Bovine Mammary Epithelium Research Articles

The impact of kit, environment, and sampling contamination on the observed microbiome of bovine milk.

In low-microbial biomass samples such as bovine milk, contaminants can outnumber endogenous bacteria. Because of this, milk microbiome research suffers from a critical knowledge gap, namely, does non-mastitis bovine milk contain a native microbiome? In this study, we sampled external and internal mammary epithelia and stripped and cisternal milk and used numerous negative controls, including air and sampling controls and extraction and library preparation blanks, to identify the potential sources of contamination. Two algorithms were used to mathematically remove contaminants and track the potential movement of microbes among samples. Results suggest that the majority (i.e., >75%) of sequence data generated from bovine milk and mammary epithelium samples represents contaminating DNA. Contaminants in milk samples were primarily sourced from DNA extraction kits and the internal and external skin of the teat, while teat canal and apex samples were mainly contaminated during the sampling process. After decontamination, the milk microbiome displayed a more dispersed, less diverse, and compositionally distinct bacterial profile compared with epithelial samples. Similar microbial compositions were observed between cisternal and stripped milk samples, as well as between teat apex and canal samples. Staphylococcus and Acinetobacter were the predominant genera detected in milk sample sequences, and bacterial culture showed growth of Staphylococcus and Corynebacterium spp. in 50% (7/14) of stripped milk samples and growth of Staphylococcus spp. in 7% (1/14) of cisternal milk samples. Our study suggests that microbiome data generated from milk samples obtained from clinically healthy bovine udders may be heavily biased by contaminants that enter the sample during sample collection and processing workflows.IMPORTANCEObtaining a non-contaminated sample of bovine milk is challenging due to the nature of the sampling environment and the route by which milk is typically extracted from the mammary gland. Furthermore, the very low bacterial biomass of bovine milk exacerbates the impacts of contaminant sequences in downstream analyses, which can lead to severe biases. Our finding showed that bovine milk contains very low bacterial biomass and each contamination event (including sampling procedure and DNA extraction process) introduces bacteria and/or DNA fragments that easily outnumber the native bacterial cells. This finding has important implications for our ability to draw robust conclusions from milk microbiome data, especially if the data have not been subjected to rigorous decontamination procedures. Based on these findings, we strongly urge researchers to include numerous negative controls into their sampling and sample processing workflows and to utilize several complementary methods for identifying potential contaminants within the resulting sequence data. These measures will improve the accuracy, reliability, reproducibility, and interpretability of milk microbiome data and research.

DNAJA1 positively regulates amino acid-stimulated milk protein and fat synthesis in bovine mammary epithelial cells.

Several cellular processes, including the recovery of misfolded proteins, the folding of polypeptide chains, transit of polypeptides across the membrane, construction and disassembly of protein complexes, and modulation of protein control, are carried out by DnaJ homologsubfamily A member 1(DNAJA1), which belongs to the DnaJ heat-shock protein family. It is unknown if DNAJA1 regulates the production of milk in bovine mammary epithelium cells (BMECs). Methionine and leucine increased DNAJA1 expression and nuclear location, as seen by us. In contrast to DNAJA1 knockdown, overexpression of DNAJA1 boosted the production of milk proteins and fats as well as mammalian target of rapamycin (mTOR) and sterol regulatory element binding protein-1c(SREBP-1c). As a result of amino acids, mTOR and SREBP-1c gene expression are stimulated, and DNAJA1 is a positive regulator of BMECs'amino acid-induced controlled milk protein and fat production.

Involvement of Pyocyanin in Promoting LPS-Induced Apoptosis, Inflammation, and Oxidative Stress in Bovine Mammary Epithelium Cells

Involvement of Pyocyanin in Promoting LPS-Induced Apoptosis, Inflammation, and Oxidative Stress in Bovine Mammary Epithelium Cells

Adipocyte-Derived Paracrine Factors Regulate the In Vitro Development of Bovine Mammary Epithelial Cells.

