HIV-infected Macrophages Research Articles

Efficient HIV-1 transmission from macrophages to T cells across transient virological synapses

AbstractMacrophages are reservoirs of HIV-1 infection, proposed to transmit virus to CD4+ T cells, the primary target of the virus. Here we report that human monocyte-derived macrophages (MDMs) rapidly spread HIV-1 to autologous CD4+ T cells resulting in productive infection. Transmission takes place across transient adhesive contacts between T cells and MDMs, which have the features of a virological synapse including copolarization of CD4 on the T cell with HIV-1 Gag and Env on the macrophage. We propose that an infected MDM can infect at least one T cell every 6 hours. Since HIV-1–infected macrophages can survive for many weeks, these results highlight the central role played by macrophages in HIV-1 infection and pathogenesis.

Evaluation of denoising algorithms for biological electron tomography

Tomograms of biological specimens derived using transmission electron microscopy can be intrinsically noisy due to the use of low electron doses, the presence of a "missing wedge" in most data collection schemes, and inaccuracies arising during 3D volume reconstruction. Before tomograms can be interpreted reliably, for example, by 3D segmentation, it is essential that the data be suitably denoised using procedures that can be individually optimized for specific data sets. Here, we implement a systematic procedure to compare various nonlinear denoising techniques on tomograms recorded at room temperature and at cryogenic temperatures, and establish quantitative criteria to select a denoising approach that is most relevant for a given tomogram. We demonstrate that using an appropriate denoising algorithm facilitates robust segmentation of tomograms of HIV-infected macrophages and Bdellovibrio bacteria obtained from specimens at room and cryogenic temperatures, respectively. We validate this strategy of automated segmentation of optimally denoised tomograms by comparing its performance with manual extraction of key features from the same tomograms.

Targeting of efavirenz loaded tuftsin conjugated poly(propyleneimine) dendrimers to HIV infected macrophages in vitro

HIV infected macrophages are considered as reservoirs for spreading the virus in AIDS patients. Tuftsin not only binds specifically to the mononuclear phagocytic cells but also enhances their natural killer activity. The purpose of this study is to explore the targeting potential and anti-HIV activity of efavirenz (EFV) loaded, tuftsin conjugated 5th generation poly(propyleneimine) dendrimers (TuPPI) in vitro. Tuftsin was chemically conjugated to 5th generation poly(propyleneimine) dendrimers (PPI). The entrapment efficiency of PPI and TuPPI were found to be 37.43 ± 0.3% and 49.31 ± 0.33%, respectively. TuPPI was found to slow down and prolong the in vitro release of EFV upto 144 h against PPI, which releases the drug completely within 24 h. TuPPI possessed negligible cytotoxicity as compared to that of PPI. The cellular uptake of TuPPI was found to be 34.5 times higher than that of the free drug in first 1 h and was significantly higher in HIV infected macrophages than that of uninfected cells. TuPPI was found to reduce the viral load by 99% at a concentration of 0.625 ng/ml, which is due to the enhanced cellular uptake, reduced toxicity and the inherent anti-HIV activity of TuPPI.

Regulatory T cells in HIV infection: pathogenic or protective participants in the immune response?

Regulatory T cells in HIV infection: pathogenic or protective participants in the immune response?

Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy

BackgroundUnlike CD4+ T cells, HIV-1 infected macrophages exhibit extended life span even upon stress, consistent with their in vivo role as long-lived HIV-1 reservoirs.ResultsHere, we demonstrate that PI3K/Akt inhibitors, including clinically available Miltefosine, dramatically reduced HIV-1 production from long-living virus-infected macrophages. These PI3K/Akt inhibitors hyper-sensitize infected macrophages to extracellular stresses that they are normally exposed to, and eventually lead to cell death of infected macrophages without harming uninfected cells. Based on the data from these Akt inhibitors, we were able to further investigate how HIV-1 infection utilizes the PI3K/Akt pathway to establish the cytoprotective effect of HIV-1 infection, which extends the lifespan of infected macrophages, a key viral reservoir. First, we found that HIV-1 infection activates the well characterized pro-survival PI3K/Akt pathway in primary human macrophages, as reflected by decreased PTEN protein expression and increased Akt kinase activity. Interestingly, the expression of HIV-1 or SIV Tat is sufficient to mediate this cytoprotective effect, which is dependent on the basic domain of Tat – a region that has previously been shown to bind p53. Next, we observed that this interaction appears to contribute to the downregulation of PTEN expression, since HIV-1 Tat was found to compete with PTEN for p53 binding; this is known to result in p53 destabilization, with a consequent reduction in PTEN protein production.ConclusionSince HIV-1 infected macrophages display highly elevated Akt activity, our results collectively show that PI3K/Akt inhibitors may be a novel therapy for interfering with the establishment of long-living HIV-1 infected reservoirs.

