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IL-9 induces IL-8 production via STIM1 activation and ERK phosphorylation in epidermal keratinocytes: A plausible mechanism of IL-9R in atopic dermatitis

  • Chien-Hui Hong
    Affiliations
    Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

    Department of Dermatology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan

    Department of Dermatology, National Yang-Ming University College of Medicine, Taipei, Taiwan
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  • Kee-Lung Chang
    Affiliations
    Department of Biochemistry, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
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  • Hung-Jen Wang
    Affiliations
    Department of Urology, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan
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  • Hsin-Su Yu
    Correspondence
    Corresponding author at: National Environmental Health Research Center, National Health Research Institutes, National Environmental Health Research Center, Kaohsiung Medical University, No. 35, Keyan Road, Zhunan Town, Miaoli County 35053, Taiwan. Tel.: +886 37 246166x31010; fax: +886 37 582 946.
    Affiliations
    Department of Dermatology, Kaohsiung Medical University, Kaohsiung, Taiwan

    National Environmental Health Research Center, National Health Research Institute, Miao-Li, Taiwan
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  • Chih-Hung Lee
    Correspondence
    Corresponding author at: Department of Dermatology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, 123 Dapi Rd, Kaohsiung 83301, Taiwan. Tel.: +886 7 3121101x2299; fax: +886 7 7337612.
    Affiliations
    Department of Dermatology, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan

    Department of Dermatology, Chang Gung University College of Medicine, Taoyuan, Taiwan
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      Highlights

      • IL-9R expression is increased in atopic skin.
      • IL-9R expression in epidermal keratinocytes is enhanced by IL-4.
      • IL-9 induces IL-8 production from keratinocyte via STIM1 and ERK activation.

      Abstract

      Background

      IL-9 and its receptor play important roles in the pathogenesis of asthma. Its role in atopic dermatitis (AD) was examined in just a few studies, including nucleotide polymorphisms, increased transcriptional levels of IL-9 and IL-9R in diseased skin, and an association of blood IL-9 levels with clinical severity.

      Objective

      Little was known about the pathophysiological regulation of IL-9/IL-9R in AD skin. We asked whether IL-9R was expressed in epidermal keratinocytes; if so, what the functional outcome, cytokine production, and signaling pathway of IL-9/IL-9R in keratinocytes are.

      Methods

      We measured and compared the expression of IL-9R in skin from AD patients and controls by immunofluorescence. We also performed in vitro studies on the IL-9-treated primary keratinocytes, including flow cytometry for IL-9R expressions, Western blotting for mTOR, S6K, ERK, p38, and STAT3 activations, ELISA for cytokine levels, and immunofluorescence for STIM1.

      Results

      We found that IL-9R was indeed expressed in keratinocytes but not in fibroblasts. Its expression in keratinocytes was enhanced by IL-4 but not by TGF-beta1. IL-9 induced a moderate production of IL-8 but not CXCL16, CCL22, TSLP, nor IL-33. IL-9 induced formation of STIM1-puncta. IL-9 induced ERK phosphorylation both dose- and time-dependently, but not mTOR, S6K, p38, or STAT3. Pretreatment with U0126 (ERK inhibitor) but not rapamycin (mTOR inhibitor) abrogated the IL-9-mediated IL-8 production. Blockage of STIM1 with BTP2 or SKF96265 abrogated ERK phosphorylation and IL-8 production induced by IL-9.

      Conclusion

      This study represents the first to show the regulation of the IL-9-STIM1-ERK-IL-8 axis in keratinocyte, and how the axis might play an important role in the pathophysiology of AD.

