TREM-2受体调节猪繁殖与呼吸综合征病毒复制的分子机制

Previous studies have shown that CD163 is necessary for porcine reproductive and respiratory syndrome virus (PRRSV) infection, CD163 knockout pigs are resistant to PRRSV. However, it is worth to understand the molecular mechanisms of target genes modulating CD163 expression. We recently found that PRRSV promoted triggering receptors expressed on myeloid cells 2 (TREM-2) expression in porcine alveolar macrophages (PAMs); the expressions of TREM-2 and CD163 were dramatically decreased, and a disintegrin and metalloprotease 17 (ADAM17) was activated, leading to the inhibition of PRRSV replication after LPS treatment in PAMs. Based on the achievements obtained, we plan to study the relationship between TREM-2 and PRRSV by TREM-2 knockdown and overexpression; then analyse whether TREM-2 regulates cytokine expression and ADAM17 via TLR signaling pathway, thereby modulating CD163 and PRRSV replication, which can reveal the mechanisms of TREM-2 regulating the replication of PRRSV; finally we further demonstrate its role to PRRSV replication in vivo. The data not only confirm the role of TREM-2 in PRRSV replication, but also provide new theoretical basis for screening susceptibility genes and breeding for disease resistance against PRRSV and contribute to further understand the pathogenesis of the virus.

研究表明CD163受体是PRRSV感染必需受体，敲除该基因猪可以抵抗病毒感染，但调控该受体表达的分子机制有待深入挖掘。本项目组发现PRRSV感染PAMs后会促进TREM-2受体表达；利用LPS刺激PAMs后，明显抑制TREM-2表达，同时激活酶ADAM17并抑制了CD163表达，结果PRRSV复制受到显著抑制。在此基础上，本项目拟通过TREM-2干扰和过表达等试验研究该受体与PRRSV复制关系；然后分析该受体是否通过 TLR信号通路调节细胞因子表达和ADAM17活性，进而调控CD163表达和PRRSV复制，揭示TREM-2受体调节该病毒复制的分子机制；最后通过动物实验，进一步验证TREM-2受体对PRRSV复制作用。预期研究结果不仅可确证TREM-2受体可调节PRRSV复制，还可为筛选PRRSV易感基因及抗病育种奠定基础，也有助于进一步理解该病毒致病机理。

TREM-2作为抗炎症受体，负调节先天性免疫应答，TREM-2主要在树突状细胞和巨噬细胞上表达，而这些细胞正是PRRSV的靶细胞。基于此，我们研究TREM-2在PRRSV感染中的作用。我们发现，猪肺泡巨噬细胞感染PRRSV后，TREM-2表达量显著上调，干扰TREM-2抑制病毒复制，而TREM-2过表达促进病毒复制。进一步发现，TREM-2的胞质尾区与PRRSV NSP2互作促进病毒感染。分析TREM-2调节PRRSV机制为：TREM-2下调激活 PI3K/NF-κB信号通路，进而使促炎细胞因子和I型干扰素上调表达，由于这些因子上调表达，ADAM17被激活，从而切割CD163，最终导致PRRSV感染的抑制。此外，我们原核表达了TREM-2的胞外域部分，并发现胞外域部分通过抑制病毒吸附抑制病毒复制，这种抑制病毒吸附，可能是由于TREM-2胞外域竞争性与病毒表面糖蛋白结合导致的。最后，通过活体实验，采集猪体内不同器官组织，发现肺、淋巴结、脾脏等免疫组织中TREM-2表达量最高，仔猪PRRSV攻毒后，免疫组织肺和淋巴结中的TREM-2表达量显著升高，与细胞体外结果一致。以上研究揭示了TREM-2通过炎症应答调节PRRSV感染，TREM-2可作为预防治疗PRRSV感染的重要靶点，同时本研究进一步解释了PRRSV致病机理。.另外，我们还发现，外源添加原核表达的TREM-2胞外域部分通过促进Heparanase酶活性，切割病毒受体硫酸乙酰肝素（HS），进而抑制病毒吸附，最终抑制病毒复制。同时，在病毒感染晚期，病毒会激活Heparanase，以切割HS受体，抑制胞内复制的病毒粒子与细胞表面HS相互黏连，最终促进病毒释放，由此可知Heparanase上调表达可促进病毒释放。基于此，我们筛选到靶向Heparanase酶活性的锌离子载体Pyrithione (PT). PT通过抑制NF-kappaB通路，进而抑制Heparanase酶活性，从而抑制病毒释放，最终抑制病毒复制。PT可作为一种有效的抗PRRSV制剂。