Animal infection and experiment design
M. vogae infection is maintained by serial passage from infected mice to a naive ICR-strain of mice at the animal facilities of the Institute of Parasitology of the Slovak Academy of Sciences under pathogen-free conditions. Metacestodes of M. vogae were collected in sterile physiological saline from the peritoneal cavity of female mice after 2–4 months of infection.
The experiment was carried out on 8-week-old male BALB/c mice purchased from Velaz (Prague, Czech Republic). The mice were infected orally (60 ± 5 tetrathyridia per animal) and randomly divided into eight groups (n = 5 per group). Healthy mice were used as a control (n=3). Peritoneal exudates as well as peritoneal exudate cells (PEC) were obtained on day 0, 3, 6, 10, 14, 19, 25, 30 and 35 post-infection (p.i.). In the subsequent experiments aimed at studying the direct effect of parasitic ES antigens, groups of mice (n=5/each) received either intraperitoneal injections of 200 µl of PBS (control) or 20 µg of ES in 200 µl of PBS and the applications of ES and PBS were performed six times, every day for a total 6 days. The antigen doses correlated approximately with the concentration of M. vogae secretion detected in vivo . In these experiments, peritoneal fluid and PEC were isolated as described below and used in further analysis..
Isolation of exudates and cell preparation
Exudates from peritoneal cavities of healthy and infected mice were collected by washing the peritoneal cavity with 1 ml of sterile PBS, and PEC were obtained by second peritoneal lavage under sterile conditions by injections of 5 mL of Roswell Park Memorial Institute-1640 (RPMI) medium (Biochrom, Berlin, Germany) containing 2mM of stable glutamine and supplemented with 10% heat-inactivated bovine fetal serum (Biochrom, Berlin, Germany), 100 U/ml of penicillin, 100 μg/ml of streptomycin, 10 μg/ml of gentamicin and 2.5 μg/ml of amphotericin B (complete medium, CM) (all from Sigma-Aldrich, St. Louis, USA). The freshly isolated cell suspension was then washed with LPS-free Dulbecco phosphate buffered saline (DPBS, Sigma-Aldrich, St. Louis, USA) and resuspended in CM. The viability and numbers of isolated cells were evaluated by trypan blue exclusion test (Sigma-Aldrich, St. Louis, USA). Then, PEC were stained with various combinations of antibodies and the cell suspension was subsequently aliquoted for phenotypic analysis by flow cytometry and an assessment of classical cell activation (LPS stimulation of PEC). The remainder of the unstained cell samples was utilized for RNA extraction.
To evaluate the suppressive capacity of the peritoneal myeloid cell subsets, Ly6G+ and Ly6C+ cells were separated from the PEC of infected mice at day 35 p.i. Ly6G+ cells were isolated by positive selection using biotin conjugated anti-mouse Ly6G antibody and magnetic streptavidin nanobeads (MojoSort Mouse Ly-6G Selection Kit, Biolegend, San Diego, CA, USA). The magnetically labelled fraction was separated on a MACS column according to the manufacturer’s instructions. Ly6C+ cells were collected from Ly6G-depleted fractions using Gr-1-biotin antibody and magnetic streptavidin nanobeads (both from Biolegend, San Diego, CA, USA). The purity of various populations was determined via FACS analysis and the viability and numbers of isolated cells were evaluated using trypan blue staining (Sigma-Aldrich, St. Louis, USA).
May-Grünwald/ Giemsa staining
To evaluate the morphology of cells, 1x105 sorted Ly6G+ and Ly6C+ cells were fixed on glass slides using the cytospin technique. Then, the cells were dried and stained with May-Grünwald/Giemsa solutions (Sigma-Aldrich, St. Louis, USA) according to the standard procedure. The stained cells were then observed under a light microscope (Olympus, Prague, Czech Republic) and an analysis of the cell types was done at 1000 x magnification.
Flow cytometric analysis
PEC were enumerated (n=5/infected group; n=3/control group) and resuspended in CM (0.5 x 106 cells/100 μl). The viability of these cells was more than 95% determined by trypan blue exclusion. Fifty microliter aliquots of cell suspension were plated into a round bottom tube and stained with monoclonal antibodies that recognize CD11b (FITC; clone M1/70; Biolegend, San Diego, CA, USA), F4/80 (APC; clone CI:A3-1; BioRad, Hercules, CA, USA), Gr1 (PE; clone RB6-8C5), MHC-II (Pe Cyanine7; clone M5/114.15.2), CD3 (PerCPeFluor710; clone 17A2), CD4 (FITC; clone GK 1.5), CD8 (PE; clone 53-6.7) and CD 49b (APC; clone DX5) from eBioscience (San Diego, CA, USA). Cells were stained for 20 min at room temperature, fixed with fixation buffer (eBioscience, San Diego, CA, USA) for 15 min and rinsed twice with FACS buffer.
