Inflammatory responses to Mycoplasma hyopneumoniae in murine alveolar macrophage cell lines

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-kappa B) and mitogen-activated protein kinase (MAPK) pathways. Nuclear translocation of NF-kappa B 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)-alpha and interleukin (IL)-1 beta 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 (I kappa B)-alpha was observed, while the luciferase gene reporter assays revealed activation of NF-kappa B after stimulation by M. hyopneumoniae. Inhibition of NF-kappa B by pyrrolidine dithiocarbamate decreased M. hyopneumoniae-induced production of NO and IL-1 beta (p<0.05), whereas no inhibitory effect was observed on concentrations of TNF-alpha, and IL-6. CONCLUSION: These findings indicate that M. hyopneumoniae induces NO and pro-inflammatory cytokines, and NF-kappa B and the three MAPK pathways are involved in the process.