NOD2/c-Jun NH(2)-Terminal Kinase Triggers Mycoplasma ovipneumoniae-Induced Macrophage Autophagy.
NOD2/c-Jun NH(2)-Terminal Kinase Triggers Mycoplasma ovipneumoniae-Induced Macrophage Autophagy.
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NOD2/JNK触发绵羊肺炎支原体诱导的巨噬细胞自噬
DOI：
10.1128/jb.00689-19
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发表时间：
2020-09-23
影响因子：
3.2
通讯作者：
Li M
中科院分区：
文献类型：
--
作者：
Luo H;Wu X;Xu Z;Hao X;Wang Y;Li M
绵羊肺炎支原体缺乏细胞壁，可引起山羊和绵羊的传染性胸膜肺炎。在本研究中，我们重点研究了NOD和肺炎支原体之间的相互作用，以及它与自噬的关系。我们首次表明，即使在不存在肽多聚糖的情况下，NOD2也能被绵羊肺炎支原体激活。我们还观察到NOD2和JNK途径的激活都促进了绵羊肺炎支原体诱导的自噬。绵羊肺炎支原体属于支原体属，是最小的自我复制微生物属，可引起山羊和绵羊的传染性胸膜肺炎。核苷酸结合的寡聚化结构域含蛋白(NOD2)是一种细胞内模式识别受体，它与胞壁二肽(MDP)相互作用识别细菌的肽聚糖，并参与自噬诱导。然而，还没有关于支原体或肺炎支原体诱导的自噬的NOD识别的报道。在这项研究中，我们试图通过Western blotting、免疫荧光、实时聚合酶链式反应(RT-PCR)和变色单位(CCU)分析来确定NOD2在绵羊肺炎支原体诱导的自噬中的作用。绵羊肺炎支原体感染RAW 264.7细胞后，NOD2的表达明显增加，而NOD1的表达无明显变化。用MDP处理RAW 264.7细胞可显著增加绵羊肺炎支原体与Lc3的共定位，而NOD抑制剂NOD-IN-1处理可降低绵羊肺炎支原体与Lc3的共定位。此外，用小干扰RNA(SiRNA)-NOD2抑制NOD2的表达不能通过检测自噬标记物ATG5、BECLIN1和LC3-II来触发绵羊肺炎支原体诱导的自噬。此外，绵羊肺炎支原体感染显著增加RAW 264.7细胞的磷酸化c-jun氨基末端激酶(p-JNK)/JNK、p-Bcl2/Bcl2、Beclin1、ATG5和Lc3-II比值。用JNK抑制剂SP600126或siRNA-NOD2处理不会增加这一反应。这些结果表明，绵羊肺炎支原体感染激活了NOD2，NOD2和JNK途径激活促进了绵羊肺炎支原体诱导的自噬。本研究为探讨NOD2重组机制和绵羊肺炎支原体感染的发病机制提供了新的思路。缺乏细胞壁的绵羊肺炎分枝杆菌可引起山羊和绵羊的传染性胸膜肺炎。在本研究中，我们重点研究了NOD和肺炎支原体之间的相互作用，以及它与自噬的关系。我们首次表明，即使在不存在肽多聚糖的情况下，NOD2也能被绵羊肺炎支原体激活。我们还观察到NOD2和JNK途径的激活都促进了绵羊肺炎支原体诱导的自噬。
M. ovipneumoniae, which lacks a cell wall, causes infectious pleuropneumonia in goats and sheep. In the present study, we focused on the interaction between NOD and M. ovipneumoniae, as well as its association with autophagy. We showed for the first time that NOD2 was activated by M. ovipneumoniae even when peptidoglycans were not present. We also observed that both NOD2 and JNK pathway activation promoted M. ovipneumoniae-induced autophagy. Mycoplasma ovipneumoniae belongs to Mycoplasma, a genus containing the smallest self-replicating microorganisms, and causes infectious pleuropneumonia in goats and sheep. Nucleotide-binding oligomerization domain-containing protein (NOD2), an intracellular pattern recognition receptor, interacts with muramyl dipeptide (MDP) to recognize bacterial peptidoglycans and is involved in autophagy induction. However, there have been no reports about NOD recognition of mycoplasmas or M. ovipneumoniae-induced autophagy. In this study, we sought to determine the role of NOD2 in M. ovipneumoniae-induced autophagy using Western blotting, immunofluorescence, real-time PCR (RT-PCR), and color-changing unit (CCU) analysis. M. ovipneumoniae infection markedly increased NOD2 but did not increase NOD1 expression in RAW 264.7 cells. Treating RAW 264.7 cells with MDP significantly increased colocalization of M. ovipneumoniae and LC3, whereas treatment with NOD inhibitor, NOD-IN-1, decreased colocalization of M. ovipneumoniae and LC3. Furthermore, suppressing NOD2 expression with small interfering RNA (siRNA)-NOD2 failed to trigger M. ovipneumoniae-induced autophagy by detecting autophagy markers Atg5, beclin1, and LC3-II. In addition, M. ovipneumoniae infection significantly increased the phosphorylated c-Jun NH2-terminal kinase (p-JNK)/JNK, p-Bcl-2/Bcl-2, beclin1, Atg5, and LC3-II ratios in RAW 264.7 cells. Treatment with JNK inhibitor, SP600126, or siRNA-NOD2 did not increase this reaction. These findings suggested that M. ovipneumoniae infection activated NOD2, and both NOD2 and JNK pathway activation promoted M. ovipneumoniae-induced autophagy. This study provides new insight into the NOD2 reorganization mechanism and the pathogenesis of M. ovipneumoniae infection. IMPORTANCE M. ovipneumoniae, which lacks a cell wall, causes infectious pleuropneumonia in goats and sheep. In the present study, we focused on the interaction between NOD and M. ovipneumoniae, as well as its association with autophagy. We showed for the first time that NOD2 was activated by M. ovipneumoniae even when peptidoglycans were not present. We also observed that both NOD2 and JNK pathway activation promoted M. ovipneumoniae-induced autophagy.