Comparison between conjugated linoleic acid and essential fatty acids in preventing oxidative stress in bovine mammary epithelial cells

Some in vitro and in vivo studies have demonstrated protective effects of conjugated linoleic acid (CLA) isomers against oxidative stress and lipid peroxidation. However, only a few and conflicting studies have been conducted showing the antioxidant potential of essential fatty acids. The objectives of the study were to compare the effects of CLA to other essential fatty acids on the thiol redox status of bovine mammary epithelia cells (BME-UV1) and their protective role against oxidative damage on the mammary gland by an in vitro study. The BME-UV1 cells were treated with complete medium containing 50 μM of cis-9,trans-11 CLA, trans-10,cis-12 CLA, α-linolenic acid, γ-linolenic acid, and linoleic acid. To assess the cellular antioxidant response, glutathione, NADPH, and γ-glutamyl-cysteine ligase activity were measured 48 h after addition of fatty acids (FA). Intracellular reactive oxygen species and malondialdehyde production were also assessed in cells supplemented with FA. Reactive oxygen species production after 3 h of H₂O₂ exposure was assessed to evaluate and to compare the potential protection of different FA against H₂O₂-induced oxidative stress. All FA treatments induced an intracellular GSH increase, matched by high concentrations of NADPH and an increase of γ-glutamyl-cysteine ligase activity. Cells supplemented with FA showed a reduction in intracellular malondialdehyde levels. In particular, CLA isomers and linoleic acid supplementation showed a better antioxidant cellular response against oxidative damage induced by H₂O₂ compared with other FA.     

Staphylococcus aureus induces autophagy in bovine mammary epithelial cells and the formation of autophagosomes facilitates intracellular replication of Staph. aureus

Staphylococcus aureus is an important pathogen causing chronic and subclinical mastitis of cows. Autophagy is an important regulatory mechanism that participates in the elimination of invading pathogenic organisms. Here, we hypothesize that autophagy is involved in the process of Staph. aureus survival in bovine mammary epithelial cells (BMEC). In this study, we detected the expression of autophagy-related proteins during infection and assessed the effect of autophagosome formation and degradation on the proliferation of intracellular Staph. aureus. Infection with Staph. aureus increased the protein expression of microtubule-associated protein 1 light chain 3-II (MAP1LC3, also called LC3-II) and sequestosome-1 (SQSTM1, also called p62) in BMEC. After infection, the formation of the autophagosomes increased but the autophagosomes and lysosomes could not fuse normally to form autolysosomes. When the formation of the autophagosomes was enhanced or the degradation of the autolysosomes was inhibited, the number of Staph. aureus in the BMEC increased. However, the intracellular proliferation of Staph. aureus was slowed when formation of autophagosomes was inhibited. Therefore, autophagy was induced in BMEC challenged by Staph. aureus but the autophagic flux was obstructed. Inhibiting the formation of autophagosomes in BMEC facilitated the clearance of intracellular Staph. aureus, which may offer a new strategy for the treatment of mastitis in cows.     

Zearalenone induces apoptosis in bovine mammary epithelial cells by activating endoplasmic reticulum stress

