Atopic dermatitis keratinocytes exhibit normal TH17 cytokine responses
2010; Elsevier BV; Volume: 125; Issue: 3 Linguagem: Inglês
10.1016/j.jaci.2009.12.934
ISSN1097-6825
AutoresKristine Nograles, Mayte Suárez‐Fariñas, Avner Shemer, Judilyn Fuentes‐Duculan, Andrea Chiricozzi, Irma Cardinale, Lisa C. Zaba, Toyoko Kikuchi, Michal Ramon, Richard N. Bergman, James G. Krueger, Emma Guttman‐Yassky,
Tópico(s)Allergic Rhinitis and Sensitization
ResumoTo the Editor: Host response against infectious challenge is typically mediated by specialized subsets of CD4+ effector TH cells. TH1 cells producing IFN-γ mediate cellular immunity against intracellular pathogens, whereas TH2 cells producing IL-4 and IL-13 mediate humoral immunity against parasites and helminths. TH17 cells producing IL-17 confer early protection against pathogenic insult in epithelial surfaces by inducing neutrophil-mediated immune responses and enhanced production of antimicrobial peptides (AMPs) such as β-defensins, lipocalin, and S100 proteins.1Iwakura Y. Nakae S. Saijo S. Ishigame H. The roles of IL-17A in inflammatory immune responses and host defense against pathogens.Immunol Rev. 2008; 226: 57-79Crossref PubMed Scopus (392) Google Scholar, 2Nograles 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 Atopic dermatitis (AD) and psoriasis vulgaris are inflammatory skin diseases that share some similarities—acanthotic epidermis and abundant dermal mononuclear infiltrate—in the chronic phase. However, 1 striking difference between these 2 diseases is the high frequency of recurrent skin infections in AD3Leung D.Y. Bieber T. Atopic dermatitis.Lancet. 2003; 361: 151-160Abstract Full Text Full Text PDF PubMed Scopus (1207) Google Scholar versus an increased resistance to infections in psoriasis.4Henseler T. Christophers E. Disease concomitance in psoriasis.J Am Acad Dermatol. 1995; 32: 982-986Abstract Full Text PDF PubMed Scopus (609) Google Scholar AD skin is heavily colonized by toxin-producing Staphylococcus aureus5Breuer K. S Ha Kapp A, Werfel T. Staphylococcus aureus: colonizing features and influence of an antibacterial treatment in adults with atopic dermatitis.Br J Dermatol. 2002; 147: 55-61Crossref PubMed Scopus (279) Google Scholar and is more prone to cutaneous viral infections such as eczema vaccinatum and eczema herpeticum.6Beck 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: 260-269Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar This has been attributed to the relative deficiency of innate defense molecules, including AMPs,7Ong P.Y. Ohtake T. Brandt C. Strickland I. Boguniewicz M. Ganz T. et al.Endogenous antimicrobial peptides and skin infections in atopic dermatitis.N Engl J Med. 2002; 347: 1151-1160Crossref PubMed Scopus (1693) Google Scholar primarily regulated by IL-17.2Nograles 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 The role of TH17 cells and IL-17 in AD is still unclear. Increased IL-17+ T cells have been identified in tissue sections of acute AD lesions relative to normal skin.8Toda M. Leung D.Y. Molet S. Boguniewicz M. Taha R. Christodoulopoulos P. et al.Polarized in vivo expression of IL-11 and IL-17 between acute and chronic skin lesions.J Allergy Clin Immunol. 2003; 111: 875-881Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar Our previous work on chronic AD lesions also demonstrated increased IL-17 expression compared with normal skin, but the overall expression of the IL-23/TH17 pathway is much reduced compared with psoriasis.9Guttman-Yassky E. Lowes M.A. Fuentes-Duculan J. Zaba L.C. Cardinale I. Nograles K.E. et al.Low expression of the IL-23/Th17 pathway in atopic dermatitis compared to psoriasis.J Immunol. 2008; 181: 7420-7427PubMed Google Scholar To clarify whether AMP deficiency in AD was a result of (1) absence of cognate IL-17 receptors, (2) defective signaling, or (3) TH2 suppression, we obtained lesional and nonlesional skin from 18 patients with chronic AD and 15 healthy volunteers under a Rockefeller University institutional review board–approved protocol. Biopsy samples were processed for histology, RT-PCR analysis, or generation of primary keratinocyte cultures. Normal human keratinocytes were also obtained as previously described2Nograles 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 (see this article's Methods in the Online Repository