Signal Transduction Pathways Involved In Mycoplasma Genitalium LAMPs-induced Proinflammatory Cytokines Production In THP-1 Cells

Objective: This study was carried out to investigate the production of proinflam- matory cytokines (CKs) TNF-α, IL-1βand IL-6 and the activation of mitogen- activated protein kinases (MAPKs), nuclear factor-κB (NF-κB) and activator protein- 1 (AP-1) induced by Mycoplasma genitalium (Mg) lipid-associated membrane proteins (LAMPs) in THP-1 cells, and then elucidate whether these signal tran- sduction pathways were involved in LAMPs induced CKs production.Methods: The human monocyte cell line THP-1 was incubated with various concentrations of LAMPs preparations derived from the cultured Mg G37 strain. The proinflammatory cytokines TNF-α, IL-1βor IL-6 level induced by LAMPs was detected by the quantitative"sandwich"ELISA technique; Phosphorylate level of stress activated protein kinase/c-Jun- NH2-terminal kinase (SAPK/JNK), extracellular signal-regulated kinase (ERK1/2) or p38 was demonstrated by Western blot; and the DNA-binding activity of NF-κB and AP-1 was assessed by electrophoretic mobility shift assay (EMSA); Then THP-1 cells were preincubated with different concentra- tions of SAPK/JNK inhibitor SP600125, ERK1/2 inhibitor PD98059, p38 inhibitor SB203580 or pyrrolidine dithiocarbamate (PDTC) for 30min, and then 3μg/ml LAMPs was added into the medium for another 24h, the proinflammatory cytokines level were detected by ELISA.Results:⑴LAMPs induced THP-1 cells production of significant amount of TNF-α, IL-1βand IL-6. Cytokine production by LAMPs-treated cells was found to be dose dependent in a concentration range from 0.5μg/ml to 3μg/ml of lipoproteins, However, when the concentration of LAMPs was more than 3μg/ml, the CKs production level decreased.⑵Western blot clearly showed that the intensity of the signals to phosphorylated SAPK/JNK, p38 and ERK1/2 increased as the incubation time proceeds, the phosphorylated level occurred after stimulated with 15min, the peak arrived at 30min, and lasted for more than 60min, then decreased. However, the intensity of the total MAPKs (both phosphorylated and non-phosphorylated) signals didn't change. The DNA binding activity of NF-κB and AP-1 was also changed in THP-1 cells, the kinetics of these two nuclear transcription factors was identical: the DNA binding activities were maximal by 2h of stimulation and then declined.⑶PD98059 inhibited LAMPs induced TNF-αand IL-βproduction except IL-6, SP600125 slightly suppressed IL-6 production but no evident effect was obtained for TNF-αand IL-1β, while SB203580 and PDTC abrogated TNF-α, IL-1βand IL-6 production.Conclusion:⑴LAMPs could induce THP-1 cells production of TNF-α, IL-1βand IL-6.⑵MAPKs, NF-κB and AP-1 were activated after LAMPs stimulation.⑶Activation of MAPKs and NF-κB were involved in LAMPs induced proinflammatory cytokines production.