Modeling HIV-1 Infection in CNS via Infected Monocytes Using Immunocompetent Brain Organoids.

The development of 3D-organoid models has revolutionized the way diseases are studied. Recently, our brain organoid model has been shown to recapitulate in in vitro the human brain cytoarchitecture originally encountered in HIV-1 neuropathogenesis, allowing downstream applications. Infected monocytes, macrophages, and microglia are critically important immune cells for infection and dissemination of HIV-1 throughout brain during acute and chronic phase of the disease. Once in the brain parenchyma, long-lived infected monocytes/macrophages along with resident microglia contribute to the establishment of CNS latency in people with HIV (PWH). Hence, it is important to better understand how HIV-1 enters and establishes infection and latency in CNS to further develop cure strategies. Here we detailed an accessible protocol to incorporate monocytes (infected and/or labeled) as a model of transmigration of peripheral monocytes into brain organoids that can be applied to characterize HIV-1 neuroinvasion and virus dissemination.

Neuroinflammation driven by human immunodeficiency virus-1 (HIV-1) directs the expression of long noncoding RNA RP11-677M14.2 resulting in dysregulation of neurogranin in vivo and in vitro.

Neuroinflammation and synaptodendritic damage represent the pathological hallmarks of HIV-1 associated cognitive disorders (HAND). The post-synaptic protein neurogranin (Nrgn) is significantly reduced in the frontal cortex of postmortem brains from people with HIV (PWH) and it is associated with inflammatory factors released by infected microglia/macrophages. However, the mechanism involved in synaptic loss have yet to be elucidated. In this study, we characterized a newly identified long non-coding RNA (lncRNA) transcript (RP11-677M14.2), which is antisense to the NRGN locus and is highly expressed in the frontal cortex of HIV-1 individuals. Further analysis indicates an inverse correlation between the expression of RP11-677M14.2 RNA and Nrgn mRNA. Additionally, the Nrgn-lncRNA axis is dysregulated in neurons exposed to HIV-1 infected microglia conditioned medium enriched with IL-1ß. Moreover, in vitro overexpression of this lncRNA impacts Nrgn expression at both mRNA and protein levels. Finally, we modeled the Nrgn-lncRNA dysregulation within an HIV-1-induced inflammatory environment using brain organoids, thereby corroborating our in vivo and in vitro findings. Together, our study implicates a plausible role for lncRNA RP11-677M14.2 in modulating Nrgn expression that might serve as the mechanistic link between Nrgn loss and cognitive dysfunction in HAND, thus shedding new light on the mechanisms underlying synaptodendritic damage.

Neuroinflammation driven by Human Immunodeficiency Virus-1 (HIV-1) directs the expression of long noncoding RNA RP11-677M14.2 resulting in dysregulation of Neurogranin in vivo and in vitro.

Neuroinflammation and synaptodendritic damage represent the pathological hallmarks of HIV-1 associated cognitive disorders (HAND). The post-synaptic protein neurogranin (Nrgn) is significantly reduced in the frontal cortex of postmortem brains from people with HIV (PWH) and it is associated with inflammatory factors released by infected microglia/macrophages. However, the mechanism involved in synaptic loss have yet to be elucidated. In this study, we characterized a newly identified long non-coding RNA (lncRNA) transcript (RP11-677M14.2), which is antisense to the NRGN locus and is highly expressed in the frontal cortex of HIV-1 individuals. Further analysis indicates an inverse correlation between the expression of RP11-677M14.2 RNA and Nrgn mRNA. Additionally, the Nrgn-lncRNA axis is dysregulated in neurons exposed to HIV-1 infected microglia conditioned medium enriched with IL-1b. Moreover, in vitro overexpression of this lncRNA impact Nrgn expression at both mRNA and protein levels. Finally, we modeled the Nrgn-lncRNA dysregulation within an HIV-1-induced neuroinflammatory environment using brain organoids, thereby corroborating our in vivo and in vitro findings. Together, our study implicates a plausible role for lncRNA RP11-677M14.2 in modulating Nrgn expression that might serve as the mechanistic link between Nrgn loss and cognitive dysfunction in HAND, thus shedding new light on the mechanisms underlying synaptodendritic damage.

Three-Dimensional Human Brain Organoids to Model HIV-1 Neuropathogenesis.

