Staphylococcus aureus impairs dermal fibroblast functions with deleterious effects on wound healing

The FASEB JournalVolume 35, Issue 7 e21695 RESEARCH ARTICLEOpen Access Staphylococcus aureus impairs dermal fibroblast functions with deleterious effects on wound healing Masaya Yokota, Masaya Yokota Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Zurich, Switzerland Department of Allergy and Clinical Immunology, Graduate School of Medicine, Chiba University, Chiba, JapanSearch for more papers by this authorNicola Häffner, Nicola Häffner Department of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, Zurich, SwitzerlandSearch for more papers by this authorMatthew Kassier, Matthew Kassier Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Zurich, SwitzerlandSearch for more papers by this authorMatthias Brunner, Matthias Brunner Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Zurich, SwitzerlandSearch for more papers by this authorSrikanth Mairpady Shambat, Srikanth Mairpady Shambat Department of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, Zurich, SwitzerlandSearch for more papers by this authorFabian Brennecke, Fabian Brennecke Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Zurich, SwitzerlandSearch for more papers by this authorJanine Schniering, Janine Schniering Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Zurich, SwitzerlandSearch for more papers by this authorEwerton Marques Maggio, Ewerton Marques Maggio Institute of Pathology and Molecular Pathology, University Hospital Zurich, Zurich, SwitzerlandSearch for more papers by this authorOliver Distler, Oliver Distler Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Zurich, SwitzerlandSearch for more papers by this authorAnnelies Sophie Zinkernagel, Annelies Sophie Zinkernagel Department of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, Zurich, SwitzerlandSearch for more papers by this authorBritta Maurer, Corresponding Author Britta Maurer Britta.Maurer@usz.ch Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Zurich, Switzerland Department of Rheumatology and Immunology, University Hospital Bern, University Bern, Bern, Switzerland Correspondence Britta Maurer, Center of Experimental Rheumatology, Department of Rheumatology, Gloriastrasse 25, 8091 Zurich, Switzerland. Email: Britta.Maurer@usz.chSearch for more papers by this author Masaya Yokota, Masaya Yokota Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Zurich, Switzerland Department of Allergy and Clinical Immunology, Graduate School of Medicine, Chiba University, Chiba, JapanSearch for more papers by this authorNicola Häffner, Nicola Häffner Department of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, Zurich, SwitzerlandSearch for more papers by this authorMatthew Kassier, Matthew Kassier Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Zurich, SwitzerlandSearch for more papers by this authorMatthias Brunner, Matthias Brunner Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Zurich, SwitzerlandSearch for more papers by this authorSrikanth Mairpady Shambat, Srikanth Mairpady Shambat Department of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, Zurich, SwitzerlandSearch for more papers by this authorFabian Brennecke, Fabian Brennecke Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Zurich, SwitzerlandSearch for more papers by this authorJanine Schniering, Janine Schniering Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Zurich, SwitzerlandSearch for more papers by this authorEwerton Marques Maggio, Ewerton Marques Maggio Institute of Pathology and Molecular Pathology, University Hospital Zurich, Zurich, SwitzerlandSearch for more papers by this authorOliver Distler, Oliver Distler Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Zurich, SwitzerlandSearch for more papers by this authorAnnelies Sophie Zinkernagel, Annelies Sophie Zinkernagel Department of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, Zurich, SwitzerlandSearch for more papers by this authorBritta Maurer, Corresponding Author