Shah V.
      • Molyneaux M.
      • Carter M.J.
      Macrophages: a review of their role in wound healing and their therapeutic use.
      ). SD is often accompanied by wound and chronic venous ulcers (
      • Sundaresan S.
      • Migden M.R.
      • Silapunt S.
      Stasis dermatitis: pathophysiology, evaluation, and management.
      ). The inhibition of M2-skewing might result in delayed wound healing. This point should be examined in future studies.
      The key limitation of this study was the lack of patients’ numerical rating scale scores for itch, and we were not able to calculate the correlation between protein expression and itch severity. However, this study provides insights into the pathophysiological mechanisms of itch in SD. IL-31 and IL-31RA can be useful therapeutic targets for itch in SD.

      Materials and Methods

       Samples

      Skin lesional biopsy specimens and clinical data were obtained from 20 patients with SD (age, 42-93 years old; 5 males and 15 females) and from six non-itchy healthy control subjects (age, 25-62; four males and two females) at Tokyo Medical and Dental University Hospital and University of Miami Hospital. Their diagnoses were confirmed based on clinical and histological findings. This study was approved by the ethical committees of Tokyo Medical and Dental University (#M2018-033; informed written consent was not required because the specimens were de-identified) and the University of Miami (no IRB number; informed written consent was not required as tissue was collected from a de-identified repository and considered non-human research). Based on the presence of itch according to their medical information, the subjects were divided into two groups: with severe itch (n=9; ages, 44-93; three males and six females) and without severe itch (n=11; ages, 42-85; two males and nine females). Severe itch was defined as scale 4 in the Likert scale from zero (no itch) to 4 (the worst), and without severe itch was defined as scales zero to three. All patients had no other pruritic skin disorders.

       Mice

      Seven-week-old female C57BL/6 mice were obtained from Sankyo Lab Service (Tokyo, Japan). The mice were maintained under specific-pathogen-free conditions in our animal facility. All animal experiments were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (Protocols A2018-299A, A2018-199A, A2018-298A, and A2018-237A) and performed at the Tokyo Medical and Dental University.

       Antibodies

      Antibodies (Abs) used in this study are listed in the Supplementary Material.

       Immunofluorescence and iron staining

      Formalin-fixed paraffin-embedded samples (5-μm) were deparaffinized and pretreated with DAKO target retrieval solution (Dako, Glostrup, Denmark) at 60 °C overnight. The slides were then treated with phosphate buffer solution with 5% normal donkey serum and 0.2% Triton X-100 for 2 hours at room temperature. Next, sections were incubated with primary Abs at 4 °C overnight followed by reaction with Alexa Fluor 488- or 594-conjugated secondary Abs (Molecular Probes, Eugene, OR). The samples were then mounted with Vectashield with DAPI (Vector Laboratories, Burlingame, CA). For hemosiderin detection, Prussian blue staining was used.

       Quantification

      Photomicrographs were captured with a CTR6000 microscope (Leica Microsystems, Wetzlar, Germany) at original magnification ×20, and three images from each subject were analyzed. For periostin detection, whole scanning images were analyzed. The epidermal expression and dermal periostin deposition were measured as fluorescence intensity in arbitrary units normalized by area and background fluorescence using Image J software (NIH, Bethesda, MD). The dermal deposition of hemosiderin was calculated by dividing Prussian blue stain-reactive area by dermal area in each photomicrograph. The number of dermal infiltrating cells was manually quantified. The IENF density was calculated by dividing the number of neuron specific marker β-tubulin III(+) nerves crossing the dermo-epidermal junction by the length of the epidermis (
      • Sanders K.M.
      • Nattkemper L.A.
      • Rosen J.D.
      • Andersen H.H.
      • Hsiang J.
      • Romanelli P.
      • et al.
      Non-histaminergic itch mediators elevated in the skin of a porcine model of scabies and of human scabies patients.
      ).

       Preparation of murine peritoneal macrophages

      Peritoneal cells were collected from C57BL/6N mice, seeded at the concentration of 5×105/well in plates in RPMI-1640 complete medium supplemented with 10% fetal bovine serum and 100 IU/mL penicillin-streptomycin and incubated for 2 hours at 37 °C and 5% CO2. The non-adherent cells were washed out, and the remaining adherent cells (>80% of macrophages) were incubated with or without substance P (1 μM; Peptide institute, Osaka, Japan), recombinant periostin (20 ng/mL; eBioscience, San Diego, CA), and/or RBC lysates (5% volume of total medium volume). After 24 hours, the cells were subjected to total RNA extraction or flow cytometric analysis. The RBC lysates were prepared as follows: murine whole blood was collected and centrifuged, and serum were removed. The blood was frosted and thawed twice.

