Mice and topical application
Female ApoE−/− mice were purchased from Taconic (Ry, Denmark), model no APO-F (B6.129P2-ApoE
tm1Unc N11). Two separate, but similar, studies were conducted, i.e., a pilot study with n = 5–7 mice/group (study 1) followed by a full-scale study with n = 15 mice/group (study 2). Mice had access to water and standard diet ad libitum (Altromin 1314, Brogaarden, Gentofte, Denmark) and were housed with 12 h light/dark cycles in a temperature- and humidity-controlled room at 21–23 °C at the University of Copenhagen.
At the age of 11 weeks, mice received 2 topical applications/week (20 μl/ear) of either vehicle (acetone) or TPA (Sigma-Aldrich, Brøndby, Denmark; dissolved in acetone at a 0.1 μg/μl concentration). Applications were given on both ears and the mice received 16–17 applications during a total of 8 weeks. Mice were terminated either 3–4 (study 1) or 2 (study 2) days after the last TPA application. Ear thickness was measured prior to each TPA application using a digimatic thickness gauge (Mitutoyo, Illinois, US). All measurements were performed by the same investigator. At study termination, mice were anaesthetized subcutaneously with a 0.1 ml/10 g mouse dose of either a mixture of fentanyl (0.079 mg/mL), fluanisone (2.5 mg/mL), and midazolam (1.25 mg/mL) (study 1), or a mixture of tiletamine (1.63 mg/mL), zolazepam (1.63 mg/mL), xylazin (2.61 mg/mL), and butorphanol tartrate (0.065 mg/mL) (study 2). Subsequently, blood was collected and mice were perfused with ice-cold saline.
Half of an 8 mm biopsy of the right ear was prepared for histology by fixation for one week at room temperature in 10 % neutral buffered formalin (“Lillie” formaldehyde solution 4 %, Hounisen, Skanderborg, Denmark) and embedded in paraffin. Cross-sections of 4 μm were deparaffinized and rehydrated prior to staining with Mayer’s hematoxylin and eosin (Rigshospitalet, Copenhagen, Denmark), rinsing and dehydration. Digital images were obtained with a light microscope (Leica Microsystems, Ballerup, Denmark).
Protein analysis from serum and skin samples
Blood was collected in heparinized microtubes (capiject; Terumo Medical Coorporation, Elkton, US) prior to the first TPA/acetone application (baseline sample, submandibular vein) and again at study termination (retro-orbital vein). Plasma was collected after centrifugation for 10 min at 1000 × g at 4 °C, aliquoted, and stored at −80 °C until use. Plasma cholesterol was measured in duplicates using the CHOD-PAP reagent from Roche (Roche Diagnostics, Denmark). For protein analyses of ear lysates, an 8 mm biopsy of the left ear was snap-frozen in liquid nitrogen. Using a tissue homogenizer (Precellys 24, Bertin Technologies, Montigny le Bretonneux, France), the biopsies were crushed in cell lysis buffer (Cell Signaling Technology, The Netherlands) containing freshly added protease inhibitors (complete protease inhibitor with Halt, Thermo Scientific, Rockford, US). Tissue lysates were collected after 15 min of centrifugation at 15,000 × g and total protein concentration was measured with the Pierce BCA protein assay kit (Thermo Scientific), according to the manufacturer’s instructions. Murine IL-22 and IL-17F (R&D Systems, Minneapolis, US) and serum amyloid A (SAA) (Tridelta, Kildare, Ireland) were measured by commercial ELISA according to the manufacturer’s instructions. Mouse interferon-γ (IFNγ), tumor necrosis factor- α (TNFα), keratinocyte-derived cytokine (KC), IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, and total IL-12 were measured with the ProInflammatory 7-Plex and Th1/Th2 9-Plex MSD MULTI-spot Assay Systems (Meso Scale Discovery, Rockville, US) according to the manufacturer’s instructions. For each assay, a volume of 1.7–5 μl heparinized plasma or a total protein amount of 12–200 μg of ear lysate was used.
Aortic arch atherosclerosis (en face) and aortic arch mRNA
The relative amount of atherosclerosis was measured en face in the aortic arch (from the heart to the 7th rib), and the same tissue was used for RNA extraction and quantitative real-time PCR. The aortic arch (from the heart to the 7th rib) was snap-frozen in liquid nitrogen. For en face analysis, the aortic arch was opened longitudinally, and images of the luminal surface were acquired with a digital camera connected to a dissecting microscope and analysed using the Leica IM50 software (Leica Microsystems). For mRNA analysis, total RNA was extracted from the aortic arch using TRIzol (Life Technologies, Naerum, Denmark) and examined on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, US). RNA concentration was measured using a NanoDrop 1000 Spectrophotometer (Thermo Scientific) before cDNA synthesis of 250 ng RNA/aorta using the High Capacity cDNA Reverse Transcription Kit (Life Technologies). Real-time quantitative PCR was performed on a TaqMan (Life Technologies). Primer and probe information can be found in Additional file 1.
