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S100A8 and S100A9 Are Induced by Decreased Hydration in the Epidermis and Promote Fibroblast Activation and Fibrosis in the Dermis

2015; Elsevier BV; Volume: 186; Issue: 1 Linguagem: Inglês

10.1016/j.ajpath.2015.09.005

ISSN

1525-2191

Autores

Aimei Zhong, Wei Xu, Jingling Zhao, Ping Xie, Shengxian Jia, Jiaming Sun, Robert D. Galiano, Thomas A. Mustoe, Seok Jong Hong,

Tópico(s)

Immune Response and Inflammation

Resumo

The most critical function of the epidermis is to prevent water loss and maintain skin homeostasis. Disruption of the functional skin barrier causes delayed wound healing, hypertrophic scarring, and many skin diseases. Herein, we show that reduced hydration increases the expression of S100 protein family members, S100A8/S100A9, in stratified keratinocyte culture and human ex vivo skin culture. Immunohistological analyses show that S100A8/A9 are highly expressed in the epidermis of human hypertrophic scar and keloid tissues. Reduced hydration demonstrates activation of fibroblasts in the keratinocyte-fibroblast co-culture. In contrast, knockdown of S100A8 or S100A9 by RNA interference in keratinocytes failed to activate fibroblasts. Pretreatment with pharmacological blockers of S100A8/A9 receptors, Toll-like receptor 4 and receptor for advanced glycation end products, inhibits fibroblast activation induced by recombinant S100A8/A9 proteins. Moreover, we observe that local delivery of S100A8 protein results in a marked increase in hypertrophic scarring in the in vivo rabbit ear scar model. Our results indicate that hydration status promotes fibroblast activation and fibrosis by directly affecting the expression of inflammatory signaling in keratinocytes, thereby strongly suggesting S100A8/A9 to be novel targets in preventing scarring. The most critical function of the epidermis is to prevent water loss and maintain skin homeostasis. Disruption of the functional skin barrier causes delayed wound healing, hypertrophic scarring, and many skin diseases. Herein, we show that reduced hydration increases the expression of S100 protein family members, S100A8/S100A9, in stratified keratinocyte culture and human ex vivo skin culture. Immunohistological analyses show that S100A8/A9 are highly expressed in the epidermis of human hypertrophic scar and keloid tissues. Reduced hydration demonstrates activation of fibroblasts in the keratinocyte-fibroblast co-culture. In contrast, knockdown of S100A8 or S100A9 by RNA interference in keratinocytes failed to activate fibroblasts. Pretreatment with pharmacological blockers of S100A8/A9 receptors, Toll-like receptor 4 and receptor for advanced glycation end products, inhibits fibroblast activation induced by recombinant S100A8/A9 proteins. Moreover, we observe that local delivery of S100A8 protein results in a marked increase in hypertrophic scarring in the in vivo rabbit ear scar model. Our results indicate that hydration status promotes fibroblast activation and fibrosis by directly affecting the expression of inflammatory signaling in keratinocytes, thereby strongly suggesting S100A8/A9 to be novel targets in preventing scarring. Scarring is an inevitable result of the wound healing process. However, excess fibrosis results in hypertrophic scarring, causing cosmetic disfiguration and disruption of the skin barrier function. Hypertrophic scarring is commonly associated with a state of chronic inflammation caused by a variety of stimuli, including persistent tension, foreign body retention, and infection. The sustained release of many proinflammatory cytokines and chemokines, which contributes to the activation of fibroblasts and the excessive production of collagen in the dermis, is found in hypertrophic scars.1Ueha S. Shand F.H. Matsushima K. Cellular and molecular mechanisms of chronic inflammation-associated organ fibrosis.Front Immunol. 