miRNA profiles in unstimulated and TLR stimulated MDMs

Macrophages play a crucial role in HIV-1 pathogenesis. Toll-like receptors (TLRs) are fundamental for innate and adaptive immune responses, but their role in HIV-1 infection is still incompletely understood. The TLR3 and TLR4 ligands poly(I:C) and LPS are known to modulate HIV-1 infection of and replication in monocyte-derived macrophages (MDMs), but the mechanism is incompletely understood. We found that MDMs stimulation with poly(I:C) or LPS abrogated infection by CCR5-using, macrophage-tropic HIV-1, or by VSV-G-pseudotyped HIV-1 virions, while TLR7 and TLR9 agonists Imiquimod and CpG only reduced infection to varying extent. Suppression of infection, or lack thereof, did not correlate with differential effects on CD4 or CCR5 expression, type I interferon induction, or production of pro-inflammatory cytokines. Furthermore, integrated pro-viruses were readily detected in unstimulated, TLR7- and TLR9-stimulated cells, but not in TLR3- or TLR4-stimulated MDMs, suggesting the alteration of post-entry, pre-integration event(s). MicroRNA (miRNA) microarray and real time PCR demonstrated increased miR-155 levels in MDMs upon TLR3/4, but not TLR7, stimulation, and a miR-155 inhibitor partially restored infectivity in poly(I:C)-stimulated MDMs. Finally, miR-155 over-expression in MDMs and cell lines remarkably diminished HIV-1 infection, inducing an accumulation of late reverse transcription products, concurrently with a decrease in mRNA levels of several HIV-1 dependency factors involved in nuclear import of pre-integration complexes. Our results suggest that miR-155 may target mRNA(s) for host cell protein(s) that either participate in or facilitate post-entry, pre-integration events, resulting in severely diminished HIV-1 infection. miRNA profiles were investigated in total RNA isolated from unstimulated and TLR3-, TLR4- and TLR7-stimulated human MDMs from a single normal donor

巨噬细胞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。Toll样受体（Toll-like receptors, TLRs）是固有免疫与适应性免疫应答的核心调控分子，但其在HIV-1感染过程中的作用仍未完全阐明。已知TLR3与TLR4的配体聚肌胞苷酸（poly(I:C)）和脂多糖（LPS）可调控单核细胞来源巨噬细胞（monocyte-derived macrophages, MDMs）的HIV-1感染与复制，但具体分子机制尚不明确。
我们的研究发现，用poly(I:C)或LPS刺激MDMs后，可完全阻断利用CCR5受体的嗜巨噬细胞HIV-1，以及水泡性口炎病毒G蛋白（VSV-G）假型HIV-1病毒颗粒的感染；而TLR7激动剂咪喹莫特与TLR9激动剂CpG仅能不同程度降低感染效率。感染抑制与否与CD4或CCR5的表达变化、I型干扰素的诱导，以及促炎细胞因子的产生均无相关性。
进一步研究显示，未刺激组、TLR7与TLR9刺激组的细胞中均可稳定检测到整合型前病毒，但TLR3或TLR4刺激的MDMs中未检测到整合型前病毒，这提示该过程可能影响了病毒进入宿主细胞后、整合前的事件。
MicroRNA（miRNA）芯片与实时荧光定量PCR结果表明，TLR3/4（而非TLR7）刺激MDMs后，细胞内miR-155的表达水平显著升高；而miR-155抑制剂可部分恢复poly(I:C)刺激的MDMs中的HIV-1感染能力。
最终实验证实，在MDMs及细胞系中过表达miR-155可显著抑制HIV-1感染，导致晚期逆转录产物积累，同时降低多种参与整合前复合物核输入的HIV-1宿主依赖因子的mRNA水平。
本研究结果提示，miR-155可能靶向调控参与或促进病毒进入后、整合前事件的宿主蛋白编码mRNA，从而显著抑制HIV-1感染。本研究对单名健康供体来源的未刺激、TLR3、TLR4及TLR7刺激的人单核细胞来源巨噬细胞的总RNA进行了miRNA表达谱分析。