【作者】 王超；

【导师】 张改平；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2015， 博士

【摘要】 猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)是严重危害养猪业的重大病毒性传染病,其致病原猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)抑制宿主的天然免疫和特异性免疫反应,引起机体免疫抑制,造成持续性感染,从而给该病的防控带来困难。PRRSV感染可以引起机体强烈的间质性肺炎,诱导肺部许多促炎性细胞因子的产生。最近的研究表明PRRSV可以激活NLRP3介导的炎症反应,诱导促炎性细胞因子IL-1β的分泌。炎症反应是机体天然免疫系统中抵抗病毒入侵的坚固防线,但是对PRRSV如何调控炎症反应从而实现免疫逃逸的研究还未见报道。因此,本篇论文对PRRSV调控NLRP3介导的炎症反应的分子机制加以研究。其主要内容和结果如下:1.PRRSV激活NLRP3炎症小体的动态变化情况。用PRRSV BJ-4株在不同时间点(12 h、24 h、48 h、72 h)感染猪肺泡巨噬细胞(Porcine alveolar macrophages,PAMs),利用荧光定量PCR和ELISA方法检测NLRP3介导的炎症反应信号通路中重要组分的mRNA水平和IL-1β的蛋白水平,发现NLRP3、ASC、procaspase-1和pro-IL-1β的mRNA水平和细胞上清中的IL-1β在PRRSV感染早期都呈现上升趋势,并分别在24 h和48 h达到峰值,但是在感染晚期NLRP3、ASC、procaspase-1和pro-IL-1β的mRNA水平急剧下降,并与空白对照组水平相当,IL-1β的蛋白水平在之后的阶段也呈现出下降趋势。而不同剂量PRRSV感染PAMs的实验发现,PRRSV上调的procaspase-1、pro-IL-1β的mRNA水平和IL-1β的蛋白水平呈明显的剂量依赖效应。这些实验结果表明PRRSV通过上调NLRP3信号通路蛋白而促进早期炎症反应,但在感染晚期,PRRSV可能通过某种机制而抑制了NLRP3介导的炎症反应。因此接下来本研究首先确证PRRSV激活的炎症反应是NLRP3炎症小体所依赖的,然后筛选鉴定PRRSV编码抑制炎症反应的组分,最后探究PRRSV感染早期促进炎症反应的分子机制。2.PRRSV诱导IL-1β的分泌依赖于NLRP3炎症小体。分别利用NLRP3的特异性抑制剂Glyburide以及针对NLRP3和ASC的特异性siRNAs对NLRP3炎症小体进行抑制和干扰,结果发现,Glyburide能够显著抑制由PRRSV BJ-4株诱导的IL-1β的分泌,而且,针对NLRP3和ASC的特异性siRNAs可以显著下调该基因的表达,并且也能够显著抑制由PRRSV诱导的IL-1β的分泌。3.PRRSV非结构蛋白nsp11和nsp1α是抑制NLRP3炎症小体介导的IL-1β分泌的病毒组分。分别将nsp11及其核酸内切酶活性缺失的突变体和nsp1α及其缺失或突变N端锌指(ZF)结构的突变体转染PAMs细胞,结果发现,nsp11和nsp1α都能够抑制由LPS引起的pro-IL-1βmRNA水平的升高以及由LPS和尼日利亚菌素(Nigericin)引起的IL-1β蛋白水平的升高。进一步的突变实验结果显示nsp11的核酸内切酶活性以及nsp1α的N端锌指结构对于其抑制作用是必需的。然后,在已重构NLRP3炎症小体模型的HEK293T细胞上转染PRRSV nsp11和nsp1α及其突变体的真核表达质粒,得到与PAMs细胞上相一致的实验结果,进一步确定nsp11和nsp1α对NLRP3炎症小体介导的IL-1β表达的抑制作用。4.PRRSV上调的miR-373能促进炎症反应。我们的前期实验发现PRRSV感染MARC-145细胞后上调宿主miR-373的表达,而进一步的实验发现,在LPS和Nigericin共同刺激的PAMs细胞上过表达miR-373,可以显著增加NLRP3炎症小体介导的IL-1β的分泌以及促进NLRP3、procaspase-1和pro-IL-1βmRNA水平的升高,从而促进炎症反应的发生。另外,PRRSV感染PAMs细胞后可以显著下调miR-373的靶基因TGFBR2(免疫抑制性受体)的表达。综上所述,PRRSV感染激活NLRP3炎症小体并产生IL-1β等促炎性细胞因子来抵御病毒入侵和保护机体的同时,病毒自身又编码能拮抗NLRP3炎症小体活化的蛋白——nsp11和nsp1α,抑制炎症反应的发生,从而使其能够在宿主体内持续增殖。另外,首次发现miR-373可以促进炎症反应的发生,可能参与调控PRRSV引起的天然免疫反应过程。PRRSV感染可以显著上调miR-373并下调其靶基因TGFBR2的表达,促进感染早期炎症反应的发生。因此,本研究进一步发现了PRRSV抵抗天然免疫的新机制——拮抗NLRP3炎症小体活化,为PRRSV的防控提供了潜在的分子靶点和理论基础。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome(PRRS) is an important viral infectious disease in swine industry worldwide. The causative agent is porcine reproductive and respiratory syndrome virus(PRRSV), which can inhibit host innate and adaptive immune response, cause immunosuppression, and lead to persistent infection. So it is difficult to control and eradicate PRRS. PRRSV can cause a strong interstitial pneumonia in infected pigs, and induce proinflammatory cytokines in lung. Recent studies have shown that PRRSV activated the NLRP3 inflammsome and induced IL-1β secretion. The inflammatory response is the solid line in host anti-viral immunity; however, how PRRSV regulate the inflammatory response to escape the immunologic surveillance remains unknown. Therefore, this study focused on the molecular mechanism of how PRRSV regulated the NLRP3-mediated the inflammatory response. The results of this study were as follows:1. The dynamic changes of NLRP3 inflammasome during PRRSV infection. PAMs were infected with the PRRSV strain BJ-4 at different times(12 h, 24 h, 48 h, 72 h), then, NLRP3, ASC, procaspase-1 and pro-IL-1β mRNA expression and IL-1β production were detected by qPCR and ELISA. The obtained results showed that PRRSV could induce NLRP3, ASC, procaspase-1 and pro-IL-1β mRNA expression and secretion of IL-1β in early infection in porcine alveolar macrophages(PAMs), but the levels of