HIV-1 Infected T-cells Research Articles

Cocaine augments neuro-inflammation via modulating extracellular vesicle release in HIV-1 infected immune cells

BackgroundExtracellular Vesicles (EV) recently have been implicated in the pathogenesis of HIV-1 syndromes, including neuroinflammation and HIV-1 associated neurological disorder (HAND). Cocaine, an illicit stimulant drug used worldwide is known to exacerbate these HIV-1 associated neurological syndromes. However, the effects of cocaine on EV biogenesis and roles of EVs in enhancing HIV-1 pathogenesis are not yet well defined.ResultsHere, we investigated the effects of cocaine on EV biogenesis and release in HIV-1 infected immune cells and explored their roles in elicitation of neuroinflammation. We found that cocaine significantly augmented the release of EVs from uninfected and HIV-1 infected T-cells, DCs and macrophages. Further analysis of the molecular components of EVs revealed enhanced expression of adhesion molecules integrin β1 and LFA-1 in those EVs derived from cocaine treated cells. Intriguingly, in EVs derived from HIV-1 infected cells, cocaine treatment significantly increased the levels of viral genes in EVs released from macrophages and DCs, but not in T-cells. Exploring the molecular mechanism to account for this, we found that DCs and macrophages showed enhanced expression of the cocaine receptor Sigma 1-Receptor compared to T-cells. In addition, we found that cocaine significantly altered the integrity of the RNA-induced silencing complex (RISC) in HIV-1 infected macrophages and DCs compared to untreated HIV-1 infected cells. Characterizing further the molecular mechanisms involved in how cocaine increased EV release, we found that cocaine decreased the expression of the interferon-inducible protein BST-2; this resulted in altered trafficking of intracellular virus containing vesicles and EV biogenesis and release. We also observed EVs released from cocaine treated HIV-1 infected macrophages and DCs enhanced HIV-1 trans-infection to T-cells compared to those from untreated and HIV-1 infected cells. These EVs triggered release of proinflammatory cytokines in human brain microvascular endothelial cells (HBMECs) and altered monolayer integrity.ConclusionsTaken together, our results provide a novel mechanism which helps to elucidate the enhanced prevalence of neurological disorders in cocaine using HIV-1 infected individuals and offers insights into developing novel therapeutic strategies against HAND in these hosts.

Latent HIV‐1 Exosomes Induce Mitochondrial Hyperfusion due to Loss of Phosphorylated Dynamin‐related Protein 1 in Brain Endothelium

Background Damage to the cerebral vascular endothelium is a critical initiating event in the development of HIV-1-associated neurocognitive disorders (HAND). To study the role of mitochondria in cerebral endothelial dysfunction, we investigated how exosomes, isolated from both cell lines with integrated provirus and HIV-1 infected primary cells (HIV-exosomes), accelerate the dysfunction of either primary human brain microvascular endothelial cells (HBMVECs) in vitro or mice brain microvessels (MVs), ex vivo. Methods Exosomes were isolated by either ultracentrifugation from uninfected and latent HIV-1 infected T-cells (Jurkat Exo and J-Lat(9.2) Exo, respectively) and monocytes (U937 Exo and U1 Exo, respectively) or by using QIAGEN exoEasy Maxi kit from HIV-1 infected human peripheral blood mononuclear cells (HIV+PBMCs Exo). Exosomes were characterized by the qNano-IZON system. Initially, the assessment of mitochondrial characteristics was performed in primary HBMVECs, and this study was further extended in extracted cerebral MVs from mice brain expressing mito-Dendra2+ fluorescence protein (Green) in endothelial mitochondria. Results Quantitative analysis of the extracellular vesicles (EVs) indicates that the isolated EVs were predominantly exosomes. This was further supported by the detection of exosomal markers, e.g. CD63, CD81, Alix, TSG101 and the absence of large EV-related protein, GRP94. These exosomes were readily taken up by both primary HBMVECs and mice cerebral MVs. HIV-exosomes induced cellular and mitochondrial superoxide production but reduced mitochondrial membrane potential in HBMVECs. HIV-exosomes increased mitochondrial hyperfusion, possibly due to the loss of phosphorylated dynamin-related protein 1 (p-DRP1). We further confirmed that HIV-exosomes, containing the HIV-Tat protein, and viral Tat protein accelerate brain endothelial dysfunction due to reduction in the expressions of (a) p-DRP1, (b) phosphorylated endothelial nitric oxide synthase (p-eNOS), and (c) blood brain barrier related tight junction proteins. Finally, HIV-1 infected primary human PBMCs produced more exosomes than uninfected controls, and exposure to HIV+PBMCs Exo also reduced both p-DRP1 and p-eNOS expressions in primary HBMVECs. Conclusions Our novel findings reveal the significant role of HIV-exosomes on dysregulation of mitochondrial function, which induces adverse changes in the function of the brain microvascular endothelium. These mechanistic studies using HIV-exosomes may elucidate the etiology of HAND and provide novel therapeutic strategies.

