Toll-like receptor 7-adapter complex modulates interferon-α production in HIV-stimulated plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDCs) and their production of interferon-alpha (IFN-α) are believed to play an important role in human immunodeficiency virus, type I (HIV-1) pathogenesis. PDCs produce IFN-α and other proinflammatory cytokines through stimulation of Toll-like receptor 7 (TLR7) and TLR9 present in endosomal compartments. TLR7 recognizes single-stranded viral RNA, while TLR9 recognizes unmethylated DNA. In this study, we examined the mechanisms that may underlie variations in IFN-α production in response to HIV, and the impact of these variations on HIV pathogenesis. In four distinct cohorts, we examined PDC production of IFN-α upon stimulation with inactivated HIV-1 particles and unmethylated DNA. The signaling cascade of TLR7 bifurcates at the myeloid differentiation protein 88 (MyD88) adaptor protein to induce expression of either IFN-α or TNF-α. To determine whether variations in IFN-α production are modulated at the level of the receptor complex or downstream of it, we correlated production of IFN-α and TNF-α following stimulation of TLR7 or TLR9 receptors. Flow cytometry detection of intracellular cytokines showed strong, direct correlations between IFN-α and TNF-α expression in all four cohorts, suggesting that variations in IFN-α production are not due to variations downstream of the receptor complex. We then investigated the events upstream of TLR binding by using lipid-like vesicles to deliver TLR ligands directly to the TLR receptors, bypassing the need for CD4 binding and endocytosis. Similar tight correlations were found in IFN-α and TNF-α production in response to the TLR ligands. Taken together, these results strongly suggest that differences in IFN-α production depend on the regulatory processes at the level of the TLR7 receptor complex. Additionally, we found no association between IFN-α production before HIV infection and disease progression.

浆细胞样树突状细胞（Plasmacytoid dendritic cells, PDCs）及其分泌的干扰素-α（IFN-α）被认为在人类免疫缺陷病毒1型（HIV-1）的致病机制中发挥关键作用。PDCs可通过激活内体区室中表达的Toll样受体7（TLR7）与Toll样受体9（TLR9），合成并分泌IFN-α及其他促炎细胞因子。其中，TLR7可识别单链病毒RNA，TLR9则识别未甲基化DNA。本研究聚焦于HIV刺激下IFN-α分泌差异的潜在调控机制，以及此类差异对HIV-1致病进程的影响。我们在4个独立研究队列中，分别以灭活HIV-1病毒颗粒与未甲基化DNA刺激PDCs，检测其IFN-α的分泌水平。TLR7的信号转导级联可在髓系分化蛋白88（myeloid differentiation protein 88, MyD88）衔接蛋白处发生分支，分别诱导IFN-α与肿瘤坏死因子-α（TNF-α）的基因表达。为明确IFN-α分泌的差异是发生于受体复合物层面，还是其下游信号通路，我们对TLR7或TLR9受体激活后，IFN-α与TNF-α的分泌水平进行了相关性分析。流式细胞术检测细胞内细胞因子的结果显示，在全部4个队列中，IFN-α与TNF-α的表达均呈现显著的直接相关性，这表明IFN-α分泌的差异并非源于受体复合物下游的信号通路变化。随后，我们采用脂质样囊泡将TLR配体直接递送至TLR受体，绕过CD4结合与内吞作用的步骤，探究TLR结合上游的调控事件。在使用TLR配体刺激后，IFN-α与TNF-α的分泌仍呈现类似的紧密相关性。综合上述结果，本研究有力提示IFN-α分泌的差异取决于TLR7受体复合物层面的调控过程。此外，我们未发现HIV感染前的IFN-α分泌水平与疾病进展存在显著关联。