The mammary gland is composed of epithelial tissue forming ducts and lobules, and the stroma, composed of adipocytes, connective tissue, and other cell types. The stromal microenvironment regulates mammary gland development by paracrine and cell-cell interactions. In the present study, primary cultures of bovine mammary epithelial cells (bMEC) and bovine adipose-derived stem cells (bASC) subjected to adipogenic differentiation were used to investigate the influence of paracrine factors secreted by preadipocytes and adipocytes on bMEC development. Four types of conditioned media (CM) were collected from undifferentiated preadipocytes (preA) and adipocytes on days: 8, 12, 14 of differentiation. Next, bMEC were cultured for 24 h in CM and cell viability, apoptosis, migratory activity, ability to form spheroids on Matrigel, and secretory activity (alpha S1-casein concentration) were evaluated. CM derived from fully differentiated adipocytes (12 d and 14 d) significantly decreased the number of apoptotic cells in bMEC population and increased the size of spheroids formed by bMEC on Matrigel. CM collected from preadipocytes significantly enhanced bMEC's migration, and stimulated bMEC to produce alpha S1-casein, but only in the presence of prolactin. These results confirm that preadipocytes and adipocytes are important components of the stroma, providing paracrine factors that actively regulate the development of bovine mammary epithelium.

Identification of key genes in bovine mammary epithelial cells challenged with Escherichia coli and Staphylococcus aureus by integrated bioinformatics analysis

This study integrated four microarray datasets by Robust Rank Aggregation (RRA) method to identify the differentially expressed genes (DEG) in bovine mammary epithelial (BME) cells in response to Escherichia coli and Staphylococcus aureus infection. Furthermore, the GO function and KEGG pathway enrichment analysis of the integrated DEG were performed. Finally, the protein-protein interaction (PPI) network was constructed. A total of 72 integrated DEG were identified from the four datasets. The most significantly enriched terms within the integrated DEG were [...]

Traditional and emerging Fusarium mycotoxins disrupt homeostasis of bovine mammary cells by altering cell permeability and innate immune function.

High incidence of traditional and emerging Fusarium mycotoxins in cereal grains and silages can be a potential threat to feed safety and ruminants. Inadequate biodegradation of Fusarium mycotoxins by rumen microflora following ingestion of mycotoxin-contaminated feeds can lead to their circulatory transport to target tissues such as mammary gland. The bovine udder plays a pivotal role in maintaining milk yield and composition, thus, human health. However, toxic effects of Fusarium mycotoxins on bovine mammary gland are rarely studied. In this study, the bovine mammary epithelial cell line was used as an in-vitro model of bovine mammary epithelium to investigate effects of deoxynivalenol (DON), enniatin B (ENB) and beauvericin (BEA) on bovine mammary gland homeostasis. Results indicated that exposure to DON, ENB and BEA for 48h significantly decreased cell viability in a concentration-dependent manner (P<0.001). Exposure to DON at 0.39μmol/L and BEA at 2.5μmol/L for 48h also decreased paracellular flux of FITC-40 kDa dextran (P<0.05), whereas none of the mycotoxins affected transepithelial electrical resistance after 48h exposure. The qPCR was performed for assessment of expression of gene coding tight junction (TJ) proteins, toll-like receptor 4 (TLR4) and cytokines after 4, 24 and 48h of exposure. DON, ENB and BEA significantly upregulated the TJ protein zonula occludens-1, whereas markedly downregulated claudin 3 (P<0.05). Exposure to DON at 1.35μmol/L for 4h significantly increased expression of occludin (P<0.01). DON, ENB and BEA significant downregulated TLR4 (P<0.05). In contrast, ENB markedly increased expression of cytokines interleukin-6 (IL-6) (P<0.001), tumor necrosis factor α (TNF-a) (P<0.05) and transforming growth factor-β (TGF-β) (P<0.01). BEA significantly upregulated IL- 6 (P<0.001) and TGF-β (P=0.01), but downregulated TNF-α (P<0.001). These results suggest that DON, ENB and BEA can disrupt mammary gland homeostasis by inducing cell death as well as altering its paracellular permeability and expression of genes involved in innate immune function.

Comparative Assessment of Different Yeast Cell Wall-Based Mycotoxin Adsorbents Using a Model- and Bioassay-Based In Vitro Approach.

