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

• Gondaira S
• Higuchi H
• Iwano H
• Nishi K
• Nebu T
• Nakajima K
• Nagahata H

• KMID: 2402698
• DOI: http://doi.org/10.4142/jvs.2018.19.1.79

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.

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