MAC-T Cells Research Articles

Development of a model describing regulation of casein synthesis by the mammalian target of rapamycin (mTOR) signaling pathway in response to insulin, amino acids, and acetate

To improve dietary protein use efficiency in lactating cows, mammary protein synthesis responses to AA, energy substrates, and hormones must be better understood. These entities exert their effects through stimulation of mRNA translation via control of initiation and elongation rates at the cellular level. A central protein kinase of this phenomenon is the mammalian target of rapamycin (mTOR), which transfers the nutritional and hormonal stimuli onto a series of proteins downstream through a cascade of phosphorylation reactions that ultimately affect protein synthesis. The objective of this work was to further develop an existing mechanistic model of mTOR phosphorylation responses to insulin and total essential AA to include the effects of specific essential AA and acetate mediated by signaling proteins including protein kinase B (Akt), adenosine monophosphate activated protein kinase (AMPK), and mTOR and to add a representation of milk protein synthesis. Data from 6 experiments in MAC-T cells and mammary tissue slices previously conducted in our laboratory were assembled and used to parameterize the dynamic system of differential equations representing Akt, AMPK, and mTOR in their phosphorylated and dephosphorylated states and the resulting regulation of milk protein synthesis. The model predicted phosphorylated Akt, mTOR, AMPK, and casein synthesis rates with root mean square prediction errors of 16.8, 28.4, 33.0, and 54.9%, respectively. All other dependent variables were free of mean and slope bias, indicating an adequate representation of the data. Whereas mTOR was not very sensitive to changes in insulin or acetate levels, it was highly sensitive to leucine and isoleucine, and this signal appeared to be effectively transduced to casein synthesis. Although prior work had observed a relationship with additional essential AA, and data supporting those conclusions were present in the data set, we were unable to derive significant relationships with any essential AA other than leucine and isoleucine. The signaling properties and dynamics of AMPK under nutrient depletion and sufficiency, the responses to additional essential AA, and the consequent effects on protein synthesis remain to be better understood.

Citrus-derived oil inhibits Staphylococcus aureus growth and alters its interactions with bovine mammary cells

This experiment examined the effects of cold-pressed, terpeneless citrus-derived oil (CDO) on growth of Staphylococcus aureus, which a major cause of contagious bovine mastitis, and invasion of bovine mammary cells (MAC-T). To determine minimum inhibitory concentration, we used the broth dilution method, using CDO concentrations range from 0.0125 to 0.4% with 2-fold dilutions. Growth inhibition was examined by adding 0.00, 0.05, 0.025, 0.0125, and 0.00625% CDO to 105 cfu/mL S. aureus in nutrient broth and enumerating colonies after serial dilution. In a 96-well plate, S. aureus (107 cfu/mL) was allowed to form a biofilm, treated with 0, 0.025, 0.5, or 1% CDO, and then was measured using a spectrophotometer. Cytotoxic effect on immortalized MAC-T cells was also examined at various concentrations of CDO using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. We observed that the minimum inhibitory concentration of CDO to inhibit the growth of S. aureus in vitro was 0.025% CDO. A time kill curve for CDO action on S. aureus over 4h was generated. The CDO completely eliminated S. aureus after 3h of incubation at a concentration of 0.25%, or after 2h of incubation at concentrations of 0.05%. It was also observed that CDO had no effect on preformed biofilms except at a concentration of 0.05%, in which a significant reduction in the measured absorbance was noted. In addition, the association and invasion of S. aureus to MAC-T cells were significantly inhibited after 1h of treatment with CDO. Citrus-derived oil was also able to increase cellular proliferation of MAC-T cells at concentrations up 0.05% and had no effect at a concentration of 0.1% after 1 h. Our data suggests that CDO should be considered for further research as a preventive and therapeutic against bovine mastitis.

Assessment of yeast Saccharomyces cerevisiae component binding to Mycobacterium avium subspecies paratuberculosis using bovine epithelial cells.