      Keywords

      1. Introduction

      Atopic dermatitis (AD) is a common chronic inflammatory skin disease usually associated with personal or family history of allergic diseases, including AD, asthma, and allergic rhinitis [
      • Lee C.H.
      • Chuang H.Y.
      • Shih C.C.
      • Jong S.B.
      • Chang C.H.
      • Yu H.S.
      Transepidermal water loss, serum IgE and beta-endorphin as important and independent biological markers for development of itch intensity in atopic dermatitis.
      ,
      • Leung D.Y.
      • Bieber T.
      Atopic dermatitis.
      ]. Clinically, it occurs with eczematous changes and intense itching [
      • Tokura Y.
      Extrinsic and intrinsic types of atopic dermatitis.
      ]. AD skin is characterized microscopically by spongiotic dermatitis with moderate dermal immune cell infiltrates. The immune abnormalities of AD have been found to involve aberrant T helper cell polarization, imbalances in cytokines and chemokines, and dysregulation of innate immunity. Filaggrin mutations [
      • Irvine A.D.
      • McLean W.H.
      • Leung D.Y.
      Filaggrin mutations associated with skin and allergic diseases.
      ] and thymus stromal lymphopoietin (TSLP) [
      • Siracusa M.C.
      • Saenz S.A.
      • Hill D.A.
      • Kim B.S.
      • Headley M.B.
      • Doering T.A.
      • et al.
      TSLP promotes interleukin-3-independent basophil haematopoiesis and type 2 inflammation.
      ] from epidermal keratinocytes are reported to mediate the development of asthma, suggesting that epidermal keratinocytes play a major role in the pathogenesis of AD and bronchial asthma. In fact, one of our previous studies found that IL-31, a pruritus-related cytokine, activated IL-31R, induced calcium propagation, activated STAT3, and released beta-endorphin in keratinocytes [
      • Lee C.H.
      • Hong C.H.
      • Yu W.T.
      • Chuang H.Y.
      • Huang S.K.
      • Chen G.S.
      • et al.
      Mechanistic correlations between two itch biomarkers, cytokine interleukin-31 and neuropeptide beta-endorphin, via STAT3/calcium axis in atopic dermatitis.
      ].
      In AD skin, a significant portion of T cells is polarized to Th2 subsets. IL-9 was previously thought to be a Th2 cytokine, but it is now known that TGF-beta1 is able to reprogram Th2 cells to produce IL-9 instead of Th2 cytokines [
      • Veldhoen M.
      • Uyttenhove C.
      • van Snick J.
      • Helmby H.
      • Westendorf A.
      • Buer J.
      • et al.
      Transforming growth factor-beta ‘reprograms’ the differentiation of T helper 2 cells and promotes an interleukin 9-producing subset.
      ]. The transcriptional factors of IL-9, which is produced by mast cells, T cells, and NKT cells [
      • Soroosh P.
      • Doherty T.A.
      Th9 and allergic disease.
      ], include PU.1 [
      • Chang H.C.
      • Sehra S.
      • Goswami R.
      • Yao W.
      • Yu Q.
      • Stritesky G.L.
      • et al.
      The transcription factor PU.1 is required for the development of IL-9-producing T cells and allergic inflammation.
      ] and IRF4 [
      • Staudt V.
      • Bothur E.
      • Klein M.
      • Lingnau K.
      • Reuter S.
      • Grebe N.
      • et al.
      Interferon-regulatory factor 4 is essential for the developmental program of T helper 9 cells.
      ]. Its receptor, IL-9R, is expressed in T cells as well as bronchial epithelial cells [
      • Little F.F.
      • Cruikshank W.W.
      • Center D.M.
      Il-9 stimulates release of chemotactic factors from human bronchial epithelial cells.
      ], neutrophils [
      • Dragon S.
      • Takhar M.K.
      • Shan L.
      • Hayglass K.T.
      • Simons F.E.
      • Gounni A.S.
      T(H)2 cytokines modulate the IL-9R expression on human neutrophils.
      ], mast cells [
      • Sismanopoulos N.
      • Delivanis D.A.
      • Alysandratos K.D.
      • Angelidou A.
      • Vasiadi M.
      • Therianou A.
      • et al.
      IL-9 induces VEGF secretion from human mast cells and IL-9/IL-9 receptor genes are overexpressed in atopic dermatitis.
      ], B cells [
      • Fawaz L.M.
      • Sharif-Askari E.
      • Hajoui O.
      • Soussi-Gounni A.
      • Hamid Q.
      • Mazer B.D.
      Expression of IL-9 receptor alpha chain on human germinal center B cells modulates IgE secretion.
      ], and human airway smooth muscle cells [
      • Gounni A.S.
      • Hamid Q.
      • Rahman S.M.
      • Hoeck J.
      • Yang J.
      • Shan L.
      IL-9-mediated induction of eotaxin1/CCL11 in human airway smooth muscle cells.
      ]. The activation of IL-9R causes different physiological functions in different cells. One recent mouse study has described the biological role of IL-9 secreting cells in the mediation of tumor immunity to melanoma [
      • Purwar R.
      • Schlapbach C.
      • Xiao S.
      • Kang H.S.
      • Elyaman W.
      • Jiang X.
      • et al.
      Robust tumor immunity to melanoma mediated by interleukin-9-producing T cells.
      ]. Only few studies examined the role of IL-9R of keratinocytes in AD. One recent study showed that K5.hTGF-β1 transgenic mice exhibiting a psoriasis-like phenotype with increased expressions of IL-9 and IL-9R in skin [
      • Singh T.P.
      • Schon M.P.
      • Wallbrecht K.
      • Gruber-Wackernagel A.
      • Wang X.J.
      • Wolf P.
      Involvement of IL-9 in Th17-associated inflammation and angiogenesis of psoriasis.
      ]. In the same study, they found IL-9R is increased in the basal layer of epidermis in psoriatic skin [
      • Singh T.P.
      • Schon M.P.
      • Wallbrecht K.
      • Gruber-Wackernagel A.
      • Wang X.J.
      • Wolf P.
      Involvement of IL-9 in Th17-associated inflammation and angiogenesis of psoriasis.
      ]. It is not known, however, what the functional outcome and the regulatory mechanisms of IL-9R in AD skin are.
      Previous translational studies of IL-9 have mainly focused on asthma. In mice, transgenic expression of IL-9 causes allergic inflammation [
      • Temann U.A.
      • Geba G.P.
      • Rankin J.A.
      • Flavell R.A.
      Expression of interleukin 9 in the lungs of transgenic mice causes airway inflammation, mast cell hyperplasia, and bronchial hyperresponsiveness.
      ,
      • Temann U.A.
      • Ray P.
      • Flavell R.A.
      Pulmonary overexpression of IL-9 induces Th2 cytokine expression, leading to immune pathology.
      ] while antibodies against IL-9 ameliorates its development [
      • Cheng G.
      • Arima M.
      • Honda K.
      • Hirata H.
      • Eda F.
      • Yoshida N.
      • et al.
      Anti-interleukin-9 antibody treatment inhibits airway inflammation and hyperreactivity in mouse asthma model.
      ]. In humans, IL-9 induces mucus production by lung epithelial cells [
      • Vermeer P.D.
      • Harson R.
      • Einwalter L.A.
      • Moninger T.
      • Zabner J.
      Interleukin-9 induces goblet cell hyperplasia during repair of human airway epithelia.
      ]. The expression of IL-9 and IL-9R has also been found to be higher in the bronchial epithelium of asthmatic patients compared to controls [
      • Toda M.
      • Tulic M.K.
      • Levitt R.C.
      • Hamid Q.
      A calcium-activated chloride channel (HCLCA1) is strongly related to IL-9 expression and mucus production in bronchial epithelium of patients with asthma.
      ,
      • Tsicopoulos A.
      • Shimbara A.
      • de Nadai P.
      • Aldewachi O.
      • Lamblin C.
      • Lassalle P.
      • et al.
      Involvement of IL-9 in the bronchial phenotype of patients with nasal polyposis.
      ]. In asthmatic families, IL-9 gene polymorphism and environmental exposures synergistically affect the clinical aggravation of asthma [
      • Wang T.N.
      • Chen W.Y.
      • Huang Y.F.
      • Shih N.H.
      • Feng W.W.
      • Tseng H.I.
      • et al.
      The synergistic effects of the IL-9 gene and environmental exposures on asthmatic Taiwanese families as determined by the transmission/disequilibrium test.
      ]. Compared with the large number studies addressing the role of IL-9/IL-9R in asthma, there are only a limited number of studies investigating its role in AD. One of these studies, reported that transcriptional level of IL-9 and IL-9 receptor is significantly increased in lesional skin areas of AD patients as compared to normal control skin [
      • Sismanopoulos N.
      • Delivanis D.A.
      • Alysandratos K.D.
      • Angelidou A.
      • Vasiadi M.
      • Therianou A.
      • et al.
      IL-9 induces VEGF secretion from human mast cells and IL-9/IL-9 receptor genes are overexpressed in atopic dermatitis.
      ], and another has reported a correlation between IL-9 level in the blood and severity of AD [
      • Ciprandi G.
      • De Amici M.
      • Giunta V.
      • Marseglia A.
      • Marseglia G.
      Serum interleukin-9 levels are associated with clinical severity in children with atopic dermatitis.
      ]. In addition, IL-9 single nucleotide polymorphisms have been associated with the susceptibility of AD [
      • Namkung J.H.
      • Lee J.E.
      • Kim E.
      • Park G.T.
      • Yang H.S.
      • Jang H.Y.
      • et al.
      An association between IL-9 and IL-9 receptor gene polymorphisms and atopic dermatitis in a Korean population.
      ]. Since keratinocytes make up the majority of cells in the skin, we sought to investigate whether IL-9R is expressed in keratinocytes and, if so, what functional outcomes and pathophysiological regulations are in AD.

      2. Materials and methods

      2.1 Reagents and ELISAs

      IL-9 was purchased from R&D (Minneapolis, MN, USA). ELISA kits for IL-8, CXCL16, IL-33, CXCL1, and CCL22 were also purchased from R&D to measure their levels in conditioned media from IL-9-treated keratinocytes. ELISA kit for TSLP was obtained from BioLegend (San Diego, CA). Multiplex cytokine array kits, including Human cytokine array panel A (ARY005) and Human chemokine array kit (ARY017), were purchased from R&D (Minneapolis, MN, USA). The mTOR inhibitor rapamycin was obtained from Sigma–Aldrich (St. Louis, MO) and ERK inhibitor (U0126) from Promega (Madison, WI, USA).

