IntroductionAtopic dermatitis (AD), a common chronic inflammatory skin disease, is characterized by type-2-mediated inflammation, along with the detection of type-1 and type-3 cytokines in lesional skin. The skin microbiome of lesional skin is dominated by the pathogen Staphylococcus aureus, which can aggravate the disease via pathogenicity factors. To elucidate the impact of the adaptive immune response on inflammation in AD, this study focused on staphylococcal serine-like proteases (Spl) of S. aureus, a family of secreted pathogenicity factors with the potential to induce type-2 responses.MethodsSpecific serum IgE against Spl family members was quantified, and SplB-specific CD4+ T cells were identified by surface expression of CD154 after in vitro stimulation with recombinant SplB. Immunodominant epitopes within the SplB primary structure were predicted to generate MHC multimers for staining, sorting, and cytokine analysis of SplB-specific T cells. TCRB sequencing was applied to identify SplB-specific T cells in AD skin lesions.ResultsWe observed significantly elevated levels of IgE antibodies specific for Spl family proteins in patients with AD compared to healthy controls. In vitro, recombinant SplB was sufficient to induce T cell activation and cytokine secretion in PBMCs from patients with AD and healthy controls. SplB-specific T helper cells, which were cell-sorted from patients’ blood by MHC-II multimers, showed the capacity to produce IFN-γ and IL-13 ex vivo. Clonal propagation of specific T cells was confirmed by TCR sequencing, and SplB-specific TCR sequences were re-identified in autologous lesional skin biopsy material.DiscussionThe presence of clonally propagated SplB-specific T cells in the skin of patients with AD strongly suggests an impact on inflammation. This type of cellular immune response, which is not exclusively polarized towards type 2, reflects the AD phenotype. This suggests that the adaptive immune response to S. aureus contributes to this phenotype.

Background: Impaired virus clearance in a subgroup of atopic dermatitis (AD) patients can lead to severe herpes simplex virus (HSV) infections called eczema herpeticum (EH). We recently identified a type 2 skewed viral immune response in EH patients. Clinical data suggest a reduced incidence of EH in AD patients treated with dupilumab, although immunologic investigations of this phenomenon are still lacking. Objective: We examined the impact of dupilumab on the HSV type 1 (HSV-1) specific immune response in AD, focusing on patients with (ADEH+) and without (ADEH-) a history of EH. Methods: Sera and peripheral blood mononuclear cells were collected from ADEH+ and ADEH- patients, a subgroup of whom was receiving dupilumab treatment, and healthy controls. Serum samples were tested for IgE against HSV-1 glycoprotein D (n = 85). Peripheral blood mononuclear cells were stimulated with HSV peptides, and activated CD4+ and CD8+ cells were characterized by flow cytometry after magnetic enrichment via CD154 or CD137 (n = 60). Cytokine production of HSV-1- reactive T-cell lines (n = 33) and MHC-I tetramer+ (HSV-1- UL25) CD8+ T cells was investigated by bead assay and intracellular cytokine staining (n = 21). Results: We confirmed that HSV-1-specific IgE is elevated in ADEH+ patients. During dupilumab treatment, the IgE levels were significantly decreased, reaching levels of healthy controls. HSV-1-specific TC1 frequencies were elevated in ADEH- patients treated with dupilumab compared to dupilumabnegative patients. There were no changes in the frequencies of HSV-1-specific TH cells while receiving dupilumab therapy. AD patients receiving dupilumab exhibited elevated IFN-y and reduced IL-4 production in HSV-1-UL25-epitope-specific T cells compared to dupilumab-negative patients. Conclusion: Dupilumab may improve the HSV-1-specific immune response in AD as a result of an increased type I immune response and a reduction of HSV-1-specific IgE. (J Allergy Clin Immunol 2023;152:1460-9.)

Eczema herpeticum (EH), a disseminated and potentially life-threatening herpes simplex virus (HSV) infection, poses a significant risk to patients with atopic dermatitis (AD). Our recent findings have highlighted a type-2 biased immune response to viral infections in individuals with EH. Dupilumab, a monoclonal antibody targeting the IL-4Rα chain, offers a direct intervention against type-2 inflammation. However, there remains a lack of comprehensive molecular investigations into the influence of dupilumab on antiviral immune defense. This study aims to assess the impact of dupilumab on the adaptive cellular antiviral response in AD patients with a history of EH (ADEH+) and those without (ADEH-). Blood samples were collected from 56 AD patients, including 25 under dupilumab treatment, along with 10 healthy controls. Peripheral blood mononuclear cells (PBMCs) were stimulated with HSV-1 protein and peptides. HSV-reactive CD4+ and CD8+ T cells were enriched by targeting CD154 and CD137, respectively, and analyzed using flow cytometry to determine polarization. Cytokine profiles of HSV-reactive T cell lines were assessed using cytokine bead assays. MHC-I tetramer+ CD8+ T cells specific to the HSV-UL25 epitope were stained for intracellular IL-4 and IFN-γ. HSV-specific IgE levels in the serum were elevated in ADEH+ compared to ADEH- patients and significantly reduced in those undergoing dupilumab treatment. ADEH- patients treated with dupilumab exhibited increased frequencies of HSV-specific Tc1 cells compared to untreated counterparts. HSV-specific Th cell frequencies remained stable during dupilumab therapy. Notably, HSV-UL25-specific T cells from dupilumab-treated AD patients displayed increased frequency of IFN-γ and reduced IL-4 positivity, indicating a reinforcement of the specific type-1 immune response and attenuation of type-2-associated reactions, including HSV-specific IgE production. In conclusion, this study suggests that dupilumab enhances HSV-specific defense mechanisms in AD patients, promoting an antiviral type-1 immune response while dampening type-2-mediated reactions, shedding light on potential therapeutic avenues for managing EH in AD.

A subgroup of patients suffering from atopic dermatitis (AD) does not respond to biologics therapy targeting the key players of type-2 inflammation, and it is an ongoing discussion whether skin-infiltrating Th17 cells may underlie this phenomenon. This study aimed to investigate the potential of allergen-induced, immune-cell derived IL-17 on the induction of inflammatory processes in keratinocytes. Peripheral blood mononuclear cells derived from respectively sensitized AD patients were stimulated with house dust mite (HDM) extract and cell culture supernatants were applied subsequently in absence or presence of secukinumab to primary human keratinocytes. Hereby we confirm that the immune response of sensitized AD patients to HDM contains aside from type-2 cytokines significant amounts of IL-17. Blocking IL-17 efficiently reduced the stimulation-induced changes in keratinocyte gene expression. IL-17-dependent transcriptional changes included increased expression of the cytokines IL-20 and IL-24 as well as Suppressor of Cytokine Siganling 3 (SOCS3), a negative feedback-regulator of the STAT3/IL-17/IL-24 immune response. We conclude that the immune response to HDM can induce pro-inflammatory cytokines from keratinocytes in AD, which in part is mediated via IL-17. Targeting IL-17 may turn out to be a reasonable alternative therapy in a subgroup of patients with moderate to severe AD and HDM sensitization.