BackgroundSince yeast Saccharomyces cerevisiae and its components are being used for the prevention and treatment of enteric diseases in different species, they may also be useful for preventing Johne’s disease, a chronic inflammatory bowel disease of ruminants caused by Mycobacterium avium spp. paratuberculosis (MAP). This study aimed to identify potential yeast derivatives that may be used to help prevent MAP infection. The adherence of mCherry-labeled MAP to bovine mammary epithelial cell line (MAC-T cells) and bovine primary epithelial cells (BECs) co-cultured with yeast cell wall components (CWCs) from four different yeast strains (A, B, C and D) and two forms of dead yeast from strain A was investigated.ResultsThe CWCs from all four yeast strains and the other two forms of dead yeast from strain A reduced MAP adhesion to MAC-T cells and BECs in a concentration-dependent manner after 6-h of exposure, with the dead yeast having the greatest effect.ConclusionsThe following in vitro binding studies suggest that dead yeast and its’ CWCs may be useful for reducing risk of MAP infection.

Effects of Chinese Propolis in Protecting Bovine Mammary Epithelial Cells against Mastitis Pathogens-Induced Cell Damage.

Chinese propolis (CP), an important hive product, can alleviate inflammatory responses. However, little is known regarding the potential of propolis treatment for mastitis control. To investigate the anti-inflammatory effects of CP on bovine mammary epithelial cells (MAC-T), we used a range of pathogens to induce cellular inflammatory damage. Cell viability was determined and expressions of inflammatory/antioxidant genes were measured. Using a cell-based reporter assay system, we evaluated CP and its primary constituents on the NF-κB and Nrf2-ARE transcription activation. MAC-T cells treated with bacterial endotoxin (lipopolysaccharide, LPS), heat-inactivated Escherichia coli, and Staphylococcus aureus exhibited significant decreases in cell viability while TNF-α and lipoteichoic acid (LTA) did not. Pretreatment with CP prevented losses in cell viability associated with the addition of killed bacteria or bacterial endotoxins. There were also corresponding decreases in expressions of proinflammatory IL-6 and TNF-α mRNA. Compared with the mastitis challenged cells, enhanced expressions of antioxidant genes HO-1, Txnrd-1, and GCLM were observed in CP-treated cells. CP and its polyphenolic active components (primarily caffeic acid phenethyl ester and quercetin) had strong inhibitive effects against NF-κB activation and increased the transcriptional activity of Nrf2-ARE. These findings suggest that propolis may be valuable in the control of bovine mastitis.

Protection of Bovine Mammary Epithelial Cells from Hydrogen Peroxide-Induced Oxidative Cell Damage by Resveratrol.

The mammary epithelial cells (MECs) of high-producing dairy cows are likely to be subject to oxidative stress (OS) due to the intensive cell metabolism. The objectives of this study were to investigate the cytoprotective effects of resveratrol against hydrogen peroxide- (H2O2-) induced OS in cultured bovine MECs (MAC-T). Pretreatment of MAC-T cells with resveratrol could rescue the decrease in cell viability and resulted in lower intracellular reactive oxygen species (ROS) accumulation after H2O2 exposure. Resveratrol helped MAC-T cells to prevent H2O2-induced endoplasmic reticulum stress and mitochondria-related cell apoptosis. Moreover, resveratrol induced mRNA expression of multiple antioxidant defense genes in MAC-T cells under normal/oxidative conditions. Nuclear factor erythroid 2-related factor 2 (Nrf2) was required for the cytoprotective effects on MAC-T cells by resveratrol, as knockdown of Nrf2 significantly abolished resveratrol-induced cytoprotective effects against OS. In addition, by using selective inhibitors, we further confirmed that the induction of Nrf2 by resveratrol was mediated through the prolonged activation of PI3K/Akt and ERK/MAPK pathways but negatively regulated by p38/MAPK pathway. Overall, resveratrol has beneficial effects on bovine MECs redox balance and may be potentially used as a therapeutic medicine against oxidative insult in lactating animals.