Frequently reported occurrences of deoxynivalenol (DON), beauvericin (BEA), and, to a lesser extent, ochratoxin A (OTA) and citrinin (CIT) in ruminant feed or feedstuff could represent a significant concern regarding feed safety, animal health, and productivity. Inclusion of yeast cell wall-based mycotoxin adsorbents in animal feeds has been a common strategy to mitigate adverse effects of mycotoxins. In the present study, an in vitro approach combining adsorption isotherm models and bioassays was designed to assess the efficacy of yeast cell wall (YCW), yeast cell wall extract (YCWE), and a postbiotic yeast cell wall-based blend (PYCW) products at the inclusion rate of 0.5% (w/v) (ratio of adsorbent mass to buffer solution volume). The Hill's adsorption isotherm model was found to best describe the adsorption processes of DON, BEA, and CIT. Calculated binding potential for YCW and YCWE using the Hill's model exhibited the same ranking for mycotoxin adsorption, indicating that BEA had the highest adsorption rate, followed by DON and CIT, which was the least adsorbed. PYCW had the highest binding potential for BEA compared with YCW and YCWE. In contrast, the Freundlich isotherm model presented a good fit for OTA adsorption by all adsorbents and CIT adsorption by PYCW. Results indicated that YCW was the most efficacious for sequestering OTA, whereas YCWE was the least efficacious. PYCW showed greater efficacy at adsorbing OTA than CIT. All adsorbents exhibited high adsorption efficacy for BEA, with an overall percentage average of bound mycotoxin exceeding 60%, whereas moderate efficacies for the other mycotoxins were observed (up to 37%). Differences in adsorbent efficacy of each adsorbent significantly varied according to experimental concentrations tested for each given mycotoxin (p < 0.05). The cell viability results from the bioassay using a bovine mammary epithelial cell line (MAC-T) indicated that all tested adsorbents could potentially mitigate mycotoxin-related damage to bovine mammary epithelium. Results from our studies suggested that all tested adsorbents had the capacity to adsorb selected mycotoxins in vitro, which could support their use to mitigate their effects in vivo.

Ochratoxin A and Citrinin Differentially Modulate Bovine Mammary Epithelial Cell Permeability and Innate Immune Function.

Frequent detection of mycotoxins ochratoxin A (OTA) and citrinin (CIT) in ruminant feed and feedstuff can be a potential threat to feed safety, animal performance and health. Ineffective biodegradation of these mycotoxins by rumen microflora following ingestion of contaminated feeds can lead to their circulatory transport to tissues such as mammary gland as the result of their biodistribution throughout the body. The bovine mammary epithelium plays a pivotal role in maintaining milk yield and composition and contributes to innate immune defense of the udder. The present study is the first to investigate individual effects of OTA and CIT on barrier and innate immune functions of the bovine mammary epithelium using a bovine mammary epithelial cell line (MAC-T). Results indicated that OTA and CIT exposure for 48 h significantly decreased cell viability in a concentration-dependent manner (p < 0.05). A decrease in transepithelial electrical resistance and increase in paracellular flux of FITC-40 kDa dextran was significantly induced by OTA treatment (p < 0.05), but not by CIT after 48 h exposure. qPCR was performed for assessment of expression of tight-junction proteins, Toll-like receptor 4 (TLR4) and cytokines after 4, 24 and 48 h of exposure. Both OTA and CIT markedly downregulated expression of claudin 3 and occludin (p < 0.05), whereas CIT did not affect zonula occludens-1 expression. Expression of TLR4 was significantly upregulated by OTA (p < 0.001) but downregulated by CIT (p < 0.05) at 48 h. Expression of IL-6, TNF-a and TGF-β was significantly upregulated by OTA (p < 0.05), whereas IL-6 and TGF-β expression was downregulated by CIT (p < 0.01). These results suggest that OTA and CIT could potentially differentially modulate barrier and innate immune functions of mammary epithelium. The present study not only throws light on the individual toxicity of each mycotoxin on bovine mammary epithelium but also lays the foundation for future studies on the combined effects of the two mycotoxins.

The protective roles of tea tree oil extracts in bovine mammary epithelial cells and polymorphonuclear leukocytes

BackgroundTea tree oil (TTO) plays an important role in antibacterial activity and alleviating the inflammatory responses. Bovine mammary epithelium and polymorphonuclear leukocytes (PMNL) can actively respond to bovine mastitis infection. However, regulatory effects of TTO extracts on the innate immune response of bovine mammary epithelial cells (BMECs) and PMNL remain not reported. Therefore, aim of the study was to evaluate the effects of TTO extracts on the mRNA levels of the genes involved in the innate immune response of BMECs and PMNL.ResultsOur results demonstrated that addition of 0.025% and 0.05% TTO increased the proliferation of BMECs, and significantly enhanced (P < 0.05) the viability of BMECs exposed to Staphylococcus aureus (S. aureus). An inhibitory effect was observed against the growth of S. aureus by TTO incubation. The 0.05% TTO reduced S. aureus biofilm formation, association and invasion of S. aureus to BMECs, and changed the morphological and structural features of S. aureus. The proinflammatory cytokines IL-1β, IL-6, and TNF-α were decreased (P < 0.001) by the incubation of TTO. Interestingly, the expression of IL-8 known for PMNL chemotactic function was elevated (P < 0.05) by 0.05% TTO treatment. Consistently, 0.05% TTO increased the migration of PMNL in S. aureus-exposed BMECs when compared with S. aureus treatment alone (P < 0.05). In addition, PMNL incubated with 0.05% TTO decreased the levels of NFKB inhibitor alpha (NFKBIA) and TNF-α.ConclusionsOur results indicate that use of TTO can relieve the BMECs pro-inflammatory response caused by S. aureus and promote the migration of PMNL to mount the innate immune responses, and it may be novel strategy for the treatment of bovine mastitis caused by S. aureus.

