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Amphiregulin from Basophils Amplifies Basophil-Mediated Chronic Skin Inflammation

Open ArchivePublished:March 13, 2019DOI:https://doi.org/10.1016/j.jid.2019.02.023

      Abbreviations:

      AD (atopic dermatitis), AREG (amphiregulin), BMBas (bone marrow-derived basophils), DT (diphtheria toxin), DTR (diphtheria toxin receptor), OVA (ovalbumin), rAREG (recombinant amphiregulin), siRNA (small interfering RNA), TNP (2,4,6-trinitrophenyl), WT (wild type)
      To the Editor
      A large number of basophils are observed in the lesions of chronic allergic skin diseases such as atopic dermatitis (AD) and prurigo (
      • Ito Y.
      • Satoh T.
      • Takayama K.
      • Miyagishi C.
      • Walls A.F.
      • Yokozeki H.
      Basophil recruitment and activation in inflammatory skin diseases.
      ). Evidence suggests that basophils are capable of provoking T helper type 2-mediated inflammation, and thus, basophils are inferred to play a pivotal role in chronic allergic skin inflammation (
      • Karasuyama H.
      • Miyake K.
      • Yoshikawa S.
      • Yamanishi Y.
      Multifaceted roles of basophils in health and disease.
      ). However, the molecules/mediators involved in basophil-dependent skin inflammation have yet to be elucidated.
      A murine model of prurigo reaction, one of the models of basophil-dependent chronic skin inflammation, features marked eosinophil and basophil infiltration and increased local production of amphiregulin (AREG), an epidermal growth factor-like molecule (
      • Hashimoto T.
      • Satoh T.
      • Yokozeki H.
      Protective role of STAT6 in basophil-dependent prurigo-like allergic skin inflammation.
      ). AREG is produced principally by epithelial cells, but the factor is also generated by various immune cells, including basophils (
      • Meulenbroeks C.
      • Van Weelden H.
      • Schwartz C.
      • Voehringer D.
      • Redegeld F.A.M.
      • Rutten V.P.M.G.
      • et al.
      Basophil-derived amphiregulin is essential for UVB irradiation-induced immune suppression.
      ,
      • Qi Y.
      • Operario D.J.
      • Oberholzer C.M.
      • Kobie J.J.
      • Looney R.J.
      • Georas S.N.
      • et al.
      Human basophils express amphiregulin in response to T cell-derived IL-3.
      ), and has been implicated in T helper type 2 inflammation (
      • Zaiss D.M.W.
      • Gause W.C.
      • Osborne L.C.
      • Artis D.
      Emerging functions of amphiregulin in orchestrating immunity, inflammation, and tissue repair.
      ). Here, we assessed the contribution of basophil-derived AREG in murine models of prurigo-like skin inflammation and AD. All animal experiments were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University.
      A mouse model of prurigo-like skin inflammation has been described previously (
      • Hashimoto T.
      • Satoh T.
      • Yokozeki H.
      Protective role of STAT6 in basophil-dependent prurigo-like allergic skin inflammation.
      ); see also the Supplementary Materials online. Repeated local injection of 2,4,6-trinitrophenyl-ovalbumin (TNP-OVA) into the dorsal skin of mice passively immunized with TNP-IgE has been shown to induce prurigo-like nodules. In this study, however, skin inflammation was induced on the ear lobes to permit objective assessment of the magnitude of changes in inflammation. We initially assessed the in vivo effects of AREG small interfering RNA (siRNA) on prurigo-like skin inflammation. Inhibition of local AREG production by repeated administration of the siRNA resulted in a partial, but significant, attenuation of skin responses. Histological findings showed ameliorated infiltration of inflammatory cells and improvement of acanthosis (Figure 1a). We then administered recombinant AREG (rAREG) intradermally into the inflamed ear lobes. rAREG enhanced ear swelling responses in prurigo-like skin inflammation (Figure 1b). In contrast, rAREG failed to elicit inflammation in naive skin (see Supplementary Figure S1 online).
      Figure thumbnail gr1