BACKGROUND Immediate as well as delayed-type hypersensitivity immune reactions to pet-borne allergens are commonly observed in atopic diseases. Further on in atopic dermatitis (AD), cross-reactivity to self-proteins is discussed to contribute to the disease. Human cystatin A and the cat allergen Fel d 3 belong to the cystatin family, an evolutionary conserved protein family. The objective of the present study was to assess cross-reactivity between mammalian cystatins and to analyze T cell responses to cystatin in AD patients sensitized to pet dander. MATERIAL AND METHODS cDNA coding for dog cystatin was cloned from dog skin. Sera of 245 patients with IgE-sensitization to cat and dog dander were tested for IgE-binding to recombinantly expressed feline, canine, and human cystatin, respectively. Of these, 141 were also diagnosed for AD. RESULTS Cystatin-specific IgE was detected in 14.7 %(36) of patients, of which 19 suffered from AD. Within the AD patients, 9 carried measurable IgE against all three cystatins. Cystatin-sensitized AD patients did not differ from non-cystatin sensitized patients in terms of disease severity, age or total IgE levels. T cell cytokine measurements showed elevated IL-4 levels after stimulation with feline and human cystatin. CONCLUSION The humoral response suggests that next to Fel d3 also the homologous protein from dog might play a role in allergy. Further on, the human cystatin appears to be capable of driving a type2 immune response in sensitized AD patients and may therefore be considered a so-called autoallergen, as it has been proposed for other evolutionary conserved proteins.

atopic dermatitis (AD) patients are commonly sensitized to house dust mite (HDM). The current consensus-based European guideline underlines the impact of allergens inducing AD flares and the importance of allergen reduction in AD patients1,2 . With regard to preventive measures in patients with AD, the use of mattress encasings aiming to reduce skin contacts to mite allergens in bed may be considered in HDM-sensitized patients.

Human thioredoxin (hTrx), which can be secreted from cells upon stress, functions in allergic skin inflammation as a T cell antigen due to homology and cross-reactivity with the fungal allergen Mala s13 of the skin-colonizing yeast Malassezia sympodialis . Recent studies have shown that cell wall polysaccharides of Malassezia are detected by the immune system via the C-type lectin receptors Dectin-1 and Dectin-2, which are expressed on myeloid cells. Therefore, this study aimed to investigate a putative interaction between Dectin-1, Dectin-2 and the allergens Mala s13 and hTrx. Stimulation of human monocyte-derived dendritic cells or macrophages with Mala s13 or hTrx resulted in remarkable secretion of IL-1β and IL-23. Blocking experiments suggest that hTrx induces IL-23 by Dectin-1 binding and IL-1β by binding to either Dectin-1 or Dectin-2. Regarding Mala s13, Dectin-1 appears to be involved in IL-1β signaling. Interference of Syk kinase function was performed to investigate downstream signaling, which led to diminished hTrx responses. In our experiments, we observed rapid internalization of Mala s13 and hTrx upon cell contact and we were able to confirm direct interaction with Dectin-1 as well as Dectin-2 applying a fusion protein screening platform. We hypothesize that this cytokine response may result in a Th2/Th17-polarizing milieu, which may play a key role during the allergic sensitization in the skin, where allergen presentation to T cells is accompanied by microbial colonization and skin inflammation.

Atopic dermatitis (AD) is an inflammatory, relapsing skin disorder that affects 15% to 25% of children worldwide and persists in adulthood in around 25% of these cases.1Werfel T. Allam J.P. Biedermann T. Eyerich K. Gilles S. Guttman-Yassky E. et al.Cellular and molecular immunologic mechanisms in patients with atopic dermatitis.J Allergy Clin Immunol. 2016; 138: 336-349Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar About 3% to 8% of patients with AD seem to have a disturbance in viral clearance, manifesting severe forms of molluscum contagiosum, papilloma virus, and most prominently, the generalized cutaneous infection with herpes simplex virus 1 (HSV1), called eczema herpeticum (EH),2Bin L. Edwards M.G. Heiser R. Streib J.E. Richers B. Hall C.F. et al.Identification of novel gene signatures in patients with atopic dermatitis complicated by eczema herpeticum.J Allergy Clin Immunol. 2014; 134: 848-855Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, E1Morgan T.K. Hanifin J. Mahmood M. Larson B. Baig-Lewis S. Long T. et al.Atopic dermatitis is associated with cervical high risk human papillomavirus infection.J Low Genit Tract Dis. 2015; 19: 345-349Crossref PubMed Scopus (13) Google Scholar, E2Olsen J.R. Piguet V. Gallacher J. Francis N.A. Molluscum contagiosum and associations with atopic eczema in children: a retrospective longitudinal study in primary care.Br J Gen Pract. 2016; 66: e53-e58Crossref PubMed Scopus (17) Google Scholar which can lead to life-threatening complications.E3Wollenberg A. Eczema herpeticum.Chem Immunol Allergy. 2012; 96: 89-95Crossref PubMed Scopus (25) Google Scholar The occurrence of EH is associated with a more severe AD disease according to disease scoring systems, total serum IgE, polyallergen sensitization, and other measures.3Wollenberg A. Wetzel S. Burgdorf W.H. Haas J. Viral infections in atopic dermatitis: pathogenic aspects and clinical management.J Allergy Clin Immunol. 2003; 112: 667-674Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 4Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269.e7Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar In addition, not only skin-affecting viral diseases but also respiratory tract infections, such as influenza, are more prevalent in patients with AD.5Silverberg J.I. Silverberg N.B. Childhood atopic dermatitis and warts are associated with increased risk of infection: a US population-based study.J Allergy Clin Immunol. 2014; 133: 1041-1047Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, E4Brunner P.M. Silverberg J.I. Guttman-Yassky E. Paller A.S. Kabashima K. Amagai M. et al.Increasing comorbidities suggest that atopic dermatitis is a systemic disorder.J Invest Dermatol. 2017; 137: 18-25Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar Several studies point to a defect in the antiviral IFN-γ response and the skin barrier in patients with AD with a history of EH (ADEH+).6Mathias R.A. Weinberg A. Boguniewicz M. Zaccaro D.J. Armstrong B. Schneider L.C. et al.Atopic dermatitis complicated by eczema herpeticum is associated with HLA B7 and reduced interferon-g-producing CD8+ T cells.Br J Dermatol. 2013; 169: 700-703Crossref PubMed Scopus (17) Google Scholar, 7Gao P.S. Rafaels N.M. Hand T. Murray T. Boguniewicz M. Hata T. et al.Filaggrin mutations that confer risk of atopic dermatitis confer greater risk for eczema herpeticum.J Allergy Clin Immunol. 2009; 124 (513.e1-7): 507-513Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 8De Benedetto A. Slifka M.K. Rafaels N.M. Kuo I.H. Georas S.N. Boguniewicz M. et al.Reductions in claudin-1 may enhance susceptibility to herpes simplex virus 1 infections in atopic dermatitis.J Allergy Clin Immunol. 