Evaluation of the Immunomodulatory Ability of Lactic Acid Bacteria Isolated from Feedlot Cattle Against Mastitis Using a Bovine Mammary Epithelial Cells In Vitro Assay.

Bovine mastitis, the inflammation of the mammary gland, affects the quality and quantity of milk yield. Mastitis control relies on single or multiple combinations of antibiotic therapy. Due to increasing antibiotic resistance in pathogens, the intramammary infusion of lactic acid bacteria (LAB) has been considered as a potential alternative to antibiotics for treating and preventing bovine mastitis through the improvement of the host immunity. Probiotic effects are a strain-dependent characteristic; therefore, candidate LAB strains have to be evaluated efficiently to find out the ones with the best potential. Here, we investigated LAB strains originally isolated from feedlot cattle’s environment regarding their ability in inducing the Toll-like receptor (TLR)-triggered inflammatory responses in bovine mammary epithelial (BME) cells in vitro. The BME cells were pre-stimulated with the LAB strains individually for 12, 24, and 48 h and then challenged with Escherichia coli-derived lipopolysaccharide (LPS) for 12 h. The mRNA expression of selected immune genes—interleukin 1 alpha (IL-1α), IL-1β, monocyte chemotactic protein 1 (MCP-1), IL-8, chemokine (C-X-C motif) ligand 2 (CXCL2), and CXCL3 were quantified by real-time quantitative PCR (RT-qPCR). Results indicated that pretreatment with some Lactobacillus strains were able to differentially regulate the LPS inflammatory response in BME cells; however, strain-dependent differences were found. The most remarkable effects were found for Lactobacillus acidophilus CRL2074, which reduced the expression of IL-1α, IL-1β, MCP-1, IL-8, and CXCL3, whereas Lactobacillus rhamnosus CRL2084 diminished IL-1β, MCP-1, and IL-8 expression. The pre-stimulation of BME cells with the CRL2074 strain resulted in the upregulated expression of three negative regulators of the TLRs, including the ubiquitin-editing enzyme A20 (also called tumor necrosis factor alpha-induced protein 3, TNFAIP3), single immunoglobin IL-1 single receptor (SIGIRR), and Toll interacting protein (Tollip) after the LPS challenge. The CRL2084 pre-stimulation upregulated only Tollip expression. Our results demonstrated that the L. acidophilus CRL2074 strain possess remarkable immunomodulatory abilities against LPS-induced inflammation in BME cells. This Lactobacillus strain could be used as candidate for in vivo testing due to its beneficial effects in bovine mastitis through intramammary infusion. Our findings also suggest that the BME cells immunoassay system could be of value for the in vitro evaluation of the immunomodulatory abilities of LAB against the inflammation resulting from the intramammary infection with mastitis-related pathogens.

Effect of folic acid supplementation on proliferation and apoptosis in bovine mammary epithelial (MAC-T) cells

Folic acid (FA) is known to be an important micronutrient in humans; however, information regarding the effect of FA supplementation on bovine mammary epithelial (BME) cells is insufficient. FA supplementation is reported to increase milk production in dairy cows, but the underlying molecular mechanisms are unknown. This study examined the effects of FA supplementation on the proliferation and apoptosis of a BME cell line (MAC-T). MAC-T cells were treated with various concentrations (deficient in FA (DF) < 0.01 ng/mL; low-level FA (LF) 3.1 ng/mL; normal FA (NF) 15.4 ng/mL; and high-level FA (HF) 30.8 ng/mL) based on serum folate (10–20 ng/mL) in milking cows. HF treatment significantly increased the proliferation of MAC-T cells. Cellular apoptosis was observed mainly in the DF group. The number of apoptotic cells in DF media was significantly higher than that in NF media. The bcl-2/bax mRNA expression ratio was significantly increased in the HF group compared to that in the DF group. FA supplementation significantly increased the ratio of Bcl-2/Bax protein levels in MAC-T cells. FA supplementation increases proliferation and decreases apoptosis in these cells. This study might provide information regarding the molecular mechanism through which FA supplementation is associated with increased milk yield.

