HIV-1 Tat Protein Induces Production of Proinflammatory Cytokines by Human Dendritic Cells and Monocytes/Macrophages through Engagement of TLR4-MD2-CD14 Complex and Activation of NF-κB Pathway

We recently reported that the human immunodeficiency virus type-1 (HIV-1) Tat protein induced the expression of programmed death ligand-1 (PD-L1) on dendritic cells (DCs) through a TLR4 pathway. However, the underlying mechanisms by which HIV-1 Tat protein induces the abnormal hyper-activation of the immune system seen in HIV-1 infected patients remain to be fully elucidated. In the present study, we report that HIV-1 Tat protein induced the production of significant amounts of the pro-inflammatory IL-6 and IL-8 cytokines by DCs and monocytes from both healthy and HIV-1 infected patients. Such production was abrogated in the presence of anti-TLR4 blocking antibodies or soluble recombinant TLR4-MD2 as a decoy receptor, suggesting TLR4 was recruited by Tat protein. Tat-induced murine IL-6 and CXCL1/KC a functional homologue of human IL-8 was abolished in peritoneal macrophages derived from TLR4 KO but not from Wt mice, confirming the involvement of the TLR4 pathway. Furthermore, the recruitment of TLR4-MD2-CD14 complex by Tat protein was demonstrated by the activation of TLR4 downstream pathways including NF-κB and SOCS-1 and by down-modulation of cell surface TLR4 by endocytosis in dynamin and lipid-raft-dependent manners. Collectively, these findings demonstrate, for the first time, that HIV-1 Tat interacts with TLR4-MD2-CD14 complex and activates the NF-κB pathway, leading to overproduction of IL-6 and IL-8 pro-inflammatory cytokines by myeloid cells from both healthy and HIV-1 infected patients. This study reveals a novel mechanism by which HIV-1, via its early expressed Tat protein, hijacks the TLR4 pathway, hence establishing abnormal hyper-activation of the immune system.本课题组此前曾报道，1型人类免疫缺陷病毒（human immunodeficiency virus type-1, HIV-1）的Tat蛋白可通过Toll样受体4（Toll-like receptor 4, TLR4）通路诱导树突状细胞（dendritic cells, DCs）表达程序性死亡受体配体1（programmed death ligand-1, PD-L1）。然而，HIV-1 Tat蛋白诱导HIV-1感染者体内出现免疫系统异常过度激活的具体分子机制，仍有待全面阐明。 本研究中，我们证实HIV-1 Tat蛋白可诱导健康个体及HIV-1感染者来源的树突状细胞与单核细胞，分泌大量促炎性细胞因子IL-6与IL-8。当体系中加入抗TLR4阻断抗体或可溶性重组TLR4-MD2作为诱饵受体时，该细胞因子的分泌会被完全阻断，提示Tat蛋白可招募TLR4。 在Toll样受体4基因敲除（TLR4 KO）小鼠的腹腔巨噬细胞中，Tat诱导产生的小鼠IL-6以及人IL-8的功能同源物CXCL1/KC的分泌会被完全消除；而野生型（Wt）小鼠的腹腔巨噬细胞则无此现象，这进一步证实了TLR4通路的参与。 此外，通过检测TLR4下游通路（包括NF-κB与SOCS-1）的激活情况，以及动力蛋白（dynamin）和脂筏依赖的内吞作用介导的细胞表面TLR4下调，我们证实了Tat蛋白可招募TLR4-MD2-CD14复合物。 综上，本研究首次证实：HIV-1 Tat蛋白可与TLR4-MD2-CD14复合物结合并激活NF-κB通路，进而导致健康个体及HIV-1感染者来源的髓系细胞过量分泌IL-6与IL-8等促炎性细胞因子。本研究揭示了一种全新的分子机制：HIV-1可通过其早期表达的Tat蛋白劫持TLR4通路，最终引发免疫系统的异常过度激活。