Detection of Staphylococcus aureus adhesion and biofilm-producing genes and their expression during internalization in bovine mammary epithelial cells

Staphylococcus aureus is one of the most prevalent pathogens isolated from bovine mastitis, causing chronic intramammary infections (IMI) that limit profitable dairying. The course of infection is often associated with factors both related to the host and the bacterium. Aims of this study were to select S. aureus isolates from bovine IMI with different genotypic profiles harboring genes involved in adherence and biofilm production, to determine the behavior of these strains in contact with bovine mammary epithelial cells (MAC-T) and the expression of those genes during bacterial-cell early interactions. The genetic diversity of 20 S. aureus strains that were isolated from milk samples taken from cows with persistent-P and non-persistent-NP IMI was high, discriminated into 13 fingerprint groups. The occurrence of genes coding for S. aureus surface proteins (clfA, clfB, fnbA, fnbB, fib, cna) and biofilm formation (icaA, icaD, icaC, bap) and in vitro biofilm-forming ability was not related to strain clinical origin (NP or P). Internalization of S. aureus into MAC-T cells was strain-dependent and internalized bacteria overexpressed adherence and biofilm-forming genes compared with those that remained in the supernatant of co-cultures; particularly those genes encoding FnBPs and IcaD. Strains yielding highest invasion percentages were those able to overexpress fnBP, irrespectively of the presence of other evaluated genes. Strains from NP IMI showed a greater multiplication capacity in vitro compared with strains from P IMI. These results provide new insights about S. aureus differential gene expression of adhesion-internalization factors during early interaction with mammary epithelial cells.

239 MAC-T CELLS AS A TOOL TO EXAMINE GENOME EDITING USING CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)

In 2050, the expected size of the human population is 9 billion, the demand for food will increase, and the demand for milk will increase along with it. Genetically modifying animals is a tool that can be used to meet this growing demand. In the United States, Holstein is the leading breed for milk production and Holsteins produce on average 24 291 pounds of milk per year, whereas Jerseys, the other major dairy breed, produce on average 16 997 pounds. Their ability to produce large quantities of milk is linked to 2 mutations. These mutations are on the α-lactalbumin (α-lac) gene; the α-lac exon (+1) corresponds to the transcription start point of α-lac, (+15) and (–1689) are the positions corresponding to the single nucleotide polymorphism associated with increased milk production. Holstein cows have an adenine at both of these positions in contrast to the other cattle breeds with lower milk production, which have either a cytosine or guanine at either position. Inserting an adenine at position (+15) and (–1689) in cows without this mutation could lead to increased milk production and a better response to market demands. The purpose of this experiment was to test the cutting efficiency of candidate clustered regularly interspaced short palindromic repeats (CRISPR) that will later be used in knock-in experiments. CRISPRs were used because the CRISPR-Cas9 system is inexpensive, easily programmed, and efficient. In this preliminary study, we worked with Holstein MAC-T cells, which already contain the mutation at both positions. CRISPRs were used on this cell line to cut the DNA at a site near the mutation. Based on the genomic DNA sequence of these MAC-T cells, 3 guide RNAs were designed. Cells were then transfected with the designed CRISPRs by a variety of transfection methods, including Fugene™ (Promega, Madison, WI, USA), electroporation, and Lipofectamine (ThermoFisher Scientific, Waltham, MA, USA). Green fluorescent protein was used to determine the efficiency of transfection; 30% efficiency was seen for Fugene™, whereas electroporation and Lipofectamine™ had 70% efficiency. To select for successfully transfected cells, puromycin selection was applied. The DNA was later extracted and sent in for sequencing. Next, the website TIDE was used to compare the transfected MAC-T cells to normal MAC-T cells. The TIDE software measures the editing efficiency and looks for major insertions or deletions in pools of DNA by comparing 2 sequences to quantify the editing efficacy of CRISPR-Cas9. Our results showed that CRISPRs successfully cut the DNA near the α-lac mutation region with a total efficiency of 13.8%. The desired next step will be to insert a single-strand oligonucleotide (ssODN) donor to make a single basepair mutation. The ultimate aim of this research would be to insert these mutations into other cattle species in order to increase their milk production.

Phytoncide Extracted from Pinecone Decreases LPS-Induced Inflammatory Responses in Bovine Mammary Epithelial Cells.

