Differential cytokine induction by the human skin–associated autoallergen thioredoxin in sensitized patients with atopic dermatitis and healthy control subjects
2014; Elsevier BV; Volume: 135; Issue: 5 Linguagem: Inglês
10.1016/j.jaci.2014.10.038
ISSN1097-6825
AutoresSusanne Hradetzky, Lennart M. Roesner, Annice Heratizadeh, Reto Crameri, Mattia Garbani, Annika Scheynius, Thomas Werfel,
Tópico(s)Allergic Rhinitis and Sensitization
ResumoThe term autoallergy describes IgE reactivity to autoantigens, which can be found in a subgroup of patients with atopic dermatitis (AD).1Valenta R. Mittermann I. Werfel T. Garn H. Renz H. Linking allergy to autoimmune disease.Trends Immunol. 2009; 30: 109-116Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar Thioredoxin belongs to the group of autoallergens that are also described as panallergens because these evolutionary highly conserved proteins are present in many allergenic sources. Human thioredoxin (hTrx) is a homologue of the Malassezia sympodialis allergen Mala s 13, and both proteins have been shown to cross-react at the IgE level in vitro, as well as to induce immediate and late-phase skin reactions in vivo.2Limacher A. Glaser A.G. Meier C. Schmid-Grendelmeier P. Zeller S. Scapozza L. et al.Cross-reactivity and 1.4-A crystal structure of Malassezia sympodialis thioredoxin (Mala s 13), a member of a new pan-allergen family.J Immunol. 2007; 178: 389-396Crossref PubMed Google Scholar Because as many as 50% of adults with AD are sensitized to M sympodialis and this sensitization has been associated with more severe phenotypes of AD,3Gaitanis G. Magiatis P. Hantschke M. Bassukas I.D. Velegraki A. The Malassezia genus in skin and systemic diseases.Clin Microbiol Rev. 2012; 25: 106-141Crossref PubMed Scopus (444) Google Scholar autosensitization to hTrx might have a significant effect on exacerbation of the disease. We have recently shown cross-reactivity between Mala s 13 and hTrx at the T-cell level.4Balaji H. Heratizadeh A. Wichmann K. Niebuhr M. Crameri R. Scheynius A. et al.Malassezia sympodialis thioredoxin-specific T cells are highly cross-reactive to human thioredoxin in atopic dermatitis.J Allergy Clin Immunol. 2011; 128: 92-99.e4Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar Proliferation to Mala s 13 or hTrx could only be induced in T-cell lines generated in the presence of Mala s 13 from PBMCs of patients with AD who were sensitized to Malassezia species but not from nonsensitized patients with AD, patients with psoriasis, or healthy control subjects. The cross-reactive T-cell lines were mainly CD4+, and all were positive for the skin-homing receptor cutaneous lymphocyte antigen. They could be assigned to the T-cell subsets TH1, TH2, TH17, and TH22. Furthermore, release of hTrx from primary keratinocytes was induced by stimulation with IFN-γ, which might exacerbate atopic skin inflammation in sensitized patients through activation of hTrx-specific T cells.4Balaji H. Heratizadeh A. Wichmann K. Niebuhr M. Crameri R. Scheynius A. et al.Malassezia sympodialis thioredoxin-specific T cells are highly cross-reactive to human thioredoxin in atopic dermatitis.J Allergy Clin Immunol. 2011; 128: 92-99.e4Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar The present study aimed to compare cytokine induction by hTrx and Mala s 13 in circulating mononuclear cells from patients with AD who were sensitized or not to Malassezia species and from healthy donors. Patients' characteristics are listed in Table E1 in this article's Online Repository at www.jacionline.org. The TH2 cytokine IL-13 was detected at significantly higher levels in supernatants of hTrx-stimulated PMBCs from patients with AD who were sensitized to Malassezia species compared with those from healthy donors, which was not the case for patients with AD who were not sensitized to Malassezia species (Fig 1, A). Levels of the TH1 cytokine IFN-γ were significantly increased in supernatants from control PBMCs stimulated with Mala s 13, and a similar trend was observed for hTrx stimulation compared with levels seen in patients with AD who were not sensitized to Malassezia species (Fig 1, A). A number of other innate or T-cell cytokines (eg, IL-12p40 and IL-17) were induced efficiently both by hTrx and Mala s 13 in PBMCs from both patients with AD and healthy donors (Hradetzky et al, manuscript in preparation). Compared with α-chain of the nascent polypeptide-associated complex (α-NAC), another autoallergen whose cytokine effects we have studied before,E1Hradetzky S. Balaji H. Roesner L.M. Heratizadeh A. Mittermann I. Valenta R. et al.The human skin-associated autoantigen α-NAC activates monocytes and dendritic cells via TLR-2 and primes an IL-12-dependent Th1 response.J Invest Dermatol. 