2011; 128: 242-246.e5Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar In this study, we aimed to characterize virus-specific T-cell responses in patients suffering from AD with or without history of EH applying state-of-the-art detection systems, namely, measurements of IL-4 and IFN-γ cytokine expression in stimulated T-cell lines (TCLs), direct ex vivo enrichment and characterization of virus-specific CD4+ and CD8+ T cells via CD154 (CD40L) or CD137 (4-1BB), respectively, as well as specific detection of virus-specific CD8+ T cells with MHC class I tetramers in combination with intracellular cytokine staining. For a detailed delineation of material and methods, please see this article's Online Repository at www.jacionline.org. The analysis of the cytokines IL-4 and IFN-γ in supernatants of TCLs generated in the presence of HSV1 and influenza antigens and peptides revealed a significantly reduced antiviral IFN-γ response of patients with AD compared with TCLs from healthy controls. The significantly reduced IFN-γ production by TCLs from patients with AD grown in the presence of HSV1 protein glycoprotein D (gD), HSV1 immunodominant peptides, or the immunodominant peptides from influenza hemagglutinin was more pronounced in ADEH+ subjects than in patients with AD without a history of EH. No differences between the groups were observed when tetanus toxoid was used as a control antigen under similar experimental conditions (Fig 1, A and B, left). Interestingly, we found the secretion of the type 2 cytokine IL-4 to be significantly elevated in HSV1 gD-stimulated TCLs generated from patients with ADEH+ compared with healthy controls (Fig 1, A and B, right). To investigate T cells directly ex vivo, we detected the upregulated expression of CD154 (CD40L) on CD4+ cells after stimulation for analyzing virus-specific T cells in combination with a panel of surface markers to define T-cell subpopulations (see Fig E1, A and B, in this article's Online Repository at www.jacionline.org). By this, we observed that the HSV1-specific cell numbers expressing the surface marker panel CD4+CD154+CXCR3+CCR4−CCR6−CCR10−, described as TH1 in literature,9Mahnke Y.D. Beddall M.H. Roederer M. OMIP-017: human CD4(+) helper T-cell subsets including follicular helper cells.Cytometry A. 2013; 83: 439-440Crossref PubMed Scopus (46) Google Scholar were not different from healthy controls. Interestingly, a pronounced increase in CD4+CD154+CXCR3−CCR4+CCR6−CCR10− polarized cells, mentioned as TH2,9Mahnke Y.D. Beddall M.H. Roederer M. OMIP-017: human CD4(+) helper T-cell subsets including follicular helper cells.Cytometry A. 2013; 83: 439-440Crossref PubMed Scopus (46) Google Scholar in response to HSV1 was displayed (Fig 1, C). Although this effect was consistent among all stimuli in ADEH+ patients, it was less pronounced but observable by trend in ADEH− patients (Fig 1, D, right). The antigen response to influenza displayed differences in both investigated subsets. We detected a significantly reduced frequency of influenza hemagglutinin-specific CXCR3+CCR4−CCR6−CCR10−TH cells in patients with AD (Fig 1, C, left); however, no differences are apparent in the AD subgroups (Fig 1, D, left). Similar to CD4+ T cells, TCR-stimulated CD8+ T cells can be identified by the expression of a surface marker, namely CD137 (4-1BB) (see Fig E1, C and D).E5Wolfl M. Kuball J. Eyrich M. Schlegel P.G. Greenberg P.D. Use of CD137 to study the full repertoire of CD8+ T cells without the need to know epitope specificities.Cytometry A. 2008; 73: 1043-1049Crossref PubMed Scopus (52) Google Scholar Further on, cytotoxic T cells have been subgrouped into TC subsets, with TC1 expressing CXCR3,E13Cosmi L. Annunziato F. Galli M.I.G. Maggi R.M.E. Nagata K. Romagnani S. CRTH2 is the most reliable marker for the detection of circulating human type 2 Th and type 2 T cytotoxic cells in health and disease.Eur J Immunol. 2000; 30: 2972-2979Crossref PubMed Scopus (222) Google Scholar TC2 expressing CCR4 but not CXCR3,E13Cosmi L. Annunziato F. Galli M.I.G. Maggi R.M.E. Nagata K. Romagnani S. CRTH2 is the most reliable marker for the detection of circulating human type 2 Th and type 2 T cytotoxic cells in health and disease.Eur J Immunol. 2000; 30: 2972-2979Crossref PubMed Scopus (222) Google Scholar and TC17 expressing CCR6.E14Kondo T. Takata H. Matsuki F. Takiguchi M. Cutting edge: phenotypic characterization and differentiation of human CD8+ T cells producing IL-17.J Immunol. 2009; 182: 1794-1798Crossref PubMed Scopus (147) Google Scholar In our hands, differences in the expression of these markers between patients and controls were detected specifically after stimulation with HSV1 but not after stimulation with influenza antigens (Fig 2, A). More precise, in patients with AD significantly less HSV1 gD and HSV1 peptide-specific cytotoxic T cells expressed the bona fide Tc1 marker panel CD8+CD137+CXCR3+CCR4−CCR6− (Fig 2, A, left). This effect was comparable in ADEH− and ADEH+ subjects (Fig 2, B, left). More interestingly, an increase in cells expressing the marker set described for TC2 T cells (CD8+CD137+CXCR3−CCR4+CCR6−) was observed in the response of ADEH+ patients to HSV1 peptides compared with healthy subjects (Fig 2, A and B, right). Furthermore, we applied HSV1-specific MHC class I tetramers, harboring an immunodominant peptide from UL25.E6Laing K.J. Magaret A.S. Mueller D.E. Zhao L. Johnston C. De Rosa S.C. et al.Diversity in CD8(+) T cell function and epitope breadth among persons with genital herpes.J Clin Immunol. 2010; 30: 703-722Crossref PubMed Scopus (46) Google Scholar To gain adequate cells numbers, these were propagated in vitro for 14 days in the presence of the respective peptide before the staining (see Fig E2, A, in this article's Online Repository at www.jacionline.org). Detection of cytokines by intracellular staining revealed that UL25-specific T cells of healthy subjects respond nearly exclusively with IFN-γ and not with IL-4 production, whereas ADEH+ patients bear a substantial amount of T cells (median, 10.16% ± 15.11%) expressing IL-4. In parallel, tetramer+ IFN-γ+ cells were found to be reduced in ADEH+ patients by trend. Patients of the ADEH− group displayed an intermediate phenotype: we found slightly less IFN-γ and more IL-4–producing specific T cells compared with healthy individuals (Fig 2, C; for exemplary scatter plots see Fig E2, B). Importantly, these findings were observed by trend also directly ex vivo without in vitro T-cell proliferation (Fig E2, C). Taken together, this is the first study indicating that a type 2 response by virus-specific T cells could be part of a complex pathology of EH and the susceptibility of patients with AD to HSV1. Applying different techniques, we were able to show differences in the response to viral antigens between healthy donors and patients suffering from AD. Within the patient cohort, the largest differences were consistently detectable within the subgroup of patients with a history of EH. The observed type 2–skewed phenotype leads to presumably inappropriate cytokine responses and eventually ineffective expansion.E7Oran A.E. Robinson H.L. DNA vaccines: influenza virus challenge of a Th2/Tc2 immune response results in a Th2/Tc1 response in the lung.J Virol. 