      2.2 Primary culture of epidermal keratinocytes and dermal fibroblasts

      Normal human keratinocytes were obtained from adult foreskins through routine circumcision. The keratinocytes were harvested and cultured as described previously [
      • Lee C.H.
      • Wu S.B.
      • Hong C.H.
      • Chen G.S.
      • Wei Y.H.
      • Yu H.S.
      Involvement of mtDNA damage elicited by oxidative stress in the arsenical skin cancers.
      ]. Briefly, skin specimens were washed with PBS (pH 7.2), cut into small pieces, and harvested in a medium containing 0.25% trypsin (Gibco, Grand Island, NY) overnight at 4 °C. The epidermal sheet was lifted from the dermis by a fine forceps. The epidermal cells were spun down by centrifugation (500 × g, 10 min) and then were dispersed into individual cells by repeated aspiration. The keratinocytes were gently resuspended in 5 ml of keratinocyte–serum-free medium (Gibco), which contained 25 μg/ml bovine pituitary extract and 5 ng/ml recombinant human epidermal growth factor. Keratinocytes at the third passage were then grown in a keratinocyte–serum-free medium without bovine pituitary extract and recombinant human epidermal growth factor for 24 h before experimentation.
      For fibroblasts, the primary fibroblasts were harvested as described previously [
      • Lee C.H.
      • Hong C.H.
      • Chen Y.T.
      • Chen Y.C.
      • Shen M.R.
      TGF-beta1 increases cell rigidity by enhancing expression of smooth muscle actin: keloid-derived fibroblasts as a model for cellular mechanics.
      ]. Briefly, dermal parts after lifting of epidermis were cut to 1–2 mm3 and incubated with Dulbecco's Modified Eagle Medium (DMEM; Invitrogen, Grand Island, NY) supplemented with 10% FBS (Invitrogen), 2 mM l-glutamine (Invitrogen) and 0.1 mM 2-mercaptoethanol (Sigma–Aldrich), along with 50 units/ml of penicillin and 50 g/ml of streptomycin (Invitrogen). The 3rd passage of human fibroblasts were used for the experiments.

      2.3 Cytokine stimulations and blocking experiments

      In preparation for the measurement of IL-9R, we treated cells with IL-4 (up to 10 ng/ml) and/or TGF-beta1 (up to 50 ng/ml) for 24 h and stained cells with 1:100 mouse anti-IL-9R (BioLegend, San Diego, CA) overnight at 4 °C. We then measured the expressions of IL-9R by flow cytometry (BD Biosciences, San Jose, CA, USA). To measure what cytokines/chemokines would be induced by IL-9 treatment, we treated keratinocytes with IL-9 at 0, 5, 10, and 20 ng/ml for 12 or 24 h and then measured the expressions of individual cytokines by ELISA. To determine the intracellular signaling pathways in keratinocytes by IL-9, we treated cells with IL-9 at 10 ng/ml for 0, 5, 10, 15, 20, and 30 min or IL-9 at 0, 1, 5, 10, and 20 ng/ml for 10 min and then evaluated the signaling pathways by Western blot. To determine whether IL-9 might induce the production of IL-8 through ERK or mTOR pathways, we pretreated keratinocytes with U0126 (an ERK inhibitor) or rapamycin (an mTOR inhibitor) at indicated concentrations for 2 h prior to their treatment with IL-9. Finally, for STIM1 blocking, we pretreated keratinocytes with STIM1 inhibitors (BTP-2 up to 0.1 nM or SKF96365 up to 50 nM, both from Sigma–Aldrich) for 24 h before IL-9 treatments.

      2.4 Immunoflorescent study for IL-9R in skin

      Immunofluorescent studies for IL-9R were performed on 5-μm serial tissue sections obtained from skin of AD patients (active and stable), patients with psoriasis, and controls (n = 3, 3, 2, and 5, respectively). The protocol for these studies were approved by IRB from the affiliated hospital. All participants provided written informed consent. To perform these studies, all sections were blocked with 3% bovine serum albumin at room temperature for 2 h. After the blocking, the sections were incubated with rabbit polyclonal antihuman IL-9R (1:200; GeneTex, GTX87356, Irvine, CA) at 4 °C overnight. Image analysis was performed using NIH IMAGEJ (http://rsbweb.nih.gov/ij/). Fluorescent intensity index (0–255) was calculated in five random mid-power fields above the dermoepidermal junction.
      For animals, female C57BL/6JNarl mice (8–12 weeks old) were obtained from the National Laboratory Animal Center-Tainan Facility (Tainan, Taiwan). Mice were painted with benzopyrene at 0.5 ppm/mice/day for 5 days. Control mice received a similar treatment but patched with acetone, the solvent control. At day 8, mice were sacrificed and their samples of skin were incubated with the same IL-9R antibody, that crossly reacts with mouse tissue, at 4 °C overnight.

      2.5 Immunofluorescent staining of IL-9R and STIM1

      In preparation for the measurement of IL-9R, we stained keratinocytes with 1:100 mouse anti-IL-9R (BioLegend, San Diego, CA) overnight at 4 °C. For the measurement of STIM1, we incubated keratinocytes with mouse anti-STIM1 (1:100, Abnova, Walnut, CA) followed by Alexa Fluor® 568-rabbit anti-mouse IgG (Invitrogen, Carlsbad, CA). STIM1 puncta were analyzed using the feature of “Analyze Particles” in ImageJ. Briefly, following background subtraction, fluorescent images were converted into a binary mask and puncta were identified according to their relative intensity, compared with background, size, and circularity.

      2.6 Western blotting

      Methods for Western blotting have been described previously [
      • Lee C.H.
      • Wu S.B.
      • Hong C.H.
      • Liao W.T.
      • Wei Y.H.
      • Yu H.S.
      Aberrant cell proliferation by mitochondrial biogenesis and mtTFA in arsenical cancers.
      ]. Antibodies for STAT3 (1:1000), ERK p38 (1:1000), mTOR (1:1000), and S6K (1:1000) were obtained from Cell Signaling Technology (Boston, MA, USA). Specific proteins were detected using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and visualized films recorded on a digital imaging system (Alpha Imager 2000; Alpha Innotech Corp., San Leandro, CA, USA).

      2.7 Statistical analyses

      The numeric variables between two groups were compared by nonparametric Mann–Whitney U test, while ratio variables were compared by Chi-square test using SPSS ver 14 (Chicago, IL). A p-value less than 0.05 was considered significant.

