Name: dissecting innate immune signaling in viral evasion of cytokine production
Uploaded: 2014-03-02T14:00:00
Description: Watch this Scientific Journal Video about Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production at JoVE.com

野生型（ 小牛+ / +）和基因敲除（ 小牛- / - ）小鼠，野生型（ 小牛+ / +）和基因敲除（ 小牛- / - ）的MEF由李志坚陈军大学博士（友情提供得克萨斯大学西南医学中心）13。本出版物是基于由美国国立卫生研究院（R01 CA134241和R01 DE021445）和美国癌症协会（RSG-11-162-01-MPC）赠款资助的工作。

在严格按照指南中的美国国立卫生研究院实验动物的护理和使用的建议进行的所有动物工作：操守准则 。该方案经南加州大学的机构动物护理和使用委员会（IACUC）。 1。通过实时定量PCR和分泌的ELISA方法γHV68感染小鼠细胞因子基因表达<ol><li>通过腹腔注射1.5毫克ketamine/0.12毫克甲苯噻嗪麻醉性别匹配，6-8周龄的C57BL / 6（B6）窝（8-12只/组），并与40 PFUγHV68的滴鼻?...

<ol>
	<li>Akira, S., Uematsu, S., Takeuchi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen+recognition+and+innate+immunity.">Pathogen recognition and innate immunity.</a> Cell. 124, 783-801 (2006).</li><li>Kato, H., Takahasi, K., Fujita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RIG-I-like+receptors:+cytoplasmic+sensors+for+non-self+RNA.">RIG-I-like receptors: cytoplasmic sensors for non-self RNA.</a> Immunol. Rev. 243, 91-98 (2011).</li><li>Kawai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IPS-1,+an+adaptor+triggering+RIG-I-+and+Mda5-mediated+type+I+interferon+induction.">IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction.</a> Nat. Immunol. 6, 981-988 (2005).</li><li>Seth, R. B., Sun, L., Ea, C. K., Chen, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+MAVS,+a+mitochondrial+antiviral+signaling+protein+that+activates+NF-kappaB+and+IRF+3.">Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3.</a> Cell. 122, 669-682 (2005).</li><li>Xu, L. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VISA+is+an+adapter+protein+required+for+virus-triggered+IFN-beta+signaling.">VISA is an adapter protein required for virus-triggered IFN-beta signaling.</a> Mol. Cell. 19, 727-740 (2005).</li><li>Meylan, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardif+is+an+adaptor+protein+in+the+RIG-I+antiviral+pathway+and+is+targeted+by+hepatitis+C+virus.">Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus.</a> Nature. 437, 1167-1172 (2005).</li><li>Speck, S. H., Virgin, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+and+viral+genetics+of+chronic+infection:+a+mouse+model+of+gamma-herpesvirus+pathogenesis.">Host and viral genetics of chronic infection: a mouse model of gamma-herpesvirus pathogenesis.</a> Curr. Opin. Microbiol. 2, 403-409 (1999).</li><li>Dong, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+gamma-herpesvirus+68+hijacks+MAVS+and+IKKbeta+to+initiate+lytic+replication.">Murine gamma-herpesvirus 68 hijacks MAVS and IKKbeta to initiate lytic replication.</a> PLoS Pathog. , (2010).</li><li>Dong, X., Feng, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+gamma+herpesvirus+68+hijacks+MAVS+and+IKKbeta+to+abrogate+NFkappaB+activation+and+antiviral+cytokine+production.">Murine gamma herpesvirus 68 hijacks MAVS and IKKbeta to abrogate NFkappaB activation and antiviral cytokine production.</a> PLoS Pathog. 7, (2011).</li><li>Dong, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+gammaherpesvirus+68+evades+host+cytokine+production+via+replication+transactivator-induced+RelA+degradation.">Murine gammaherpesvirus 68 evades host cytokine production via replication transactivator-induced RelA degradation.</a> J. Virol. 86, 1930-1941 (2012).</li><li>Dong, X., Feng, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+Host-virus+Interaction+in+Lytic+Replication+of+a+Model+Herpesvirus.">Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus.</a> J. Vis. Exp. , (2011).</li><li>He, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+interacting+protein+kinase-3+determines+cellular+necrotic+response+to+TNF-alpha.">Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha.</a> Cell. 137, 1100-1111 (2009).</li><li>Sun, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+specific+and+essential+role+of+MAVS+in+antiviral+innate+immune+responses.">The specific and essential role of MAVS in antiviral innate immune responses.</a> Immunity. 24, 633-642 (2006).</li></ol>

