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 D(3) [25(OH)D(3), 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)D(3), stimulant, and interactions between 25(OH)D(3) and stimulant. Significant interactions between 25(OH)D(3) and stimulant were observed for dROM and AOP of culture supernatants. In unstimulated cultures, 25(OH)D(3) 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)D(3) 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)D(3) (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)D(3) increased transcripts of genes encoding metallothionein 1A and metallothionein 2A in the presence of IFN-γ but not LPS. Furthermore, 25(OH)D(3) increased transcripts of genes encoding thioredoxin and thioredoxin reductase, but the effect of 25(OH)D(3) did not depend on IFN-γ or LPS stimulation. In conclusion, 25(OH)D(3) increased antioxidant capacity of IFN-γ–stimulated bovine monocytes, potentially by increasing metallothionein and thioredoxin activities in monocytes.