Mycoplasma Pneumoniae Lipids Induce Murine Macrophages Secretion Of Pro-inflammatory Cytokines Via TLR4 And Autophagy Pathways

Objective: Mycoplasma pneumoniae is frequently considered the causative agent for community acquired pneumonia.We previously demonstrated that M.pneumoniae was recognized by Toll-like receptor(TLR)2/6 and TLR1/2 by the innate immune system and thus resulted in inflammatory response.However,recent studies indicated that the TLR4 is also involved in the cytokines secretion in macrophages via a TLR4-dependent autophagy-associated pathway.Mycoplasma species lack cell walls and thus do not possess LPS,and other ligands for TLR4 in mycoplasma emain unclear.Moreover,mycoplasma species could express some unique components like glycolipids,phosphoglycolipids,and polysaccharides on the membrane.These molecules may be potential TLR4 ligands,and further studies are needed to determine the exact ligands of M.pneumoniae for TLR4.In this study,we explore whether M.pneumoniae lipids induce secretion of pro-inflammatory cytokines in RAW264.7 cells and clarify the exact mechanism.Method: M.pneumoniae was cultured in vitro and its lipid was extracted.Different concentrations of lipids were used to stimulate mouse RAW264.7 macrophages in vitro,followed by procedures as below: 1.Stimulating RAW264.7 cells with 0~120?g/mL lipid for 24 h,and detecting the levels of IL-1? and TNF-? from supernatant.2.Pre-incubating cells with TLR2 or TLR4 neutralizing antibody(10?g/mL)for 1 h,or with TLR2 or TLR4 siRNA designated to block the expression of TLR2 or TLR4 and then to confirm wheather lipid induce the secretion of TNF-? and IL-1? through TLR2 or TLR4.3.The nuclear translocation of NF-?B was detected by immunofluorescence,and the expression of I?B was detected by Western blot,after RAW264.7 cells were treated with 120?g/mL lipid for 12 h.To observe the role of TLR2 and TLR4 in lipid-activated NF-?b pathway,cells were pretreated with TLR2 or TLR4 neutralizing antibody.Finally,cells were pretreated with NF-?B inhibitor for 40 min,and the secretion levels of TNF-? and IL-1? were detected by ELISA to determine the role of NF-?B in mediating the secretion of pro-inflammatory cytokines.4.RAW264.7 cells were treated with 120?g/mL lipid for 18 h,or treated wih NF-?B inhibitor BAY 11-7082 before lipid treatment.The role of NF-?B in mediating NLRP3 mRNA expression was detected by qRT-PCR and the level of reactive oxygen species(ROS)in macrophages was tested by molecular probes.Moreover,TLR2 or TLR4 inhibitors were pretreated with lipid to confirm the effect of TLR2 and TLR4 on ROS secretion.Meanwhile,ROS scavenger NAC was used to observe the role of ROS in activation of NLRP3 and secretion of IL-1? during lipid stimulation.5.RAW264.7 cells were stimulated with lipid,or pretreat with TLR2 or TLR4 inhibitors before lipid stimulation,changes of LC3 ? spots were detected by immunofluorescence method to observe the effect of TLR2 or TLR4 inhibitiors on LC3? spots.Expressions of autophagy-related molecules LC3?,Beclin-1 and p62 were measured by Western blot to clarify the role of TLR2 or TLR4 in mediating lipid-induced autophagy.Stimutaneously,the cells were pretreatment with autophagy inhibitor 3-MA,or silencing agt5 and beclin1 before lipid stimulation,and effects of autophagy on lipid-induced TNF-? and IL-1? were observed.6.3-MA was used to reduce the transcriptional levels of agt5 and beclin1,and then observe the effect on nuclear translocation of NF-?B in lipid-stimulated cells.Or treat with NF-?B inhibitor,LC3? protein expression or spot formation were detected by using Western blot or Immunofluorescence to identify the asscociateion of autophagy and NF-?B.Results: 1.When the lipid concentration was lower than 30?g/mL,RAW264.7 cells could not induce the secretion of TNF-? and IL-1?.When the concerntration was among 30~120?g/mL,the expressions of TNF-? and IL-1? were significantly increased,and lipids have no significant toxic effect on cells in this concentration range.2.Transfection of TLR2 siRNA or pretreatment of cells with TLR2 neutralizing antibody had no significant effect on secretion of lipid-induced IL-1? and TNF-?,whereas transfection of TLR4 siRNA or pretreatment with TLR4 neutralizing antibody,TNF-? and IL-1? were significantly reduced.3.Immunofluorescence results shows that treatment of RAW264.7 cells with 120?g/mL lipid for 12 h could induce nuclear translocation of NF-?B p65 subunit,accompanied by a decrease in I?B levels.TLR2 neutralizing antibody treatment did not affect p65 nuclear translocation and I?B expression,but treatment with TLR4 neutralizing antibody remarkably inhibited lipid translocation of p65 and degradation of I?B.In addition,TNF-? and IL-1? secretion was significantly reduced after treatment with the NF-?B inhibitor BAY11-7082.4.After lipid stimulation of RAW264.7 cells for 18 h,qRT-PCR results shows that NLRP3 mRNA transcriptional level increases,NF-?B inhibitor pretreatment could down-regulate NLRP3 mRNA transcriptional level;in the meanwhile,lipid can up-regulate ROS in RAW264.7 cells.The ROS level after TLR4 neutralizing antibody treatment was markedly decreased,while the TLR2 antibody treatment has no significant effect on it;the lipid also up-regulate the expression of the caspase-1 p10 subunit,and the p10 level is notably decreased after treatment with the ROS inhibitor NAC.ASC spots and IL-1? secretion are also dramatically reduced.5.After lipid treatment,LC3 spots increased significantly.TLR2 neutralizing antibodies can not affect the formation of LC3?,but the spots are obviously reduced after inhibition of TLR4.Western blot results shows that the expression of LC3?,beclin1 and ATG5 all are up-regulated after lipid treatment.Through treatment with the lysosomal fusion inhibitor chloroquine,the expression of LC3? is clearly decreased but the expression of p62 is increased.Lipid-induced TNF-? and IL-1? are reduced after treatment with autophagy inhibitor 3-MA,and similar results are obtained after interference with agt5 or beclin1.6.NF-?B nuclear translocation is inhibited after pretreatment of cells with autophagy inhibitor 3-MA or interference with Beclin-1 or ATG5 by siRNA.After treatment with the NF-?B inhibitor BAY11-7082,the expression of LC3 ? is down-regulated,while the formation of spots is reduced as well.Conclusion: 1.M.pneumoniae lipids induced autophagy and activated NF-B and NLRP3 by TLR4 in mouse macrophages.2.Autophagy can form a positive feedback loop with NF-?B to further promote the secretion of TNF-? and IL-1?.