Regulation of Human Immunodeficiency Virus Type 1 Infection, β-Chemokine Production, and CCR5 Expression in CD40L-Stimulated Macrophages: Immune Control of Viral Entry

Mononuclear phagocytes (MP) and T lymphocytes play a pivotal role in the host immune response to human immunodeficiency virus type 1 (HIV-1) infection. Regulation of such immune responses can be mediated, in part, through the interaction of the T-lymphocyte-expressed molecule CD40 ligand (CD40L) with its receptor on MP, CD40. Upregulation of CD40L on CD4+ peripheral blood mononuclear cells during advanced HIV-1 disease has previously been reported. Based on this observation, we studied the influence of CD40L-CD40 interactions on MP effector function and viral regulation in vitro. We monitored productive viral infection, cytokine and β -chemokine production, and β-chemokine receptor expression in monocyte-derived macrophages (MDM) after treatment with soluble CD40L. Beginning 1 day after infection and continuing at 3-day intervals, treatment with CD40L inhibited productive HIV-1 infection in MDM in a dose-dependent manner. A concomitant and marked upregulation of β-chemokines (macrophage inhibitory proteins 1α and 1β and RANTES [regulated upon activation normal T-cell expressed and secreted]) and the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) was observed in HIV-1-infected and CD40L-treated MDM relative to either infected or activated MDM alone. The addition of antibodies to RANTES or TNF-α led to a partial reversal of the CD40L-mediated inhibition of HIV-1 infection. Surface expression of CD4 and the b-chemokine receptor CCR5 was reduced on MDM in response to treatment with CD40L. In addition, treatment of CCR5- and CD4-transfected 293T cells with secretory products from CD40L-stimulated MDM prior to infection with a CCR5-tropic HIV-1 reporter virus led to inhibition of viral entry. In conclusion, we demonstrate that CD40L-mediated inhibition of viral entry coincides with a broad range of MDM immune effector responses and the down-modulation of CCR5 and CD4 expression

Dopamine Neuron Stimulating Actions of a GDNF Propeptide

BACKGROUND: Neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), have shown great promise for protection and restoration of damaged or dying dopamine neurons in animal models and in some Parkinson's disease (PD) clinical trials. However, the delivery of neurotrophic factors to the brain is difficult due to their large size and poor bio-distribution. In addition, developing more efficacious trophic factors is hampered by the difficulty of synthesis and structural modification. Small molecules with neurotrophic actions that are easy to synthesize and modify to improve bioavailability are needed. METHODS AND FINDINGS: Here we present the neurobiological actions of dopamine neuron stimulating peptide-11 (DNSP-11), an 11-mer peptide from the proGDNF domain. In vitro, DNSP-11 supports the survival of fetal mesencephalic neurons, increasing both the number of surviving cells and neuritic outgrowth. In MN9D cells, DNSP-11 protects against dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA)-induced cell death, significantly decreasing TUNEL-positive cells and levels of caspase-3 activity. In vivo, a single injection of DNSP-11 into the normal adult rat substantia nigra is taken up rapidly into neurons and increases resting levels of dopamine and its metabolites for up to 28 days. Of particular note, DNSP-11 significantly improves apomorphine-induced rotational behavior, and increases dopamine and dopamine metabolite tissue levels in the substantia nigra in a rat model of PD. Unlike GDNF, DNSP-11 was found to block staurosporine- and gramicidin-induced cytotoxicity in nutrient-deprived dopaminergic B65 cells, and its neuroprotective effects included preventing the release of cytochrome c from mitochondria. CONCLUSIONS: Collectively, these data support that DNSP-11 exhibits potent neurotrophic actions analogous to GDNF, making it a viable candidate for a PD therapeutic. However, it likely signals through pathways that do not directly involve the GFRalpha1 receptor

HIV-1-Infected and Immune-Activated Macrophages Induce Astrocytic Differentiation of Human Cortical Neural Progenitor Cells via the STAT3 Pathway