      Figure 1Amphiregulin amplifies prurigo-like skin inflammation. (a) siRNA inhibition of AREG expression attenuated ear swelling responses (upper right panel) concomitant with reduced local production of AREG (lower left panel). Histological findings showed alleviation of dermal inflammation and decreased epidermal thickening (lower right panels, Giemsa staining). Scale bar = 100 μm. P < 0.05 versus scramble siRNA-treated mice. (b) Daily administration of rAREG into the ear lobes enhanced ear swelling responses. P < 0.05. (c) Immunofluorescence staining. TUG8+ basophils expressed AREG in the lesional skin. Scale bar = 10 μm. (d) Quantitative real-time reverse transcription–PCR assays showed that IL-3–elicited BMBas and TSLP-elicited BMBas accumulated AREG mRNA to high levels. P < 0.05 versus medium-treated BMBas. (e) DT-induced basophil depletion attenuated prurigo-like skin inflammation in Mcpt8DTR mice. Adaptive transfer of scramble siRNA-treated basophils into the ear lobes, but not that of AREG siRNA-treated basophils, restored ear swelling responses. P < 0.05. The data shown are representative of the results of at least two independent experiments with at least four mice per group. Vertical bars indicate standard deviation. AREG, amphiregulin; BMBas, bone marrow-derived basophils; DT, diphtheria toxin; OVA, ovalbumin; rAREG, recombinant amphiregulin; S.D., standard deviation; siRNA, small interfering RNA; TNP, 2,4,6-trinitrophenyl.
      We next focused on basophils as a potential cellular source of AREG. In the lesional skin of prurigo-like skin inflammation, basophils expressed AREG (Figure 1c). To confirm the ability of basophils to secrete AREG, we investigated the mRNA expression of AREG in IL-3– and TSLP-elicited ex vivo bone marrow-derived basophils (BMBas) (
      • 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.
      ). Both IL-3– and TSLP-elicited BMBas showed higher expression of AREG after exposure to IL-33 or when stimulated with IgE plus IL-3 or TSLP (Figure 1d).
      To assess the contribution of basophil-derived AREG to prurigo-like skin inflammation, basophil-depleted mice were reconstituted with basophils derived from wild-type (WT) mice that had been treated with AREG siRNA. Basophils from Mcpt8DTR mice (BALB/c background) specifically express diphtheria toxin (DT) receptors, and thus DT treatment selectively and inducibly abolishes basophils in those mice (see Supplementary Figure S2 online) (
      • Wada T.
      • Ishiwata K.
      • Koseki H.
      • Ishikura T.
      • Ugajin T.
      • Ohnuma N.
      • et al.
      Selective ablation of basophils in mice reveals their nonredundant role in acquired immunity against ticks.
      ), which were used as the recipient animals in this experiment. DT-induced basophil depletion attenuated prurigo-like skin inflammation in Mcpt8DTR mice (Figure 1e), indicating that basophils contribute to this inflammation. The CD49b+ basophil-enriched cell fraction of spleen cells (primary basophils) from WT mice was adoptively transferred into DT-treated Mcpt8DTR mice on day –1, and prurigo-like skin inflammation was elicited starting on day 0. Reconstitution of basophil-depleted Mcpt8DTR mice with scramble siRNA-treated wild-type (WT) primary basophils restored the inflammation. However, this restoration was abolished when basophil-depleted Mcpt8DTR mice were reconstituted with AREG siRNA-treated primary basophils. Thus, AREG derived from basophils appears to contribute, at least in part, to prurigo-like skin inflammation in mice.
      We next examined the involvement of basophil-derived AREG in an MC903-induced murine model of AD, in which basophils mediate inflammation (
      • Li M.
      • Hener P.
      • Zhang Z.
      • Kato S.
      • Metzger D.
      • Chambon P.
      Topical vitamin D3 and low-calcemic analogs induce thymic stromal lymphopoietin in mouse keratinocytes and trigger an atopic dermatitis.
      ) (see Supplementary Materials). Administration of AREG siRNA attenuated ear-swelling responses concomitantly with the inhibition of AREG generation (Figure 2a). These effects were accompanied by decreased numbers of dermal infiltrating cells and reduced epidermal thickness (Figure 2a). On the other hand, lesional administration of rAREG in the inflamed skin exacerbated ear swelling responses (Figure 2b). We observed AREG-expressing basophils in the skin lesions of the MC903-induced murine AD model (Figure 2c).
