Anti-Inflammatory Action of Keratinocyte-Derived Vaspin
2016; Elsevier BV; Volume: 186; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2015.10.030
ISSN1525-2191
AutoresAnja Saalbach, Jenny Tremel, Diana Herbert, Katharina Schwede, Elke Wandel, Christine Schirmer, Ulf Anderegg, Annette G. Beck‐Sickinger, John T. Heiker, Stephan Schultz, Thomas M. Magin, Jan C. Simon,
Tópico(s)Adipokines, Inflammation, and Metabolic Diseases
ResumoImpaired cross talk between keratinocytes (KCs) and immune cells is believed to contribute to the pathogenesis of chronic inflammatory skin diseases, such as psoriasis. We have previously identified KCs as a rich source of the serpin protease inhibitor vaspin (serpinA12), originally described as an adipokine in adipose tissue. Herein, we studied whether dysregulated vaspin expression in KCs contributes to the pathogenesis of psoriasis. We found vaspin expression to be closely associated to epidermal differentiation, with low levels in proliferating KCs and high levels in differentiated cells. Consistently, in human psoriasis and in a mouse model of a psoriasis-like skin inflammation, epidermal vaspin expression was significantly down-regulated. Down-regulation of vaspin in KCs resulted in decreased expression of differentiation-associated genes and up-regulation of interferon-inducible and inflammation-associated psoriasis signature genes. Vaspin was also shown to modulate the communication between KCs and inflammatory cells under co-culture conditions. A decrease in vaspin expression in KCs stimulated the secretion of tumor necrosis factor-α, IL-1β, IL-6, IL-8, and monocyte chemoattractant protein-1 by co-cultured dendritic cells, macrophages, monocytes, and neutrophils. Consequently, the application of vaspin inhibited myeloid cell infiltration in a mouse model of a psoriasis-like skin inflammation. In conclusion, vaspin expression by maturing KCs modulates cutaneous immune responses and may be involved in the pathogenesis of psoriasis. Impaired cross talk between keratinocytes (KCs) and immune cells is believed to contribute to the pathogenesis of chronic inflammatory skin diseases, such as psoriasis. We have previously identified KCs as a rich source of the serpin protease inhibitor vaspin (serpinA12), originally described as an adipokine in adipose tissue. Herein, we studied whether dysregulated vaspin expression in KCs contributes to the pathogenesis of psoriasis. We found vaspin expression to be closely associated to epidermal differentiation, with low levels in proliferating KCs and high levels in differentiated cells. Consistently, in human psoriasis and in a mouse model of a psoriasis-like skin inflammation, epidermal vaspin expression was significantly down-regulated. Down-regulation of vaspin in KCs resulted in decreased expression of differentiation-associated genes and up-regulation of interferon-inducible and inflammation-associated psoriasis signature genes. Vaspin was also shown to modulate the communication between KCs and inflammatory cells under co-culture conditions. A decrease in vaspin expression in KCs stimulated the secretion of tumor necrosis factor-α, IL-1β, IL-6, IL-8, and monocyte chemoattractant protein-1 by co-cultured dendritic cells, macrophages, monocytes, and neutrophils. Consequently, the application of vaspin inhibited myeloid cell infiltration in a mouse model of a psoriasis-like skin inflammation. In conclusion, vaspin expression by maturing KCs modulates cutaneous immune responses and may be involved in the pathogenesis of psoriasis. The epidermis provides a defense against external challenges by both acting as a physicochemical barrier and coordinating the local immune response. Impaired cross talk between keratinocytes (KCs) and immune cells is believed to contribute to chronic inflammatory skin diseases like psoriasis.1Stefansson K. Brattsand M. Roosterman D. Kempkes C. Bocheva G. Steinhoff M. Egelrud T. Activation of proteinase-activated receptor-2 by human kallikrein-related peptidases.J Invest Dermatol. 