Short communication: Inhibition of DNA methyltransferase and histone deacetylase increases ß-defensin expression but not the effects of lipopolysaccharide or 1,25-dihydroxyvitamin D3 in bovine mammary epithelial cells.

Antimicrobial peptides are a common defense against bacterial infections in many species and a significant part of the innate immune response of the bovine mammary gland. The objective of this study was to investigate the influence of epigenetic factors on vitamin D and toll-like receptor-mediated induction of ß-defensins in mammary epithelial cells. Primary bovine mammary epithelial cells were treated with lipopolysaccharide (LPS, 0 or 100 ng/mL), 1,25-dihydroxyvitamin D3 [1,25(OH)2D3, 0 or 10 nM], and 5-aza-2'-deoxycytidine (5-Aza, inhibitor of DNA methyltransferase, 0 or 5 µM) or trichostatin A (TSA, inhibitor of histone deacetylase, 0 or 80 nM) in a factorial arrangement. Effects of treatments on ß-defensin gene expression along with genes for cytokines and enzymes known to be induced by LPS or 1,25(OH)2D3 were evaluated by quantitative PCR. The LPS treatment induced expression of ß-defensin (DEFB)3, DEFB5, DEFB7, DEFB10, enteric ß-defensin (EBD), lingual antimicrobial peptide (LAP), and tracheal antimicrobial peptide (TAP); whereas, the 1,25(OH)2D3 treatment increased DEFB5 and DEFB7 expression and decreased LAP. The 5-Aza treatment increased expression of DEFB3, DEFB5, DEFB10, EBD, LAP, and TAP in the presence and absence of LPS. The TSA treatment increased expression of DEFB3, DEFB4, DEFB5, DEFB7, and DEFB10 in the absence of LPS but decreased LPS-induced expression of and LAP and TAP. Together these results indicate that ß-defensin expression in bovine mammary epithelial cells is likely influenced by DNA methylation and histone acetylation. Investigation of environmental and nutritional factors that influence epigenetic control of ß-defensins in the mammary gland may be beneficial for improving resistance to intramammary infections.

Neutrophil ß-defensin gene expression of postpartum dairy cows is altered by prepartum dietary cation-anion difference.

The objective of this study was to evaluate expression of a cluster of genes encoding ß-defensin antimicrobial peptides in neutrophils of postpartum cows in relation to prepartum dietary cation-anion difference (DCAD), vitamin D, and postpartum disease. Pregnant dry Holstein cows (28 nulliparous and 51 parous) at 255 d gestation were blocked by parity and randomly assigned to 4 prepartum diets of positive (+130 mEq/kg) or negative (-130 mEq/kg) DCAD and either 3 mg vitamin D3 or 3 mg of 25-hydroxyvitamin D3 per 11 kg of dry matter/d. Treatment diets were fed from 255 d of gestation until calving. Peripheral blood neutrophils of 35 parous cows were collected at 0 and 3 d after calving and stimulated with 0 or 100 ng/mL of lipopolysaccharide (LPS). Furthermore, serum Ca and incidences of postpartum diseases were recorded for all cows. The mRNA transcripts of ß-defensin genes were quantified by real-time PCR, and data were analyzed with a general linear mixed model to test for fixed effects and interactions of day, level of DCAD, source of vitamin D, and incidence of disease. Effects of DCAD and vitamin D on neutrophil oxidative burst and phagocytosis were previously reported but were analyzed for effects of disease in the present study. Transcripts for DEFB1, DEFB3, DEFB4, DEFB5, DEFB7, DEFB10, and lingual antimicrobial peptide (LAP) in neutrophils were upregulated by LPS at 0 d but not at 3 d. Transcripts for DEFB4 and DEFB7 in LPS-stimulated neutrophils were greater in cows fed negative DCAD diets compared with positive DCAD. Source of vitamin D (vitamin D3 vs. 25-hydroxyvitamin D3) did not affect expression of ß-defensins in neutrophils. Cows with postpartum subclinical hypocalcemia (serum Ca <2.0 mM) had decreased DEFB3, DEFB4, DEFB6, DEFB7, DEFB10, and LAP expression in LPS-stimulated neutrophils compared with cows that did not experience subclinical hypocalcemia. Likewise, DEFB4, DEFB6, DEFB7, DEFB10, and LAP in LPS-stimulated neutrophils at 3 d postpartum were positively associated with serum Ca at 0 d postpartum. Transcripts for DEFB7, DEFB10 and LAP also were less abundant in neutrophils from cows with metritis compared with healthy cows. In conclusion, feeding a prepartum negative DCAD to improve postpartum serum Ca resulted in greater neutrophil ß-defensin expression, and greater neutrophil ß-defensin expression was positively associated with postpartum health.

