Study design
The overall objective of this study was to determine the efficacy and mechanism of action of CAP in the context of bacterial wound infection. Experimental approaches comprised in vivo studies in mice, in vitro analyses in human bioengineered 3D models of staphylococcal wound infection, and in vitro studies in 2D models of infection using mBMDM and RAW264.7 macrophages.
The efficacy of CAP was investigated using a murine model of infected full-thickness burn wound reconstructed with allogeneic skin graft. To monitor bacterial burden, wounds were infected with a bioluminescent strain of S. aureus. Two series of in vivo experiments were performed each consisting of three groups, namely, (1) sham or S. aureus-infected mice treated with placebo gas (2) S. aureus-infected mice treated with CAP, and (3) positive control or S. aureus-infected mice treated with anti-staphylococcal antibiotic. Each treatment was administered locally 6 h after infection, and once daily thereafter. Real-time monitoring of wound infection in living animals was performed once every 48 h over 14 days via bioluminescence imaging. Bioluminescent measurements were correlated with bacterial enumeration by calculating the number of living bacteria in a homogenized sample of wound biopsy. The experiments were arbitrarily ended after 3-, 7- and 14-days post-infection to recapitulate the main phases of wound healing: inflammatory, proliferative and remodeling. Longitudinal assessment of microscopic wound size, length of neoepidermis and new extracellular matrix formation was based on histological wound staining, quantitative RT-qPCR, chromogenic detection of protein expression, confocal and two-photon microscopy techniques.
Following acclimation period of 7 days prior to use in surgery, age-matched animals were randomly assigned to one of three experimental groups. Prior power calculations were performed to predetermine the sample size using an open-access software (https://shade.pasteur.fr/) and based on previous experience with similar wound healing models. Placebo controls were used, and no bias was applied during husbandry or during tissue harvesting. Blinded assessment of experimental outcomes was not always applicable; however, it was used whenever possible. Animals that did not reach experimental end point of a study due to failure to recover from general anesthesia were removed from the study. Animals that reached a predefined humane end-point were excluded from the study. Histological samples were randomized using a computer-based random number generator. Histological slide assessment and data acquisition were performed by two assessors. Only one of the two assessors was formally blinded. For representative images, at least six fields of view were examined microscopically in at least five biological replicates; the numbers of biological replicates for each experiment are in the figure legends. Sources of variability and conditions that could potentially bias results were controlled where possible. All surgeries were performed by the same surgeon. All mice that received placebo control, topical antibiotic or cold atmospheric plasma were treated by the same operator. All animal studies were approved by relevant Animal Care and Use Committee. Mice were maintained and humanely euthanized by overdose of inhalant anesthetic followed by cervical dislocation at predefined study end points. All procedures were in accordance with the European and French Code of Practice for the Care and the Use of Animals for Scientific Purposes.
To identify the mechanism of action, we hypothesized that antibacterial efficacy of CAP is NOX2-dependent, and therefore, signaling pathway activation and inhibition studies were performed in 3D and 2D in vitro models of wound infection using pharmacological inhibitors and genetic ablation. To delineate underlying mechanisms, a range of cellular and molecular biology techniques were performed to evaluate cellular proliferation, intracellular ROS production, gene expression, post-transcriptional modification of proteins and assembly of phagocyte NADPH oxidase components at phagolysosomal membrane of infected macrophages. Bioengineered wounds were fabricated using automated 3D printing technology with high precision and reproducibility. Inhibition studies using 3D bioengineered human wound equivalents further strengthened the argument that the antibacterial action of CAP is NOX2 dependent. Multi-cellular 3D wounds consisted of human primary keratinocytes, fibroblasts and peripheral blood monocytes collected from the same human donor. Human tissue samples were obtained from patients undergoing elective surgery after written informed consent and approval by relevant human ethics committees of “Le Ministère de l’Enseignement supérieur, de la Recherche et de l’Innovation” (number AC-2018-3243 and DC-2018-3242). The number of experimental replicates is indicated in the figure legends.