Contrasting effects of transforming growth factor β1 on programmed cell death of bovine mammary epithelial cell lines MAC-T and BME-UV1

A previous study in the bovine mammary epithelial cell line BME-UV1 demonstrated that suppression of the phosphatidylinositol-4,5-biphosphate 3 kinase (PI3K)/AKT (somatotropic) signaling pathway was required for transforming growth factor β1 (TGFβ1)-induced programmed cell death (PCD). To investigate whether this is a universal mechanism for TGFβ1 to induce PCD in bovine mammary epithelium, we compared TGFβ1 modulation of PI3K/AKT and its role in PCD in 2 bovine mammary epithelial cell lines: MAC-T and BME-UV1. In MAC-T cells, TGFβ1 promoted cell survival, and this paralleled a reduction in PI3K/AKT activity, rather than an increase. In BME-UV1 cells, TGFβ1 induced PCD, and this was accompanied by a time-dependent effect on PI3K/AKT activity, including an initial significant increase in the phosphorylation of AKT at 3 h, followed by a reduction between 12 and 24 h, and then an increase at 48 h. Inhibition of AKT activity enhanced TGFβ1-induced PCD in BME-UV1 cells but had no effect on MAC-T cells, suggesting that TGFβ1 mediates PCD in BME-UV1 cells through suppression of AKT activity. Inhibition of TGFβ receptor type I (TβRI) kinase activity completely abrogated TGFβ1-induced PCD in BME-UV1 cells but had no effect on TGFβ1-induced suppression of PCD in MAC-T cells, demonstrating that TGFβ1-induced PCD in BME-UV1 cells is dependent on TβRI/SMAD signaling. These and previous observations suggest that the different effects of TGFβ1 on PCD in these cell lines might involve noncanonical signaling pathways other than PI3K/AKT, and may reflect their different lineages. Future studies should address this finding, taking into consideration the effect that different culture conditions might have on cell phenotype.

Transcriptome Analysis of The Inflammatory Responses of Bovine Mammary Epithelial Cells: Exploring Immunomodulatory Target Genes for Bovine Mastitis.

Bovine mastitis is the inflammatory reaction of the mammary gland and is commonly caused by bacterial infections in high-yielding dairy cows. The detailed investigation of the immunotranscriptomic response of bovine mammary epithelial (BME) cells to pattern recognition receptors (PRRs) activation by microbial-associated molecular patterns (MAMPs) can be of great importance for understanding the innate immune defense mechanisms, and for exploring the immunomodulatory candidate genes. In this work, we investigated the transcriptome modifications of BME cells after the in vitro stimulation with Escherichia coli derived lipopolysaccharide (LPS) and heat-killed Staphylococcus aureus JE2 and S. aureus SA003. In addition, the effect of Pam3CSK4 (a synthetic triacylated lipopeptide that activates Toll-like receptor 2 (TLR2)), and the intracellular chemotactic protein cyclophilin A (CyPA), which is secreted by BME cells during mastitis, in the expression changes of selected cytokines and chemokines were evaluated by qPCR. Microarray analysis identified 447, 465 and 520 differentially expressed genes (DEGs) in the BME cells after LPS, S. aureus JE2 and S. aureus SA003 stimulation, respectively. A major differential response in the inflammatory gene expression was noticed between the stimulation of LPS and S. aureus strains. Unlike the S. aureus strains, LPS stimulation resulted in significant upregulation of CCL2, CXCL2, CXCL3, CXCL8, IL1α and IL1β, which were confirmed by qPCR analysis. Pam3CSK4 was not able to induce significant changes in the expression of cytokines and chemokines in challenged BME cells. The exogenous CyPA administration was able to upregulate CXCL2, CXCL3, CXCL8, IL1α and IL1β expression in BME cells indicating its ability to promote inflammation. The identification of transcriptional markers of mastitis specific for individual inflammatory factors such as LPS, Pam3CSK4 or CyPA, which can be evaluated in vitro in BME cells, may enable the development of novel diagnostics and/or immunomodulatory treatments, providing new tools for the effective management of mastitis in dairy cows. The results of this work are an advance in this regard.