2012; 3: 71Crossref PubMed Scopus (119) Google Scholar, 2Wynn T.A. Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases.J Clin Invest. 2007; 117: 524-529Crossref PubMed Scopus (1107) Google Scholar Although scarring has been characterized as fibroblast activation in the dermis, recent evidence suggests that keratinocytes in the epidermis also play an essential role in this process.3Bellemare J. Roberge C.J. Bergeron D. Lopez-Valle C.A. Roy M. Moulin V.J. Epidermis promotes dermal fibrosis: role in the pathogenesis of hypertrophic scars.J Pathol. 2005; 206: 1-8Crossref PubMed Scopus (112) Google Scholar, 4Machesney M. Tidman N. Waseem A. Kirby L. Leigh I. Activated keratinocytes in the epidermis of hypertrophic scars.Am J Pathol. 1998; 152: 1133-1141PubMed Google Scholar, 5Tandara A.A. Mustoe T.A. The role of the epidermis in the control of scarring: evidence for mechanism of action for silicone gel.J Plast Reconstr Aesthet Surg. 2008; 61: 1219-1225Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar Unrecovered barrier function of the epidermis results in increased transepithelial water loss (TEWL) after wound repair in the skin. Many reports suggest that TEWL remains high in pathological scars, such as hypertrophic scars and keloids, and demonstrates that TEWL is implicated in dermal fibrosis after wounds are healed.6Suetake T. Sasai S. Zhen Y.X. Ohi T. Tagami H. Functional analyses of the stratum corneum in scars: sequential studies after injury and comparison among keloids, hypertrophic scars, and atrophic scars.Arch Dermatol. 1996; 132: 1453-1458Crossref PubMed Google Scholar It has been widely accepted that occlusion improves the appearance and symptoms of scars by regulating cytokines and growth factors in the epidermis.5Tandara A.A. Mustoe T.A. The role of the epidermis in the control of scarring: evidence for mechanism of action for silicone gel.J Plast Reconstr Aesthet Surg. 2008; 61: 1219-1225Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 7Mustoe T.A. Evolution of silicone therapy and mechanism of action in scar management.Aesthetic Plast Surg. 2008; 32: 82-92Crossref PubMed Scopus (209) Google Scholar, 8O'Shaughnessy K.D. De La Garza M. Roy N.K. Mustoe T.A. Homeostasis of the epidermal barrier layer: a theory of how occlusion reduces hypertrophic scarring.Wound Repair Regen. 2009; 17: 700-708Crossref PubMed Scopus (50) Google Scholar However, the mechanisms are not well charted yet. It is also known that mucosal wounds heal faster and have less scarring compared with skin wounds, perhaps, in part, because of the moist characteristics of mucosal surfaces.9Gallant-Behm C.L. Mustoe T.A. Occlusion regulates epidermal cytokine production and inhibits scar formation.Wound Repair Regen. 2010; 18: 235-244Crossref PubMed Scopus (52) Google Scholar, 10Gallant-Behm C.L. Du P. Lin S.M. Marucha P.T. DiPietro L.A. Mustoe T.A. Epithelial regulation of mesenchymal tissue behavior.J Invest Dermatol. 2011; 131: 892-899Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 11Chen L. Arbieva Z.H. Guo S. Marucha P.T. Mustoe T.A. DiPietro L.A. Positional differences in the wound transcriptome of skin and oral mucosa.BMC Genomics. 2010; 11: 471Crossref PubMed Google Scholar By using the rabbit partial-thickness incisional wounding scar model, we reveal that maintaining hydration enhances the wound repair process and down-regulates the expression of many cytokines compared with wounds in which hydration is not maintained. Interestingly, the global gene expression of skin wounds is also changed compared with that of mucosal wounds when hydration of the skin wounds is preserved.12Xu W. Jia S. Xie P. Zhong A. Galiano R.D. Mustoe T.A. Hong S.J. The expression of proinflammatory genes in epidermal keratinocytes is regulated by hydration status.J Invest Dermatol. 