2008; 128: 18-25Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar KCs sense external challenges via a wide repertoire of danger and pathogen recognition receptors.2Gutowska-Owsiak D. Ogg G.S. The epidermis as an adjuvant.J Invest Dermatol. 2012; 132: 940-948Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar As a result, they secrete cytokines and chemokines that recruit and activate various subsets of immune cells. The activation of immune cells, in turn, leads to the production of cytokines that support KC activation, proliferation, and differentiation. Disturbances in the balance of this cross talk can lead to erroneous positive feedback reactions promoting chronic inflammatory skin conditions, such as psoriasis.3Lowes M.A. Russell C.B. Martin D.A. Towne J.E. Krueger J.G. The IL-23/T17 pathogenic axis in psoriasis is amplified by keratinocyte responses.Trends Immunol. 2013; 34: 174-181Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar Psoriasis is characterized by inflammation accompanied by hyperproliferation and disturbed differentiation of KCs. Current paradigms suggest that it is based mainly on a T-cell–driven immune response targeting KCs.4Davidovici B.B. Sattar N. Prinz J.C. Puig L. Emery P. Barker J.N. van de K.P. Stahle M. Nestle F.O. Girolomoni G. Krueger J.G. Psoriasis and systemic inflammatory diseases: potential mechanistic links between skin disease and co-morbid conditions.J Invest Dermatol. 2010; 130: 1785-1796Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar, 5Lowes M.A. Bowcock A.M. Krueger J.G. Pathogenesis and therapy of psoriasis.Nature. 2007; 445: 866-873Crossref PubMed Scopus (1396) Google Scholar, 6Nickoloff B.J. Qin J.Z. Nestle F.O. Immunopathogenesis of psoriasis.Clin Rev Allergy Immunol. 2007; 33: 45-56Crossref PubMed Scopus (148) Google Scholar, 7Di C.A. Di M.P. Nestle F.O. The IL-23/Th17 axis in the immunopathogenesis of psoriasis.J Invest Dermatol. 2009; 129: 1339-1350Abstract Full Text Full Text PDF PubMed Scopus (830) Google Scholar, 8Albanesi C. Scarponi C. Pallotta S. Daniele R. Bosisio D. Madonna S. Fortugno P. Gonzalvo-Feo S. Franssen J.D. Parmentier M. De P.O. Girolomoni G. Sozzani S. Chemerin expression marks early psoriatic skin lesions and correlates with plasmacytoid dendritic cell recruitment.J Exp Med. 2009; 206: 249-258Crossref PubMed Scopus (250) Google Scholar, 9Nograles K.E. Davidovici B. Krueger J.G. New insights in the immunologic basis of psoriasis.Semin Cutan Med Surg. 2010; 29: 3-9Crossref PubMed Scopus (162) Google Scholar However, there are numerous indications that intrinsic alterations in epidermal KCs also play an important role in the disease. First, many intracellular signaling pathways have been found to be altered in psoriatic KCs.4Davidovici B.B. Sattar N. Prinz J.C. Puig L. Emery P. Barker J.N. van de K.P. Stahle M. Nestle F.O. Girolomoni G. Krueger J.G. Psoriasis and systemic inflammatory diseases: potential mechanistic links between skin disease and co-morbid conditions.J Invest Dermatol. 2010; 130: 1785-1796Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar, 5Lowes M.A. Bowcock A.M. Krueger J.G. Pathogenesis and therapy of psoriasis.Nature. 2007; 445: 866-873Crossref PubMed Scopus (1396) Google Scholar, 6Nickoloff B.J. Qin J.Z. Nestle F.O. Immunopathogenesis of psoriasis.Clin Rev Allergy Immunol. 