pro-IL-1β mRNA and IL-1β protein decreased to a degree that was similar to the level of the mock-infected group in later infection. And different dose of PRRSV infection induced procaspase-1 and pro-IL-1β mRNA expression and secretion of IL-1β at a dose dependent manner. The experiment results showed that PRRSV promoted early inflammatory response through up-regulating NLRP3 signaling pathway proteins, but inhibited NLRP3-mediated inflammatory response through some unknown mechanisms in later infection. So the following studies first confirmed whether the inflammatory response induced by PRRSV was dependent on the NLRP3 signaling pathway, and then screened the components of PRRSV which inhibited the inflammatory response. Finally, this study explored the molecular mechanism on how PRRSV promoted the inflammatory response in early infection.2. The secretion of IL-1β induced by PRRSV is dependent on the NLRP3 inflammasome pathway. NLRP3 inhibitor-glyburide and specific siRNA targeting NLRP3 or ASC were used to inhibit the NLRP3 inflammasome activation. The results showed that glyburide could inhibit the secretion of IL-1β induced by PRRSV significantly. And the siRNA targeting NLRP3 or ASC could down-regulated the gene expression and the IL-1β induction.3. PRRSV nsp11 and nsp1α are the components to inhibit NLRP3 inflammasome mediated IL-1β secretion. PAMs were transfected with PRRSV nsp11 and the inactivated endoribonuclease mutants or nsp1α and the deletion or mutation of Zinc-Finger(ZF) domain mutants. The results showed that nsp11 and nsp1α could inhibit the expression of pro-IL-1β mRNA induced by lipopolysaccharide(LPS) and the secretion of IL-1β induced by LPS plus nigericin. Furthermore, the mutation studies showed that the endoribonuclease activity and ZF domain were essential for nsp11 and nsp1α to inhibit the secretion of IL-1β. Then, the NLRP3 inflammasome was reconstructed in HEK293 T cells which were then transfected with expression plasmid encoding nsp11, nsp1α and the mutants. The results were consistent with that in PAMs, which further determined the inhibition of nsp11 and nsp1α to NLRP3 inflammasome mediated IL-1β secretion.4. MiR-373 is up-regulated by PRRSV and it can promote the inflammatory response. Our previous study showed that PRRSV could up-regulate mi R-373 expression in MARC-145 cells. And further study found overexpression of miR-373 in PAMs stimulated by LPS plus nigericin could increase secretion of IL-1β and NLRP3, procaspase-1 and pro-IL-1β mRNA expression, which could promote the inflammatory response. In addition, PRRSV infection could down-regulated the expression of target gene TGFBR2(immuno-inhibitory receptor) of mi R-373 significantly.In conclusion, PRRSV infection could activate the NLRP3 inflammasome and induced the proinflammatory cytokine IL-1β secretion which can protect host from the virus invasion. Meanwhile, PRRSV could encode nsp11 and nsp1α to antagonize the activation of NLRP3 inflammasome and inhibit the inflammatory response, which were in favor of the virus proliferation. In addition, we for the first time found that miR-373 could promote the inflammatory response and regulate the innate immune response induced by PRRSV. PRRSV infection could up-regulated miR-373 and down-regulated TGFBR2 expression significantly, which promoted the inflammatory response in early infection. Our study reveals a new mechanism that PRRSV antagonize host innate immune responses and may provide some insights into the research on molecular targets of anti-PRRSV drugs and prevention of PRRS.更多还原