Aortic root histology
The apex of the heart was cut off and the remaining part fixed in Lillie’s formalin at 4 °C overnight prior to being snap-frozen in Tissue-Tek O.C.T. (Sakura Finetek, Leiden, Netherlands) in ice-cold isopentane. The aortic root was sectioned on a cryostat (Leica) at −18 to −25 °C. Ten μm sections were collected on SuperFrost Plus slides (Menzel-Gläser; Thermo Scientific) for a total of 900 μm starting from where an aortic valve cusp was first visible. The atherosclerotic plaque area was measured, where all three aortic valve cusps were visible to ensure that quantifications were performed at the same anatomical site in each mouse. Masson’s Trichrome staining was performed according to the manufacturer’s instructions (Sigma-Aldrich), and was used to detect collagen/fibrosis. Immunohistochemical staining was performed with monoclonal rat anti-mouse macrophages/monocytes (MOMA-2 MCA519, 1:500; AbD Serotec, Kidlington, UK). Corresponding antibody isotype control was run with monoclonal rat IgG2b (MAB0061, 1:500, R&D systems). For detection, we used a biotinylated secondary antibody rabbit anti-rat (E0468, 1:2000; Dako, Glostrup, Denmark). The staining procedure included blocking of endogenous peroxidase with 0.5 % H2O2, blocking of unspecific antibody binding with 2 % BSA, brown positive staining using a horse-raddish peroxidase approach (Vectastain Elite ABC kit; VectorLab) followed by diaminobenzidine (DAB+, Dako), and counterstaining with Mayer’s hematoxylin (Sigma-Aldrich). Digital photos of histological sections were acquired using a slide scanner (Pannoramic, 3DHISTECH, Budapest, Hungary or Axio Scan.Z1, Zeiss, Birkerød, Denmark), and quantified using the Visiomorph software (Visiopharm, Hørsholm, Denmark).
Single-cell splenocyte preparations were made by gently forcing splenic tissue through a 70 μm mesh using a 3-ml syringe plunger and ice-cold Hanks Buffered Salt Solution (HBSS, Panum, Denmark). Splenocytes were pelleted at 300×g for 8 min, washed once in HBSS, and counted using methylene violet and the ‘Countess’ (Invitrogen). Half of the mice were euthanized in one day and the other half the following day, and each day we made a pool of splenocytes from control mice and from TPA mice. These pools were used for setup and for making ‘fluorescence minus one’ (FMO)-controls. Four different flow cytometry analyses were carried out (see Additional files 2 and 3 for more information on antibodies applied together with the corresponding representative figures for gating strategies). Cell surface staining was accomplished using standard techniques in 100 μl in V-bottom 96-well microplates (TPP Techno Plastic Products, Trasadingen, Switzerland). Briefly, 1–2 × 106 splenocytes were pelleted and blocked with 50 μl FACS buffer (0.1 % sodium azide and 2 % bovine serum albumin in phosphate-buffered saline, PBS) containing FcBlock (1:100; Cat. n° 101302, BioLegend) for 5 min to block Fcγ receptors on the splenocytes. Without washing, staining antibodies were added in 50 μl FACS buffer and incubated for an additional 20 min at 4 °C in the dark. Next, splenocytes were washed, fixed with paraformaldehyde in PBS, and analysed within 24 h using LSRII flow cytometer (BD Biosciences, Albertslund, Denmark). For intracellular staining of Foxp3 (regulatory T-cells), we followed eBioscience’s protocol for staining of intracellular/nuclear proteins after cell surface markers (CD4 and CD25) had been stained using the above protocol. To assess changes in CD4+ helper T-cell bias due to the TPA application, we followed the manufacturer’s protocol for the Mouse Th1/Th2/Th17 Phenotyping Kit (Cat. n° 560758, BD Biosciences). In order to investigate parallel changes in CD8+ cytotoxic T-cell bias, an anti-CD8 antibody was added to splenocytes as described in the manufacturer’s protocol. Briefly, for individual mice, two cultures with 10 × 106 splenocytes were seeded in RP-10 media (RPMI-1640 media containing 2 mM L-glutamine, 10 % heat-inactivated fetal bovine serum, 10 mM HEPES buffer, 0.1 mM non-essential amino acids, 100 U/ml penicillin, and 100 μg/ml streptomycin) containing the BD GolgiStop reagent. Splenocytes in one culture were stimulated with 50 ng/ml TPA and 1 μg/ml Ionomycin for 4 h at 37 °C, whereas the second culture was left unstimulated. Splenocytes were harvested, washed, counted, and 1.2 × 106 splenocytes were fixed using BD Cytofix buffer, washed, permeabilized using BD Perm/Wash buffer, and stained using the kit’s antibody cocktail, followed by staining with the anti-CD8 antibody. Stimulated and unstimulated cells were then washed in FACS buffer prior to flow cytometric analysis.
Results are shown as mean ± SEM or mean ± SD for normally distributed data or median [interquartile range (IQR)] for non-normally distributed data. Differences between groups were analysed with parametric or non-parametric t-tests, and multiple t-tests with correction for multiple comparisons were used when appropriate. A p-value <0.05 was considered significant. Data were analysed using the Graphpad Prism version 6.05 (GraphPad Software, California, US).