Studying neurological diseases have long been hampered by the lack of physiologically relevant models to resemble the complex human brain and the associated pathologies. Three-dimensional brain organoids have emerged as cutting-edge technology providing an alternative in vitro model to study healthy neural development and function as well as pathogenesis of neurological disorders and neuropathologies induced by pathogens. Nonetheless,  the absence of immune cells in current models poses a barrier to fully recapitulate brain microenvironment during the onset of HIV-1-associated neuropathogenesis. To address this and to further the brain organoid technology, we have incorporated HIV-target microglia into brain organoids, generating a complex multicellular interaction, which mimics the HIV-1-infected brain environment. Here we describe the method to generate a brain organoid consisting on neurons, astrocytes, and microglia (with and without HIV infection) that recapitulate the HIV-associated neuropathology. This model has tremendous potential to expand our knowledge on neuronal dysfunction associated with HIV-1 infection of glia.

Modeling HIV-1 neuropathogenesis using three-dimensional human brain organoids (hBORGs) with HIV-1 infected microglia.

HIV-1 associated neurocognitive disorder (HAND) is characterized by neuroinflammation and glial activation that, together with the release of viral proteins, trigger a pathogenic cascade resulting in synaptodendritic damage and neurodegeneration that lead to cognitive impairment. However, the molecular events underlying HIV neuropathogenesis remain elusive, mainly due to lack of brain-representative experimental systems to study HIV-CNS pathology. To fill this gap, we developed a three-dimensional (3D) human brain organoid (hBORG) model containing major cell types important for HIV-1 neuropathogenesis; neurons and astrocytes along with incorporation of HIV-infected microglia. Both infected and uninfected microglia infiltrated into hBORGs resulting in a triculture system (MG-hBORG) that mirrors the multicellular network observed in HIV-infected human brain. Moreover, the MG-hBORG model supported productive viral infection and exhibited increased inflammatory response by HIV-infected MG-hBORGs, releasing tumor necrosis factor (TNF-α) and interleukin-1 (IL-1ß) and thereby mimicking the chronic neuroinflammatory environment observed in HIV-infected individuals. This model offers great promise for basic understanding of how HIV-1 infection alters the CNS compartment and induces pathological changes, paving the way for discovery of biomarkers and new therapeutic targets.

Human immunodeficiency virus type 1 (HIV-1)-mediated neuroinflammation dysregulates neurogranin and induces synaptodendritic injury.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1)-associated neurocognitive disorder (HAND) is a common outcome of a majority of HIV-1-infected subjects and is associated with synaptodendritic damage. Neurogranin (Ng), a postsynaptic protein, and calmodulin (CaM) are two important players of synaptic integrity/functions. The biological role of Ng in the context of HAND is unknown. METHODS: We compared the expression of Ng in frontal cortex (FC) tissues from control and HIV-1-positive subjects with and without HAND by immunohistochemistry, western blot, and qRT-PCR. The interaction between Ng and CaM was analyzed by co-immunoprecipitation. Ng, microtubule-associated protein 2 (MAP2), CaM, CaM-dependent protein kinase II (CaMKII), CREB, synaptophysin (Syp), and synapsin I (Syn I) expressions were evaluated by western blot using FC tissue lysates and differentiated SH-SY5Y (dSH-SY5Y) cells. Identification of inflammatory factors related to Ng loss was accomplished by exposing dSH-SY5Y cells to HIV-1 and mock-infected monocyte-derived macrophage (MDM) supernatants or HIV-1 NLYU2 pseudotyped with VSV-G-Env. Levels of interleukin (IL)-1ß, IL-8, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, MCP-2, and CXCL5 in MDM supernatants were measured by ELISA. Association of IL-1ß and IL-8 to Ng expression in context of HIV-1 infection was evaluated in the presence or absence of neutralizing antibodies against these cytokines. RESULTS: Expression level of Ng was reduced significantly in FC of HAND-positive (HAND+) patients compared to uninfected individuals. Although no difference was found in CaM expression, interaction between Ng and CaM was reduced in HAND+ patients, which was associated with decreased level of CaMKII, a downstream signaling molecule of CaM pathway. This in turn resulted in reduction of synaptic markers, Syp and Syn I. HIV-1 infection directly had no considerable effect on dysregulation of Ng expression in dSH-SY5Y cells, whereas high amount of pro-inflammatory IL-1ß and IL-8 in HIV-1-infected MDM supernatants was associated with significant reduction in Ng expression. CONCLUSIONS: Synaptic damage in HAND+ patients could be a result of abrogation of Ng through HIV-1-induced inflammation that dysregulates Ng-CaM interaction and downstream signaling cascades associated with synaptodendritic functions. This is the first study evaluating the potential role of Ng in the context of HIV-1 neuropathogenesis.

