TLR9/MyD88信号通路在HIV-1不易感中的作用及机制研究

So far the mechanism(s) of insusceptibility to HIV infection in HIV highly exposed persistently seronegative (HEPS) individuals has not been clearly understood. Previous studies have shown that the expression of two types of HIV inhibitor: MIP-1α/β is elevated in (HEPS) individuals. MIP-1α/β are the competitive ligands binging to CCR5 that is the receptor of HIV, while MX1/2 have the avility to inhibit the HIV infection and replication. Our preliminary data indicated that the expression of TLR9, MIP-1α/β, and MX1 decreased in HIV-1-infected individuals. It is well known that TLR9 recognizes HIV infection and activation of TLR9 pathway leads to production of MIP-1α/β and MX1/2. TLR9 has a direct relationship with all three factors. Thus, we hypothesize: TLR9/MyD88 signaling pathway plays a key role in HIV-1 insusceptibility through up-regulation of MIP-1α/β and MX1/2 expression, which can control HIV infection/replication in host cells. To verify our hypothesis, we will utilize HEPS population investigation and in vitro studies to detect the the expression of key factors in TLR9 pathway under the conditions of in vitro activating or blocking TLR9 pathway, thus illuminate the association of these factors with insusceptibility to HIV infection. This study, for the first time, explore the mechanism of HIV insusceptiblily from a new perspective- innate TLR9 signaling pathway. We expect this study will reveal the role of TLR9/MyD88 signaling pathway in HIV-1 insusceptibility. Our study, if successful, will lay the foundation for revealing the mechanism of HEPS and provide new strategy for HIV clinical treatment.

HIV高暴露持续血清阴性者（HEPS）的HIV不易感机理尚未清楚。研究表明HIV抑制因子MIP-1α/β在HEPS中高表达，MX1/2可抑制HIV的感染和复制。我们前期发现TLR9、MIP-1α/β、MX1在HIV感染者中表达下降。TLR9通路可识别HIV感染，并可诱导产生MIP-1α/β和MX1/2，与三者都有直接关系。据此推测：TLR9/MyD88通路可上调MIP-1α/β、MX1/2的表达，阻止HIV在体内感染复制，从而在HEPS的HIV-1不易感中起关键作用。本项目利用HEPS人群调查和病毒-细胞系统研究，在体外激活或封闭TLR9通路，通过RT-PCR、WB等方法检测该通路关键因子的表达，分析其与HIV不易感的关系。本研究首次从免疫通路切入，体内外结合，从正反两面探讨TLR9/MyD88通路对HIV不易感的影响作用和机制，为揭示HEPS人群HIV不易感机理奠定基础。

HIV-1高暴露血清持续阴性人群（HESN，也叫HEPS）是研究宿主免疫抗HIV的重要人群，其作用机制的阐明将有助于发现新的药物靶点和免疫佐剂。HESN人群的抗病毒机制被认为与天然免疫相关，但目前研究结果不一致。针对上述问题，本研究从天然免疫的角度切入，通过人群和体外细胞实验相结合，探讨HESN人群对HIV不易感的机制。本研究首先筛选了HESN人群与健康对照组之间PPRs的差异表达谱，发现 HESN人群的TLR9/MyD88信号通路关键因子（TLR9、IRF7、NF-κB）、主要抗病毒因子（IFN-α/β）的表达高于健康对照组。结果表明，TLR9/MyD88信号通路可能参与了HESN人群对HIV-1感染的抵抗。因此我们进一步在人群验证该信号通路的表达，发现HESN人群的TLR9、IRF7、NF-κB等关键因子的表达显著高于健康对照组。为了明确其具体机制，我们建立了体外PBMCs-病毒模型，在体外ODN2216激活条件下，检测TLR9/MyD88信号通路的表达，发现在TLR9激活的条件下，HESN人群的TLR9、IRF7、IFN-α、MIP-1α/β的表达显著高于健康对照组。表明在TLR9被激活后，HESN人群在PBMCs中显示出更高应答的TLR9信号通路。另外，为了确认TLR9对HESN人群的作用机制，我们进一步建立了体外巨噬细胞-病毒模型，在体外TLR9激活条件下，检测TLR9/MyD88信号通路发现，HESN人群的TLR9、IRF7、NF-κB、IFN-α/β、MIP-1α/β、RANTES的表达显著高于健康对照组，与人群体内结果一致。同时，为了明确HESN人群的TLR9对HIV的作用，我们检测了巨噬细胞系统P24的表达，与没有激活TLR9通路相比，不管是HESN人群还是健康对照人群，激活后均能抑制HIV的感染，但HESN人群能更好的抑制HIV感染。上述结果表明HESN人群不易感的机制可能是通过TLR9/MyD88信号通路产生IFN-α/β、MIP-1α/β、RANTES来抑制HIV的感染。本研究首次从体内外证实TLR9对HESN人群的HIV不易感具有重要的作用，并阐释了可能通过依赖MyD88信号通路，激活抗病毒因子和趋化因子发挥关键作用。该研究为全面揭示HESN人群HIV不易感的机制奠定基础，对于艾滋病临床治疗和疫苗研究具有重要的理论意义和实用价值。