In vivo mouse model
All experiments were approved by the Institut Pasteur Animal Ethics Committee (approval number APAFIS#15346-2018053111431668 v1) following European and French Code of Practice for the Care and the Use of Animals for Scientific Purposes (Council Directive 86/609/EEC). Six-weeks-old (20–25 g), inbred, immunocompetent hairless female Crl:SKH1-Hrhr mice (Charles River Laboratories, Germany) were randomly assigned to one of three groups of six to twelve animals per group as predetermined by statistical power calculations. Two series of experiments were performed each involving 45 mice. The total number of 90 mice were used in the study. Not all mice reached the final time-point. Intraperitoneal injection of ketamine (100 µg/g) and xylazine (10 µg/g) was administered to induce general anesthesia, and subcutaneous injection of lidocaine (5 µg/g) was administered to induce local anesthesia. Buprenorphine (0.05 µg/g) was administered subcutaneously, and paracetamol (3 mg/ml) was added to drinking water to control intra and post-operative pain. A 79-mm2 burn wound was created on the dorsal surface using a brass block as previously described28. Twenty-four hours after the wound induction, necrotic tissue was excised to fascia and reconstructed with a full-thickness allogenic skin graft originating from the donor’s tail and secured with a Leukosan surgical adhesive (BSN medical, Hamburg, Germany). S. aureus Xen36 overnight cultures were diluted in BHI and bacteria were grown to an optical density at 600 nm (OD600) of 1. Bacterial cultures were centrifuged at 3500 × g for 15 min and washed three times in PBS. Following skin grafting, mouse wounds were infected topically with 1 × 108 bacteria diluted in 50 µl of PBS. Serial dilutions of the inoculum were plated to control the number of bacteria inoculated. Wounds were treated once daily (in the morning) with either (1) placebo control (topical treatment with inert, helium gas), (2) topical treatment with CAP (2 min, 24 kV) or (3) topical application of 200 µl of mupirocin at 2% w/v (Bactroban, GlaxoSmithKline, Middlesex, UK). Low-adherent paraffin gauze (Adaptic, North Yorkshire, UK) wound dressing and Micropore™ Surgical Tape (3 M, Cergy-Pontoise, France) were changed daily. CAP treatment and wound dressings were performed under anesthesia, which was induced by inhalation of isoflurane (4% induction at 2 L/min and 2% maintenance at 500 ml/min).
BLI was performed on alternate days to assess bacterial wound burden in living animals. Bacterial wound burden was assessed by measuring bioluminescent signal in anesthetised animals using IVIS Spectrum Imaging system and Living Image 4.5.5 software (Perkin Elmer, Boston, MA, USA). Two wound biopsies were collected at 3, 7 and 14 d. p. i. using a sterile 6 mm biopsy punch (Acuderm Inc. FL, USA). The first wound biopsy was homogenized, serially diluted on BHI plates and grown over 24 h at 37 °C. Colonies were counted to assess bacterial load per biopsy. The second wound biopsy was bisected with one half fixed in 10% buffered formalin and processed for histology and immunohistochemistry. The other half was fast frozen in liquid nitrogen for RNA extraction, MPO and NAG quantitation.
Cold atmospheric plasma
Biocompatible helium gas was used to produce plasma as previously described28,30. The plasma source consisted of a dielectric polylactic acid capillary surrounded by a high-voltage electrode, through which flows helium gas. CAP was propagated from the source to the target in a single channel plasma jet (Supplementary Movie 1). Voltage was set to 24 kV during in vivo and 32 kV during in vitro experiments corresponding to an operating power of 50 ± 10 mW and 90 ± 10 mW respectively. Helium mass flow was regulated by mass flow controller and set to 500 standard cubic centimetres per minute (sccm). The gap between CAP nozzle and the target was set to 4–6 mm. The duration of treatment in all experiments was 2 min. In the in vivo experiments, mouse skin temperature was monitored after CAP treatment with an infrared thermal imaging camera (Testo Ltd, Alton, Hampshire, UK). CAP had no effect on mouse skin temperature. The optical emission spectra of ultra violet radiation produced by the CAP device was not measured in this study. The concentration of chemical species (H2O2, NO2− and NO3−) in plasma-activated medium was indicated in our previous work30.
Biofabrication of three-dimensional human skin
Primary human keratinocytes and dermal fibroblasts were isolated from male and female adult skin through enzymatic dissociation. The skin specimens were sampled by a surgeon during sessions of elective abdominoplasty or reduction mammoplasty. The study was conducted in accordance with the 1975 Declaration of Helsinki. Informed written consent was obtained from all donors. All protocols were performed in accordance with national laws and guidelines for the collection, use and storage of human tissue. Keratinocytes were cultured in EpiLife medium (Gibco Paisley, PA, USA) supplemented with Human Keratinocytes Growth Supplement (Gibco Paisley, PA, USA). Fibroblasts were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco Paisley, PA, USA) supplemented with 15% FetalClone III (GE Healthcare Bio-Sciences, Uppsala, Sweden). All cells were used at passages 2–3. Peripheral blood mononuclear cells (PBMC) were isolated from buffy coat using Ficoll (GE Healthcare Bio-Sciences, Uppsala, Sweden). CD14+ monocytes were isolated by magnetic selection (Miltenyi Biotech, Bergish Gladbach, Germany) and differentiated using M1-Macrophage Generation medium DXF (PromoCell GmbH, Heidelberg, Germany). Three-dimensional modeling computer software, including open-source 3D printing toolbox (Slic3r https://slic3r.org/) and SketchUp (Trimble Inc., Sunnyvale, CA, USA), were used to design the pattern of 3D structures. Computer-aided transfer of prescribed organization was performed through deposition of bioink formulation (CTI BIOTECH, Lyon, France) containing single-cell suspensions. Three-dimensional skin models were built through the layering of printed filaments with the assistance of a BIO XTM bioprinter (CELLINK, Gothenburg, Sweden). Following bioprinting, skin models were cultured for 21 days. During this period, 3D models were cultured in medium (CTI Biotech, Lyon, France), which aided dermal maturation, epidermis differentiation, and air-liquid interphase cornification. Each sample measured 1 cm (length) × 1 cm (width) × 200 µm (depth). Cellular viability was controlled at day 21 after bioprinting with an alamarBlue™ Cell Viability Reagent (Life Technologies Corporation, Eugene, OR, USA) and Invitrogen™ LIVE/DEAD™ Viability/Cytotoxicity Kit for mammalian cells (Life Technologies Corporation, Eugene, OR, USA).
