State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, China.
Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, Hubei, China.
J Neuroinflammation. 2021 May 13;18(1):114. doi: 10.1186/s12974-021-02165-4.
Escherichia coli is the most common Gram-negative bacterium causing meningitis, and E. coli meningitis is associated with high mortality and morbidity throughout the world. Our previous study showed that E. coli can colonize the brain and cause neuroinflammation. Increasing evidence supports the involvement of miRNAs as key regulators of neuroinflammation. However, it is not clear whether these molecules participate in the regulation of meningitic E. coli-mediated neuroinflammation.
The levels of miR-155 and miR-146a, as well as their precursors, in E. coli-infected astrocytes were measured using quantitative real-time PCR (qPCR). Overexpression and knockdown studies of miR-155 and miR-146a were performed to observe the effects on bacterial loads, cytokines, chemokines, and NF-κB signaling pathways. Bioinformatics methods were utilized to predict the target genes, and these target genes were validated using qPCR, Western blotting, and luciferase reporter system. In vivo knockdown of miR-155 and miR-146a was carried out to observe the effects on bacterial loads, inflammatory genes, astrocyte activation, microglia activation, and survival in a mouse model.
The levels of miR-155, miR-146a, and their precursors were significantly increased in astrocytes during E. coli infection. miR-155 and miR-146a were induced by the NF-κB-p65 signaling pathway upon infection. Overexpressing and inhibiting miR-155 and miR-146a in astrocytes did not affect the bacterial loads. Further, the in vitro overexpression of miR-155 and miR-146a suppressed the E. coli-induced inflammatory response, whereas the inhibition of miR-155 and miR-146a enhanced it. Mechanistically, miR-155 inhibited TAB2, and miR-146a targeted IRAK1 and TRAF6; therefore, they functioned collaboratively to modulate TLR-mediated NF-κB signaling. In addition, both miR-155 and miR-146a could regulate the EGFR-NF-κB signaling pathway. Finally, the in vivo suppression of E. coli-induced miR-155 and miR-146a further promoted the production of inflammatory cytokines, aggravated astrocyte and microglia activation, and decreased mouse survival time, without affecting the bacterial loads in the blood and brain.
E. coli infection induced miR-155 and miR-146a, which collectively regulated bacteria-triggered neuroinflammatory responses through negative feedback regulation involving the TLR-mediated NF-κB and EGFR-NF-κB signaling pathways, thus protecting the central nervous system from further neuroinflammatory damage.
大肠杆菌是引起脑膜炎的最常见革兰氏阴性菌，大肠杆菌性脑膜炎在全球范围内具有较高的死亡率和发病率。我们之前的研究表明，大肠杆菌可以定植于大脑并引发神经炎症。越来越多的证据支持 miRNA 作为神经炎症关键调节因子的作用。然而，目前尚不清楚这些分子是否参与调节大肠杆菌性脑膜炎介导的神经炎症。
采用实时定量 PCR（qPCR）检测大肠杆菌感染星形胶质细胞中 miR-155 和 miR-146a 及其前体的水平。通过过表达和敲低 miR-155 和 miR-146a 研究其对细菌载量、细胞因子、趋化因子和 NF-κB 信号通路的影响。利用生物信息学方法预测靶基因，并通过 qPCR、Western blot 和荧光素酶报告系统对这些靶基因进行验证。在体内敲低 miR-155 和 miR-146a，观察其对细菌载量、炎症基因、星形胶质细胞激活、小胶质细胞激活和存活的影响。
在大肠杆菌感染期间，星形胶质细胞中 miR-155、miR-146a 及其前体的水平显著升高。miR-155 和 miR-146a 是由感染后 NF-κB-p65 信号通路诱导的。在星形胶质细胞中过表达和抑制 miR-155 和 miR-146a 并不影响细菌载量。此外，体外过表达 miR-155 和 miR-146a 可抑制大肠杆菌诱导的炎症反应，而抑制 miR-155 和 miR-146a 则增强了该反应。机制上，miR-155 抑制 TAB2，miR-146a 靶向 IRAK1 和 TRAF6，因此它们协同作用调节 TLR 介导的 NF-κB 信号。此外，miR-155 和 miR-146a 均可调节 EGFR-NF-κB 信号通路。最后，体内抑制大肠杆菌诱导的 miR-155 和 miR-146a 进一步促进了炎症细胞因子的产生，加重了星形胶质细胞和小胶质细胞的激活，减少了小鼠的存活时间，而对血液和大脑中的细菌载量没有影响。
大肠杆菌感染诱导 miR-155 和 miR-146a，通过 TLR 介导的 NF-κB 和 EGFR-NF-κB 信号通路的负反馈调节，共同调节细菌触发的神经炎症反应，从而保护中枢神经系统免受进一步的神经炎症损伤。