Zearalenone (ZEA) is a common mycotoxin produced by fungi within the genus Fusarium. However, few studies have examined the direct effects of the toxin on the mammary glands. In the present study, the effects of ZEA treatment on bovine mammary epithelial cells (MAC-T) from dairy cows were investigated. The cells were treated with different concentrations of ZEA to evaluate the effect of the toxin on cell viability, intracellular reactive oxygen species (ROS) concentrations, mitochondrial membrane potential, endoplasmic reticulum (ER) stress, and the expression of apoptosis-related genes. The results indicated that different concentrations (5, 10, 15, 20, 25, 30, 50, 60, or 100 μM) of ZEA were able to inhibit growth of MAC-T cells. After exposing the MAC-T cells to 30 μM ZEA, compared with the control group, ROS levels increased, mitochondrial membrane potential decreased, and mRNA expression of the ER-specific stress-related genes GRP78, HSP70, ATF6, EIF2A, ASK1, and CHOP was upregulated in the ZEA-treated group. Further, we analyzed the increase in apoptotic rate by flow cytometry. At the mRNA level, compared with the control group, the expression of the apoptosis-promoting gene BAX was increased in the ZEA-treated group, the expression of the inhibitory gene BCL2 decreased, and the expression of the gene CASP3 increased. We observed a significant increase in caspase-3 activity in ZEA-treated MAC-T cells. Furthermore, the apoptotic rate of the cells in the ZEA group treated with 4-phenylbutyric acid (ER stress inhibitor) decreased and the mRNA expression levels of ER stress markers GRP78 and CHOP decreased. Compared with the ZEA treatment group, the mRNA expression level of the apoptosis-related gene BAX was decreased and the expression level of BCL2 was increased in the ZEA + 4-phenylbutyric acid cotreatment group. These findings indicate that ZEA-induced ER stress increases apoptosis in MAC-T cells. The treatment of MAC-T cells with ZEA reduced cell viability, increased ROS content, decreased mitochondrial membrane potential, increased ER stress marker expression, and induced apoptosis.     

Selenomethionine stimulates expression of glutathione peroxidase 1 and 3 and growth of bovine mammary epithelial cells in primary culture

This study examined the localization of cellular glutathione peroxidase (GPx1) and extracellular glutathione peroxidase (GPx3) in lactating mammary tissue and in primary cultures of bovine mammary epithelial cells (BMEC). The effect of selenium as selenomethionine (SeMet) on the growth and viability of BMEC and GPx protein expression and activity were also studied. Single mammary epithelial cells were recovered by serial collagenase/hyaluronidase digestion from lactating bovine mammary tissue and cultured in a low-serum collagen gel system enriched with lactogenic hormones and 0, 10, 20, or 50 nM SeMet. Positive immunostaining with anti-cytokeratin and bovine anti-casein confirmed the epithelial nature and differentiated state of BMEC. Addition of SeMet to media facilitated rapid confluence of BMEC and formation of dome structures. Immunohistochemical and immunocytochemical staining revealed that both GPx1 and GPx3 are synthesized by BMEC and localized in the cytoplasm and nucleus. Up to 50 nM SeMet linearly increased BMEC number and viability over 5 d of culture. Bovine mammary epithelial cells cultured in SeMet-supplemented medium also exhibited markedly elevated GPx activity and linear increases in abundance of GPx1 and GPx3 proteins. It is apparent that SeMet degradation to release Se for synthesis of selenoproteins is carried out by BMEC. Results indicate that bovine mammary epithelial cells express GPx1 and GPx3 in vivo and in vitro; SeMet enhances expression of these selenoproteins in vitro and the growth and viability of BMEC.     

Evidence for a local effect of leptin in bovine mammary gland

On average, high-energy diets promoting body growth rates above 1 kg/d before puberty impair mammary development by 15 to 20% in cattle. We hypothesized that leptin, a protein produced by adipocytes, mediates the inhibitory effect of high-energy diets on mammary development. Therefore, our objectives were to determine the effect of leptin on mammary epithelial cell proliferation, and the distribution of mRNA for two leptin receptor isoforms in prepubertal bovine mammary glands and other peripheral tissues. Addition of leptin to culture media containing either 5 ng/ml of insulin-like growth factor-I (IGF-I) or 1% fetal bovine serum decreased DNA synthesis of a bovine mammary epithelial cell line (MAC-T) in a dose-dependent manner. The minimal doses of leptin that decreased IGF-I- and fetal bovine serum-stimulated cell proliferation were 64 and 1 ng/ml, respectively. In addition, we determined that MAC-T cells and isolated bovine mammary epithelial cells express the long form of leptin receptor (Ob-Rb) mRNA. Ob-Rb mRNA was detected in all bovine tissues examined. In contrast with reports on other species, mRNA expression of the short form of leptin receptor (Ob-Ra) was detected only in bovine liver, pituitary body, and spleen. These results support the concept that leptin mediates the inhibitory effect of high-energy diets on mammary development.     