       Real-time PCR

      Total cellular RNA was extracted from cells using ISOGEN II (Nippon Gene Co., Tokyo, Japan), reverse-transcribed with SuperScript IV VILO Master Mix (Thermo Fisher Scientific), and quantitative reverse transcriptase-PCR was performed by real-time monitoring of the increase in fluorescence of SYBR Green dye (Brilliant SYBR Green QPCR Master Mix; Agilent Technologies Japan, Ltd., Tokyo, Japan) using the AriaMx Real-Time PCR System (Agilent Technologies). The primers used for PCR were 5′-TCGGTCATCATAGCACATCTGGAG-3′ and 5′-GCACAGTCCCTTTGGAGTTAAGTC-3′ for mouse IL-31 and 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′ for mouse glyceraldehyde-3-phosphate dehydrogenase. The mRNA expression levels were calculated by the comparative relative fold gene method relative to glyceraldehyde-3-phosphate dehydrogenase.

       Flow cytometric analyses

      Single cell-suspensions of cultured pM were obtained using cell scrapers after fixation. They were pretreated with anti-CD16/32 Ab (BioLegend, San Diego, CA) and labeled with monocyte/macrophage-2 PE conjugated Ab (BioRad, Hercules, CA), anti-ariginase-1 Ab (Abcam plc, Cambridge, UK: ab92274), and anti-IL-31 Ab (abcam: ab102750) followed by reaction with Alexa Fluor 647 anti-goat IgG- and Alexa Fluor 488 anti-rabbit IgG- secondary Abs (Abcam plc, ab150131 and ab150061, respectively) using Intracellular Fix & Perm set (eBioscience). They were then analyzed with FACSCalibur cell analyzer (BD Biosciences, San Jose, CA).

       Statistical analysis

      All data are reported as the mean + Standard deviation. In order to compare the differences between two groups, two-tailed, unpaired t-tests were used. For detecting correlation, we calculated Spearman’s rank correlation coefficient (r) using a statistical software “EZR” (
      • Kanda Y.
      Investigation of the freely available easy-to-use software “EZR” for medical statistics.
      ). Statistical significance was set at P < 0.05.

       Data Availability Statement

      Datasets related to this article can be found at https://doi.org/10.17632/bmmkj2psfz.1, an open-source online data repository hosted at Mendeley Data.

      ORCIDs

      Christina Dorothy Kursewicz: https://orcid.org/0000-0001-5135-6865

      Conflict of Interest

      GY serves on the Scientific Boards of Menlo, Trevi, Sienna, Sanofi, Regeneron, Galderma, Pfizer, Novartis, Bayer, Kiniksa, Eli Lilly, and Ortho. Research support was provided by Pfizer, Sun Pharma, Leo, Menlo, and Kiniksa. The other authors state no conflicts of interest.

      Acknowledgments

      The authors thank Ms. Chiyako Miyagishi at Tokyo Medical and Dental University for technical assistance. This work was supported by a Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Young Scientists (B) ( #17K16328 ) and by an unrestricted fellowship grant from Menlo Therapeutics .

      Author Contributions

      Conceptualization: TH, GY. Data curation: TH. Formal analysis: TH. Funding acquisition: TH, HY, GY. Investigation, TH, CK, RF, SN, SS. Methodology: TH, LN. Project administration: TH, LN, HY, GY. Resources: TH, LN, HY, GY. Supervision: LN, HY, GY. Validation: TH, GY. Visualization: TH, GY. Writing – Original Draft Preparation: TH, Writing – Review and Editing: TH, CK, RF, SN, SS, LN, HY, and GY. All the authors have read the manuscript and have approved this submission.

