Evaluation of the Effects of Vitamin D and Toll-Like Receptor Signaling Pathways on Expression of Antibacterial β-Defensin Genes in Bovine Neutrophils and Mammary Epithelial Cells
Bacterial infection in the udder stimulates synthesis of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) from 25-dihydroxyvitamin D3 in macrophages that are in the mammary glands. The 1,25(OH)2D3, along with toll-like receptor (TLR) recognition of pathogen associated molecules, enhances the expression of β-defensin 3 (DEFB3), DEFB6, DEFB7, and DEFB10 genes in bovine macrophages. The β-defensin genes encode for small cationic peptides that have potent bactericidal and immunomodulatory activity. Neutrophils and mammary epithelial cells (MEC) are additional sources of β-defensin peptides in the udder. It was hypothesized that 1,25(OH)2D3 and TLR agonists also would promote expression of the β-defensins in neutrophils and MEC. Therefore, the objective of this study was to determine the contribution of vitamin D and TLR signaling pathways on expression of DEFB3, DEFB4, DEFB6, DEFB7, and DEFB10 genes in bovine neutrophils and MEC. Peripheral blood neutrophils from cattle and primary bovine MEC cultures were treated with 0 or 1 μg/mL lipopolysaccharide (LPS) in combination with 0 or 10 nM 1,25(OH)2D3. The mRNA transcripts of DEFB3, DEFB4, DEFB6, DEFB7, and DEFB10 genes were quantified by real-time PCR. The threshold cycle (Ct) for each gene was normalized to ribosomal protein S9 transcript abundance and the normalized Ct values for each treatment were analyzed with a general linear model to test for effects of 1,25(OH)2D3 and LPS treatments. In contrast to macrophages, 1,25(OH)2D3 did not upregulate expression of the DEFB3, DEFB6, DEFB7, or DEFB10 genes in either non-stimulated or LPS-stimulated neutrophils or MEC (P>0.05). However, the 1,25(OH)2D3 treatment increased DEFB4 gene expression 5±1-fold (P<0.05) in MEC not treated with LPS, and 3±1-fold (P<0.05) in MEC treated with LPS. Furthermore, LPS combined with 1,25(OH)2D3 upregulated DEFB4 30-fold (P<0.05) compared to MEC cultured in the absence of LPS and 1,25(OH)2D3. The LPS treatment alone upregulated DEFB3, DEFB4, and DEFB7 in MEC (P<0.05; 3±1, 11±3, and 8±3 fold change ± SE, respectively), and DEFB3, DEFB4, DEFB6, DEFB7, and DEFB10 in neutrophils (P<0.05; 121±44, 10±7, 144±85, 112±51, and 56±22, fold change ± SE, respectively). In conclusion, 1,25(OH)2D3 does not enhance β-defensin gene expression in bovine MEC or neutrophils as it does in macrophages. However, LPS does strongly enhance several of the β-defensins in MEC and neutrophils. Therefore, activation of the TLR pathway in neutrophils and MEC, combined with activation of the vitamin D pathway in macrophages, may serve to boost the innate defense system of the udder.
Keywords: Vitamin D, Innate Immunity, Mammary