Bovine leukemia virus alters growth properties and casein synthesis in mammary epithelial cells

Bovine leukemia virus (BLV) is widespread in US dairy herds, yet only about 1% of infected cattle develop bovine leukosis and are culled from the herd. A major concern is whether BLV infection of dairy cows alters milk yield. Although several studies have examined the effect of BLV on milk production in vivo, the results were inconclusive. No in vitro studies have been done. The discovery of BLV in mammary epithelial cells (MEC) of infected cows raises the possibility that the virus could affect these cells directly. The purpose of this study was to use an in vitro system to determine if BLV could alter milk yield by altering cell number and/or milk production per cell. A short-term cell line established from the MEC of a BLV-negative cow, and a proven casein-producer mouse cell line, Comma D, were stably transfected with a plasmid containing the entire BLV genome. Untransfected parental lines served as negative controls. The BLV-containing bovine MEC line has a reduced population-doubling time, higher saturation density, and increased longevity. The Comma D line is an already-transformed cell line, and growth properties did not change after transfection with BLV. Under appropriate differentiation conditions, both the bovine and mouse MEC transfected with BLV displayed decreased casein production and mRNA synthesis compared with control cell lines without BLV. Our results suggest that effects of BLV infection on milk production may not be related solely to overall animal health but may also be mediated directly at a cellular level.     

miR-27a controls triacylglycerol synthesis in bovine mammary epithelial cells by targeting peroxisome proliferator-activated receptor gamma

Growing evidence has revealed that microRNA are central elements in milk fat synthesis in mammary epithelial cells. A negative regulator of adipocyte fat synthesis, miR-27a has been reported to be involved in the regulation of milk fat synthesis in goat mammary epithelial cells; however, the regulatory role of miR-27a in bovine milk fat synthesis remains unclear. In the present study, primary bovine mammary epithelial cells (BMEC) were harvested from mid-lactation cows and cultured in Dulbecco's modified Eagle's medium/F-12 medium with 10% fetal bovine serum, 5 μg/mL of insulin, 1 μg/mL of hydrocortisone, 2 μg/mL of prolactin, 1 μg/mL of progesterone, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. We found that the overexpression of miR-27a significantly suppressed lipid droplet formation and decreased the cellular triacylglycerol (TAG) levels, whereas inhibition of miR-27a resulted in a greater lipid droplet formation and TAG accumulation in BMEC. Meanwhile, overexpression of miR-27a inhibited mRNA expression of peroxisome proliferator-activated receptor gamma (PPARG), CCAAT/enhancer-binding protein beta (C/EBPβ), perilipin 2 (PLIN2), and fatty acid binding protein 3 (FABP3), whereas miR-27a downregulation increased PPARG, C/EBPβ, FABP3, and CCAAT enhancer binding protein alpha (C/EBPα) mRNA expression. Furthermore, Western blot analysis revealed the protein level of PPARG in miR-27a mimic and inhibitor transfection groups to be consistent with the mRNA expression response. Moreover, luciferase reporter assays verified that PPARG was the direct target of miR-27a. In summary, these results indicate that miR-27a has the ability to control TAG synthesis in BMEC via targeting PPARG, suggesting that miR-27a could potentially be used to improve beneficial milk components in dairy cows.     

Bovine miR-146a regulates inflammatory cytokines of bovine mammary epithelial cells via targeting the TRAF6 gene

It has been reported previously that bovine miR-146a (bta-miR-146a) is significantly differentially expressed in mammary glands infected with mastitis, compared with healthy udders. This suggests that bta-miR-146a plays an important role in the regulation of mammary inflammation. However, the specifics of this function have yet to be elucidated. Bovine mammary epithelial cells (bMEC) represent the first line of defense against pathogens and have important roles in initiating and regulating inflammatory responses and innate immunity during infection. In this study, a double luciferase reporter assay was used to confirm that bta-miR-146a directly targets the 3′ UTR of the tumor-necrosis factor receptor-associated factor 6 (TRAF6) gene. To elucidate the role of bta-miR-146a in innate immune responses, either a mimic or inhibitor of bta-miR-146a was transfected into bMEC stimulated with lipopolysaccharide, which activates the innate immune response through the toll-like receptor (TLR) 4/nuclear factor (NF)-κB signaling pathway. Forty-eight hours posttransfection, quantitative real-time PCR and Western blots were used to detect the expressions of the related genes and proteins, respectively. An ELISA was used to measure the quantity of inflammatory factors in culture supernatants. The results showed that bta-miR-146a significantly inhibits both mRNA and protein expression levels of bovine TRAF6, and ultimately suppresses downstream expression of NF-κB mRNA and protein. As a result, production of NF-κB-dependent inflammatory mediators such as tumor necrosis factor α, IL-6, and IL-8 are suppressed following lipopolysaccharide stimulation of bMEC. Thus, we concluded that bta-miR-146a acts as a negative feedback regulator of bovine inflammation and innate immunity through downregulation of the TLR4/TRAF6/NF-κB pathway. This study presents a potential regulatory mechanism of bta-miR-146a on immune responses in bovine mammary infection and may provide a potential therapeutic target for mastitis.     

