Table_1_A Label-Free Quantitative Proteomic Analysis of Mouse Neutrophil Extracellular Trap Formation Induced by Streptococcus suis or Phorbol Myristate Acetate (PMA).DOC

Streptococcus suis (S. suis) ranks among the five most important porcine pathogens worldwide and occasionally threatens human health, particularly in people who come into close contact with pigs or pork products. An S. suis infection induces the formation of neutrophil extracellular traps (NETs) in vitro and in vivo, and the NET structure plays an essential role in S. suis clearance. However, the signaling pathway by which S. suis induces NET formation remains to be elucidated. In the present study, we used a label-free quantitative proteomic analysis of mouse NET formation induced by S. suis or phorbol myristate acetate (PMA), a robust NET inducer. Greater than 50% of the differentially expressed proteins in neutrophils infected by S. suis showed similar changes as observed following PMA stimulation, and PKC, NADPH oxidase, and MPO were required for NET formation induced by both stimuli. Because PMA induced robust NET formation while S. suis (MOI = 2) induced only weak NET formation, the association between the inducer and NET formation was worth considering. Interestingly, proteins involved in peptidase activity showed significant differential changes in response to each inducer. Of these peptidases, MMP-8 expression was obviously decreased in response to PMA, but it was not significantly changed in response to S. suis. A subsequent study further confirmed that MMP-8 activity was inversely correlated with NET formation induced by both stimuli. Therefore, the present study provides potentially important information about the manner by which neutrophils responded to the inducers to form NETs.

链球菌 suis (S. suis) 位列全球五大重要猪类病原体之列，偶尔威胁人类健康，特别是与猪或猪肉制品密切接触的人群。S. suis 感染可诱导体外和体内中性粒细胞胞外陷阱（NETs）的形成，而NET结构在S. suis 清除过程中发挥着至关重要的作用。然而，S. suis 诱导NET形成的信号通路尚待阐明。在本研究中，我们利用标记物免染的定量蛋白质组学方法分析了由S. suis 或佛波醇肉豆蔻酸酯（PMA，一种强效的NET诱导剂）诱导的小鼠NET形成。在S. suis 感染的中性粒细胞中，超过50%的差异表达蛋白在PMA刺激后表现出相似的变化，PKC、NADPH氧化酶和MPO对于两种刺激诱导的NET形成均至关重要。由于PMA诱导了强大的NET形成，而S. suis（MOI = 2）仅诱导了微弱的NET形成，因此考虑诱导剂与NET形成之间的关联具有重要意义。有趣的是，参与肽酶活性的蛋白质在响应每种诱导剂时均表现出显著差异。在这些肽酶中，MMP-8的表达在PMA刺激下明显降低，但在S. suis 刺激下则没有显著变化。随后的研究进一步证实了MMP-8活性与两种刺激诱导的NET形成呈负相关。因此，本研究为中性粒细胞如何响应诱导剂形成NET提供了潜在的重要信息。