2007; 33: 45-56Crossref PubMed Scopus (148) Google Scholar, 7Di C.A. Di M.P. Nestle F.O. The IL-23/Th17 axis in the immunopathogenesis of psoriasis.J Invest Dermatol. 2009; 129: 1339-1350Abstract Full Text Full Text PDF PubMed Scopus (830) Google Scholar, 8Albanesi C. Scarponi C. Pallotta S. Daniele R. Bosisio D. Madonna S. Fortugno P. Gonzalvo-Feo S. Franssen J.D. Parmentier M. De P.O. Girolomoni G. Sozzani S. Chemerin expression marks early psoriatic skin lesions and correlates with plasmacytoid dendritic cell recruitment.J Exp Med. 2009; 206: 249-258Crossref PubMed Scopus (250) Google Scholar, 9Nograles K.E. Davidovici B. Krueger J.G. New insights in the immunologic basis of psoriasis.Semin Cutan Med Surg. 2010; 29: 3-9Crossref PubMed Scopus (162) Google Scholar Second, genome-wide association studies have shown that 80% of differentially expressed genes in psoriatic skin lesions are linked to KC activity and infiltration of T cells and macrophages. The group of epidermal genes that is elevated in psoriasis shares a significant overlap with those stimulated by IL-1, IL-17, and IL-20 in cultured KCs.10Swindell W.R. Johnston A. Voorhees J.J. Elder J.T. Gudjonsson J.E. Dissecting the psoriasis transcriptome: inflammatory- and cytokine-driven gene expression in lesions from 163 patients.BMC Genomics. 2013; 14: 527Crossref PubMed Scopus (91) Google Scholar The balance between proteolytic enzymes and their inhibitors plays an important role in many aspects of skin biology, including epidermal differentiation, barrier formation, shedding, inflammation, immune responses, host defense, and wound healing.11Hovnanian A. Netherton syndrome: skin inflammation and allergy by loss of protease inhibition.Cell Tissue Res. 2013; 351: 289-300Crossref PubMed Scopus (151) Google Scholar, 12Meyer-Hoffert U. Reddish, scaly, and itchy: how proteases and their inhibitors contribute to inflammatory skin diseases.Arch Immunol Ther Exp (Warsz). 2009; 57: 345-354Crossref PubMed Scopus (110) Google Scholar, 13Meyer-Hoffert U. Neutrophil-derived serine proteases modulate innate immune responses.Front Biosci (Landmark Ed). 2009; 14: 3409-3418Crossref PubMed Google Scholar Proteolytic enzymes exert their regulatory functions through the control of cell-cell contacts, the activation of receptors (proteinase-activated receptors), and the regulation of cytokine and growth factor activity.1Stefansson K. Brattsand M. Roosterman D. Kempkes C. Bocheva G. Steinhoff M. Egelrud T. Activation of proteinase-activated receptor-2 by human kallikrein-related peptidases.J Invest Dermatol. 2008; 128: 18-25Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 14Sharony R. Yu P.J. Park J. Galloway A.C. Mignatti P. Pintucci G. Protein targets of inflammatory serine proteases and cardiovascular disease.J Inflamm (Lond). 2010; 7: 45Crossref PubMed Scopus (53) Google Scholar Changes in specific proteolytic activities in this context can result in inflammation with the typical clinical signs of redness, scaling, and itching.12Meyer-Hoffert U. Reddish, scaly, and itchy: how proteases and their inhibitors contribute to inflammatory skin diseases.Arch Immunol Ther Exp (Warsz). 