2014; 134: 1044-1055Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar The expression of S100A8/A9 is significantly elevated in the epidermis when the skin is exposed to reduced hydration conditions. The low-molecular-weight proteins S100A8/A9 belong to the S100 protein family and are involved in diverse cellular processes, such as cell cycle regulation and differentiation.13Eckert R.L. Broome A.M. Ruse M. Robinson N. Ryan D. Lee K. S100 proteins in the epidermis.J Invest Dermatol. 2004; 123: 23-33Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 14Vogl T. Gharibyan A.L. Morozova-Roche L.A. Pro-inflammatory S100A8 and S100A9 proteins: self-assembly into multifunctional native and amyloid complexes.Int J Mol Sci. 2012; 13: 2893-2917Crossref PubMed Scopus (125) Google Scholar, 15Korndorfer I.P. Brueckner F. Skerra A. The crystal structure of the human (S100A8/S100A9)2 heterotetramer, calprotectin, illustrates how conformational changes of interacting alpha-helices can determine specific association of two EF-hand proteins.J Mol Biol. 2007; 370: 887-898Crossref PubMed Scopus (207) Google Scholar S100A8/A9 are abundantly expressed in myeloid cells and have been extensively characterized as major players involved in both acute and chronic inflammation.16Vogl T. Tenbrock K. Ludwig S. Leukert N. Ehrhardt C. van Zoelen M.A. Nacken W. Foell D. van der Poll T. Sorg C. Roth J. Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock.Nat Med. 2007; 13: 1042-1049Crossref PubMed Scopus (1016) Google Scholar, 17van Lent P.L. Grevers L. Blom A.B. Sloetjes A. Mort J.S. Vogl T. Nacken W. van den Berg W.B. Roth J. Myeloid-related proteins S100A8/S100A9 regulate joint inflammation and cartilage destruction during antigen-induced arthritis.Ann Rheum Dis. 2008; 67: 1750-1758Crossref PubMed Scopus (147) Google Scholar, 18Foell D. Wittkowski H. Vogl T. Roth J. S100 proteins expressed in phagocytes: a novel group of damage-associated molecular pattern molecules.J Leukoc Biol. 2007; 81: 28-37Crossref PubMed Scopus (664) Google Scholar S100A8/A9 (alias calprotectin) have antifungal, antibacterial, and immunomodulating effects reflecting their protective properties.15Korndorfer I.P. Brueckner F. Skerra A. The crystal structure of the human (S100A8/S100A9)2 heterotetramer, calprotectin, illustrates how conformational changes of interacting alpha-helices can determine specific association of two EF-hand proteins.J Mol Biol. 2007; 370: 887-898Crossref PubMed Scopus (207) Google Scholar, 19Sorenson B.S. Khammanivong A. Guenther B.D. Ross K.F. Herzberg M.C. IL-1 receptor regulates S100A8/A9-dependent keratinocyte resistance to bacterial invasion.Mucosal Immunol. 2012; 5: 66-75Crossref PubMed Scopus (32) Google Scholar, 20Abtin A. Eckhart L. Glaser R. Gmeiner R. Mildner M. Tschachler E. The antimicrobial heterodimer S100A8/S100A9 (calprotectin) is upregulated by bacterial flagellin in human epidermal keratinocytes.J Invest Dermatol. 