at www.jacionline.org for details). We have previously shown that IL-17 strongly induces keratinocyte expression of innate defense molecules. To determine whether functional IL-17 receptors were essential for the epidermal response to IL-17, we cultured pooled human keratinocytes with 1 μg/mL or 10 μg/mL monoclonal IL-17 Receptor A blocking antibody before stimulation with IL-17. After 24 hours, keratinocytes were harvested and examined for expression of known IL-17–induced innate defense genes. Fig 1 demonstrates that functional blockade of IL-17RA substantially inhibits expression of β-defensin 2 (DEFB4), IL-8, chemokine C-C motif ligand 20 (CCL20), and lipocalin2 (LCN2). Although all 4 genes were substantially inhibited by 10 μg/mL IL-17RA blocking antibody, 1 μg/mL IL-17RA blocking antibody was sufficient to suppress keratinocyte mRNA expression of DEFB4 and IL-8, with less effective inhibition of CCL20 and LCN2 expression, compared with isotype-blocked keratinocytes (Fig 1). The housekeeping gene human acidic ribosomal protein was used to control for general keratinocyte gene expression effects. These results demonstrate that fully functional IL-17 receptors are necessary for IL-17–mediated barrier defense. The differential sensitivity of AMP expression to IL-17RA blockade necessitates further investigation. One possibility for residual expression of AMP despite IL-17RA blockade is signaling through human IL-17RC, another receptor from the IL-17R family that can bind IL-17. We noted strong cell-surface expression of IL-17R in lesional AD keratinocytes (n = 5) by immunohistochemistry, with a staining pattern that is consistent with normal keratinocytes (n = 5; Fig 2, A). In addition to keratinocytes, scattered dermal cells were noted to express IL-17R as previously described.2Nograles 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 Consistent with immunohistochemical findings, gene expression levels for IL-17R measured by quantitative RT-PCR were similar between AD and normal keratinocytes (see this article's Fig E1 in the Online Repository at www.jacionline.org). After establishing surface expression of IL-17R in AD keratinocytes, we assessed whether these cells can sufficiently respond to IL-17 stimulation by genomic analysis (see Methods in the Online Repository). Genes with fold change >1.5 and false discovery rate <0.1 in IL-17–treated versus untreated AD keratinocytes were considered differentially expressed (see this article's Table E1 in the Online Repository at www.jacionline.org). The AD gene set was subsequently compared against previously published IL-17–modulated genes in normal keratinocytes.2Nograles 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 We found strong positive correlation between IL-17–modulated gene sets generated from AD keratinocytes and normal keratinocytes (r = 0.91; Fig 2, B). The close similarity between AD and normal keratinocyte gene sets was further established by the computed connectivity score10Lamb J. Crawford E.D. Peck D. Modell J.W. Blat I.C. Wrobel M.J. et al.The Connectivity Map: using gene-expression signatures to connect small molecules, genes, and disease.Science. 2006; 313: 1929-1935Crossref PubMed Scopus (938) Google Scholar (0.904; P < 10−16), wherein a perfect agreement between the 2 gene sets would yield a value of 1, whereas a perfect disagreement would be –1. Importantly, AD keratinocytes stimulated with IL-17 had robust upregulation of AMPs DEFB4 (517-fold from control) and LCN2 (5.7-fold from control), similar to normal keratinocytes (Fig 2, B). Proinflammatory neutrophil chemoattractants, CXCL 1, 3, 5, 6, and 8, as well as CCR6+ lymphocyte chemoattractant CCL20 were likewise strongly induced in both AD and normal keratinocytes (Fig 2, B). This was verified by quantitative RT-PCR analysis showing that IL-17 significantly upregulated DEFB4, LCN2, IL-8, and CCL20 expression in AD keratinocytes (P < .005) as it would in normal keratinocytes (Fig 2, C). Despite increased IL-17 levels compared with normal skin8Toda M. Leung D.Y. Molet S. Boguniewicz M. Taha R. Christodoulopoulos P. et al.Polarized in vivo expression of IL-11 and IL-17 between acute and chronic skin lesions.J Allergy Clin Immunol. 