Hitting HIV where it hides

The recent finding that inhibitors of PI3/Akt can sensitize HIV infected macrophages to oxidative stress-induced cell death suggest a potential new therapeutic approach to targeting HIV reservoirs.

HIV-infected macrophages mediate neuronal apoptosis through mitochondrial glutaminase

HIV-infected macrophages mediate neuronal apoptosis through mitochondrial glutaminase

HIV‐infected macrophages mediate neuronal apoptosis through mitochondrial glutaminase

A significant number of patients infected with human immunodeficiency virus-1 (HIV-1) suffer cognitive impairment ranging from mild to severe HIV-associated dementia (HAD), a result of neuronal degeneration in the basal ganglia, cerebral cortex and hippocampus. Mononuclear phagocyte dysfunction is thought to play an important role in the pathogenesis of HAD. Glutamate neurotoxicity is triggered primarily by massive Ca2+ influx arising from over-stimulation of the NMDA subtype of glutamate receptors. The underlying mechanisms, however, remain elusive. We have tested the hypothesis that mitochondrial glutaminase in HIV-infected macrophages is involved in converting glutamine to glutamate. Our results demonstrate that the concentration of glutamate in HIV-1 infected conditioned media was dependent on glutamine dose, and HIV-1 infected conditioned medium mediated glutamine-dependent neurotoxicity. These results indicate HIV-infection mediates neurotoxicity through glutamate production. In addition, glutamate-mediated neurotoxicity correlated with caspase activation and neuronal cell cycle re-activation. Inhibition of mitochondrial glutaminase diminished the HIV-induced glutamate production, and attenuated NMDA over-stimulation and subsequent neuronal apoptosis. These data implicate mitochondrial glutaminase in the induction of glutamate-mediated neuronal apoptosis during HIV-associated dementia, and provides a possible therapeutic strategy for HAD treatment.

Long-Distance Tunneling Nanotubules Shuttle Viral Immunoglobulin Class Switch-Suppressing Factors from HIV-Infected Macrophages to B Cells.

Class switching from immunoglobulin M (IgM) to IgG, IgA or IgE is central to immunity against viruses and requires activation of B cells by CD4+ T cells through CD40 ligand (CD40L). Human immunodeficiency virus (HIV) evades protective T cell-dependent IgG and IgA responses against viral proteins and vaccines through mechanisms that remain unknown. Recent findings from our lab suggested the involvement of negative factor (Nef), an HIV accessory protein that suppresses CD40 signaling in B cells. The present work addressed the mechanism by which Nef invades B cells. Fluorescence microscopy-based studies showed that accumulation of Nef in infected lymphoid follicles was associated with severe loss of activation-induced cytidine deaminase (AID), a Nef-targeted DNA-editing enzyme essential for class switching. Loss of follicular AID correlated with in vivo inactivation of CD40 signaling and formation of long-distance membrane tethers connecting Nef-positive B cells with HIV-infected macrophages. By combining confocal microscopy, videomicroscopy and electron microscopy with standard molecular biology techniques, we demonstrated that macrophages harboring HIV or expressing a chimeric Nef-green-fluorescent protein were capable of transferring Nef-containing cargos to B cells via long-distance, actin-propelled and energy-dependent membrane channels, including tunneling nanotubules. Transfer of Nef to B cells involved a clathrin-dependent pathway that required the guanine nucleotide exchange factor Vav as well as downstream Rho, Rac and Ccd42 small GTPases. This pathway led to Nef-mediated inhibition of CD40-induced AID expression and IgG and IgA class switching. In summary, our findings indicate that HIV evades T cell-dependent IgG and IgA responses by shuttling virus-encoded Ig class switch-suppressing factors from infected macrophages to non-infected B cells via long-range membrane tethers, including tunneling nanotubules.