Transcriptome analyses identify key cellular factors associated with HIV-1-associated neuropathogenesis in infected men.

OBJECTIVE: HIV-1 viral proteins and host inflammatory factors have a direct role in neuronal toxicity in vitro; however, the contribution of these factors in vivo in HIV-1-associated neurocognitive disorder (HAND) is not fully understood. We applied novel Systems Biology approaches to identify specific cellular and viral factors and their related pathways that are associated with different stages of HAND. DESIGN: A cross-sectional study of individuals enrolled in the Multicenter AIDS Cohort Study including HIV-1-seronegative (N = 36) and HIV-1-seropositive individuals without neurocognitive symptoms (N = 16) or with mild neurocognitive disorder (MND) (N = 8) or HIV-associated dementia (HAD) (N = 16). METHODS: A systematic evaluation of global transcriptome of peripheral blood mononuclear cells (PBMCs) obtained from HIV-1-seronegative individuals and from HIV-1-positive men without neurocognitive symptoms, or MND or HAD was performed. RESULTS: MND and HAD were associated with specific changes in mRNA transcripts and microRNAs in PBMCs. Comparison of upstream regulators and TimePath analyses identified specific cellular factors associated with MND and HAD, whereas HIV-1 viral proteins played a greater role in HAD. In addition, expression of specific microRNAs - miR-let-7a, miR-124, miR-15a and others - were found to correlate with mRNA gene expression and may have a potential protective role in asymptomatic HIV-1-seropositive individuals by regulating cellular signal transduction pathways downstream of chemokines and cytokines. CONCLUSION: These results identify signature transcriptome changes in PBMCs associated with stages of HAND and shed light on the potential contribution of host cellular factors and viral proteins in HAND development.

Reconstructing the temporal progression of HIV-1 immune response pathways.

MOTIVATION: Most methods for reconstructing response networks from high throughput data generate static models which cannot distinguish between early and late response stages. RESULTS: We present TimePath, a new method that integrates time series and static datasets to reconstruct dynamic models of host response to stimulus. TimePath uses an Integer Programming formulation to select a subset of pathways that, together, explain the observed dynamic responses. Applying TimePath to study human response to HIV-1 led to accurate reconstruction of several known regulatory and signaling pathways and to novel mechanistic insights. We experimentally validated several of TimePaths' predictions highlighting the usefulness of temporal models. AVAILABILITY AND IMPLEMENTATION: Data, Supplementary text and the TimePath software are available from http://sb.cs.cmu.edu/timepath CONTACT: zivbj@cs.cmu.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

HIV-1 Infection Dysregulates Cell Cycle Regulatory Protein p21 in CD4+ T Cells Through miR-20a and miR-106b Regulation.

Both CD4+ T lymphocytes and macrophages are the major targets of human immunodeficiency virus type 1 (HIV-1); however, they respond differently to HIV-1 infection. We hypothesized that HIV-1 infection alters gene expression in CD4+ T cells and monocyte-derived macrophages (MDMs) in a cell specific manner and microRNAs (miRNAs) in part play a role in cell-specific gene expression. Results indicate that 183 and 31 genes were differentially regulated in HIV-1 infected CD4+ T cells and MDMs, respectively, compared to their mock-infected counterparts. Among the differentially expressed genes, cell cycle regulatory gene, p21 (CDKN1A) was upregulated in virus infected CD4+ T cells both at the mRNA and protein level in CD4+ T cells, whereas no consistent change was observed in MDMs. Productively infected CD4+ T cells express higher amount of p21 compared to bystander cells. In determining the mechanism(s) of cell type specific regulation of p21, we found that the miRNAs miR-106b and miR-20a that target p21 were specifically downregulated in HIV-1 infected CD4+ T cells. Overexpression of these two miRNAs reduced p21 expression significantly in HIV-1 infected CD4+ T cells. These findings provide a potential mechanism, by which, HIV-1 could exploit host cellular machineries to regulate selective gene expression in target cells. J. Cell. Biochem. 117: 1902-1912, 2016. © 2016 Wiley Periodicals, Inc.

