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. 2012 Jun 22:9:138.
doi: 10.1186/1742-2094-9-138.

Neuronal apoptosis by HIV-1 Vpr: contribution of proinflammatory molecular networks from infected target cells

Debjani Guha  1 , Pruthvi Nagilla, Carrie Redinger, Alagarsamy Srinivasan, Gerald P Schatten, Velpandi Ayyavoo
Affiliations

Neuronal apoptosis by HIV-1 Vpr: contribution of proinflammatory molecular networks from infected target cells

Debjani Guha et al. J Neuroinflammation. .

Abstract

Background: Human immunodeficiency virus type 1 (HIV-1) induces neuronal dysfunction through host cellular factors and viral proteins including viral protein R (Vpr) released from infected macrophages/microglia. Vpr is important for infection of terminally differentiated cells such as macrophages. The objective of this study was to assess the effect of Vpr in the context of infectious virus particles on neuronal death through proinflammatory cytokines released from macrophages.

Methods: Monocyte-derived macrophages (MDM) were infected with either HIV-1 wild type (HIV-1wt), Vpr deleted mutant (HIV-1∆Vpr) or mock. Cell lysates and culture supernatants from MDMs were analyzed for the expression and release of proinflammatory cytokines by quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay respectively. Mitogen-activated protein kinases (MAPK) were analyzed in activated MDMs by western blots. Further, the effect of Vpr on neuronal apoptosis was examined using primary neurons exposed to culture supernatants from HIV-1wt, HIV-1∆Vpr or mock-infected MDMs by Annexin-V staining, MTT and Caspase - Glo® 3/7 assays. The role of interleukin (IL)-1β, IL-8 and tumor necrosis factor (TNF)-α on neuronal apoptosis was also evaluated in the presence or absence of neutralizing antibodies against these cytokines.

Results: HIV-1∆Vpr-infected MDMs exhibited reduced infection over time and specifically a significant downregulation of IL-1β, IL-8 and TNF-α at the transcriptional and/or protein levels compared to HIV-1wt-infected cultures. This downregulation was due to impaired activation of p38 and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) in HIV-1∆Vpr-infected MDMs. The association of SAPK/JNK and p38 to IL-1β and IL-8 production was confirmed by blocking MAPKs that prevented the elevation of IL-1β and IL-8 in HIV-1wt more than in HIV-1∆Vpr-infected cultures. Supernatants from HIV-1∆Vpr-infected MDMs containing lower concentrations of IL-1β, IL-8 and TNF-α as well as viral proteins showed a reduced neurotoxicity compared to HIV-1wt-infected MDM supernatants. Reduction of neuronal death in the presence of anti-IL-1β and anti-IL-8 antibodies only in HIV-1wt-infected culture implies that the effect of Vpr on neuronal death is in part mediated through released proinflammatory factors.

Conclusion: Collectively, these results demonstrate the ability of HIV-1∆Vpr to restrict neuronal apoptosis through dysregulation of multiple proinflammatory cytokines in the infected target cells either directly or indirectly by suppressing viral replication.

