Methamphetamine increases HIV infectivity in neural progenitor cells

Abstract

HIV-1 infection and methamphetamine (METH) abuse frequently occur simultaneously and may have synergistic pathological effects. Although HIV-positive/active METH users have been shown to have higher HIV viral loads and experience more severe neurological complications than non-users, the direct impact of METH on HIV infection and its link to the development of neurocognitive alternations are still poorly understood. In the present study, we hypothesized that METH impacts HIV infection of neural progenitor cells (NPCs) by a mechanism encompassing NFκB/SP1-mediated HIV LTR activation. Mouse and human NPCs were infected with EcoHIV (modified HIV virus infectious to mice) and HIV, respectively, in the presence or absence of METH (50 or 100 μm). Pretreatment with METH, but not simultaneous exposure, significantly increased HIV production in both mouse and human NPCs. To determine the mechanisms underlying these effects, cells were transfected with different variants of HIV LTR promoters and then exposed to METH. METH treatment induced transcriptional activity of the HIV LTR promotor, an effect that required both NFκB and SP1 signaling. Pretreatment with METH also decreased neuronal differentiation of HIV-infected NPCs in both in vitro and in vivo settings. Importantly, NPC-derived daughter cells appeared to be latently infected with HIV. This study indicates that METH increases HIV infectivity of NPCs, through the NFκB/SP1-dependent activation of the HIV LTR and with the subsequent alterations of NPC neurogenesis. Such events may underlie METH- exacerbated neurocognitive dysfunction in HIV-infected patients.

Keywords: NFkappaB transcription factor; drug abuse; human immunodeficiency virus (HIV); methamphetamine; neurocognitive disorders; neurogenesis; neuroprogenitor cell; neurotoxin; viral transcription.

Figure 1.

Productive HIV infection in NPCs. Mouse (NE4C cells) and human (ReNcell VM cells) NPCs were plated at identical densities onto 12-well plates and kept in an undifferentiated state. Cells were treated with the indicated doses of METH simultaneously (A and C) or 24 h before (B and D) infection. EcoHIV (A and B) or HIV (C and D) was added for 12 h and washed out, and samples of culture medium were taken every 24 h, replaced with fresh medium containing METH to maintain the same volume and constant METH levels. HIV production was measured by the assessment of p24 antigen in the medium. Results are mean ± S.D. (error bars) from triplicates for each condition at each time point. Consistent data were obtained from 2–3 independent experiments. *, p < 0.05, HIV versus 100 μm METH + HIV; **, p < 0.05, HIV versus 50 μm METH + HIV or 100 μm METH + HIV. A, 24 h: p < 0.0001, EcoHIV versus 100 μm METH + EcoHIV; 48 h: p < 0.0001, EcoHIV versus 100 μm METH + EcoHIV. B, 72 h: p = 0.0064, EcoHIV versus 50 μm METH + EcoHIV; p = 0.0077, EcoHIV versus 100 μm METH + EcoHIV. D, 24 h: p = 0.0002, HIV versus 50 μm METH + HIV; p = 0.0004, HIV versus 100 μm METH + HIV; 48 h: p = 0.0002, HIV versus 50 μm METH + HIV; p = 0.0001, HIV versus 100 μm METH; 72 h, p < 0.0001, HIV versus 50 μm METH + HIV; p < 0.0001, HIV versus 100 μm METH + HIV.

Figure 2.

Integration of HIV DNA with the host NPC genome. Mouse (NE4C cells) and human (ReNcell VM cells) NPCs were pretreated with 100 μm METH for 24 h and infected with EcoHIV or HIV, respectively, as in Fig. 1. HIV DNA integrated into the host genome was measured by digital droplet PCR. Results are mean ± S.D. (error bars) and expressed as number of copies/100,000 cells; n = 3. *, p < 0.05, HIV versus 100 μm METH + HIV. A, 72 h: p = 0.0225, EcoHIV versus 100 μm METH + EcoHIV. B, 48 h: p = 0.0007, HIV versus 100 μm METH + HIV; 72 h: p = 0.0075, HIV versus 100 μm METH + HIV.

