Cells and viruses
MARC-145 cells, an African green monkey kidney cell line that is highly permissive to PRRSV [17], were used for virus propagation and assays. These cells were maintained in RPMI-1640 medium (Gibco® RPMI-1640, Life Technologies, Carlsbad, CA, USA) supplemented with 10% heat-inactivated foetal bovine serum (FBS, Life Technologies), 2 mM l-glutamine, and an antibiotic–antimycotic cocktail (Anti-Anti, Life Technologies) containing 100 IU/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL Fungizone® [amphotericin B] in a humidified chamber with 5% CO2 at 37 °C. In this manuscript, this medium is designated RPMI growth medium. The North American PRRSV-2 strain JA142 (GenBank: AY424271.1) was used in the present study.
Animal study
One hundred 4-week-old piglets purchased from a PRRSV-seronegative farm were randomly assigned to two groups and housed in separate animal rooms. After 3 days of acclimatization, the pigs in the infected group (n = 75) were intramuscularly inoculated with 2 mL of the PRRSV-JA142 strain (1 × 103 TCID50/mL) diluted in sterile PBS. The control pigs (n = 25) remained uninfected. Feed and water were provided ad libitum to all the pigs. Five pigs from the control group and 12, 16, 14, 18 and 12 pigs from the infected group were humanely euthanized on days 3, 10, 21, 28 and 35 post-challenge (dpc), respectively. Euthanasia was performed by electrocution after the intramuscular injection of 2 mL of azaperone (40 mg/mL, StressGuard®, Dong Bang Inc., Seoul, South Korea). Three infected pigs died during the course of the experiment due to high fever and reduced body growth. An overview of the animal study is presented in Figure 1. After euthanasia, the lungs, trachea and bronchi were aseptically extracted and lavaged with 75 mL of sterile PBS. The collected lavage fluid was centrifuged at 1000 × g and room temperature for 10 min to separate the bronchoalveolar lavage fluid (BALF) and cells (BAL), and these samples, along with the lung parenchyma and BLN, were also used for immune cell analysis. These tissues and BALF were collected in tubes, snap-frozen using liquid nitrogen and stored immediately at −80 °C for RNA extraction and cytokine analysis, respectively. For histopathology, the lung tissues were also collected in 10% neutral-buffered formalin.
Blood was collected from the euthanized pigs at 3, 10, 21, 28 and 35 dpc, and the serum and PBMC were separated. In addition, blood samples from 42 pigs, including uninfected and infected pigs that were going to be euthanized on 28 and 35 dpc, were also collected at 0, 3, 7, 10, 14, 21 and 28 dpc, and the serum and PBMC were separated. The body weights of all the pigs were measured at 0 dpc, and the body weight gains of the euthanized pigs were measured at 3, 10, 21, 28 and 35 dpc.
Quantification of the virus in serum and lung tissue
Serum viremia was measured at 0, 3, 7, 10, 14, 21, 28 and 35 dpc, and the viral load in the lungs was quantified in the euthanized pigs at 3, 10, 21, 28 and 35 dpc. Viral RNA was extracted from serum using a MagMAX viral RNA isolation kit (Ambion; Applied Biosystems, Life Technologies, Inc.) according to the manufacturer’s instructions. Real-time reverse transcription-polymerase chain reaction (RT-qPCR) employing the Prime-Q PCV2 PRRSV Detection Kit (Genet Bio, Daejeon, South Korea) was performed for the quantification of serum viremia. One-step RT-qPCR was performed in accordance with the manufacturer’s instructions. PCR amplification was performed using a model 7500 fast real-time PCR system (Applied Biosystems, Foster City, CA, USA). The cycling conditions were as follows: (i) cDNA synthesis for 20 min at 50 °C; (ii) 10-min predenaturation step at 95 °C; and (iii) 40 cycles of denaturation for 10 s at 95 °C and annealing/extension for 30 s at 60 °C. To calculate the amount of PRRSV in each sample, the Cq values were converted to virus titres (TCID50/mL) by generating a standard curve through the titration of PRRSV-2 strain JA142.
In addition, MARC-145 cells were used to quantify the virus titres in lung tissues using a microtitration infectivity assay [18]. Briefly, tissue homogenates (10% [weight/volume]) of finely chopped lung pieces were prepared in Dulbecco modified Eagle medium (DMEM) with antibiotics, and these mixtures were vortexed vigorously for 10–15 min and then centrifuged at ~4000 × g and 4 °C for 1 h. The collected supernatant was filtered through a 0.20-μm sterile syringe filter and used as an inoculum for the measurement of virus titres. The virus titres were calculated at 5 to 6 days post-inoculation based on the cytopathic effect (CPE) and are expressed as TCID50/mL [19].
Pathological evaluation of the lungs
The lungs of the necropsied pigs in both groups were subjected to pathological evaluation on each day of necropsy. The microscopic lung lesions were given a score on a scale from 0 to 3 to reflect no lesion, mild interstitial pneumonia, moderate multifocal interstitial pneumonia, and severe interstitial pneumonia, respectively. The microscopic lesions were examined from five different lobes of the lungs, and the average value was ultimately utilized for scoring purposes.