Differences in Transcriptional Dynamics Between T-cells and Macrophages as Determined by a Three-State Mathematical Model

HIV-1 viral transcription persists in patients despite antiretroviral treatment, potentially due to intermittent HIV-1 LTR activation. While several mathematical models have been explored in the context of LTR-protein interactions, in this work for the first time HIV-1 LTR model featuring repressed, intermediate, and activated LTR states is integrated with generation of long (env) and short (TAR) RNAs and proteins (Tat, Pr55, and p24) in T-cells and macrophages using both cell lines and infected primary cells. This type of extended modeling framework allows us to compare and contrast behavior of these two cell types. We demonstrate that they exhibit unique LTR dynamics, which ultimately results in differences in the magnitude of viral products generated. One of the distinctive features of this work is that it relies on experimental data in reaction rate computations. Two RNA transcription rates from the activated promoter states are fit by comparison of experimental data to model predictions. Fitting to the data also provides estimates for the degradation/exit rates for long and short viral RNA. Our experimentally generated data is in reasonable agreement for the T-cell as well macrophage population and gives strong evidence in support of using the proposed integrated modeling paradigm. Sensitivity analysis performed using Latin hypercube sampling method confirms robustness of the model with respect to small parameter perturbations. Finally, incorporation of a transcription inhibitor (F07#13) into the governing equations demonstrates how the model can be used to assess drug efficacy. Collectively, our model indicates transcriptional differences between latently HIV-1 infected T-cells and macrophages and provides a novel platform to study various transcriptional dynamics leading to latency or activation in numerous cell types and physiological conditions.

Proteomic profiling of HIV-infected T-cells by SWATH mass spectrometry

Viral pathogenesis results from changes in host cells due to virus usurpation of the host cell and the innate cellular responses to thwart infection. We measured global changes in protein expression and localization in HIV-1 infected T-cells using subcellular fractionation and the Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS) proteomic platform. Eight biological replicates were performed in two independent experimental series. In silico merging of both experiments identified 287 proteins with altered expression (p < .05) between control and infected cells- 172 in the cytoplasm, 84 in the membrane, and 31 in nuclei. 170 of the proteins are components of the NIH HIV interaction database. Multiple Reaction Monitoring and traditional immunoblotting validated the altered expression of several factors during infection. Numerous factors were found to affect HIV infection in gain- and loss-of-expression infection assays, including the intermediate filament vimentin which was found to be required for efficient infection.

Exosomes Derived from HIV-1 Infected DCs Mediate Viral trans-Infection via Fibronectin and Galectin-3

Exosomes are membrane enclosed nano-sized vesicles actively released into the extracellular milieu that can harbor genomic, proteomic and lipid cargos. Functionally, they are shown to regulate cell-cell communication and transmission of pathogens. Though studies have implicated a role for exosomes in HIV-1 pathogenesis, their mechanisms are not well defined. Here, we characterized exosomes derived from uninfected or HIV-1 infected T-cells and DCs. We demonstrate substantial differences in morphological, molecular and biogenesis machinery between exosomes derived from these two immune cell types. In addition, exosomes derived from HIV-1 infected DCs were 4 fold more infective than either cell free HIV-1 or exosomes derived from T-cells. Molecular analysis of exosomes detected the presence of fibronectin and galectin-3 in those derived from DCs, whereas T-cell exosomes lacked these molecules. Addition of anti-fibronectin antibody and β-lactose, a galectin-3 antagonist, significantly blocked DC exosome-mediated HIV-1 infection of T-cells. We also observed increased gene expression of the pro-inflammatory cytokines IFN-γ, TNF-α, IL-1β and RANTES and activation of p38/Stat pathways in T-cells exposed to exosomes derived from HIV-1 infected DCs. Our study provides insight into the role of exosomes in HIV pathogenesis and suggests they can be a target in development of novel therapeutic strategies against viral infection.

