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The attenuation of Th1 and Th17 responses via autophagy protects against methicillin-resistant staphylococcus aureus-induced sepsis

Shufang Zhang  Xiaofang Huang  Huiqing Xiu  Zhongheng Zhang  Kai Zhang  Jiachang Cai  Zhijian Cai  Zhanghui Chen  Zhaocai Zhang  Wei Cui  Gensheng Zhang  Meixiang Xiang  
【摘要】:Objective:Whether autophagy affects methicillin-resistant Staphylococcus aureus(MRSA)-induced sepsis and associated mechanisms are largely unknown.The purpose of the study was to investigate the role of autophagy and the contribution of autophagy to T helper(Th) 1 and Th17 responses in MRSA-induced sepsis.Methods:MRSA-induced sepsis model was established by intravenous injection of USA3000 in C57 BL/6 mice.Autophagy-related genes knockout(LC3 B~(-/-) and Beclin-1~(+/-)) mice and autophagy-enhanced agent rapamycin were used.Protein levels of microtubule-associated protein 1 light chain 3(LC3)-Ⅱ/Ⅰ,Beclin-1 and p62 after USA300 infection were examined by western blot and immunohistochemical staining.Bacterial burden,hematoxylineosin staining,and Kaplan-Meier analysis were performed to evaluate the effect of autophagy on MRSA-induced sepsis.In addition,cytokine productions of interferon-γ(IFN-γ) and interleukin-17(IL-17) were analyzed by enzyme-linked immunosorbent assay,and CD4+T cells differentiation was assessed by flow cytometry to observe the contribution of autophagy on Thl and Th17 polarization after USA3000 infection.Results:Compared with baseline level,LC3-Ⅱ/Ⅰ levels were increased at 3 h,peaked at 6 h,and declined at 12 h after USA300 infection in lung,liver,kidney,and peripheral blood mononuclear cells;Beclin-1 protein levels were also significantly increased,while p62 protein levels were significantly decreased at 6 h after infection.Meanwhile,increased LC3-II aggregation in CD4+T cells was also observed at 6 h after injection.Compared with sepsis model group,survival rates were significantly decreased in LC3 B~(-/-) and Beclin-1~(+/-) groups(67% vs.25%;67% vs.33%,both P0.01),accompanied with aggravated organ injuries likes lung,liver,and kidney;whereas enhancing autophagy with rapamycin alleviated these organ damages,and improved survival rate(67% vs.92%,P0.01).However,LC3-II and Beclin-1 knockout or enhancing autophagy by rapamycin had no significant effects on bacterial burden in these organs of lung,liver,kidney,spleen and blood.In addition,compared with sepsis model group,cytokines of IFN-γand IL-17 in the serum were significantly increased in LC3 B~(-/-) and Beclin-1~(+/-) group,whereas rapamycin treatment decreased their production.Flow cytometric analysis showed that in the peripheral blood mononuclear cell,rapamycin treatment decreased the frequencies of Thl and Th17;whereas they were significantly up-regulated in LC3 B~(-/-) group and in Beclin-1~(+/-) group.Conclusion:Autophagy plays a protective role in the pathogenesis of MRSA-induced sepsis,which may be partly associated with the alleviation of organ injuries via the down-regulation of Thl and Th17 response.

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