Immune relevant gene expression of mammary epithelial cells and their influence on leukocyte chemotaxis in response to different mastitis pathogens

Different mastitis pathogens induce different courses of infection, i.e. more or less severe. Mammary epithelial cells play an important role in the initial combat against microorganisms by expression of cytokines and acute phase proteins that regulate the immune response. The objective of the present study was to investigate the involvement of the epithelial cells into the outcome of mastitis induced by different pathogens. Primary epithelial cell cultures isolated from milk were used to test the immune response by measuring the mRNA expression of immunomodulators and their influence on polymorph nuclear chemotaxis. Because the cells showed different responses to isolated bacterial endotoxins (lipopolysaccharide, lipoteichoic acid, and peptidoglycans) compared to whole bacteria, they were treated with heat inactivated (10 MOI) gram-negative Escherichia coli, a very common pathogen causing acute intra-mammary infections, with Staphylococcus aureus, a prevalent cause of chronic, and, Streptococcus uberis, an inducer of acute and chronic mastitis. E. coli induced an increased mRNA expression of interleukin (IL)-8 within a 1 h treatment. A treatment for 6 h with E. coli and S. aureus induced increased mRNA expression of IL-6, IL-8, TNF-á and serum amyloid A (SAA). After a 24 h treatment the expression of these immunomodulators was still elevated, except in the E. coli treatment the SAA expression showed no differences to control cells anymore. Interestingly, Str. uberis in the same concentration did only induce the expression of IL-8 after a 6 h treatment but had no influence on other immunomodulator mRNA expression. Cell culture supernatants of E. coli and S. aureus treated cells for 12 h increased leukocyte chemotaxis in a 96-well MultiScreen<sup>TM</sup>MIC-plate. S. aureus seemed to induce increased chemotaxis after shorter treatments than E. coli. In conclusion, mammary epithelial cells are involved in the different immune response to various mastitis pathogens, and the induction of chemotaxis of leukocytes from blood to milk during mastitis. Therefore, most likely epithelial cells play a role in the differential pattern of immunomediators stimulated by different pathogens.

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Effects of Sodium Octanoate on Innate Immune Response of Mammary Epithelial Cells duringStaphylococcus aureusInternalization

BioMed Research International ◽

10.1155/2013/927643 ◽

2013 ◽

Vol 2013 ◽

pp. 1-8 ◽

Cited By ~ 6

Author(s):

Nayeli Alva-Murillo ◽

Alejandra Ochoa-Zarzosa ◽

Joel E. López-Meza

Keyword(s):

Immune Response ◽

Epithelial Cells ◽

Mrna Expression ◽

Innate Immune Response ◽

Mammary Epithelial Cells ◽

Short Chain Fatty Acids ◽

Mammary Epithelial ◽

Innate Immune ◽

Sodium Octanoate ◽

Bovine Mammary Epithelial Cells

Bovine mammary epithelial cells (bMECs) are capable of initiating an innate immune response to invading bacteria. Short chain fatty acids can reduceStaphylococcus aureusinternalization into bMEC, but it has not been evaluated if octanoic acid (sodium octanoate, NaO), a medium chain fatty acid (MCFA), has similar effects. In this study we determined the effect of NaO onS. aureusinternalization into bMEC and on the modulation of innate immune elements. NaO (0.25–2 mM) did not affectS. aureusgrowth and bMEC viability, but it differentially modulated bacterial internalization into bMEC, which was induced at 0.25–0.5 mM (~60%) but inhibited at 1-2 mM (~40%). Also, bMEC showed a basal expression of all the innate immune genes evaluated, which were induced byS. aureus. NaO induced BNBD4, LAP, and BNBD10 mRNA expression, but BNBD5 and TNF-αwere inhibited. Additionally, the pretreatment of bMEC with NaO inhibited the mRNA expression induction generated by bacteria which coincides with the increase in internalization; only TAP and BNDB10 showed an increase in their expression; it coincides with the greatest effect on the reduction of bacterial internalization. In conclusion, NaO exerts a dual effect onS. aureusinternalization in bMEC and modulates elements of innate immune response.

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Microfluidic high-throughput RT-qPCR measurements of the immune response of primary bovine mammary epithelial cells cultured from milk to mastitis pathogens

animal ◽

10.1017/s1751731112002315 ◽

2013 ◽

Vol 7 (5) ◽

pp. 799-805 ◽

Cited By ~ 10

Author(s):

D. Sorg ◽

K. Danowski ◽

V. Korenkova ◽

V. Rusnakova ◽

R. Küffner ◽

...