Inhibition of HIV-1 Tat-Induced Interleukin-10 Expression by Mononuclear Cells at 3% O2.

The production of IL-10, which counteracts the effect of pro-inflammatory cytokines such as TNF-alpha, is induced by HIV-1 Tat in monocytes/macrophages by a process regulated by CREB-1 transcription factors [1]. IL-10 has been reported to suppress HIV-1 replication in macrophages by inhibiting HIV-1 transcription [2] at a, but it has also been reported to contribute to cell-mediated immunity in the setting of HIV infection [3]. We compared Tat-induced expression of IL-10 by THP-1 monocytes at 3% O2 (20 mmHg), the tension that monocytes see in certain tissues, and atmospheric 21% O2 (150 mmHg), the conventional in vitro culture condition. THP-1 cells were treated with recombinant HIV-Tat protein in combination with or without lipopolysaccharide (LPS) and IL-10 expression was analyzed by RT-PCR and ELISA. Treatment of THP-1 cells with HIV-Tat and LPS resulted in a dose- and time-dependent increase in IL-10 expression in the cells cultured at 21% O2. In contrast under 3% O2 the expression of IL-10 was reduced by 3-fold. Treatment with tautomycin, a protein phosphatase-1 (PP1) inhibitor, prevented IL-10 expression in LPS-stimulated THP-1 cells cultured at 21% O2. Thus, PP1 is likely to participate in Tat-mediated IL-10 production. PP1 has shown to affect CREB activity [4]; therefore we propose that PP1 might be involved in the CREB-mediated IL-10 gene expression. The inability of Tat to induce IL-10 production under 3% O2 might reflect the inadequate response of HIV-1 infected macrophages in vivo that might permit viral replication in infected macrophages. Further investigation of the role of PP1 in IL-10 expression could lead to new therapeutic opportunities for HIV-1 infection.

Cognitive dysfunction in HIV encephalitic SCID mice correlates with levels of Interferon-α in the brain

Interferon alpha (IFNalpha) is an antiviral cytokine produced in response to viral infection. IFNalpha also acts as a neuromodulatory molecule in the central nervous system (CNS). Elevated IFNalpha in the CNS causes cognitive deficits. To determine if elevated levels of IFNalpha in an HIV encephalitis mouse model correlate with cognitive deficits. C57BL/6J SCID mice were inoculated intracerebrally (i.c.) with HIV infected or uninfected (control) macrophages and cognitively tested in a water escape radial arm maze. After behavioral testing was completed, immunohistochemistry and ELISA were used to examine brain pathology and IFNalpha expression. Mice injected i.c. with HIV infected macrophages exhibited significantly more working memory errors, particularly in trials with the highest memory load. Immunohistochemistry indicated increased mouse IFNalpha staining prevalent on neurons and glial cells in the brains of mice with HIV infected macrophages compared to mice with uninfected control macrophages. In addition, IFNalpha levels in the brain correlated directly with working memory errors for mice with HIV infected macrophages. These data suggest that the cognitive deficit noted for the C57BL/6J SCID mice with HIV infected macrophages is mediated by the infection induced increase in IFNalpha.

Apoptotic Killing of HIV-1–Infected Macrophages Is Subverted by the Viral Envelope Glycoprotein

Viruses have evolved strategies to protect infected cells from apoptotic clearance. We present evidence that HIV-1 possesses a mechanism to protect infected macrophages from the apoptotic effects of the death ligand TRAIL (tumor necrosis factor–related apoptosis-inducing ligand). In HIV-1–infected macrophages, the viral envelope protein induced macrophage colony-stimulating factor (M-CSF). This pro-survival cytokine downregulated the TRAIL receptor TRAIL-R1/DR4 and upregulated the anti-apoptotic genes Bfl-1 and Mcl-1. Inhibition of M-CSF activity or silencing of Bfl-1 and Mcl-1 rendered infected macrophages highly susceptible to TRAIL. The anti-cancer agent Imatinib inhibited M-CSF receptor activation and restored the apoptotic sensitivity of HIV-1–infected macrophages, suggesting a novel strategy to curtail viral persistence in the macrophage reservoir.