      Supplementary Materials and Methods

       Antibodies

      Antibodies (Abs) obtained from Abcam plc (Cambridge, UK) included: Anti-mast cell tryptase (AAI; ab2378), IL-31 (ab102750), IL-31RA (ab113498), C-X-C chemokine receptor type 3 (ab64714), C-C chemokine receptor type 4 (ab1669), IL-17 (ab79056), CD68 (KP1; ab955), CD163 (ab182422), basophil (2D7; ab155577), Substance P (ab106291), thymic stromal lymphopoietin (ab188766), periostin (ab14041), transient receptor potential vanilloid 1 (ab3487), transient receptor potential ankyrin 1 (ab62053), and arginase-1 (ab92274) Abs. Anti-neurokinin 1 receptor (PA3-301), protease-activated receptor 2 (sc-5597), β-tubulin III (Tuj1; Mo15013), major basic protein (MBP) (NBP1-42140-1ml), oncostatin M receptor β (LS-B11477) Abs were obtained from ThermoFisher Scientifics (Waltham, MA), Santa Cruz Biotechnology (Dallas, TX), Neuromics (Edina, MN), Novus biologicals (Centennial, CO), and LifeSpan BioSciences (Seattle, WA), respectively. R-Phycoerythrin-conjugated anti-monocyte/macrophage-2 Ab (MCA519PE) was purchased from Bio Rad Laboratories (Hercules, CA). Alexa Fluor 647 anti-goat IgG- (ab150131) and Alexa Fluor 488 anti-rabbit IgG- (ab150061) secondary Abs were obtained from Abcam plc.
      Figure thumbnail fx2
      Supplementary Figure S1Epidermal nerve fibers, mast cells, PAR-2, and TRPA1 in lesional skin of stasis dermatitis. Representative images of SD lesions and healthy skin with the quantification of staining. Intraepidermal nerve fiber (IENF) density was reduced in the SD lesions but did not correlate with itch. The number of dermal mast cells was not increased and did not correlate with itch in the SD lesions. The epidermal expression of PAR-2, TRPA1, and TRPV1 was not enhanced in the SD lesions. Bar = 100 μm. Dotted lines indicate the dermo-epidermal junction. *P < 0.05, unpaired t-test. Vertical bars indicate standard deviation. AU, arbitrary unit; IENF, intraepidermal nerve fiber; NS, not significant; PAR-2, protease-activated receptor-2; SD, statis dermatitis; TRPA1, transient receptor potential ankyrin-1; TRPV1, transient receptor potential vanilloid-1; w/, with; w/o, without.
      Figure thumbnail fx3
      Supplementary Figure S2IL-31 mRNA expression of murine peritoneal macrophages in response to substance P. Representative results of two independent experiments are shown. Values represent mean + SD of three samples. NS, not significant, unpaired t-test, compared with non-treated macrophages.