Mastitis is a prevalent inflammatory disease that remains one of the main causes of poor quality of milk. Phytoncides are naturally occurring anti-inflammatory compounds derived from plants and trees. To determine if treatment with phytoncide could decrease the severity of lipopolysaccharide (LPS)-induced inflammatory responses, mammary alveolar epithelial cells (MAC-T) were pretreated with phytoncide (0.02% and 0.04% (v/v)) followed by LPS treatment (1 and 25 μg/ml). The results demonstrated that phytoncide downregulated LPS-induced pro-inflammatory cyclooxygenase-2 (COX-2) expression. Additionally, LPS-induced activation of ERK1/2, p38, and Akt was attenuated by phytoncide. Treatment of cells with known pharmacological inhibitors of ERK1/2 (PD98059), p38 (SB203580), and Akt (LY294002) confirmed the association of these signaling pathways with the observed alterations in COX-2 expression. Moreover, phytoncide attenuated LPS-induced NF-κB activation and superoxide production, and, finally, treatment with phytoncide increased Nrf2 activation. Results suggest that phytoncide can decrease LPS-induced inflammation in MAC-T cells.

Chemerin is a novel regulator of lactogenesis in bovine mammary epithelial cells

Chemerin is a chemoattractant cytokine (chemokine) produced by adipocytes and hepatocytes; it regulates insulin sensitivity and adipocyte differentiation. The objective of this study was to investigate the effect of chemerin on the expression of genes related to lactogenesis and the regulators of chemerin signaling in a bovine mammary epithelial cell line (MAC-T). Two types of chemerin receptors, chemokine like-receptor 1 (CMKLR1) and chemokine (C–C motif) receptor-like 2 (CCRL2), were detected in cultured MAC-T cells, whereas chemerin was not detected. G protein-coupled receptor 1 (GPR1), another receptor of chemerin, was undetectable in MAC-T cells. Chemerin upregulated transcript expression of CMKLR1, CCRL2, and genes associated with fatty acid synthesis, glucose uptake, insulin signaling, and casein synthesis in MAC-T cells. Lactogenic hormones (insulin, growth hormone, and prolactin) downregulated the expression of CMKLR1 in MAC-T cells. Adiponectin suppressed CMKLR1 expression. TNF-α suppressed CMKLR1, but induced CCRL2 expression. These data suggest chemerin is a novel regulator of lactogenesis via its own receptor in bovine mammary epithelial cells.

Proteomic Analysis Reveals PGAM1 Altering cis-9, trans-11 Conjugated Linoleic Acid Synthesis in Bovine Mammary Gland.

cis-9, trans-11 Conjugated linoleic acid (CLA) is one of the most extensively studied CLA isomers due to its multiple isomer-specific effects. However, the molecular mechanisms of cis-9,trans-11 CLA synthesis in ruminant mammary gland are still not clearly understood. This process may be mediated, to a certain extent, by trans-11 C18:1 regulated by stearoyl-CoA desaturase-1 (SCD1) and/or its syntrophic proteins. This study aimed to investigate the effects of TVA on SCD1-mediated cis-9,trans-11 CLA synthesis in MAC-T cells and its potential molecular mechanism. Results showed that trans-11 C18:1 was continually taken up and converted into cis-9,trans-11 CLA in MAC-T cells during the 4-h incubation of 50μM trans-11 C18:1. SCD1 protein expression increased more than twofold at 2h (P<0.01) and 2.5h (P<0.05) before decreasing to less than half of the normal level at 4h (P<0.05). One up-regulated (RAS guanyl releasing protein 4 isoform 1 [RASGRP4]) and six down-regulated proteins (glucosamine-6-phosphate deaminase 1 [GNPDA1], triosephosphate isomerase [TPI1], phosphoglycerate mutase 1 [PGAM1], heat shock protein beta-1 [HSPB1], annexin A3 [ANXA3], thiopurine S-methyltransferase [TPMT]) were found in MAC-T cells treated with trans-11 C18:1. Of these seven identified proteins, the presence of GNPDA1 and PGAM1 was verified in several models. More trans-11 C18:1 was taken up after PGAM1 knockdown by small interfering RNA (siRNA). In conclusion, our data suggested that PGAM1 may have a negative relationship with SCD1 and seemed to be involved in cis-9, trans-11 CLA synthesis by facilitating the absorption of trans-11 C18:1 in the bovine mammary gland.