Britta Maurer Britta.Maurer@usz.ch Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Zurich, Switzerland Department of Rheumatology and Immunology, University Hospital Bern, University Bern, Bern, Switzerland Correspondence Britta Maurer, Center of Experimental Rheumatology, Department of Rheumatology, Gloriastrasse 25, 8091 Zurich, Switzerland. Email: Britta.Maurer@usz.chSearch for more papers by this author First published: 23 June 2021 https://doi.org/10.1096/fj.201902836RAboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Chronic wounds are a major disease burden worldwide. The breach of the epithelial barrier facilitates transition of skin commensals to invasive facultative pathogens. Therefore, we investigated the potential effects of Staphylococcus aureus (SA) on dermal fibroblasts as key cells for tissue repair. In co-culture systems combining live or heat-killed SA with dermal fibroblasts derived from the BJ-5ta cell line, healthy individuals, and patients with systemic sclerosis, we assessed tissue repair including pro-inflammatory cytokines, matrix metalloproteases (MMPs), myofibroblast functions, and host defense responses. Only live SA induced the upregulation of IL-1β/-6/-8 and MMP1/3 as co-factors of tissue degradation. Additionally, the increased cell death reduced collagen production, proliferation, migration, and contractility, prerequisite mechanisms for wound closure. Intracellular SA triggered inflammatory and type I IFN responses via intracellular dsDNA sensor molecules and MyD88 and STING signaling pathways. In conclusion, live SA affected various key tissue repair functions of dermal fibroblasts from different sources to a similar extent. Thus, SA infection of dermal fibroblasts should be taken into account for future wound management strategies. Abbreviations CFU colony-forming unit DU digital ulcers dcSSc diffuse cutaneous systemic sclerosis HC healthy control HKSA heat-killed Staphylococcus aureus MMP matrix metalloprotease MyD88 myeloid differentiation factor 88 qPCR quantitative real-time PCR SA Staphylococcus aureus siRNA small interfering RNA SSc systemic sclerosis STING stimulator of interferon genes TLR Toll-like receptor 1 INTRODUCTION Chronic wounds have become a problem of epidemic proportions worldwide.1, 2 Disturbed circulation due to macroangiopathy as in peripheral arterial occlusive disease or resulting from microangiopathy such as in diabetes mellitus or certain autoimmune disorders is a major contributing factor.3 For example in systemic sclerosis (SSc), a multi-systemic autoimmune connective tissue disease, up to two thirds of patients suffer from chronic digital ulcers (DU),4 which are often complicated by necrosis, soft tissue infection, and osteomyelitis.5 Chronic non-healing wounds are characterized by persistent inflammation, defective re-epithelialization, and impaired matrix remodeling.3, 6 Chronic wounds are always colonized by bacteria.1, 7 In dermal ulcers, the breach of the basement membrane8 exposes dermal tissue and cells to commensal skin bacteria, facilitating invasion.9 Staphylococcus aureus (SA) is a common colonizer of the human skin and one of the most prevalent opportunistic pathogens in chronic wounds1, 10, 11 as well as a cause of serious local and systemic infections.12 SA produces various virulent factors such as α-toxin, enterotoxins, and coagulase, which damage cell membranes and induce cell death.13 In addition, cell surface molecules such as lipopeptides, peptidoglycan and lipoprotein can activate immune cells via pattern (=pathogen) recognition receptors (PRRs) resulting in an enhanced pro-inflammatory response, which also delays wound healing.14 However, recent research points toward novel mechanisms of SA pathogenicity with respect to chronic and/or recurrent wound infections. While long considered a strictly extracellular pathogen, recent studies showed that SA survived phagocytosis by macrophages and neutrophils, and induced its intracellular uptake even in non-professional phagocytes including, for