      3. Results

      3.1 IL-9R was increased in atopic skin

      We performed immunofluorescent studies to determine whether IL-9R would be more expressed in AD skin than control skin. IL-9R was barely expressed in control skin. In psoriatic skin, there were some dermal expressions of IL-9R in psoriatic skin, however, the epidermal expressions of IL-9R were limited. In AD skin, IL-9R expressions were highly increased in active AD epidermal skin and slightly increased in stable AD epidermal skin (Fig. 1A) . In animals, IL-9R was found to be localized in the epidermis and in the hair follicle. The expressions of IL-9R in the epidermis from benzopyrene-painted mice were higher than those from acetone-painted mice. Next, to find out whether normal epidermal keratinocytes and normal dermal fibroblasts, two major primary skin cells, expressed IL-9R, we performed flow cytometry to measure the surface expressions of IL-9R of normal primary keratinocytes and fibroblasts harvested from adult foreskin. We found a small but appreciable expression of IL-9R in epidermal keratinocytes but not in dermal fibroblasts (Fig. 1A).
      Figure thumbnail gr1
      Fig. 1Increased expressions of IL-9R in atopic skin and IL-4 induced IL-9R expressions in epidermal keratinocytes. (A) Skin from normal controls (n = 5), patients with active AD (n = 3), patients with stable AD (n = 3), and patients with psoriasis (n = 2), were stained by anti-IL-9R antibody for immunohistochemistry (200X). Skin from mice painted with benzopyrene or acetone was stained by the same antibody. Keratinocytes and fibroblasts were harvested from adult foreskin. The IL-9R expressions were measured on those cells by flow cytometry (n = 3 each). (B) Normal human primary keratinocytes were harvested from foreskin. Keratinocytes were treated with IL-4 at 5 or 10 ng/ml and/or TGF-beta 1 at 20 or 50 ng/ml for 24 h. The expressions of IL-9R in keratinocytes were measured by flow cytometry.

      3.2 IL-4 treatment induced the expression of IL-9R in keratinocytes

      We next asked what cytokine might induce the expression of IL-9R in keratinocytes. Because Th2 skewing is prominent in AD skin, we first added IL-4, a representative Th2 cytokine, to investigate its effect on expression of IL-9R. Furthermore, because IL-9R is increased in the skin of K5-TGF-β1 transgenic mice [
      • Singh T.P.
      • Schon M.P.
      • Wallbrecht K.
      • Gruber-Wackernagel A.
      • Wang X.J.
      • Wolf P.
      Involvement of IL-9 in Th17-associated inflammation and angiogenesis of psoriasis.
      ], we also investigated the effect of TGF-β1 on IL-9R expression. We found that IL-4 treatment (10 ng/ml for 24 h) significantly increased the expression of IL-9R in keratinocytes (Fig. 1B). However, while TGF-beta1 (50 ng/ml for 24 h) promoted the expression of IL-9R to some extent, it did not increase the expression of IL-9 expressions as much as IL-4 did. Combining both IL-4 and TGF-beta1 did not further increase the expression of IL-9R (Fig. 1B).

      3.3 IL-9 treatment increased production of IL-8, but not CCL22, CXCL16, TSLP, CXCL1, nor IL-33, from primary keratinocytes

      To the best of our knowledge, no study has investigated the immunological effect of IL-9R activation and its mechansitic regulations in keratinocytes. To screen for what cytokines and/or chemokines would be increased by IL-9 treatment, we measured a panel of cytokines and chemokines using a multiplex ELISA that profiles the expressions of several dozen cytokines and chemokines. Among these cytokines and chemokines, CCL22, CXCL16, and IL-8 levels were increased mostly (Fig. 2A) . Measuring the individual expressions of those cytokines by standard ELISAs, we found that IL-9 dose-dependently and significantly increased the expression of IL-8 in keratinocytes (Fig. 2B) but only minimally increased CCL22 and CXCL16. We also tested whether IL-9 treatment would increase TSLP, CXCL1, and IL-33, three important keratinocyte-derived cytokines in AD by standard ELISAs and found that it did not (Fig. 2B).
      Figure thumbnail gr2
      Fig. 2IL-9 induced production of IL-8 in a dose- and time-dependent manner. (A) The profiles of cytokines and chemokines from supernatants of IL-9-treated keratinocytes were measured by multiplex ELISA. The expressions of CXCL16, CCL22, and IL-8 were greatly enhanced by IL-9 treatment (n = 2). (B) To confirm the results from the multiplex ELISA, we measured the expressions of CXCL16, CCL22, IL-8, IL-33, CXCL1, as well as TSLP, in condition medium from IL-9-treated keratinocytes by standard ELISA. Keratinocytes were treated with IL-9 for 24 h at indicated concentrations. The experiments were repeated 3 times. *p < 0.05.

      3.4 IL-9 activated ERK phosphorylation both dose-dependently and time-dependently

      Next, we asked what signaling pathways were activated in keratinocytes treated with IL-9. To find out, we used Western blot to measure the expression of total and phosphorylated forms of mTOR, S6K, ERK, STAT3, and p38 in IL-9-treated keratinocytes. Kinetic analysis revealed that IL-9 (10 ng/ml) activated ERK phosphorylation at 5, 10, and 15 min (Fig. 3A) , but not mTOR, S6K, STAT3, or p38 (from 5 to 30 min). Ten-minute treatment with IL-9 at different concentrations (0, 1, 5, 10, and 20 ng/ml) induced phosphorylation of ERK but not mTOR, S6K, STAT3, or p38.
      Figure thumbnail gr3
      Fig. 3IL-9 induced IL-8 production through ERK in keratinocytes. (A) Human primary epidermal keratinocytes were treated with IL-9 for indicated time at 10 ng/ml (left panel). They were treated with IL-9 for 10 min at indicated concentrations (right panel). The total and phosphorylated forms of mTOR, S6K, ERK, STAT3, and p38 were measured by Western blot (n = 3, a representative blot is shown). (B) Keratinocytes were pretreated with U0126 or rapamycin for 2 h before treatment with IL-9. The IL-8 production was measured by ELISA after IL-9 treatment for 24 h (three repeated experiments). *p < 0.05.

      3.5 IL-9 induced IL-8 production in keratinocytes through ERK phosphorylation

      Since we have found that IL-9 induces the phosphorylation of ERK along with the production IL-8, we hypothesized that IL-9 might induce the production of IL-8 via phosphorylation of ERK. We investigated this possibility by pretreating keratinocytes with various concentrations of U0126 (an ERK inhibitor) or rapamycin (an mTOR inhibitor) for two hours prior to their treatment with IL-9. IL-8 production was measured by ELISA. IL-9 consistently induced approximately a 2-fold increase in IL-8 level. Pretreatment with U0126 (0.5, 1, and 5 ng/ml) abrogated increases of IL-8 in IL-9-treated keratinocytes but pretreatment with rapamycin has no such effect (Fig. 3B), indicating that indeed IL-9 induced the production of IL-8 production through its activation of ERK.