2003; 111: 875-881Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 9Guttman-Yassky E. Lowes M.A. Fuentes-Duculan J. Zaba L.C. Cardinale I. Nograles K.E. et al.Low expression of the IL-23/Th17 pathway in atopic dermatitis compared to psoriasis.J Immunol. 2008; 181: 7420-7427PubMed Google Scholar, 11Nograles K.E. Zaba L.C. Shemer A. Fuentes-Duculan J. Cardinale I. Kikuchi T. et al.IL-22-producing “T22” T cells account for upregulated IL-22 in atopic dermatitis despite reduced IL-17-producing TH17 T cells.J Allergy Clin Immunol. 2009; 123: 1244-1252Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar and equivalent capacity to respond to cytokine stimulation, AD lesions still have minimal DEFB4 and LCN2 gene expression.9Guttman-Yassky E. Lowes M.A. Fuentes-Duculan J. Zaba L.C. Cardinale I. Nograles K.E. et al.Low expression of the IL-23/Th17 pathway in atopic dermatitis compared to psoriasis.J Immunol. 2008; 181: 7420-7427PubMed Google Scholar To determine whether the TH2 cytokine milieu within AD lesions may have an effect toward IL-17–induced AMP expression, we cultured normal keratinocytes with IL-17 (200 ng/mL) alone or in combination with IL-4 (200 ng/mL) or IL-13 (200 ng/mL) and examined for gene expression of the aforementioned AMPs. We found that both IL-4 and IL-13 can significantly antagonize IL-17 induction of DEFB4 and LCN2 in keratinocytes (Fig 2, D) as previously reported.12Eyerich K. Pennino D. Scarponi C. Foerster S. Nasorri F. Behrendt H. et al.IL-17 in atopic eczema: linking allergen-specific adaptive and microbial-triggered innate immune response.J Allergy Clin Immunol. 2009; 123: 59-66Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar Thus, decreased innate defense molecule expression in AD is derived not from an inherent inability to respond to TH17 cytokine stimulation but likely from low ligand levels, compounded by further suppressive action from the TH2 microenvironment. Our findings have interesting implications for translational research because future therapeutics directed at increasing signaling through intact IL-17 receptors, despite low ligand expression, may potentially reverse this relative AMP deficiency in AD. One such approach might be TH2 blockade, allowing existing IL-17 to function fully without suppression, potentially restoring AMP expression and alleviating microbial load in AD. Lesional and nonlesional skin punch biopsies (5 mm diameter) were obtained from 18 adult patients with chronic AD and 15 healthy volunteers under a Rockefeller University institutional review board–approved protocol. Biopsy specimens were either frozen in OCT (Sakura, Torrance, Calif) and stored at –80°C for immunohistochemistry, snap-frozen in liquid nitrogen for RNA extraction, or used to generate primary keratinocyte cultures. To generate AD keratinocyte cultures, skin biopsies were incubated at 4°C overnight in dispase (Invitrogen Life Technologies). Epidermis was peeled off, then cultured in Epilife media (Cascade Biologics, Portland, Ore) with EDGS (Cascade Biologics) and penicillin-streptomycin (100 IU/mL and 0.1 mg/mL, respectively; Gibco, Carlsbad, Calif) at 37°C. Normal keratinocytes were obtained from the Yale Skin Diseases Research Center core facility as previously described.13 When cultured keratinocytes were 80% confluent, the culture media was supplemented with recombinant human (rh) IL-17 (200 ng/mL; R&D Systems, Minneapolis, Minn) with or without anti-IL17RA mouse mAb (clone M202; Amgen, Thousand Oaks, Calif), rhIL-4 (200 ng/mL; R&D Systems) or rhIL-13 (200 ng/mL; R&D Systems). RNA was extracted using the RNeasy Mini Kit (Qiagen) with on-column DNase digestion and used for either gene array or RT-PCR. Tissue sections were stained with IL-17RA mouse mAb (clone M202; Amgen) followed by biotin-labeled horse antimouse antibody (Vector Laboratories, Burlingame, Calif). Avidin-biotin complex (Vector Laboratories) was used for amplication and developed with chromogen 3-amino-9-ethylcarbazole (Sigma Aldrich, St Louis, Mo). Appropriate negative controls were used. For each Affymetrix genechip, 2 μg total RNA was reverse-transcribed, amplified, and labeled as described previously by using the BioArray High Yield RNA Transcription Labeling Kit (Enzo Biochem Inc, Farmingdale, NY).E1Nograles K.E. Zaba L.C. Guttman-Yassky E. 