Bacterial strains and culture conditions
The bioluminescent Staphylococcus aureus Xen36 strain (PerkinElmer, Waltham, Massachusetts, USA) was used in this study. This MSSA strain was derived from a clinical isolate of bacteremic patient and has been genetically engineered to express a stable copy of the modified Photorhabdus luminescens luxABCDE operon at a single integration site on a native plasmid. A highly virulent MRSA strain, Staphylococcus aureus USA300, was obtained from ATCC (reference BAA-1717). Bacteria were grown aerobically, as previously described25, in brain heart infusion (BHI) broth (BD Biosciences, San Jose, CA) with shaking at 200 rpm at 37 °C or on BHI agar plates. Overnight cultures were collected or diluted 1:50 in fresh BHI and grown at 37 °C until exponential phase (optical density at 600 nm (OD600) of 1.0). OD600 values were measured using a Biochrom Libra S22 spectrophotometer (Biochrom Ltd., Cambridge, UK).
Histology, immunohistochemistry and image analysis
Histological sections (4 μm thickness) were prepared from formalin-fixed and paraffin-embedded tissue, which were stained with Haematoxylin and Eosin (H&E) or subjected to immunohistochemistry using a Leica Bond III stainer (Leica Biosystems, Nanterre, France). Primary antibodies were applied and incubated for 1 h. Detection was performed by species-specific horseradish peroxidase (HRP) or alkaline phosphatase (AP)-conjugated secondary antibodies. Sections were reacted with one of two substrates: (1) for HRP, 3,3’-diaminobenzidine (DAB) with Bond enhancer (AR9432, Leica Biosystems, Wetzlar, Germany), which produced a brown to black color or (2) for AP, Bond Polymer Refine Red (DS9390, Leica Biosystems, Wetzlar, Germany), which yielded a bright red color. For identification of non-specific binding and other experimental artefacts, negative controls were used. These consisted of omission of primary antibodies with a non-immune immunoglobulin of the same isotype and concentration as the primary antibody or incubation with antibody diluent. All control sections showed negligible staining. The list of antibodies and dilution factors is provided in Table 1. Stained sections were scanned using a Lamina instrument (Perkin Elmer, Waltham, MA, USA), visualized with the CaseViewer digital microscope application (3Dhistech, Budapest, Hungary), and analyzed with ImageJ software.
Histological wound assessment
Histological slides stained with H&E were evaluated for the microscopic wound length. The public domain software ImageJ was used to determine the microscopic wound length by drawing a straight line between the dermal wound margins. To estimate the length of neoepidermis, the area of the wound that was covered with newly formed epidermis was measured and expressed in µm.
Quantification of immunohistochemical staining was by color deconvolution as described previously74,75. A total of six microscopic fields of view were used for the immunohistochemical data analysis. Out of the six microscopic images, two represented the region around the left border of the wound lesion, two additional images included the graft region, and the two remaining images included the area around the right border of the wound. Both the epidermal and dermal regions were assessed. Control average was normalized to 1. For each time point, the value of integrated density (arbitrary units) is shown relative to the value of the integrated density in control.
Gram staining
Sections (4 µm) of formalin-fixed paraffin embedded tissue were de-waxed and taken through a decreasing series of graded alcohols to water. Gram staining was performed using a Gram Stain Kit (Sigma-Aldrich, St Louis, MO, USA) to visualize S. aureus in tissue sections according to the manufacturer’s instructions. S. aureus are stained in blue and the remaining biological tissue is visible in varying shades of magenta.
Masson’s trichrome and May Grunwald-Giemsa staining
For histological assessment of collagen deposition in the dermis, trichrome staining was performed using a Masson Trichrome Staining Kit according to the manufacturer’s instructions (Sigma-Aldrich, St Louis, MO, USA). May Grunwald-Giemsa staining was performed on formalin-fixed and paraffin-embedded sections to visualize cellular components of human bioengineered skin (Sigma-Aldrich, St Louis, MO, USA).
