Immune activation of human brain microvascular endothelial cells inhibits HIV replication in macrophages

Abstract

There is limited information about the role of blood-brain barrier (BBB) endothelial cells (ECs) in the central nervous system (CNS) and their innate immunity against HIV. We examined whether brain ECs can be immunologically activated to produce antiviral factors that inhibit HIV replication in macrophages. Human brain microvascular ECs expressed functional toll-like receptor 3 (TLR3) that could be activated by polyinosinic-polycytidylic acid (PolyI:C), resulting in the induction of endogenous interferon-β (IFN-β) and IFN-λ. The TLR3 activation of ECs also induced the phosphorylation of interferon regulatory transcription factor 3 (IRF3) and IRF7, the key regulators of IFN signaling pathway. When supernatant (SN) of PolyI:C-activated EC cultures was applied to infected macrophage cultures, HIV replication was significantly suppressed. This SN action of ECs on HIV was mediated through both IFN-β and IFN-λ because antibodies to their receptors could neutralize the SN-mediated anti-HIV effect. The role of IFNs in EC-mediated anti-HIV activity is further supported by the observation that treatment with SN from EC cultures induced the expression of IFN-stimulated genes (ISGs: ISG56, OAS-1, and MxA) in macrophages. These observations indicate that brain microvascular ECs may be a key regulatory bystander, playing a crucial role in the BBB innate immunity against HIV infection.

Figure 1

TLR3 activation induced IFN-β and IFN-λ expression. (A) hCMEC/D3 cells were stimulated with 0.1 µg/mL PolyI:C-Rhodamine for 16 hours and cultured for 48 hours poststimulation. Cells were fixed and stained with antibody to ZO-1 tight junction protein (mouse; 1:100; green). After nuclear counterstaining with Hoechst dyes, cells were observed under a fluorescence microscope. White arrows indicate the internalization of PolyI:C (magnification ×100). (B) Effect of PolyI:C on IFN expression of brain ECs. RNA extracted from the cells was subjected to real-time polymerase chain reaction (RT-PCR) for IFNs as indicated. (C,D) Dose-dependent effect of PolyI:C on IFN induction of hCMEC/D3 cells at (C) mRNA and (D) protein levels. Data were the mean ± standard deviation (SD) of 3 independent experiments. Asterisks indicate that the differences between the indicated groups are statistically significant (*P < .05; **P < .01). FITC, fluorescein isothiocyanate; Lyo, LyoVec (transfection reagent).

Figure 2

Role of TLR3 and RIG-I in PolyI:C-mediated IFN induction. (A) Effect of disruption of TLR3 function by Bafi A1 on the induction of IFN-β and IFN-λ by PolyI:C stimulation. hCMEC/D3 cells were pretreated with 100 nM Bafi A1 for 1 hour and then stimulated with 1 µg/mL PolyI:C for 16 hours. (B) hCMEC/D3 cells were stimulated with 1 µg/mL 5′ppp dsRNA or 5′ppp dsRNA control for 16 hours, washed with plain medium 3 times, and then cultured for 48 hours poststimulation. (C) hCMEC/D3 cells were pretransfected with RIG-I siRNA or control siRNA for 24 hours prior to stimulation with 1 µg/mL PolyI:C. RNA was extracted and IFN expression was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Data were the mean ± SD of 3 independent experiments. Asterisks indicate that the differences between the indicated groups are statistically significant (*P < .05; **P < .01).

Figure 3

Effect of TLR3 activation on the nuclear translocation of IRF3 and IRF7. (A) hCMEC/D3 cells were pretreated with 100 nM Bafi A1 for 1 hour and then stimulated with the indicated concentrations of PolyI:C. Nuclear lysates were prepared, and expression of IRF3 and IRF7 was examined by immunoblot analysis. (B,C) Densitometry analysis of relative IRF3 and IRF7 levels compared with histone was performed by using ImageJ 1.44 software. The data are the average of at least 3 independent experiments and are expressed as mean ± SD. Asterisks indicate that the differences are statistically significant (*P < .05; **P < .01).

Figure 4

Effect of SN from hCMEC/D3 cultures on HIV replication in macrophages. Macrophages were derived from human primary monocytes after 7-day culture and then pretreated with SN from control (Control/SN), LyoVec (LyoVec/SN) or PolyI:C (PolyI:C/SN) -stimulated hCMEC/D3 cultures for 24 hours. Macrophages were then infected with HIV Jago for overnight incubation, and after washing 3 times, cells were maintained in fresh medium for 8 days postinfection. (A) Morphologic observation of HIV-infected macrophages with no pretreatment, LyoVec/SN, or PolyI:C/SN pretreatment under phase contrast microscope (arrows indicate syncytium). (B,C) Intracellular HIV GAG gene expression in macrophages (B) or extracellular HIV reverse transcriptase (RT) activity in macrophage cultures (C) with 10% (volume to volume ratio [v/v]) of indicated SN pretreatments. For PolyI:C/SN, the SNs were collected from 0.1, 0.25, 1, or 2.5 μg/mL PolyI:C-stimulated hCMEC/D3 cultures. (D) HIV GAG gene expression at 8 days postinfection in macrophages pretreated with indicated volumes of 1 μg/mL PolyI:C-stimulated hCMEC/D3 cultures for 24 hours. Representative data from at least 5 donor macrophages were shown and were expressed as mean ± SD from triplicate wells. Asterisks indicate that the differences between indicated groups are statistically significant (*P < .05; **P < .01). GAPDH, glyceraldehyde phosphate dehydrogenase.