Symposium review: Determinants of milk production: Understanding population dynamics in the bovine mammary epithelium

The mammary gland undergoes distinct periods of growth, development, and secretory activity. During bovine lactation, a gradual decrease in the number of mammary epithelial cells largely accounts for the decline in milk production with advancing lactation. The net decline in cell number (approx. 50%) is due to cell death but is simultaneously accompanied by cell renewal. Although the rate of cell proliferation is slow, by the end of lactation most cells in the gland were formed after calving. Typically milking is terminated when cows are in the final 2 mo of pregnancy. This causes regenerative involution, wherein extensive cell replacement and mammary growth occurs. We hypothesized that replacement of senescent secretory cells and progenitor cells during the dry period increases milk yield in the next lactation. Analysis of global gene expression revealed networks and canonical pathways during regenerative involution that support cell turnover and mammary growth, and reflect oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress. Immune responses consistent with influx of neutrophils, macrophages, and lymphocytes, and processes that support mammary differentiation and lactogenesis were also evident. Data also suggest that replication of stem and progenitor cells occurs during the dry period. Relying on long-term retention of bromodeoxyuridine-labeled DNA, we identified putative bovine mammary stem cells. These label-retaining epithelial cells (LREC) are in low abundance within mammary epithelium (<1%), predominantly estrogen receptor-negative, and localized in a basal or suprabasal layer of the epithelium. Analyses of gene expression in laser-microdissected LREC are consistent with the concept that LREC represent stem cells and progenitor cells, which differ in properties and location within the epithelial layer. We identified potential markers for these cells and have increased their number by infusing xanthosine through the teat canal of prepubertal heifers. Altering population dynamics of mammary stem and progenitor cells during the mammary cycle may be a means to increase efficiency of milk production.

Biofilm formation and adhesion to bovine udder epithelium of potentially probiotic lactic acid bacteria.

Mastitis is one of the most important diseases threatening modern dairy herds. The idea of fighting the disease through colonising the udder with lactic acid bacteria (LAB), thereby building a beneficial biofilm, is the base for a probiotic approach towards mastitis control. The purpose of this study was to screen 13 LAB strains (eleven wild strains, two ATCC strains) inhibitory to the growth of mastitis-causing pathogens for their in vitro ability to form a biofilm and to adhere to bovine glandular mammary epithelium in order to assess their probiotic potential. Furthermore, we aimed to gain knowledge about the chemical nature of the adhesins involved by subjecting the bacteria to various chemical and enzymatical pre-treatments. The biofilms were grown on hydrophilic glass and on hydrophobic polypropylene in de Man, Rogosa and Sharpe (MRS) broth and afterwards quantified with a crystal violet assay. Biofilm formation was observed in all strains. However, the extent strongly depended on the strain, surface charge and medium. The adhesion assay also revealed a strong strain dependency, but this trait was also present in all of the investigated LAB isolates. Depending on the strain, chemical or enzymatical pre-treatment revealed carbohydrate molecules as well as proteins and lipids to be crucial for the adhesion of LAB to epithelial cells. The seven strains showing the strongest biofilm formation and/or adhesion represent promising candidates for further investigation in order to develop a probiotic remedy for the treatment of mastitis. Still, their safety for consumers and patients as well as their capability to colonise the udder remain to be investigated in in vivo studies.

Development and validation of immortalized bovine mammary epithelial cell line as an in vitro model for the study of mammary gland functions.

This study aimed to develop a bovine mammary epithelial (BME) cell line model, which provides a possibility to determine functional properties of the bovine mammary gland. The primary cell culture was derived from bovine mammary gland tissues and processed enzymatically to obtain cell colonies with epithelial-like morphology. The cultures of BME cells were purified and optimally cultured at 37°C in DMEM/F12 medium supplemented with 10% fetal bovine serum. The BME cells were identified as epithelial cell line by the evaluating the expression of keratin-18 using immunofluorescence staining. A novel gene expression system strongly enhances the expression of telomerase, has been used to immortalize BME cell line termed hTBME cell line. Interestingly, telomerase remained active even after over 60 passages of hTBME cell line, required for immortalization of BME cells. In addition, the hTBME cell line was continuously subcultured with a spontaneous epithelial-like morphology, with a great proliferation activity, and without evidence of apoptotic and necrotic effects. Further characterization showed that hTBME cell line can be continuously propagated in culture with constant chromosomal features and without tumorigenic properties. Finally, established hTBME cell line was evaluated for mammary gland specific functions. Our results demonstrated that the hTBME cell line was able to retain functional-morphological structure, and functional differentiation by expression of beta (β)-casein as in the bovine mammary gland in vivo. Taken together, our findings suggest that the established hTBME cell line can serve as a valuable tool for the study of bovine mammary gland functions.