      References

        • Azimi E.
        • Reddy V.B.
        • Pereira P.J.S.
        • Talbot S.
        • Woolf C.J.
        • Lerner E.A.
        Substance P activates Mas-related G protein–coupled receptors to induce itch.
        J Allergy Clin Immunol. 2017; 140: 447-453.e3
        • Caggiati A.
        • Rosi C.
        • Casini A.
        • Cirenza M.
        • Petrozza V.
        • Acconcia M.C.
        • et al.
        Skin iron deposition characterises lipodermatosclerosis and leg ulcer.
        Eur J Vasc Endovasc Surg. 2010; 40: 777-782
        • Cevikbas F.
        • Wang X.
        • Akiyama T.
        • Kempkes C.
        • Savinko T.
        • Antal A.
        • et al.
        A sensory neuron-expressed IL-31 receptor mediates T helper cell-dependent itch: involvement of TRPV1 and TRPA1.
        J Allergy Clin Immunol. 2014; 133: 448-460
        • Cornelissen C.
        • Brans R.
        • Czaja K.
        • Skazik C.
        • Marquardt Y.
        • Zwadlo-Klarwasser G.
        • et al.
        Ultraviolet B radiation and reactive oxygen species modulate interleukin-31 expression in T lymphocytes, monocytes and dendritic cells.
        Br J Dermatol. 2011; 165: 966-975
        • Dillon S.R.
        • Sprecher C.
        • Hammond A.
        • Bilsborough J.
        • Rosenfeld-Franklin M.
        • Presnell S.R.
        • et al.
        Interleukin 31, a cytokine produced by activated T cells, induces dermatitis in mice.
        Nat Immunol. 2004; 5: 752-760
        • Furudate S.
        • Fujimura T.
        • Kakizaki A.
        • Kambayashi Y.
        • Asano M.
        • Watabe A.
        • et al.
        The possible interaction between periostin expressed by cancer stroma and tumor-associated macrophages in developing mycosis fungoides.
        Exp Dermatol. 2016; 25: 107-112
        • Furue M.
        • Yamamura K.
        • Kido-Nakahara M.
        • Nakahara T.
        • Fukui Y.
        Emerging role of interleukin-31 and interleukin-31 receptor in pruritus in atopic dermatitis.
        Allergy. 2018; 73: 29-36
        • Han H.
        • Headley M.B.
        • Xu W.
        • Comeau M.R.
        • Zhou B.
        • Ziegler S.F.
        Thymic stromal lymphopoietin amplifies the differentiation of alternatively activated macrophages.
        J Immunol. 2013; 190: 904-912
        • Haribhai D.
        • Ziegelbauer J.
        • Jia S.
        • Upchurch K.
        • Yan K.
        • Schmitt E.G.
        • et al.
        Alternatively activated macrophages boost induced regulatory T and Th17 cell responses during immunotherapy for colitis.
        J Immunol. 2016; 196: 3305-3317
        • Hashimoto T.
        • Kursewicz C.D.
        • Fayne R.A.
        • Nanda S.
        • Shah S.M.
        • Nattkemper L.
        • et al.
        Pathophysiological mechanisms of itch in bullous pemphigoid.
        J Am Acad Dermatol. 2019; (accessed 26 October 2019)https://doi.org/10.1016/j.jaad.2019.07.060
        • Hashimoto T.
        • Satoh T.
        Immunological Perspectives: Th2 Cells/Mast Cells/Basophils/Eosinophils.
        in: Katayama I. Murota H. Satoh T. Evolution of Atopic Dermatitis in the 21st century. Springer, Berlin2018: 69-82
        • Hashimoto T.
        • Satoh T.
        • Yokozeki H.
        Pruritus in ordinary scabies: il-31 from macrophages induced by overexpression of thymic stromal lymphopoietin and periostin.
        Allergy. 2019; 74: 1727-1737
        • Kabashima K.
        • Furue M.
        • Hanifin J.M.
        • Pulka G.
        • Wollenberg A.
        • Galus R.
        • et al.
        Nemolizumab in patients with moderate-to-severe atopic dermatitis: randomized, phase II, long-term extension study.
        J Allergy Clin Immunol. 2018; 142: 1121-1130.e7
        • Kanda Y.
        Investigation of the freely available easy-to-use software “EZR” for medical statistics.
        Bone Marrow Transplant. 2013; 48: 452-458
        • Kato A.
        • Fujii E.
        • Watanabe T.
        • Takashima Y.
        • Matsushita H.
        • Furuhashi T.
        • et al.
        Distribution of IL-31 and its receptor expressing cells in skin of atopic dermatitis.
        J Dermatol Sci. 2014; 74: 229-235
        • Kim S.Y.
        • Nair M.G.
        Macrophages in wound healing: activation and plasticity.
        Immunol Cell Biol. 2019; 97: 258-267
        • Kittaka H.
        • Tominaga M.
        The molecular and cellular mechanisms of itch and the involvement of TRP channels in the peripheral sensory nervous system and skin.