Figure 5

Role of IFN-β and IFN-λ in PolyI:C-mediated anti-HIV activity. (A) Effect of PolyI:C-stimulated hCMEC/D3 culture SN on ISG expression of macrophages. HCMEC/D3 cells were stimulated with 1 µg/mL PolyI:C for 16 hours. Culture SN was collected for treatment of macrophages (10% v/v) for 6 hours. RNA was extracted, and the expression of ISGs (ISG56, MxA, OAS-1, and PKR) was measured by qRT-PCR. The results shown are mean ± SD of triplicate cultures, representative of 3 separate experiments. (B) Effect of neutralization antibodies (Abs) to IFNs or IFN-λ receptor on PolyI:C-stimulated hCMEC/D3 culture SN-mediated anti-HIV activity. PolyI:C-stimulated hCMEC/D3 culture SN was preincubated with anti-IFN-α (10 µg/mL), anti-IFN-β (10 µg/mL), or anti-IFN-γ (10 µg/mL) for 1 hour and then used to treat macrophages 24 hours prior to HIV Jago infection. For IFN-λ receptor pretreatment, the anti–IL-10Rβ neutralization antibody (10 µg/mL) was added to treat macrophages for 1 hour prior to the addition of SN. Eight days postinfection, RNA was extracted, and HIV GAG expression was measured by qRT-PCR. The results shown are mean ± SD of triplicate separate experiments. Asterisks indicate that the differences between the indicated groups are statistically significant (*P < .05; **P < .01). n.s., not significant.

Figure 6

Effect of PolyI:C-stimulated hCMEC/D3 culture SN on the activation of ISGF3, STAT3, and MAPK in macrophages. (A) hCMEC/D3 cells were stimulated with 0.1, 1, and 2.5 µg/mL PolyI:C for 16 hours, then washed 3 times, and further incubated in fresh medium to 48 hours poststimulation. SN was collected to treat macrophages for 6 hours (10% v/v). (B) Macrophages were treated with 10% (v/v) of SN from 1 µg/mL PolyI:C-stimulated hCMEC/D3 culture for the indicated time periods. (C) Macrophages were treated with indicated percentage (v/v) of SN from 1 µg/mL PolyI:C-stimulated hCMEC/D3 culture for 6 hours. Protein was extracted and western blot was performed to examine the expression of ISGF-3γp48. Representative blots from 3 independent experiments ae shown. Densitometry analysis of the blot was performed by using ImageJ 1.44 software (National Institutes of Health) and plotted into graphs using data collected from triplicate experiments. Asterisks indicate that the differences between the SN-treated and control cells are statistically significant (*P < .05; **P < .01). (D) Macrophages were treated with 10% (v/v) of SN from indicated concentrations of PolyI:C stimulated hCMEC/D3 cultures for 6 hours. (E) Macrophages were treated with 10% (v/v) of SN from 1 μg/ml PolyI:C-stimulated hCMEC/D3 culture for indicated time points or with indicated percentage (v/v) of SN from 1 μg/ml PolyI:C stimulated hCMEC/D3 cutlure for 6 hours. Protein was extracted after treatment and western blot was performed to examine the expression of STAT3, p-STAT3, p-JNK, p-Erk1/2 amd GAPDH. Representative data from three times independent experiments was shown. Vertical lines have been inserted to indicate repositioned blot lanes.

Figure 7

Hypothetic anti-HIV mechanism of TLR3 signaling of ECs. Stimulation of brain ECs with dsRNA activates TLR3 and/or RIG-I pathways, which facilitates phosphorylation and translocation of IRF3 and IRF7, initiating the transcription of IFN-β and IFN-λ in the ECs. When released from the ECs, IFN-β and IFN-λ bind to their receptors in macrophages and activate ISGF3, STAT3, and MAPK, inducing anti-HIV ISGs in macrophages. GAF, gamma-interferon activation factor; JAK1, Janus kinase 1; MAVS, mitochondrial antiviral-signaling protein; P, phosphorylation; TRIF, TIR-domain-containing adapter-inducing interferon-β; TYK2, Tyrosine kinase 2.