Effects of niacin and betaine on bovine mammary and uterine cells exposed to thermal shock in vitro

The objective of this study was to investigate the direct effects of feed supplements niacin and betaine on the heat shock responses of in vitro cultured cells derived from bovine mammary and uterine tissues. First, we determined the mRNA expression profiles of the niacin receptor (GPR109A) in bovine tissues (liver, skin, uterus, udder, and ovary) and in cells derived from bovine mammary epithelium (mammary alveolar cells, MAC-T; bovine mammary epithelial cells, BMEC) and endometrium (bovine endometrial cells, BEND). We found that GPR109A was distributed in all examined tissues and cells, and the highest expression was in cells from skin and udder. Second, we evaluated the effects of niacin treatment on the mRNA abundance of heat shock proteins 70 and 27 (HSP70 and HSP27) in MAC-T, BMEC, and BEND under thermoneutral conditions and heat stress, and whether these effects were associated with alterations in the mRNA expression of prostaglandin E2 synthesis-related genes, including cyclooxygenase 1 and 2 (COX-1 and COX-2) and microsomal prostaglandin E synthase 1 and 2 (mPGES-1 and mPGES-2). Quantitative PCR data indicated that niacin suppressed HSP70 mRNA expression in BMEC and both HSP70 and HSP27 in BEND under thermoneutral conditions. Only COX-2 expression was downregulated by niacin in BMEC; other prostaglandin E2 synthesis-related genes stayed unaltered in BMEC and BEND. The mRNA abundance of HSP70, COX-1, COX-2, and mPGES-1 were elevated in niacin-treated MAC-T. During heat stress, niacin increased mRNA levels of HSP70 and HSP27 in MAC-T and HSP27 in BEND, but decreased HSP70 in BMEC. Although mPGES-2 was stimulated by niacin in BEND, the mRNA expression of prostaglandin E2 synthesis-related genes were consistent with neither HSP70 nor HSP27 expression patterns in niacin-treated BMEC and MAC-T. These data suggest that the effects of niacin on heat shock protein expression and prostaglandin E2 synthesis were not well coupled in these cells. Finally, we tested the effects of betaine treatment on viability and apoptosis in BMEC. Compared with control cultures, viability was higher in betaine-treated cells at 8 h under thermoneutral conditions and at 16 h in heat stress, and apoptotic rates were lower at 8 h. Our data support a dual role for niacin in regulating heat shock protein expression in normal and heat-shocked cells derived from mammary and uterine tissues, and positive effects of betaine in regulating mammary cell viability during heat stress.

Sterol regulatory element-binding proteins are regulators of the sodium/iodide symporter in mammary epithelial cells

The sodium/iodide symporter (NIS), which is essential for iodide concentration in the thyroid, is reported to be transcriptionally regulated by sterol regulatory element-binding proteins (SREBP) in rat FRTL-5 thyrocytes. The SREBP are strongly activated after parturition and throughout lactation in the mammary gland of cattle and are important for mammary epithelial cell synthesis of milk lipids. In this study, we tested the hypothesis that the NIS gene is regulated also by SREBP in mammary epithelial cells, in which NIS is functionally expressed during lactation. Regulation of NIS expression and iodide uptake was investigated by means of inhibition, silencing, and overexpression of SREBP and by reporter gene and DNA-binding assays. As a mammary epithelial cell model, the human MCF-7 cell line, a breast adenocarcinoma cell line, which shows inducible expression of NIS by all-trans retinoic acid (ATRA), and unlike bovine mammary epithelial cells, is widely used to investigate the regulation of mammary gland NIS and NIS-specific iodide uptake, was used. Inhibition of SREBP maturation by treatment with 25-hydroxycholesterol (5 µM) for 48h reduced ATRA (1 µM)-induced mRNA concentration of NIS and iodide uptake in MCF-7 cells by approximately 20%. Knockdown of SREBP-1c and SREBP-2 by RNA interference decreased the mRNA and protein concentration of NIS by 30 to 50% 48h after initiating knockdown, whereas overexpression of nuclear SREBP (nSREBP)-1c and nSREBP-2 increased the expression of NIS in MCF-7 cells by 45 to 60%, respectively, 48h after initiating overexpression. Reporter gene experiments with varying length of NIS promoter reporter constructs revealed that the NIS 5'-flanking region is activated by nSREBP-1c and nSREBP-2 approximately 1.5- and 4.5-fold, respectively, and activation involves a SREBP-binding motif (SRE) at -38 relative to the transcription start site of the NIS gene. Gel shift assays using oligonucleotides spanning either the wild-type or the mutated SRE at -38 of the NIS 5'-flanking region showed that in vitro-translated nSREBP-1c and nSREBP-2 bind only the wild-type but not the mutated SRE at -38 of NIS. Collectively, the present results from cell culture experiments with human mammary epithelial MCF-7 cells and from genetic studies show for the first time that the NIS gene and iodide uptake are regulated by SREBP in cultured human mammary epithelial cells. Future studies are necessary to clarify if the regulation of NIS expression and iodide uptake by SREBP also applies to the lactating bovine mammary epithelium.

