人腺病毒7型感染激活NLRP3炎症小体的机制研究

Mechanism of NLRP3 activation by human adenovirus 7 infection

目的探讨人腺病毒7型(human adenovirus type 7,HAdV-7)感染对于炎症小体激活的影响及作用机制。方法采用免疫荧光检测HAdV-7对于THP-1分化的巨噬细胞的感染;应用ELISA方法检测HAdV-7感染对于IL-1β的激活;实时荧光定量PCR检测病毒感染对于NLRP3、pro-IL-1β、caspase-1等mRNA表达的影响;蛋白质免疫印迹实验(western blot,WB)检测病毒感染后细胞内NLRP3、pro-IL-1β的表达及上清中caspase-1和IL-1β蛋白表达情况。结果免疫荧光检测显示HAdV-7可感染THP-1分化的巨噬细胞,WB显示HAdV-7感染诱导细胞内pro-IL-1β蛋白表达上调,ELISA检测表明HAdV-7感染可诱导IL-1β的分泌表达(MOI=0.5,P=0.0008)。使用Ac-YVDK-cmk抑制caspase-1表达后,HAdV-7感染上清中IL-1β的表达被明显抑制(P=0.0025);HAdV-7感染caspase-1缺陷的THP-1分化的巨噬细胞,IL-1β的表达同样被显著抑制(P=0.0191)。荧光定量PCR检测HAdV-7感染可以诱导细胞内pro-IL-1β和NLRP3 mRNA表达上调(NLRP3:P=0.0004;pro-IL-1β:P=0.0007),同时WB显示HAdV-7可以诱导细胞内pro-IL-1β和NLRP3蛋白表达上调;使用NLRP3特异性抑制剂MCC950处理细胞,可抑制HAdV-7感染的细胞上清中IL-1β的表达(P=0.0027);HAdV-7感染NLRP3缺陷的THP-1分化的巨噬细胞,IL-1β的表达被明显抑制(P=0.0189)。分别使用TLR2、TLR4、TLR7/9信号传导抑制剂,抑制HAdV-7感染THP-1分化的巨噬细胞,结果显示抑制TLR4信号传导,细胞上清中IL-1β的表达水平有明显下调(P=0.0122);使用ROS抑制剂、组织蛋白酶B抑制剂,或抑制K+外流,分别处理HAdV-7感染THP-1分化的巨噬细胞,结果显示抑制组织蛋白酶B(P=0.0292),或抑制K+外流时(KCl,P=0.0022;Glibenclamide,P=0.0275),细胞上清中IL-1β的表达水平有明显下调。结论HAdV-7感染THP-1细胞激活NLRP3炎症小体,NLRP3炎症小体激活的第一信号通过Toll样受体4(TLR4)信号传导,第二信号主要通过半通道模型介导K+外流的和溶酶体破坏模型释放组织蛋白酶B的激活NLRP3炎症小体。

Objective To investigate the mechanism of activation of NOD-like receptors protein(NLRP)3 in the process of human adenovirus type 7(HAdV-7)infection.Methods THP-1 cells were treated with adenosine triphosphate(ATP),an NLRP3 inhibitor MCC950,and a caspase-1 inhibitor Ac-YVDK-cmk,ammonium pyrrolidinedithiocarbamate(APDC),N-acetyl-L-cysteine(NAC),cathepsin B-selective inhibitor CA-074-me,glibenclamide and potassium chloride(KCl)respectively.THP-1 cells without infection were used as the control group and those infected with HAdV-7 were used as the experimental group.The expression of NLRP3,caspase-1 and IL-1βin the supernatant was detected by enzyme-linked immunosorbent assay(ELISA),Q-PCR and western blot(WB).Results Immunofluorescence detection showed that HAdV-7 infected THP-1,while WB showed that HAdV-7 infection could induce the pro-IL-1βprotein expression in cells,and caspase-1 cleavage induce IL-1βmaturation and release to the outside of cells.The expression of IL-1βin the supernatant of cells was significantly increased as detected by ELISA(MOI=0.5,P=0.0008).After inhibition of caspase-1 expression,the expression of IL-1βin the supernatant was significantly inhibited(P=0.0025).The expression of pro-IL-1βand NLRP3 mRNA in HAdV-7 infected cells was up-regulated(NLRP3:P=0.0004;pro-IL-1β:P=0.0007),and WB showed that HAdV-7 could up regulate the expression of pro-IL-1βand NLRP3 protein in the cells.After THP-1 was treated with NLRP3 specific inhibitor MCC950 the expression of IL-1βin the supernatant of HAdV-7 infected cells were significantly inhibited(P=0.002)7).Toll-like receptor(TLR)2,TLR4 and TLR7/9 signal transduction inhibitors were used respectively to inhibit HAdV-7 infection of THP-1.The result showed that TLR4 signal transduction was inhibited,and the expression level of IL-1βin cell supernatant was significantly decreased(P=0.0122).Using reactive oxygen species(ROS)inhibitor,cathepsin B inhibitor,or K+outflow inhibitor to inhibit the infection of THP-1 differentiated macrophages by HAdV-7,the result showed that when cathepsin B was inhibited(P=0.0292),or K+outflow was inhibited(KCl,P=0.0022;glibenclamide,P=0.0275),the expression level of IL-1βin the supernatant was significantly decreased.Conclusions HAdV-7 infection of THP-1 cells can activate NLRP3 inflammasome.The first signal of NLRP3 activation is transmitted by TLR4,and the second signal is mainly activated by K+outflow and cathepsin B release.