example, epithelial and endothelial cells, osteoblasts, and murine fibroblasts.9, 15 Intracellular SA causes cytotoxicity, but also may progress to persistence and then represents a mechanism of immune evasion leading to recurrence or chronicity of infections, and further bacterial dissemination.9, 16 Dermal fibroblasts are cellular key players in physiologic wound healing3 since they proliferate and produce extracellular matrix proteins to fill up the wound.1 Although the diverse effects of SA on the immune system have been described, its effect on fibroblast functions in the context of dermal wound healing, and especially its contribution to non-healing DUs in SSc, has not yet been investigated. Therefore, we evaluated the interaction of SA and dermal fibroblasts with respect to key tissue repair functions including potential signaling pathways. 2 MATERIALS AND METHODS 2.1 Study subjects Human primary dermal fibroblasts were obtained from lesional skin of patients with diffuse cutaneous (dc) SSc (n = 3) and from skin biopsies from healthy controls (HC) (n = 3) as previously described.17 Wound swabs of DU were subjected to routine microbiology diagnostics in 30 SSc patients. The main characteristics of the analyzed SSc patients (n = 30) at the time of the microbial sampling are provided in Table 1. Written informed consent was obtained. The study was approved by the Zurich ethics committee (pre-BASEC-EK-839, BASEC KEK-no. 016-01515, BASEC-no. 2014-0197, KEK-no. 2018-0187). Patients/public were not involved in the study. TABLE 1. Patients' main characteristics Parameters Numbers Age (years) 42 (20-79) Gender (m/f) 3/27 (10/90%) Disease duration (years) 8 (0-49) Skin involvement Limited cutaneous 12 (40%) Diffuse cutaneous 15 (50%) Only sclerodactyly 3 (10%) Raynaud's phenomenon 30 (100%) Presence of active digital ulcers 30 (100%) Number of digital ulcers 3 (1-20) Auto-antibody status ANA 28 (93%) Anti-Centromere 18 (60%) Anti Scl-70 14 (47%) Anti RNA-Polymerase 6 (20%) Anti-U1nRNP 3 (10%) Organ involvement Pulmonary arterial hypertension 4 (13%) Lung fibrosis (HRCT scan) 16 (53%) Esophageal symptoms 23 (77%) Intestinal symptoms 17 (57%) Stomach symptoms 17 (57%) Renal crisis 0 (0%) Immunosuppressive therapy 10 (30%) Note Data are presented as absolute numbers (percentages) or medians (ranges). 2.2 Cell culture Human primary dermal fibroblasts were obtained from forearms of dcSSc patients and HCs at the department of Rheumatology, University Hospital Zurich. Skin was minced into small pieces with a sterile scalpel. The pieces were transferred to a 6-well plate with the dermis facing the bottom of the plate. The skin pieces were incubated with complete cell culture medium (DMEM containing 10% fetal calf serum (FCS), 50 units/mL of penicillin, 50 μg/mL of streptomycin and 100 μM of 2-mercaptoethanol) to let fibroblasts outgrow from the skin for at least two weeks. Cells were passed 3 times to get rid of other cell types and expand the culture. The purity of the obtained fibroblasts was confirmed by FACS analysis and immunofluorescence. FACS analysis revealed that more than 90% of cells were CD90+CD45-CD31-. Immunofluorescence showed robust expression of alpha-smooth muscle actin and stress fiber upon TGF-beta stimulation (Figure S1). These results suggested that our primary fibroblasts consisted of a sufficiently pure cell population. Primary dermal fibroblasts were cultured in the complete cell culture medium. Human neonatal fibroblasts immortalized with hTERT (BJ-5ta, ATCC CRL-4001) were cultured in DMEM and Medium 199 (mix ratio 4:1; Sigma-Aldrich, St Louis, MO) containing 10% FCS and 0.01 mg/mL of hygromycin (Sigma-Aldrich) at 37°C in 5% carbon dioxide. Cells were stimulated with 1 × 105 or 1 × 107 CFU/mL of live SA (ATCC 6538; kindly provided by A. S. Zinkernagel) or 1 × 108/mL of HKSA (InvivoGen, San Diego, CA), and then were used for each subsequent analysis. The maximum bacterial load was determined by cytotoxicity assay using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Rockville, MD). Treatment of both primary human dermal fibroblasts and BJ-5ta cells with SA at a bacterial load within a range of 1 × 104-5 × 107 was found to have no cytotoxic effect (>95% viability) on such cells. 