Use and relevance of a bovine mammary gland explant model to study infection responses in bovine mammary tissue

2006 ◽  
Vol 74 (1) ◽  
pp. 93-99 ◽  
Author(s):  
Aline Rabot ◽  
Olga Wellnitz ◽  
Heinrich H D Meyer ◽  
Rupert M Bruckmaier
Keyword(s):  
Mammary Gland ◽  
Mrna Expression ◽  
Early Response ◽  
Mammary Epithelial ◽  
Mammary Tissue ◽  
Bovine Mammary Gland ◽  
Bovine Mammary Epithelial Cells ◽  
Lps Challenge ◽  
Tnf Α ◽  
Immunoglobulin Receptors

Our aim was to develop an explant model to define more precisely the early response of bovine mammary epithelial cells to infection. Therefore we investigated the mRNA expression encoding for some soluble immunological factors in lipopolysaccharide (LPS)-treated bovine mammary gland explants. Explants were taken out from the mammary gland of eight lactating cows after slaughter then incubated with LPS (10 μg/ml) for 6 h. The mRNA expression of α-lactalbumin (α-la), various cytokines, tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, and two immunoglobulin receptors, the neonatal Fc receptor (FcRn) and polymeric immunoglobulin receptor (pIGR), were assessed with qPCR before and after 3 h and 6 h of LPS challenge. Both immunoglobulin receptors and α-la increased at 3 h then recovered their initial level at 6 h whereas IL-1β, IL-6 and IL-8 increased only after 6 h (P<0·05). Surprisingly, TNF-α transcripts did not show any regulation in response to the LPS treatment. We nevertheless concluded that our model was valid to examine the short-term response of mammary epithelial cell challenged with LPS.

Bovine mammary epithelial cells, initiators of innate immune responses to mastitis

2005 ◽  
Vol 45 (8) ◽  
pp. 757 ◽  
Author(s):  
C. Gray ◽  
Y. Strandberg ◽  
L. Donaldson ◽  
R. L. Tellam
Keyword(s):  
Immune Response ◽  
Mammary Gland ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Vital Role ◽  
Mammary Epithelial ◽  
Innate Immune ◽  
Mammary Tissue ◽  
Effector Cells ◽  
Bovine Mammary Epithelial Cells

Innate immunity plays a vital role in the protection of the bovine mammary gland against mastitis. Until recently, the migration of effector cells such as neutrophils and monocytes into the mammary gland was thought to provide the only defence against invading pathogens. However, mammary epithelial cells may also play an important role in the immune response, contributing to the innate defence of the mammary tissue through secretion of antimicrobial peptides and attraction of circulating immune effector cells. This paper reviews the innate immune pathways in mammary epithelial cells and examines their role in the initiation of an innate immune response to Gram-positive and Gram-negative bacteria.

scholarly journals Expression and localisation of oestrogen and progesterone receptors in the bovine mammary gland during development, function and involution

2003 ◽  
Vol 177 (2) ◽  
pp. 305-317 ◽  
Author(s):  
D Schams ◽  
S Kohlenberg ◽  
W Amselgruber ◽  
B Berisha ◽  
MW Pfaffl ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Epithelial Cells ◽  
Mrna Expression ◽  
Western Blotting ◽  
Mammary Gland Development ◽  
Mammary Tissue ◽  
Positive Staining ◽  
Bovine Mammary Gland ◽  
Total Rna ◽  
Gland Development