2013; 133: 2289-2292Crossref PubMed Scopus (14) Google Scholar, E2Hradetzky S. Roesner L.M. Balaji H. Heratizadeh A. Mittermann I. Valenta R. et al.Cytokine effects induced by the human autoallergen α-NAC.J Invest Dermatol. 2014; 134: 1570-1578Crossref PubMed Scopus (27) Google Scholar hTrx induced secretion of IFN-γ and IL-10 more efficiently (see Fig E1 in this article's Online Repository at www.jacionline.org). The most prominent difference regarding hTrx-induced cytokine secretion between patients with AD and control donors was observed for IL-10: PBMCs from patients with AD who were sensitized to Malassezia species secreted significantly lower levels of this anti-inflammatory cytokine in response to hTrx than those from the healthy donors, whereas there was no significant difference for non–Malassezia species–sensitized patients (Fig 1, B). The same was also true when patients were grouped according to IgE sensitization to Mala s 13 or hTrx (see Fig E2 in this article's Online Repository at www.jacionline.org). Compared with the autoantigen hTrx, the exogenous allergen Mala s 13 induced lower levels of IL-10 in PBMCs from patients with AD who were sensitized to Malassezia species, as well as from healthy donors. Because Mala s 13 and hTrx share about 40% sequence identity,2Limacher A. Glaser A.G. Meier C. Schmid-Grendelmeier P. Zeller S. Scapozza L. et al.Cross-reactivity and 1.4-A crystal structure of Malassezia sympodialis thioredoxin (Mala s 13), a member of a new pan-allergen family.J Immunol. 2007; 178: 389-396Crossref PubMed Google Scholar the recognition of non-hTrx–cross-reactive epitopes on Mala s 13 might promote inflammation instead of tolerance induction. IL-10 was mainly secreted by monocytes, as shown in Fig 1, C. Because it is known that monocytes from donors with the extrinsic form of AD express higher levels of the high-affinity IgE receptor than those from nonatopic donors,5Maurer D. Fiebiger E. Reininger B. Wolff-Winiski B. Jouvin M.H. Kilgus O. et al.Expression of functional high affinity immunoglobulin E receptors (Fc epsilon RI) on monocytes of atopic individuals.J Exp Med. 1994; 179: 745-750Crossref PubMed Scopus (334) Google Scholar we further assessed whether specific IgE to Mala s 13 and hTrx was directly involved in IL-10 and IL-13 regulation in monocytes. Therefore we treated PBMCs from patients with AD who were sensitized to Mala s 13 with lactic acid to remove surface-bound IgE.E3Pruzansky J.J. Grammer L.C. Patterson R. Roberts M. Dissociation of IgE from receptors on human basophils. I. Enhanced passive sensitization for histamine release.J Immunol. 1983; 131: 1949-1953PubMed Google Scholar, E4Maurer D. Ebner C. Reininger B. Fiebiger E. Kraft D. Kinet J.P. et al.The high affinity IgE receptor (Fc epsilon RI) mediates IgE-dependent allergen presentation.J Immunol. 1995; 154: 6285-6290PubMed Google Scholar By using this treatment, surface levels of IgE on monocytes were reduced (Fig 1, D), and IL-10 secretion was significantly increased after Mala s 13 stimulation but not after hTrx stimulation (Fig 1, E). In contrast, IL-13 secretion could be reduced after lactic acid treatment followed by hTrx stimulation but not by Mala s 13 stimulation (Fig 1, E). An explanation for these discrepancies might be the small fraction of Mala s 13– and hTrx-specific IgE in relation to the patient's total serum IgE levels. In fact, the increase in IL-10 levels after IgE stripping and stimulation with Mala s 13 correlated with the Malassezia species–specific IgE/total IgE ratio (Spearman r = 0.536, P = .048). In turn, this can be explained by the correlation of monocyte surface levels of FcεRI with total serum IgE levels.6Sihra B.S. Kon O.M. Grant J.A. Kay A.B. Expression of high-affinity IgE receptors (FcεRI) on peripheral blood basophils, monocytes, and eosinophils in atopic and nonatopic subjects: relationship to total serum IgE concentrations.J Allergy Clin Immunol. 