2010; 130: 2423-2430Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Interestingly, their expression is up-regulated in skin injury and during the wound healing process.21Thorey I.S. Roth J. Regenbogen J. Halle J.P. Bittner M. Vogl T. Kaesler S. Bugnon P. Reitmaier B. Durka S. Graf A. Wockner M. Rieger N. Konstantinow A. Wolf E. Goppelt A. Werner S. The Ca2+-binding proteins S100A8 and S100A9 are encoded by novel injury-regulated genes.J Biol Chem. 2001; 276: 35818-35825Crossref PubMed Scopus (192) Google Scholar, 22Eming S.A. Koch M. Krieger A. Brachvogel B. Kreft S. Bruckner-Tuderman L. Krieg T. Shannon J.D. Fox J.W. Differential proteomic analysis distinguishes tissue repair biomarker signatures in wound exudates obtained from normal healing and chronic wounds.J Proteome Res. 2010; 9: 4758-4766Crossref PubMed Scopus (166) Google Scholar Moreover, higher expression of S100A8/A9 is found in cancer, intestinal diseases, and lesions of psoriasis patients.23Madsen P. Rasmussen H.H. Leffers H. Honore B. Celis J.E. Molecular cloning and expression of a novel keratinocyte protein (psoriasis-associated fatty acid-binding protein [PA-FABP]) that is highly up-regulated in psoriatic skin and that shares similarity to fatty acid-binding proteins.J Invest Dermatol. 1992; 99: 299-305Abstract Full Text PDF PubMed Scopus (220) Google Scholar Herein, we hypothesize that decreased hydration (ie, increased TEWL) up-regulates proinflammatory cytokine expression, which further causes fibrosis in the dermis. In this study, we used human ex vivo skin culture (HESC) and stratified keratinocyte culture (SKC) to show that decreased hydration leads to up-regulation of S100A8/A9 in the epidermis. We then establish that S100A8/A9 from epidermal keratinocytes regulate dermal fibroblast activation through Toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE) with the loss-of-function study using RNA interference (RNAi) and gain-of-function study using recombinant proteins. Finally, we demonstrate that delivery of exogenous S100A8 protein causes increased hypertrophic scar formation in the rabbit ear model. HESC was performed following the protocol described previously.24Xu W. Jong Hong S. Jia S. Zhao Y. Galiano R.D. Mustoe T.A. Application of a partial-thickness human ex vivo skin culture model in cutaneous wound healing study.Lab Invest. 2012; 92: 584-599Crossref PubMed Scopus (61) Google Scholar Briefly, skin from elective abdominoplasties was processed to make partial-thickness skin grafts that were cultured in growth medium: Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (Life Technologies, Grand Island, NY). The stratum corneum of the skin was removed by tape stripping to remove the functional skin barrier. SKC was performed following the standard protocol.25Tandara A.A. Kloeters O. Mogford J.E. Mustoe T.A. Hydrated keratinocytes reduce collagen synthesis by fibroblasts via paracrine mechanisms.Wound Repair Regen. 2007; 15: 497-504Crossref PubMed Scopus (37) Google Scholar, 26Xu W. Hong S.J. Zeitchek M. Cooper G. Jia S. Xie P. Qureshi H.A. Zhong A. Porterfield M.D. Galiano R.D. Surmeier D.J. Mustoe T.A. Hydration status regulates sodium flux and inflammatory pathways through epithelial sodium channel (ENaC) in skin.J Invest Dermatol. 2015; 135: 796-806Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar HaCaT cells, a spontaneously immortalized human keratinocyte cell line, were cultured in DMEM containing 10% FBS. Human primary foreskin keratinocytes were obtained from Northwestern University Skin Disease Research Center (Chicago, IL) and cultured in keratinocyte-serum free medium (Life Technologies). A total of 5 × 105 HaCaT cells or primary keratinocytes were cultured in a 12-well plate insert (0.4-μm pore size) for 1 to 2 days in their growth medium. The inserts were switched to E-medium and cultured for 2 weeks to make the stratified epidermis. E-medium contained 50:50 (v/v) DMEM and Ham's F-12 supplemented with 18 μmol/L adenine, 500 ng/mL bovine pancreatic insulin, 500 ng/mL human apo-transferrin, 500 ng/mL triiodothyronine, 4 mmol/L l-glutamine, 0.4 μg/mL hydrocortisone, 10 ng/mL cholera toxin, 5 ng/mL epidermal growth factor, 5% FBS, and antibiotics (penicillin/streptomycin). When the keratinocyte and fibroblast co-culture was