It is now well established that oestrogen and progesterone are absolutely essential for mammary gland development. Lactation can be induced in non-pregnant animals by sex steroid hormone treatment. Most of the genomic actions of oestrogens are mediated by two oestrogen receptors (ER)-alpha and ERbeta, and for gestagens in ruminants by the progesterone receptor (PR). Our aim was the evaluation of mRNA expression and protein (localisation and Western blotting) during mammogenesis, lactogenesis, galactopoiesis (early, middle and late) and involution (8, 24, 28, 96-108 h and 14-28 days after the end of milking) in the bovine mammary gland (total no. 53). During these stages, the mRNA was assessed by means of real-time RT-PCR (LightCycler). The protein for ERalpha, ERbeta and PR was localised by immunohistochemistry and Western blotting. The mRNA expression results indicated the existence of ERalpha, ERbeta and PR in bovine mammary gland. Both ERalpha and PR are expressed in fg/ micro g total RNA range. The highest mRNA expression was found for ERalpha and PR in the tIssue of non-pregnant heifers, followed by a significant decrease to a lower level at the time of lactogenesis with low concentrations remaining during lactation and the first 4 weeks of involution. In contrast, the expression of ERbeta was about 1000-fold lower (ag/ micro g total RNA) and showed no clear difference during the stages examined, with a significant increase only 2-4 weeks after the end of milking. Immunolocalisation for ERalpha revealed a strong positive staining in nuclei of lactocytes in non-pregnant heifers, became undetectable during pregnancy, lactogenesis and lactation, and was again detectable 14-28 days after the end of milking. In contrast, PR was localised in the nuclei of epithelial cells in the mammary tIssue of non-pregnant heifers, in primigravid animals, and during late lactation and involution. During lactogenesis, peak and mid lactation, fewer nuclei of epithelial cells were positive, but increased staining of the cytoplasm of epithelial cells was obvious. ERalpha and ERbeta protein was found in all mammary gland stages examined by Western blotting. In contrast to mRNA expression, the protein signal for ERalpha was weaker in the tIssue of non-pregnant heifers and during involution (4 weeks). ERbeta protein showed a stronger signal (two isoform bands) in non-pregnant heifers and 4 weeks after the end of milking. This correlated with the mRNA expression data. Three isoforms of PR (A, B and C) were found by Western blotting in the tIssue of non-pregnant heifers, but only isoform B remained during the following stages (lactogenesis, galactopoiesis and involution). In conclusion, the mRNA expression and protein data for ER and PR showed clear regulatory changes, suggesting involvement of these receptors in bovine mammary gland development and involution.

scholarly journals The bovine mammary gland expresses multiple functional isoforms of serotonin receptors

2009 ◽  
Vol 203 (1) ◽  
pp. 123-131 ◽  
Author(s):  
Laura L Hernandez ◽  
Sean W Limesand ◽  
Jayne L Collier ◽  
Nelson D Horseman ◽  
Robert J Collier
Keyword(s):  
Mammary Gland ◽  
Epithelial Cell ◽  
Mrna Expression ◽  
Mammary Epithelial Cell ◽  
Caspase 3 ◽  
Mammary Epithelial ◽  
Bovine Mammary Gland ◽  
Functional Activities ◽  
Receptor Isoforms ◽  
Human And Mouse

Recent studies in dairy cows have demonstrated that serotonergic ligands affect milk yield and composition. Correspondingly, serotonin (5-HT) has been demonstrated to be an important local regulator of lactational homeostasis and involution in mouse and human mammary cells. We determined the mRNA expression of bovine 5-HT receptor (HTR) subtypes in bovine mammary tissue (BMT) and used pharmacological agents to evaluate functional activities of 5-HT receptors. The mRNAs for five receptor isoforms (HTR1B, 2A, 2B, 4, and 7) were identified by conventional real-time (RT)-PCR, RT quantitative PCR, and in situ hybridization in BMT. In addition to luminal mammary epithelial cell expression, HTR4 was expressed in myoepithelium, and HTR1B, 2A, and 2B were expressed in small mammary blood vessels. Serotonin suppressed milk protein mRNA expression (α-lactalbumin and β-casein mRNA) in lactogen-treated primary bovine mammary epithelial cell (BMEC) cultures. To probe the functional activities of individual receptors, caspase-3 activity and expression of α-lactalbumin and β-casein were measured. Both SB22489 (1B antagonist) and ritanserin (2A antagonist) increased caspase-3 activity. Expression of α-lactalbumin and β-casein mRNA levels in BMEC were stimulated by low concentrations of SB224289, ritanserin, or pimozide. These results demonstrate that there are multiple 5-HT receptor isoforms in the bovine mammary gland, and point to profound differences between serotonergic systems of the bovine mammary gland and the human and mouse mammary glands. Whereas human and mouse mammary epithelial cells express predominately the protein for the 5-HT7 receptor, cow mammary epithelium expresses multiple receptors that have overlapping, but not identical, functional activities.