Multiphoton microscopy
TriM Scope™ Matrix multiphoton microscope (LaVision BioTec GmbH Miltenyi Biotec Company, Bielefeld, Germany) equipped with a 25 × water immersion objective with numerical aperture of 0.95 (XLPLN25XWMP2; Olympus Corporation, Tokyo, Japan) was used to assess cellular viability in live bioengineered skin. Following bioprinting and in vitro maturation (i.e., 21 days after 3D bioprinting), bioengineered skin samples were incubated (1 h, 37 °C) with reagents provided in Invitrogen™ LIVE/DEAD™ Viability/Cytotoxicity kit. Bioengineered skin was scanned in 1024 × 1024 pixel/frames, which represents a scanned area of 393 µm × 393 µm. The InSight® X3+™ laser (Spectra-Physics®, Milpitas, CA, USA) was tuned at 910 nm to capture calcein-positive cells (pseudocolored green) and 1100 nm for ethidium homodimer-1-positive cells (pseudocolored red). Signals were collected using non-descanned GaAsP detectors (Hamamatsu Corporation, Hamamatsu City Japan). Sequentially, live cells were detected through a bandwidth filter 500 nm to 520 nm and dead cell signal was detected through a bandwidth filter 568 to 593 nm of two non-descanned detector in a backscattering geometry. Maximum intensity projection images were generated using Fiji > Image > Stack > Zproject tools.
Collagen second harmonic generation
Multiphoton inverted stand Leica SP5 microscope (Leica Microsystems Gmbh, Wetzlar, Germany) was used to assess mouse wounds ex vivo as previously described30. A Ti:Sapphire Chameleon Ultra (Coherent, Saclay, France) with a center wavelength at 810 nm was used as the laser source to generate second harmonic and two-photon excited fluorescence signals (TPEF). The laser beam was circularly polarized and equipped with a Leica Microsystems HCX IRAPO 4x/0.95 W objective, which was used to collect and excite second harmonic generation (SHG) and TPEF. Signals were detected in epi-collection through a 405/15-nm and a 525/50 bandpass filters respectively, by NDD PMT detectors (Leica Microsystems, Wetzlar, Germany). LAS software (Leica Microsystems, Wetzlar, Germany) was used for laser scanning control and image acquisition. SHG signal (magenta) is emitted from collagen I fibers and TPEF (green) is due to the signal emitted by cellular and tissue constituents of the skin. Combined SHG and TPEF backscattering microscopy technique provides complementary information and allows non-invasive, spatial characterization of skin tissue and wounds.
SHG and TPEF images were acquired using detectors with a constant voltage supply and constant laser excitation power allowing direct comparison of SHG intensity values. Analyses were performed using homemade Image J routine (http://imagej.nih.gov/ij/). Two fixed thresholds were chosen to distinguish biological material from the background signal (TPEF images) and specific collagen fibers were imaged with SHG images. Collagen SHG score was then established by comparing the area occupied by the collagen relative to the sample surface. TPEF (green) and SHG (magenta) images were pseudocolored and overlaid for publication using Image J. Several microscopic fields of view were captured with a 4 × objective lens. A montage scan of the entire section was reconstructed and wound edges determined on either side of the graft. The SHG signal was quantified over the area between the left and right borders of the wound, which included the region covered by the skin graft.
TUNEL assay and DNase I treatment
The Calbiochem® TdT-FragEL™ DNA Fragmentation Detection Kit was purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Formalin-embedded samples of mouse skin tissue were sectioned (4 µm) and placed on a glass slide pre-coated with Biobond tissue section adhesive (vWR International, Radnor, PA, USA). Skin tissue sections were treated according to the manufacturer’s protocol. Briefly, all sections were deparaffinised by immersion in HistoClear (Euromedex, Souffelweyersheim, France) and hydrated by transferring the slides through a graduated ethanol series to 1× Tris-buffered saline (TBS) solution. Specimens were stripped of proteins by incubation with 100 µl of 20 µg/ml proteinase K per section. A positive control was generated by using deparaffinised sections of mouse skin that were initially incubated with 20 µg/ml proteinase K, and then treated with 1 µg/µl DNase I (Sigma-Aldrich, Saint-Quentin Fallavier, France) in 1 × TBS/1 mM MgSO4. Terminal deoxynucleotidyl transferase (TdT) binds to exposed 3′-OH ends of DNA fragments generated in response to apoptotic signals and catalyses the addition of fluorescein-labeled and unlabeled deoxynucleotides. When excited, fluorescein generates an intense signal at the site of DNA fragmentation of apoptotic cells. DAPI staining was used to visualize normal and apoptotic cells. TUNEL-stained sections were scanned using a Lamina instrument (Perkin Elmer, Waltham, MA, USA), and analyzed with the CaseViewer digital microscope application (3DHISTECH).
Infection of bioengineered three-dimensional human skin
At day 21 after bioprinting, 3D bioengineered skin was inoculated by injecting a total of 1 × 108 of S. aureus Xen36 bacteria diluted in 50 µl of PBS. To maximize the even distribution, the intradermal inoculum was administered through five entry points (10 µl at 12, 3, 6 and 9 o’clock plus one in the center). Serial dilutions of the inoculum were plated to control the number of bacteria inoculated. Culture medium was not supplemented with antibiotics. Each model was treated for the duration of 2 min with either placebo control (helium) or CAP at 1 and 4 h. p. i. All skin models were collected at 24 h. p. i. and homogenized in 2 ml of 1 x PBS. Homogenized skin was serially diluted, plated onto BHI plates and grown overnight at 37°C. CFU were enumerated to assess bacterial load. CFU average in control group was normalized to 1.