2004; 78: 4376-4380Crossref PubMed Scopus (10) Google Scholar Noteworthy, these observed effects were not directly related to a more severe AD disease burden, an increased atopic predisposition, or higher age because the 3 groups were matched for age, total IgE, severity of AD (SCORing Atopic Dermatitis), and Sx1 positivity (see Fig E3 in this article's Online Repository at www.jacionline.org). Our finding of elevated type 2 and reduced type 1 T cell immune response to HSV1, in parallel with the discovery of increased IL-4 levels in specific CD8+ cells in patients with AD and in particular ADEH, requires further investigation concerning their ability of viral defense. In summary, we demonstrate a type 2 immune profile in virus-specific T cells in AD that is more pronounced in ADEH+ patients, which may contribute to a susceptibility to viral infections. We kindly thank Annice Heratizadeh, MD, and Paulina Syryca, MD, for patient recruitment and discussion. Furthermore, we thank Beate Sodeik, PhD, Katinka Döhner, PhD, and Anna Buch, PhD, for fruitful discussion on HSV-1 immunology. PBMCs were isolated from whole blood of 28 adult patients with AD fulfilling the criteria of Hanifin and RajkaE8Hanifin J.M. Rajka G. Diagnostic features of atopic dermatitis.Acta Dermato-Venereologica. 1980; 92: 44-47Google Scholar and 16 healthy donors without skin disease. Fourteen of the patients with AD had at least 1 episode of EH in the last 5 years (ADEH+) diagnosed at the Department of Dermatology and Allergy, Hanover Medical School. ADEH− and ADEH+ patients were matched for age, total IgE, and SCORing Atopic Dermatitis to reduce confounding factors (see Fig E3). No patient was under systemic immunosuppressive treatment and all blood donors provided their informed written consent. The study was conducted according to the Declaration of Helsinki Protocols and approved by the ethics committee of the Hannover Medical School (MHH). For T-cell stimulation, recombinant proteins as well as peptides with previously described immunodominance (MHC class I and II) were applied. The HSV1 immunodominant peptides HSV1 ICP0642-651 encoded by RL2, HSV1 ICP81096-1105 encoded by UL29,E9Van Velzen M. Jing L. Osterhaus A.D. Sette A. Koelle D.M. Verjans G.M. Local CD4 and CD8 T-cell reactivity to HSV-1 antigens documents broad viral protein expression and immune competence in latently infected human trigeminal ganglia.PLoS Pathog. 2013; 9: e1003547Crossref PubMed Scopus (70) Google Scholar HSV1 gD49-82, and HSV1 gD121-152E10Zhang X. Castelli F.A. Zhu X. Wu M. Maillere B. BenMohamed L. Gender-dependent HLA-DR-restricted epitopes identified from herpes simplex virus type 1 glycoprotein D.Clin Vaccine Immunol. 2008; 15: 1436-1449Crossref PubMed Scopus (48) Google Scholar as well as the influenza immunodominant peptides of Matrix protein M159-73 and M1101-113E11Ge X. Tan V. Bollyky P.L. Standifer N.E. James E.A. Kwok W.W. Assessment of seasonal influenza A virus-specific CD4 T-cell responses to 2009 pandemic H1N1 swine-origin influenza A virus.J Virol. 2010; 84: 3312-3319Crossref PubMed Scopus (92) Google Scholar were synthesized at ProImmune (Oxford, UK). HSV1 gD protein recombinant expressed in Pichia pastoris was purchased at biorbyt (Cambridge, UK). Influenza A H1N1 (A/New Caledonia/20/99) Hemagglutinin Protein recombinantly expressed in human cells was purchased at Sino Biological Inc (Bejing, China). The HSV1 UL25/MHC class I tetramer was generated as described elsewhere.E6Laing K.J. Magaret A.S. Mueller D.E. Zhao L. Johnston C. De Rosa S.C. et al.Diversity in CD8(+) T cell function and epitope breadth among persons with genital herpes.J Clin Immunol. 2010; 30: 703-722Crossref PubMed Scopus (46) Google Scholar Fresh heparinized blood was taken and PBMCs were isolated by density-gradient centrifugation on Ficoll (Pan Biotech, Aidenbach, Germany). Cells were cultured as described beforeE12Roesner L.M. Heratizadeh A. Begemann G. Kienlin P. Hradetzky S. Niebuhr M. et al.Der p1 and Der p2-specific T cells display a Th2, Th17, and Th2/Th17 phenotype in atopic dermatitis.J Invest Dermatol. 2015; 135: 2324-2327Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar in Iscove medium (Biochrom KG, Berlin, Germany) supplemented with 4% human AB serum (Pan Biotech), 2 mM glutamine, 50 mg/mL gentamicin, 100 mg/mL penicillin and streptomycin, and nonessential amino acids (all from Biochrom KG). 1 × 106 cells at a density of 1 × 106/mL cells were stimulated with 2 μg/mL HSV1 gD protein, HSV1 peptide mix (each 10 μg/mL), 10 μg/mL influenza hemagglutinin protein, influenza peptide mix (each 10 μg/mL), or tetanus toxoid (1:2000, Mérieux, Sanofi Pasteur MSD, Lyon, France). After 7 days, 10 U/mL rhIL-2 (Roche Diagnostics GmbH, Mannheim, Germany) was added; after 14 days, the culture was expanded with 1 × 106 allogenic irradiated (55Gy) PBMCs with 10 μg/mL PHA (Sigma Aldrich, St Louis, Mo) and 10 U/mL rhIL-2. Restimulation testing was performed after 3 weeks as described.E12Roesner L.M. Heratizadeh A. Begemann G. Kienlin P. Hradetzky S. Niebuhr M. et al.Der p1 and Der p2-specific T cells display a Th2, Th17, and Th2/Th17 phenotype in atopic dermatitis.J Invest Dermatol. 2015; 135: 2324-2327Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Cell culture supernatants for ELISA were harvested after 48-hour stimulation with 10 μg/mL Concanavalin A (Sigma Aldrich). ELISAs for IL-4 (eBioscience, San Diego, Calif), IFN-γ, IL-17, and IL-22 (R&D, Minneapolis, Minn) were performed according to manufacturer's recommendations and detected by a FluoStar optima plate reader (BMG Labtech, Ortenberg, Germany). Freshly isolated PBMCs of 20 patients with AD and 10 healthy donors were cultured overnight (1 × 107/mL). Subsequently, cells were stimulated for 7 to 24 hours9Mahnke Y.D. Beddall M.H. Roederer M. OMIP-017: human CD4(+) helper T-cell subsets including follicular helper cells.Cytometry A. 2013; 83: 439-440Crossref PubMed Scopus (46) Google Scholar with HSV1 protein, HSV1 peptide mix, influenza protein, and influenza peptide mix, respectively, as described above plus 1 μg/mL anti-CD40 (Miltenyi Biotec, Bergisch Gladbach, Germany) and 1 μg/mL anti-CD28 (Becton Dickinson, Franklin Lakes, NJ). CD154+ cells were enriched by specific magnetic beads (Miltenyi Biotec) and stained in RMPI 1640 (Biochrom KG) with 4% FBS (Pan Biotech) with an allophycocyanin (APC)-conjugated anti-CXCR3 antibody (R&D) for 30 minutes at room temperature, followed by anti-CD4 phycoerythrin (PE)-Cy5 (Beckton Dickinson), anti-CD14 brilliant violet (BV)-510, anti-CD19 BV510, anti-CD154 fluorescein isothiocyanate (FITC), anti-CCR4 BV421, anti-CCR6 PE-Cy7, anti-CCR10 PE (BioLegend, San Diego, Calif), and Fixable Viability Dye eFluor 506 (eBioscience) for 30 minutes on ice. Cells were detected by flow cytometry (FACS Canto II, Becton Dickinson). 