Lycium barbarum polysaccharides alleviate LPS-induced inflammatory responses through PPARγ/MAPK/NF-κB pathway in bovine mammary epithelial cells

2021 ◽  
Author(s):  
Tianle Xu ◽  
Run Liu ◽  
Xubin Lu ◽  
Xinyue Wu ◽  
Petr Heneberg ◽  
...  
Keyword(s):  
Protective Effect ◽  
Inflammatory Responses ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Protein Abundance ◽  
Lycium Barbarum ◽  
Bovine Mammary Epithelial Cells ◽  
Lps Challenge ◽  
Tnf Α ◽  
Cox 2

Abstract As the main component of the gram-negative bacterial cell wall, lipopolysaccharide (LPS) is well-documented as an inducer of inflammation in bovine mammary cells. Lycium barbarum (goji) polysaccharides (LBP) have been used in non-ruminants as prebiotics to improve growth performance, immune ability and antioxidant capacity. We aimed to investigate the underlying effects of LBPs on pro-inflammatory responses in LPS-stimulated primary bovine mammary epithelial cells (bMECs). Cells were isolated from mammary tissue of 3 lactating Holstein cows without clinical disease (30.26 ± 3.1 kg/d of milk yield; 175 ± 6 DIM). For the pre-experimental treatment, bMECs were precultured with serum-free medium for 12 h. Treatments were as follows: pretreatment with culture medium devoid of LPS or LBP for 30 h (CON); CON for 24 h followed by challenge with 2 μg/mL LPS for 6 h (LPS); pretreatment with 100 μg/mL or 300 μg/mL LBP for 24 h followed by LPS challenge (2 μg/mL) for 6 h (LBP(100)+LPS; LBP(300)+LPS). To further determine if the effect of LBP on immune-regulation is PPARγ activation-dependent, an inhibitor of PPARγ, GW9662, at a concentration of 1 μM was used. Cells treated with LBP at 100, 300 and 500 μg/mL had upregulated protein abundance of PPARγ, while PGC1α had a higher expression only at 300 μg/mL of LBP treatment. Compared with CON, cells pretreated with LBP at 100 and 300 μg/mL had greater protein abundance of SCD1 and SREBP1. EdU staining and cell wound healing assays showed that the negative effect of LPS alone on cell proliferation was reversed by pretreatment with LBP at both 100 and 300 μg/mL. Upregulation of gene and protein abundance of proinflammatory factors and cytokines (COX-2, NLRP3, TNF-α, IL-1β and IL-6) induced by LPS stimulation were alleviated by LBP pretreatment at 300 μg/mL (more than 2-fold decrease). Compared with LPS challenge alone, phosphorylation of proteins involved in NF-κB (IκBα and p65) and MAPK (p38, JNK and ERK) pathways was downregulated following LBP treatment. Additionally, inhibition of PPARγ by GW9662 weakened the protective effect of LBP on LPS-induced protein abundance of phosphorylated p65, COX-2, IL-1β and TNF-α. These results indicated that the protective effect of LBP on LPS-induced bMECs inflammatory responses is PPARγ activation-dependent. As such, this knowledge might help design strategies for intervening against the detrimental effects of bovine mastitis.

