J Vet Sci.  2018 Jan;19(1):79-87. 10.4142/jvs.2018.19.1.79.

Innate immune response of bovine mammary epithelial cells to Mycoplasma bovis

Affiliations
  • 1Animal Health Laboratory, Graduate School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu 069-8501, Japan. higuchi@rakuno.ac.jp
  • 2Department of Veterinary Biochemistry, Graduate School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu 069-8501, Japan.
  • 3National Agricultural Research Center for Hokkaido Region, National Agriculture and Food Research Organization, Sapporo 062-8555, Japan.

Abstract

Mycoplasma spp. are contagious bacteria, and mycoplasmal mastitis is a serious productivity problem on dairy farms. Bovine mammary epithelial cells (bMECs) have an important role in the elimination of pathogens, but the effect of Mycoplasma bovis on bMECs has not been fully described. To elucidate the immune response against intramammary infection by M. bovis, we undertook microarray analysis to examine and profile mRNA expression in bMECs after stimulation with M. bovis. We also compared the effects of M. bovis, Staphylococcus aureus, and Escherichia coli on immune-related mRNA expression in bMECs. Transcriptome analysis indicated a significant decrease in the level of mRNA-encoding lysine-specific demethylase 4D, suggesting that the immune response is suppressed by a decrease in histone demethylase activity. Interleukin (IL)-1β, IL-6, tumor necrosis factor alpha, toll-like receptor (TLR) 2, and TLR4 mRNA expression levels were significantly increased in bMECs stimulated with heat-killed M. bovis, but the expression levels were lower than those following stimulation by heat-killed S. aureus or E. coli. Our results suggest that M. bovis weakly affects mRNA expression in bMECs compared to the effects of E. coli or S. aureus. Moreover, live M. bovis may induce suppression of the immune response in bMECs.

Keyword

Mycoplasma bovis; bovine mammary epithelial cells; cytokines; innate immunity; microarray analysis

MeSH Terms

Agriculture
Bacteria
Cytokines
Efficiency
Epithelial Cells*
Escherichia coli
Female
Gene Expression Profiling
Histones
Immunity, Innate*
Interleukin-6
Interleukins
Mastitis
Microarray Analysis
Mycoplasma bovis*
Mycoplasma*
RNA, Messenger
Staphylococcus aureus
Toll-Like Receptors
Tumor Necrosis Factor-alpha
Cytokines
Histones
Interleukin-6
Interleukins
RNA, Messenger
Toll-Like Receptors
Tumor Necrosis Factor-alpha

Figure

  • Fig. 1 Microarray analysis and validation of KDM4D mRNA expression in bMECs stimulated with Mycoplasma bovis; bovine mammary epithelial cells (bMECs) were incubated with live M. bovis at an multiplicity of infection of 1,000 for 6 h. The data are expressed from three (microarray) or four (real-time polymerase chain reaction [PCR]) independent experiments. Significant difference (*p<0.05) compared with unstimulated bMECs.

  • Fig. 2 Expressions of cytokine mRNA in bovine mammary epithelial cells (bMECs) stimulated with Mycoplasma bovis, Staphylococcus aureus, or Escherichia coli; bMECs were incubated with M. bovis, S. aureus, or E. coli, live or heat-killed, at multiplicities of infection of 10 (△), 100 (□), and 1,000 (●) for 6, 12, and 24 h. Expressions of interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α) mRNA determined by real-time polymerase chain reaction and expressed as fold increases, as described in Materials and Methods section. The data are expressed as mean ± SE values from five independent experiments. Significant differences (*p<0.05 or **p<0.01) compared with unstimulated bMECs.

  • Fig. 3 Expressions of chemokine mRNA in bovine mammary epithelial cells (bMECs) stimulated with Mycoplasma bovis, Staphylococcus aureus, or Escherichia coli; bMECs were incubated with M. bovis, S. aureus, or E. coli, live or heat-killed, at multiplicities of infection of 10 (△), 100 (□), and 1,000 (●) for 6, 12, and 24 h. Expressions of interleukin (IL)-8 mRNA were determined by real-time polymerase chain reaction and are expressed as fold increases, as described in Materials and Methods section. The data are expressed as mean ± SE values from five independent experiments. Significant differences (*p<0.05 or **p<0.01) compared with unstimulated bMECs.