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 A total of 15 μg biotinylated complimentary RNA was then hybridized to the Affymetrix Human Genome U133A 2.0 Array (14,500 probe sets; Affymetrix, Santa Clara, Calif). Chips were washed, stained with streptavidin-phycoerythrin, and scanned with a Hewlett-Packard HP GeneArray Scanner (Hewlett-Packard, Palo Alto, Calif). Preprocessing and statistical analysis was conducted in R (http://www.rproject.org/). GeneChip CEL files were scrutinized for spatial artifacts using the Harshlight packageE2Suarez-Farinas M. Pellegrino M. Wittkowski K.M. Magnasco M.O. Harshlight: a “corrective make-up” program for microarray chips.BMC Bioinformatics. 2005; 6: 294Crossref PubMed Scopus (67) Google Scholar (http://asterion.rockefeller.edu/Harshlight/index2.html). Row intensities values (CEL files) were preprocessed to obtain expression values using the GCRMA algorithm. RT-PCR was performed by using EZ PCR core reagents, primers, and probles (Applied Biosystems, Foster City, Calif), as previously published.E1Nograles K.E. Zaba L.C. Guttman-Yassky E. 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 Primers and probes used in this study were as follows: DEFB4 (Hs00175474_m1), LCN2 (Hs00194353_m1), CCL20 (Hs00171125_m1), IL-8 (forward: GCTGGCCGTGGCTCTCT; reverse: TTTAGCACTCCTTGGCAAAACTG; probe: FAM-CCTTCCTGATTTCTGCAGCTCTGTGTGA-TAMRA). Data were analyzed and quantified by using the Applied Biosystems PRISM 7700 (Sequence Detection Systems, version 1.7), then normalized to the human acidic ribosomal protein housekeeping gene. For gene array analysis, expression values in log2-scale were obtained by GCRMA algorithm and probesets, then filtered to eliminate genes with low range intensities (values smaller than 2 in all samples) and low variation across samples (SD < 0.1). A moderated 2-tailed paired t test was performed with the Limma package from the Bioconductor project (http://www.bioconductor.org/). The P values of the moderated t test were adjusted for multiple hypotheses testing, controlling the false discovery rate with the Benjamini-Hochberg procedure. Results generated from AD keratinocytes were compared against those previously generated from normal keratinocytes.E1Nograles K.E. Zaba L.C. Guttman-Yassky E. 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 The connectivity score (CS),E3Lamb J. Crawford E.D. Peck D. et al.The connectivity map: using gene-expression signatures to connect small molecules, genes, and disease.Science. 2006; 313: 1929-1935Crossref PubMed Scopus (3777) Google Scholar introduced in the context of the connectivity map, quantifies how closely upregulated or downregulated genes in IL-17–treated normal keratinocytes1Iwakura Y. Nakae S. Saijo S. Ishigame H. The roles of IL-17A in inflammatory immune responses and host defense against pathogens.Immunol Rev. 2008; 226: 57-79Crossref PubMed Scopus (392) Google Scholar rank in IL-17–treated AD keratinocytes. The CS was generated as follows: An enrichment score (ES)E4Subramanian A. Tamayo P. Mootha V.K. et al.Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.Proc Natl Acad Sci U S A. 2005; 102: 15545-15550Crossref PubMed Scopus (30403) Google Scholar was first calculated for both upregulated and downregulated normal KC genes. If upregulated genes in normal keratinocytes rank high in AD, the ES (up) score will be near 1; if the opposite effect happens, the value approaches –1. An ES (down) score was likewise generated. The CS was then calculated as ES (up) minus ES (down), leading to a value between –2 and 2 followed by normalization to the unit interval. A CS value near 1 would indicate perfect agreement between normal and AD keratinocytes, 0 no agreement, and –1 a negative correlation. The distribution of the CS under the null hypothesis was obtained by simulation (number of simulations = 10,000). For each simulation, a random permutation in the AD fold change list was produced and the connectivity score calculated (CSn). The P value was obtained as 1 minus frequency of cases, where CSn is bigger in absolute value than CS for normal KC genes. The 2-tailed t test was used in the analysis of log-normalized RT-PCR data from patient samples. The 1-tailed paired t test was used in the analysis of log-normalized RT-PCR values from keratinocyte cultures. P values were designated as ∗P < .05, ∗∗P < .01, and ∗∗∗P < .005. Download .doc (.21 MB) Help with doc files
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