Exogenous phospholipase A2 affects inflammatory gene expression in primary bovine mammary epithelial cells

2019 ◽  
Vol 86 (2) ◽  
pp. 177-180
Author(s):  
Jacqueline P. Kurz ◽  
Mark P. Richards ◽  
Matthew Garcia ◽  
Zhongde Wang
Keyword(s):  
Epithelial Cells ◽  
Phospholipase A2 ◽  
Inflammatory Responses ◽  
Mammary Epithelial Cells ◽  
Constitutive Expression ◽  
Mammary Epithelial ◽  
Inflammatory Factors ◽  
Bovine Mammary Epithelial Cells ◽  
Lps Challenge

AbstractThis Research Communication addresses the hypothesis that exogenously administered phospholipase A2 (PLA2) affects the inflammatory responses of bovine mammary epithelial cells (bMEC) in vitro with the aim of providing preliminary justification of investigation into the uses of exogenously administered PLA2 to manage or treat bovine mastitis. Primary bMEC lines from 11 lactating Holstein dairy cows were established and the expression of 14 pro-inflammatory genes compared under unchallenged and lipopolysaccharide (LPS)-challenged conditions, with and without concurrent treatment with bovine pancreatic PLA2G1B, a secreted form of PLA2. No differences in the expression of these genes were noted between PLA2-treated and untreated bMEC under unchallenged conditions. Following LPS challenge, untreated bMEC exhibited significant downregulation of CXCL8, IL1B, CCL20, and CXCL1. In contrast, PLA2-treated bMEC exhibited significant downregulation of IL1B and CCL20 only. These findings indicate that exogenous PLA2 affects the expression of some pro-inflammatory factors in immune-stimulated bMEC, but does not influence the constitutive expression of these factors. Further investigation of the influence of exogenous PLA2 in the bovine mammary gland is justified.

scholarly journals MiR-125b regulates inflammation in bovine mammary epithelial cells by targeting the NKIRAS2 gene

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Zhuo-Ma Luoreng ◽  
Da-Wei Wei ◽  
Xing-Ping Wang
Keyword(s):  
T Cells ◽  
Epithelial Cells ◽  
Dairy Cows ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Luciferase Reporter ◽  
Regulation Mechanism ◽  
Bovine Mammary Epithelial Cells ◽  
Tnf Α

AbstractMastitis is a complex inflammatory disease caused by pathogenic infection of mammary tissue in dairy cows. The molecular mechanism behind its occurrence, development, and regulation consists of a multi-gene network including microRNA (miRNA). Until now, there is no report on the role of miR-125b in regulating mastitis in dairy cows. This study found that miR-125b expression is significantly decreased in lipopolysaccharide (LPS)-induced MAC-T bovine mammary epithelial cells. Also, its expression is negatively correlated with the expression of NF-κB inhibitor interacting Ras-like 2 (NKIRAS2) gene. MiR-125b target genes were identified using a double luciferase reporter gene assay, which showed that miR-125b can bind to the 3′ untranslated region (3′ UTR) of the NKIRAS2, but not the 3′UTR of the TNF-α induced protein 3 (TNFAIP3). In addition, miR-125b overexpression and silencing were used to investigate the role of miR-125b on inflammation in LPS-induced MAC-T. The results demonstrate that a reduction in miR-125b expression in LPS-induced MAC-T cells increases NKIRAS2 expression, which then reduces NF-κB activity, leading to low expression of the inflammatory factors IL-6 and TNF-α. Ultimately, this reduces the inflammatory response in MAC-T cells. These results indicate that miR-125b is a pro-inflammatory regulator and that its silencing can alleviate bovine mastitis. These findings lay a foundation for elucidating the molecular regulation mechanism of cow mastitis.