2009; 57: 345-354Crossref PubMed Scopus (110) Google Scholar Thus, transgenic mice overexpressing the serine protease kallikrein 7 (KLK7) developed psoriasis-like skin alterations with a thickened epidermis, hyperkeratosis, a defective epidermal barrier, and chronic itchy dermatitis.15Ovaere P. Lippens S. Vandenabeele P. Declercq W. The emerging roles of serine protease cascades in the epidermis.Trends Biochem Sci. 2009; 34: 453-463Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar Analyses of psoriatic skin lesions have shown many proteases to be elevated.15Ovaere P. Lippens S. Vandenabeele P. Declercq W. The emerging roles of serine protease cascades in the epidermis.Trends Biochem Sci. 2009; 34: 453-463Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 16Komatsu N. Saijoh K. Toyama T. Ohka R. Otsuki N. Hussack G. Takehara K. Diamandis E.P. Multiple tissue kallikrein mRNA and protein expression in normal skin and skin diseases.Br J Dermatol. 2005; 153: 274-281Crossref PubMed Scopus (121) Google Scholar, 17Komatsu N. Saijoh K. Kuk C. Shirasaki F. Takehara K. Diamandis E.P. Aberrant human tissue kallikrein levels in the stratum corneum and serum of patients with psoriasis: dependence on phenotype, severity and therapy.Br J Dermatol. 2007; 156: 875-883Crossref PubMed Scopus (77) Google Scholar, 18Li W. Danilenko D.M. Bunting S. Ganesan R. Sa S. Ferrando R. Wu T.D. Kolumam G.A. Ouyang W. Kirchhofer D. The serine protease marapsin is expressed in stratified squamous epithelia and is up-regulated in the hyperproliferative epidermis of psoriasis and regenerating wounds.J Biol Chem. 2009; 284: 218-228Crossref PubMed Scopus (29) Google Scholar Vaspin is a serine protease inhibitor of the serpin family. Originally identified in white adipose tissue, vaspin has been suggested to function as an adipokine19Hida K. Wada J. Eguchi J. Zhang H. Baba M. Seida A. Hashimoto I. Okada T. Yasuhara A. Nakatsuka A. Shikata K. Hourai S. Futami J. Watanabe E. Matsuki Y. Hiramatsu R. Akagi S. Makino H. Kanwar Y.S. 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Visceral adipose tissue-derived serine protease inhibitor: a unique insulin-sensitizing adipocytokine in obesity.Proc Natl Acad Sci U S A. 2005; 102: 10610-10615Crossref PubMed Scopus (590) Google Scholar KLK7 has been identified as the primary target protease of vaspin and is abundantly expressed in human epidermis.19Hida K. Wada J. Eguchi J. Zhang H. Baba M. Seida A. Hashimoto I. Okada T. Yasuhara A. Nakatsuka A. Shikata K. Hourai S. Futami J. Watanabe E. Matsuki Y. Hiramatsu R. Akagi S. Makino H. Kanwar Y.S. Visceral adipose tissue-derived serine protease inhibitor: a unique insulin-sensitizing adipocytokine in obesity.Proc Natl Acad Sci U S A. 2005; 102: 10610-10615Crossref PubMed Scopus (590) Google Scholar, 20Klöting N. Berndt J. Kralisch S. Kovacs P. Fasshauer M. Schon M.R. Stumvoll M. Blüher M. Vaspin gene expression in human adipose tissue: association with obesity and type 2 diabetes.Biochem Biophys Res Commun. 2006; 339: 430-436Crossref PubMed Scopus (295) Google Scholar, 21Klöting N. Kovacs P. Kern M. Heiker J.T. Fasshauer M. Schon M.R. Stumvoll M. Beck-Sickinger A.G. Bluher M. Central vaspin administration acutely reduces food intake and has sustained blood glucose-lowering effects.Diabetologia. 2011; 54: 1819-1823Crossref PubMed Scopus (121) Google Scholar, 22Heiker J.T. Kloting N. Kovacs P. Kuettner E.B. Strater N. Schultz S. Kern M. Stumvoll M. Bluher M. Beck-Sickinger A.G. Vaspin inhibits kallikrein 7 by serpin mechanism.Cell Mol Life Sci. 2013; 70: 2569-2583Crossref PubMed Scopus (102) Google Scholar KLK7 controls the process of KC desquamation and activation of proinflammatory IL-1β and prochemerin, all of which are involved in the pathogenesis of psoriasis.23Borgono C.A. Michael I.P. Komatsu N. Jayakumar A. Kapadia R. Clayman G.L. Sotiropoulou G. Diamandis E.P. A potential role for multiple tissue kallikrein serine proteases in epidermal desquamation.J Biol Chem. 2007; 282: 3640-3652Crossref PubMed Scopus (224) Google Scholar, 24Lundstrom A. Egelrud T. Stratum corneum chymotryptic enzyme: a proteinase which may be generally present in the stratum corneum and with a possible involvement in desquamation.Acta Derm Venereol. 