金属蛋白酶ADAM17对猪繁殖与呼吸综合征病毒感染的抑制作用及机制研究

A disintegrin and metalloprotease 17（ADAM17） is a key metalloprotease involved in cleaving ectodomain of various thransmembrane proteins and producing a soluble extracellular domain fragment. This process provides a mechanism for protein downregulation on the cell surface, which directs both instantaneous and prolonged alternations in the activity of various cytokines, cytokines receptors, and adhesion molecules, and serves to initiate or inhibit autocrine and paracrine signaling. Thus, ADAM17 can act as a molecular switch to control immune responses, tissue regeneration and cancer development. Currently drugs targeted to ADAM17 are getting more attention on therapeutic interventions. However, there is no report about the inhibition role of ADAM17 on viral infection. PRRSV is one of the most severe porcine pathogens in recent years, which caused economic loss in swine production in China. Our preliminary experiment showed that inhibitor to ADAM17 can improve PRRSV infection through modifying its substrates, which indicates that ADAM17 can inhibit PRRSV infection.Therefore, in this study we are going to use PRRSV as a virus infection model to explore the inhibition mechanisms of ADAM17. First, we will generate porcine ADAM17 genetic expression mapping and establish ADAM17 knockout cell line using lentivirus-drived shRNA. Second, we will examine porcine ADAM17 immune response-related substrates (such as tumor necrosis factor-alpha、IgG Fc receptors, L-selectin, p75TNFalphaRⅡ, IL6R, and VACM-Ⅰ et. al.) and PRRSV receptor CD163. Third, in vitro co-stimulation model will be used to investigate how ADAM17 activity switch inhibits PRRSV infection, thus, based on in vitro results we will inspect the inhibition role of ADAM17 on PRRSV infection in vivo. Our goal is to manipulate the cell response using ADAM17-based modifiaction during PRRSV infection in order to decrease viral infection efficiency and attenuate viral damaging effects. This proposed basic research project will build on the foundation to advance our understanding the inhibition role of ADAM17 in virus infection so that evaluate ADAM17 as a novel therapeutic target in virus-induced diseases.

细胞表面的金属蛋白酶ADAM17作为免疫调节分子，可对细胞膜蛋白剪切修饰，调节机体免疫应答，在多种疫病治疗中备受关注。但有关ADAM17在病毒感染中的免疫调节作用和机制未见报道。PRRSV是国内近几年危害养猪业的重要病原之一，前期实验表明ADAM17具有抑制PRRSV感染的作用，所以本课题以PRRSV为感染模型，首次探索ADAM17在抑制病毒感染中的作用及机制。研究内容包括绘制猪源ADAM17基因表达图谱；阐述ADAM17猪源底物，如肿瘤坏死因子、IgG Fc受体等免疫分子及PRRSV受体CD163；体内外实验探讨ADAM17在抑制PRRSV感染的分子机制。该项目主要目的是拟通过修饰ADAM17生物学活性，降低PRRSV感染效率，减少病毒对机体造成的损伤。该基础研究将为ADAM17调控其他病毒感染奠定基础，为拮抗病毒感染提供新思路。

猪繁殖与呼吸综合征病毒（PRRSV）体内感染动物体，造成一系列的炎症反应，TNFa为炎症应答中最为重要的炎性因子。金属蛋白酶家族（ADAM）通过其特有的蛋白酶活性，对细胞膜蛋白进行剪切修饰，成为重要的免疫调节分子，在机体感染与免疫过程中发挥重要作用。体外感染实验表明，猪肺泡巨噬细胞（PAMs）感染PRRSV后，产生大量TNFa。研究表明，可溶性TNFa可发挥抗PRRSV感染的作用。通过运用抑制剂或基因修饰的实验手段，我们揭示猪源TNFa的产生是由ADAM17介导产生的。.进一步研究发现，特异性抑制ADAM17活性可提高病毒感染入侵细胞的能力。具体机制为：过表达ADAM17可下调PRRSV细胞受体CD163的表达水平进而抑制PRRSV感染；siRNA敲低内源性ADAM17时可上调CD163的表达进而促进PRRSV感染。这种由ADAM17介导的影响PRRSV感染的效应在PRRSV非易感细胞（瞬时转染表达CD163）也得到验证。这些发现表明PPRSV感染活化ADAM17进而剪切细胞受体CD163限制了PRRSV的入侵。.然而，由于PRRSV感染存在抗体依赖性增强作用（ADE），致使病毒在免疫预防中存在一定困难。我们研究发现，亚中和活性的血清在PAM细胞上可产生PRRSV感染的ADE效应。CD16功能抑制性抗体研究证实，CD16参与了PRRSV感染的ADE效应。由于我们的研究结果表明CD16是PAM细胞上高表达的Fcγ受体，CD16的ADE效应在Fcγ受体非表达细胞中进行。结果表明，CD16可介导PRRSV-抗体复合物的吸附与内化，进而产生具有感染性的子代病毒。这种CD16介导的ADE效应可促进PRRSV感染入侵表达的CD16免疫细胞进而促进病毒的扩散。.最后，我们测定PRRSV感染动物体的血清，结果发现可溶性CD16在PRRSV阳性猪群中水平显著高于对照、PEDV, PCV-2和PRV感染的猪只。其中，PRRSV可非特异吸附于中性粒细胞进而下调CD16的细胞表达水平。体外实验进一步验证PRRSV感染促进CD16的剪切。基因修饰试验方法表明PRRSV活化的ADAM17介导了CD16的剪切。提示可溶性CD16水平的升高提示急性病毒感染，如PRRSV感染。. 我们的研究结果已经归纳总结并发表在研究论文，如JVI，JGV (2篇)，Imm Res, 中文核心期刊。