      Figure thumbnail gr2
      Figure 2Amphiregulin amplifies MC903-induced skin inflammation. (a) siRNA inhibition of AREG ameliorated MC903-induced skin inflammation as assessed by ear swelling responses (upper right panel) concomitant with decreased local production of AREG (lower left panel). Histological findings showed alleviation of dermal inflammation and decreased epidermal thickening (lower right panels, Giemsa staining). Scale bar = 100 μm. P < 0.05 versus scramble siRNA-treated mice. (b) Daily administration of rAREG into the ear lobes enhanced ear swelling responses. P < 0.05. (c) Immunofluorescence staining. TUG8+ basophils expressed AREG. Scale bar = 10 μm. (d) DT-induced basophil depletion attenuated skin inflammation. Adaptive transfer of scramble siRNA-treated cells into the ear lobes amplified ear-swelling responses compared with AREG siRNA-treated cells. Vertical bars indicate standard deviation. P < 0.05. The data shown are representative of the results of at least two independent experiments with at least four mice per group. Vertical bars indicate standard deviation. AREG, amphiregulin; DT, diphtheria toxin; rAREG, recombinant amphiregulin; siRNA, small interfering RNA.
      We then attempted to elicit MC903-induced inflammation in basophil-depleted Mcpt8DTR mice reconstituted with AREG-siRNA–treated WT primary basophils. Basophil depletion with DT in Mcpt8DTR mice (on day –1) ameliorated MC903-induced inflammation (Figure 2d). Adoptive transfer of the scramble siRNA-treated, but not AREG siRNA-treated, primary basophils partially restored MC903-induced inflammation (Figure 2d). Collectively, these results indicate that basophil-derived AREG seems to play a role in MC903-induced basophil-mediated skin inflammation, as seen in prurigo-like skin inflammation.
      In this work, we showed that basophils contribute to inflammation in murine models of prurigo and AD via the release of AREG. Previous research has shown that IL-3–elicited basophils are activated by FcεRI crosslinking to release cytokines/chemokines, and TSLP-elicited basophils respond to IL-18 and IL-33 (
      • 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.
      ). In this study, IL-3–elicited BMBas showed higher levels of AREG when stimulated with IgE than did TSLP-elicited BMBas. In contrast, TSLP-elicited BMBas were more responsive to IL-33 than were IL-3–elicited BMBas. The types of basophils that develop in the murine models of prurigo and AD are uncertain. However, the immunological environment in these models appeared to be ideal for AREG generation irrespective of the types of basophil, because both of these models showed increased local production of IL-33 and TSLP, along with elevated serum IgE (
      • Hashimoto T.
      • Satoh T.
      • Yokozeki H.
      Protective role of STAT6 in basophil-dependent prurigo-like allergic skin inflammation.
      ,
      • Li M.
      • Hener P.
      • Zhang Z.
      • Kato S.
      • Metzger D.
      • Chambon P.
      Topical vitamin D3 and low-calcemic analogs induce thymic stromal lymphopoietin in mouse keratinocytes and trigger an atopic dermatitis.
      ,
      • Li C.
      • Maillet I.
      • Mackowiak C.
      • Viala C.
      • Di Padova F.
      • Li M.
      • et al.
      Experimental atopic dermatitis depends on IL-33R signaling via MyD88 in dendritic cells.
      ).
      The relationship between AREG and immunological processes in chronic skin inflammation is still uncertain. Epidermal proliferation induced by AREG (
      • Pastore S.
      • Mascia F.
      • Mariani V.
      • Girolomoni G.
      The epidermal growth factor receptor system in skin repair and inflammation.
      ) may contribute to increased generation of IL-33 and TSLP from epidermal cells. Nonetheless, our results indicate that basophil-derived AREG may be a therapeutic target for chronic skin inflammation, including that occurring in prurigo and AD.