Anti-PRRSV-specific antibody detection
The serum samples from uninfected and challenged pigs were tested for anti-PRRSV antibody (IgG) using a commercially available ELISA kit (PRRS Ab ELISA 4.0; BioNote Inc., Hwaseong-si, Republic of Korea) according to the manufacturer’s instructions. Samples with an S/P ratio (the ratio of the net optical density of the test samples to the net optical density of the positive controls) ≥ 0.4 were considered to be positive for the PRRSV antibody.
The serum virus-neutralizing (SVN) antibodies were measured through a fluorescent focus neutralization (FFN)-based SVN assay with MARC-145 cells as described previously [20], with some modifications. After heat inactivation at 56 °C for 1 h, the serum samples were serially (twofold) diluted using RPMI-1640 growth medium. Two-hundred-microlitre mixtures were prepared by mixing each diluted serum sample with 103 fluorescent focus-forming units per mL (FFU/mL) of PRRSV-JA142 at a ratio of 1:1 and were then incubated for 1 h at 37 °C in a humidified atmosphere with 5% CO2. Each mixture was transferred onto a monolayer of MARC-145 cells in 96-well plates and incubated for another 1 h at 37 °C. The medium was replaced with 200 µL of fresh RPMI growth medium per well and further incubated for 20 h at 37 °C. The cells were later fixed using ice-cold 80% (v/v) acetone, air-dried, and stained with mouse anti-PRRS NC Mab 4A5 (Median Diagnostic, Gangwondo, Korea) and FITC-conjugated goat anti-mouse IgG (h + l) (Bethyl Laboratories, TX, USA). Subsequently, the plates were washed at least three times with PBS and observed under a fluorescence microscope to examine the PRRSV-specific FFU. The SVN titre is expressed as the reciprocal of the highest dilution at which a 90% or higher reduction in the number of FFU was observed.
Isolation of PBMC, BAL cells, and mononuclear cells from lymph nodes and lung tissues
PBMC were isolated from the blood samples (6 mL) by the density gradient method using Leucosep™ Centrifuge Tubes (Greiner Bio-One North America Inc., NC, USA) and Leucoprep™ Lymphocyte Separation Media (Greiner Bio-One North America Inc.) according to the manufacturers’ instructions. The blood samples were briefly stratified on Leucoprep™ solution at a ratio of 2:1 (blood:Leucoprep) and centrifuged at 1000 × g for 10 min. The purified PBMC were collected, washed twice with sterile PBS (pH 7.0) and resuspended in 0.5 mL of sterile PBS supplemented with 1% heat-inactivated FBS (Gibco, Carlsbad, CA, USA). Contaminating red blood cells (RBC) were removed by treatment with RBC lysis buffer (eBioscience, CA, USA).
For pathological evaluation, the right-sided lobes were clamped to collect specimens for RNA extraction and histopathology, and the left lobes of the lungs were used for BAL collection according to a previous study [21]. The lungs were lavaged with 50–75 mL of PBS containing 100 µg/mL ampicillin (USB Corporation Cleveland, OH, USA) and an antibiotic–antimycotic cocktail (Anti-Anti, Life Technologies), and the harvested fluid was centrifuged for 10 min at 1000 × g. The resulting supernatant was collected as BAL fluid (BALF), whereas the cell pellet (BAL cells) was washed three times with PBS after RBC lysis. The cells were resuspended in FACS buffer (3% FBS in phosphate-buffered saline and 0.02% sodium azide).
The BLN were passed through a 40-μm cell strainer (SPL Life Sciences, Pocheon, Korea) in PBS and then washed with FACS buffer according to a previous study [22]. The single cell suspension obtained was used for flow cytometric analysis.
Mononuclear cells from lung parenchyma were prepared based on a previous study [23], with few modifications. Briefly, lung tissue was collected, washed in sterile ice-cold PBS and suspended in serum-free RPMI media containing DNase I (25 U/mL, Sigma, St. Louis, MO, USA) and collagenase D (2 mg/mL, Roche Diagnostics, Mannheim, Germany). Single-cell suspensions were prepared using the gentleMACS Octo Dissociator (Miltenyi Biotec, San Diego, CA, USA) and incubated at 37 °C for 30 min. Subsequently, the cells were passed through a 40-μm cell strainer, washed, and resuspended in FACS buffer for flow cytometry analysis after RBC lysis.
Finally, the cells were counted with a Countess™ Automated Cell Counter (Invitrogen, Carlsbad, CA, USA), and their viability was tested by trypan blue (Sigma-Aldrich, St. Louis, MO, USA) exclusion [24].
Flow cytometry
For cell surface staining, single-cell suspensions were incubated on ice for 30 min with specific antibodies as listed in Additional file 1, and the cells were then washed three times with FACS buffer. When necessary, secondary antibodies conjugated with fluorochrome were used. Natural killer (NK) cells, DC and macrophages required only cell surface staining, whereas the different subsets of T cells required intranuclear and intracellular staining.
