Intramammary 1,25-dihydroxyvitamin D3 treatment increases expression of host-defense genes in mammary immune cells of lactating dairy cattle

•Mammary glands of dairy cattle were injected with 10μg 1,25-dihydroxyvitamin D3.•A single injection increased expression of iNOS and DEFB7 genes within 4h.•Repeated injections of cows with subclinical mastitis increased expression of iNOS, DEFB4, and DEFB7 genes in cells of infected glands. Bacterial infection of the mammary gland activates an intracrine vitamin D pathway in macrophages of dairy cows. The active hormone of the vitamin D pathway, 1,25-dihydroxyvitamin D3 (1,25D), stimulates nitric oxide and β-defensin responses in bovine monocyte cultures, but the effect of 1,25D on innate immune genes in the mammary gland remained unknown. Therefore, the objective of this study was to determine the effects intramammary 1,25D treatment on expression of vitamin D associated host-defenses of the bovine mammary gland. Intramammary treatment of normal, healthy mammary glands of lactating dairy cows (n=14) with 10μg 1,25D increased inducible nitric oxide synthase (iNOS) and β-defensin 7 (DEFB7) gene expression in total milk somatic cells more than two-fold relative to placebo-treated glands within 8h after treatment. The vitamin D 24-hydroxylase gene (CYP24A1) also was increased nearly 100-fold in 1,25D-treated glands within 4h after treatment but was not affected in placebo-treated glands. Both macrophages and neutrophils isolated from milk had increased CYP24A1 expression in response to 1,25D treatment but only macrophages had increased iNOS expression. Repeated intramammary 1,25D treatment, every 12h for 48h, of infected mammary glands of cows diagnosed with subclinical mastitis resulted in increased expression of CYP24A1, DEFB4, DEFB7 and iNOS genes compared to placebo-treated glands. The 1,25D treatment resulted in elevated serum 1,25D concentrations (55 vs 33pg/mL) compared to placebo but it did not change serum calcium concentrations or bacteria counts in milk of infected mammary glands. In conclusion, 1,25D upregulates iNOS and β-defensin genes in vivo in cattle and affirms earlier reports that vitamin D supports innate immune functions of cattle.