病毒免疫逃避是最有活力和迷人的交互接口病毒进攻和宿主防御9之一。宿主先天免疫成分的结构，使得信号转导被有效地启动和忠实传送。划定信号级联的层次结构和调控是先天免疫的一个主要话题。在这里，我们介绍一个协议，以确定一种天生的免疫成分，MAVS，在细胞因子产生的病毒逃避的调控作用。该协议包括一种方法确定细胞因子产生的病毒感染的小鼠和评估的MEF细胞因子基因表...

最近的研究已经安装在宿主先天免疫反应中概述的整体信号级联。不同的隔间内居住，模式识别受体（的PRRs）检测病原体相关分子模式不同来源的（的PAMP）来触发先天免疫信号1。视黄酸诱导基因I（RIG-I）和黑色素瘤分化抗原5（MDA5）蛋白是细胞内的传感器能够识别RNA分子具有特定的结构特征2。在激活时，RIG-I与其下游MAVS（也称为IPS-1，VISA和CARDIF）适配器，反过来，激活IKK（IKKαβγ）和IKK-相关激酶（TBK1和IKKε，也称为一骑交互）配合物3-6。激活先天免疫激酶磷酸化的基因表达的关键调节剂，包括转录因子和其抑制剂，以及使宿主的抗病毒基因（ 如 IL6，TNF＆＃转录激活945，CCL5和IFNβ）。这些信号级联构成了建立抗微生物状态，在感染的早期阶段，以限制病原体传播烈性固有先天免疫反应。 鼠γHV68是密切相关的人类致癌KSHV和EB病毒。因此，γHV68感染的小鼠提供了一种易于处理的小动物模型，研究对γ疱疹病毒感染的体内7的宿主免疫应答。使用γHV68，我们的实验室已经发现了一个复杂的策略，即γHV68劫持宿主先天免疫信号，使病毒感染。一方面，γHV68激活MAVS-IKKβ途径通过指导激活IKKβ磷酸化的复制式激活（RTA），该病毒的转录因子密钥γHV68复制8，以促进病毒的转录激活。在另一方面，本IKKβ介导的磷酸化素数的RelA降解和术语inatesNFκB激活9。因此，γHV68感染有效地避免了抗病毒细胞因子的产生。有趣的是，利用γHV68的表达文库筛选鉴定RTA作为一个E3连接酶诱导的RelA退化和废除NFκB激活10。这些发现揭示了复杂的免疫逃避策略，即上游免疫信号事件是由γHV68利用，以否定的终极抗病毒细胞因子的产生。 在这里，我们描述了一个协议来衡量抗病毒细胞因子的产生在体内和前VIVØ感染γHV68小鼠。在协议中，我们通过慢病毒转导，从而精确定位特定的固有免疫分子在调节抗病毒细胞因子产生的作用进一步探讨在淘汰赛胚胎成纤维细胞的天然免疫成分的“复原”的表情。这个协议可以很容易地适应其它维鲁SES和信号通路。

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			<td height="20" style="height:20px;width:251px;">Mouse CCL5 ELISA kit</td>
			<td style="width:211px;">R&#38;D systems</td>
			<td style="width:223px;"><a href="http://www.rndsystems.com/Products/DY478">DY478</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:251px;">1.0 mm Zirconia/Silica beads</td>
			<td style="width:211px;">BioSpec Products</td>
			<td style="width:223px;">11079110z</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:251px;">TRIzol</td>
			<td style="width:211px;">Invitrogen</td>
			<td style="width:223px;"><a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=15596018">15596-018</a></td>
		</tr>
		<tr height="79">
			<td height="98" rowspan="2" style="height:98px;width:251px;">SuperScript II Reverse Transcriptase</td>
			<td rowspan="2" style="width:211px;">Invitrogen</td>
			<td rowspan="2" style="width:223px;">18064-014</td>
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			<td height="20" style="height:20px;"> </td>
			<td></td>
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			<td colspan="2" height="21" style="height:21px;">CCL5 quantitative real-time PCR primers</td>
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			<td height="20" style="height:20px;">CCTGCTGCTTTGCCTACCTCTC</td>
			<td></td>
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			<td height="20" style="height:20px;">ACACACTTGGCGGTTCCTTCGA</td>
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dissecting innate immune signaling in viral evasion of cytokine production