  • Fig. 4 Expressions of antimicrobial peptide mRNA in bovine mammary epithelial cells (bMECs) stimulated with Mycoplasma bovis, Staphylococcus aureus, or Escherichia coli; bMECs were incubated with M. bovis, S. aureus, or E. coli, live or heat-killed, at multiplicities of infection of 10 (△), 100 (□), and 1,000 (●) for 6, 12, and 24 h. Expressions of lactoferrin (Lf) and β-defensin mRNA were determined by real-time polymerase chain reaction and expressed as fold increases, as described in Materials and Methods section. The data are expressed as mean ± SE values from five independent experiments. Significant differences (*p<0.05 or **p<0.01) compared with unstimulated bMECs.

  • Fig. 5 Expressions of TLR mRNA in bovine mammary epithelial cells (bMECs) stimulated with Mycoplasma bovis, Staphylococcus aureus, or Escherichia coli; bMECs were incubated with M. bovis, S. aureus, or E. coli, live or heat-killed, at multiplicities of infection of 10 (△), 100 (□), and 1,000 (●) for 6, 12, and 24 h. Expressions of toll-like receptor (TLR) 2 and TLR4 mRNA were determined by real-time polymerase chain reaction and expressed as fold increases, as described in Materials and Methods section. The data are expressed as mean ± SE values from five independent experiments. Significant differences (*p<0.05 or **p<0.01) compared with unstimulated bMECs.