      3.6 IL-9 induced ERK phosphorylation through STIM1 activation

      Since the activation of several cytokine receptors requires the propagation of calcium, we next asked whether IL-9 activation of ERK required calcium propagation and whether that occurred through its activation of STIM1. STIM1 is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane (so called store-operated channel). We first measured the ERK activation by IL-9 with or without STIM1 inhibitors, either BTP2 or SKF96365 (Fig. 4A and B , respectively). The increases in the phosphorylation of ERK were abolished by pretreatment with both STIM1 inhibitors, indicating the IL-9-mediated ERK phosphorylation occurred through the activation of STIM1. We then wanted to determine whether IL-9 activated STIM1. To find out, we treated the keratinocytes with IL-9 (10 ng/ml) and measured the expression of STIM1 by immunofluorescent exam (Fig. 4C). STIM1 expressions were diffuse in the cytosol at baseline, while punctate aggregation of STIM1 was apparent in cells treated with IL-9 at 30 s and 2 min. At 30 min after IL-9 treatment, the punctate aggregation of STIM1 in the cytosol resumed to baseline diffuse distributions. Finally, we asked whether the IL-9 induced the phosphorylation of ERK and the production of IL-8 through activation of STIM1 in keratinocytes. We pretreated IL-9-incubated keratinocytes with the STIM1 inhibitors of BTP2 or SKF96365, and measured the expressions of IL-8 in the condition media and the phophorylation of ERK in keratinocytes (Fig. 4A and B). Both the production of IL-8 and phosphorylation of ERK by IL-9 in keratinocytes were inhibited by the STIM1 inhibitors, indicating IL-9 induced the production of IL-8 through its activation of STIM1 followed by phosphorylation of ERK.
      Figure thumbnail gr4
      Fig. 4IL-9 induced IL-8 production through STIM1 activation followed by ERK phosphorylation. (A) Keratinocytes were pre-treated with BTP2 using indicated concentrations for 24 h before IL-9 was added. Total and phosphorylated forms of mTOR, S6K, and ERK were measured by Western blot (n = 3, one representative blot was shown). IL-8 level in the condition media was measured by ELISA (n = 3). *p < 0.05. (B) Keratinocytes were pre-treated with SKF96365 using indicated concentrations for 24 h before IL-9 was added. Total and phosphorylated forms of mTOR, S6K, and ERK were measured by Western blot (n = 3, one representative blot was shown). IL-8 level in the condition media was measured by ELISA (n = 3). *p < 0.05. (C) Keratinocytes were treated with IL-9 (10 ng/ml) for 0, 0.5, 2, and 30 min. The expressions of STIM1 were measured by immunofluorescent exams (n = 3). The values inside (C) represent the STIM1 puncta fluorescent values as analyzed by the feature of “Analyze Particle” in Image J.