Temporal transcriptional response to latency reversing agents identifies specific factors regulating HIV-1 viral transcriptional switch.

BACKGROUND: Latent HIV-1 reservoirs are identified as one of the major challenges to achieve HIV-1 cure. Currently available strategies are associated with wide variability in outcomes both in patients and CD4(+) T cell models. This underlines the critical need to develop innovative strategies to predict and recognize ways that could result in better reactivation and eventual elimination of latent HIV-1 reservoirs. RESULTS AND DISCUSSION: In this study, we combined genome wide transcriptome datasets post activation with Systems Biology approach (Signaling and Dynamic Regulatory Events Miner, SDREM analyses) to reconstruct a dynamic signaling and regulatory network involved in reactivation mediated by specific activators using a latent cell line. This approach identified several critical regulators for each treatment, which were confirmed in follow-up validation studies using small molecule inhibitors. Results indicate that signaling pathways involving JNK and related factors as predicted by SDREM are essential for virus reactivation by suberoylanilide hydroxamic acid. ERK1/2 and NF-κB pathways have the foremost role in reactivation with prostratin and TNF-α, respectively. JAK-STAT pathway has a central role in HIV-1 transcription. Additional evaluation, using other latent J-Lat cell clones and primary T cell model, also confirmed that many of the cellular factors associated with latency reversing agents are similar, though minor differences are identified. JAK-STAT and NF-κB related pathways are critical for reversal of HIV-1 latency in primary resting T cells. CONCLUSION: These results validate our combinatorial approach to predict the regulatory cellular factors and pathways responsible for HIV-1 reactivation in latent HIV-1 harboring cell line models. JAK-STAT have a role in reversal of latency in all the HIV-1 latency models tested, including primary CD4(+) T cells, with additional cellular pathways such as NF-κB, JNK and ERK 1/2 that may have complementary role in reversal of HIV-1 latency.

Transcriptional Regulation of CXCL5 in HIV-1-Infected Macrophages and Its Functional Consequences on CNS Pathology.

Human immunodeficiency virus-1 (HIV-1)-infected monocytes/macrophages and microglia release increased levels of proinflammatory cytokines and chemokines, including ELR+ (containing glutamic acid-leucine-arginine motif) chemokines. To investigate the role of HIV-1 infection on chemokine regulation, monocyte-derived macrophages (MDMs) from normal donors were infected with HIV-1 and the expression of chemokines and their downstream biological functions were evaluated. Among the tested chemokines, CXCL5 was upregulated significantly both at the mRNA and protein level in the HIV-1-infected MDMs compared with mock-infected cultures. Upregulation of CXCL5 in the HIV-1-infected MDMs is, in part, regulated by increased interleukin-1ß (IL-1ß) production and phosphorylation of ERK1/2. Functional analyses indicate that HIV-1-induced overexpression of CXCL5 has enhanced the ability to attract neutrophils, as observed by chemotaxis assay. However, exposure of NT2, SH-SY5Y cells, and primary neurons to HIV-1-infected MDM supernatants resulted in cell death that was not rescued by anti-CXCL5 antibody suggesting that CXCL5 does not have direct effect on neuronal death. Together, these results suggest that the increased level of CXCL5 in tissue compartments, including the central nervous system of HIV-1-infected individuals might alter the inflammatory response through the infiltration of neutrophils into tissue compartment, thus causing secondary effects on resident cells.

CD40L induces functional tunneling nanotube networks exclusively in dendritic cells programmed by mediators of type 1 immunity.

The ability of dendritic cells (DC) to mediate CD4(+) T cell help for cellular immunity is guided by instructive signals received during DC maturation, as well as the resulting pattern of DC responsiveness to the Th signal, CD40L. Furthermore, the professional transfer of antigenic information from migratory DC to lymph node-residing DC is critical for the effective induction of cellular immune responses. In this study we report that, in addition to their enhanced IL-12p70 producing capacity, human DC matured in the presence of inflammatory mediators of type 1 immunity are uniquely programmed to form networks of tunneling nanotube-like structures in response to CD40L-expressing Th cells or rCD40L. This immunologic process of DC reticulation facilitates intercellular trafficking of endosome-associated vesicles and Ag, but also pathogens such HIV-1, and is regulated by the opposing roles of IFN-γ and IL-4. The initiation of DC reticulation represents a novel helper function of CD40L and a superior mechanism of intercellular communication possessed by type 1 polarized DC, as well as a target for exploitation by pathogens to enhance direct cell-to-cell spread.