performed, stratified HaCaT cells were placed in the upper well, and human foreskin dermal fibroblasts were placed in the bottom plates. Human foreskin fibroblasts and E-medium were purchased from Northwestern University Skin Disease Research Center. To maintain hydration conditions, HESC and SKC were cultured in a closed chamber with a humid environment (Figure 1A). To generate increased water loss conditions (reduced hydration), HESC and SKC were cultured while exposed to dry air flow (Figure 1A). The two major S100A8/A9 receptors, TLR4 and RAGE, were blocked by adding 400 nmol/L TAK-242, a specific inhibitor of TLR4 (EMD Millipore, Billerica, MA), and 500 nmol/L FPS-ZM1, a specific antagonist of RAGE (EMD Millipore), to the cell culture medium. The human skin three-dimensional culture model was established on the basis of previous studies with some modifications.27Ridky T.W. Chow J.M. Wong D.J. Khavari P.A. Invasive three-dimensional organotypic neoplasia from multiple normal human epithelia.Nat Med. 2010; 16: 1450-1455Crossref PubMed Scopus (164) Google Scholar Briefly, human skin specimens were obtained from elective abdominoplasties. By using a dermatome, specifically a Weck knife, a partial-thickness skin specimen (approximately 10% of full thickness) was generated and then placed in 2 mol/L NaCl solution at 37°C for 24 hours to completely remove the epidermis. Thereafter, the dermis was snap frozen in liquid nitrogen, followed by an immediate thaw at 37°C. The freeze-thaw process was repeated three times. The dermal tissue was incubated in phosphate-buffered saline at 4°C for 3 weeks to completely remove the stromal cells in the dermis. HaCaT cells were then seeded onto the basement membrane of the decellularized dermal matrix set in a cell culture insert. After 2 days, the growth medium, DMEM supplemented with 10% FBS, was drained from the top of the cell culture to generate a liquid-air interface environment for the seeded keratinocytes, whereas the growth medium in the surrounding area was replaced with E-medium. HaCaT cells were fully differentiated between days 10 and 14. Hemidesmosome protein BP180- and BP230-specific antibodies (gifts from Dr. Jonathan Jones, Northwestern University Feinberg School of Medicine, Chicago, IL) were used to analyze the integrity of the epidermal basement membrane and dermal-epidermal junction.24Xu W. Jong Hong S. Jia S. Zhao Y. Galiano R.D. Mustoe T.A. Application of a partial-thickness human ex vivo skin culture model in cutaneous wound healing study.Lab Invest. 2012; 92: 584-599Crossref PubMed Scopus (61) Google Scholar The epidermis of the HESC samples was separated from the dermis by treating the sample with 0.5 mol/L ammonium thiocyanate for 30 minutes at room temperature.12Xu W. Jia S. Xie P. Zhong A. Galiano R.D. Mustoe T.A. Hong S.J. The expression of proinflammatory genes in epidermal keratinocytes is regulated by hydration status.J Invest Dermatol. 2014; 134: 1044-1055Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 26Xu W. Hong S.J. Zeitchek M. Cooper G. Jia S. Xie P. Qureshi H.A. Zhong A. Porterfield M.D. Galiano R.D. Surmeier D.J. Mustoe T.A. Hydration status regulates sodium flux and inflammatory pathways through epithelial sodium channel (ENaC) in skin.J Invest Dermatol. 2015; 135: 796-806Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 28Trost A. Bauer J.W. Lanschutzer C. Laimer M. Emberger M. Hintner H. Onder K. Rapid, high-quality and epidermal-specific isolation of RNA from human skin.Exp Dermatol. 