Cells and culture conditions
Murine macrophage-like cell line RAW 264.7 (ATCC®, Molsheim, France) was grown in DMEM high glucose supplement pyruvate (Gibco Paisley, PA, USA) and 10% fetal calf serum (BioWest, Nuaillé, France) at 37 °C in 5% CO2.
Primary macrophages were obtained from flushed bone marrow originating from femurs and tibias of female C57BL/6J. Mouse BMDM were cultured for 7 days in complete medium containing RPMI 1640 Medium (Gibco Paisley, PA, USA) supplemented with 10% fetal calf serum (BioWest, Nuaillé, France), 2 mM glutamine (Gibco Paisley, PA, USA), 1 mM sodium pyruvate (Gibco Paisley, PA, USA), 10 mM HEPES (Sigma-Aldrich, MO, USA), 50 μM β-mercaptoethanol, 100 U ml−1 penicillin/streptomycin (Gibco Paisley, PA, USA) and 25 ng/ml mouse macrophage colony-stimulating factor (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany).
Primary dermal fibroblasts were isolated from patients undergoing mammoplasty and/or abdominoplasty as previously described30. Informed consent was obtained from all the patients and the study protocol is conformed to the ethical guidelines of the 1975 Declaration of Helsinki. Dermis and epidermis were separated and dermis was dissociated in an enzymatic bath containing dispase II (2.4 UI/ml) and collagenase II (2.4 mg/ml) (Gibco, Paisley, PA, USA) during 2 h under agitation at 37 °C. After filtration, the suspension was centrifuged at 450G during 5 min and the fibroblasts were counted and seeded at 20,000 cell/cm2 in the growing medium made of DMEM Glutamax, supplemented with 1% antibiotic (Gibco, Paisley, PA, USA) and 10% HyClone FetalClone II serum (GE Healthcare Life Sciences, Tewksbury, MA, USA).
Scratch wounds in human dermal fibroblasts
To examine the direct effect of CAP on collagen synthesis, primary human dermal fibroblasts were suspended in 500 μL of in DMEM Glutamax, supplemented with 1% antibiotic (Gibco, Paisley, PA, USA) and 10% HyClone FetalClone II serum (GE Healthcare Life Sciences, Tewksbury, MA, USA). Fibroblasts were seeded into a 24-well plate at 3 × 105 cells/ml. Confluent monolayers of fibroblasts were scratched with a P200 pipette tip producing a wound of approximately 2 mm × 1 cm. Scratch wounded fibroblasts were rinsed once with complete medium to remove detached cells. Unwounded and scratch-wounded fibroblasts were treated either with helium (2 min, distance = 1 cm) or CAP (2 min, distance = 1 cm). Helium and CAP-treated fibroblasts were harvested at 6 and 24 h post-CAP treatment and processed for RNA extraction. All experiments were based on fibroblasts isolated from the skin originating from four different donors. Three independent experiments with at least three biological replicates were used to represent the data.
Bacterial infection of macrophages
RAW 264.7 cells were seeded onto a 24-well plate at a density of 105 cells per well. Cells were infected with S. aureus (MOI of 10), and incubated for 20 min at 37 °C. The samples were incubated for another 30 min, but this time with gentamicin-containing medium at 20 µg/mL (Sigma-Aldrich, MO, USA) to kill extracellular bacteria. After a washing step, 1 ml of complete medium was added and macrophages were treated either with placebo control (inert helium gas) or CAP for 2 min (distance 4–6 mm). Infected cells were lysed at 0, 2 and 5 h post infection with 0.1% Triton X-100. For enumeration of intracellular bacteria, dilution series of lysed macrophages were plated onto BHI agar.
Primary BMDMs were plated at 5 × 105 cells/ml onto a 92 × 16 mm Petri dish and cultured in the presence of complete medium. At day 5, medium was replaced with fresh complete medium. At day 7, cells were washed and seeded onto a 24-well plate at a density of 105 cells per well. Cells were cultured in complete medium without antibiotics and incubated for 24 h before bacterial challenge. For infection of BMDMs, bacteria were grown in BHI overnight, then regrown to exponential phase and added to the cells at a MOI of 10, centrifuged at 900 × g for 1 min and incubated at 37 °C for 20 min. To follow a synchronized population of bacteria, extracellular bacteria were killed through incubation of BMDM in RPMI containing 20 μg/mL gentamicin (Sigma-Aldrich, MO, USA) for 30 min. To compare the intracellular growth profiles of control and CAP-treated macrophages, the samples were then either lysed immediately, to measure the initial number of intracellular bacteria, or replaced with fresh media containing gentamicin (20 µg/mL), thus permitting intracellular bacteria to replicate for 2 or 5 h. p. i. For enumeration of intracellular bacteria, infected cells were lysed with 0.1% Triton X-100 for 5 min and dilution series were plated onto BHI agar. The number of living intracellular bacteria was monitored by counting CFU. The initial number of intracellular bacteria (0 h. p. i.) was averaged was normalized to 100. The data reported are the means and standard error of means of triplicate determinations and are representative of at least three experiments.