1991; 71: 471-474PubMed Google Scholar, 25Schultz S. Saalbach A. Heiker J.T. Meier R. Zellmann T. Simon J.C. Beck-Sickinger A.G. Proteolytic activation of prochemerin by kallikrein 7 breaks an ionic linkage and results in C-terminal rearrangement.Biochem J. 2013; 452: 271-280Crossref PubMed Scopus (43) Google Scholar Colocalization of vaspin and KLK7 in skin has been demonstrated.25Schultz S. Saalbach A. Heiker J.T. Meier R. Zellmann T. Simon J.C. Beck-Sickinger A.G. Proteolytic activation of prochemerin by kallikrein 7 breaks an ionic linkage and results in C-terminal rearrangement.Biochem J. 2013; 452: 271-280Crossref PubMed Scopus (43) Google Scholar We recently reported a high expression of vaspin in KCs and down-regulation in psoriatic skin lesions.26Saalbach A. Vester K. Rall K. Tremel J. Anderegg U. Beck-Sickinger A.G. Bluher M. Simon J.C. Vaspin: a link of obesity and psoriasis?.Exp Dermatol. 2012; 21: 309-312Crossref PubMed Scopus (31) Google Scholar Because the activity of proteases and their inhibitors influences a wide range of biological functions in skin, the regulation of vaspin expression in skin and its relevance to the control of cutaneous inflammation were investigated. Our data suggest that expression of vaspin in KCs serves to restrict skin inflammation and may be of clinical relevance to the pathogenesis of chronic inflammatory skin diseases, such as psoriasis. A total of 13 paired nonlesional and lesional skin biopsy specimens from psoriasis patients (all plaque-type) were analyzed for vaspin expression by real-time PCR or immunofluorescence staining. Psoriatic skin lesions were defined by erythematous, scaly plaques with the typical appearance of plaque-type psoriasis. Patient characteristics and biopsy localization are summarized in Table 1. Nonlesional skin was collected from the contralateral side. The study was approved by the local ethics committee (005-12-23012012). Healthy human skin was obtained after informed consent from patients undergoing cosmetic surgery (local ethics committee approval 025-14-27012014).Table 1Patient CharacteristicsAge (years)SexBMIDiabetes type IIPASIBiopsy specimenTreatment50M20N19.2Right thighNo therapy45M22.7N12.6Left thighNo therapy29M20.2N16.6Gluteal regionMometasone57F39.1Y19.7Lower backNo therapy67M25.2N3.9Left thighNo therapy56M30N28.8Right thighNo therapy78M24.8N24.3Right thighBetamethasone55M25.6N24Gluteal regionTriamcinolone68M44.1Y19.6Left thighNo therapy49M19.2N7.1Lower abdomenNo therapy42M37.6N3.8Left thighMometasone69F26.7Y37Lower backBetamethasone and calcipotriol69F35.2Y5.7Right thighBetamethasoneTopical treatment at the biopsy area was finished 4 weeks before the sampling was done. There was no systemic treatment.F, female; M, male; BMI, body mass index; N, no; PASI, Psoriasis Area Severity Index; Y, yes. Open table in a new tab Topical treatment at the biopsy area was finished 4 weeks before the sampling was done. There was no systemic treatment. F, female; M, male; BMI, body mass index; N, no; PASI, Psoriasis Area Severity Index; Y, yes. Human dendritic cells (DCs), monocytes, and M1 macrophages were generated from CD14-positive monocytes from whole blood using the CD14+ Cell Isolation Kit (Miltenyi, Bergisch Gladbach, Germany), as described.27Franz S. Allenstein F. Kajahn J. Forstreuter I. Hintze V. Moller S. Simon J.C. Artificial extracellular matrices composed of collagen I and high-sulfated hyaluronan promote phenotypic and functional modulation of human pro-inflammatory M1 macrophages.Acta Biomater. 