DDX19A識別PRRSV基因組RNA并激活NLRP3炎癥小體
發(fā)布時間：2018-09-12 12:29
【摘要】：豬繁殖與呼吸綜合征(porcine reproductive and respiratory syndrome,PRRS)是由豬繁殖與呼吸綜合征病毒(PRRS virus,PRRSV)引起的一類嚴(yán)重接觸性傳染病,主要引起懷孕母豬流產(chǎn)、早產(chǎn)、死胎、木乃伊胎及仔豬呼吸道疾病等癥狀。2006年爆發(fā)的由高致病PRRSV(highly pathogenic PRRSV,HP-PRRSV)引起的以高熱、高發(fā)病率和高死亡率為特征的高致病PRRS給我國的生豬產(chǎn)業(yè)造成了難以估量的經(jīng)濟(jì)損失。HP-PRRSV感染豬肺泡巨噬細(xì)胞(pulmonary alveolar macrophages,PAMs),誘導(dǎo)促炎性細(xì)胞因子的大量分泌,引起嚴(yán)重的肺部炎癥癥狀。我們實驗室前期研究結(jié)果證明弱毒株CH1a、CH1R和疫苗毒株Hu N4-F112均不能誘導(dǎo)IL-1β和TNF-α的分泌,而HP-PRRSV毒株Hu N4和SDA2卻均能誘導(dǎo)這些細(xì)胞因子的高水平表達(dá)和分泌,表明強(qiáng)弱毒株誘導(dǎo)炎癥反應(yīng)的能力不同。IL-1β作為最重要的炎癥介質(zhì)之一,在啟動和放大炎癥反應(yīng),清除感染,加速組織修復(fù),活化保護(hù)性免疫反應(yīng)等過程中發(fā)揮重要作用。大量研究表明,HP-PRRSV感染可以誘導(dǎo)IL-1β的分泌,但是,具體的分子機(jī)制還不清楚。本研究從分子水平探究HP-PRRSV感染誘導(dǎo)IL-1β分泌的機(jī)理。IL-1β的成熟需要兩個信號:第一信號由細(xì)胞編碼的模式識別受體(Pattern recognition receptor,PRRs)啟動NF-κB信號通路活化,促進(jìn)pro-IL-1β的產(chǎn)生;第二信號由病原相關(guān)分子模式(Pathogen-associated molecular patterns,PAMPs)或危險相關(guān)分子模式(Damage Associated Molecular Patterns,DAMPs)介導(dǎo)炎癥小體激活,引起pro-caspase-1活化及pro-IL-1β成熟分泌。本研究證明,HP-PRRSV感染可以誘導(dǎo)pro-caspase-1的活化以及pro-IL-1β的切割成熟。深入研究發(fā)現(xiàn)HP-PRRSV感染可以誘導(dǎo)NLRP3炎癥小體復(fù)合物的形成。敲低NLRP3的表達(dá)顯著抑制PRRSV感染誘導(dǎo)的pro-caspase-1活化和IL-1β分泌。隨后的研究發(fā)現(xiàn)HP-PRRSV基因組RNA可以誘導(dǎo)較高水平的IL-1β分泌,證明HP-PRRSV基因組RNA及其5?UTR轉(zhuǎn)錄物均可以誘導(dǎo)NLRP3炎癥小體的激活。敲低NLRP3的表達(dá)顯著抑制HP-PRRSV基因組RNA及其5?UTR轉(zhuǎn)錄物誘導(dǎo)的IL-1β分泌。本研究用大量數(shù)據(jù)證明了HP-PRRSV感染可以激活NLRP3炎癥小體,且發(fā)現(xiàn)HP-PRRSV基因組RNA就足以激活NLRP3炎癥小體,其誘導(dǎo)IL-1β分泌與病毒的復(fù)制過程無關(guān)。病毒RNA可以誘導(dǎo)NLRP3炎癥小體的活化,但是目前還沒有任何證據(jù)證明NLRP3炎癥小體的組分可以直接識別病毒的RNA。因此,我們推測細(xì)胞內(nèi)可能存在未知的病毒RNA受體分子。利用HP-PRRSV感染豬PAMs為模型,通過抗ASC抗體免疫共沉淀試驗和串聯(lián)質(zhì)譜分析鑒定出了DDX19A。我們發(fā)現(xiàn)DDX19A與NLRP3炎癥小體復(fù)合物中的NLRP3相互作用,而不與ASC和caspase-1相互作用。進(jìn)一步證明DDX19A能直接結(jié)合HP-PRRSV基因組RNA及其5?UTR和3?UTR轉(zhuǎn)錄物、poly I:C。敲低DDX19A的表達(dá)顯著抑制HP-PRRSV基因組RNA及其5?UTR轉(zhuǎn)錄物誘導(dǎo)的NLRP3炎癥小體活化和IL-1β分泌,但不影響poly I:C和LPS/ATP誘導(dǎo)的IL-1β分泌。綜上所述,本研究從分子水平揭示了HP-PRRSV感染PAMs誘導(dǎo)NLRP3炎癥小體激活的機(jī)理,證明宿主蛋白DDX19A可以識別HP-PRRSV基因組RNA,招募NLRP3,激活NLRP3炎癥小體,誘導(dǎo)IL-1β分泌。本研究的創(chuàng)新性結(jié)果為防控PRRS和開發(fā)抗炎藥物提供重要參考。
[Abstract]:Porcine reproductive and respiratory syndrome (PRRS) is a serious * * contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRS virus), which mainly causes pregnancy, sow abortion, premature birth, stillbirth, mummification * and piglet respiratory diseases. V (highly pathogenic PRRSV, HP-PRRSV), a highly pathogenic PRRS characterized by high fever, high morbidity and high mortality, has caused incalculable economic losses to the pig industry in China..HP-PRRSV infection of * * porcine alveolar macrophages (pulmonary alveolar macrophages, PAMs) has induced severe secretion of proinflammatory cytokines, causing severe morbidity. Previous studies in our laboratory showed that attenuated strains CH1a, CH1R and vaccine strains Hu N4-F112 could not induce the secretion of IL-1beta and TNF-alpha, while HP-PRRSV strains Hu N4 and SDA2 could induce the high level expression and secretion of these cytokines, indicating that the ability of strong and weak strains to induce inflammation was different. One of the important inflammatory mediators plays an important role in initiating and amplifying inflammatory response, clearing infection, accelerating tissue repair and activating protective immune response. A large number of studies have shown that HP-PRRSV infection