Cell proliferation assay
Cellular proliferation was determined using tetrazolium salt WST-1 reagent (Roche Applied Science, Rotkreuz, Switzerland), which is cleaved to soluble formazan by cellular mitochondrial dehydrogenases. Infected RAW 264.7 and BMDM were treated with either placebo control or CAP and supernatants were collected from cultured macrophages at 0, 2 and 5 h. p. i. Colorimetric assay (WST-1 based) for the quantification of cell proliferation was performed using the 96-well-plate format according to the manufacturer’s instructions. Absorbance was measured using a microplate reader (GloMax® Discover Microplate reader, Promega, MI, USA) at 555 nm.
ROS measurement
RAW 264.7 cells were seeded onto 24-well plates at a density of 105 cells per well. Cells were infected with S. aureus at a MOI of 10, gently centrifuged and incubated for 20 min at 37 °C to synchronize phagocytosis. The extracellular bacteria were eliminated by the addition 20 µg/mL gentamicin-containing medium and incubation for 30 min. Cells were treated by placebo control (helium) or CAP for 2 min. Fluorometric intracellular ROS probe (Catalog Number MAK142, Sigma-Aldrich, MO, USA) was added and incubated for 30 min at 37 °C. Fluorescence was measured using a microplate reader (GloMax® Discover Microplate reader, Promega, MI, USA) at 640 nm.
Pharmacological inhibition of NOX2
RAW 264.7 cells and bioengineered human skin were pre-incubated with two different NOX2 inhibitors: (1) GSK2795039 (MedChemExpress, NJ, USA) at 30 and 50 µM respectively, and (2) gp91 ds-tat (Anaspec, CA, USA) at 50 and 100 µM respectively. Following a 30-min incubation with GSK2795039 and gp91 ds-tat, samples were treated either with placebo control (helium) or CAP.
Transfection of small interfering RNAs (siRNAs)
RAW 264.7 cells were transfected with non-targeting negative control siRNA (ON-TARGETplus Non-Targeting Control Pool, Dharmacon, CO, USA) or endogenous positive control siRNA (ON-TARGETplus GAPD Control Pool, Dharmacon, CO, USA) at final concentration of 25 nM. ON-TARGETplus Mouse Cybb siRNA (Dharmacon, CO, USA) was transfected at 50 nM final concentration using DharmaFECT™ transfection Reagent (Dharmacon, CO, USA). Transfection of negative control, positive control and target siRNA was performed in serum-free medium in triplicates according to manufacturer’s instructions. mRNA extraction and cell viability analysis were performed at 24 h after transfection. Samples with target mRNA knockdown of >80% and cell viability >80% were used for subsequent experimentation. siRNA-mediated silencing of Cybb was monitored and assessed using a ΔΔCq method to determine relative gene expression from qPCR data with an endogenous reference gene and non-targeting siRNA (ON-TARGET plus Non-Targeting Control Pool, Dharmacon, CO, USA). RAW 264.7 cells exhibited siRNA knockdown of Cybb message with mRNA reduction of ≥80% when cells were treated with 50 nM final concentration of the targeting Cybb siRNA.
RNA isolation
RNA isolation from RAW 264.7 cells and primary human fibroblasts was carried out using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) and RNeasy Mini Extraction Kit (Qiagen, Courtaboeuf, France) as described by the manufacturer. Total RNA was isolated from mouse wounds, which were homogenized in TRIzolTM Reagent (Thermo Fisher Scientific, Waltham, MA, USA) using GentleMACS M-tubes (Miltenyi Biotec, Bergisch Gladbach, Germany) and gentle MACSTM dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). After homogenizing the sample with TRIzol™ Reagent, chloroform was added. RNA was precipitated with isopropanol, washed to remove impurities, and then resuspended for use in downstream applications. DNA elimination was performed using Invitrogen™Ambion™TURBO DNA-free kit (Invitrogen Life Technologies Corporation, Oregon, USA). Total RNA concentration was quantified using Nanodrop 2000 (Thermo Scientific, Waltham, MA, USA) or Qubit® RNA BR Assay Kit (Invitrogen Life Technologies Corporation, Oregon, USA). The RNA quality was determined by analyzing the proportion between 28S to 18S ribosomal RNA electropherogram peak using an Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). Samples with an RNA integrity number >8 were used for cDNA synthesis.