Nanotechnology-based approaches for the development of diagnostics, therapeutics, and vaccines.

The architecture of nanoparticles of biological origin, generally also known as bionanoparticles, presents several features that are ideal for their use in developing diagnostics, therapeutics, and vaccines. In this regard, particles formed by viral proteins using recombinant DNA technology resemble authentic virus particles. However, they lack infectivity due to the absence of genetic components such as DNA or RNA. Hence, they are designated as virus-like particles (VLP). VLPs possess the following characteristics: (1) they can be generated by either a single or a few viral proteins; (2) their size, formed by viral proteins, is in the range of 20 to100 nm; (3) the number of protein molecules required for particle assembly is from hundreds to thousands, depending on the VLP; (4) the protein(s) responsible for their assembly are amenable for manipulation; and (5) multiple proteins/peptides can be incorporated into a VLP. The potential advantages of VLPs directed by retroviral proteins are discussed in this review.

Serum microRNAs in HIV-infected individuals as pre-diagnosis biomarkers for AIDS-NHL.

OBJECTIVE: To determine if changes in levels of serum microRNAs (miRNAs) were seen preceding the diagnosis of AIDS-related non-Hodgkin lymphoma (AIDS-NHL). DESIGN: Serum miRNA levels were compared in 3 subject groups from the Multicenter AIDS Cohort Study: HIV-negative men (n = 43), HIV-positive men who did not develop NHL (n = 45), and HIV-positive men before AIDS-NHL diagnosis (n = 62, median time before diagnosis, 8.8 months). METHODS: A total of 175 serum-enriched miRNAs were initially screened to identify differentially expressed miRNAs among these groups and the results validated by quantitative polymerase chain reaction. Receiver-operating characteristic analysis was then performed to assess biomarker utility. RESULTS: Higher levels of miR-21 and miR-122, and a lower level of miR-223, were able to discriminate HIV-infected from the HIV-uninfected groups, suggesting that these miRNAs are biomarkers for HIV infection but are not AIDS-NHL specific. Among the HIV-infected groups, a higher level of miR-222 was able to discriminate diffuse large B-cell lymphoma (DLBCL) and primary central nervous system lymphoma (PCNSL) subjects from HIV-infected subjects who did not develop NHL, with area under the receiver-operating characteristic curve of 0.777 and 0.792, respectively. At miR-222 cutoff values of 0.105 for DLBCL and 0.109 for PCNSL, the sensitivity and specificity were 75% and 77%, and 80% and 82%, respectively. CONCLUSIONS: Altered serum levels of miR-21, miR-122, and miR-223 are seen in HIV-infected individuals. Higher serum level of miR-222 has clear potential as a serum biomarker for earlier detection of DLBCL and PCNSL among HIV-infected individuals.

Human immunodeficiency virus type 1 Vpr polymorphisms associated with progressor and nonprogressor individuals alter Vpr-associated functions.

Following infection with Human immunodeficiency virus 1 (HIV-1) there is a remarkable variation in virus replication and disease progression. Both host and viral factors have been implicated in the observed differences in disease status. Here, we focus on understanding the contribution of HIV-1 viral protein R (Vpr) by evaluating the disease-associated Vpr polymorphism and its biological functions from HIV-1 positive rapid progressor (RP) and long-term nonprogressor (LTNP) subjects. Results presented here show distinct variation in phenotypes of Vpr alleles from LTNP and RP subjects. Most notably, the polymorphism of Vpr at R36W and L68M associated with RP shows higher levels of oligomerization, and increased virus replication, whereas R77Q exhibits poor replication kinetics. Interestingly, we did not observe correlation with cell cycle arrest function. Together these results indicate that polymorphisms in Vpr in part may contribute to altered virus replication kinetics leading to the observed differences in disease progression in LTNP and RP groups.