Figure 3.

METH- and/or HIV-mediated activation of NFκB and SP1 in NPCs. mNPCs (NE-4C cells) were pretreated with METH (100 μm) for 1 h (NFκB experiments) or 0.5 h (SP1 experiments) and/or infected with HIV as in Fig. 1. NFκB activation was evaluated by measuring the fluorescence intensities of NFκB acetyl-Lys-310 staining (A) and activation of SP1 by phospho-Thr-453 foci (B) in cell nuclei. A confocal laser-scanning microscope was used to obtain fluorescence imaging. Approximately 300 randomly chosen cells were analyzed. Images show representative results, and the bar graphs reflect quantitative results. Results are mean ± S.D. (error bars), expressed as a percentage of control, n = 3 (3 independent experiments). *, p < 0.05 versus control. A, p = 0.0026, control versus EcoHIV; p = 0.0005, control versus 100 μm METH; p < 0.0001, control versus 100 μm METH + EcoHIV. B, p = 0.0004, control versus EcoHIV; p < 0.0001, control versus 100 μm METH; p < 0.0001, control versus 100 μm METH + EcoHIV.

Figure 4.

Transcriptional regulation of METH-induced modulation of HIV replication. A, schematic representation of the HIV LTR constructs used in the experiment. Mutated SP1-binding sites are represented by crossed circles. B, HIV LTR activities were detected by measuring the luciferase activity in the cell lysates. NPCs were transfected with construct from B for 24 h. Cells were left untreated or stimulated with 100 μm METH with or without infection with EcoHIV for 12 h, followed by a luciferase assay. Results are mean ± S.D. (error bars) from 3–5 cell culture replicates, and each experiment was done in quadruplicates. *, p < 0.05 (p = 0.0003, control versus METH; p < 0.0001, control versus METH + EcoHIV). SP1 mut, mutated SP1 binding site; ΔNFκB, deleted NFκB enhancer.

Figure 5.

METH acting together with HIV impairs differentiation of NPCs. Primary mouse NPCs were induced to differentiate in the presence or absence of 100 μm METH with or without EcoHIV (20 ng) for 1, 5, and 10 days. For each time point, cells were fixed and then immunostained for DCX (marker for immature neurons) and NeuN (marker for mature neurons). DRAQ5 was used to counterstain nuclei. At least three coverslips were analyzed for each experimental group, and three pictures were taken per coverslip. Upper panels, representative fluorescence images of differentiated NPCs at day 10 showing reduced DCX (red) and NeuN (green) immunoreactivity in cells exposed to 100 μm METH with or without EcoHIV. Lower panels, data obtained from counting DCX- and NeuN-positive cells. Results are expressed as a percentage of DCX- or NeuN-positive cells of the total number of cells. n = 4. *, p < 0.05 versus control. For DCX data, p = 0.0259, control versus 100 μm METH + EcoHIV. For NeuN data, p = 0.0313, control versus EcoHIV; p = 0.0274, control versus 100 μm METH + EcoHIV. Error bars, S.D.

Figure 6.

METH and/or HIV affects morphology of newly differentiated neurons. Primary mouse NPCs were induced to differentiate in the presence or absence of 100 μm METH with or without EcoHIV. Cells were immunostained for MAP2 to assess changes in morphology. A, the percentage ratio of different types of neurons was analyzed and compared between the groups. Results are mean ± S.D. (error bars), n = 3. For multipolar neurons: *, p < 0.05 (p = 0.0034, control versus EcoHIV; p = 0.001, control versus METH; p = 0.0016, control versus METH + EcoHIV); for anaxonic neurons: #, p < 0.05 (p = 0.0103, control versus EcoHIV; p = 0.0065, control versus METH; p = 0.0039, control versus METH + EcoHIV). B, multipolar neurons were next subjected to Scholl analysis. Branching complexity and axonic and dendritic lengths were analyzed. Results are mean ± S.E. (error bars); n = 14–19. *, p < 0.05 (for point 0: p = 0.0083, control versus EcoHIV; p = 0.0038, control versus METH; p = 0.0126, control versus METH + EcoHIV; for point 10: p < 0.0001, control versus EcoHIV; p < 0.0001, control versus METH; p = 0.0037, control versus METH + EcoHIV).

