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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.

The aim of this experiment was to demonstrate the ability of feeding serotonin (5-HT; 5-hydroxytryptamine) precursors to increase 5-HT production during the transition from pregnancy to lactation and the effects this has on maternal energy metabolism in the liver and mammary gland. Pregnant rats (n = 45) were fed one of three diets: I) control (CON), II) CON supplemented with 0.2% 5-hydroxytryptophan (5-HTP) or III) CON supplemented with 1.35% L-tryptophan (L-TRP), beginning on d13 of pregnancy through d9 of lactation (d9). Serum (pre and post-partum), milk (daily), liver and mammary gland tissue (d9) were collected. Serum 5-HT was increased in the 5-HTP fed dams beginning on d20 of gestation and remained elevated through d9, while it was only increased on d9 in the L-TRP fed dams. 5-HT levels were increased in mammary gland and liver of both groups. Additionally, 5-HTP fed dams had serum and milk glucose levels similar to the CON, while L-TRP had decreased serum (d9) and milk glucose (all dates evaluated). Feeding 5-HTP resulted in increased mRNA expression of key gluconeogenic and glycolytic enzymes in liver and glucose transporters 1 and 8 (GLUT-1, -8) in the mammary gland. We demonstrated the location of GLUT-8 in the mammary gland both in the epithelial and vascular endothelial cells. Finally, phosphorylated 59 AMP-activated protein kinase (pAMPK), a known regulator of intracellular energy status, was elevated in mammary glands of 5-HTP fed dams. Our results suggest that increasing 5-HT production during the transition from pregnancy to lactation increases mRNA expression of enzymes involved in energy metabolism in the liver, and mRNA abundance and distribution of glucose transporters within the mammary gland. This suggests the possibility that 5-HT may be involved in regulating energy metabolism during the transition from pregnancy to lactation.