Effects of trans fatty acids on markers of inflammation in bovine mammary epithelial cells

Trans fatty acids (tFA) contribute to inflammation. The objective was to investigate the effects of tFA on mRNA expression of proinflammatory markers in cultured bovine mammary epithelial cells (MAC-T cell line). Bovine mammary epithelial cells were grown in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum. Cells were then subcultured in a medium lacking fetal bovine serum, to which incremental concentrations (up to 90 μM) of elaidic acid (trans-9 C18:1) or linoleidic acid (trans-9, trans-12 C18:2) were added. Bovine serum albumin (fatty acid-free) solutions were added and cells were collected at specific time points over 48 h. Then, RNA was extracted and converted to complementary DNA for quantitative real-time PCR analysis of proinflammatory gene expression. Presence of elaidic acid caused increases in mRNA expression of interleukin (IL)-1β (3.4-fold; dose-independently over a 6-h period) and intercellular adhesion molecule (ICAM)-1 (up to 1.4-fold) relative to that for cells treated with no tFA, whereas expression of IL-6 and IL-8 was reduced 0.75- and 0.85-fold, respectively. Presence of linoleidic acid reduced mRNA expression of IL-6 and IL-8 relative to that for control (0.95- and 0.87-fold, respectively). Trans mono- and dienoic fatty acids upregulated mRNA expression of IL-1β and ICAM-1, whereas expression of IL-6 and IL-8 was downregulated in MAC-T cells. Because these genes are ultimately involved in inflammation, elaidic or linoleidic acid, either directly fed or formed in the rumen during biohydrogenation, may alter the risk for mastitis in vivo.     

Hepatocyte growth factor exerts multiple biological functions on bovine mammary epithelial cells

The met proto-oncogene product Met is a member of the family of tyrosine kinase growth factor receptors, and hepatocyte growth factor/scatter factor (HGF/SF) has been identified as its only ligand. Bovine Met and HGF/SF have been recently cloned and their expression has been characterized in the mammary gland, but no data regarding the biological effects of this ligand/receptor couple in bovine mammary cells are yet available. We examined the role of HGF/SF and its receptor in a bovine mammary epithelial cell line (BME-UV). Expression of Met at the mRNA level in BME-UV mammary epithelial cells evaluated by real-time PCR was similar to the expression in MDCK cells, a widely used model for Met biology. Met expression in BME-UV at the protein level was confirmed by western blot. The analysis of some signal transductional pathways downstream from the Met receptor revealed that HGF/SF addition to BME-UV cells induced activation of the extracellular signal-regulated kinase ½ proliferative pathway and the Akt antiapoptotic pathway. The BME-UV cells treated with HGF responded with increased proliferation, cell scatter, and motility. Met activation by HGF induced degradation of the extracellular matrix and migration through matrigel coated transwells. Moreover, BME-UV cells included in a 3-dimensional matrix of collagen and treated with HGF developed tubular structures, reminiscent of the mammary gland ducts. These data indicate that HGF and Met might be important regulators of mammary gland growth, morphogenesis, and development in the bovine.     

