Animals and selection criteria
The study was performed as a prospective, in vitro, cohort study. The study protocol was approved by the Midwestern University Animal Care and Use Committee (protocol # 2932). Ten randomly selected dogs from the Arizona Humane Society were eligible for enrollment in this study. Inclusion criteria consisted of being housed in the shelter for ≥ 7 days and tested negative for Dirofilaria immitis antigen, and antibodies to Anaplasma phagocytophilum, Anaplasma platys, Ehrlichia canis, Ehrlichia ewingii, and Borrelia burgdorferi C6 peptide using a commercial ELISA-based kit (SNAP 4DX, Plus Test kit, IDEXX Laboratories Inc, Westbrook, ME). Exclusion criteria included detection of ³1 of the aforementioned pathogens. Furthermore, dogs were excluded if pregnant, lactating, had a surgical procedure performed, or had any illness since shelter admission. Furthermore, dogs were excluded if they had been administered medications, with the exception of routine parasitic prevention or fenbendazole.
Calcitriol (Sigma-Aldrich, St. Louis, MO) was dissolved in 75% ethanol (Sigma-Aldrich, St. Louis, MO) to make a stock solution of calcitriol at 24 nmol/mL and stored light protected at 4°C as previously described (11).
Blood sample collection and calcitriol treatment
A blood sample (8 mL) was collected from each dog via jugular venipuncture into tubes containing sodium heparin as an anticoagulant and processed within 1 h. Four mLs of blood was allocated into 2 separate 15 mL conical tubes for each dog. Blood samples in each conical tube were then diluted 1:2 with RPMI 1640 culture medium (Thermo Fisher Scientific, Carlsbad, CA) containing 200 U of penicillin/mL and 200 mg of streptomycin/mL. The blood-RPMI mixture was then incubated with calcitriol (final concentration, 10-7 M) or ethanol negative control substance (final concentration, 6 x 10-2 M for 24 h at 37°C in 5% CO2 in the dark as previously described (11).
Leukocyte cytokine production
After incubation with calcitriol or control, blood-RPMI mixture was transferred to 96-well plates and stimulated with lipopolysaccharide (LPS) from Escherichia coli O127:B8 (final concentration, 100 ng/mL, Sigma Aldrich, St Louis, MO), lipoteichoic acid (LTA) from Streptococcus faecalis (final concentration, 1 µg/mL, Sigma Aldrich, St Louis, MO,), or phosphate-buffered saline (PBS) control substance. Plates were incubated for 24 h at 37°C in 5% CO2 in the dark. Following incubation, plates were centrifuged (400 X g for 7 minutes) at 21°C as previously described (11). The supernatant was collected and stored at -80°C for batch analysis. For analysis, samples were thawed, and then TNF-α, IL-6, and IL-10 were measured in supernatant with a canine cytokine-specific multiplex bead-based assay (Milliplex MAP canine cytokine-chemokine panel, EMD Millipore Corp, Billerica, MA)(11). The median fluorescence intensity and cytokine concentration in each sample was measured in duplicate with appropriate controls and associated data analysis software (Milliplex Analyst version 5.1, EMD Millipore Corp, Billerica, MA). The lower limit of detection for TNF-α, IL-10 was 195 rg/mL and IL-6 was 48.8 rg/mL.
Phagocytosis of E. coli
Phagocytic function of GM was determined with the PhagoTest commercial test kit (Orpegen Pharma, Heidelberg, Germany), validated for use in canines, according to manufacturer’s instructions. Briefly, 100 mL of blood-RPMI mixture post 24 h of incubation with calcitriol or control was then incubated in a 37°C water bath for 10 minutes with 20 mL FITC-labeled, opsonized-E. coli bacteria or washing solution (negative control). The samples were placed on ice to arrest phagocytosis and 100 mL of quenching solution was added to remove the FITC-fluorescence of surface bound bacteria. The cells were washed, erythrocytes lysed, all cells were washed again, and 200 mL of DNA staining solution (R-phycoerythrin) was added to facilitate exclusion of aggregated artifacts of bacteria or cells.
E coli-induced oxidative burst
Oxidative burst function in GM was determined with a PhagoBurst kit, validated for use in canines, according to manufacturer’s instructions (Orpegen Pharma, Heidelberg, Germany). Briefly, 100 mL of blood-RPMI mixture post 24 h of incubation with calcitriol or control was then incubated with 20 mL of opsonized-E. coli or control solution for 10 minutes at 37°C in a water bath. Next, samples were incubated with 20 mL of dihydrorhodamine-123 as a fluorogenic substrate for oxygen intermediates for 10 minutes at 37°C in a water bath. After cessation of this reaction, erythrocytes were lysed, the cells were washed, and 200 mL of DNA staining solution (R-phycoerythrin) was added to facilitate exclusion of aggregated artifacts of bacteria or cells.
Flow cytometry was performed at the Midwestern University College of Veterinary Medicine Immunology Laboratory using a flow cytometer (Guava easyCyte HT, Luminex Corporation, Austin, TX) and associated data analysis software (GuavaSoft 3.2, Luminex Corporation, Austin, TX). A minimum of 20,000 events/sample were recorded. These events were then applied to a forward scatter versus side scatter plot to identify and gate GM populations concurrently (Fig. 1). For assessment of phagocytosis, data were recorded as the percentage of GM cells having internalized FITC-labeled E. coli as well as their mean fluorescent intensity (MFI), a method of quantifying the phagocytosed bacteria per cell. Data for assessment of oxidative burst were recorded as the percentage of GM cells having produced reactive oxygen metabolites and the MFI, the relative robustness of oxidative burst reaction produced per cell.
Statistical analysis was performed by commercial software (SigmaStat, Systat Software Inc). Normality was determined using the Shapiro-Wilk test. Normally distributed data was presented as mean and standard deviation (SD), while data that was not normally distributed was presented as median and interquartile range (IQR). For between-treatment (i.e., calcitriol or control) comparisons (factor 1) for leukocyte production of each TNF-α, IL-6, and IL-10 after exposure with LPS, LTA, or PBS (factor 2), a two-way repeated measures analysis of variance (ANOVA) was performed with post hoc Bonferroni t-test making pairwise multiple comparisons. Granulocyte/monocyte oxidative burst and phagocytic function data were compared between treatment types (i.e., calcitriol or control) using paired t-tests. When the measured TNF-α, IL-6, and IL-10 concentrations fell below the lower limit of detection data were recorded at the lower limit of detection for statistical purposes. A p-value of <0.05 was considered significant.