can induce IL-1 beta secretion, but the specific molecular mechanism is still unclear. Mechanisms of IL-1 beta secretion. The maturation of IL-1 beta requires two signals: the first signal is activated by cell-encoded pattern recognition receptors (PRRs) to activate the NF-kappa B signaling pathway and promote pro-IL-1 beta production; the second signal is either Pathogen-associated molecular patterns (PAMPs) or risk-related. Damage Associated Molecular Patterns (DAMPs) mediate the activation of inflammatory corpuscles, induce the activation of pro-caspase-1 and the maturation and secretion of pro-IL-1 beta. This study demonstrates that HP-PRRSV infection can induce the activation of pro-caspase-1 and the cleavage and maturation of pro-IL-1 beta. Inhibition of NLRP3 expression significantly inhibited pro-caspase-1 activation and IL-1 beta secretion induced by PRRSV infection. Subsequent studies found that HP-PRRSV genomic RNA could induce a higher level of IL-1 beta secretion, suggesting that both HP-PRRSV genomic RNA and its 5?UTR transcripts could induce the activation of NLRP3 inflammatory bodies. P-PRRSV genomic RNA and its 5?UTR transcripts induce IL-1 beta secretion. This study demonstrated that HP-PRRSV infection can activate NLRP3 inflammation bodies, and found that HP-PRRSV genomic RNA is sufficient to activate NLRP3 inflammation bodies, and its induction of IL-1 beta secretion has nothing to do with the replication process of the virus. Viral RNA can induce the activation of NLRP3 inflammation bodies. However, there is no evidence that the components of NLRP3 inflammable bodies can directly recognize the RNA. of the virus. Therefore, we speculate that there may be unknown RNA receptor molecules in the cell. * using HP-PRRSV infected pig PAMs as a model, we identified DDX19A. by anti ASC antibody immunoprecipitation and tandem mass spectrometry, and we found DDX. It is further demonstrated that DDX19A can bind directly to HP-PRRSV genomic RNA and its 5?UTR and 3?UTR transcripts, and poly I:C. knockdown of DDX19A significantly inhibits the activation of NLRP3 inflammatory bodies induced by HP-PRRSV genomic RNA and its 5?UTR transcripts and IL-1. In conclusion, this study revealed the mechanism of activation of NLRP3 inflammatory bodies induced by HP-PRRSV infection with PAMs at the molecular level, and demonstrated that host protein DDX19A could recognize HP-PRRSV genomic RNA, recruit NLRP3, activate NLRP3 inflammatory bodies and induce IL-1 beta secretion. The results provide important reference for preventing and controlling PRRS and developing anti-inflammatory drugs.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.28
本文編號：2239010
[Abstract]:Porcine reproductive and respiratory syndrome (PRRS) is a serious * * contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRS virus), which mainly causes pregnancy, sow abortion, premature birth, stillbirth, mummification * and piglet respiratory diseases. V (highly pathogenic PRRSV, HP-PRRSV), a highly pathogenic PRRS characterized by high fever, high morbidity and high mortality, has caused incalculable economic losses to the pig industry in China..HP-PRRSV infection of * * porcine alveolar macrophages (pulmonary alveolar macrophages, PAMs) has induced severe secretion of proinflammatory cytokines, causing severe morbidity. Previous studies in our laboratory showed that attenuated strains CH1a, CH1R and