MAC-T Cells as a Tool to Evaluate Lentiviral Vector Construction Targeting Recombinant Protein Expression in Milk

Prior to generating transgenic animals for bioreactors, it is important to evaluate the vector constructed to avoid poor protein expression. Mammary epithelial cells cultured in vitro have been proposed as a model to reproduce the biology of the mammary gland. In the present work, three lentiviral vectors were constructed for the human growth hormone (GH), interleukin 2 (IL2), and granulocyte colony-stimulating factor 3 (CSF3) genes driven by the bovine β-casein promoter. The lentiviruses were used to transduce mammary epithelial cells (MAC-T), and the transformed cells were cultured on polystyrene in culture medium with and without prolactin. The gene expression of transgenes was evaluated by PCR using cDNA, and recombinant protein expression was evaluated by Western-blotting using concentrated medium and cellular extracts. The gene expression, of the three introduced genes, was detected in both induced and non induced MAC-T cells. The human GH protein was detected in the concentrated medium, whereas CSF3 was detected in the cellular extract. Apparently, the cellular extract is more appropriate than the concentrated medium to detect recombinant protein, principally because concentrated medium has a high concentration of bovine serum albumin. The results suggest that MAC-T cells may be a good system to evaluate vector construction targeting recombinant protein expression in milk.

Functional role of antibodies generated in heifers through immunization with Staphylococcus aureus vaccines in invasion and phagocytosis assays.

A successful Staphylococcus aureus vaccine should elicit a long-term antibody response that prevents establishment of the infection. The aim of the present study was to evaluate the functional role of antibodies raised against different S. aureus CP5 vaccines in invasion to bovine mammary epithelial cells (MAC-T) and phagocytosis by bovine milk macrophages in vitro. Sera and whey from cows immunized with a whole-cell S. aureus CP5 vaccine adjuvanted with Al(OH)3 or with ISCOM Matrix, significantly reduced internalization of S. aureus in MAC-T cells without significant differences between both groups. The effect of antibodies generated by a S. aureus whole-cell and a lysate vaccine formulated with ISCOM Matrix was also evaluated. Sera and whey from both immunized groups significantly reduced S. aureus internalization in MAC-T cells without significant differences between both groups. Whey antibodies against whole-cell and lysate vaccines were also able to inhibit internalization in MAC-T cells of a heterologous S. aureus strain. In addition, sera from animals vaccinated with S. aureus lysate or bacterin promoted milk macrophage phagocytosis. These results provide an insight into the potential mechanisms by which these vaccines can afford protection to the mammary gland against S. aureus intramammary infection.

Biofilm formation on biotic and abiotic surfaces in the presence of antimicrobials by Escherichia coli Isolates from cases of bovine mastitis.

Escherichia coli is a highly adaptive microorganism, and its ability to form biofilms under certain conditions can be critical for antimicrobial resistance. The adhesion of four E. coli isolates from bovine mastitis to bovine mammary alveolar (MAC-T) cells, biofilm production on a polystyrene surface, and the expression profiles of the genes fliC, csgA, fimA, and luxS in the presence of enrofloxacin, gentamicin, co-trimoxazole, and ampicillin at half of the MIC were investigated. Increased adhesion of E. coli isolates in the presence of antimicrobials was not observed; however, increased internalization of some isolates was observed by confocal microscopy. All of the antimicrobials induced the formation of biofilms by at least one isolate, whereas enrofloxacin and co-trimoxazole decreased biofilm formation by at least one isolate. Quantitative PCR analysis revealed that all four genes were differentially expressed when bacteria were exposed to subinhibitory concentrations of antimicrobials, with expression altered on the order of 1.5- to 22-fold. However, it was not possible to associate gene expression with induction or reduction of biofilm formation in the presence of the antimicrobials. Taken together, the results demonstrate that antimicrobials could induce biofilm formation by some isolates, in addition to inducing MAC-T cell invasion, a situation that might occur in vivo, potentially resulting in a bacterial reservoir in the udder, which might explain some cases of persistent mastitis in herds.