2.3 Bacterial strain and bacterial co-culture stimulation system SA (ATCC 6538) was stored in Tryptic soy broth (TSB; BD Biosciences, San Jose, CA) medium containing 20% glycerol at −20°C. Bacteria were cultured in 3 mL of TSB medium in a 10 mL Falcon round bottom tube for 18 h at 37°C in an incubator shaker to reach the early stationary phase for use in the co-culture system. The bacterial concentration was estimated by measuring the optic density of bacterial suspension with an optical densitometer (BioRad, Hercules, CA). Bacteria were harvested by centrifugation at 4200 g for 10 min. The bacterial pellet was washed with phosphate-buffered saline (PBS) twice and then suspended in DMEM supplemented with 1% human albumin (Sigma-Aldrich) and 25 mM of HEPES (Thermo Fisher Scientific, Waltham, MA) (invasion medium). The bacteria containing medium was then added to a monolayer of cultured fibroblasts,16, 18 plated as per assay requirements. Stimulation of fibroblasts was unopposed by antibiotics for 3 h. Thereafter DMEM with 1% FCS supplemented with the antibiotic flucloxacillin sodium (0.1 mg/mL) (Sigma-Aldrich) was added. The concentration was determined by bacteria killing assay to minimize undesirable effects on fibroblasts. 2.4 ELISA One hundred thousand cells were cultured in a 6-well plate. After 24 h starvation with 1% FCS, cells were washed with PBS and treated with the indicated amount of SA as described above. After 72 h incubation, the supernatants were collected. The levels of IL-1β, IL-6, IL-8, IFN-β and pro-collagen Iα1 in the co-culture supernatants were quantified using ELISA assays (R&D Systems, Minneapolis, MN) and a microplate reader (Biotek Instruments, Winooski, VT) according to the manufacturer's protocols. 2.5 Quantitative real-time PCR (qPCR) analysis Fifty thousand cells were cultured in a 12-well plate. After a 24 h starvation with 1% FCS, cells were washed with PBS and treated with the indicated amount of SA as described above. After 24 h incubation, cells were washed with PBS and total RNA samples were prepared using a Quick-RNA MicroPrep Kit (Zymo research, Irvine, CA). Synthesis of cDNA was performed using a Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). Quantitative PCR was performed using a Stratagene Mx3005P Quantative PCR System (Agilent Technologies, Santa Clara, CA) making use of a SYBR green reagent (Promega, Madison, WI). The levels of each gene were normalized to the levels of RPLP0. The sequences of primers used are presented in Table 2. TABLE 2. qPCR primer sequences Gene symbol Primer sequence RPLP0 Forward primer: 5′-ACA CTG GTC TCG GAC CTG AGA A-3′ Reverse primer: 5′-AGC TGC ACA TCA CTC AGA ATT TCA-3′ MMP1 Forward primer: 5′-CTC TGG AGT AAT GTC ACA CCT CT-3′ Reverse primer: 5′-TGT TGG TCC ACC TTT CAT CTT C-3′ MMP3 Forward primer: 5′-TCA CTC ACA GAC CTG ACT CG-3′ Reverse primer: 5′-AAA GCA GGA TCA CAG TTG GC-3′ TLR2 Forward primer: 5′-CTG TGC TCT GTT CCT GCT GA-3′ Reverse primer: 5′-GAT GTT CCT GCT GGG AGC TT-3′ TLR9 Forward primer: 5′-AAT CCC TCA TAT CCC TGT CCC-3′ Reverse primer: 5′-GTT GCC GTC CAT GAA TAG GAA G-3′ MYD88 Forward primer: 5′-GCA CAT GGG CAC ATA CAG AC-3′ Reverse primer: 5′-GAC ATG GTT AGG CTC CCT CA-3′ ZBP1 Forward primer: 5′-AAC ATG CAG CTA CAA TTC CAG A-3′ Reverse primer: 5′-AGT CTC GGT TCA CAT CTT TTG C-3′ CGAS Forward primer: 5′-ACA TGG CGG CTA TCC TTC TCT-3′ Reverse primer: 5′-GGG TTC TGG GTA CAT ACG TGA AA-3′ IFI16 Forward primer: 5′-TAG AAG TGC CAG CGT AAC TCC-3′ Reverse primer: 5′-TGA TTG TGG TCA GTC GTC CAT-3′ TMEM173 Forward primer: 5′-CAC TTG GAT GCT TGC CCT C-3′ Reverse primer: 5′-GCC ACG TTG AAA TTC CCT TTT T-3′ IL1B Forward primer: 5′-ATG CAC CTG TAC GAT CAC TG-3′ Reverse primer: 5′-ACA AAG GAC ATG GAG AAC ACC-3′ IL6 Forward primer: 5′-CTC TTC AGA ACG AAT TGA CAA ACA A-3′ Reverse primer: 5′-GAG ATG CCG TCG AGG ATG TAC-3′ IL8 Forward primer: 5′-TTG GCA GCC TTC CTG ATT TC-3′ Reverse primer: 5′-TGG CAA AAC TGC ACC TTC AC-3′ IFNB1 Forward primer: 5′-GTC ACT GTG CCT GGA CCA TAG-3′ Reverse primer: 5′-GTT TCG GAG GTA ACC TGT AAG TC-3′ 2.6 Migration assay Migration assays were performed as described19 using a 2-well cell culture insert (Ibidi, Planegg, Germany) allowing for an equal gap down the center of a cellular monolayer. After bacterial co-culture, serial imaging of fibroblast migration was performed using an imaging system (IX81; Olympus, Tokyo, Japan) equipped with excellencePro software (Olympus). Fibroblast migration was measured by assessing the percentage of the gap still uncovered by fibroblasts at various time intervals using ImageJ software (NIH, Bethesda, MD). 2.7 Cell contraction assay A collagen solution (400 μL) was prepared from the cell contraction assay kit (Cell Biolabs, San Diego, CA) and mixed with 100 μL of cell-bacteria solution. The solution was plated in a 24-well culture plate and incubated for 1 h to allow polymerization of the collagen gel. Thereafter, 1000 μL of the DMEM (Sigma-Aldrich) containing 1% FCS was added to each well, followed by flucloxacillin after 2 h. After 48 h, the gel was gently released from the sides of the well and after further 6 h, the area of each gel was measured using ImageJ software. 2.8 Proliferation assay Cells were seeded into a 96-well plate. After 24 h starvation, the cells were cultured with SA as described above. After 72 h, cell proliferation was assessed by CCK-8 according to the manufacturer's protocol. The absorbance was then read at 450 nm using a microplate reader (Biotek Instruments). 2.9 Apoptosis and necrosis assay Cells were seeded into a white 96-well plate. After overnight culture, the cells were cultured with SA as described above. After 3 h of bacterial co-culture, apoptosis and necrosis were assessed at 7 h (4 h after flucloxacillin was added) using RealTime-Glo Annexin V Apoptosis and Necrosis Assay (Promega). Chemiluminescence and fluorescence were measured using a microplate reader. 2.10 Invasion assay One hundred thousand cells were cultured in a 6-well plate. After 24 h starvation, cells were washed twice with PBS and treated with the indicated amount of SA in invasion medium for 3 h. Hereafter, the supernatant was aspirated and the cells were treated with lysostaphin (Sigma-Aldrich), which is an antibacterial endopeptidase, for 1 h to eradicate extracellular bacteria. Cells were washed once with PBS and then lysed with distilled water. The lysate containing intracellular bacteria was diluted and seeded on agar plates. Bacterial invasion was quantified by counting the number of bacterial colonies on the plates.20 2.11 Immunofluorescence and confocal microscopy Glass cover slips with a diameter of 12 mm were placed in 24-well plates. Thereafter, 5 × 104 fibroblasts (in 500 µL/well) in DMEM with 1% FCS (1% FCS-DMEM) were added and starved for 24 h. SA cultures were grown overnight in 5 mL TSB and then adjusted to reach 2 × 108 CFU/mL. Cells were infected with MOI (multiplicity of infection) 1 (1 × 105 CFU/mL) or 100 (1 × 107 CFU/mL) in 1% FCS-DMEM. After 3 h of bacterial invasion, the supernatants were aspirated and cells were fixed with 4% paraformaldehyde. Nucleic acids (eukaryotic nuclei and bacteria) were then stained with 20 µM Hoechst reagent, washed once with PBS, and then blocked with PBS supplemented with 1% bovine serum albumin (1% BSA-PBS). Thereafter, 1:100 rhodamine phalloidin (Invitrogen, Waltham, MA) was added to stain actin. After the removal of the blocking buffer and the actin dye, a mouse monoclonal primary antibody against SA (clone 704; Abcam, Cambridge, MA) was added in 1% BSA-PBS at a final concentration of 2 µg/mL. After washing once with PBS, a goat anti-mouse antibody Alexa 488 (Invitrogen) was added in 1% BSA-PBS at a final concentration of 4 µg/mL. To stain late endosomes, a mouse primary antibody against LAMP2 (BD Biosciences) and a goat anti-mouse antibody Alexa 488 (Invitrogen) were used. For identification of intracellular SA in Figure 3, CFSE (carboxyfluorescein succinimidyl ester)-labeled SA or HKSA were used. After washing once with PBS, cells were mounted in ProLong Gold (Invitrogen) on glass slides. Images were obtained with an inverted SP8 confocal laser scanning microscope (Leica, Wetzlar, Germany). 