vaccine strains Hu N4-F112 could not induce the secretion of IL-1beta and TNF-alpha, while HP-PRRSV strains Hu N4 and SDA2 could induce the high level expression and secretion of these cytokines, indicating that the ability of strong and weak strains to induce inflammation was different. One of the important inflammatory mediators plays an important role in initiating and amplifying inflammatory response, clearing infection, accelerating tissue repair and activating protective immune response. A large number of studies have shown that HP-PRRSV infection can induce IL-1 beta secretion, but the specific molecular mechanism is still unclear. Mechanisms of IL-1 beta secretion. The maturation of IL-1 beta requires two signals: the first signal is activated by cell-encoded pattern recognition receptors (PRRs) to activate the NF-kappa B signaling pathway and promote pro-IL-1 beta production; the second signal is either Pathogen-associated molecular patterns (PAMPs) or risk-related. Damage Associated Molecular Patterns (DAMPs) mediate the activation of inflammatory corpuscles, induce the activation of pro-caspase-1 and the maturation and secretion of pro-IL-1 beta. This study demonstrates that HP-PRRSV infection can induce the activation of pro-caspase-1 and the cleavage and maturation of pro-IL-1 beta. Inhibition of NLRP3 expression significantly inhibited pro-caspase-1 activation and IL-1 beta secretion induced by PRRSV infection. Subsequent studies found that HP-PRRSV genomic RNA could induce a higher level of IL-1 beta secretion, suggesting that both HP-PRRSV genomic RNA and its 5?UTR transcripts could induce the activation of NLRP3 inflammatory bodies. P-PRRSV genomic RNA and its 5?UTR transcripts induce IL-1 beta secretion. This study demonstrated that HP-PRRSV infection can activate NLRP3 inflammation bodies, and found that HP-PRRSV genomic RNA is sufficient to activate NLRP3 inflammation bodies, and its induction of IL-1 beta secretion has nothing to do with the replication process of the virus. Viral RNA can induce the activation of NLRP3 inflammation bodies. However, there is no evidence that the components of NLRP3 inflammable bodies can directly recognize the RNA. of the virus. Therefore, we speculate that there may be unknown RNA receptor molecules in the cell. * using HP-PRRSV infected pig PAMs as a model, we identified DDX19A. by anti ASC antibody immunoprecipitation and tandem mass spectrometry, and we found DDX. It is further demonstrated that DDX19A can bind directly to HP-PRRSV genomic RNA and its 5?UTR and 3?UTR transcripts, and poly I:C. knockdown of DDX19A significantly inhibits the activation of NLRP3 inflammatory bodies induced by HP-PRRSV genomic RNA and its 5?UTR transcripts and IL-1. In conclusion, this study revealed the mechanism of activation of NLRP3 inflammatory bodies induced by HP-PRRSV infection with PAMs at the molecular level, and demonstrated that host protein DDX19A could recognize HP-PRRSV genomic RNA, recruit NLRP3, activate NLRP3 inflammatory bodies and induce IL-1 beta secretion. The results provide important reference for preventing and controlling PRRS and developing anti-inflammatory drugs.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.28
【參考文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 劉園園;豬繁殖與呼吸綜合征病毒激活NLRP3炎癥小體的研究[D];西北農(nóng)林科技大學(xué);2014年
，本文編號：2239010
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