Keyword(s):

Immune Response ◽

Epithelial Cells ◽

High Throughput ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Bovine Mammary Epithelial Cells ◽

Mastitis Pathogens ◽

Cells Cultured

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Transcriptome Profiling of m6A mRNA Modification in Bovine Mammary Epithelial Cells Treated with Escherichia coli

International Journal of Molecular Sciences ◽

10.3390/ijms22126254 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6254

Author(s):

Ting Li ◽

Changjie Lin ◽

Yifan Zhu ◽

Haojun Xu ◽

Yiya Yin ◽

...

Keyword(s):

Epithelial Cells ◽

Mrna Expression ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Future Research ◽

Infection Model ◽

Common Disease ◽

Bovine Mammary Epithelial Cells ◽

E Coli

Mastitis is a common disease in dairy cows that is mostly caused by E. coli, and it brings massive losses to the dairy industry. N6-Methyladenosine (m6A), a methylation at the N6 position of RNA adenine, is a type of modification strongly associated with many diseases. However, the role of m6A in mastitis has not been investigated. In this study, we used MeRIP-seq to sequence the RNA of bovine mammary epithelial cells treated with inactivated E. coli for 24 h. In this in vitro infection model, there were 16,691 m6A peaks within 7066 mRNA transcripts in the Con group and 10,029 peaks within 4891 transcripts in the E. coli group. Compared with the Con group, 474 mRNAs were hypermethylated and 2101 mRNAs were hypomethylated in the E. coli group. Biological function analyses revealed differential m6A-modified genes mainly enriched in the MAPK, NF-κB, and TGF-β signaling pathways. In order to explore the relationship between m6A and mRNA expression, combined MeRIP-seq and mRNA-seq analyses revealed 212 genes with concomitant changes in the mRNA expression and m6A modification. This study is the first to present a map of RNA m6A modification in mastitis treated with E. coli, providing a basis for future research.

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Escherichia coli and Staphylococcus aureus Differentially Regulate Nrf2 Pathway in Bovine Mammary Epithelial Cells: Relation to Distinct Innate Immune Response

Cells ◽

10.3390/cells10123426 ◽

2021 ◽

Vol 10 (12) ◽

pp. 3426

Author(s):

Yi-Tian Ying ◽

Jing Yang ◽

Xun Tan ◽

Rui Liu ◽

Ying Zhuang ◽

...

Keyword(s):

Immune Response ◽

Epithelial Cells ◽

Inflammatory Response ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Innate Immune ◽

Bovine Mammary Epithelial Cells ◽

E Coli ◽

Nrf2 Activation ◽

And Staphylococcus Aureus

Escherichia coli and Staphylococcus aureus are major mastitis causing pathogens in dairy cattle but elicit distinct immune and an inflammatory response in the udder. However, the host determinants responsible for this difference remains largely unknown. Our initial studies focused on the global transcriptomic response of primary bovine mammary epithelial cells (pbMECs) to heat-killed E. coli and S. aureus. RNA-sequencing transcriptome analysis demonstrates a significant difference in expression profiles induced by E. coli compared with S. aureus. A major differential response was the activation of innate immune response by E. coli, but not by S. aureus. Interestingly, E. coli stimulation increased transcript abundance of several genes downstream of Nrf2 (nuclear factor erythroid 2-related factor 2) that were enriched in gene sets with a focus on metabolism and immune system. However, none of these genes was dysregulated by S. aureus. Western blot analysis confirms that S. aureus impairs Nrf2 activation as compared to E. coli. Using Nrf2-knockdown cells we demonstrate that Nrf2 is necessary for bpMECs to mount an effective innate defensive response. In support of this notion, nuclear Nrf2 overexpression augmented S. aureus-stimulated inflammatory response. We also show that, unlike E. coli, S. aureus disrupts the non-canonical p62/SQSTM1-Keap1 pathway responsible for Nrf2 activation through inhibiting p62/SQSTM1 phosphorylation at S349. Collectively, our findings provide important insights into the contribution of the Nrf2 pathway to the pathogen-species specific immune response in bovine mammary epithelial cells and raise a possibility that impairment of Nrf2 activation contributes to, at least in part, the weak inflammatory response in S. aureus mastitis.

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

Australian Journal of Experimental Agriculture ◽

10.1071/ea05046 ◽

2005 ◽

Vol 45 (8) ◽

pp. 757 ◽

Cited By ~ 15

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.

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

Toxins ◽

10.3390/toxins11050298 ◽

2019 ◽

Vol 11 (5) ◽

pp. 298 ◽

Cited By ~ 9

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.

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