2.12 Blockade of endocytosis Cells were treated with 10 µg/mL of cytochalasin D (Sigma-Aldrich) for 1 h. After the cells were washed with PBS, bacteria containing medium were added to the wells to examine the effect of bacterial invasion on cell death or expression of IFN-β and inflammatory mediators. 2.13 Gene knockdown by siRNA Human MYD88 siRNA, human TMEM173 siRNA, and AllStars Negative Control siRNA were purchased from Qiagen (Venlo, Netherlands). The sequences of the siRNAs are shown in Table 3 below. TABLE 3. siRNA sequences for TME173 and MYD88 siRNA Sequence TMEM173 Target Sequence: 5′-CCG CAC GGA TTT CTC TTG AGA-3′ Sense strand: 5′-GCA CGG AUU UCU CUU GAG ATT-3′ Antisense strand: 5′-UCU CAA GAG AAA UCC GUG CGG-3′ MYD88 Target Sequence: 5′-CAG GAC CAG CTG AGA CTA AGA-3′ Sense strand: 5′-GGA CCA GCU GAG ACU AAG ATT-3′ Antisense strand: 5′-UCU UAG UCU CAG CUG GUC CTG-3′ Fifty thousand cells were cultured without antibiotics in a 12-well plate. After overnight culture, siRNA-Lipofectamine2000 (Thermo Fisher Scientific) complexes in Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific) were prepared for each transfection sample. The siRNA-Lipofectamine complexes were added to each well, and the cells were incubated in a humidified CO2 incubator. The final concentration of each siRNA was 20 nM. After 6 h incubation, the cell culture medium was changed to fresh DMEM with 1% FCS. After further 18 h incubation, the cells were used for each gene knockdown assay. 2.14 Statistical analysis Data are summarized as medians or means depending on the experimental setting. The statistical analysis was performed using Prism 8.1 (GraphPad Software, San Diego, CA). The respective statistical analyses are specified in each figure legend. P values <.05 were considered significant. 2.15 FACS analysis Cells were co-cultured with the indicated amount of SA for 24, 48, or 72 h. Cells were treated with 2 μM of monensin (Sigma-Aldrich) for the final 6 h. Cells were washed and incubated with LIVE/DEAD Fixable Near-IR Dead Cell Stain (Thermo Fisher Scientific) for 30 min. Thereafter, anti-CD45-eFluor 450 (clone HI30), anti-CD31-Super Bright 600 (clone WM-59) (Thermo-Fischer Scientific), and anti-CD90-APC (clone 5E10; BD Biosciences) antibody for 30 min at 4°C. Cells were then fixed with Cytofix/Cytoperm Cell Permeabilization/Fixation Solution (BD) for 15 min at 4°C. Cells were then washed and incubated with anti-IL-6-PE (clone MQ2-6A3, BD), anti-IL-8-PE (Clone 8CH, Thermo Fisher) or anti-IFN-β-Fluorescein (Clone MMHB-3, R&D Systems) for 30 min at 4°C. Purity of primary fibroblasts and level of intracellular cytokine expression were analyzed on a flow cytometer (Attune NxT, Thermo Fisher). Flow cytometry data were analyzed with FlowJo (v10.2, BD). 2.16 Immunofluorescence of human primary dermal fibroblasts Cells were cultured on an 8-well chamber slide (Thermo Scientific) with 10 ng/mL of TGF-β1 for 72 h. Cells were then washed and fixed with 4% paraformaldehyde for 5 min. Cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 5 min. After blocking with 10% FCS for 20 min, cells were incubated with anti-actin, α-smooth muscle (clone 1A4; Sigma-Aldrich) for 1 h at room temperature. Cells were then washed and incubated with anti-mouse IgG-Alexa Flour 488 (Thermo Fischer Scientific) and phalloidin-TRITC (Sigma-Aldrich) for 45 min. Nucleic acids were then stained with DAPI (4′,6-diamidino-2-phenylindole; Roche). After washing with PBS, cells were mounted in fluorescence mounting medium (Dako) and images were obtained with a fluorescence microscope (DP80, Olympus). 