响应于病毒感染，宿主先天免疫反应被激活，上调抗病毒细胞因子的基因表达和生产。相反，病毒已经进化复杂的策略，以规避和利用宿主的免疫信号传导的生存和繁殖。病毒免疫逃避，将会导致宿主防御和病毒逃避，提供了最迷人的和动态的接口之一辨别宿主 - 病毒相互作用。这些研究推进我们理解先天免疫调节和我们铺平道路，以开发新的抗病毒治疗。 鼠γHV68鼠是啮齿动物的天然病原体。 γHV68感染小鼠提供了一个听话的小动物模型，研究抗病毒对人类社会的KSHV和EB病毒的体内病毒-宿主相互作用的扰动并不适用。在这里，我们描述了一个协议来确定抗病毒细胞因子的产生。这个协议可以适于其它vir区使用和信号通路。 最近，我们已发现，γHV68劫持MAVS与IKKβ，胞质RIG-I和MDA5的下游键先天免疫信号分量，废除NFκB活化和抗病毒细胞因子的产生。具体来说，γHV68感染激活IKKβ和激活IKKβ磷酸化的RelA加速的RelA退化。因此，γHV68高效解耦从上游激活IKKβNFκB活化，否定了抗病毒细胞因子基因表达。这项研究阐明了一个复杂的策略，即上游先天免疫激活被病毒病原体截获请求撤销的直接下游转录激活和逃避抗病毒细胞因子的产生。

三个有代表性的人物都在这里显示，包括细胞因子产生的γHV68感染小牛肺+ / +和小牛 -/ - / - - γHV68感染的小牛 + / +和小牛的鼠标，细胞因子的分泌和基因表达水平的MEF和γHV68感染小牛的细胞因子mRNA水平- / - MEFs细胞“再造”与MAVS。这些代表性实验利用基因敲除小鼠的研究抗病毒药细胞因子的产生在体内和探索基因敲除...

Watch this Scientific Journal Video about Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production at JoVE.com

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

我们描述了一种协议来测量抗病毒细胞因子的产生在感染了模型疱疹病毒，鼠γ-疱疹病毒68（γHV68）小鼠是密切相关的人类卡波西肉瘤相关疱疹病毒（KSHV）和Epstein-Barr病毒（EBV）。利用转基因小鼠品系和小鼠胚胎成纤维细胞（MEFs细胞）中，我们评估了抗病毒细胞因子的产生在体内和离体 。 “重构”的淘汰赛中胚胎成纤维细胞通过慢病毒转导的天然免疫成分的表达，我们进一步确定具体的固有免疫分子和剖析关键的信号事件差异调节抗病毒细胞因子的产生。

细胞因子产生的病毒逃避解剖先天免疫信号

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

Research

JoVE Journal

Immunology and Infection

Production and Titering of Recombinant Adeno-associated Viral Vectors

重组腺相关病毒载体的生产和滴定

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

科研

免疫与感染

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

呼吸爆发的响应为先天免疫健康的斑马鱼中的指标量化

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic Poly(I:C)

Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic PolyI:C

母体免疫激活诱导小鼠的病毒性模仿聚妊娠中期阶段（I：C）

Maternal immune activation (MIA) model is increasingly well appreciated as a rodent model for the environmental risk factor of various psychiatric ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

分子互补亲和纯化 (BiCAP) 对多蛋白信号络合物的解剖

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

大鼠外周全血细胞免疫表型和因子生成的单细胞分析

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

一种评价金黄色葡萄球菌感染先天免疫反应的小鼠模型

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

一种研究食细胞诱导免疫应答的 mellonella 口服液模型

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

单核细胞分离研究人类粘膜淋巴组织中先天免疫反应

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...