铜绿假单胞菌的信号分子3-oxo-C12-HSL促进小鼠MH-S肺泡巨噬细胞自噬
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摘要
目的探讨3-oxo-C12-HSL对MH-S小鼠肺泡巨噬细胞自噬的影响。方法用Las I基因(3-oxo-C12-HSL合成基因)缺失型和野生型的铜绿假单胞菌(PA)菌株的培养物上清液以及化学合成的3-oxo-C12-HSL信号分子处理MH-S细胞,激光共聚焦荧光显微镜观察LC3-GFP荧光斑点以及Western blot检测LC3Ⅱ/LC3Ⅰ比值来监测自噬的形成,同时检测p62的降解以及氯喹对LC3Ⅱ/LC3Ⅰ比值的影响来监测自噬流(autophagic flux)的变化。结果野生型PA菌株的培养物上清液使MH-S细胞LC3-GFP荧光斑点增多(P<0.05),LC3Ⅱ/LC3Ⅰ比值增加(P<0.01),LasⅠ基因缺失型PA菌株不能引起上述自噬相关指标的改变。化学合成的3-oxo-C12-HSL信号分子能够明显增加自噬体数量,上调LC3Ⅱ的表达(P<0.01);自噬底物p62随着3-oxo-C12-HSL作用时间的延长而进行性降解,溶酶体抑制剂氯喹可增强3-oxoC12-HSL引起的LC3Ⅱ累积(P<0.05,P<0.01)。结论 3-oxo-C12-HSL可增加MH-S细胞的自噬水平。
Objective To investigate the effect of 3-oxo-C12-HSL on autophagy in mouse alveolar macrophages MH-S cells. Methods MH-S cells were treated with culture supernatants of the mutant and wild type Pseudomonas aeruginosa( PA) strains of Las I gene( 3-oxo-C12-HSL synthetic gene) and chemically synthesized 3-oxo-C12-HSL signaling molecules. GFP puncta was observed by laser confocal fluorescence microscopy and the ratio of LC3 Ⅱ/LC3Ⅰ was detected by Western blot to detect the formation of autophagic. Autophagic flux was also detected by monitoring the degradation of p62 and the change of chloroquine to LC3Ⅱ/LC3Ⅰ ratio. Results The supernatant of the culture medium of the wild type PA strain increased the GFP puncta of the MH-S cells( P < 0. 05) and the ratio of LC3Ⅱ/LC3Ⅰ( P < 0. 01),The mutant PA strain of Las I gene could not cause the above changes related to autophagy. The chemically synthesized 3-oxo-C12-HSL signal molecules could increase the number of autophagic bodies and the expression of LC3 Ⅱ( P < 0. 01). Autophagic substrate p62 was degraded by 3-oxo-C12-HSL.Chloroquine,a lysosomal inhibitor,enhanced LC3Ⅱ accumulation caused by 3-oxo-C12-HSL( P<0. 05,P<0. 01).Conclusions 3-oxo-C12-HSL increases the level of autophagy in MH-S cells.
Objective To investigate the effect of 3-oxo-C12-HSL on autophagy in mouse alveolar macrophages MH-S cells. Methods MH-S cells were treated with culture supernatants of the mutant and wild type Pseudomonas aeruginosa( PA) strains of Las I gene( 3-oxo-C12-HSL synthetic gene) and chemically synthesized 3-oxo-C12-HSL signaling molecules. GFP puncta was observed by laser confocal fluorescence microscopy and the ratio of LC3 Ⅱ/LC3Ⅰ was detected by Western blot to detect the formation of autophagic. Autophagic flux was also detected by monitoring the degradation of p62 and the change of chloroquine to LC3Ⅱ/LC3Ⅰ ratio. Results The supernatant of the culture medium of the wild type PA strain increased the GFP puncta of the MH-S cells( P < 0. 05) and the ratio of LC3Ⅱ/LC3Ⅰ( P < 0. 01),The mutant PA strain of Las I gene could not cause the above changes related to autophagy. The chemically synthesized 3-oxo-C12-HSL signal molecules could increase the number of autophagic bodies and the expression of LC3 Ⅱ( P < 0. 01). Autophagic substrate p62 was degraded by 3-oxo-C12-HSL.Chloroquine,a lysosomal inhibitor,enhanced LC3Ⅱ accumulation caused by 3-oxo-C12-HSL( P<0. 05,P<0. 01).Conclusions 3-oxo-C12-HSL increases the level of autophagy in MH-S cells.
引文
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[2]Yuan K,Huang C,Fox J,et al.Autophagy plays an essential role in the clearance of Pseudomonas aeruginosa by alveolar macrophages[J].J Cell Sci,2012,125:507-515.
[3]Junkins RD,Shen A,Rosen K,et al.Autophagy enhances bacterial clearance during P.aeruginosa lung infection[J].PLo S One,2013,8:e72263.doi:10.1371/journal.pone.0072263.
[4]Kariminik A,Baseri-Salehi M,Kheirkhah B.Pseudo-monas aeruginosa quorum sensing modulates immune responses:An updated review article[J].Immunol Lett,2017,190:1-6.
[5]von Hoven G,Kloft N,Neukirch C,et al.Modulation of translation and induction of autophagy by bacterial exoproducts[J].Med Microbiol Immunol,2012,201:409-418.
[6]Zeng J,Zhang N,Huang B,et al.Mechanism of azithromycin inhibition of HSL synthesis in Pseudomonas aeruginosa[J].Sci Rep,2016,6:24299.doi:10.1038/srep24299.
[7]Reilly R,Mroz MS,Dempsey E,et al.Targeting the PI3K/Akt/m TOR signalling pathway in cystic fibrosis[J].Sci Rep,2017,7:7642.doi:10.1038/s41598-017-06588-z.
[8]Brockman SM,Bodas M,Silverberg D,et al.Dendrimerbased selective autophagy-induction rescuesΔF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis[J].PLo S One,2017,12:e0184793.doi:10.1371/journal.pone.0184793.
[9]Kopf M,Schneider C,Nobs SP.The development and function of lung-resident macrophages and dendritic cells[J].Nat Immunol,2015,16:36-44.
[10]Cadwell K.Crosstalk between autophagy and inflammatory signalling pathways:balancing defence and homeostasis[J].Nat Rev Immunol,2016,16:661-675.
[11]Weindel CG,Richey LJ,Mehta AJ,et al.Autophagy in dendritic cells and B cells is critical for the inflammatory state of TLR7-mediated autoimmunity[J].J Immunol,2017,198:1081-1092.
[12]Kimmey JM,Stallings CL.Bacterial pathogens versus autophagy:implications for therapeutic interventions[J].Trends Mol Med,2016,22:1060-1076.