Quantitative reverse-transcription–PCR
cDNA was synthesized from 1 μg of RNA using reverse Transcriptase Core Kit (Eurogentec, Seraing, Belgium). Quantitative reverse-transcription–PCR (qRT–PCR) was performed using a QuantiTect SYBR Green PCR Kit (Qiagen, Courtaboeuf, France) and C1000 CFX384 Touch Real-Time PCR System (Bio-Rad Laboratories, Hercules, California, USA). Gene expression assays were performed with primer sequences purchased from Qiagen (Qiagen, Courtaboeuf, France). The list of primers is provided in Table 2. In experiments involving primary human fibroblasts, qRT-PCR was performed as follows. A final volume of 10 μl was prepared, which consisted of 1 μl of cDNA (10 ng/μL), 1 μl of primer (10 mM) and 5 μl of SsoAdvancedTM Universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, California, USA) and ultrapure water. The C1000 CFX384 Touch Real-Time PCR System (Bio-Rad Laboratories, Hercules, California, USA) was programmed using the following parameters: 3 min at 95 °C and 40 cycles of three steps (30 s at 98 °C; 15 s at 98 °C and 30 s at 60 °C).
In all experiments at least three reference genes were tested to establish the stability value. Two housekeeping genes with the highest stability value were used to normalize cDNA within each sample. Differences were calculated using the Ct and comparative Ct methods for relative quantification. The relative level of gene expression was normalized with a housekeeping gene and used as a reference to calculate the relative level of gene expression according to the following formula: 2−ΔΔCt, where −ΔΔCt = Δ Ct gene − Δ Ct Average where Δ Ct gene = Ct gene − Ct (housekeeping gene) and Δ Ct Average = Average Δ Ct control. For all biological replicates, two technical replicates were performed. All samples were evaluated in at least three independent experiments.
Fluorescence microscopy
Eight-mm round glass coverslips (Electron Microscopy Sciences, Hatfield, PA, USA) were sterilized, coated with poly-L-lysine (Sigma-Aldrich, MO, USA) and placed into the wells of a 24-well plate. RAW 264.7 cells were seeded onto coverslips at a density of 105 cells/cm2, incubated in complete medium without antibiotics at 37 °C overnight, and infected with S. aureus Xen36 at MOI of 10. Infected RAW 264.7 cells were incubated in antibiotic-free culture medium for 30 min, after which the medium was supplemented with 20 µg/mL of gentamicin to ensure the killing of extracellular bacteria. At 5 h. p. i., all samples were washed in PBS, fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, USA) in PBS for 60 min and washed in 0.5% bovine serum albumin in PBS. Paraformaldehyde-fixed cells were permeabilised with 0.1% Triton X-100 in PBS for 4 min. Cells were blocked with 5% goat serum (Sigma-Aldrich, Saint-Louis, MO, USA) in PBS for 30 min, incubated with primary antibody diluted in 0.5% BSA in PBS for 60 min, and then washed with 0.5% BSA in PBS. Cells were incubated with the appropriate fluorophore-tagged secondary antibodies for 60 min, washed with 0.5% BSA in PBS. The list with antibody names and dilution factors is provided in Table 1. Coverslips were mounted in VECTASHIELD HardSet Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA) on glass slides. Cell imaging was performed with an inverted Eclipse TiE Nikon microscope equipped with a CSU-X1 spinning disk confocal scanning unit (Yokogawa, Ishikawa, Japan) and with an Evolve 512 Delta EMCCD Camera (Photometrics, AZ, USA). Images were acquired with a x100 1.4 numerical aperture oil objective and MetaMorph software (Molecular Devices, CA, USA). Images were converted to 8-bit images using ImageJ software, and then exported as .tif files. Analyses were performed with an open source CellProfiler 4.1.3. (https://cellprofiler.org/) software76. Briefly, the pipeline segmented channel intensity using “IdentifyPrimaryObjects” module followed by “MeasureImageIntensity”. Next, colocalization was pre-formed with the “MeasureColocalization” module for each channel within the identified objects (Bacteria, Cell, Protein target of interest) with a minimum threshold of 15. The correlation overlap was then classified to the Bacteria or Cell regions using “ClassifyObjects” module. The data of the colocalization analysis are expressed as the values for 50–100 S. aureus-containing phagosomes, which were counted and scored for the presence or absence of markers. All samples were evaluated in triplicate and in at least three independent experiments. Results were expressed as the mean percentage of marker colocalization on S. aureus-containing phagosomes ± SEM.