2007; 16: 185-190Crossref PubMed Scopus (33) Google Scholar The epidermis was placed in Trizol Reagent (Sigma-Aldrich, St. Louis, MO) and homogenized using a MagNA Lyser (Roche, Indianapolis, IN) in the presence of Zirconia beads (2.0-mm diameter; Biospec Products Inc., Bartlesville, OK). Stratified or monolayer HaCaT cells were treated with Trizol Reagent alone. Total RNA was isolated according to the manufacturer's protocol. Contaminating genomic DNA during RNA preparation was removed using the Turbo DNA-free kit (Life Technologies). cDNA was made using Superscript II from 2 μg of total RNA, and quantitative PCR analyses using SYBR Green I were performed using an ABI Fast Real-Time PCR System (Life Technologies). Glyceraldehyde-3-phosphate dehydrogenase was used as a normalization control, ΔCt. The 2−ΔΔCt formula was used to calculate the gene expression difference between samples. Primers used for quantitative PCR are listed in Table 1.Table 1Primer Pairs Used for qPCRTarget genePrimer typeSequenceCXCL8Forward5′-TTTTGCCAAGGAGTGCTAAAGA-3′Reverse5′-AACCCTCTGCACCCAGTTTTC-3′IL1BForward5′-CTCGCCAGTGAAATGATGGCT-3′Reverse5′-GTCGGAGATTCGTAGCTGGAT-3′TNFForward5′-ATGAGCACTGAAAGCATGATCC-3′Reverse5′-GAGGGCTGATTAGAGAGAGGTC-3′PTGS2 (Cox-2)Forward5′-GTGCAACACTTGAGTGGCTAT-3′Reverse5′-AGCAATTTGCCTGGTGAATGAT-3′S100A8Forward5′-ATGCCGTCTACAGGGATGAC-3′Reverse5′-ACGCCCATCTTTATCACCAG-3′S100A9Forward5′-GGTCATAGAACACATCATGGAGG-3′Reverse5′-GGCCTGGCTTATGGTGGTG-3′GAPDHForward5′-TGTTGCCATCAATGACCCCTT-3′Reverse5′-CTCCACGACGTACTCAGCG-3′β-ActinForward5′-CATGTACGTTGCTATCCAGGC-3′Reverse5′-CTCCTTAATGTCACGCACGAT-3′qPCR, quantitative PCR Open table in a new tab qPCR, quantitative PCR Slides divided into sections from paraffin-embedded tissues and fixed cell culture were used for immunostaining. For the tissues, sections (4 μm thick) were deparaffinized and treated with antigen retrieval solution (10 mmol/L sodium citrate and 0.05% Tween 20, pH 6.0). After primary antibody treatment, fluorescence-labeled secondary antibodies were used for immunofluorescence staining. The primary antibodies used for immunostaining include S100A8 (rabbit anti-human; a gift from Dr. Philippe Tessier, Université Laval, Quebec City, QC, Canada; 1:1000 dilution), S100A9 (rabbit anti-human; a gift from Dr. Philippe Tessier; 1:1000 dilution), procollagen-I (mouse anti-human; Developmental Studies Hybridoma Bank at University of Iowa, Iowa City, IA; 1:1000 dilution), cytokeratin 14 (CK14; rabbit anti-human; Covance, Princeton, NJ; 1:5000 dilution), CK10 (mouse anti-human; Dako, Carpinteria, CA; 1:5000 dilution), and α-smooth muscle actin (α-SMA; rabbit anti-human; Santa Cruz Biotechnology, Santa Cruz, CA; 1:1000 dilution). For immunohistochemistry, biotin-conjugated secondary antibodies (Vector Laboratories, Burlingame, CA) were treated after Ki-67 (mouse anti-human; BD Biosciences, San Jose, CA; 1:1000) or caspase-3 (Cell Signaling, Beverly, MA) specific antibody treatment. The signal was detected using the Vectastain kit (Vector Laboratories) and visualized using 3,3′-diaminobenzidine. For Western blot analysis, whole cell extracts were prepared using radioimmunoprecipitation assay buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1% Triton X-100, 0.1% SDS, and 0.5% sodium deoxycholate, pH 7.5). Trichloroacetic acid/acetone/medium (1:8:1 volume) was used to concentrate proteins in culture medium. Proteins were separated onto SDS polyacrylamide gels and transferred onto polyvinylidene difluoride–nitrocellulose membranes. After primary antibody treatment, horseradish peroxidase–conjugated secondary antibody was applied and the signal was visualized using an Enhanced Chemiluminescence detection kit (GE Healthcare Bio-Sciences, Piscataway, NJ). β-Actin was used as a normalization control. The density of the signal was quantified by the ImageJ software version 1.46r (NIH, Bethesda, MD; http://imagej.nih.gov/ij). The primary antibodies used for immunoblot