Figure 7.

METH acting together with HIV decreases NPC differentiation to glial cells and neurons. Primary mNPCs were differentiated for 2 weeks in the presence or absence of 100 μm METH and/or EcoHIV, followed by FACS analysis. A, sequential cell sorting strategy of differentiated NPCs based on live/dead sorting (left two images), followed by CD24, CD184, and CD44. The obtained population of CD24⁺/CD184⁺/CD44⁺ cells is considered to be glial cells, and the CD24⁺/CD184⁻/CD44⁻ population is considered to be neurons. B, examples of intensity distributions of cell cultures stained with surface markers defined for isolation of NPC-derived neuronal and glia populations. C and D, quantitative data from the sorting procedure for glial and neuronal populations, respectively. Results are mean ± S.D. (error bars) from three independent experiments. *, p < 0.05 versus control. C, p = 0.008, control versus 100 μm METH; p = 0.0177, control versus 100 μm METH + EcoHIV. D, p = 0.0125, control versus 100 μm METH + EcoHIV. E, sorted cells were plated, cultured for 5 days, and stained for GFAP (marker of astrocytes) and Tuj-1 (marker of neurons). Left, CD24⁺/CD184⁺/CD44⁺ cells positive for GFAP; right, CD24⁺/CD184⁻/CD44⁻ cells positive for Tuj-1. Note that there is no cross-contamination in sorted cell populations.

Figure 8.

Latent infection of differentiated mNPCs. A, integrated HIV DNA in NPC-derived daughter neuronal and glial populations. Primary mouse NPCs, not infected or infected with EcoHIV, were differentiated for 2 weeks, followed by sorting for neuronal and glial population as in Fig. 7. After sorting, both cell populations were collected, and HIV DNA integrated into the host genome was assessed. Results are mean ± S.D. (error bars) and expressed as number of copies/100,000 cells. B and C, reactivation of EcoHIV from latently infected differentiated mNPCs. Primary mNPCs were infected with EcoHIV and differentiated for 2 weeks. Then cells were treated with potential latency-reversing agents: METH (100 and 300 μm), TNF-α (10 ng/ml), and/or SAHA (5 and 10 μm) alone or in combinations (as indicated) for up to 3 days. The effect of treatments on p24 production (B) and HIV RNA expression (C) was examined. Results are mean ± S.D. B, n = 8–16, from four independent experiments. *, p = 0.0335, EcoHIV versus EcoHIV + 5 μm SAHA; **, p = 0.0476, EcoHIV + 10 μm SAHA; ***, p < 0.0001, EcoHIV versus EcoHIV + TNF (10 ng/ml) + SAHA (5 μm) and EcoHIV versus EcoHIV + TNF (10 ng/ml) + SAHA (10 μm). C, n = 6–9, from three independent experiments. *, p = 0.0054, EcoHIV versus SAHA (10 μm). **, p < 0.0001 EcoHIV versus EcoHIV + TNF (10 ng/ml) + SAHA (5 μm) and EcoHIV + TNF (10 ng/ml) + SAHA (10 μm).

Figure 9.

METH and EcoHIV impair neurogenesis in mouse hippocampus. Mice were exposed to METH ± EcoHIV as described under “Experimental procedures.” Two weeks after, mice were sacrificed, and brain sections were immunostained for NeuN (red) and BrdU (green) to detect newly differentiated neurons within the DG area. Representative images are shown on the left. Results are mean ± S.D. The number of co-stained NeuN⁺/BrdU⁺ cells/μm³ of DG is shown on the right; n = 5–6 animals/group. *, p = 0.0003, control versus METH; **, p < 0.0001, control versus METH + EcoHIV; #, p = 0.0012, EcoHIV versus METH + EcoHIV.