        Allergol Int. 2017; 66: 22-30
        • Kunsleben N.
        • Rüdrich U.
        • Gehring M.
        • Novak N.
        • Kapp A.
        • Raap U.
        IL-31 induces chemotaxis, calcium mobilization, release of reactive oxygen species, and CCL26 in eosinophils, which are capable to release IL-31.
        J Invest Dermatol. 2015; 135: 1908-1911
        • Lai J.P.
        • Douglas S.D.
        • Ho W.Z.
        Human lymphocytes express substance P and its receptor.
        J Neuroimmunol. 1998; 86: 80-86
        • Leal E.C.
        • Carvalho E.
        • Tellechea A.
        • Kafanas A.
        • Tecilazich F.
        • Kearney C.
        • et al.
        Substance P promotes wound healing in diabetes by modulating inflammation and macrophage phenotype.
        Am J Pathol. 2015; 185: 1638-1648
        • Lim J.E.
        • Chung E.
        • Son Y.
        A neuropeptide, substance-P, directly induces tissue-repairing M2 like macrophages by activating the PI3K/Akt/mTOR pathway even in the presence of IFNγ.
        Sci Rep. 2017; 7: 9417
        • Mao H.
        • Pan F.
        • Guo H.
        • Bu F.
        • Xin T.
        • Chen S.
        • et al.
        Feedback mechanisms between M2 macrophages and Th17 cells in colorectal cancer patients.
        Tumour Biol. 2016; 37: 12223-12230
        • Marriott I.
        • Bost K.L.
        IL-4 and IFN-gamma up-regulate substance P receptor expression in murine peritoneal macrophages.
        J Immunol. 2000; 165: 182-191
        • Masuoka M.
        • Shiraishi H.
        • Ohta S.
        • Suzuki S.
        • Arima K.
        • Aoki S.
        • et al.
        Periostin promotes chronic allergic inflammation in response to Th2 cytokines.
        J Clin Invest. 2012; 122: 2590-2600
        • Nakai K.
        • He Y.Y.
        • Nishiyama F.
        • Naruse F.
        • Haba R.
        • Kushida Y.
        • et al.
        IL-17A induces heterogeneous macrophages, and it does not alter the effects of lipopolysaccharides on macrophage activation in the skin of mice.
        Sci Rep. 2017; 7: 12473
        • Nakashima C.
        • Otsuka A.
        • Kabashima K.
        Interleukin-31 and interleukin-31 receptor: new therapeutic targets for atopic dermatitis.
        Exp Dermatol. 2018; 27: 327-331
        • Nattkemper L.A.
        • Martinez-Escala M.E.
        • Gelman A.B.
        • Singer E.M.
        • Rook A.H.
        • Guitart J.
        • et al.
        Cutaneous T-cell lymphoma and pruritus: the expression of IL-31 and its receptors in the skin.
        Acta Derm Venereol. 2016; 96: 894-898
        • Nattkemper L.A.
        • Tey H.L.
        • Valdes-Rodriguez R.
        • Lee H.
        • Mollanazar N.K.
        • Albornoz C.
        • et al.
        The genetics of chronic itch: gene expression in the skin of patients with atopic dermatitis and psoriasis with severe itch.
        J Invest Dermatol. 2018; 138: 1311-1317
        • Niyonsaba F.
        • Ushio H.
        • Hara M.
        • Yokoi H.
        • Tominaga M.
        • Takamori K.
        • et al.
        Antimicrobial peptides human beta-Defensins and cathelicidin LL-37 induce the secretion of a pruritogenic cytokine IL-31 by human mast cells.
        J Immunol. 2010; 184: 3526-3534
        • Patra V.
        • Strobl J.
        • Gruber-Wackernagel A.
        • Vieyra-Garcia P.
        • Stary G.
        • Wolf P.
        CD11b+ cells markedly express the itch cytokine interleukin 31 in polymorphic light eruption.
        Br J Dermatol. 2019; (accessed 26 October 2019)https://doi.org/10.1111/bjd.18092
        • Pereira M.P.
        • Mühl S.
        • Pogatzki-Zahn E.M.
        • Agelopoulos K.
        • Ständer S.
        Intraepidermal nerve fiber density: diagnostic and therapeutic relevance in the management of chronic pruritus: a review.
        Dermatol Ther (Heidelb). 2016; 6: 509-517
        • Raap U.
        • Gehring M.
        • Kleiner S.
        • Rüdrich U.
        • Eiz-Vesper B.
        • Haas H.
        • et al.
        Human basophils are a source of - and are differentially activated by - IL-31.
        Clin Exp Allergy. 2017; 47: 499-508
        • Rubio-Navarro A.
        • Amaro Villalobos J.M.
        • Lindholt J.S.
        • Buendía I.
        • Egido J.
        • Blanco-Colio L.M.
        • et al.
        Hemoglobin induces monocyte recruitment and CD163-macrophage polarization in abdominal aortic aneurysm.
        Int J Cardiol. 2015; 201: 66-78
        • Saharay M.
        • Shields D.A.
        • Porter J.B.
        • Scurr J.H.