2013; 9: 5621-5629Crossref PubMed Scopus (74) Google Scholar, 28Schirmer C. Klein C. von B.M. Simon J.C. Saalbach A. Human fibroblasts support the expansion of IL-17-producing T cells via up-regulation of IL-23 production by dendritic cells.Blood. 2010; 116: 1715-1725Crossref PubMed Scopus (67) Google Scholar Human in vitro–generated Langerhans cells were generated by cultivation of CD14-selected monocytes in DC medium [RPMI 1640 medium (Invitrogen, Darmstadt, Germany), 2% fetal calf serum (Promocell, Heidelberg, Germany), 1% penicillin/streptomycin (Invitrogen), 1% l-glutamine (Invitrogen), 0.1 mmol/L nonessential amino acids (Invitrogen), 10 mmol/L HEPES (Invitrogen), 100 U/mL IL-4 (Peprotech, Hamburg, Germany), and 1000 U/mL granulocyte-macrophage colony-stimulating factor (Leukine; Berlex, Richmond, VA)] and 20 ng/mL transforming growth factor-β (Peprotech) for 4 days. Normal human epidermal KCs were isolated from healthy skin biopsy specimens. First, the epidermal layer of the skin was separated by overnight dispase (2.2 U/mL Dispase II; Roche, Mannheim, Germany) digestion at 4°C. Single cells were then isolated from the epidermis by trypsin digestion (Trypsin/EDTA; Biochrom, Berlin, Germany) for 9 minutes at 37°C, followed by vortex mixing, and were cultured in KGM-Gold KC medium (Lonza, Cologne, Germany). HaCaT cells were cultured in Dulbecco's modified Eagle's medium (Biochrom) containing 10% fetal calf serum and 1% penicillin/streptomycin (Biochrom). For 3D epidermis equivalents, outer root sheath cells were isolated by outgrowth from anagen hairs, which were plucked from the temporal scalp region. After treatment with antibiotics, the hair follicles were placed on 24-mm Transwell Nylon meshes (Corning, Darmstadt, Germany), seeded with mitomycin-treated fibroblasts on the lower site, and grown at air-liquid interface for 3 days, in K-medium (Euroderm GmbH, Leipzig, Germany) exposed to 5% O2, 5% CO2, and 90% N2. At 40% confluence (days 10 ± 2), the cells were covered with K-medium. At 90% confluence (days 18 ± 2), the cells were removed from the mesh by treatment with TrypZean solution (Sigma, Deisenhofen, Germany) and seeded at a density of 0.7 × 106/mL on 12-mm Transwell Nylon meshes prepared with fibroblasts, as described above, and cultivated under submerged conditions in K0-medium (Euroderm GmbH). After 3 days, cultures were lifted and cells were maintained at the air-liquid interface for a further 15 days. Cryostat sections of human healthy skin, lesional and nonlesional psoriatic skin, or murine healthy and lesional skin were fixed with acetone and incubated with indicated antibodies for immunofluorescence staining (Table 2). Nuclei were stained with DAPI (Merck, Berlin, Germany). Microscopy was performed with Keyence BZ-9000E and corresponding software BZ-II Viewer version 1.41, BZ-II Analyser version 2.2 (Keyence, Leipzig, Germany).Table 2Antibodies for ImmunohistochemistryTargetReactivityCompanyVaspinHumanEnzo LifeSciences (Lörrach, Germany)Cytokeratin 10HumanDako (Hamburg, Germany)Cytokeratin 14HumanAbcam (Cambridge, UK)VaspinMouseEnzo LifeSciencesCD11b-PEMouseMiltenyi Biotech (Bergisch-Gladbach, Germany)Gr-1–PEMouseBeckmann Coulter (Krefeld, Germany)F4/80MouseBioLegend (Fell, Germany)Anti-rat Alexa 546, anti-mouse Alexa 546, and anti-rabbit Alexa 546Invitrogen Life Technologies (Darmstadt, Germany)All primers were purchased from Metabion (Munich, Germany), and siRNA was purchased from Riboxx (Radebeul, Germany).PE, phycoerythrin. Open table in a new tab All primers were purchased from Metabion (Munich, Germany), and siRNA was purchased from Riboxx (Radebeul, Germany). PE, phycoerythrin. Skin biopsy specimens were homogenized