ABC- and SLC-Transporters in Murine and Bovine Mammary Epithelium--Effects of Prochloraz.

Some chemicals are ligands to efflux transporters which may result in high concentrations in milk. Limited knowledge is available on the influence of maternal exposure to chemicals on the expression and function of transporters in the lactating mammary gland. We determined gene expression of ABC and SLC transporters in murine mammary tissue of different gestation and lactation stages, in murine mammary cells (HC11) featuring resting and secreting phenotypes and in bovine mammary tissue and cells (BME-UV). Effects on transporter expression and function of the imidazole fungicide prochloraz, previously reported to influence BCRP in mammary cells, was investigated on transporter expression and function in the two cell lines. Transporters studied were BCRP, MDR1, MRP1, OATP1A5/OATP1A2, OCTN1 and OCT1. Gene expressions of BCRP and OCT1 in murine mammary glands were increased during gestation and lactation, whereas MDR1, MRP1, OATP1A5 and OCTN1 were decreased, compared to expressions in virgins. All transporters measured in mammary glands of mice were detected in bovine mammary tissue and in HC11 cells, while only MDR1 and MRP1 were detected in BME-UV cells. Prochloraz treatment induced MDR1 gene and protein expression in both differentiated HC11 and BME-UV cells and increased protein function in HC11 cells, resulting in decreased accumulation of the MDR1 substrate digoxin. In conclusion, our results demonstrate that murine (HC11) and bovine (BME-UV) mammary epithelial cells can be applied to characterize expression and function of transporters as well as effects of contaminants on the mammary transporters. An altered expression, induced by a drug or toxic chemical, on any of the transporters expressed in the mammary epithelial cells during lactation may modulate the well-balanced composition of nutrients and/or secretion of contaminants in milk with potential adverse effects on breast-fed infants and dairy consumers.

Staphylococcus aureus and Escherichia coli elicit different innate immune responses from bovine mammary epithelial cells

Escherichia coli and Staphylococcus aureus are the most important pathogenic bacteria causing bovine clinical mastitis and subclinical mastitis, respectively. However, little is known about the molecular mechanisms underlying the different host response patterns caused by these bacteria. The aim of this study was to characterize the different innate immune responses of bovine mammary epithelium cells (MECs) to heat-inactivated E. coli and S. aureus. Gene expression of Toll-like receptor 2 (TLR2) and TLR4 was compared. The activation of nuclear factor kappa B (NF-κB) and the kinetics and levels of cytokine production were analyzed. The results show that the mRNA for TLR2 and TLR4 was up-regulated when the bovine MECs were stimulated with heat-inactivated E. coli, while only TLR2 mRNA was up-regulated when the bovine MECs were stimulated with heat-inactivated S. aureus. The expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and IL-8 increased more rapidly and higher when the bovine MECs were stimulated with heat-inactivated E. coli than when they were stimulated with heat-inactivated S. aureus. E. coli strongly activated NF-κB in the bovine MECs, while S. aureus failed to activate NF-κB. Heat-inactivated S. aureus could induce NF-κB activation when bovine MECs cultured in medium without fetal calf serum. These results were confirmed using TLR2- and TLR4/MD2-transfected HEK293 cells and suggested that differential TLR recognition and the lack of NF-κB activation account for the impaired immune response elicited by heat-inactivated S. aureus.