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

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 D3 [25(OH)D3] would augment expression of vitamin D-associated genes. The objective of this experiment was to determine the effects of intramammary LPS and 25(OH)D3 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)D3, 5 µg of LPS, or a combination of 100 µg of 25(OH)D3 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)D3 treatments, treatment interactions, and simple effects of 25(OH)D3 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)D3, LPS, and LPS plus 25(OH)D3 glands, respectively, such that the interaction of LPS and 25(OH)D3 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)D3 and LPS treatments. The inducible nitric oxide synthase (iNOS), chemokine (C-C-motif) ligand 5 (CCL5), ß-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)D3, such that 25(OH)D3 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)D3. In conclusion, intramammary endotoxin challenge activates the vitamin D pathway in mammary macrophages and neutrophils, and intramammary 25(OH)D3 treatment alters the percentage of neutrophils and expression of immune genes in milk somatic cells.

Symposium review: Targeting antimicrobial defenses of the udder through an intrinsic cellular pathway.

The bovine innate immune system has a strong repertoire of antimicrobial defenses to rapidly attack infectious pathogens that evade physical barriers of the udder. Exploration of the intracrine vitamin D pathway of bovine macrophages has improved understanding of the signals that initiate antimicrobial defenses that protect the udder. In the intracrine vitamin D pathway, pathogen recognition receptors upregulate CYP27B1 mRNA that encodes for the enzyme that converts 25-hydroxyvitamin D [25(OH)D3] to the active vitamin D hormone, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The 1,25(OH)2D3, in turn, is generally known to increase antimicrobial activity and decrease inflammatory responses of immune cells. In cattle specifically, 1,25(OH)2D3 increases nitric oxide and ß-defensin antimicrobial responses of bovine monocytes. Immune activation of the intracrine vitamin D pathway, including induction of inducible nitric oxide synthase and ß-defensin gene expression by 1,25(OH)2D3, has been documented in the mammary glands of lactating dairy cows. Furthermore, intramammary 25(OH)D3 treatment decreased bacteria counts and indicators of mastitis severity in cows experimentally infected with Streptococcus uberis. We propose that vitamin D signaling in the udder contributes to containment of bacterial pathogens and inflammatory responses of the udder. Verification of vitamin D-mediated defenses of the mammary gland potentially provides a path for development of alternative solutions (i.e., nutritional, genetic, therapeutic) to increase mastitis resistance of dairy cows. Continued exploration of the intrinsic cellular pathways that specifically promote antimicrobial defenses of the udder, such as the vitamin D pathway, is needed to support mastitis control efforts for dairy cows.

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 D3 [25(OH)D3, 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)D3, stimulant, and interactions between 25(OH)D3 and stimulant. Significant interactions between 25(OH)D3 and stimulant were observed for dROM and AOP of culture supernatants. In unstimulated cultures, 25(OH)D3 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)D3 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)D3 (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)D3 increased transcripts of genes encoding metallothionein 1A and metallothionein 2A in the presence of IFN-γ but not LPS. Furthermore, 25(OH)D3 increased transcripts of genes encoding thioredoxin and thioredoxin reductase, but the effect of 25(OH)D3 did not depend on IFN-γ or LPS stimulation. In conclusion, 25(OH)D3 increased antioxidant capacity of IFN-γ-stimulated bovine monocytes, potentially by increasing metallothionein and thioredoxin activities in monocytes.

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

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 ß-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 ß-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 ß-defensin genes in vivo in cattle and affirms earlier reports that vitamin D supports innate immune functions of cattle.

Multiple ß-defensin genes are upregulated by the vitamin D pathway in cattle.

Experimental models of bacterial and viral infections in cattle have suggested vitamin D has a role in innate immunity of cattle. The intracrine vitamin D pathway of bovine macrophages, however, has only been shown to activate a nitric oxide-mediated defense mechanism, as opposed to cathelicidin and ß-defensin antimicrobial peptides in human macrophages. In this study we have investigated the actions of 1,25-dihydroxyvitamin D3 (1,25D) on a cluster of eleven bovine ß-defensin genes on the basis of RNAseq data indicating they were targets of 1,25D in cattle. Treatment of bovine monocyte cultures with 1,25D (10 nM, 18 h) in the absence and presence of LPS stimulation increased the expression of bovine ß-defensin 3 (BNBD3), BNBD4, BNBD6, BNBD7, and BNBD10 genes 5 to 10-fold compared to control (P<0.05). Treatment of lipopolysaccharide (LPS)-stimulated monocytes with 0-100 ng/mL 25-hydroxyvitamin D3 also increased BNBD3, BNBD4, BNBD7, and BNBD10 in a dose-dependent manner. Treatment of monocytes with the protein translation inhibitor, cycloheximide, however, blocked upregulation of the ß-defensins in response to 1,25D suggesting the ß-defensins in cattle are not direct targets of the vitamin D receptor. Furthermore, preliminary investigation of vitamin D's contribution to ß-defensin expression in vivo revealed that intramammary 1,25D treatment of lactating cows increased BNBD7 expression in mammary macrophages. In conclusion, our data demonstrate that multiple ß-defensin genes are upregulated by 1,25D in cattle, providing further indication that vitamin D contributes to bovine innate immunity.

Serotonin (5-HT) affects expression of liver metabolic enzymes and mammary gland glucose transporters during the transition from pregnancy to lactation.

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 5' 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.