Inhibition of growth and alteration of host cell interactions of Pasteurella multocida with natural byproducts

Pasteurella multocida is a leading cause of fowl cholera in both free-range pasture and conventional/commercially raised poultry. Its infection is a serious threat to poultry health and overall flock viability. Organic poultry is comparatively more vulnerable to this pathogen. It is a significant cause of production loss and price increase of poultry products, specifically organic poultry products. Some plant products are well documented as sources of natural antimicrobials such as polyphenols found in different berry pomaces and citrus oil. Pomace, a byproduct (primarily of seeds and skins) of fruits used for juice and wine production, and citrus oil, the byproduct of citrus juice production, show promising antimicrobial activity against various pathogens. Here, we showed for the first time that blackberry and blueberry pomace extracts and citrus oil inhibited P. multocida growth. Minimum bactericidal concentrations were determined as 0.3 and 0.4 mg/mL gallic acid equivalent for blackberry and blueberry pomace extracts, respectively. Similarly, only 0.05% citrus oil (vol/vol) completely inhibited P. multocida growth. Under shaking conditions, the antimicrobial activity of both pomace extracts and citrus oil was more intensive. Even citrus oil vapor also significantly reduced the growth of P. multocida. In addition, cell surface hydrophobicity of P. multocida was increased by 2- to 3-fold and its adherence to chicken fibroblast (DF1) and bovine mammary gland (MacT) cells was reduced significantly in the presence of pomace extracts only. This study indicates that these natural products might be good alternatives to conventional antimicrobial agents, and hence, may be used as feed or water supplements to control fowl cholera and reduce production loss caused by P. multocida.

Gene expression profiling of bovine mammary gland epithelial cells stimulated with lipoteichoic acid plus peptidoglycan from Staphylococcus aureus.

A Gram-positive bacterium, Staphylococcus aureus is known to be one of the major pathogenic bacteria responsible for causing bovine mastitis. Among the various cell wall components of S. aureus, lipoteichoic acid (LTA) and peptidoglycan (PGN) are closely associated with inflammatory responses. However, the role of LTA and PGN derived from S. aureus in bovine mastitis has not been clearly elucidated. In this study, we characterized the gene expression profile of a bovine mammary gland epithelial cell line, MAC-T cells, in the presence of LTA and PGN from S. aureus. LTA plus PGN, but not LTA or PGN alone, activated MAC-T cells. The analysis of transcriptional profiles using an Affymetrix genechip microarray showed that stimulation with LTA plus PGN produced a total of 2019 (fold change >1.2) differentially expressed genes (DEGs), with 801 up-regulated genes and 1218 down-regulated genes. Of the up-regulated genes, 14 inflammatory mediator-related DEGs, 22 intra-cellular signaling molecule-related DEGs, and 15 transcription factor-related DEGs were observed, whereas among the down-regulated DEGs 17 inflammation-related DEGs were found. The microarray results were confirmed using real-time RT-PCR of 18 genes with substantial changes in expression (9 each from the up-regulated and down-regulated DEGs). These results provide a comprehensive analysis of gene-expression profiles elicited by S. aureus LTA and PGN in MAC-T cells, contributing to an understanding of the pathogenesis for S. aureus-induced bovine mastitis.

Short communication: Expression of T-box 2 and 3 in the bovine mammary gland

To increase our understanding of the mechanisms by which growth hormone (GH) and insulin-like growth factor (IGF)-I influence bovine mammary gland development, the potential roles of T-box2 (TBX2) and T-box3 (TBX3) were investigated. Although no information regarding expression of either transcription factor in the bovine mammary gland exists, it is known that TBX3 and its closely related family member, TBX2, are required for mammary gland development in humans and mice. Additionally, TBX3 mutations in humans and mice lead to ulnar mammary syndrome. Evidence is present in bone that TBX3 is required for proliferation and its expression is regulated by GH, an important regulator of mammary gland development and milk production. We hypothesized that TBX2 and TBX3 are expressed in the bovine mammary gland and that GH, IGF-I, or both increase TBX2 and TBX3 expression in bovine mammary epithelial cells (MEC). Bovine mammary gland tissue, MAC-T cells, primary MEC, and fibroblasts were obtained and TBX2 and TBX3 expression was determined by real-time reverse transcription PCR. In addition, TBX2 and TBX3 expression was examined in cells treated with 100 or 500ng/mL of GH or 100 or 200ng/mL of IGF-I for 24 or 48h. Both TBX2 and TBX3 were expressed in bovine mammary tissue. Surprisingly, expression of TBX2 was only detected in mammary fibroblast cells, whereas TBX3 was expressed in all 3 cell types. Growth hormone did not alter TBX3 expression in MAC-T cells or MEC. However, IGF-I increased TBX3 expression in MAC-T, but not in primary MEC. We did not observe a change in TBX2 or TBX3 expression in fibroblasts treated with GH and IGF. Therefore, we concluded that (1) TBX2 and TBX3 are expressed in bovine mammary gland, (2) their expression is cell-type specific, and (3) IGF-I stimulates TBX3 expression in MAC-T cells.

