背景:先前的研究已经阐明miR-155在耐甲氧西林金黄色葡萄球菌(MRSA)肺炎中增加,并调节Th9分化。像Th9细胞一样,Th17细胞也是CD4+T细胞的一个亚群,参与MRSA肺炎的进展。本研究旨在探讨miR-155在Th17分化中的作用及机制。
方法:收集MRSA肺炎和支气管异物患儿的支气管肺泡灌洗液(BALF)。建立MRSA感染的小鼠模型,然后收集BALF和肺组织。qRT-PCR,ELISA和流式细胞术检测上述样品中IL-17的mRNA表达和浓度以及Th17细胞的数量。HE和ELISA用于评估肺部的炎症反应。此外,从儿童的BALF中分离CD4+T细胞用于体外实验。用miR-155模拟物/抑制剂治疗后,确定了miR-155在Th17/IL-17调节中的作用。通过qRT-PCR探索miR-155的下游,西方印迹,双荧光素酶报告分析和RIP测定。
结果:MRSA肺炎患儿IL-17水平和Th17细胞比例升高。在MRSA感染的小鼠中观察到类似的模式。相反,IL-17中和消除了MRSA感染诱导的Th17/IL-17的激活。此外,IL-17阻断减少了由MRSA引起的炎症。体外实验证明miR-155正调节IL-17表达和Th17分化。机械上,FOXP3是miR-155的直接靶标。miR-155通过FOXP3和Argonaute2(AGO2)之间的结合抑制FOXP3水平,RNA诱导沉默复合物(RISC)的关键成分。FOXP3过表达逆转了miR-155诱导的IL-17水平升高和Th17分化。
结论:miR-155通过AGO2和FOXP3相互作用降低FOXP3促进Th17分化,从而促进MRSA肺炎的发病机制。IL-17阻断减弱了MRSA引起的炎症,这为MRSA肺炎提供了非抗生素治疗策略.
BACKGROUND: Previous researches have clarified that miR-155 is increased in methicillin-resistant Staphylococcus aureus (MRSA) pneumonia, and modulates Th9 differentiation. Like Th9 cells, Th17 cells were also a subset of CD4+ T cells and involved in MRSA pneumonia progression. This work aimed to investigate the role and mechanism of miR-155 in Th17 differentiation.
METHODS: Bronchoalveolar lavage fluid (BALF) was collected from children with MRSA pneumonia and bronchial foreign bodies. MRSA-infected murine model was established followed by collecting BALF and lung tissues. qRT-PCR, ELISA and flow cytometry were performed to examine the mRNA expression and concentration of IL-17 and the number of Th17 cells in above samples. HE and ELISA were used to evaluate inflammatory responses in lung. Furthermore, CD4+ T cells were isolated from BALF of children for in vitro experiments. After treatments with miR-155 mimic/inhibitor, the roles of miR-155 in Th17/IL-17 regulation were determined. The downstream of miR-155 was explored by qRT-PCR, western blotting, dual luciferase reporter analysis and RIP assay.
RESULTS: The levels of IL-17 and the proportion of Th17 cells were increased in children with MRSA pneumonia. A similar pattern was observed in MRSA-infected mice. On the contrary, IL-17 neutralization abolished the activation of Th17/IL-17 induced by MRSA infection. Furthermore, IL-17 blockade diminished the inflammation caused by MRSA. In vitro experiments demonstrated miR-155 positively regulated IL-17 expression and Th17 differentiation. Mechanistically, FOXP3 was a direct target of miR-155. miR-155 inhibited FOXP3 level via binding between FOXP3 and Argonaute 2 (AGO2), the key component of RNA-induced silencing complex (RISC). FOXP3 overexpression reversed elevated IL-17 levels and Th17 differentiation induced by miR-155.
CONCLUSIONS: miR-155 facilitates Th17 differentiation by reducing FOXP3 through interaction of AGO2 and FOXP3 to promote the pathogenesis of MRSA pneumonia. IL-17 blockade weakens the inflammation due to MRSA, which provides a nonantibiotic treatment strategy for MRSA pneumonia.