        • Coleridge Smith P.D.
        Leukocyte activity in the microcirculation of the leg in patients with chronic venous disease.
        J Vasc Surg. 1997; 26: 265-273
        • Sanders K.M.
        • Nattkemper L.A.
        • Rosen J.D.
        • Andersen H.H.
        • Hsiang J.
        • Romanelli P.
        • et al.
        Non-histaminergic itch mediators elevated in the skin of a porcine model of scabies and of human scabies patients.
        J Invest Dermatol. 2019; 139: 971-973
        • Schuhknecht B.
        • Marziniak M.
        • Wissel A.
        • Phan N.Q.
        • Pappai D.
        • Dangelmaier J.
        • et al.
        Reduced intraepidermal nerve fibre density in lesional and nonlesional prurigo nodularis skin as a potential sign of subclinical cutaneous neuropathy.
        Br J Dermatol. 2011; 165: 85-91
        • Siebenhaar F.
        • Sharov A.A.
        • Peters E.M.J.
        • Sharova T.Y.
        • Syska W.
        • Mardaryev A.N.
        • et al.
        Substance P as an immunomodulatory neuropeptide in a mouse model for autoimmune hair loss (alopecia areata).
        J Invest Dermatol. 2007; 127: 1489-1497
        • Snyder R.J.
        • Lantis J.
        • Kirsner R.S.
        • Shah V.
        • Molyneaux M.
        • Carter M.J.
        Macrophages: a review of their role in wound healing and their therapeutic use.
        Wound Repair Regen. 2016; 24: 613-629
        • Sonkoly E.
        • Muller A.
        • Lauerma A.I.
        • Pivarcsi A.
        • Soto H.
        • Kemeny L.
        • et al.
        IL-31: A new link between T cells and pruritus in atopic skin inflammation.
        J Allergy Clin Immunol. 2006; 117: 411-417
        • Ständer S.
        • Yosipovitch G.
        Substance P and neurokinin 1 receptor are new targets for the treatment of chronic pruritus.
        Br J Dermatol. 2019; (accessed 26 October 2019)https://doi.org/10.1111/bjd.18025
        • Steinhoff M.
        • Buddenkotte J.
        • Lerner E.A.
        Role of mast cells and basophils in pruritus.
        Immunol Rev. 2018; 282: 248-264
        • Steinhoff M.
        • Neisius U.
        • Ikoma A.
        • Fartasch M.
        • Heyer G.
        • Skov P.S.
        • et al.
        Proteinase-activated receptor-2 mediates itch: a novel pathway for pruritus in human skin.
        J Neurosci. 2003; 23: 6176-6180
        • Sundaresan S.
        • Migden M.R.
        • Silapunt S.
        Stasis dermatitis: pathophysiology, evaluation, and management.
        Am J Clin Dermatol. 2017; 18: 383-390
        • Tan Y.
        • Ng W.J.
        • Lee S.Z.X.
        • Lee B.T.K.
        • Nattkemper L.A.
        • Yosipovitch G.
        • et al.
        3-Dimensional optical clearing and imaging of pruritic atopic dermatitis and psoriasis skin reveals downregulation of epidermal innervation.
        J Invest Dermatol. 2019; 139: 1201-1204
        • Thomas P.R.
        • Nash G.B.
        • Dormandy J.A.
        White cell accumulation in dependent legs of patients with venous hypertension: a possible mechanism for trophic changes in the skin.
        Br Med J (Clin Res Ed). 1988; 296: 1693-1695
        • Valdes-Rodriguez R.
        • Mollanazar N.K.
        • González-Muro J.
        • Nattkemper L.
        • Torres-Alvarez B.
        • López-Esqueda F.J.
        • et al.
        Itch prevalence and characteristics in a Hispanic Geriatric population: A comprehensive study using a standardized itch questionnaire.
        Acta Derm Venereol. 2015; 95: 417-421
        • Wang N.
        • Liang H.
        • Zen K.
        Molecular mechanisms that influence the macrophage m1-m2 polarization balance.
        Front Immunol. 2014; 5: 614
        • Wenk J.
        • Foitzik A.
        • Achterberg V.
        • Sabiwalsky A.
        • Dissemond J.
        • Meewes C.
        • et al.
        Selective pick-up of increased iron by deferoxamine-coupled cellulose abrogates the iron-driven induction of matrix-degrading metalloproteinase 1 and lipid peroxidation in human dermal fibroblasts in vitro: a new dressing concept.
        J Invest Dermatol. 2001; 116: 833-839
        • Yalçin B.
        • Tamer E.
        • Toy G.G.
        • Öztaş P.
        • Hayran M.
        • Alli N.
        The prevalence of skin diseases in the elderly: analysis of 4099 geriatric patients.
        Int J Dermatol. 2006; 45: 672-676
        • Yosipovitch G.
        • Rosen J.D.
        • Hashimoto T.
        Itch: From mechanism to (novel) therapeutic approaches.
        J Allergy Clin Immunol. 2018; 142: 1375-1390