Transcriptome microRNA profiling of bovine mammary epithelial cells challenged with Escherichia coli or Staphylococcus aureusbacteria reveals pathogen directed microRNA expression profiles

BackgroundMicroRNAs (miRNAs) can post-transcriptionally regulate gene expression and have been shown to be critical regulators to the fine-tuning of epithelial immune responses. However, the role of miRNAs in bovine responses to E. coli and S. aureus, two mastitis causing pathogens, is not well understood.ResultsThe global expression of miRNAs in bovine mammary epithelial cells (MAC-T cells) challenged with and without heat-inactivated Staphylococcus aureus (S. aureus) or Escherichia coli (E. coli) bacteria at 0, 6, 12, 24, and 48 hr was profiled using RNA-Seq. A total of 231 known bovine miRNAs were identified with more than 10 counts per million in at least one of 13 libraries and 5 miRNAs including bta-miR-21-5p, miR-27b, miR-22-3p, miR-184 and let-7f represented more than 50% of the abundance. One hundred and thirteen novel miRNAs were also identified and more than one third of them belong to the bta-miR-2284 family. Seventeen miRNAs were significantly (P < 0.05) differentially regulated by the presence of pathogens. E. coli initiated an earlier regulation of miRNAs (6 miRNAs differentially regulated within the first 6 hrs post challenge as compared to 1 miRNA for S. aureus) while S. aureus presented a delayed response. Five differentially expressed miRNAs (bta-miR-184, miR-24-3p, miR-148, miR-486 and let-7a-5p) were unique to E. coli while four (bta-miR-2339, miR-499, miR-23a and miR-99b) were unique to S. aureus. In addition, our study revealed a temporal differential regulation of five miRNAs (bta-miR-193a-3p, miR-423-5p, miR-30b-5p, miR-29c and miR-un116) in unchallenged cells. Target gene predictions of pathogen differentially expressed miRNAs indicate a significant enrichment in gene ontology functional categories in development/cellular processes, biological regulation as well as cell growth and death. Furthermore, target genes were significantly enriched in several KEGG pathways including immune system, signal transduction, cellular process, nervous system, development and human diseases.ConclusionUsing next-generation sequencing, our study identified a pathogen directed differential regulation of miRNAs in MAC-T cells with roles in immunity and development. Our study provides a further confirmation of the involvement of mammary epithelia cells in contributing to the immune response to infecting pathogens and suggests the potential of miRNAs to serve as biomarkers for diagnosis and development of control measures.

Associations between properties linked with persistence in a collection of Staphylococcus aureus isolates from bovine mastitis

Staphylococcus aureus is recognized worldwide as a pathogen causing many serious diseases in humans and animals, and is one of the most important etiological agents of clinical and subclinical bovine mastitis. The aim of the present study was to investigate and correlate properties, that may be associated with persistent mastitis, of S. aureus strains isolated from milk of cows suffering from mastitis: (i) expression of capsular antigens (CP5 or CP8) by specific ELISA; (ii) intracellular survival by invasion of MAC-T cells; and (iii) biofilm production by spectrophotometry analysis after growth in TSBglc. The results showed that (i) the proportion of strains expressing capsular antigen was higher in cap8- than in cap5-positive isolates; (ii) a correlation was observed between the capsular profile and the intracellular survival as well as the biofilm production; and (iii) the capsular profile, biofilm production and intracellular survival were associated with only two agr-groups. Statistical and clustering analysis allowed us to establish different profiles that could be associated with in vivo persistence. Indeed, isolates belonging to agr group II, expressing the capsular antigen CP8 and showing low intracellular survival are probably better adapted to an extracellular niche. Conversely, isolates belonging to agr group I that do not express any capsular antigen (CP5 or CP8) but show high intracellular survival are probably better adapted to an intracellular niche.