Insulin stimulates glucose uptake via a phosphatidylinositide 3-kinase-linked signaling pathway in bovine mammary epithelial cells

The aim of this study was to investigate the effects of insulin on glucose uptake in lactating bovine mammary epithelial cells (BMEC). Primary BMEC were cultured in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 and treated with different levels of insulin (0, 5, 50, and 500 ng/mL) for 48 h after a 24-h starvation without fetal calf serum. Compared with the control cells (0 ng of insulin/mL), cell proliferation was enhanced by insulin treatment at all tested levels. Insulin significantly increased glucose uptake at a concentration of 500 ng/mL. In addition, the protein synthesis inhibitor cycloheximide (0.5 mg/mL) counteracted the insulin-elevated glucose uptake, thereby suggesting that newly synthesized transporter protein might take part in the insulin-induced glucose uptake. Furthermore, pretreatment of the cells with SB203580, an inhibitor of p38 mitogen-activated protein kinase, did not influence the insulin-induced glucose uptake, but LY294002, a specific inhibitor of phosphatidylinositide 3-kinase, significantly reduced the insulin-stimulated glucose uptake. These results indicated that insulin-induced glucose uptake in BMEC may involve the phosphatidylinositide 3-kinase- but not mitogen-activated protein kinase-mediated signaling pathways.     

Selenomethionine increases proliferation and reduces apoptosis in bovine mammary epithelial cells under oxidative stress

The decline in mammary epithelial cell number as lactation progresses may be due, in part, to oxidative stress. Selenium is an integral component of several antioxidant enzymes. The present study was conducted to examine the effect of oxidative stress and selenomethionine (SeMet) on morphology, viability, apoptosis, and proliferation of bovine mammary epithelial cells (BMEC) in primary culture. Cells were isolated from mammary glands of lactating dairy cows and grown for 3 d in a low-serum gel system containing lactogenic hormones and 0 or 100 μM H₂O₂ with 0, 10, 20, or 50 nM SeMet. Hydrogen peroxide stress increased intracellular H₂O₂ to 3 times control concentrations and induced a loss of cuboidal morphology, cell-cell contact, and viability of BMEC by 25%. Apoptotic cell number more than doubled during oxidative stress, but proliferating cell number was not affected. Supplementation with SeMet increased glutathione peroxidase activity 2-fold and restored intracellular H₂O₂ to control levels with a concomitant return of morphology and viability to normal. Apoptotic BMEC number was decreased 76% below control levels by SeMet and proliferating cell number was increased 4.2-fold. These findings suggest that SeMet modulated apoptosis and proliferation independently of a selenoprotein-mediated reduction of H₂O₂. In conclusion, SeMet supplementation protects BMEC from H₂O₂-induced apoptosis and increased proliferation and cell viability under conditions of oxidative stress.     

Investigation of the pathophysiology of bacterial mastitis using precision-cut bovine udder slices

Mastitis in cattle is a major health problem as well as incurring high costs for the dairy industry. To assess the suitability of precision-cut bovine udder slices (PCBUS) for bovine mastitis studies, we infected PCBUS with 2 different Staphylococcus aureus strains. Accordingly, we investigated both the tissue response to infection based on immune mediators at the mRNA and protein levels and the invasion of bacteria within the tissue. The studied proteins represent immune mediators of early inflammation [IL-1β, tumor necrosis factor-α (TNF-α), prostaglandin E₂ (PGE₂)] and showed a time-dependent increase in concentration. Infection of PCBUS with S. aureus resulted in increased expression of proinflammatory cytokines and chemokines such as TNF-α, C-C motif chemokine ligand 20 (CCL20), IL-1β, IL-6, and IL-10, but not C-X-C motif chemokine ligand 8 (CXCL8), lingual antimicrobial peptide (LAP), or S100 calcium binding protein A9 (S100A9) at the mRNA level. To compare the data acquired with this model, we carried out investigations on primary bovine mammary epithelial cells. Our results showed that the immune responses of both models—PCBUS and primary bovine mammary epithelial cells—were similar. In addition, investigations using PCBUS enabled us to demonstrate adherence of bacteria in the physiological cell network. These findings support the use of PCBUS in studies designed to further understand the complex pathophysiological processes of infection and inflammation in bovine mastitis and to investigate alternative therapies for mastitis.