staining include S100A8 (rabbit anti-human; a gift from Dr. Philippe Tessier; 1:5000 dilution), S100A9 (rabbit anti-human; a gift from Dr. Philippe Tessier; 1:5000 dilution), α-SMA (mouse anti-human; Santa Cruz Biotechnology; 1:1000 dilution), and β-actin (rabbit anti-human; Santa Cruz Biotechnology; 1:5000). The sequences of shRNAs were selected using the RNAi consortium database.29Moffat J. Grueneberg D.A. Yang X. Kim S.Y. Kloepfer A.M. Hinkle G. Piqani B. Eisenhaure T.M. Luo B. Grenier J.K. Carpenter A.E. Foo S.Y. Stewart S.A. Stockwell B.R. Hacohen N. Hahn W.C. Lander E.S. Sabatini D.M. Root D.E. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen.Cell. 2006; 124: 1283-1298Abstract Full Text Full Text PDF PubMed Scopus (1353) Google Scholar The double-stranded shRNA oligos were cloned to pLKO.1 puro vector (Addgene, Cambridge, MA), and lentiviruses were made following the protocol provided by Addgene. Lentivirus-transduced HaCaT cells were selected in the presence of 2 μg/mL puromycin. Knockdown of S100A8 or S100A9 was tested by Western blot analysis. The following double-stranded shRNA sequences were made (IDT, Coralville, IA) for RNAi of S100A8 or S100A9 (Table 2). The boldfaced sequences indicate the target sequences of RNAi for S100A8 and S100A9. RNAi information for IL-1β, IL-8, cyclooxygenase (Cox)-2, and tumor necrosis factor (TNF)-α was described previously.12Xu W. Jia S. Xie P. Zhong A. Galiano R.D. Mustoe T.A. Hong S.J. The expression of proinflammatory genes in epidermal keratinocytes is regulated by hydration status.J Invest Dermatol. 2014; 134: 1044-1055Abstract Full Text Full Text PDF PubMed Scopus (27) Google ScholarTable 2Sequence for shRNATarget genePrimer typeSequenceCXCL8∗Gene knockdown information described previously.12Forward5′-CCGGGCTCTGTGTGAAGGTGCAGTTCTCGAGAACTGCACCTTCACACAGAGCTTTTTG-3′Reverse5′-AATTCAAAAAGCTCTGTGTGAAGGTGCAGTTCTCGAGAACTGCACCTTCACACAGAGC-3′IL1B∗Gene knockdown information described previously.12Forward5′-CCGGATCAATAACAAGCTGGAATTTCTCGAGAAATTCCAGCTTGTTATTGATTTTTTG-3′Reverse5′-AATTCAAAAAATCAATAACAAGCTGGAATTTCTCGAGAAATTCCAGCTTGTTATTGAT-3′TNF∗Gene knockdown information described previously.12Forward5′-CCGGCCTCTCTCTAATCAGCCCTCTCTCGAGAGAGGGCTGATTAGAGAGAGGTTTTT-3′Reverse5′-AATTAAAAACCTCTCTCTAATCAGCCCTCTCTCGAGAGAGGGCTGATTAGAGAGAGG-3′PTGS2 (Cox-2)∗Gene knockdown information described previously.12Forward5′-CCGGGCTGAATTTAACACCCTCTATCTCGAGATAGAGGGTGTTAAATTCAGCTTTTTG-3′Reverse5′-AATTCAAAAAGCTGAATTTAACACCCTCTATCTCGAGATAGAGGGTGTTAAATTCAGC-3′S100A8Forward5′-CCGGCCACAAGTACTCCCTGATAAACTCGAGTTTATCAGGGAGTACTTGTGGTTTTTG-3′Reverse5′-AATTCAAAAACCACAAGTACTCCCTGATAAACTCGAGTTTATCAGGGAGTACTTGTGG-3′S100A9Forward5′-CCGGAGAGACCATCATCAACACCTTCTCGAGAAGGTGTTGATGATGGTCTCTTTTTTG-3′Reverse5′-AATTCAAAAAAGAGACCATCATCAACACCTTCTCGAGAAGGTGTTGATGATGGTCTCT-3′The bold sequences indicate the target sequences of RNA interference.∗ Gene knockdown information described previously.12Xu W. Jia S. Xie P. Zhong A. Galiano R.D. Mustoe T.A. Hong S.J. The expression of proinflammatory genes in epidermal keratinocytes is regulated by hydration status.J Invest Dermatol. 2014; 134: 1044-1055Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar Open table in a new tab The bold sequences indicate the target sequences of RNA interference. The open reading frames of human S100A8, human S100A9, and rabbit S100A8 (http://www.ncbi.nlm.nih.gov/nuccore; GenBank accession numbers NM_002964, NM_002965, and XM_002715343, respectively) genes were cloned by PCR, and their sequences were confirmed by DNA sequencing. The open reading frames of human S100A8, human S100A9, and rabbit S100A8 were cloned to pET-15b (Novagen, Madison, WI), which carries an N-terminal His Tag, using the NdeI and BamHI restriction enzyme