1997; 99: 699-706Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar Our findings are in contrast to those of previous reports showing an increased IL-10 secretion of FcεRI-activated monocytes.7Pyle D.M. Yang V.S. Gruchalla R.S. Farrar J.D. Gill M.A. IgE cross-linking critically impairs human monocyte function by blocking phagocytosis.J Allergy Clin Immunol. 2013; 131 (e1-5): 491-500Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 8Novak N. Bieber T. Katoh N. Engagement of Fc epsilon RI on human monocytes induces the production of IL-10 and prevents their differentiation in dendritic cells.J Immunol. 2001; 167: 797-804Crossref PubMed Scopus (106) Google Scholar This might be due to differences in the experimental setup, such as the use of monoclonal IgE and anti-IgE for receptor cross-linking compared with our assay by using 1 distinct allergen, as well as due to different cell-culture conditions and the time point of analysis. Using anti-IgE stimulation, we could also upregulate IL-10 in PMBCs from patients with AD who were sensitized to Malassezia species (see Fig E3 in this article's Online Repository at www.jacionline.org). Therefore this discrepancy between massive IgE receptor cross-linking with anti-IgE and the potentially weaker cross-linking resulting from application of native allergens should be considered in future studies. The major findings of this study are (1) an IgE-dependent upregulation of the TH2 cytokine IL-13 by hTrx and (2) an impaired upregulation of IL-10 by hTrx in patients with AD who were sensitized to Mala s 13 and hTrx. IL-10 promotes the development of regulatory dendritic cells and T cells and therefore tolerance, and it is involved in a number of processes involved in the downregulation of a local inflammatory response.9Palomares O. Martin-Fontecha M. Lauener R. Traidl-Hoffmann C. Cavkaytar O. Akdis M. et al.Regulatory T cells and immune regulation of allergic diseases: roles of IL-10 and TGF-β.Genes Immun. 2014; 15: 511-520PubMed Google Scholar The relative lack of antigen-specific IL-10 secretion in PBMCs from patients with AD who were sensitized to Malassezia species might contribute to the perpetuation of the local cutaneous inflammation in patients with AD and to further development of TH2- and IgE-mediated immune responses. Moreover, specific sensitization against hTrx can directly be involved in disease exacerbation in patients with AD because hTrx can be released from dermal or epidermal cells and might then promote inflammatory responses by activating specific T cells and cells expressing receptors for IgE. We thank Mrs Gabriele Begemann, Petra Kienlin, and Ute Staar for their excellent technical assistance. Recombinant Mala s 13 and hTrx were produced as [His]6-tagged fusion proteins in Escherichia coli and purified by means of Ni2+ affinity chromatography, as previously described.E5Limacher A. Glaser A.G. Meier C. Schmid-Grendelmeier P. Zeller S. Scapozza L. et al.Cross-reactivity and 1.4-A crystal structure of Malassezia sympodialis thioredoxin (Mala s 13), a member of a new pan-allergen family.J Immunol. 2007; 178: 389-396Crossref PubMed Scopus (70) Google Scholar Recombinant α-NAC was expressed in E coli as a soluble protein and purified by means of Ni2+ affinity chromatography, as described previously.E6Mittermann I. Reininger R. Zimmermann M. Gangl K. Reisinger J. Aichberger K.J. et al.The IgE-reactive autoantigen Hom s 2 induces damage of respiratory epithelial cells and keratinocytes via induction of IFN-gamma.J Invest Dermatol. 2008; 128: 1451-1459Crossref PubMed Scopus (44) Google Scholar The LPS concentration of hTrx was 10 ng/mg protein, corresponding to 25 pg/mL in the working concentration, and the LPS content of Mala s 13 was 1.5 ng/mg protein, corresponding to 3.75 pg/mL in the working concentration. The highest LPS concentration of 25 pg/mL was used as a control in all experiments. Blood was obtained from patients with AD who provided informed consent and were recruited from the outpatient clinic of the Department of Dermatology and Allergy, Hannover Medical School, Germany. Only patients without any immunosuppressive treatment were admitted. Sensitization to Malassezia species mix (m227) was analyzed by using Phadia with ImmunoCAP (Phadia AB, Uppsala, Sweden). Values of greater than 3.5 kU/L were regarded as positive for Malassezia species–specific IgE. Patients were tested for sensitization to Mala s 13 or