2.17 Cell count Fifty thousand cells were co-cultured with the indicated amount of SA for 24, 48, or 72 h on 12-well plates. Cells were stained with trypan blue. Live and dead cells were then determined using an automated cell counter (TC10, Bio-Rad). 3 RESULTS 3.1 Chronic DU of SSc patients are colonized by SA To assess which bacteria are predominantly found in chronic DU, we analyzed wound swabs of 30 SSc patients from our department. Most patients with DU showed positive results with mostly moderate (CFU = 104-105/mL, ie, critically colonized; 30%) or plentiful bacterial growth (CFU >105-106/mL, ie, infected; 37%), while only 5 (17%) did not show any growth at all. In 53% of dermal wounds, SA was detected. Other detected bacterial strains included Pseudomonas aeruginosa, Haemophilus parainfluenzae, Enterobacter cloacae, Enterococcus faecalis, Streptococcus pyogenes, Klebsiella pneumoniae, Proteus vulgaris, Staphylococcus lugdunensis, and Staphylococcus epidermidis. In SA-positive wounds, deep soft tissue infections (n = 7, 43.8%) and osteomyelitis (n = 5, 31.3%) occurred more often. These findings are in accordance with previous reports.10, 11 3.2 SA affects key functions of dermal fibroblasts To investigate the impact of SA-fibroblast interactions on tissue repair we first co-cultured dermal fibroblasts (BJ-5ta) with live SA. We deliberately chose a cell line for the first evaluation step in order to avoid the potential heterogeneity of results that might arise from the use of primary cells. A lower inoculum representing colonization (1 × 105 CFU/mL) and a higher inoculum reflecting infection (1 × 107 CFU/mL) were used.21 We confirmed dose-dependent effects of SA between 1 × 105 and 1 × 107 CFU/mL on some fibroblast functions (Figure S2). Exposure to the higher inoculum influenced several important fibroblast functions. The secretion of IL-6 and IL-8 by dermal fibroblasts was increased to 1340 pg/mL (P < .05) and to 120 pg/mL (P = .12), respectively (Figure 1A). IL-1β was not detected by ELISA (data not shown) at 72 h. The gene expression was likewise upregulated for IL-6/-8 (Figure 1B). Although the gene expression of IL-1β was also upregulated (Figure 1B), the expression level relative to ribosomal protein lateral stalk subunit P0 (RPLP0) was much lower than those of IL6 and IL8 (data not shown) consisting with no detection of IL-1β protein. The increased secretion and gene expression of the cytokines were already observed at 24 h (Figure S3). In addition, we observed an upregulated gene expression of MMP1 by 2.8-fold (P < .05) and MMP3 by 1.9-fold (P < .05) (Figure 1C). Wound closure as assessed by migration was reduced by 34% at 16 h (P = .64) (Figure 1D). Type 1 collagen synthesis was significantly decreased in 1 × 107 CFU/mL of SA-challenged BJ-5ta cells (Figure 1E). Collagen gel contraction as measure of fibroblast contractility was impaired by 26% (P = .05) (Figure 1F). Proliferation was reduced by 57% (P = .26) (Figure 1G). Live cell numbers and proportions of live cells at 24 h and 72 h were also assessed. Live cells were decreased to around 50% compared with controls without SA-exposure at 72 h (Figure S4). Given that the cell number was reduced by exposure to higher dosages of SA, we performed intracellular cytokine staining to assess the production of cytokines while controlling for the cell number. The FACS analyses revealed that the higher dosages of SA increased the production of the cytokines by human dermal fibroblast at the single cell level (Figure S5). FIGURE 1Open in figure viewer S aureus (SA) affects key functions of dermal fibroblasts. A, The concentration of IL-6 and IL-8 in the culture supernatants was quantified at 72 h by ELISA. B, The gene expression of IL-6, IL-8, and IL-1B was assessed at 72 h by qPCR analysis. C, MMP1 and MMP3 mRNA levels were evaluated at 24 h by qPCR analysis. D, Migration assay was performed at 16 h (BJ-5ta cells) or 28 h (primary cells). E, Pro-collagen 1α concentration in the culture supernatants was quantified at 72 h by ELISA. F, Cell contractility was evaluated at 48 h by collagen gel