Cell line and viruses
Madin-Darby Canine Kidney (MDCK) cell lines used for this study were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and grown on Minimum Essential Medium (MEM) with 10% fetal bovine serum (FBS), 100 µg/ml streptomycin and 100 U/ml penicillin. The influenza A virus, A/WSN/1933 (H1N1), was obtained from Professor Man-Seong Park (Korea University, Seoul, Korea). The virus was amplified using specific-pathogen-free (SPF) embryonated eggs followed by infecting the MDCK cell lines. MDCK cells (2 x 105/well) were cultured in 6-well plates using MEM media containing 10% FBS at 37oC overnight in a CO2 incubator. After the overnight culture, the cells were washed with PBS, and each well was infected with the influenza A virus at MOI 0.01 in MEM media containing 1 µg/ml L-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin and then incubated at 37oC in CO2 incubator. After 1 h incubation, the supernatants were removed and then cultured for 3 days in MEM media containing 0.3% BSA at 37oC in a CO2 incubator. The virus culture supernatants were collected and centrifuged at 2,000 rpm for 10 min at 4oC to remove the cell debris. The quantification of the amplified viruses was performed by plaque assay. MDCK cells (6 x 105/well) were cultured in 6-well plates. The cells were cultured, washed with PBS, and infected with the amplified influenza A virus culture supernatants after a ten-fold serial dilution. After 1 h incubation, the supernatants were removed and overlaid with 2 ml of DMEM/F12 media containing 2 mM glutamine, 4% BSA, 10 mM HEPES, 2.5% sodium bicarbonate, 50 mg/ml DEAE dextran, 1 µg/ml L-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin, 100 U/ml penicillin, 100 mg/ml streptomycin and 0.6% immunodiffusion-grade agar. After 72 h incubation, the plates were stained with crystal violet (0.1% crystal violet in 20% methanol) for 1 h. The plaques were counted to determine the virus titers. The entire process for the virus preparation and cell culture procedure was conducted in a biosafety level 2 laboratory.
Eight-week-old female C57BL/6J mice were purchased from NARA Biotech, Inc. (Seoul, Korea), and C57BL/6J CD83 KO mice (B6.129S4-Cd83tm1Tft/J, JAX stock #017703) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were maintained at the Animal Center of Hallym University under specific pathogen-free conditions (20–25°C, 40–45% humidity, 12 h light/dark cycle, and food and water access, ad libitum). Mice experiments were performed in a biosafety level 2 facility in the Hallym Clinical and Translational Science Institute.
A/WSN/1933 virus inactivation was performed by 254 nm UV light exposure with 1,500 mW/s/cm2 UV for 15 min from a height of 5 cm. The virus inactivation was verified by a plaque assay with MDCK cells . The live A/WSN/1933 virus or UV-inactivated A/WSN/1933 virus was intraperitoneally infected at a dose of 5 × 106 pfu per mouse and CD83 expression in the peritoneal cavity, bone marrow and spleen cells was observed at 1 h, 3 h, 6 h, and 24 h after infection. Changes in lymphocyte population were observed at 5, 7 and 14 days after live A/WSN/1933 virus infection. The production of virus-specific antibody was measured at 14 days after live A/WSN/1933 virus infection.
C57BL/6J wild type or C57BL/6J CD83 KO mice were infected intraperitoneally with 5 × 106 pfu of A/WSN/1933 virus. After the virus infection, cells of the peritoneal cavity, splenocytes, and cells of the bone marrow were harvested in RPMI 1640 media containing 5% FBS. The cells were centrifuged at 1,200 rpm and 4°C for 5 minutes, and the pellets were treated with RBC lysis buffer (20 mM Tris HCl and 140 mM NH4Cl) for 5 min. After washing with RPMI media, total cell numbers of peritoneal cavity, spleen and bone marrow were counted and analyses of cell populations were performed as described previously (26). The cells were washed with FACS buffer (1% FBS in PBS) and transferred into Falcon tubes and then treated with 10 µg/ml anti-FcγRII/III antibody (Catalogue No: 553142, BD Biosciences, San Jose, CA, USA). To analyze lymphoid population, PerCP Cy5.5-conjugated anti-CD3 antibody (Catalog No: 551163, BD Biosciences), Pacific blue ef450-conjugated anti-CD4 antibody (Catalog No: 558107, BD Biosciences), FITC-conjugated anti-CD8 antibody (Catalog No: 553031, BD Biosciences), APC-Cy7-conjugated anti-CD19 antibody (Catalog No: 557655, BD Biosciences), PE-conjugated anti-CD23 antibody (Catalog No: 12-0232-81, eBioscience, San Diego, CA, USA), and APC-conjugated anti-CD83 antibody (Catalog No: 558208, BD Biosciences) were used. We pre-gated peritoneal cells, bone marrow cells, splenocytes (FSClowSSClow) for the enriched lymphoid population and then analyzed specific cell population with a FACSCantoTM II Flow Cytometry (BD Biosciences).
For the detection of A/WSN/1933 virus-specific IgG, the subclasses of IgG, ninety-six well Immuno plates (NuncTM, Roskilde, Denmark) were coated with the A/WSN/1933 virus. After overnight incubation at 4°C, the plates were washed three times with PBST (0.1% Tween-20 in PBS) and blocked with 1% BSA for 1 h and then loaded with three-fold dilutions of sera and peritoneal supernatants in PBST. After incubation at room temperature for 2 h, the plates were washed three times with PBST and goat anti-mouse IgG/IgG1/IgG2a/IgG2b/IgG3 antibodies labeled with Horseradish peroxidase (Southern Biotechnology Associates, Inc. Birmingham, AL, USA) with dilutions 1:500 were used to bind total IgG and each subclasses antibody. The plates were incubated for 1 h at room temperature, and then, TMB substrate solution was added (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA). The absorbance was measured by a Spectra Max 250 microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 450 nm
Results are shown as the mean ± standard deviation. The statistical significance of the differences between the two samples was evaluated using Student’s t-test with P < 0.05 as the threshold for statistical significance.
Mice experimental procedures were undertaken following the guidelines of Laboratory Animals of the National Veterinary Research and Quarantine Service of Korea. Animal use and related experimental procedures were approved by the Institutional Animal Care and Use Committee of Hallym University (Permit Number: Hallym R2/2018-25). Mice were anesthetized with 3% to 5% isoflurane (Pharmaceutical, Seoul, Korea) inhalation to minimize any pain. The mice were sacrificed by CO2 inhalation after the experiments and euthanized by CO2 inhalation if they lost 25% of their baseline adult body weight or if they revealed evidence of debilitation, pain or distress such as a hunched posture, rough hair coat, reduced food consumption, emaciation, inactivity, ambulation difficulty, and respiratory problems, and all efforts were made to limit suffering.