      Data availability statement

      Data sets related to this article can be found at https://doi.org/10.17632/yy5nkxkftr.1, an open-source online data repository hosted at Mendeley Data.

      ORCIDs

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      The authors thank Chiyako Miyagishi for technical assistance. This work was supported by the Japan Society for the Promotion of Science KAKENHI Grant-in-Aid for Young Scientists (B) (no. 17K16328) and by a GlaxoSmithKline Japan Research Grant 2016.

      Author Contributions

      TH: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, validation, visualization, and writing (the draft). TS: methodology, project administration, supervision, and writing (review and editing). HK: methodology, resources, supervision, and writing (review and editing). HY: funding acquisition, project administration, supervision, and writing (review and editing). All authors have read the manuscript and approved this submission.

      Supplementary Materials and Methods

      Mice

      Six- to 9-week-old male and female C57BL/6 mice, BALB/c mice, and BALB/c-nu/nu mice were obtained from Sankyo Lab Service (Tokyo, Japan). Mast cell protease 8-diphtheria toxin receptor (Mcpt8DTR) mice (BALB/c background) were generated as described previously (
      • Wada T.
      • Ishiwata K.
      • Koseki H.
      • Ishikura T.
      • Ugajin T.
      • Ohnuma N.
      • et al.
      Selective ablation of basophils in mice reveals their nonredundant role in acquired immunity against ticks.
      ). Both male and female mice showed similar results in the experiments of this work. 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).

      Basophil depletion

      Mcpt8DTR mice were administered DT intravenously (750 ng/20 g body weight) (Sigma-Aldrich, St. Louis, MO) as described previously (
      • Wada T.
      • Ishiwata K.
      • Koseki H.
      • Ishikura T.
      • Ugajin T.
      • Ohnuma N.
      • et al.
      Selective ablation of basophils in mice reveals their nonredundant role in acquired immunity against ticks.
      ).

      Preparation of TNP-specific IgE (TNP-IgE)

      TNP-IgE was collected from ascites of BALB/c-nu/nu mice by intraperitoneal injection of IgELb4 cell hybridoma (American Type Culture Collection, Manassas, VA). Supernatants were precipitated with 55% saturated ammonium sulfate, followed by phosphate buffered saline(–) dialysis. The concentration of IgE was determined with an ELISA kit (Yamasa Co., Tokyo, Japan) as described previously (
      • Hashimoto T.
      • Satoh T.
      • Yokozeki H.
      Protective role of STAT6 in basophil-dependent prurigo-like allergic skin inflammation.
      ).

      Induction of prurigo-like skin inflammation

      Mice were passively immunized with TNP-IgE (120 μg/mouse) on days –1, 2, and 5, followed by subcutaneous administration of TNP(12)-OVA (100 μg/site; Biosearch Technologies, Petaluma, CA) into the ear lobes on days 0, 3, and 6 (
      • Hashimoto T.
      • Satoh T.
      • Yokozeki H.
      Protective role of STAT6 in basophil-dependent prurigo-like allergic skin inflammation.
      ).

      MC903-induced murine model of AD

      MC903 (calcipotriol) (AdooQ Bioscience, Irvine, CA) was dissolved in ethanol (2 nmol/25 μL) and applied to the ear lobes once per day for 7 days (
      • Li M.
      • Hener P.
      • Zhang Z.
      • Kato S.
      • Metzger D.
      • Chambon P.
      Topical vitamin D3 and low-calcemic analogs induce thymic stromal lymphopoietin in mouse keratinocytes and trigger an atopic dermatitis.
      ). On day 7, animals were euthanized, and ear samples were collected.

      Antibodies

      Anti-amphiregulin antibody (H-155) was obtained from Santa Cruz Biotechnology (Dallas, TX). Anti-mouse basophil-specific antibody (TUG8) was purchased from BioLegend (San Diego, CA)

      Immunofluorescence staining

      Formalin-fixed paraffin-embedded samples (5-μm-thick sections) were pretreated with proteinase K (Dako, Glostrup, Denmark). The treated sections then were incubated with a combination of the indicated antibodies, followed by a reaction with Alexa Fluor 488- or 568-conjugated secondary antibodies (Abcam) and mounted with Fluoroshield with DAPI (GeneTex, TX). Photomicrographs were captured with a TCS SP8 confocal microscope (Leica Microsystems, Tokyo, Japan).

      Measurement of cytokines

      The 8-mm-diameter skin punch samples were homogenized with 200 μl of phosphate buffered saline supplemented with 1% proteinase inhibitor cocktail (Sigma-Aldrich) and then centrifuged at 10,000g for 10 minutes at 4 °C. Levels of AREG in the supernatants of homogenates or of cell cultures were quantified by sandwich ELISA (R&D Systems, Minneapolis, MN).

      Preparation of BMBas

      BMBas were prepared by culturing bone marrow cells collected from 8-week-old C57BL/6 N mice in RPMI 1640 complete medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin-streptomycin, and 10 ng/ml recombinant IL-3, or 500 ng/ml recombinant TSLP (eBioscience, San Diego, CA), and incubated for 10 days at 37 °C and 5% CO2. Then, CD49b+ cells were isolated by the use of a magnetically activated cell sorter system with biotinylated anti-CD49b antibody and streptavidin microbeads (Miltenyi Biotec, Auburn, CA).