Candidate prioritization for low-abundant differentially expressed proteins in 2D-DIGE datasets

BackgroundTwo-dimensional differential gel electrophoresis (2D-DIGE) provides a powerful technique to separate proteins on their isoelectric point and apparent molecular mass and quantify changes in protein expression. Abundantly available proteins in spots can be identified using mass spectrometry-based approaches. However, identification is often not possible for low-abundant proteins.ResultsWe present a novel computational approach to prioritize candidate proteins for unidentified spots. Our approach exploits noisy information on the isoelectric point and apparent molecular mass of a protein spot in combination with functional similarities of candidate proteins to already identified proteins to select and rank candidates. We evaluated our method on a 2D-DIGE dataset comparing protein expression in uninfected and HIV-1 infected T-cells. Using leave-one-out cross-validation, we show that the true-positive rate for the top-5 ranked proteins is 43.8%.ConclusionsOur approach shows good performance on a 2D-DIGE dataset comparing protein expression in uninfected and HIV-1 infected T-cells. We expect our method to be highly useful in (re-)mining other 2D-DIGE experiments in which especially the low-abundant protein spots remain to be identified.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0455-x) contains supplementary material, which is available to authorized users.

Incorporation of podoplanin into HIV released from HEK-293T cells, but not PBMC, is required for efficient binding to the attachment factor CLEC-2

BackgroundPlatelets are associated with HIV in the blood of infected individuals and might modulate viral dissemination, particularly if the virus is directly transmitted into the bloodstream. The C-type lectin DC-SIGN and the novel HIV attachment factor CLEC-2 are expressed by platelets and facilitate HIV transmission from platelets to T-cells. Here, we studied the molecular mechanisms behind CLEC-2-mediated HIV-1 transmission.ResultsBinding studies with soluble proteins indicated that CLEC-2, in contrast to DC-SIGN, does not recognize the viral envelope protein, but a cellular factor expressed on kidney-derived 293T cells. Subsequent analyses revealed that the cellular mucin-like membranous glycoprotein podoplanin, a CLEC-2 ligand, was expressed on 293T cells and incorporated into virions released from these cells. Knock-down of podoplanin in 293T cells by shRNA showed that virion incorporation of podoplanin was required for efficient CLEC-2-dependent HIV-1 interactions with cell lines and platelets. Flow cytometry revealed no evidence for podoplanin expression on viable T-cells and peripheral blood mononuclear cells (PBMC). Podoplanin was also not detected on HIV-1 infected T-cells. However, apoptotic bystander cells in HIV-1 infected cultures reacted with anti-podoplanin antibodies, and similar results were obtained upon induction of apoptosis in a cell line and in PBMCs suggesting an unexpected link between apoptosis and podoplanin expression. Despite the absence of detectable podoplanin expression, HIV-1 produced in PBMC was transmitted to T-cells in a CLEC-2-dependent manner, indicating that T-cells might express an as yet unidentified CLEC-2 ligand.ConclusionsVirion incorporation of podoplanin mediates CLEC-2 interactions of HIV-1 derived from 293T cells, while incorporation of a different cellular factor seems to be responsible for CLEC-2-dependent capture of PBMC-derived viruses. Furthermore, evidence was obtained that podoplanin expression is connected to apoptosis, a finding that deserves further investigation.

Reduction of the HIV-1-infected T-cell reservoir by immune activation treatment is dose-dependent and restricted by the potency of antiretroviral drugs.

Treatments combining T-cell activating agents and potent antiretroviral drugs have been proposed as a possible means of reducing the reservoir of long-lived HIV-1 infected quiescent CD4 T-cells. To analyse the effect of such therapies on HIV-1 dynamics and T-cell homeostasis. A mathematical framework describing HIV-1 dynamics and T-cell homeostasis was developed. Three patients who were kept on a particularly potent course of highly active antiretroviral therapy (HAART) were treated with the anti-CD3 monoclonal antibody OKT3 and interleukin (IL)-2. Plasma HIV-RNA, and HIV-RNA and DNA in peripheral blood mononuclear cells and lymph node mononuclear cells were measured. These results and other published studies on the use of IL-2 alone were assessed using our mathematical framework. We show that outcome of treatment is determined by the relative rates of depletion of the infected quiescent T-cell population by activation and of its replenishment through new infection. Which of these two processes dominates is critically dependent on both the potency of HAART and also the degree of T-cell activation induced. We demonstrate that high-level T-cell stimulation is likely to produce negative outcomes, both by failing to reduce viral reservoirs and by depleting the CD4 T-cell pool and disrupting CD4/CD8 T-cell homeostasis. In contrast, repeated low-level stimulation may both aid CD4 T-cell pool expansion and achieve a substantial reduction in the long-lived HIV-1 reservoir. Our analysis suggests that although treatment that activates T-cells can reduce the long-lived HIV-1 reservoir, caution should be used as high-level stimulation may result in a negative outcome.