Differential effects of HIV infected macrophages on dorsal root ganglia neurons and axons

Human immunodeficiency virus-associated distal-symmetric neuropathy (HIV-DSP) is the most common neurological complication of HIV infection. The pathophysiology of HIV-DSP is poorly understood and no treatment is available for this entity. The dorsal root ganglia (DRG) are the principal sites of neuronal damage and are associated with reactive mononuclear phagocytes as well as HIV-infected macrophages. To determine the role of HIV-infected macrophages in the pathogenesis of HIV-DSP, we developed a technique for culturing human DRG's. When the dissociated DRG neurons were exposed to supernatants from macrophages infected with CXCR4 or CCR5 tropic HIV-1 strains axonal retraction was observed without neuronal cell death but there was mitochondrial dysfunction in the neuronal cell body. Even though CXCR4 and CCR5 were expressed on the DRG neurons, the effects were independent of these receptors. Antioxidants rescued the neuronal cell body but not the axon from the toxic effects of the culture supernatants. Further, peripheral nerves of HIV-infected patients obtained at autopsy did not show evidence of increased oxidative stress. These observations suggest a differential effect on the axon and cell body. Different mechanisms of injury may be operative in these two structures.

Activation of cyclin‐dependent kinase 5 by calpains contributes to human immunodeficiency virus‐induced neurotoxicity

Although the specific mechanism of neuronal damage in human immunodeficiency virus (HIV) -associated dementia is not known, a prominent role for NMDA receptor (NMDAR)-induced excitotoxicity has been demonstrated in neurons exposed to HIV-infected/activated macrophages. We hypothesized NMDAR-mediated activation of the calcium-dependent protease, calpain, would contribute to cell death by induction of cyclin-dependent kinase 5 (CDK5) activity. Using an in vitro model of HIV neurotoxicity, in which primary rat cortical cultures are exposed to supernatants from primary human HIV-infected macrophages, we have observed increased calpain-dependent cleavage of the CDK5 regulatory subunit, p35, to the constitutively active isoform, p25. Formation of p25 is dependent upon NMDAR activation and calpain activity and is coincident with increased CDK5 activity in this model. Further, inhibition of CDK5 by roscovitine provided neuroprotection in our in vitro model. Consistent with our observations in vitro, we have observed a significant increase in calpain activity and p25 levels in midfrontal cortex of patients infected with HIV, particularly those with HIV-associated cognitive impairment. Taken together, our data suggest calpain activation of CDK5, a pathway activated in HIV-infected individuals, can mediate neuronal damage and death in a model of HIV-induced neurotoxicity.

HIV-1–specific CTLs effectively suppress replication of HIV-1 in HIV-1–infected macrophages

AbstractBoth CD4+ T cells and macrophages are major reservoirs of HIV-1. Previous study showed that HIV-1–specific cytolytic T lymphocytes (CTLs) hardly recognize HIV-1–infected CD4+ T cells because of Nef-mediated HLA class I down-regulation, suggesting that HIV-1 escapes from HIV-1–specific CTLs and continues to replicate in HIV-1–infected donors. On the other hand, the CTL recognition of HIV-1–infected macrophages and the effect of Nef-mediated HLA class I down-regulation on this recognition still remain unclear. We show a strong HIV-1 antigen presentation by HIV-1–infected macrophages. HIV-1–specific CTLs had strong abilities to suppress HIV-1R5 virus replication in HIV-1–infected macrophages and to kill HIV-1R5–infected macrophages. Nef-mediated HLA class I down-regulation minimally influenced the recognition of HIV-1–infected macrophages by HIV-1–specific CTLs. In addition, HIV-1–infected macrophages had a stronger ability to stimulate the proliferation of HIV-1–specific CTLs than HIV-1–infected CD4+ T cells. Thus, the effect of Nef-mediated HLA class I down-regulation was less critical with respect to the recognition by HIV-1–specific CTLs of HIV-infected macrophages than that of HIV-1–infected CD4+ T cells. These findings support the idea that the strong HIV-1 antigen presentation by HIV-1–infected macrophages is one of the mechanisms mediating effective induction of HIV-1–specific CTLs in the acute and early chronic phases of HIV-1 infection.