Diminished adult neurogenesis is considered a potential mechanism in the pathogenesis of HIV-1-associated dementia (HAD). In HAD, HIV-1-infected and immune-activated brain mononuclear phagocytes (MP; perivascular macrophages and microglia) drive central nervous system (CNS) inflammation and may alter normal neurogenesis. We previously demonstrated HIV-1-infected and lipopolysaccharide (LPS) activated monocyte-derived macrophages (MDM) inhibit human neural progenitor cell (NPC) neurogenesis, while enhancing astrogliogenesis through the secretion of the inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), in vitro and in vivo. Here we further test the hypothesis that HIV-1-infected/activated MDM promote NPC astrogliogenesis via activation of the transcription factor signal transducer and activator of transcription 3 (STAT3), a critical factor for astrogliogenesis. Our results show that LPS-activated MDM-conditioned medium (LPS-MCM) and HIV-infected/LPS-activated MDM-conditioned medium (LPS+HIV-MCM) induced Janus kinase 1 (Jak1) and STAT3 activation. Induction of the Jak-STAT3 activation correlated with increased glia fibrillary acidic protein (GFAP) expression, demonstrating an induction of astrogliogenesis. Moreover, STAT3-targeting siRNA (siSTAT3) decreased MCM-induced STAT3 activation and NPC astrogliogenesis. Furthermore, inflammatory cytokines (including IL-6, IL-1β and TNF-α) produced by LPS-activated and/or HIV-1-infected MDM may contribute to MCM-induced STAT3 activation and astrocytic differentiation. These observations were confirmed in severe combined immunodeficient (SCID) mice with HIV-1 encephalitis (HIVE). In HIVE mice, siRNA control (without target sequence, sicon) pre-transfected NPCs injected with HIV-1-infected MDM showed more astrocytic differentiation and less neuronal differentiation of NPCs as compared to NPC injection alone. siSTAT3 abrogated HIV-1-infected MDM-induced astrogliogenesis of injected NPCs. Collectively, these observations demonstrate that HIV-1-infected/activated MDM induces NPC astrogliogenesis through the STAT3 pathway. This study generates important data elucidating the role of brain inflammation in neurogenesis and may provide insight into new therapeutic strategies for HAD

Interferon-Alpha Mediates Restriction of Human Immunodeficiency Virus Type-1 Replication in Primary Human Macrophages at an Early Stage of Replication

Type I interferons (IFNα and β) are induced directly in response to viral infection, resulting in an antiviral state for the cell. In vitro studies have shown that IFNα is a potent inhibitor of viral replication; however, its role in HIV-1 infection is incompletely understood. In this study we describe the ability of IFNα to restrict HIV-1 infection in primary human macrophages in contrast to peripheral blood mononuclear cells and monocyte-derived dendritic cells. Inhibition to HIV-1 replication in cells pretreated with IFNα occurred at an early stage in the virus life cycle. Late viral events such as budding and subsequent rounds of infection were not affected by IFNα treatment. Analysis of early and late HIV-1 reverse transcripts and integrated proviral DNA confirmed an early post entry role for IFNα. First strand cDNA synthesis was slightly reduced but late and integrated products were severely depleted, suggesting that initiation or the nucleic acid intermediates of reverse transcription are targeted. The depletion of integrated provirus is disproportionally greater than that of viral cDNA synthesis suggesting the possibility of a least an additional later target. A role for either cellular protein APOBEC3G or tetherin in this IFNα mediated restriction has been excluded. Vpu, previously shown by others to rescue a viral budding restriction by tetherin, could not overcome this IFNα induced effect. Determining both the viral determinants and cellular proteins involved may lead to novel therapeutic approaches. Our results add to the understanding of HIV-1 restriction by IFNα

Proteomic Modeling for HIV-1 Infected Microglia-Astrocyte Crosstalk

Background: HIV-1-infected and immune competent brain mononuclear phagocytes (MP; macrophages and microglia) secrete cellular and viral toxins that affect neuronal damage during advanced disease. In contrast, astrocytes can affect disease by modulating the nervous system’s microenvironment. Interestingly, little is known how astrocytes communicate with MP to influence disease. Methods and Findings: MP-astrocyte crosstalk was investigated by a proteomic platform analysis using vesicular stomatitis virus pseudotyped HIV infected murine microglia. The microglial-astrocyte dialogue was significant and affected microglial cytoskeleton by modulation of cell death and migratory pathways. These were mediated, in part, through F-actin polymerization and filament formation. Astrocyte secretions attenuated HIV-1 infected microglia neurotoxicity and viral growth linked to the regulation of reactive oxygen species. Conclusions: These observations provide unique insights into glial crosstalk during disease by supporting astrocytemediated regulation of microglial function and its influence on the onset and progression of neuroAIDS. The results open new insights into previously undisclosed pathogenic mechanisms and open the potential for biomarker discovery an

