肠道病毒71型（EV71）感染通常导致婴幼儿的手足口病（HFMD），少数重症患儿可发生无菌性脑膜炎、脑干脑炎、神经源性肺水肿等并发症，极少数患者病情危重、甚至死亡。但是，EV71引起炎症反应、免疫应答及导致疾病的机制尚未完全阐明。武汉大学病毒学国家重点实验室吴建国教授团队于2017年1月6日在PLoS Pathogens杂志上发表文章“EV71 3D protein binds with NLRP3 and enhances the assembly of inflammasome complex”（PLoS Pathog 2017 Jan 6; 13(1):e1006123. doi:10.1371/journal.ppat.1006123.），报道了EV71的3D蛋白（RNA聚合酶）在病毒感染导致宿主炎症反应中发挥了重要作用，并阐述了EV71感染激活NLRP3炎性小体的新机制。该论文的共同通讯作者为吴建国教授和刘映乐副教授，共同第一作者为王文标博士和肖风博士生。
2017年8月30日，吴建国团队再一次在最新一期的PLoS Pathogens杂志上在线发表文章“HRS plays an important role for TLR7 signaling to orchestrate inflammation and innate immunity upon EV71 infection”（PLoS Pathog 2017 Aug 30; 13(8):e1006585. doi:10.1371/journal.ppat.1006585. [Epub ahead of print]），进一步揭示了EV71感染诱导宿主炎症反应和免疫应答的新机制。该论文的共同通讯作者为吴建国教授、刘映乐副教授和刘芳副教授，第一作者为罗震博士。
该研究首先通过临床标本分析发现在EV71感染的病人血清中，炎症因子IL-1b及IL-6的表达水平显著高于正常组；并且在EV71感染的小鼠模型和巨噬细胞中证明EV71感染能激活炎症因子产生。由于Toll样受体（TLRs）介导的病原体识别能诱导宿主免疫应答和炎症反应，该团队通过信号通路抑制剂筛选及基因敲除小鼠研究，证实了EV71通过调控TLR7激活NF-κB/p38 MAPK信号通路、从而促进炎症因子表达。进一步利用siRNA干扰技术筛选及其他研究，发现了肝细胞生长调节因子酪氨酸激酶蛋白底物（hepatocyte growth factor-regulated tyrosine kinase substrate，HRS）在EV71激活TLR7信号通路中起关键作用。HRS是一种重要的胞内体（endosome）蛋白，在调控细胞内蛋白的组装和分选中起重要作用。该团队进一步发现EV71能促进HRS表达，且证实HRS与TLR7直接相互作用，促进TLR7复合体在早期胞内体和晚期胞内体中的组装与激活。研究结果证明：在感染过程中，EV71上调HRS表达，HRS接下来通过激活TLR7/NF-κB/p38 MAPK信号通路，促进炎症因子表达，导致宿主炎症反应；同时HRS也能通过上调TLR7/NF-κB/IRF3信号通路，诱导干扰素产生，导致抗病毒免疫应答。因此，揭示了EV71感染引起宿主炎症反应和免疫应答的新机制。
HRS interacts with TLR7 and TAB1 in the TLR7 complex during viral infection. (A and B) Macrophages derived from THP-1 cells were stimulated with R848 (100 ng/ml) (A) or EV71 (MOI=5) (B) for various time periods as indicated. Cell lysates were prepared and immunoprecipitated with rabbit anti-TLR7 or rabbit anti-IgG antibodies. Immunoprecipitates were assayed using Western blotting analysis with specific antibodies as indicated. (C and D) Macrophages were stimulated with R848 for 15 min, or infected with EV71 or SeV for 4 h, and then probed with indicated antibodies before confocal microscopy. Bar = 20 μm. (E) Macrophages were treated with R848 (100 ng/ml) for 30 min. Cells were probed with indicated antibodies and DAPI stain before confocal microscopy. Bar=20 μm. (F) Mice were infected with EV71 and sacrificed at indicated period. Mice spleens were subjected to immunofluorescence (IF) staining with HRS (Red), TLR7 (Green), and CD68 (Blue) antibodies. Bar=20 μm.
HRS is important for TLR7-mediated proinflammatory cytokines production during viral infection. (A and B) Macrophages were transfected with siR-HRS or siR-NC and then stimulated with R848 (A) or infected with EV71 (B) for various time periods as indicated. Cell extracts were prepared and the proteins in the cell lysates were detected using Western blotting analyses with corresponding antibodies. The indicated band intensity represents as fold changes to internal control GAPDH. (C and D) Macrophages were transfected with siR-NC or siR-HRS and stimulated with R848 or infected with EV71 or SeV. The levels of CSF3, IL-1β, and IL-6 mRNAs and proteins were determined using qPCR (C) and ELISA (D), respectively. **, P<0.01. Graphs show means ± SD, n=3. (E–H) Mice were treated with 5’-cholesterol-modified siRNA duplex targeted to HRS (siR-HRS-m) and its control (siR-NC-m) via tail vein injection and injected intraperitoneally with R848; PBMCs and splenocytes were isolated from the mice. IL-6 mRNA levels expressed in mouse PBMCs (F) and splenocytes (G) were determined using qPCR (each group, n=3). The proinflammatory cytokines proteins in mouse serum (H) were determined using ELISA (n=2–4). ns, not significant; nd., not detected. Data show means ± SEM (standard error of the mean).
A proposed mechanism underlying HRS regulates TLR7-mediated inflammatory and immune responses.