1 × 107 PBMCs of 20 patients with AD and 10 healthy donors were stimulated as described beforeE5Wolfl M. Kuball J. Eyrich M. Schlegel P.G. Greenberg P.D. Use of CD137 to study the full repertoire of CD8+ T cells without the need to know epitope specificities.Cytometry A. 2008; 73: 1043-1049Crossref PubMed Scopus (52) Google Scholar for 24 to 28 hours and CD137+ cells were enriched after staining with anti–CD137-PE by anti-PE magnetic beads (Miltenyi Biotec). Cells were stained for flow cytometry as described above. Study subjects were typed for HLA-A*02 via PCR or antibody staining (BioLegend). For in vitro proliferation, 4 × 106 PBMCs were stimulated with 100 nM HSV1 UL25367-375 peptide (Peptides & Elephants, Nuthetal, Germany) in 100 μL culture medium for 1 hour at 37°C before cultivation at 1 × 106/mL containing 20 ng/mL rhIL-7 (PeproTech, Rocky Hill, NJ). At day 3, 20 U/mL rhIL-2 was added to the culture and half medium was changed at day 5, 8, and 11. At day 14, 3 × 106 cells were stained with APC-conjugated HSV1-UL25/MHC class I tetramers in 100 μL culture medium for 1 hour at room temperature. Anti-CD8 FITC, anti-CD45R0 (Becton Dickinson), and Fixable Viability Dye eFluor 506 were stained subsequently for 30 minutes on ice to visualize successful specific proliferation. At day 15, 3 × 106/mL cells were stimulated for 4 hours with 50 ng/mL phorbol-12-myristate-13-acetate and 1 μg/mL ionomycin (Sigma Aldrich) and monensin (Becton Dickinson). Cells were stained for 1 hour with APC-conjugated HSV1 UL25/MHC class I tetramer as before. Anti-CD8 FITC and Fixable Viability Dye eFluor 506 were stained subsequently on ice for 30 minutes. Fixed and permeabilized cells (eBioscience) were stained for 30 minutes with PE-conjugated anti–IFN-γ (R&D) or anti–IL-4 (Miltenyi Biotec) on ice. Alternatively, 3 × 106 CD8+ cells isolated by magnetic bead separation (Miltenyi Biotec) from whole uncultured PBMCs were subjected to APC-conjugated HSV1-UL25/MHC class I tetramer and cytokine staining as described above. Statistical analysis was performed using Mann-Whitney rank sum test for unpaired samples and Wilcoxon matched pairs test for paired samples when values were Gaussian distributed (controlled by D'Agostino and Pearson omnibus normality test and Shapiro-Wilk normality test) in GraphPad Prism 5.02 software (GraphPad Software Inc, San Diego, Calif). *P < .05; **P < .01; ***P < .001.Fig E2HSV1 UL25 MHC class I tetramer+ cells were analyzed for intracellular IL-4 and IFN-γ. A, Increase in tetramer+ cells after 14-day stimulation with the UL25 peptide compared with nonstimulated T cells at day 14. B, Intracellular cytokine staining of healthy, ADEH−, and ADEH+ donors for IFN-γ and IL-4. C, Direct ex vivo tetramer staining in combination with intracellular cytokine staining.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Patient's characteristics. Age in years and total IgE in kU/L for healthy, ADEH−, and ADEH+ patients. SCORing Atopic Dermatitis values were measured for patients with AD. Absence of significant differences between ADEH− and ADEH+ patients in the displayed characteristics. NA, Nonatopic.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Psoriasis is a common, chronic, relapsing/remitting, inflammatory skin disease and in many cases also considered a systemic disease with the greatest prevalence of 2% to 3% in northern Europe.E1Parisi R. Symmons D.P. Griffiths C.E. Ashcroft D.M. Identification and Management of Psoriasis and Associated ComorbidiTy (IMPACT) project team. Global epidemiology of psoriasis: a systematic review of incidence and prevalence.J Invest Dermatol. 2013; 133: 377-385Abstract Full Text Full Text PDF PubMed Scopus (1626) Google Scholar The clinical picture is shaped by the inflamed skin, in particular the observed hyperplasia and a thickened epidermis.E2Bata-Csorgo Z. Hammerberg C. Voorhees J.J. Cooper K.D. Flow cytometric identification of proliferative subpopulations within normal human epidermis and the localization of the primary hyperproliferative population in psoriasis.J Exp Med. 1993; 178: 1271-1281Crossref PubMed Scopus (117) Google Scholar Proinflammatory cytokines such as IL-17E3Res P.C. Piskin G. de Boer O.J. van der Loos C.M. Teeling P. Bos J.D. et al.Overrepresentation of IL-17A and IL-22 producing CD8 T cells in lesional skin suggests their involvement in the pathogenesis of psoriasis.PLoS One. 2010; 5: e14108Crossref PubMed Scopus (249) Google Scholar or IL-22E4Nograles K.E. Zaba L.C. Guttman-Yassky E. Fuentes-Duculan J. Suarez-Farinas M. Cardinale I. et al.Th17 cytokines interleukin (IL)-17 and IL-22 modulate distinct inflammatory and keratinocyte-response pathways.Br J Dermatol. 2008; 159: 1092-1102PubMed Google Scholar produced by inflammatory cells such as CD4 and CD8 T cells,E5Cheuk S. Wiken M. Blomqvist L. Nylen S. Talme T. Stahle M. et al.Epidermal Th22 and Tc17 cells form a localized disease memory in clinically healed psoriasis.J Immunol. 2014; 192: 3111-3120Crossref PubMed Scopus (230) Google Scholar, E6Lowes M.A. Kikuchi T. Fuentes-Duculan J. Cardinale I. Zaba L.C. Haider A.S. et al.Psoriasis vulgaris lesions contain discrete populations of Th1 and Th17 T cells.J Invest Dermatol. 2008; 128: 1207-1211Abstract Full Text Full Text PDF PubMed Scopus (847) Google Scholar neutrophils,E7Lin A.M. Rubin C.J. Khandpur R. Wang J.Y. Riblett M. Yalavarthi S. et al.Mast cells and neutrophils release IL-17 through extracellular trap formation in psoriasis.J Immunol. 2011; 187: 490-500Crossref PubMed Scopus (663) Google Scholar monocytes, macrophages, and dendritic cells are supposed to be a driving force of the hyperproliferation. Existing treatment options including immunosuppressants, corticosteroids, vitamin D analogues, and retinoids are all associated with different side effects.1Wittmann M. Helliwell P.S. Phosphodiesterase 4 inhibition in the treatment of psoriasis, psoriatic arthritis and other chronic inflammatory diseases.Dermatol Ther. 2013; 3: 1-15Crossref PubMed Scopus (95) Google Scholar Inhibitors of the phosphodiesterase isoenzyme 4 (PDE4i)2Torphy T.J. Phosphodiesterase isozymes: molecular targets for novel antiasthma agents.Am J Respir Crit Care Med. 1998; 157: 351-370Crossref PubMed Scopus (656) Google Scholar have been shown to ameliorate symptoms in patients with psoriasis.3Moustafa F. Feldman S.R. A review of phosphodiesterase-inhibition and the potential role for phosphodiesterase 4-inhibitors in clinical dermatology.Dermatol Online J. 2014; 20: 22608PubMed Google Scholar The next generation of PDE4i in skin diseases is topical formulations, delivering the active component to the site of inflammation and thereby avoiding side effects such as nausea that appear with oral PDE4i.4Nazarian R. Weinberg J.M. AN-2728, a PDE4 inhibitor for the potential topical treatment of psoriasis and atopic dermatitis.Curr Opin Investig Drugs. 2009; 10: 1236-1242PubMed Google Scholar In this study, the effect of 2 topical PDE4i, namely, roflumilast5Gavalda A. Roberts R.S. Phosphodiesterase-4 inhibitors: a review of current developments (2010-2012).Expert Opin Ther Pat. 2013; 23: 997-1016Crossref PubMed Scopus (39) Google Scholar and TAK-084, on inflamed psoriatic skin lesions was investigated. While roflumilast is approved worldwide for the treatment of chronic obstructive pulmonary disease, TAK-084 (Takeda) is a novel nonsteroid PDE4i currently under development. The study was performed as an additional exploratory end point of the randomized, single-center, observer-blind, vehicle- and active comparator–controlled study (registered as EUDRACT no. 2012-002998-62).6Snape S.D. Wigger-Alberti W. Goehring U.M. A phase I randomized trial to assess the effect on skin infiltrate thickness and tolerability of topical phosphodiesterase inhibitors in the treatment of psoriasis vulgaris using a modified psoriasis plaque test.Br J Dermatol. 