      Stimulation of BMBas

      For IgE-mediated stimulation, cells were sensitized with 1 μg/ml TNP-IgE for 1 hour with or without 10 ng/ml IL-3 or 500 ng/mL TSLP, then washed, and stimulated with 20 ng/ml TNP(12)-OVA for 10 hours. BMBas also were incubated with recombinant mouse IL-3 (10 ng/ml), TSLP (500 ng/ml), or IL-33 (10 ng/ml) (eBioscience) for 10 hours. Then, the cells were subjected to total RNA extraction as described below.

      Preparation of the CD49b+ basophil-enriched cell fraction from splenocytes

      Spleen cells were collected from 8-week-old WT BALB/c mice, and CD49b+ cells were isolated by the use of a magnetically activated cell sorter system as described.

      In vivo and ex vivo transfection with AREG siRNA

      For in vivo transfection with AREG or scramble siRNA, siRNAs were prepared for transfection using cationic liposomes (Lipofectamine 3000, Life Technologies, Carlsbad, CA), according to the manufacturer’s instructions. Mice were transfected with siRNA by injection into each ear lobe of 0.5 nmol/30 μl/ear diluted with Life Technologies Opti-MEM I (Invitrogen, Waltham, MA)/Lipofectamine 3000 at a ratio of 50:1
      For ex vivo transfection, siRNA was transfected into CD49b+ cells by using GenomOne-Si (Ishihara Sangyo Kaisha, Osaka, Japan) in RPMI 1640 complete medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin-streptomycin, and IL-3 (10 ng/ml) in conjunction with TSLP (500 ng/ml) for 24 hours.
      The nucleotide sequences of the sense and antisense strands of mouse AREG siRNA were 5′-GGACCUAUCCAAGAUUGCA-3′ and 5′-UGCAAUCUUGGAUAGGUCC-3′, respectively. The scramble siRNA used as a negative control was constituted from the following two primers: 5′-CCUACGCCACCAAUUUCGU-3′ (sense strand) and 5′-ACGAAAUUGGUGGCGUAGG-3′ (antisense strand) (
      • Wada T.
      • Ishiwata K.
      • Koseki H.
      • Ishikura T.
      • Ugajin T.
      • Ohnuma N.
      • et al.
      Selective ablation of basophils in mice reveals their nonredundant role in acquired immunity against ticks.
      ).

      Real-time PCR

      Total cellular RNA was extracted from cells using ISOGEN II (Nippon Gene Co., Tokyo, Japan) and reverse-transcribed with SuperScript IV VILO Master Mix with ezDNase enzyme (Thermo Fisher Scientific, Waltham MA). Then quantitative real-time PCR was performed by real-time monitoring of the increase in fluorescence of SYBR Green dye (Brilliant SYBR Green QPCR Master Mix) by using an AriaMx Real-Time PCR System (both from Agilent Technologies Japan, Tokyo, Japan). The primers used for PCR were 5′-GCCATTATGCAGCTGCTTTGGAGC-3′ and 5′- TGTTTTTCTTGGGCTTAATCACCT-3′ for mouse AREG, and 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′ for mouse GAPDH. The levels of mRNA expression were calculated by the comparative ΔΔCt method relative to GAPDH, a housekeeping gene.

      Statistics

      The Student t test was used to determine the statistical significance of differences between means. Results were considered significant at P < 0.05.
      Figure thumbnail fx1
      Supplementary Figure S1Effects of rAREG on uninflamed skin. Injection of rAREG subcutaneously into the ear lobes of naive mice did not provoke inflammation. Each group contains at least five mice. Results are shown as mean ± standard deviation. Statistical analysis was performed with a Student t test. No significant differences were observed. rAREG, recombinant amphiregulin; S.D., standard deviation.
      Figure thumbnail fx2
      Supplemental Figure S2Immunofluorescence staining for basophils of the prurigo-like skin lesion in Mcpt8DTR mice. TUG8+ basophils in (a) vehicle-treated mice were completely abrogated by the (b) DT treatment. Dotted line indicates the basement membrane. Scale bars = 5 μm (left panel) and 25 μm (right panel). DT, diphtheria.

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