Reference

1. Boulanger D, Bureau F, Mélotte D, Mainil J, Lekeux P. Increased nuclear factor kappaB activity in milk cells of mastitis-affected cows. J Dairy Sci. 2003; 86:1259–1267.
Article
2. Brand B, Hartmann A, Repsilber D, Griesbeck-Zilch B, Wellnitz O, Kühn C, Ponsuksili S, Meyer HH, Schwerin M. Comparative expression profiling of E. coli and S. aureus inoculated primary mammary gland cells sampled from cows with different genetic predispositions for somatic cell score. Genet Sel Evol. 2011; 43:24.
3. Bürki S, Frey J, Pilo P. Virulence, persistence and dissemination of Mycoplasma bovis. Vet Microbiol. 2015; 179:15–22.
4. Chain B, Bowen H, Hammond J, Posch W, Rasaiyaah J, Tsang J, Noursadeghi M. Error, reproducibility and sensitivity: a pipeline for data processing of Agilent oligonucleotide expression arrays. BMC Bioinformatics. 2010; 11:344.
Article
5. Fox LK. Mycoplasma mastitis: causes, transmission, and control. Vet Clin North Am Food Anim Pract. 2012; 28:225–237.
6. Fu Y, Zhou E, Liu Z, Li F, Liang D, Liu B, Song X, Zhao F, Fen X, Li D, Cao Y, Zhang X, Zhang N, Yang Z. Staphylococcus aureus and Escherichia coli elicit different innate immune responses from bovine mammary epithelial cells. Vet Immunol Immunopathol. 2013; 155:245–252.
Article
7. Gilbert FB, Cunha P, Jensen K, Glass EJ, Foucras G, Robert-Granié C, Rupp R, Rainard P. Differential response of bovine mammary epithelial cells to Staphylococcus aureus or Escherichia coli agonists of the innate immune system. Vet Res. 2013; 44:40.
8. Gondaira S, Higuchi H, Iwano H, Nakajima K, Kawai K, Hashiguchi S, Konnai S, Nagahata H. Cytokine mRNA profiling and the proliferative response of bovine peripheral blood mononuclear cells to Mycoplasma bovis. Vet Immunol Immunopathol. 2015; 165:45–53.
Article
9. Griesbeck-Zilch B, Meyer HH, Kühn CH, Schwerin M, Wellnitz O. Staphylococcus aureus and Escherichia coli cause deviating expression profiles of cytokines and lactoferrin messenger ribonucleic acid in mammary epithelial cells. J Dairy Sci. 2008; 91:2215–2224.
Article
10. Günther J, Esch K, Poschadel N, Petzl W, Zerbe H, Mitterhuemer S, Blum H, Seyfert HM. Comparative kinetics of Escherichia coli- and Staphylococcus aureus-specific activation of key immune pathways in mammary epithelial cells demonstrates that S. aureus elicits a delayed response dominated by interleukin-6 (IL-6) but not by IL-1A or tumor necrosis factor alpha. Infect Immun. 2011; 79:695–707.
Article
11. Kauf AC, Rosenbusch RF, Paape MJ, Bannerman DD. Innate immune response to intramammary Mycoplasma bovis infection. J Dairy Sci. 2007; 90:3336–3348.
12. Krishnan S, Trievel RC. Structural and functional analysis of JMJD2D reveals molecular basis for site-specific demethylation among JMJD2 demethylases. Structure. 2013; 21:98–108.
Article
13. Lahouassa H, Moussay E, Rainard P, Riollet C. Differential cytokine and chemokine responses of bovine mammary epithelial cells to Staphylococcus aureus and Escherichia coli. Cytokine. 2007; 38:12–21.
Article
14. Maunsell FP, Woolums AR, Francoz D, Rosenbusch RF, Step DL, Wilson DJ, Janzen ED. Mycoplasma bovis infections in cattle. J Vet Intern Med. 2011; 25:772–783.
15. Nakajima K, Nakamura M, Gao XD, Kozakai T. Possible involvement of prolactin in the synthesis of lactoferrin in bovine mammary epithelial cells. Biosci Biotechnol Biochem. 2008; 72:1103–1106.
Article
16. Nicholas RA. Bovine mycoplasmosis: silent and deadly. Vet Rec. 2011; 168:459–462.
Article
17. Nicholas RA, Ayling RD. Mycoplasma bovis: disease, diagnosis, and control. Res Vet Sci. 2003; 74:105–112.
18. Rainard P, Riollet C. Innate immunity of the bovine mammary gland. Vet Res. 2006; 37:369–400.
Article
19. Razin S, Yogev D, Naot Y. Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev. 1998; 62:1094–1156.
Article
20. Robinson TL, Sutherland IA, Sutherland J. Validation of candidate bovine reference genes for use with real-time PCR. Vet Immunol Immunopathol. 2007; 115:160–165.
Article
21. Selsted ME, Tang YQ, Morris WL, McGuire PA, Novotny MJ, Smith W, Henschen AH, Cullor JS. Purification, primary structures, and antibacterial activities of beta-defensins, a new family of antimicrobial peptides from bovine neutrophils. J Biol Chem. 1993; 268:6641–6648.
Article
22. Shimizu T. Inflammation-inducing factors of Mycoplasma pneumoniae. Front Microbiol. 2016; 7:414.
23. Shio MT, Hassan GS, Shah WA, Nadiri A, El Fakhry Y, Li H, Mourad W. Coexpression of TLR2 or TLR4 with HLA-DR potentiates the superantigenic activities of Mycoplasma arthritidis-derived mitogen. J Immunol. 2014; 192:2543–2550.
Article
24. Spalenza V, Girolami F, Bevilacqua C, Riondato F, Rasero R, Nebbia C, Sacchi P, Martin P. Identification of internal control genes for quantitative expression analysis by real-time PCR in bovine peripheral lymphocytes. Vet J. 2011; 189:278–283.
Article
25. Ulvatne H, Vorland LH. Bactericidal kinetics of 3 lactoferricins against Staphylococcus aureus and Escherichia coli. Scand J Infect Dis. 2001; 33:507–511.
26. Zhu Y, van Essen D, Saccani S. Cell-type-specific control of enhancer activity by H3K9 trimethylation. Mol Cell. 2012; 46:408–423.
Article
Full Text Links
  • JVS
Actions
Cited
CITED
export Copy
Close
Share
  • Twitter
  • Facebook
Similar articles
Copyright © 2024 by Korean Association of Medical Journal Editors. All rights reserved.     E-mail: koreamed@kamje.or.kr