2016; 175: 479-486Crossref PubMed Scopus (11) Google Scholar Skin punch biopsies of 7 patients with chronic plaque psoriasis were taken before and after treatment with roflumilast 0.5% cream, TAK-084 5% cream, and vehicle cream, respectively, and were analyzed for inflammatory biomarkers by immunostaining and mRNA microarray. For inclusion criteria as well as for materials and methods, see this article's Online Repository at www.jacionline.org. To address the hallmark of psoriasis, hyperproliferation of keratinocytes, Ki-67 and cytokeratin expression was determined by immunostaining. In healthy skin, the expression of the proliferation marker Ki-67 is restricted to basal keratinocytes, whereas in psoriatic lesions large amounts of basal and suprabasal keratinocytes express this marker protein. When compared with vehicle control samples, we detected a significant decrease in Ki-67+ cell numbers in samples from lesions treated with 5% TAK-084 and 0.5% roflumilast. This effect on pushing the proliferation toward normal is also reflected by the upregulation of cytokeratin 14 by roflumilast: cytokeratin 14 is a marker of basal proliferating keratinocytes and not of suprabasal, hyperproliferating cells. On investigating the composition of the inflammatory cell skin infiltrate, a significant decrease in CD4+ cell numbers in samples treated with 0.5% roflumilast was observed (Fig 1). Interestingly, this decrease was detectable only in the dermal compartment, while the ratio of dermal/epidermal CD4+ cells is significantly decreased after treatment (see Fig E1 in this article's Online Repository at www.jacionline.org). We believe that this effect may be due to the reimmigration of Langerhans cells into the skin, which had emigrated to the lymph nodes during the inflammation. Further on, there was a trend for less marker expression of chemokine receptor (C-C-motif) 6 (CCR6), CD4, and cytokeratin 10 in 5% TAK-084–treated samples and CCR6, CD83, CD68, CD1c, and CD123 in 0.5% roflumilast–treated samples when compared with vehicle control (Fig 1); however, these did not reach statistical significance. Especially CD4+ T helper cells of the TH17 subtype are believed to play a major role in psoriasis. The surface marker to discriminate TH17-polarized T cells from TH1, TH2, TH9, or regulatory T cells, CCR6, was reduced by trend in samples treated with both PDE4i's when compared with vehicle-treated samples. CCR6 is also expressed by the IL-17–producing neutrophils and may therefore be seen as a strong marker for psoriasis. The large and diverse set of putatively positive influences on protein expression reflects the positive results from the clinical end points of the trial6Snape S.D. Wigger-Alberti W. Goehring U.M. A phase I randomized trial to assess the effect on skin infiltrate thickness and tolerability of topical phosphodiesterase inhibitors in the treatment of psoriasis vulgaris using a modified psoriasis plaque test.Br J Dermatol. 2016; 175: 479-486Crossref PubMed Scopus (11) Google Scholar: all patient samples showed a positive clinical response regarding skin infiltrate thickness, assessed at the end of treatment and before the biopsy samples were taken.6Snape S.D. Wigger-Alberti W. Goehring U.M. A phase I randomized trial to assess the effect on skin infiltrate thickness and tolerability of topical phosphodiesterase inhibitors in the treatment of psoriasis vulgaris using a modified psoriasis plaque test.Br J Dermatol. 2016; 175: 479-486Crossref PubMed Scopus (11) Google Scholar To perform transcriptome analysis, RNA was extracted from biopsy material (see Table E1 in this article's Online Repository at www.jacionline.org). Healthy control skin was obtained from otherwise discarded material from surgery of 4 anonymous donors. On comparing the transcriptome of healthy skin samples to the vehicle-treated biopsies from psoriatic lesions, a large set of targets was found to be differentially regulated in psoriasis (see Fig E2 in this article's Online Repository at www.jacionline.org; Fig 2). Assessment of mean changes in gene expression revealed that 48 targets were upregulated 10-fold or higher in vehicle-treated psoriatic skin lesions compared with healthy skin (see Table E2 in this article's Online Repository at www.jacionline.org). Of these, 26 were downregulated by at least 5-fold following treatment with 0.5% roflumilast cream, whereas 4 targets were downregulated at least 5-fold with 5% TAK-084 cream. The S100A-family members S100A7A and S100A12, the neutrophil-associated marker transcobalamin 1, the transmembrane serine protease 11D, and human beta-defensin 2 (DEFB4A, HBD-2) are upregulated most strongly. Treatment with the PDE4i's led to the greatest reduction in the upregulated markers antimicrobial peptide human beta-defensin 2 (HBD-2, DEFB4A), the S100-family members S100A7A, S100A12, S100A9, and S100A7, the proinflammatory monocyte cytokine IL-19, the marker of suprabasal keratinocyte hyperproliferation cytokeratin16 (KRT16), the skin barrier proteins hornerin (HRNR), and late cornified envelope 3A; as well as the neutrophil-associated markers transcobalamin 1 and lipocalin 2 (Table E2). HBD-2 and IL-36-γ, both strongly upregulated compared with vehicle skin and downregulated upon PDE4i treatment, are described as biomarkers for psoriasis skin lesions.7D'Erme A.M. Wilsmann-Theis D. Wagenpfeil J. Holzel M. Ferring-Schmitt S. Sternberg S. et al.IL-36gamma (IL-1F9) is a biomarker for psoriasis skin lesions.J Invest Dermatol. 2015; 135: 1025-1032Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 8Kolbinger F. Loesche C. Valentin M.A. Jiang X. Cheng Y. Jarvis P. et al.β-Defensin-2 is a responsive biomarker of IL-17A-driven skin pathology in psoriasis.J Allergy Clin Immunol. 2017; 139: 923-932.e8Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 9Jansen P.A. Rodijk-Olthuis D. Hollox E.J. Kamsteeg M. Tjabringa G.S. de Jongh G.J. et al.Beta-defensin-2 protein is a serum biomarker for disease activity in psoriasis and reaches biologically relevant concentrations in lesional skin.PLoS One. 2009; 4: e4725Crossref PubMed Scopus (143) Google Scholar Changes in cytokine expression are depicted separately in Fig E3 in this article's Online Repository at www.jacionline.org. Gene ontology software analysis was performed on targets displayed in Table E2. Thereby, several biological processes were found to be significantly altered, including keratinocyte differentiation and keratinization, chronic inflammatory response, neutrophil aggregation, and leukocyte migration involved in inflammatory response (see Fig E4 in this article's Online Repository at www.jacionline.org). Thus, the action of topical PDE4i on several key biological processes of psoriasis was confirmed. Taken together, a reduction in pathological transcription alterations can be seen within TH1/TH17 cytokines, skin hyperproliferation, as well as neutrophil infiltration. Both immunohistological and transcriptome analyses were able to reflect the positive results from the clinical end points of the clinical trial.6Snape S.D. Wigger-Alberti W. Goehring U.M. A phase I randomized trial to assess the effect on skin infiltrate thickness and tolerability of topical phosphodiesterase inhibitors in the treatment of psoriasis vulgaris using a modified psoriasis plaque test.Br J Dermatol. 2016; 175: 479-486Crossref PubMed Scopus (11) Google Scholar Analyzing a large set of proinflammatory targets with relevance in psoriasis on the protein and the transcript levels, this study underlines the complex molecular pathology of psoriasis.E10Guttman-Yassky E. Nograles K.E. Krueger J.G. Contrasting pathogenesis of atopic dermatitis and psoriasis–part I: clinical and pathologic concepts.J Allergy Clin Immunol. 2011; 127: 1110-1118Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, E11Guttman-Yassky E. Nograles K.E. Krueger J.G. Contrasting pathogenesis of atopic dermatitis and psoriasis–part II: immune cell subsets and therapeutic concepts.J Allergy Clin Immunol. 