      4. Discussion

      This study found IL-9R to be increased in AD skin and its expression in keratinocytes to be increased by IL-4. IL-9 treatment in keratinocytes leads to the production of IL-8 through its activation of STIM1 and subsequently phosphorylation of ERK. This is the first study to show how the regulatory role IL-9/IL-9R plays in epidermal keratinocytes.
      The regulation of IL-9 activation has been investigated in different cells. For example, IL-9/IL-9R interactions could result in T cell activation and IL-9R could regulate the production of IgE from germinal center B cells [
      • Fawaz L.M.
      • Sharif-Askari E.
      • Hajoui O.
      • Soussi-Gounni A.
      • Hamid Q.
      • Mazer B.D.
      Expression of IL-9 receptor alpha chain on human germinal center B cells modulates IgE secretion.
      ]. In mast cells, IL-9 and IL-9R are over expressed in atopic skin and they induce the production of VEGF [
      • Sismanopoulos N.
      • Delivanis D.A.
      • Alysandratos K.D.
      • Angelidou A.
      • Vasiadi M.
      • Therianou A.
      • et al.
      IL-9 induces VEGF secretion from human mast cells and IL-9/IL-9 receptor genes are overexpressed in atopic dermatitis.
      ]. In smooth muscle cells, IL-9R activation upregulates the expression of eotaxin-1 [
      • Gounni A.S.
      • Hamid Q.
      • Rahman S.M.
      • Hoeck J.
      • Yang J.
      • Shan L.
      IL-9-mediated induction of eotaxin1/CCL11 in human airway smooth muscle cells.
      ]. IL-9 induces the expression of CCL11 via STAT3 signaling [
      • Yamasaki A.
      • Saleh A.
      • Koussih L.
      • Muro S.
      • Halayko A.J.
      • Gounni A.S.
      IL-9 induces CCL11 expression via STAT3 signalling in human airway smooth muscle cells.
      ]. The current study provides first evidence that IL-9R is expressed in normal human primary keratinocytes and that the expression of IL-9R expression is increased by IL-4, a Th2 cytokine. In the Th2 immunological milieu of AD, increased expressions of IL-9R by IL-4 may further reinforce the role of IL-9R in AD.
      In AD, one study demonstrated that plasma levels of IL-8 were detectable in most cases of AD but not in controls, patients with allergic rhinitis or asthma [
      • Kimata H.
      • Lindley I.
      Detection of plasma interleukin-8 in atopic dermatitis.
      ]. In skin, IL-8 in the stratum corneum might act as an indicator of the severity of inflammation in AD lesions. Comparing IL-18, VEGF, and TGF-α, one study has shown IL-8 blood levels is associated with severity scores in AD [
      • Amarbayasgalan T.
      • Takahashi H.
      • Dekio I.
      • Morita E.
      Interleukin-8 content in the stratum corneum as an indicator of the severity of inflammation in the lesions of atopic dermatitis.
      ]. Improvements in clinical severity have been found to be in parallel to the reductions in IL-8 in human skin [
      • Casas C.
      • Ribet V.
      • Alvarez-Georges S.
      • Sibaud V.
      • Guerrero D.
      • Schmitt A.M.
      • et al.
      Modulation of Interleukin-8 and staphylococcal flora by Avene hydrotherapy in patients suffering from chronic inflammatory dermatoses.
      ]. In a FLG knock-down construct that was used in 3-D skin models mimicking AD, IL-8 levels were found to be elevated after SDS application [
      • Kuchler S.
      • Henkes D.
      • Eckl K.M.
      • Ackermann K.
      • Plendl J.
      • Korting H.C.
      • et al.
      Hallmarks of atopic skin mimicked in vitro by means of a skin disease model based on FLG knock-down.
      ]. One study, using a mouse model of filaggrin deficiency, found a consistent elevation of IL-8 in addition to IL-1 [
      • Kezic S.
      • O’Regan G.M.
      • Lutter R.
      • Jakasa I.
      • Koster E.S.
      • Saunders S.
      • et al.
      Filaggrin loss-of-function mutations are associated with enhanced expression of IL-1 cytokines in the stratum corneum of patients with atopic dermatitis and in a murine model of filaggrin deficiency.
      ]. It was demonstrated that IL-8 also significantly enhances the generation of LTB4 and LTC4 of neutrophils from patients with AD than those from controls [
      • Neuber K.
      • Hilger R.A.
      • Konig W.
      Interleukin-3, interleukin-8, FMLP and C5a enhance the release of leukotrienes from neutrophils of patients with atopic dermatitis.
      ]. These leukotrienes are able to attract a robust of inflammatory cells [
      • Ohnishi H.
      • Miyahara N.
      • Gelfand E.W.
      The role of leukotriene B(4) in allergic diseases.
      ]. More specifically, IL-8 contributes to the accumulation of dendritic cells, macrophages, and monocytes in the Th2 microenvironment [
      • Bonecchi R.
      • Facchetti F.
      • Dusi S.
      • Luini W.
      • Lissandrini D.
      • Simmelink M.
      • et al.
      Induction of functional IL-8 receptors by IL-4 and IL-13 in human monocytes.
      ]. IL-13 and IL-4, two Th2 cytokines, convert IL-8 into monocyte chemotactic agonists, by up-regulating the expression of IL-8 receptor, contributing to the accumulation and positioning of mononuclear phagocytes in Th2-dominated responses [
      • Bonecchi R.
      • Facchetti F.
      • Dusi S.
      • Luini W.
      • Lissandrini D.
      • Simmelink M.
      • et al.
      Induction of functional IL-8 receptors by IL-4 and IL-13 in human monocytes.
      ]. In contrast to the multifaceted role of IL-8 in AD, the neutrophil migration and activation are impaired in AD, possibly due to the ligand-binding or ligand-signaling defects [
      • McGirt L.Y.
      • Beck L.A.
      Innate immune defects in atopic dermatitis.
      ].
      In this study, IL-9 induced IL-8 production through STIM1 activation followed by ERK phosphorylation in epidermal keratinocytes. Cell type specificity may affect the regulatory mechanisms of IL-9 activation. It has been shown in neuron cells that antiapoptotic effects of IL-9 effects were mediated by the JAK/STAT but not ERK pathway [
      • Fontaine R.H.
      • Cases O.
      • Lelievre V.
      • Mesples B.
      • Renauld J.C.
      • Loron G.
      • et al.
      IL-9/IL-9 receptor signaling selectively protects cortical neurons against developmental apoptosis.
      ]. The signaling pathways of IL-8 production in keratinocytes have been reported in several studies. Cells from anaplastic large cell lymphoma induce HMGB-1-dependent IL-8 production by keratinocytes via NF-κB activation [
      • Dejean E.
      • Foisseau M.
      • Lagarrigue F.
      • Lamant L.
      • Prade N.
      • Marfak A.
      • et al.
      ALK+ALCLs induce cutaneous, HMGB-1-dependent IL-8/CXCL8 production by keratinocytes through NF-kappaB activation.
      ]. UVA induces IL-8 production by AP-1 transactivation in human keratinocytes [
      • Wu S.
      • Gao J.
      • Dinh Q.T.
      • Chen C.
      • Fimmel S.
      IL-8 production and AP-1 transactivation induced by UVA in human keratinocytes: roles of D-alpha-tocopherol.
      ]. Benzopyrene induces oxidative stress-mediated interleukin-8 production in human keratinocytes via the aryl hydrocarbon receptor pathway [
      • Tsuji G.
      • Takahara M.
      • Uchi H.
      • Takeuchi S.
      • Mitoma C.
      • Moroi Y.
      • et al.
      An environmental contaminant, benzo(a)pyrene, induces oxidative stress-mediated interleukin-8 production in human keratinocytes via the aryl hydrocarbon receptor signaling pathway.
      ]. Consistent with our results that showed the role of STIM1 in ERK phosphorylation and IL-8 production in keratinocytes, the ORAI1 complex is important in the production of IL-8 by human airway epithelial cells from cystic fibrosis [
      • Balghi H.
      • Robert R.
      • Rappaz B.
      • Zhang X.
      • Wohlhuter-Haddad A.
      • Evagelidis A.
      • et al.
      Enhanced Ca2+ entry due to Orai1 plasma membrane insertion increases IL-8 secretion by cystic fibrosis airways.
      ]. The role of IL-9-ERK-IL-8 axis in epidermal keratinocytes found in the current study is consistent with a previous study of airway smooth muscle cells [
      • Baraldo S.
      • Faffe D.S.
      • Moore P.E.
      • Whitehead T.
      • McKenna M.
      • Silverman E.S.
      • et al.
      Interleukin-9 influences chemokine release in airway smooth muscle: role of ERK.
      ]. The current study is the first to reveal the role of STIM1 in the IL-9 mediated ERK phosphorylation and IL-8 production.
      This study also found that IL-4, not TGF-beta1, induced the expression of IL-9R in keratinocytes. To the best of our knowledge, no study has shown how IL-9R expression is regulated, particularly in epidermal or epithelial cells. One recent study has, however, demonstrated that IL-9 acts on Th1 and promotes IL-4 secretion in allergic contact dermatitis [
      • Liu J.
      • Harberts E.
      • Tammaro A.
      • Girardi N.
      • Filler R.B.
      • Fishelevich R.
      • et al.
      IL-9 regulates allergen-specific Th1 responses in allergic contact dermatitis.
      ], suggesting a reciprocal IL-4/IL-9 cycle in the proinflammatory conditions.
      There remains no clinical application of IL-9 neutralizing in the treatment of AD to date. However, there are application of IL-9 neutralizing antibodies in mouse models of asthma and allergic contact dermatitis (ACD). For example, transgenic expression of TSLP in mouse lungs stimulated IL-9 production in vivo while the anti-IL-9 treatment attenuated TSLP-induced airway inflammation [
      • Yao W.
      • Zhang Y.
      • Jabeen R.
      • Nguyen E.T.
      • Wilkes D.S.
      • Tepper R.S.
      • et al.
      Interleukin-9 is required for allergic airway inflammation mediated by the cytokine TSLP.
      ]. In a mouse model of ACD to nickel, in vivo allergen responses and allergen-specific IFN-γ production is enhanced in both WT mice with IL-9 neutralizing or in IL-9(−/−) mice [
      • Liu J.
      • Harberts E.
      • Tammaro A.
      • Girardi N.
      • Filler R.B.
      • Fishelevich R.
      • et al.
      IL-9 regulates allergen-specific Th1 responses in allergic contact dermatitis.
      ].
      In conclusion, we found that IL-9R is increased in AD skin and that the expressions of IL-9R in keratinocytes are enhanced by IL-4. IL-9 treatment of keratinocytes leads to the production of IL-8 through its activation STIM1 and the phosphorylation ERK, making it the first study to show the regulatory process of IL-9/IL-9R in epidermal keratinocytes. We hypothesize that IL-9-STIM1-ERK-IL-8 axis might play an important role in the pathogenesis of AD.

      Funding sources

      This study is supported by research funding provided by Ministry of Science and Technology (MOST-102-2314-B-010-005-MY2), Kaohsiung Veterans General Hospital (KSC104-050), and Kaohsiung Chang Gung Memorial Hospital (CMRPG8C0821 and CMRPG8D1541).

      Acknowledgements

      This study is supported by research funding provided by Ministry of Science and Technology ( MOST102-2314-B-010-005-MY2 ), Kaohsiung Veterans General Hospital ( KSC104-050 ), and Kaohsiung Chang Gung Memorial Hospital ( CMRPG8C0821 and CMRPG8D1541 ).