Two subsets of NK cells have been phenotypically defined based on NKp46 marker expression: NKp46+ and NKp46− NK cells [25, 26]. Following PBMC staining, a similar gating hierarchy was followed by excluding the unstained cells, doublets and CD3+ cells, and the CD3− lymphocytes were further analysed for CD8α and NKp46 expression. Among CD3− cells, two populations were found in the PBMC, namely, NKp46+ and NKp46− NK cells, and both of these cells were CD8α+ (Additional file 2A).
BAL cells were subjected to cell surface staining for the CD163 surface marker, and the viability of these cells was analysed using propidium iodide (PI) staining (Additional file 2B). Additionally, DC and macrophages were segregated from the BAL cell population based on staining and gating strategies outlined previously (Additional file 2C) [10, 11]. From the MHC-II+ cell population, five phenotypically and functionally defined subpopulations were distinguished using the CD163 and CD172a (Sirpα) surface markers. Among the MHC-II+ cells, CD172a+/CD163high cells were defined as AM, whereas CD172a+/CD163int, CD172a+/CD163low, CD172a+/CD163− and CD172a−/CD163− cells were defined as monocyte-derived macrophages (moMɸs), monocyte-derived dendritic cells (moDC), conventional dendritic cells 2 (cDC2) and conventional dendritic cells 1 (cDC1), respectively, based on a previous study [10].
Regulatory T cells (Tregs), which require intranuclear staining of FoxP3 after cell surface staining, were fixed with cold fixation/permeabilization buffer (eBioscience, Thermo Fisher Scientific, Seoul, Korea) at 4 °C for 30 min and were then stained for FoxP3 at 4 °C for 30 min. Based on a previously described staining and gating strategy [27], Tregs (CD25+FoxP3+ cells) were apparent among the CD4+CD8− population (Additional file 2D).
The T-cell subsets subjected to intracellular staining were stained according to previous studies [28, 29], with few modifications. Briefly, single-cell suspensions were treated with a mixture of 1 × cell stimulation cocktail (eBioscience, Thermo Fisher Scientific, Seoul, Korea) and 1 × brefeldin A (eBioscience, Thermo Fisher Scientific, Seoul, Korea) in RPMI growth media and incubated at 37 °C in a humidified chamber with 5% CO2 for 4–5 h. The cells were then stained with antibodies for various cell surface markers in cold FACS buffer for 30 min at 4 °C, properly washed twice with cold FACS buffer, and fixed with intracellular (IC) fixation buffer (eBioscience, Thermo Fisher Scientific, Seoul, Korea) at 4 °C for 30 min. For intracellular staining, the cells were washed twice with permeabilization buffer (200 μL/well) and stained with cytokine-specific antibodies in cold permeabilization buffer at 4 °C for 30 min. Subsequently, the cells were washed twice with permeabilization buffer. The gating strategy employed for obtaining various T-cell phenotypes after the gating of singlet lymphocytes is demonstrated in Additional file 2E.
A 100-μL suspension of the stained cell populations in FACS buffer was run on an Accuri C6 flow cytometer (BD Accuri™ C6 Plus, BD Biosciences, MD, USA). BD Accuri™ C6 Plus software version 1.0.23.1 (BD Biosciences, MD, USA) was used to analyse the data after setting compensation settings according to monocolour and isotype control stains. The data are presented as percentages of all the cell subsets.
Cytokine immunoassay
The cytokine levels in the sera and BALF of the uninfected and infected pigs at 3, 10 and 28 dpc were measured using a porcine-specific ProcartaPlex™ Multiplex Immunoassay (ThermoFisher Scientific, Vienna-1030, Austria) according to the manufacturer’s instructions. Magnetic microsphere technology based on porcine cytokine/chemokine antibody-immobilized magnetic beads was employed in the immunoassay for cytokine quantification [30]. The concentration of each cytokine was measured by running the samples on the Luminex® 200™ system (Luminex Corporation, Austin, TX, USA). Appropriate standards provided in the kit were utilized to determine the concentration of each cytokine. The machine was verified and calibrated using a Luminex® 100/200™ verification kit and a Luminex® 100/200™ calibration kit (Luminex Corporation, Austin, TX, USA) prior to use.
Data analysis
Graphical presentations of the data were prepared using GraphPad Prism 7.00 (GraphPad, San Diego, CA, USA), and the data were statistically analysed using SPSS Advanced Statistics 17.0 software (SPSS, Inc., Chicago, IL, USA). A nonparametric T-test (Mann–Whitney U test) was used to compare the viral loads in the lung tissues, the average daily weight gain (ADWG), the phenotypes of various cell subsets and the cytokine responses between two groups. The normalized dead CD163+ cells were analysed by repeated ANOVA (Tukey post hoc test) to determine the overall difference, and pairwise comparisons were also performed at different days post-challenge. Spearman rank correlation and linear regression were used to determine the associations between two parameters. Differences were considered statistically significant if p < 0.05 and are indicated by asterisks and different letters over the bars.