sites, and constructed pET-hS100A8, pET-hS100A9, and pET-rS100A8 (Table 3). pET-hS100A8 was transformed to Escherichia coli BL21 (DE3) strain. pET-hS100A9 and pET-rS100A8 were transformed to E. coli BL21 (DE3) pLysS strain. Induction of human S100A8 was performed by adding 1 mmol/L isopropyl β-d-1-thiogalactopyranoside for 3 hours at 37°C. Induction of human S100A9 and rabbit S100A8 was performed by adding 1 mmol/L isopropyl β-d-1-thiogalactopyranoside for 5 hours at 25°C. His-Tagged proteins were purified using the nickel-chelating resin following the standard purification protocol. Recombinant proteins were eluted in the presence of 250 mmol/L imidazole and dialysis against phosphate-buffered saline. Recombinant proteins were further purified using the Pierce High Capacity Endotoxin Removal Resin (Life Technologies).Table 3Primer Pairs Used for the Recombinant Proteins ExpressionTarget genePrimer typeSequenceHumanForward5′-CATATGTTGACCGAGCTGGAGAAA-3′ S100A8Reverse5′-GGATCCCTACTCTTTGTGGCTTTC-3′HumanForward5′-CATATGACTTGCAAAATGTCGCAG-3′ S100A9Reverse5′-GGATCCTTAGGGGGTGCCCTCCCC-3′RabbitForward5′-CATATGCCGACTGATCTGGAAAAT-3′ S100A8Reverse5′-GGATCCCTACGCCTTGTGGCTGTCTTC-3′The underlined sequences denote the restriction enzyme sites, NdeI (forward) and BamHI (reverse). The bold sequences denote the start codon (forward) and stop codon (reverse). Open table in a new tab The underlined sequences denote the restriction enzyme sites, NdeI (forward) and BamHI (reverse). The bold sequences denote the start codon (forward) and stop codon (reverse). For the HESC, human skin was obtained from elective abdominoplasties. Human tissues were acquired under a protocol approved by the Institutional Review Board of Northwestern University. For the histological analysis of S100A8 and S100A9, human normal skin, hypertrophic scar, and keloid specimens were obtained under the protocols approved by the Institutional Review Board of Northwestern University and Tongji Medical College. Human specimens were obtained from Northwestern Memorial Hospital (Chicago, IL) and Union Hospital (Wuhan, Hubei, China). A biopsy was performed on samples from chest skin of patients who underwent scar excision (10 cases of hypertrophic scar and five cases of keloid) and patients who underwent skin grafting (five cases of normal skin). The patients were 20 to 35 years old, and the lesions were excised at least 1 year after formation. Of all 20 cases, two keloid specimens were from humans of African descent and 18 were from Asian descent. Hypertrophic scars were caused by surgery, burn, or cut; however, the cause of keloids was unclear. Tissues were fixed in formalin and embedded in paraffin. Animal experiments in this study were approved by the Northwestern University Animal Care and Use Committee. Female New Zealand white rabbits were purchased from Covance. Six excisional wounds (7 mm full thickness) were generated on the ventral surface of each rabbit ear. A semiocclusive dressing, Tegaderm, was placed onto the wounded ear to prevent desiccation. When the wounds were fully reepithelialized, approximately 14 days after wounding, recombinant rabbit S100A8 protein was injected into the wounds at postoperative days (PODs) 15, 19, and 23 (10 μg per wound, each time). Saline was injected into the wounds in the contralateral ear as a control. The rabbit wounds were harvested at POD 28, fixed in formalin, embedded in paraffin, and divided into sections (4 μm thick). The histological slides were stained for hematoxylin and eosin dyes and analyzed using a Nikon Eclipse 50i light microscope (Nikon Instruments Inc., Melville, NY). The scar elevation index (SEI) was calculated.30Reid R.

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