Role Of NOD2 Mediates Autophagy In Mycoplasma Ovipneumoniae-infected RAW264.7 Cells

Sheep contagious pleuropneumonia caused by Mycoplasma ovipneumoniae(MO)has the characteristics of high infection rate and high mortality.It has caused a serious threat to the development of the sheep farming.Autophagy is a highly conserved degradation pathway in eukaryotic cells.Yet another function of autophagy could remove invasive pathogens,it plays a crucial protective role in cell survival and in the body's anti-infective immunity.Earlier studies in our lab have confirmed that MO can induce RAW264.7 cells autophagy and a certain degree of autophagy can limit the proliferation of MO.NOD2 is an intracellular pattern recognition receptor,it can recognize invading pathogens and activate the JNK signaling pathway trigger downstream cascade reactions to resist pathogenic infection.However,whether NOD2 can recognize MO and regulate JNK pathway to participate in RAW264.7 autophagy-anti-MO mechanism are not yet clear.This study was used MO to infect mouse macrophages RAW264.7,and activated,inhibited or interfered with NOD2 respectively.Immunofluorescence,RT-PCR,Western Blot and mRFP-GFP-LC3 double fluorescent labeling proteins were used to investigate the regulation of autophagy in RAW264.7 cells infected with MO from cell morphological levels and cellular levels respectively.The results were described below:1.The expression of NOD2 proteins were detected by Western Blot,the results showed that the trend of NOD2 growth was consistent with that of MO induced RAW264.7 cells autophagy.However,the expression of NOD1 was no obvious difference in MO infection time gradient.Immunofluorescence results showed that the number of autophagosomes co-localized with MO were increased when overexpression of NOD2,the results on the contrary when inhibition ofNOD2.Therefore,it was confirmed that NOD2 was involved in MO-induced RAW264.7 autophagy and NOD1 does not participate in this process.2.After RAW264.7 cells were infected by MO,when the NOD2 was over-expressed the autophagic flux was increased,and the expression levels of autophagy-related genes and proteins Atg5,Atg7,Becllinl and LC3II/I were significantly increased.It was restrained the proliferation of intracellular MO;When NOD2 was inhibited,the autophagic flow was reduced and autophagosomes and autolysosomes were significantly reduced,the results on the contrary when inhibition of NOD2.These results indicated that NOD2 was involved in the regulation of MO-induced autophagy in RAW264.7 cells and had a significant effect on the proliferation of MO in RAW264.7 cells.3.JNK was activated after MO infection,and the expression of p-JNKl/2 and LC3-?/? were increased.Immunofluorescence showed a significant increase in autophagosomes.When sp600126 inhibited JNK,the expression of p-JNK1/2 and LC3-II/I was decreased,autophagosomes were reduced.The results demonstrated that JNK was involved in the regulation of MO-induced autophagy in RAW264.7 cells.4.JNK and Bcl-2 were activated when Overexpression of NOD2,and the expressions of p-JNK p-Bcl-2 and Beclinl were increased.After interference with the NOD2,p-JNK,p-Bcl-2,Beclinl expression level were decreased,namely that NOD2 can regulate JNK pathway.After inhibition of JNK by sp600126,NOD2 no longer regulates MO-induced autophagy in RAW264.7 cells.It further showed that NOD2-mediated MO-induced RAW264.7 autophagy was dependent on the JNK-Bcl-2/Beclin1 pathway.Conclusion:Activation of NOD2 promotes MO-induced RAW264.7 autophagy.Inhibition/interference of NOD2 reduces MO-induced RAW264.7 autophagy.NOD2 dependents on the JNK-Bcl-2/Beclinl pathway to regulate RAW264.7 autophagy-anti-MO process.