2011; 127: 1420-1432Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar We can show that the broad spectrum of PDE4i effects largely covers the pathologically dysregulated genes. Furthermore, we show for the first time that this effect can be achieved when PDE4i's are applied topically and that the epidermal as well as the dermal compartment benefit. In summary, topical PDE4i's may represent a very promising treatment option in psoriasis. We kindly thank Hermann Tenor and Sue Snape from Takeda Pharmaceuticals for the cooperation in designing the experiments. This study was conducted in accordance with the declaration of Helsinki of 1975, as revised in 1983. Skin biopsy specimens were taken within the clinical trial ROF-PSOR104. The primary end point for that study was the change from baseline in thickness of the psoriatic skin infiltrate after 3 weeks; secondary end points were defined as change in thickness of the psoriatic skin infiltrate compared with baseline after 1 and 2 weeks, area under the curve of the baseline-corrected thickness of the psoriatic skin infiltrate, as well as change from baseline in clinical severity of the psoriatic lesions within each test area. Patients included were aged 18 to 65 years with chronic stable psoriasis plaques that were large enough for application of the different test substances. Exclusion criteria were pustular, erythrodermic, exfoliative, inverse, or guttate psoriasis and/or psoriatic arthritis; un- or poor responsiveness to topical treatments; known poor metabolizers of cytochrome P450 2D6; pregnancy; usage of topical antipsoriatic drugs (except for salicylic acid in Vaseline, Unilever Wirral, London, United Kingdom; Rotterdam, The Netherlands), of potent cytochrome P450 inhibitors, of ultraviolet light therapy or of any prescription medication (excluding hormonal contraceptives) within 4 weeks; and oral antipsoriatics within 3 months or injectable protein biologics within 6 months before drug administration. Adverse effects were mild or moderate. None was serious. Subjects were asked to consent to collection of biopsy samples. Consent/nonconsent was recorded in the eCRF.6Snape S.D. Wigger-Alberti W. Goehring U.M. A phase I randomized trial to assess the effect on skin infiltrate thickness and tolerability of topical phosphodiesterase inhibitors in the treatment of psoriasis vulgaris using a modified psoriasis plaque test.Br J Dermatol. 2016; 175: 479-486Crossref PubMed Scopus (11) Google Scholar Skin punch biopsies (4 mm) were obtained from the consenting subset of subjects at bioskin GmbH, Hamburg, Germany. On day 22, biopsies were taken from 3 sites, (1) vehicle-treated test field site, (2) roflumilast 0.5%–treated test field, and (3) TAK-084 5%–treated test field. The biopsies were taken under local anesthetic using aseptic technique, snap-frozen in liquid nitrogen, and then stored in a −80°C freezer or in liquid nitrogen. In addition, skin biopsy samples were obtained from volunteer donors for assessment of mRNA expression in healthy skin. A total of 7 of the 15 subjects included in the study consented to the skin biopsy procedure (Table E1). Generation of cryosections failed for the 5% TAK-084–treated sample from subject 006 and it therefore was excluded from all measurements. Skin biopsy samples were labeled at the clinical site using the subject randomization code. Samples remained blinded during processing and assessment of staining intensities and cell numbers. Samples to be included in the microarray mRNA profiling were selected on the basis of whether the extracted RNA was of sufficient quality. For immunohistological staining, serial 5-μm cryosections (CryoStar NX70) were prepared. The histological markers were chosen to address specific cell types known to be involved in the respective disease: CD4, CD8, CD68, CD1c, CD123, Ki-67, CCR6, cytokeratin 10 (CK10), cytokeratin 14 (CK14), and CD83. These markers were chosen to target the following populations and structures (in the same order): T helper cells (TH); cytotoxic T cells; monocytes and macrophages; myeloid dendritic cells; plasmacytoid dendritic cells and mast cells; proliferating keratinocytes; neutrophils and TH17-polarized T cells; keratinocytes (differentiated); keratinocytes (proliferating); and mature dendritic cells. The markers CD4, CD8, CD68, CD1c, CD123, CCR6, Ki-67, CD83, and CRTh2 were detected using immunohistochemistry methods. More precise, after cryosection, the sections were air-dried, fixed in acetone, and air-dried again. After PBS washing, EnVision horseradish peroxidase-system, substrate K 4004 (Dako, Glostrup, Denmark), suitable for mouse primary antibodies, and 3-amino-9-ethylcarbazol (AEC) substrate were applied according to manufacturer's instructions using the following antibodies: anti–Ki-67 (BD Biosciences, Franklin Lakes, NJ, 610969), anti-CD4 (Dako M7310), anti-CD8 (Dako M7103), anti-CD1c (Biolegend, San Diego, Calif, 331502), anti-CD123 (Biolegend 306002), anti-CD68 (Dako M0876), CCR6 (ThermoScientific clone 53.103.111, ThermoFisher, Waltham, Mass), and CD83 (clone HB15e). Samples were mounted using Faramount Mounting Medium, aqueous (Dako). Documentation of staining signal was performed using Olympus sc-30 color camera and getIT software (Olympus, Shinjuku, Japan). The markers CK10 and CK14 (MAB3230 and MAB3232, respectively, Chemicon/Merck Millipore, Billerica, Mass) were detected using immunofluorescence staining methods using an Mrm b/w camera and Axiovision software (both Zeiss, Jena, Germany). Isotype controls were included in the experiments. At least 2 cryosections were stained for each biopsy. At least 2 images were taken for each stained cryosection and evaluated by at least 2 blinded experts. An epidermal/dermal area covering at least 1 mm (horizontal) × 500 μm (in depth) was evaluated. The markers CD4, CD8, CD68, CD1c, CD123, CCR6, Ki-67, CD83, and CRTh2 were evaluated as positive events per area by counting in several optical fields under the microscope by at least 2 independent experienced observers. The markers CK10 and CK14 were evaluated according to a 4-point scale (no – low – medium – strong staining, scores 0-3) by at least 2 blinded experts. The marker CCR6 was assessed exclusively in the dermis because of strong epidermal keratinocyte staining. Total RNA of biopsy samples was extracted using the RNeasy Fibrous Tissue Mini Kit (Qiagen, Hilden, Germany). The quantity and purity of the total RNA were determined by the Nanodrop ND-1000 UV/VIS spectrophotometer, whereas the integrity was assessed by capillary electrophoresis with the Agilent Bioanalyzer 2100. The RNA integrity number (RIN) and the maximum delta RIN (Δ RINmax) of pairwise comparisons of the 3 samples per subject were calculated (Table E1). Samples from subjects 003, 004, and 007 showed too high intrasubject variability in RNA quality to perform microarray analysis (Δ RIN > 1.3). In contrast, samples from subjects 001, 002, 006, and 008 turned out to be considerably more homogeneous (Δ RINmax < 0.7) and were of intermediate or good quality (mean RIN > 7). These samples were therefore considered as suitable for microarray analysis (Table E1), giving rise to 12 single-color microarray hybridizations (3 conditions per subject). The RNA quality of healthy control skin matched the quality of the subject material (mean RIN = 7.0 ± 0.4). Microarray-based mRNA expression profiling was performed using the Human Whole Genome Oligo Microarray 4x44K v2 (Agilent Technologies, Santa Clara, Calif; AMADID 026652). A detailed protocol can be obtained at