Biofilm mediates Enterococcus faecalis adhesion, invasion and survival into bovine mammary epithelial cells

We proposed in this study that during intramammary infection, biofilm formation may facilitate adherence and colonization of Enterococcus faecalis to mammary gland epithelium. This was established by comparing six different Ent.faecalis isolates with different biofilm-forming profiles for their adhesive, invasive and survival capabilities to bovine mammary epithelial cell line (MAC-T). Our results showed increased ability of the biofilm-producer Ent.faecalis strains to adhere, invade and survive inside MAC-T cells rather than nonbiofilm-producer strains. We showed that growth of bacteria in bovine milk significantly augmented the adherence and invasion of all tested strains, and this feature was abolished again when strains were subcultured in brain heart infusion broth. Moreover, growth in bovine milk significantly increased biofilm formation by all tested strains. These results indicated that biofilm formation by Ent.faecalis, especially after expressing milk-dependent induction, may have special relevance in the pathogenesis of Ent.faecalis mastitis during intramammary infection by enhancing bovine mammary epithelial adhesion and colonization. Results obtained from current work highlighted the role of biofilm in the pathogenesis of Enterococcus faecalis mastitis. Those biofilm-forming strains might be substantial as useful antigens in diagnostic assays and as future vaccine candidates to control Ent.faecalis mastitis.

Effects of AMP-activated protein kinase (AMPK) signaling and essential amino acids on mammalian target of rapamycin (mTOR) signaling and protein synthesis rates in mammary cells

Regulation of mammary protein synthesis potentially changes the relationships between AA supply and milk protein output represented in current nutrient requirement models. Glucose and AA regulate muscle protein synthesis via cellular signaling pathways involving mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK). The objective of this study was to investigate the effects of essential AA (EAA) and acetate or glucose on mTOR and AMPK signaling pathways and milk protein synthesis rates. A bovine mammary epithelial cell line, MAC-T, was subjected to different media containing 0 or 3.5mmol/L EAA concentrations with 0 or 5mmol/L acetate or 0 or 17.5mmol/L glucose in 2 separate 2×2 factorial studies. In a separate set of experiments, lactogenic bovine mammary tissue slices were subjected to the same treatments except that the low EAA treatment contained a low level of EAA (0.18mmol/L). Supplementation of EAA enhanced phosphorylation of mTOR (Ser2448) and eukaryotic initiation factor 4E binding protein 1 (4EBP1, Thr37/46), and reduced phosphorylation of eukaryotic elongation factor 2 (eEF2, Thr56) in MAC-T cells. Concentration of ATP and phosphorylation of AMPK increased and decreased, respectively, in the presence of EAA in MAC-T cells. Acetate, EAA, or glucose numerically reduced AMPK phosphorylation by about 16% in mammary tissue slices. Provision of EAA increased phosphorylation of mTOR and 4EBP1, intracellular total EAA concentration, and casein synthesis rates in mammary tissue slices, irrespective of the presence of acetate or glucose in the medium. Phosphorylation of mTOR had a marginally negative association with AMPK phosphorylation, which was positively related to eEF2 phosphorylation. Casein synthesis rates were positively and more strongly linked to mTOR phosphorylation than the negative link between eEF2 phosphorylation and casein synthesis rates. A 100% increase in mTOR phosphorylation was associated with an increase in the casein synthesis rate of 0.74%·h−1, whereas a 100% increase in eEF2 phosphorylation was related to a decline in the casein synthesis rate of 0.33%·h−1. Although AMPK phosphorylation was responsive to cellular energy status and had a negative effect on mTOR-mediated signals in bovine mammary epithelial cells, its effect on milk protein synthesis rates appeared to be marginal compared with the mTOR-mediated regulation of milk protein synthesis by EAA.