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Figures

Figure 1
Figure 1
Characterization and kinetics of HIV-1wtand HIV-1∆Vpr virus particles. (a) HEK 293 T cells were transfected with 5 μg of HIV-1 YU2wt and HIV-1 YU2∆Vpr plasmids. Viruses collected after 72 hours were filtered, concentrated by ultracentrifugation and western blot analysis was performed for Gag and Vpr. (b) Productive infection of MDMs by viruses. MDMs were infected with HIV-1wt and HIV-1∆Vpr viruses at an MOI of 0.1 or left untreated as negative control (NT). The release of virus particles from infected MDM culture was monitored by p24 ELISA using the supernatants on days as indicated. The data are representatives of one of six independent experiments.
Figure 2
Figure 2
HIV-1∆Vpr produces less proinflammatory IL-1β, IL-8 and TNF-α in MDMs than HIV-1wt. MDMs were infected with HIV-1wt or HIV-1∆Vpr virus at an MOI of 0.1 or left untreated for 1, 2, 3, 4, 8, 12, 16 and 20 days. (a) Expression of IL-1β, IL-8 and TNF-α in protein levels in supernatants of HIV-1wt, HIV-1∆Vpr and mock-infected (NT) MDMs collected at indicated time points was monitored by ELISA (N = 6). Absence of Vpr reduced the production of proinflammatory cytokines. The results presented are the mean concentration of cytokines ± SEM. *P < 0.05 (two tailed student’s t-test) compared to HIV-1∆Vpr-infected cultures. (b) qRT-PCR was carried out with the cell lysates as mentioned in Methods (N = 6). RNA fold changes were compared with ELISA data for each individual experiment. The expression of cytokines in protein levels (HIV-1wt; red lines and HIV-1∆Vpr; blue lines) was correlated with the respective RNA transcripts (fold changes in HIV-1wt compared to HIV-1∆Vpr; green lines). Figure represents one of six independent experiments.
Figure 3
Figure 3
HIV-1∆Vpr does not activate p38 and SAPK/JNK in MDMs as HIV-1wt. MDMs infected with HIV-1wt, HIV-1∆Vpr or mock (NT) were activated with 1 μg/ml of LPS for 4 hours and the cells were harvested at 6, 12, 24, 48 hours, 4, 8, 12 days. (a) Cells were lysed and 50 μg of protein of each sample was analyzed by western blot using antibodies against the active and total form of ERK1/2, p38 and antibodies against SAPK/JNK and tubulin. (b) Relative band intensities of phosphorylated products were normalized with total proteins. Densitometrical quantification of western blot data represents the ± SEM of three independent observations. *P < 0.05 (two tailed student’s t-test) compared to HIV-1∆Vpr-infected cultures. (c) MDMs were preincubated with PD98059 (10 μM), SB203580 (10 μM) and SP600125 (10 μM) for 2 hours and then infected with HIV-1wt or HIV-1∆Vpr virus at an MOI of 0.1 or mock. After 48 hours of infection IL-1β, IL-8 and TNF-α ELISA were performed. MDMs without MAPK inhibitors were considered to be the control and were reused to normalize other results. Each sample was run in triplicates, the results are ± SEM of the concentration of the cytokines and *P < 0.05 compared with HIV-1 ∆Vpr treated.
Figure 4
Figure 4
Differentiation of NP cells to primary neurons. Differentiation of the NP cells to primary neurons was performed for 2 weeks. Neuronal differentiation was confirmed by immunostaining. Neurons were fixed, permeabilized and stained with neuronal marker MAP2 (green) and β-III tubulin (green) or astrocyte specific marker GFAP (red). Nuclei were stained with Hoechst 33342 (blue). The figures are representatives of one of three independent experiments.
Figure 5
Figure 5
HIV-1-induced neuronal death is mediated in part through IL-1β and IL-8 and not TNF-α present in supernatants of HIV-1wt virus-infected MDMs. Primary neurons exposed to HIV-1wt, HIV-1∆Vpr and mock-infected (NT) MDM supernatants (10% v/v) as well as recombinant IL-1β (10 ng/ml), IL-8 (10 ng/ml) and TNF-α (100 ng/ml) for 24 – 48 hours were analyzed for neuronal apoptosis by (a) Annexin-V FITC staining, nuclei of the cells were stained with Hoechst 33342 and measured the neuronal death (%); (b) MTT assay, absorbance was measured and cell viability was normalized with negative controls. HIV-1wt-infected MDM supernatants were more neurotoxic compared to HIV-1∆Vpr culture. (c) The cells were lysed in Caspase-Glo® 3/7 substrate and protease activity was measured as relative light units (RLU). (d) Neurons were lysed and 50 μg of protein for each sample was analyzed by western blot using specific antibodies for cleaved and total PARP. (e) Neurons exposed to supernatants from HIV-1wt, HIV-1∆Vpr and mock-infected MDM (NT) were treated with and without anti-IL-1β, anti-IL-8 and anti-TNF-α and neurotoxicity was analyzed after 24–48 hours by Annexin-V staining. Results are the ± SEM of three individual experiments; *P < 0.05, **P < 0.01 in HIV-1wt compared with control or HIV-1 ∆Vpr treated.
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