Tea tree oil prevents mastitis-associated inflammation in lipopolysaccharide-stimulated bovine mammary epithelial cells

2020 ◽  
Author(s):  
Zhi Chen ◽  
Yi Zhang ◽  
Jingpeng Zhou ◽  
Yu Tian ◽  
Qiaoni Zhou ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Differentially Expressed ◽  
Quantitative Detection ◽  
Tea Tree Oil ◽  
Tea Tree ◽  
Bovine Mammary Epithelial Cells ◽  
Tnf Α ◽  
Prevention And Treatment

Abstract Background Effective prevention and treatment of cow mastitis can provide a good guarantee for the healthy growth of cows and the qualified production of dairy products. The main purpose of this study was to explore the effect of tea tree oil on lipopolysaccharide (LPS) -induced mastitis in dairy cows, and the key gene in LPS -stimulated bovine mammary epithelial cells (BMECs) was identified. Results In this study, a model of mastitis induced by LPS was constructed, to which tea tree oil and LPS were added. The protective effects of tea tree oil on LPS-induced mastitis in BMECs were verified by the CCK-8 method, flow cytometry, real-time fluorescence quantitative detection, ELISA and other methods. The results showed that LPS at a concentration of 200 μg/ml could reduce the proliferative activity of the cells, induce a high proportion of apoptosis, and promote the expression of TNF-α, IL-6 and STAT1. Upon addition of tea tree oil, the proportion of apoptosis was reduced, and the expression of NF-κB, MAPK and caspase-3 was inhibited. Mammary epithelial cells were compared under control and LPS-treatment conditions and analyzed by second-generation sequencing. A total of 1270 mRNAs were identified as differentially expressed, of which 787 genes were upregulated and 483 were downregulated. These differentially expressed genes include TNF - α, IL6, STAT1, mapk4, etc. H&E staining and immunohistochemistry were used to verify the function of candidate genes. TNF-α and IL6 were observed to play important roles in mediating the preventive effect of tea tree oil on mastitis in LPS-stimulated bovine mammary epithelial cells. Conclusions The results showed that tea tree oil had a protective effect against LPS-induced mastitis. TNF - α and IL6 may be the marker genes of LPS-induced mastitis which provided a theoretical basis and experimental support for further research to determine new strategies for the prevention and treatment of mastitis and improvement of milk quality.

scholarly journals Bacterial Lipopolysaccharide Induced Alterations of Genome-Wide DNA Methylation and Promoter Methylation of Lactation-Related Genes in Bovine Mammary Epithelial Cells

Toxins ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 298 ◽  
Author(s):  
Jingbo Chen ◽  
Yongjiang Wu ◽  
Yawang Sun ◽  
Xianwen Dong ◽  
Zili Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Immune Response ◽  
Mrna Expression ◽  
Promoter Methylation ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Bovine Mammary Epithelial Cells ◽  
Genome Wide ◽  
Immune Response Pathway ◽  
Gene Mrna

Bacterial lipopolysaccharide (LPS) could result in poor lactation performance in dairy cows. High methylation of DNA is associated with gene repression. However, it is unclear whether LPS could suppress the expression of lactation-related genes by inducing DNA methylation. Therefore, the objective of this study was to investigate the impact of LPS on genome-wide DNA methylation, using methylated DNA immunoprecipitation with high-throughput sequencing (MeDIP-seq) and on the promoter methylation of lactation-related genes using MassArray analysis in bovine mammary epithelial cells. The bovine mammary epithelial cell line MAC-T cells were treated for 48 h with LPS at different doses of 0, 1, 10, 100, and 1000 endotoxin units (EU)/mL (1 EU = 0.1 ng). The results showed that the genomic methylation levels and the number of methylated genes in the genome as well as the promoter methylation levels of milk genes increased when the LPS dose was raised from 0 to 10 EU/mL, but decreased after further increasing the LPS dose. The milk gene mRNA expression levels of the 10 EU/mL LPS treatment were significantly lower than these of untreated cells. The results also showed that the number of hypermethylated genes was greater than that of hypomethylated genes in lipid and amino acid metabolic pathways following 1 and 10 EU/mL LPS treatments as compared with control. By contrast, in the immune response pathway the number of hypomethylated genes increased with increasing LPS doses. The results indicate LPS at lower doses induced hypermethylation of the genome and promoters of lactation-related genes, affecting milk gene mRNA expression. However, LPS at higher doses induced hypomethylation of genes involved in the immune response pathway probably in favor of immune responses.

