Inflammatory responses to Mycoplasma hyopneumoniae in murine alveolar macrophage cell lines

Abstract

AIM: To investigate the mechanism by which Mycoplasma hyopneumoniae induces inflammatory responses in murine alveolar macrophage (MH-S) cells.

METHODS: A pathogenic strain of M. hyopneumoniae cultured in modified Friis medium was used to investigate the inflammatory response in MH-S cell lines. The effect of stimulation by M. hyopneumoniae on the production of nitric oxide (NO) and cytokines in MH-S cells and inhibition of their production, using specific inhibitors of signalling pathways, was investigated using the Griess reaction and ELISA respectively. A Western blot assay was used to confirm activation of the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Nuclear translocation of NF-κB was further confirmed using transient transfection and luciferase gene reporter assay.

RESULTS: The results revealed dose-dependent production of NO in MH-S cells stimulated by M. hyopneumoniae. Increased concentrations of the cytokines tumour necrosis factor (TNF)-α and interleukin (IL)-1β and IL-6 were also observed (p<0.05). Using immunoblot analysis, involvement of three MAPK pathways, extracellular signal-regulated kinase I/II (ERK1/2), p38 and Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPK) was confirmed. Specific inhibitors of signal pathways also demonstrated their effect on the NO and cytokine responses of MH-S cells. Degradation and phosphorylation of inhibitory kappa B (IκB)-alpha was observed, while the luciferase gene reporter assays revealed activation of NF-κB after stimulation by M. hyopneumoniae. Inhibition of NF-κB by pyrrolidine dithiocarbamate decreased M. hyopneumoniae-induced production of NO and IL-1β (p<0.05), whereas no inhibitory effect was observed on concentrations of TNF-α, and IL-6.

CONCLUSION: These findings indicate that M. hyopneumoniae induces NO and pro-inflammatory cytokines, and NF-κB and the three MAPK pathways are involved in the process.

KEY WORDS:

• Cytokines
• MAPK
• MH-S cells
• M. hyopneumoniae
• NO
• NF-κB

Acknowledgements

This study was supported financially in part by the Technology Development Programme for Agriculture and Forestry, Ministry for Agriculture, Forestry and Fisheries, and in part by the Korea Research Foundation (KRF) grant funded by the Korean Government (MEST) (No. 2009-0071490).