Vitamin D signaling increases nitric oxide and antioxidant defenses of bovine monocytes

Vitamin D contributes to multiple aspects of bovine immunity and is reported to decrease the effects of mastitis and metritis in dairy cows. We hypothesized that vitamin D signaling in bovine monocytes increases antioxidant responses as part of its immunomodulatory actions. Our objectives were to assess the effects of vitamin D on oxidant and antioxidant responses of bovine monocytes. Monocytes from peripheral blood of nonpregnant, lactating Holstein cows between 90 and 300 d in milk were used for in vitro cell culture experiments. To test the effects of vitamin D on reactive oxygen metabolites (dROM) and antioxidant potential (AOP), monocytes from 14 cows were cultured in replicates for 16 h with 25-hydroxyvitamin D₃ [25(OH)D₃, 0 or 75 ng/mL] in a factorial arrangement with lipopolysaccharide (LPS, 100 ng/mL) or interferon-γ (IFN-γ, 10 ng/mL) or with no stimulation. Data were analyzed by ANOVA for main effects of 25(OH)D₃, stimulant, and interactions between 25(OH)D₃ and stimulant. Significant interactions between 25(OH)D₃ and stimulant were observed for dROM and AOP of culture supernatants. In unstimulated cultures, 25(OH)D₃ tended to increase dROM, but the opposite was observed in stimulated cultures. In contrast, LPS and IFN-γ treatments alone decreased AOP of culture supernatants, but 25(OH)D₃ counteracted the decrease in AOP caused by IFN-γ. Abundances of transcripts of genes encoding antioxidant-related proteins were measured by quantitative PCR using RNA from monocytes from 4 cows treated with 25(OH)D₃ (0 or 75 ng/mL) in a factorial arrangement with increasing concentrations of LPS (0 to 1,000 ng/mL) or IFN-γ (0 to 10 ng/mL). Treatment with 25(OH)D₃ increased transcripts of genes encoding metallothionein 1A and metallothionein 2A in the presence of IFN-γ but not LPS. Furthermore, 25(OH)D₃ increased transcripts of genes encoding thioredoxin and thioredoxin reductase, but the effect of 25(OH)D₃ did not depend on IFN-γ or LPS stimulation. In conclusion, 25(OH)D₃ increased antioxidant capacity of IFN-γ-stimulated bovine monocytes, potentially by increasing metallothionein and thioredoxin activities in monocytes.