Department of Pediatrics, Division of Infectious Diseases, University of California-San Diego, La Jolla, CA, USA.
J Infect Dis. 2012 Apr 15;205(8):1258-67. doi: 10.1093/infdis/jis187. Epub 2012 Mar 6.
The mechanisms responsible for interferon α (IFN-α) production by plasmacytoid dendritic cells (pDCs) during human immunodeficiency virus type 1 (HIV-1) infection are unknown. This research examined the roles of Toll-like receptor 7 (TLR7) and autophagy in IFN-α production by pDCs during HIV-1 infection.
pDCs from human peripheral blood mononuclear cells were incubated with infectious or aldrithiol 2 (AT-2)-inactivated HIV-1 or with uridine-rich single-stranded RNA40 (ssRNA40) from the HIV-1 long terminal repeat. IFN-α was quantified by enzyme-linked immunosorbant assay. Autophagic proteins were detected by Western blot, and autophagosomes were identified using immunofluorescent and confocal microscopy. To inhibit autophagy, pDCs were treated with the phosphoinositide-3 kinase inhibitor 3-methyladenine (3-MA) or were transfected with autophagy-related protein 7 or TLR7 small interfering RNA (siRNA).
Increased levels of IFN-α were present in culture supernatants following 16-hour incubation of pDCs with infectious or AT-2-inactivated HIV-1. Treatment of pDCs with ssRNA40 but not ssRNA41 resulted in high levels of IFN-α. pDCs exposed to HIV-1 gp120, rapamycin, or 3-MA alone failed to induce IFN-α. Pretreatment of pDCs with 3-MA significantly reduced the induction of IFN-α by ssRNA40. Similarly, knock down of autophagy-related protein 7 and TLR7 by use of siRNA significantly reduced the induction of IFN-α by ssRNA40 or HIV-1.
These findings demonstrate that IFN-α production by pDCs exposed to infectious or noninfectious HIV-1 and ssRNA40 occurs through induction of autophagy following TLR7 signaling.
在人类免疫缺陷病毒 1 型（HIV-1）感染期间，浆细胞样树突状细胞（pDC）产生干扰素 α（IFN-α）的机制尚不清楚。本研究探讨了 Toll 样受体 7（TLR7）和自噬在 HIV-1 感染期间 pDC 产生 IFN-α中的作用。
用人外周血单核细胞中的 pDC 孵育感染性或二硫苏糖醇 2（AT-2）失活的 HIV-1 或来自 HIV-1 长末端重复序列的尿嘧啶丰富的单链 RNA40（ssRNA40）。通过酶联免疫吸附试验定量 IFN-α。通过 Western blot 检测自噬蛋白，通过免疫荧光和共聚焦显微镜鉴定自噬体。为了抑制自噬，用磷酸肌醇 3-激酶抑制剂 3-甲基腺嘌呤（3-MA）处理 pDC 或用自噬相关蛋白 7 或 TLR7 小干扰 RNA（siRNA）转染 pDC。
在 pDC 与感染性或 AT-2 失活的 HIV-1 孵育 16 小时后，培养上清液中 IFN-α 水平升高。pDC 暴露于 ssRNA40 而不是 ssRNA41 导致高水平的 IFN-α。pDC 单独暴露于 HIV-1 gp120、雷帕霉素或 3-MA 均不能诱导 IFN-α。3-MA 预处理显著降低了 ssRNA40 诱导的 IFN-α。同样，使用 siRNA 敲低自噬相关蛋白 7 和 TLR7 显著降低了 ssRNA40 或 HIV-1 诱导的 IFN-α。
这些发现表明，pDC 暴露于感染性或非感染性 HIV-1 和 ssRNA40 后，通过 TLR7 信号诱导自噬，产生 IFN-α。