www.mh-hannover.de/rcut-downloads.html?L=1. It should be mentioned that a possible impact of the vehicle cream cannot be ruled out in this study. The emollient properties of the cream may also have a protective, anti-inflammatory effect, resulting in some inflammatory genes that may not play a role in psoriasis being less expressed in the vehicle-treated, inflamed skin compared with the control skin. It should also be noted that 1 control skin sample, healthy skin number 1, was the only biopsy within the microarray analysis derived from a male donor (as assessed by microarray analysis, target: X Inactive Specific Transcript), which could account for some of the observed differences in gene expression between this sample and the other healthy skin samples evident in the heatmap (Fig E1). To detect differences in the marker expression between vehicle and the 2 different treatments and to exclude the multiplicity testing problem, a 2-way ANOVA with Bonferroni posttest was applied. To calculate test statistics for paired observations, subsequently the paired t test was applied in case of Gaussian distributed data (assessed by Kolmogorov-Smirnov-test; CD8, CD68, CD123, CCR6, CK10, CK14, and CD83), and the Wilcoxon signed rank test for non-Gaussian distributed data (CD4, CD1c, Ki-67). All tests were performed using GraphPad Prism for Windows, version 5.02. Gene ontology analysis was performed using the WEB-based GEne SeT AnaLysis Toolkit applying the hsapiens_agilent_wholegenome_4x44k_v2 as reference gene list.E8Wang J. Duncan D. Shi Z. Zhang B. WEB-based GEne SeT AnaLysis Toolkit (WebGestalt): update 2013.Nucleic Acids Res. 2013; 41: W77-W83Crossref PubMed Scopus (1190) Google Scholar, E9Zhang B. Kirov S. Snoddy J. WebGestalt: an integrated system for exploring gene sets in various biological contexts.Nucleic Acids Res. 2005; 33: W741-W748Crossref PubMed Scopus (1386) Google ScholarFig E2Genes strongly upregulated and downregulated in psoriasis lesions as detected by microarray analyses. Fold changes of vehicle-treated psoriatic skin lesion (PSL) samples versus healthy skin samples, 0.5% roflumilast–treated, and 5% TAK-084–treated PSL samples, as indicated (n = 4). Only top 250 upregulated and downregulated markers (healthy vs vehicle-treated skin) are depicted.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Transcriptional regulation of the cytokines IL-1 to IL-38. Fold changes of vehicle-treated psoriatic lesional skin (PSL) samples versus healthy skin samples, 0.5% roflumilast–treated, and 5% TAK-084–treated PSL samples, as indicated (n = 4).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4In total, 9 biological processes were defined as significantly changed by gene ontology software (WEB-based GEne SeT AnaLysis Toolkit). Hierarchies of biological processes are displayed from left to right; colors indicate affiliation of genes to biological processes. Adjusted P values as well as contributing genes are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Subjects included in analysis of skin biopsy samplesSubjectTreatmentSample included in IHC analysisRNA concentration nanodrop (ng/μL)RNA concentration bioanalyzer (ng/μL)Bioanalyzer RINMean RINDelta RINmaxRNA profiling001Vehicle creamYes3773887.57.10.7Yes5% TAK-084 creamYes89806.90.5% roflumilast creamYes1701726.80020.5% roflumilast creamYes1811806.97.00.2Yes5% TAK-084 creamYes1231266.9Vehicle creamYes2351867.10035% TAK-084 creamYes25128.37.21.8No0.5% roflumilast creamYes117536.5Vehicle creamYes66416.70040.5% roflumilast creamYes40277.66.72.0No5% TAK-084 creamYes1611085.6Vehicle creamYes1701276.80065% TAK-084 creamNo1911266.76.90.5Yes0.5% roflumilast creamYes1961196.7Vehicle creamYes2391857.20070.5% roflumilast creamYes105776.66.61.4NoVehicle creamYes2902607.35% TAK-084 creamYes120865.9008Vehicle creamYes1851877.98.00.3Yes5% TAK-084 creamYes2562038.20.5% roflumilast creamYes2421818 Open table in a new tab Table E2Genes strongly upregulated in psoriasis lesions as detected by microarray analysesDifferentially expressed genesGene nameVehicle-treated PSL vs healthy skin5% TAK-084 vs vehicle-treated PSL0.5% Roflumilast vs vehicle-treated PSL(geometric mean of ratio values presented as fold change)S100A7AS100 calcium binding protein A7A459.96−6.55−53.02TCN1Transcobalamin 1207.67−4.49−15.73TMPRSS11DTransmembrane protease, serine 11D201.20−2.32−6.01DEFB4AHuman beta-defensin 2 (HBD-2)185.14−8.07−51.49S100A12S100 calcium-binding protein A1282.21−4.84−30.90SPRR2BSmall proline-rich protein 2B70.30−2.46−11.39S100A9S100 calcium-binding protein A955.86−1.94−4.03AKR1B10Aldo-keto reductase family 1, member B1047.89−1.57−3.02IL-19Interleukin 1947.82−7.00−26.05HRNRHornerin45.67−3.82−16.86LCE3ALate cornified envelope 3A45.21−5.70−29.25AKR1B15Aldo-keto reductase family 1, member B1539.92−1.63−3.08CLEC3AC-type lectin domain family 3, member A39.73−1.10−1.28GDAGuanine deaminase transcript variant 238.23−1.45−3.64PI3Peptidase inhibitor 3, skin-derived36.43−4.64−24.68S100A8S100 calcium-binding protein A829.39−1.29−1.93C10orf99Homo sapiens chromosome 10 open reading frame 99 (C10orf99), mRNA25.97−1.76−3.06SERPINB3Serpin peptidase inhibitor, member 322.14−4.14−14.77HYAL4Hyaluronoglucosaminidase 421.67−2.55−11.23RHCGRh family, C glycoprotein20.30−2.69−9.45KYNUKynureninase20.28−1.88−2.86ABCG4ATP-binding cassette, subfamily G (WHITE), member 417.96−2.03−5.16PITX1Paired-like homeodomain 116.281.03−1.43SERPINB4Serpin peptidase inhibitor, member 416.27−3.38−10.06VNN1Vanin 116.26−1.68−2.19KRT16Cytokeratin 1616.01−3.74−6.74PLEKHG7Pleckstrin homology domain containing, family G, member 716.00−1.61−3.32GPR110G protein–coupled receptor 11015.71−2.00−2.86MIR146AMicroRNA 146a15.18−1.09−1.78NOS2Inducible nitric oxide synthase 214.85−3.46−18.65LCE3ELate cornified envelope 3E14.33−2.00−10.24IL-36GInterleukin 36, gamma14.15−2.28−6.04DEFB103BHuman beta-defensin 3 (HBD-3)13.29−2.20−6.42GJB2Gap junction protein beta 213.25−2.45−4.79LCE3DLate cornified envelope 3D12.88−2.34−10.25PLA2G4DCytosolic phospholipase A212.55−2.28−4.57CXCL13Chemokine (C-X-C motif) ligand 1312.28−2.65−3.97TNIP3TNFAIP3 interacting protein 311.69−4.63−6.51PGBD5Homo sapiens piggyBac transposable element derived 5 (PGBD5)11.46−1.83−3.94KCNK10Potassium channel, subfamily K, member 1011.32−1.10−1.76ANKRD12Ankyrin repeat domain 12, transcript variant 310.941.351.28LOC284219Homo sapiens cDNA FLJ37117 fis, clone BRACE202227010.861.331.16FABP5P10Homo sapiens fatty acid binding protein 5 pseudogene 1010.73−1.83−3.89S100A7S100 calcium-binding protein A710.62−1.14−1.71PRSS27Serine protease 27, Pancreasin10.41−2.43−5.50LCN2Lipocalin 210.23−2.81−9.45CLLU1OSChronic lymphocytic leukemia upregulated 1 opposite strand10.07−2.33−7.29Geometric mean of ratio values presented as fold changes of vehicle-treated psoriatic skin lesion (PSL) samples vs healthy skin samples, 0.5% roflumilast–treated, and 5%TAK-084–treated PSL samples, as indicated (n = 4). Only genes showing a 10-fold or higher fold change between healthy and vehicle-treated skin are depicted. Only targets without y-chromosome–specific expression are shown. Open table in a new tab Geometric mean of ratio values presented as fold changes of vehicle-treated psoriatic skin lesion (PSL) samples vs healthy skin samples, 0.5% roflumilast–treated, and 5%TAK-084–treated PSL samples, as indicated (n = 4). Only genes showing a 10-fold or higher fold change between healthy and vehicle-treated skin are depicted. Only targets without y-chromosome–specific expression are shown.