in lysis buffer (Qiagen quick RNA isolation kit) using a tissue homogenizer. Total RNA from homogenates and cells was isolated using the quick-RNA isolation kit (Qiagen, Hilden, Germany). RNA (1 μg) was used for first-strand cDNA synthesis with Moloney Murine Leukemia Virus Reverse Transcriptase (Promega, Mannheim, Germany), according to the manufacturer's protocol, and real-time PCR of human and mouse vaspin (SERPINA12; Serpina12) and the reference genes for human ribosomal protein S26 (RPS26) and mouse RS36B4 was performed (Table 3). Expression levels were quantified using a standard curve and were normalized to the unregulated reference gene.Table 3Details of the Primers Used for Real-Time PCRGeneAccession no.∗Available from the NCBI Nucleotide database (http://www.ncbi.nlm.nih.gov/nuccore).Region on sequenceAmplicon size (bp)Forward primerReverse primerhu_RPS26NM_001029286–4741895′-CAATGGTCGTGCCAAAAAG-3′5′-TTCACATACAGCTTGGGAAGC-3′hu_VASPIN (SERPINA12)NM_1738501626–18472225′-AGGGCAAGCTGAAGCACTTGG-3′5′-CGCCCACTTTCAGGCTGCGA-3′mu_RS36B4 (Rplp0)NM_007475464–549865′-GGACCCGAGAAGACCTCCTT-3′5′-GCACATCACTCAGAATTTCAATGG-3′mu_Vaspin (Serpina12)NM_0265351209–14642565′-CCTCACCCGGATCTCATCTC-3′5′-AAGCCAACAGGGAATGCTCC-3′All primers were purchased from Metabion (Munich, Germany), and siRNA was purchased from Riboxx (Radebeul, Germany).hu, human; mu, murine.∗ Available from the NCBI Nucleotide database (http://www.ncbi.nlm.nih.gov/nuccore). Open table in a new tab All primers were purchased from Metabion (Munich, Germany), and siRNA was purchased from Riboxx (Radebeul, Germany). hu, human; mu, murine. KCs were cultivated in 24-well plates and at 70% to 80% confluence were stimulated for 24 or 72 hours with 10 ng/mL tumor necrosis factor (TNF)-α, 5 ng/mL IL-1β, or 20 ng/mL IL-6, IL-8, IL-17, IL-22, epidermal growth factor (Miltenyi), 50 ng/mL IL-19, IL-23 (Miltenyi), or 50 ng/mL IL-20 (Roche). HaCaT cells were transfected when they reached 90% confluence with a eukaryotic expression plasmid (pcDNA3.1) coding for human vaspin (NM_173850.2; 780 to 2014 bp) (HaCaT-vaspin) or the control vector [pcDNA3.1 (Invitrogen); HaCaT-vec ctr] using the FuGENE HD transfection reagent (Roche). Positive cells were selected via antibiotic resistance (G418). Vaspin expression was analyzed by enzyme-linked immunosorbent assay (ELISA; Adipogen, Hamburg, Germany). Subconfluent KCs were transfected with vaspin siRNA [hu_VASPIN (SERPINA12); accession number NM_173850; 5′-UCUCAUCCAUCUUCAGCUCCCCC-3′] or scrambled siRNA (5′-UAAGCACGAAGCUCAGAGUCCCCC-3′) at 70% confluence using RiboxxFects, according to the manufacturer's protocol (Riboxx, Radebeul, Germany). The medium was replaced after 24 hours, and the cells were cultured for a further 3 days. KCs were transfected with vaspin siRNA, as described, cultured for 3 days, and stimulated with 10 ng/mL TNF-α/IL-17 for 24 hours before isolation of RNA. Before microarray analysis, RNA integrity and concentration were examined on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) using the RNA 6.000 LabChip Kit (Agilent Technologies), according to the manufacturer's instructions. Illumina BeadChip (Illumina, San Diego, CA) analysis was conducted at the microarray core facility of the Interdisciplinary Center for Clinical Research Leipzig (Faculty of Medicine, University of Leipzig, Leipzig, Germany). RNA (250 ng/sample) was ethanol precipitated with GlycoBlue (Invitrogen) as carrier and dissolved at a concentration of 100 to 150 ng/μL before probe synthesis using the TargetAmp-Nano Labeling Kit for Illumina Expression BeadChip (Epicentre Biotechnologies, Madison, WI). Complementary RNA (750 ng) was hybridized to