In the experiment focused on the role of the application of ES antigens in vivo on the myeloid phenotype, intracellular staining of IL-10 was performed. Briefly, isolated PEC were first stained for 30 min at 4°C with antibodies to CD11b surface marker. After two washes with PBS, intracellular staining was performed using the Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA, USA) and anti-IL-10 (PE; clone: JES5- 16E3, Sony Biotechology, San Jose, CA, USA) according to the manufacturer’s instructions. Phenotypic analysis was performed on a FACS Canto (Becton Dickinson Biosciences, USA) and the acquired data were analyzed using the FACS Diva software. The portion of unstained cells was used for RNA extraction.
T-cell suppression assay
Spleen cell preparations were prepared from naïve mice. Spleens were gently homogenized between two glasses and each sample was spun at 1500 rpm for 5 min. The supernatant was removed and the cells were resuspended in 5 ml of cold NH4Cl. Following the lysis of red blood cells, splenocytes were washed and diluted at 2 x 106/ml in 5 ml of RPMI supplemented with 5% fetal bovine serum (FBS). CFSE (Biolegend, San Diego, CA, USA) was added to reach a final concentration of 2.5 µM and incubated at room temperature for 7 min. Next, Dulbecco PBS (DPBS) supplemented with 20% FBS was added. The cells were washed three times in RPMI with 10% FBS and plated. The CFSE-labeled splenocytes (at a concentration of 1 x 106/ml and a volume of 100 µl) were plated in 96-well plates coated with 10 µg/ml of anti-CD3 and 1 µg/ml of anti-CD28 mAbs (Invitrogen, Carlsbad, CA, USA). The splenocytes were cultured for 3 h on Ab-coated plates and then 100 µl of PEC (Ly6C+ or Ly6G+ cells) were added to obtain the following ratios of PEC to splenocytes: 1:1, 1:2, 1:4, 1:8, 1:16 and 1:32. Suppression assay was performed in triplicates. The co-cultures were incubated for 72 h, then harvested and analyzed by flow cytometry. Dilution of the CFSE was evaluated as a measure of T-cell proliferation by flow cytometry. To that end, cells were additionally stained with anti-CD3-PerCP-Cy5.5 (Biolegend, San Diego, CA, USA). The percentage suppression of proliferation was calculated as (1 – (proliferation with MDSC : proliferation without MDSC)) x 100.
The concentrations of IFN-g, IL-4, TGF-β as well as IL-10 present in peritoneal fluid and culture supernatants of LPS-stimulated PEC (n=5/infected group; n=3/control group, examined in duplicates) were quantified by commercial ELISA Kits (Mouse Ready-SET-Go ELISA, all from eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. Concentrations of cytokines were calculated in pg/ml.
Determination of nitrite production ex vivo and cytokine level in culture supernatants
To measure the production of nitric oxide (NO) and level of cytokines by total PEC (n=5/infected group; n=3/control group, examined in duplicates), cell suspensions (1x 106 cells/ml) were cultured in 24-well plates (Corning) in CM in the presence or absence of LPS (1 µg/ml). The plates were incubated for 72 h at 37 °C, 5% CO2. The concentration of NO in the culture supernatants was determined as nitrite (NO2−) using Griess reagent. Briefly, 50µl of a solution containing 1% sulfanilamide/5% H3PO4 was incubated with 50 µl supernatants in a 96-well plate (in triplicate) for 10 min at room temperature. After incubation, a second solution (0.1% N-(1-Naphthyl) ethylenediamine dihydrochloride) was added to the mixture and the absorbance was measured at 550 nm using an ELISA reader. Nitrite concentration was determined from calibration curve using 0.1 M NaNO3 as standard. For determination of the levels of TNF-α, IL-6, IL-10 by ELISA Kits (Mouse Ready-SET-Go ELISA, all from eBioscience, San Diego, CA, USA), a rest of culture supernatants was collected and stored at -20 °C until use. The supernatants were diluted 1:2 with PBS before use.
