Test system
Female C57BL/6 mice, approximately 6–9 weeks of age and weighing 17–20 g, were purchased from Charles River Laboratories. The mice were housed 2–3 per cage in shoebox cages in a room with temperature maintained between 64 and 80 °F (18-29 °C) and with a 12-h light/12-h dark photoperiod. The animals had ad libitum access to Harlan Teklad Global Rodent Diet 2018 and deep well water. All study procedures were reviewed and approved by Sinclair Research Center’s Institutional Animal Care and Use Committee. Housing and animal care conformed to the guidelines of the Guide for the Care and Use of Laboratory Animals, 8th edition published by the U.S. National Institutes of Health and to applicable institutional standard operating procedures. Euthanasias were performed in accordance with the American Veterinary Medical Association’s published guidelines [
3].
After being acclimated for 3 days, mice were randomized into groups with 6 mice in the untreated group, 32 mice in a group treated with LPS, and 32 mice in a group treated with LPS plus DEX. Identification of each animal was maintained using ear notches and cage cards.
Methyl cellulose, DEX, and LPS were obtained from Sigma-Aldrich (St Louis, Missouri). Methyl cellulose was dissolved in sterile water (Hospira, Lake Forest, Illinois) overnight to form a 0.5% solution for use as the vehicle. DEX was suspended overnight in 0.5% methyl cellulose at a concentration of 0.5 mg/mL and then sonicated briefly before dosing. LPS was prepared the day before dosing in 0.9% saline for injection (Hospira) at a concentration of 0.04 mg/mL.
Mice in the untreated group were bled for plasma without any treatment. Mice in the LPS treatment group were administered 0.5% methyl cellulose at 10 mL/kg via oral gavage, and then were treated 1.5 h later with 0.2 mg/kg LPS intravenously (IV) at a volume of 5 mL/kg. The LPS plus DEX groups were administered 5 mg/kg dexamethasone via oral gavage, and then underwent the same IV LPS treatment as above 1.5 h later. Six to eight mice from each treatment group were bled for plasma at 0.5, 1, 2, 4, and 6 h following LPS challenge.
Plasma preparation and analysis
All blood samples were collected into K2EDTA tubes (0.5 mL, Greiner Bio-One North America, Inc. Monroe, North Carolina). Filled tubes were placed on wet ice and were processed within 30 min after blood collections. The samples were centrifuged at 3000 rpm for 15 min at 4 °C; plasma was then drawn off and placed into separate vials. Plasma samples were separated into two sets and placed on dry ice and stored at −70 °C before being analyzed for cytokine profiles.
One set of plasma samples were shipped on dry ice to Myriad RBM, Inc. (Austin, TX) for cytokine profiling with Mouse Cytokine Panels A & B (4-h time point) and Rodent MAP V3.0 Antigen (0.5-, 1, 2, and 6-h time points) assays (based on a Multiplexed Luminex Platform).
Seven cytokines (IL-2, IFN-γ, TNFα, IL-4, IL-6, IL-17A, and IL-10) were analyzed at the 2- and 4- h time points in the second set of collected plasma samples with a cytometric bead array (CBA) mouse Th1/Th2/Th17 cytokine kit (BD Biosciences) on a BD Accuri C6 flow cytometer. The CBA Mouse Th1/Th2/Th17 Cytokine Kit Manual (BD Biosciences) was followed for the assay procedure. Plasma samples were thawed at room temperature and then placed on wet ice for duration of analysis. One vial of mixed standards was freshly reconstituted in 2.0 mL of assay diluent, and then was serial diluted. The concentrations of standards for each cytokine were 0, 20, 40, 80, 156, 312.5, 625, 1250, 2500, and 5000 pg/mL. Seven types of cytokine capture beads were freshly mixed in equal amounts (10 μL bead per assay tube) in a master tube. To perform the assay, 50 μL of the mixed beads were incubated with 50 μL of standards or samples along with 50 μL of Phycoerythrin (PE) Detection Reagent in a MultiScreen filter plate (1.2 μm pore size, EMD Millipore, Darmstadt, Germany) at room temperature for 2 h. At the end of incubation, the plate was drained on a vacuum manifold. The beads in each of the individual wells of the plate were resuspended in 120 μL of wash buffer, and were then analyzed on the BD Accuri C6 flow cytometer. The seven distinct fluorescence beads were sorted with fluorescence signals captured in FL4 channel. PE intensity of individual beads was captured in FL2 channel. Approximately 200 events for each bead group were acquired (based on experience in generating data in previous experiments). The acquired data were subsequently analyzed for individual cytokine concentrations in each sample using the FCAP Array software (BD Biosciences).
Results for the 7 cytokines IL-2, IFN-γ, TNFα, IL-4, IL-6, IL-17A, and IL-10 were compared between BD Biosciences and Myriad RBM assays. Concentrations for individual cytokines were expressed as mean ± standard deviation. Effects of DEX on LPS-induced plasma cytokine changes were evaluated with a two-way student t-test.