Intramammary 25-hydroxyvitamin D3 treatment modulates innate immune responses to endotoxin-induced mastitis

Abstract

Vitamin D signaling in response to pathogen-associated molecules contributes to activation of innate immune responses of bovine monocytes. We hypothesized that lipopolysaccharide (LPS) of bacteria associated with mastitis in dairy cows activates the vitamin D pathway in innate immune cells of the udder and that increasing availability of 25-hydroxyvitamin D [25(OH)D] would augment expression of vitamin D-associated genes. The objective of this experiment was to determine the effects of intramammary LPS and 25(OH)D treatments on activation of the vitamin D pathway and innate immune responses of mammary immune cells. Individual mammary quarters of 5 lactating cows were treated with placebo control, 100 μg of 25(OH)D, 5 μg of LPS, or a combination of 100 μg of 25(OH)D and 5 μg of LPS. Somatic cells from milk were evaluated for percentage of neutrophil and macrophage populations and expression of genes associated with vitamin D metabolism and innate immunity. Data from samples collected from 4 to 12 h after challenge were analyzed for main effects of LPS and 25(OH)D treatments, treatment interactions, and simple effects of 25(OH)D treatment. Data from samples collected at the time of challenge were used as covariates. The percentages of neutrophils in milk at 8 h postchallenge were 58 ± 10, 82 ± 11, 89 ± 10, and 63 ± 10% of total cells in milk from control, 25(OH)D, LPS, and LPS plus 25(OH)D glands, respectively, such that the interaction of LPS and 25(OH)D was significant. Expression of the vitamin D 1α-hydroxylase (CYP27B1) and vitamin D receptor genes was upregulated by LPS treatment in total cells, macrophages, and neutrophils in milk. In addition, expression of the vitamin D 24-hydroxylase (CYP24A1) gene in milk somatic cells was upregulated by 25(OH)D and LPS treatments. The inducible nitric oxide synthase (iNOS), chemokine (C-C-motif) ligand 5 (CCL5), β-defensin 3 (DEFB3), DEFB7, and DEFB10 genes were upregulated by LPS treatment in total cells and neutrophils from milk. Expression of iNOS in milk somatic cells tended to be affected by the interaction between LPS and 25(OH)D, such that 25(OH)D tended to increase iNOS in the absence of LPS but not in the presence of LPS. Furthermore, expression of CCL5 in macrophages was downregulated by 25(OH)D. In conclusion, intramammary endotoxin challenge activates the vitamin D pathway in mammary macrophages and neutrophils, and intramammary 25(OH)D treatment alters the percentage of neutrophils and expression of immune genes in milk somatic cells.

Feeding supplemental 25-hydroxyvitamin D3 increases serum mineral concentrations and alters mammary immunity of lactating dairy cows

“…A key objective of the experiment was to investigate the effects of increased concentrations of 25(OH)D on mammary gland immunity and mammary immune response to bacterial challenge. Recent studies have provided strong evidence that vitamin D, acting through an intracrine signaling pathway of innate immune cells, activates innate defenses of the mammary gland (Merriman et al, 2017(Merriman et al, , 2018. Here, we found that supplementing 25(OH)D 3 lessened the severity of mastitis but increased milk leukocyte CD11b and CD62L protein expression and iNOS gene expression in response to S. uberis challenge.…”

“…Cells were incubated 20 min on ice with allophycocyanin (APC)-conjugated anti-CD14 (Tük4 clone, Life Technologies, Carlsbad, CA), fluorescein isothiocyanate (FITC)-conjugated mouse anti-bovine CD11b (CC126 clone, AbD Serotec, Raleigh, NC) and phycoerythrin (PE)-conjugated anti-CD62L (BAQ92A clone, Kingfisher Biotech Inc., St. Paul, MN) in 100 μL of PBS containing 0.5% BSA (Thermo Fisher) and 2 mM EDTA (Thermo Fisher). Cells were then labeled with a fluorescent viability dye (eFuor 450, Thermo Fisher) and analyzed on the bases of (1) size and granularity, (2) viability, (3) CD14 and CD11b expression, and (4) CD62L expression, as previously described (Merriman et al, 2018), using a Sony SH800 cell sorter equipped with 405-, 488-, and 633-nm lasers (Sony Biotechnology Inc., San Jose, CA). The CD14-positive cells were considered monocytes, whereas agranular, CD14-negative cells were considered lymphocytes.…”

1,25-hydroxyvitamin D3 decreases endoplasmic reticulum stress-induced inflammatory response in mammary epithelial cells

“…The present findings show that 1,25D 3 is effective in attenuating ER stress and the NF-κB-driven inflammatory response in MCF-7 cells. This indicates that attenuation of ER stress by 1,25D 3 in MECs may contribute to the recently observed inhibitory effect of intramammary treatment of dairy cows with 1,25D 3 on the inflammatory process associated with mastitis [6][7][8]. In addition, the observation that the expression of VDR decreased upon induction of ER stress and 1,25D 3 increased VDR expression in MCF-7 cells exposed to ER stress demonstrates that 1,25D 3 counter-regulates the inhibitory effect of ER stress on VDR expression in MECs.…”

Widespread basal cytochrome P450 expression in extrahepatic bovine tissues and isolated cells

“…Although extrahepatic expression was relatively low for many CYP450, local effects of CYP450 should not be overlooked. For example, infusion of LPS into the mammary gland results in isolated activation of CYP450 involved in the regulation of vitamin D without changes in activation of CYP450 noted outside of the mammary gland (Merriman et al, 2018).…”

“…The activity of CYP27B1 outside of the kidney may be critical for the immune-modulating activities of 1,25-(OH) 2 D 3 . Specifically in the mammary gland, several studies have noted the antibacterial and anti-inflammatory properties of vitamin D, as well as the importance of CYP27B1 upregulation in response to pathogen insult (Merriman et al, 2017;Merriman et al, 2018). With relatively high expression in lung and uterine tissues, which commonly face pathogen challenge, CYP27B1 may contribute to the antibacterial defenses of these organs as well.…”