      References

        • Lee C.H.
        • Chuang H.Y.
        • Shih C.C.
        • Jong S.B.
        • Chang C.H.
        • Yu H.S.
        Transepidermal water loss, serum IgE and beta-endorphin as important and independent biological markers for development of itch intensity in atopic dermatitis.
        Br J Dermatol. 2006; 154: 1100-1107
        • Leung D.Y.
        • Bieber T.
        Atopic dermatitis.
        Lancet. 2003; 361: 151-160
        • Tokura Y.
        Extrinsic and intrinsic types of atopic dermatitis.
        J Dermatol Sci. 2010; 58: 1-7
        • Irvine A.D.
        • McLean W.H.
        • Leung D.Y.
        Filaggrin mutations associated with skin and allergic diseases.
        N Engl J Med. 2011; 365:: 1315-1327
        • Siracusa M.C.
        • Saenz S.A.
        • Hill D.A.
        • Kim B.S.
        • Headley M.B.
        • Doering T.A.
        • et al.
        TSLP promotes interleukin-3-independent basophil haematopoiesis and type 2 inflammation.
        Nature. 2011; 477: 229-233
        • Lee C.H.
        • Hong C.H.
        • Yu W.T.
        • Chuang H.Y.
        • Huang S.K.
        • Chen G.S.
        • et al.
        Mechanistic correlations between two itch biomarkers, cytokine interleukin-31 and neuropeptide beta-endorphin, via STAT3/calcium axis in atopic dermatitis.
        Br J Dermatol. 2012; 167: 794-803
        • Veldhoen M.
        • Uyttenhove C.
        • van Snick J.
        • Helmby H.
        • Westendorf A.
        • Buer J.
        • et al.
        Transforming growth factor-beta ‘reprograms’ the differentiation of T helper 2 cells and promotes an interleukin 9-producing subset.
        Nat Immunol. 2008; 9: 1341-1346
        • Soroosh P.
        • Doherty T.A.
        Th9 and allergic disease.
        Immunology. 2009; 127: 450-458
        • Chang H.C.
        • Sehra S.
        • Goswami R.
        • Yao W.
        • Yu Q.
        • Stritesky G.L.
        • et al.
        The transcription factor PU.1 is required for the development of IL-9-producing T cells and allergic inflammation.
        Nat Immunol. 2010; 11: 527-534
        • Staudt V.
        • Bothur E.
        • Klein M.
        • Lingnau K.
        • Reuter S.
        • Grebe N.
        • et al.
        Interferon-regulatory factor 4 is essential for the developmental program of T helper 9 cells.
        Immunity. 2010; 33: 192-202
        • Little F.F.
        • Cruikshank W.W.
        • Center D.M.
        Il-9 stimulates release of chemotactic factors from human bronchial epithelial cells.
        Am J Respir Cell Mol Biol. 2001; 25: 347-352
        • Dragon S.
        • Takhar M.K.
        • Shan L.
        • Hayglass K.T.
        • Simons F.E.
        • Gounni A.S.
        T(H)2 cytokines modulate the IL-9R expression on human neutrophils.
        Biochem Biophys Res Commun. 2009; 384: 167-172
        • Sismanopoulos N.
        • Delivanis D.A.
        • Alysandratos K.D.
        • Angelidou A.
        • Vasiadi M.
        • Therianou A.
        • et al.
        IL-9 induces VEGF secretion from human mast cells and IL-9/IL-9 receptor genes are overexpressed in atopic dermatitis.
        PLoS ONE. 2012; 7: e33271
        • Fawaz L.M.
        • Sharif-Askari E.
        • Hajoui O.
        • Soussi-Gounni A.
        • Hamid Q.
        • Mazer B.D.
        Expression of IL-9 receptor alpha chain on human germinal center B cells modulates IgE secretion.
        J Allergy Clin Immunol. 2007; 120: 1208-1215
        • Gounni A.S.
        • Hamid Q.
        • Rahman S.M.
        • Hoeck J.
        • Yang J.
        • Shan L.
        IL-9-mediated induction of eotaxin1/CCL11 in human airway smooth muscle cells.
        J Immunol. 2004; 173: 2771-2779
        • Purwar R.
        • Schlapbach C.
        • Xiao S.
        • Kang H.S.
        • Elyaman W.
        • Jiang X.
        • et al.
        Robust tumor immunity to melanoma mediated by interleukin-9-producing T cells.
        Nat Med. 2012;
        • Singh T.P.
        • Schon M.P.
        • Wallbrecht K.
        • Gruber-Wackernagel A.
        • Wang X.J.
        • Wolf P.
        Involvement of IL-9 in Th17-associated inflammation and angiogenesis of psoriasis.
        PLoS ONE. 2013; 8: e51752
        • Temann U.A.
        • Geba G.P.
        • Rankin J.A.
        • Flavell R.A.
        Expression of interleukin 9 in the lungs of transgenic mice causes airway inflammation, mast cell hyperplasia, and bronchial hyperresponsiveness.
        J Exp Med. 1998; 188: 1307-1320
        • Temann U.A.
        • Ray P.
        • Flavell R.A.
        Pulmonary overexpression of IL-9 induces Th2 cytokine expression, leading to immune pathology.
        J Clin Invest. 2002; 109: 29-39
        • Cheng G.
        • Arima M.
        • Honda K.
        • Hirata H.
        • Eda F.
        • Yoshida N.
        • et al.
        Anti-interleukin-9 antibody treatment inhibits airway inflammation and hyperreactivity in mouse asthma model.
        Am J Respir Crit Care Med. 2002; 166: 409-416
        • Vermeer P.D.
        • Harson R.
        • Einwalter L.A.
        • Moninger T.
        • Zabner J.
        Interleukin-9 induces goblet cell hyperplasia during repair of human airway epithelia.
        Am J Respir Cell Mol Biol. 2003; 28: 286-295
        • Toda M.
        • Tulic M.K.
        • Levitt R.C.
        • Hamid Q.
        A calcium-activated chloride channel (HCLCA1) is strongly related to IL-9 expression and mucus production in bronchial epithelium of patients with asthma.
        J Allergy Clin Immunol. 2002; 109: 246-250
        • Tsicopoulos A.
        • Shimbara A.
        • de Nadai P.
        • Aldewachi O.
        • Lamblin C.
        • Lassalle P.
        • et al.
        Involvement of IL-9 in the bronchial phenotype of patients with nasal polyposis.
        J Allergy Clin Immunol. 2004; 113: 462-469
        • Wang T.N.
        • Chen W.Y.
        • Huang Y.F.
        • Shih N.H.
        • Feng W.W.
        • Tseng H.I.
        • et al.
        The synergistic effects of the IL-9 gene and environmental exposures on asthmatic Taiwanese families as determined by the transmission/disequilibrium test.
        Int J Immunogenet. 2006; 33: 105-110
        • Ciprandi G.
        • De Amici M.
        • Giunta V.
        • Marseglia A.
        • Marseglia G.