scholarly journals Dose-dependent immune response in milk cells and mammary tissue after intramammary administration of lipopolysaccharide in dairy cows

2008 ◽  
Vol 52 (No. 6) ◽  
pp. 231-244 ◽  
Author(s):  
C. Werner-Misof ◽  
M.W. Pfaffl ◽  
R.M. Bruckmaier
Keyword(s):  
Immune Response ◽  
Mrna Expression ◽  
Real Time ◽  
Polymorphonuclear Neutrophils ◽  
Mammary Tissue ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Lps Challenge ◽  
Cell Counts ◽  
Cox 2

The immune response in milk cells and the status of mammary tight junctions (TJ) in response to intramammary (IM) infusion of different doses of <i>Escherichia col</i>i lipopolysaccharide (LPS) was investigated. <i>Experiment I</i>: Seven German Braunvieh cows were IM infused into one quarter with 1 &mu;g (LPS-1) and 3 &mu;g (LPS-3) of LPS, respectively, and the contralateral control quarter with saline (9 g/l; C). Milk samples were taken immediately before and 12, 24, 36, 48, 60, 84 and 108 h after infusion and analysed for somatic cell counts (SCC), lactose, sodium (Na) and chloride (Cl) ions, and electrical conductivity (EC). Milk cell mRNA expression of various inflammatory factors was quantified by real-time RT-PCR. Blood samples were taken immediately after milking for the analysis of leukocytes (WBC), polymorphonuclear neutrophils (PMN), Na and Cl. Milk SCC, lactose, Na, Cl and EC did not differ significantly between LPS-1 and C quarters after the challenge. In LPS-3 quarters SCC levels increased within the first 12 h, reached peak levels between 12 and 36 h (<i>P</i> &le; 0.001) and decreased (<i>P</i> &le; 0.05) thereafter to reach baseline at 108 hours. Lactose in LPS-3 quarters decreased (<i>P</i> &le; 0.05) to a minimum at 24 h and increased slightly thereafter while EC, Na, and Cl increased transiently in response to LPS-3. WBC and PMN levels in both groups decreased numerically within 24 h after LPS administration. In LPS-1, WBC at 24, 48 and 108 h were significantly lower whereas in LPS-3 they were significantly higher than at time 0. TNF&alpha;-mRNA expression in both groups did not change in response to IM LPS-challenge. IL-1&beta;-mRNA expression at 12, 24 and 36 h in LPS-1 quarters increased significantly as compared to time 0. In LPS-3 quarters the mRNA expression values of all tested ILs increased significantly as compared to time 0 within 12 h after LPS-challenge. IL-1&beta;-mRNA expression decreased (<i>P</i> &le; 0.05) at 48 and 84 h in LPS quarters. IL-8 mRNA was significantly decreased at 84 h after challenge in LPS-3 quarters. COX-2-mRNA expression in LPS-1 quarters decreased significantly as compared to time 0 at 48, 84 and 108 h, with a minimum at 84 h (<i>P</i> &le; 0.05). In LPS-3 quarters COX-2-mRNA levels increased (<i>P</i> &le; 0.05) within 48 h after the LPS-challenge. <i>Experiment II</i>: Six cows (5 German Braunvieh, 1 Brown Swiss) were injected in one quarter with 100 &mu;g LPS and in the contralateral quarter with saline (9 g/l; C). Mammary biopsy samples of both quarters were taken immediately before and at 3, 6, 9 and 12 h after infusion and mRNA expression of TJ proteins occludin (OCLN) and zonula occludens (ZO-) 1, 2 and 3 were quantified by real-time RT-PCR. OCLN-mRNA expression did not change in response to the IM infusion while that of ZO-1, ZO-2 and ZO-3 decreased significantly within six hours. In conclusion, a dose of 1 &mu;g LPS did not initiate a immune response in the mammary gland. Furthermore the dose of 100 &mu;g of LPS enhanced TJ permeability by reducing TJ plaque proteins density.

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