hTrx by using ELISA. OD values at least 2.5 times greater than those of the negative control sera were regarded as positive. Buffy coats from healthy voluntary blood donors provided by the Institute of Transfusion Medicine, Hannover Medical School, were used as control samples. The study was approved by the local ethics committee of Hannover Medical School and conducted according to the Declaration of Helsinki principles. PBMCs were isolated by means of density gradient centrifugation on Ficoll (Pan-Biotech, Aidenbach, Germany). Cells were cultured in Iscove medium (Biochrom KG, Berlin, Germany) supplemented with 4% human heat-inactivated AB serum, 2 mmol/L glutamine, 50 mg/mL gentamicin, 100 mg/mL penicillin and streptomycin, and nonessential amino acids. For some experiments, monocytes and CD4+ T cells were isolated from PBMCs by means of magnetic cell sorting (Monocyte Isolation Kit II, CD4+ T Cell Isolation Kit; Miltenyi Biotec, Bergisch Gladbach, Germany). The following stimulants were used: hTrx (2.5 μg/mL), Mala s 13 (2.5 μg/mL), α-NAC (2.5 μg/mL), IL-2 (50 U/mL; Roche Applied Science, Mannheim, Germany), and LPS (25 pg/mL; Sigma-Aldrich, Munich, Germany). Cells cultured at a density of 1 × 106/mL for all experiments were stimulated for 3 days for generation of supernatants. IgE stripping was performed with a buffer of 0.13 mol/L NaCl, 0.05 mo/L KCl, and 0.01 mol/L lactate for 10 minutes at room temperature. FcεRI cross-linking was performed with human myeloma IgE (1 μg/mL; Calbiochem, San Diego, Calif) for 1 hour at 37°C, followed by application of polyclonal rabbit anti-human IgE (20 μg/mL; Dako, Glostrup, Denmark) for 1 hour at 37°C. IgE stripping was assessed by staining PBMCs with a fluorescein isothiocyanate–labeled anti-CD14 antibody (Beckman Coulter, Krefeld, Germany) and a biotin-coupled anti-human IgE antibody (BD Biosciences, Heidelberg, Germany) detected with Streptavidin-allophycocyanin (BD Biosciences). Ten thousand cells were analyzed with the FACSCalibur flow cytometer (Becton Dickinson) using Cell Quest software (Becton Dickinson). Culture supernatants were taken 3 days after stimulation, centrifuged, and stored at −20°C. ELISAs were performed according to the manufacturer's instructions and measured with a FluoStar Reader (BMG Labtech GmbH, Ortenberg, Germany). ELISA kits from eBioscience (IL-10, IL-13, and IFN-γ; eBioscience, San Diego, Calif) were used. Statistical analysis was performed with the Wilcoxon signed-rank test or Mann-Whitney test (GraphPad Prism; GraphPad Software, San Diego, Calif).Fig E2IgE sensitization to Mala s 13 or hTrx similarly affects IL-10 secretion in PBMCs from the respective sensitized patients with AD compared with control subjects. A, PBMCs from patients with AD who were sensitized to Mala s 13 (AD), healthy donors (ctrl), and patients with AD who were not sensitized to the respective antigen (AD ns) were stimulated with 2.5 μg/mL hTrx or 2.5 μg/mL Mala s 13 with or without IL-2 for 72 hours. Supernatants were analyzed for levels of IL-10 by means of ELISA. B, PBMCs from patients with AD who were sensitized to hTrx (AD) were compared with PBMCs from healthy donors (ctrl) and patients with AD who were not sensitized to the respective antigen (AD ns) under similar conditions, as described for Fig E2, A. Medians and minimums/maximums are shown. *P < .05 and **P < .01. ns, Not significant.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3IL-10 is upregulated in PBMCs after monoclonal FcεR cross-linking. PBMCs from 4 patients with AD were stimulated with human myeloma IgE for 1 hour, washed, stimulated with rabbit anti-human IgE antibody for 1 hour, washed, and incubated for 24 hours. Supernatants were taken and analyzed for levels of IL-10 by means of ELISA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Patients' characteristicsPatients with AD and positive Malassezia species–specific IgE resultsPatients with AD and negative Malassezia species–specific IgE resultsNo. (male/female)24 (15/9)9 (3/6)Median age, y (range)38 (20-75)35 (22-73)Median total IgE, kU/L (range)2,678 (119-16,740)228 (4-37,870)Median Malassezia species–specific IgE, kU/L (range)27.25 (3.86-100)0.33 (0-3.36)Mala s 13–specific IgE78%44%hTrx-specific IgE30%33%Median SCORAD score (range)34.95 (8.6-89.4)34.25 (11-57.5) Open table in a new tab
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