High-Performance Capillary Electrophoresis for Determining HIV-1 Tat Protein in Neurons

The HIV-1 protein, Tat has been implicated in AIDS pathogenesis however, the amount of circulating Tat is believed to be very low and its quantification has been difficult. We performed the quantification of Tat released from infected cells and taken up by neurons using high performance capillary electrophoresis. This is the first report to successfully measure the amount of Tat in neurons and places Tat as a key player involved in HIV-associated neurocognitive disorders

Development of Epitope-Blocking ELISA for Universal Detection of Antibodies to Human H5N1 Influenza Viruses

10.1371/journal.pone.0004566PLoS ONE4

Human Immunodeficiency Virus type 1 Endocytic Trafficking Through Macrophage Bridging Conduits Facilitates Spread of Infection

Bridging conduits (BC) sustain communication and homeostasis between distant tethered cells. These are also exploited commonly for direct cell-to-cell transfer of microbial agents. Conduits efficiently spread infection, effectively, at speeds faster than fluid phase exchange while shielding the microbe against otherwise effective humoral immunity. Our laboratory has sought to uncover the mechanism(s) for these events for human immunodeficiency virus type one (HIV-1) infection. Indeed, in our prior works HIV-1 Env and Gag antigen and fluorescent virus tracking were shown sequestered into endoplasmic reticulum-Golgi organelles but the outcomes for spreading viral infection remained poorly defined. Herein, we show that HIV-1 specifically traffics through endocytic compartments contained within BC and directing such macrophage-to-macrophage viral transfers. Following clathrin-dependent viral entry, HIV-1 constituents bypass degradation by differential sorting from early to Rab11+ recycling endosomes and multivesicular bodies. Virus-containing endocytic viral cargoes propelled by myosin II through BC spread to neighboring uninfected cells. Disruption of endosomal motility with cytochalasin D, nocodasole and blebbistatin diminish intercellular viral spread. These data lead us to propose that HIV-1 hijacks macrophage endocytic and cytoskeletal machineries for high-speed cell-to-cell spread

Type I Interferons and Interferon Regulatory Factors Regulate TNF-Related Apoptosis-Inducing Ligand (TRAIL) in HIV-1-Infected Macrophages

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family that participates in HIV-1 pathogenesis through the depletion of CD4+ T cells. TRAIL is expressed on the cell membrane of peripheral immune cells and can be cleaved into a soluble, secreted form. The regulation of TRAIL in macrophages during HIV-1 infection is not completely understood. In this study, we investigated the mechanism(s) of TRAIL expression in HIV-1-infected macrophages, an important cell type in HIV-1 pathogenesis. A human monocyte-derived macrophage (MDM) culture system was infected with macrophage-tropic HIV-1ADA, HIV-1JR-FL, or HIV-1BAL strains. TRAIL, predominantly the membrane-bound form, increased following HIV-1 infection. We found that HIV-1 infection also induced interferon regulatory factor (IRF)-1, IRF-7 gene expression and signal transducers and activators of transcription 1 (STAT1) activation. Small interfering RNA knockdown of IRF-1 or IRF-7, but not IRF-3, reduced STAT1 activation and TRAIL expression. Furthermore, the upregulation of IRF-1, IRF-7, TRAIL, and the activation of STAT1 by HIV-1 infection was reduced by the treatment of type I interferon (IFN)-neutralizing antibodies. In addition, inhibition of STAT1 by fludarabine abolished IRF-1, IRF-7, and TRAIL upregulation. We conclude that IRF-1, IRF-7, type I IFNs, and STAT1 form a signaling feedback loop that is critical in regulating TRAIL expression in HIV-1-infected macrophages

Expression profile of microRNAs in young stroke patients

10.1371/journal.pone.0007689PLoS ONE41