        Serum interleukin-9 levels are associated with clinical severity in children with atopic dermatitis.
        Pediatr Dermatol. 2012;
        • Namkung J.H.
        • Lee J.E.
        • Kim E.
        • Park G.T.
        • Yang H.S.
        • Jang H.Y.
        • et al.
        An association between IL-9 and IL-9 receptor gene polymorphisms and atopic dermatitis in a Korean population.
        J Dermatol Sci. 2011; 62: 16-21
        • Lee C.H.
        • Wu S.B.
        • Hong C.H.
        • Chen G.S.
        • Wei Y.H.
        • Yu H.S.
        Involvement of mtDNA damage elicited by oxidative stress in the arsenical skin cancers.
        J Invest Dermatol. 2013;
        • Lee C.H.
        • Hong C.H.
        • Chen Y.T.
        • Chen Y.C.
        • Shen M.R.
        TGF-beta1 increases cell rigidity by enhancing expression of smooth muscle actin: keloid-derived fibroblasts as a model for cellular mechanics.
        J Dermatol Sci. 2012; 67: 173-180
        • Lee C.H.
        • Wu S.B.
        • Hong C.H.
        • Liao W.T.
        • Wei Y.H.
        • Yu H.S.
        Aberrant cell proliferation by mitochondrial biogenesis and mtTFA in arsenical cancers.
        Am J Pathol. 2013; 133: 1890-1900
        • Yamasaki A.
        • Saleh A.
        • Koussih L.
        • Muro S.
        • Halayko A.J.
        • Gounni A.S.
        IL-9 induces CCL11 expression via STAT3 signalling in human airway smooth muscle cells.
        PLoS ONE. 2010; 5: e9178
        • Kimata H.
        • Lindley I.
        Detection of plasma interleukin-8 in atopic dermatitis.
        Arch Dis Child. 1994; 70: 119-122
        • Amarbayasgalan T.
        • Takahashi H.
        • Dekio I.
        • Morita E.
        Interleukin-8 content in the stratum corneum as an indicator of the severity of inflammation in the lesions of atopic dermatitis.
        Int Arch Allergy Immunol. 2013; 160: 63-74
        • Casas C.
        • Ribet V.
        • Alvarez-Georges S.
        • Sibaud V.
        • Guerrero D.
        • Schmitt A.M.
        • et al.
        Modulation of Interleukin-8 and staphylococcal flora by Avene hydrotherapy in patients suffering from chronic inflammatory dermatoses.
        J Eur Acad Dermatol Venereol. 2011; 25: 19-23
        • Kuchler S.
        • Henkes D.
        • Eckl K.M.
        • Ackermann K.
        • Plendl J.
        • Korting H.C.
        • et al.
        Hallmarks of atopic skin mimicked in vitro by means of a skin disease model based on FLG knock-down.
        Altern Lab Anim. 2011; 39: 471-480
        • Kezic S.
        • O’Regan G.M.
        • Lutter R.
        • Jakasa I.
        • Koster E.S.
        • Saunders S.
        • et al.
        Filaggrin loss-of-function mutations are associated with enhanced expression of IL-1 cytokines in the stratum corneum of patients with atopic dermatitis and in a murine model of filaggrin deficiency.
        J Allergy Clin Immunol. 2012; 129 (1031–9.e1)
        • Neuber K.
        • Hilger R.A.
        • Konig W.
        Interleukin-3, interleukin-8, FMLP and C5a enhance the release of leukotrienes from neutrophils of patients with atopic dermatitis.
        Immunology. 1991; 73: 83-87
        • Ohnishi H.
        • Miyahara N.
        • Gelfand E.W.
        The role of leukotriene B(4) in allergic diseases.
        Allergol Int. 2008; 57: 291-298
        • Bonecchi R.
        • Facchetti F.
        • Dusi S.
        • Luini W.
        • Lissandrini D.
        • Simmelink M.
        • et al.
        Induction of functional IL-8 receptors by IL-4 and IL-13 in human monocytes.
        J Immunol. 2000; 164: 3862-3869
        • McGirt L.Y.
        • Beck L.A.
        Innate immune defects in atopic dermatitis.
        J Allergy Clin Immunol. 2006; 118: 202-208
        • Fontaine R.H.
        • Cases O.
        • Lelievre V.
        • Mesples B.
        • Renauld J.C.
        • Loron G.
        • et al.
        IL-9/IL-9 receptor signaling selectively protects cortical neurons against developmental apoptosis.
        Cell Death Differ. 2008; 15: 1542-1552
        • Dejean E.
        • Foisseau M.
        • Lagarrigue F.
        • Lamant L.
        • Prade N.
        • Marfak A.
        • et al.
        ALK+ALCLs induce cutaneous, HMGB-1-dependent IL-8/CXCL8 production by keratinocytes through NF-kappaB activation.
        Blood. 2012; 119: 4698-4707
        • Wu S.
        • Gao J.
        • Dinh Q.T.
        • Chen C.
        • Fimmel S.
        IL-8 production and AP-1 transactivation induced by UVA in human keratinocytes: roles of D-alpha-tocopherol.
        Mol Immunol. 2008; 45: 2288-2296
        • Tsuji G.
        • Takahara M.
        • Uchi H.
        • Takeuchi S.
        • Mitoma C.
        • Moroi Y.
        • et al.
        An environmental contaminant, benzo(a)pyrene, induces oxidative stress-mediated interleukin-8 production in human keratinocytes via the aryl hydrocarbon receptor signaling pathway.
        J Dermatol Sci. 2011; 62: 42-49
        • Balghi H.
        • Robert R.
        • Rappaz B.
        • Zhang X.
        • Wohlhuter-Haddad A.
        • Evagelidis A.
        • et al.
        Enhanced Ca2+ entry due to Orai1 plasma membrane insertion increases IL-8 secretion by cystic fibrosis airways.
        FASEB J. 2011; 25: 4274-4291
        • Baraldo S.
        • Faffe D.S.
        • Moore P.E.
        • Whitehead T.
        • McKenna M.
        • Silverman E.S.
        • et al.
        Interleukin-9 influences chemokine release in airway smooth muscle: role of ERK.
        Am J Physiol Lung Cell Mol Physiol. 2003; 284: L1093-L1102
        • Liu J.
        • Harberts E.
        • Tammaro A.
        • Girardi N.
        • Filler R.B.
        • Fishelevich R.
        • et al.
        IL-9 regulates allergen-specific Th1 responses in allergic contact dermatitis.
        J Invest Dermatol. 2014;
        • Yao W.
        • Zhang Y.
        • Jabeen R.
        • Nguyen E.T.
        • Wilkes D.S.
        • Tepper R.S.
        • et al.
        Interleukin-9 is required for allergic airway inflammation mediated by the cytokine TSLP.
        Immunity. 2013; 38: 360-372
        • Liu J.
        • Harberts E.
        • Tammaro A.
        • Girardi N.
        • Filler R.B.
        • Fishelevich R.
        • et al.
        IL-9 regulates allergen-specific Th1 responses in allergic contact dermatitis.
        J Invest Dermatol. 2014; 134: 1903-1911