Human HT-12 version 4 Expression BeadChips (Illumina) and scanned on the Illumina HiScan instrument, according to the manufacturer's specifications. Raw data from 47,323 probes were extracted and quantile normalized by Illumina GenomeStudio version 1.9.0. All expression values were background subtracted. HaCaT cells were first cultured to confluence. A total of 4 × 105 freshly isolated DCs, monocytes, neutrophils, or M1 macrophages were then added in 500 μL DC-medium to the HaCaT cells. A Transwell system was used for co-culture with KCs. KCs were first cultured to confluence. Then, 4 × 105 monocytes were added in 500 μL DC-medium to the upper compartment of the Transwell system (0.4 μm; Greiner BioOne, Frickenhausen, Germany). Cells were cultured for 6 hours before 100 ng/mL lipopolysaccharide (LPS) activation. Supernatants were taken for cytokine analysis by ELISA 24 hours after LPS addition. As a control, HaCaT cells, KCs, and immune cells were cultured separately under conditions identical to those of the co-culture model. The levels of cytokines in HaCaT cells or KCs alone were subtracted from the levels of cytokines in the co-culture system. TNF-α, IL-1β, IL-6, IL-8, IL-10, IL-12, IL-23, monocyte chemoattractant protein-1 (eBioscience, Frankfurt, Germany), and vaspin (Adipogen) were detected by ELISA, according to the manufacturer's instructions. Hydrogen peroxide was detected using the Red Hydrogen Peroxidase Assay Kit, according to the manufacturer's protocol (Enzo, Lörrach, Germany). Imiquimod ointment (50 mg; Aldara; Meda Pharma GmbH, Bad Homurg, Germany) was applied topically to the shaved back of BALB/c mice for 4 consecutive days under anesthesia. On the basis of studies by Klöting et al,21Klöting N. Kovacs P. Kern M. Heiker J.T. Fasshauer M. Schon M.R. Stumvoll M. Beck-Sickinger A.G. Bluher M. Central vaspin administration acutely reduces food intake and has sustained blood glucose-lowering effects.Diabetologia. 2011; 54: 1819-1823Crossref PubMed Scopus (121) Google Scholar 250 ng human recombinant vaspin (Enzo) dissolved in 20 μL phosphate-buffered saline was injected s.c. every day. Controls received 20 μL phosphate-buffered saline. On day 5, samples of lesional skin were collected for RNA isolation and immunohistochemical staining. All animal experiments were performed according to institutional and state guidelines. The Committee on Animal Welfare of Saxony approved animal protocols used in this study (TVV19/12). Distribution of data was assessed by Shapiro-Wilk test. Depending on the normality of the data, analysis was performed using the Mann-Whitney rank-sum test or the t-test. P < 0.05 was considered to be significant. We recently reported vaspin to be strongly expressed in skin.26Saalbach A. Vester K. Rall K. Tremel J. Anderegg U. Beck-Sickinger A.G. Bluher M. Simon J.C. Vaspin: a link of obesity and psoriasis?.Exp Dermatol. 2012; 21: 309-312Crossref PubMed Scopus (31) Google Scholar Because immunostaining revealed a predominant expression in the upper layer of the epidermis, we examined whether vaspin expression was associated with KC differentiation.26Saalbach A. Vester K. Rall K. Tremel J. Anderegg U. Beck-Sickinger A.G. Bluher M. Simon J.C. Vaspin: a link of obesity and psoriasis?.Exp Dermatol. 2012; 21: 309-312Crossref PubMed Scopus (31) Google Scholar To address this question, 3D equivalents of human skin were generated. Seeding of KCs onto a feeder layer of fibroblasts and induction of differentiation by raising to the air-liquid interphase significantly up-regulated KC vaspin expression (Figure 1A). This was accompanied by an increase
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