Innate immune evasion strategies by human immunodeficiency virus type 1.

Host immune components play both beneficial and pathogenic roles in human immunodeficiency virus type 1 (HIV-1) infection. During the initial stage of viral infection, a complex network of innate immune factors are activated. For instance, the immune cells express a number of inflammatory proteins including cytokines, chemokines, and antiviral restriction factors. These factors, specifically, interferons (IFNs) play a crucial role in antiviral defense system by modulating the downstream signaling events, by inducing maturation of dendritic cells (DCs), and by activation of macrophages, natural killer (NK) cells, and B and T cells. However, HIV-1 has evolved to utilize a number of strategies to overcome the antiviral effects of the host innate immune system. This review discusses the pathways and strategies utilized by HIV-1 to establish latent and persistent infection by defeating host's innate defense system.

MicroRNA regulation and its effects on cellular transcriptome in human immunodeficiency virus-1 (HIV-1) infected individuals with distinct viral load and CD4 cell counts.

BACKGROUND: Disease progression in the absence of therapy varies significantly in HIV-1 infected individuals. Both viral and host cellular molecules are implicated; however, the exact role of these factors and/or the mechanism involved remains elusive. To understand how microRNAs (miRNAs), which are regulators of transcription and translation, influence host cellular gene expression (mRNA) during HIV-1 infection, we performed a comparative miRNA and mRNA microarray analysis using PBMCs obtained from infected individuals with distinct viral load and CD4 counts. METHODS: RNA isolated from PBMCs obtained from HIV-1 seronegative and HIV-1 positive individuals with distinct viral load and CD4 counts were assessed for miRNA and mRNA profile. Selected miRNA and mRNA transcripts were validated using in vivo and in vitro infection model. RESULTS: Our results indicate that HIV-1 positive individuals with high viral load (HVL) showed a dysregulation of 191 miRNAs and 309 mRNA transcripts compared to the uninfected age and sex matched controls. The miRNAs miR-19b, 146a, 615-3p, 382, 34a, 144 and 155, that are known to target innate and inflammatory factors, were significantly upregulated in PBMCs with high viral load, as were the inflammatory molecules CXCL5, CCL2, IL6 and IL8, whereas defensin, CD4, ALDH1, and Neurogranin (NRGN) were significantly downregulated. Using the transcriptome profile and predicted target genes, we constructed the regulatory networks of miRNA-mRNA pairs that were differentially expressed between control, LVL and HVL subjects. The regulatory network revealed an inverse correlation of several miRNA-mRNA pair expression patterns, suggesting HIV-1 mediated transcriptional regulation is in part likely through miRNA regulation. CONCLUSIONS: Results from our studies indicate that gene expression is significantly altered in PBMCs in response to virus replication. It is interesting to note that the infected individuals with low or undetectable viral load exhibit a gene expression profile very similar to control or uninfected subjects. Importantly, we identified several new mRNA targets (Defensin, Neurogranin, AIF) as well as the miRNAs that could be involved in regulating their expression through the miRNA-mRNA interaction.

Development of a robust cell-based high-throughput screening assay to identify targets of HIV-1 viral protein R dimerization.

Targeting protein-protein interactions (PPI) is an emerging field in drug discovery. Dimerization and PPI are essential properties of human immunodeficiency virus (HIV)-1 proteins, their mediated functions, and virus biology. Additionally, dimerization is required for the functional interaction of HIV-1 proteins with many host cellular components. In this study, a bimolecular fluorescence complementation (BiFC)-based screening assay was developed that can quantify changes in dimerization, using HIV-1 viral protein R (Vpr) dimerization as a "proof of concept." Results demonstrated that Venus Vpr (generated by BiFC Vpr constructs) could be competed off in a dose-dependent manner using untagged, full-length Vpr as a competitor molecule. The change in signal intensity was measured quantitatively through flow cytometry and fluorescence microscopy in a high content screening assay. High content imaging was used to screen a library of small molecules for an effect on Vpr dimerization. Among the tested molecules, a few of the small molecules demonstrate an effect on Vpr dimerization in a dose-dependent manner.

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

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)-1ß, 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-1ß, 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-1ß and IL-8 production was confirmed by blocking MAPKs that prevented the elevation of IL-1ß 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-1ß, 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-1ß 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.