Cell fractionation
RAW 264.7 cells were fractionated according to the protocol previously described77. Briefly, cells were washed and resuspended in 0.5% BSA diluted in PBS and centrifuged at 300 × g for 5 min. Cells were next resuspended in homogenization buffer (8% sucrose in imidazole 3 mM MgCl2 1 mM supplemented with EGTA 0.5 mM, gelatine 0.5%, Complete™ protease and phosphatase inhibitors (Roche, Mannheim, Germany)) and centrifuged at 300 × g for 10 min. Mechanical disruption of cells was performed in homogenization buffer using a 25G 5/8 needle. Following centrifugation at 2000 × g for 15 min, the post-nuclear fraction was collected, brought to 40% sucrose, and gently layered on top of 60% sucrose in an open-top transparent tube (Beckman Coulter GmbH, Krefeld, Germany). A 60/40%, 40/30%, 30/20%, and 20/8% sucrose gradient was prepared, and ultra-centrifugation was performed using a SW 41 Ti Swinging-Bucket Rotor (Beckman Coulter, Villepinte, France) at 100,000 × g for 1 h. The recovered fractions were adjusted at a final concentration of 10% sucrose. To precipitate protein, one volume of 100% (w/v) trichloroacetic acid was added to 4 volumes of the sample, and incubated for 10 min at 4 °C. After centrifugation at 14,000 × g for 5 min the supernatant was removed, and the pellet was washed twice with cold acetone. The pellets were heated to 95 °C for 5–10 min and the dried pellets were resuspended in 50 µl of 1% SDS. The protein concentration was assessed using the QubitTM Protein Assay (Life Technologies Corporation, Oregon, USA). The samples and standards were read with the QubitTM Flex Fluorometer (Life Technologies Corporation, Oregon, USA).
SDS-polyacrylamide gel electrophoresis (SDS-PAGE), immunoprecipitation and immunoblotting
RAW2 264.7 cells were lysed in ice-cold RIPA buffer (Cell Signaling Technology, Beverly, MA, USA) supplemented with Complete™ protease and phosphatase inhibitors (Roche, Mannheim, Germany). The lysate was centrifuged at 15,000 × g for 10 min at 4 °C, and the supernatant was assayed for protein content. Protein content was quantified using QubitTM Protein Assay (Life Technologies Corporation, Oregon, USA) and QubitTM Flex Fluorometer (Life Technologies Corporation, Oregon, USA) according to the manufacturer’s instructions. Total protein was prepared in 4× Laemmli buffer (Bio-Rad Laboratories, Hercules, CA, USA) and 80 mM DTT.
For immunoprecipitation, protein was incubated with 5 mg of antibody bound to Invitrogen DynabeadsTM Protein G (Thermo Fisher Scientific, Vilnius, Lithuania) for 2 h at 4 °C with constant mixing. Tubes containing DynabeadsTM Protein G/antibody/antigen complex were washed three times with 1 × PBS and placed on a MagnaRackTM (Thermo Fisher Scientific, Waltham, MA, USA) magnetic separation rack to remove the supernatant between each wash. Antibody and the bound proteins were solubilised in NuPAGE™ LDS Sample Buffer and NuPAGE™ Sample Reducing Agent (Invitrogen, CA, USA).
Proteins were resolved on a 4%–20% Mini-PROTEAN TGX Stain-Free Protein Gel (Bio-Rad Laboratories, Hercules, CA, USA). Forty µg of protein was subject to SDS-PAGE separation and immunoblotting. Following protein electrotransfer, Immun-Blot® PVDF (Bio-Rad Laboratories, Hercules, CA, USA) membrane was blocked for 1 h at RT in 1X TBS containing 5% (w/v) BSA. Proteins were detected by incubation with primary antibodies in blocking solution overnight at 4 °C. Following three 5 min washes of TBS with 0.1% Tween-20, membranes were incubated in the appropriate dilution of horseradish peroxidase conjugated, species-specific secondary antibodies diluted in 5% blocking solution for 1 h at RT. Chemiluminescence detection was performed using the Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA). Chemiluminescence signals were acquired using the ChemiDoc XRS+ System and Image Lab Software (Bio-Rad Laboratories, Hercules, CA, USA). Signal intensity was determined by densitometry and fold changes in protein levels was calculated from this data. The list with antibody names and dilution factors is provided in Table 1. All blots or gels derive from the same experiment and were processed in parallel.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 10 software (GraphPad Inc, San Diego, CA, USA) and statistical tests are reported in figure legends. Data normality was tested by D’Agostino–Pearson test, and appropriate parametric or non-parametric tests were performed. For parametric data, data significance was analyzed using a two-tailed unpaired Student’s t test. If the assumptions were not met, a nonparametric test was used, which does not require the assumption of normality. Nonparametric data were analyzed using Mann–Whitney test. Multiple comparisons were analyzed by one-way analysis of variance (ANOVA) with 95% confidence interval, followed by Tukey’s or Dunnett’s post hoc test. In vivo experiments were performed on age and sex-matched mice. Unless stated otherwise, the data of the in vitro experiment analyses is reported as mean ± s.e.m. of the values for repeated conditions in separate wells and are representative of at least three experiments performed. Each biological replicate had at least two technical replicates. The number of biological replicates is indicated in the figure legends. p values less than 0.05 (*), 0.01 (**), or 0.001 (***) were considered as statistically significant.
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- Source: https://www.nature.com/articles/s41536-024-00372-0