RNA isolation and real-time PCR
Total RNA was extracted from the PEC (n=5/infected group; n=3/control group) using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The RNA was quantified using a NanoSpectrophotometer AstraGene (Harston, Cambridge, UK) and 3µg was transcribed to cDNA using ReverseAid H Minus M-MuLV Reverse Transcriptase and oligodT primers (Thermo Scientific, Burlington, ON, Canada). The cDNA for each cell sample was used as a template for real-time PCR reactions. Quantitative PCR analysis of the relative abundance of mRNA species was determined using the SYBR green master mix (BioRad, Hercules, CA, USA) on a BioRad CFX thermocycler (BioRad, Hercules, CA, USA). PCR was performed in 20-μl reactions with detection primer pairs for STAT-1 (forward: 5′ - CTGAATATTTCCCTCCTGGG -3′; reverse: 5′- TCCCGTACAGATGTCCATGAT -3′), STAT-3 (forward: 5′ - GAAGCCGACCCAGGTGC -3′; reverse: 5′- GTCACGTCTCTGCAGCTTCT -3′), STAT-6 (forward: 5′ - GAGTTCCTGGTCGGTTCAGA -3′; reverse: 5′- GCTCTCCAAGGTGCTGATGT-3′), iNOS (forward: 5′-GCCTCATGCCATTGAATTCATCAACC-3′; reverse: 5′- GAGCTGTGAATTCCAGAGCCTGAA-3′), Arg-1 (forward:5′- CAGAAGAATGGAAGAGTCAG -3′; reverse: 5′- CAGATATGCAGGGAGTCACC -3′). Data were normalized to a housekeeping gene (β-actin), and relative quantification was done using the 2-∆∆Ct method.
SDS‐PAGE and Western blotting
ES products or somatic homogenate (MvH) were mixed with 5x reducing sample buffer, boiled for 5min at 95 °C and processed for SDS-PAGE. The final protein concentration in each prepared sample was 80–100 μg/100 μl, which were subsequently resolved in 12% acrylamide gels under denaturing conditions and electrophoretically transferred to Nitrocellulose membranes (0.45 μm pore size, Merck Millipore Ltd., Tullagreen, Carrigtwohill, Co. Cork, Ireland) using Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA, USA). Strips with blotted antigens were utilized for immunodetection of specific anti-tetrathyridial IgG and IgM antibodies. Nonspecific binding was blocked upon incubation in 5% fat-free milk in PBS for 1 h and strips were incubated with the sera diluted in 3% fat-free milk (1:50) overnight at 4 °C. The blots were washed and probed with the HRP-conjugated goat polyclonal anti-mouse IgG (ICN, USA) diluted to 1:500 for 1h at 37 °C. Finally, the immunoreactive bands were visualized by treating the membranes with the substrate solution (4-chloronaphthol and H2O2 in PBS).
The suspension of peritoneal cells was placed on glass slides and fixated with 4% formaldehyde for 15 min at room temperature. Nonspecific binding was blocked by incubation with 4% goat serum for 30 min at room temperature. The cells were incubated with primary antibody against iNOS or Arg-1 (1:250) (Abcam, Cambridge, UK) for 2 h at room temperature and then washed three times with PBS. After incubation with secondary antibodies (1:500, FITC-conjugated anti-rabbit IgG-F0112, PE-conjugated anti-rabbit IgG-F0110, both from R&D Systems, Minneapolis, MN., USA) in the dark for 1h at room temperature, nucleus staining was performed using Hoechst 33258 Staining Dye Solution (Abcam, Cambridge, UK). Images were obtained with a fluorescence microscope (Leica DM IRB, Germany).
Determination of Ag-specific antibodies by ELISA
Parasite products (ES products or somatic homogenate MvH) were prepared as described by Vendelova et al. [4, 9], and the protein content was assessed using Bradford protein assay reagent (BioRad, Hercules, CA, USA) and using BSA (Sigma-Aldrich, St. Louis, USA) as the standard.
To determine the specific IgG/IgM, flat-bottom 96-well plates (Nunc Maxisorp) were coated with worm antigens (2.5 μg/ml ES or MvH) in carbonate-bicarbonate coating buffer (pH 9.6) per night at 4°C. The microplates were blocked for 1 h at room temperature with 10% bovine fetal serum-PBS solution. Sera/peritoneal exudates diluted 1:100 were incubated for 1 h at 37 °C. The microplates were then washed and goat anti-mouse IgG/IgM peroxidase conjugates diluted 1:5000/1:2000 (Sigma-Aldrich, St. Louis, USA) were added for 1h at 37 °C. After further washes, O-phenylendiamine (Sigma-Aldrich, St. Louis, USA) was added as a substrate and absorbance values were recorded at 492 nm. Results were expressed as the mean optical density (OD) ± SD. The cut-off levels were determined as the mean +3.8 x SD of the antibody activity in the exudates of healthy mice after Lardeux et al. .
All data were calculated as mean ± standard deviation (SD). Statistical analyses were performed with GraphPad Prism version 5.00 and 7.00 (GraphPad Software, Inc., San Diego, CA, USA). Data were analyzed by either unpaired Student’s t-test, one-way or two-way ANOVA followed by Dunnett’s or Bonferroni's multiple comparisons test to compare different groups. Differences were regarded as significant when p < 0.05.