Cytoskeletal Protein Transformation in HIV-1-Infected Macrophage Giant Cells

The mechanisms linking HIV-1 replication, macrophage biology, and multinucleated giant cell formation are incompletely understood. With the advent of functional proteomics, the characterization, regulation, and transformation of HIV-1-infected macrophage-secreted proteins can be ascertained. To these ends, we performed proteomic analyses of culture fluids derived from HIV-1 infected monocyte-derived macrophages. Robust reorganization, phosphorylation, and exosomal secretion of the cytoskeletal proteins profilin 1 and actin were observed in conjunction with productive viral replication and giant cell formation. Actin and profilin 1 recruitment to the macrophage plasma membrane paralleled virus-induced cytopathicity, podosome formation, and cellular fusion. Poly-l-proline, an inhibitor of profilin 1-mediated actin polymerization, inhibited cytoskeletal transformations and suppressed, in part, progeny virion production. These data support the idea that actin and profilin 1 rearrangement along with exosomal secretion affect viral replication and cytopathicity. Such events favor the virus over the host cell and provide insights into macrophage defense mechanisms used to contain viral growth and how they may be affected during progressive HIV-1 infection.

Novel Delivery System Enhances Efficacy of Antiretroviral Therapy in Animal Model for HIV-1 Encephalitis

Most potent antiretroviral drugs (e.g., HIV-1 protease inhibitors) poorly penetrate the blood-brain barrier. Brain distribution can be limited by the efflux transporter, P-glycoprotein (P-gp). The ability of a novel drug delivery system (block co-polymer P85) that inhibits P-gp, to increase the efficacy of antiretroviral drugs in brain was examined using a severe combined immunodeficiency (SCID) mouse model of HIV-1 encephalitis (HIVE). Severe combined immunodeficiency mice inoculated with HIV-1 infected human monocyte-derived macrophages (MDM) into the basal ganglia were treated with P85, antiretroviral therapy (ART) (zidovudine, lamivudine and nelfinavir (NEL)), or P85 and ART. Mice were killed on days 7 and 14, and brains were evaluated for levels of viral infection. Antiviral effects of NEL, P85, or their combination were evaluated in vitro using HIV-1 infected MDM and showed antiretroviral effects of P85 alone. In SCID mice injected with virus-infected MDM, the combination of ART-P85 and ART alone showed a significant decrease of HIV-1 p24 expressing MDM (25% and 33% of controls, respectively) at day 7 while P85 alone group was not different from control. At day 14, all treatment groups showed a significant decrease in percentage of HIV-1 infected MDM as compared with control. P85 alone and combined ART-P85 groups showed the most significant reduction in percentage of HIV-1 p24 expressing MDM (8% to 22% of control) that were superior to the ART alone group (38% of control). Our findings indicate major antiretroviral effects of P85 and enhanced in vivo efficacy of antiretroviral drugs when combined with P85 in a SCID mouse model of HIVE.

Glutamate production by HIV‐1 infected human macrophage is blocked by the inhibition of glutaminase

Mononuclear phagocyte (macrophages and microglia) dysfunction plays a significant role in the pathogenesis of human immunodeficiency virus (HIV) associated dementia (HAD) through the production and release of soluble neurotoxic factors including glutamate. The mechanism of glutamate regulation by HIV-1 infection remains unclear. In this report, we investigated whether the enzyme glutaminase is responsible for glutamate generation by HIV-1 infected monocyte-derived macrophages. We tested the functionality of novel small molecule inhibitors designed to specifically block the activity of glutaminase. Glutaminase inhibitors were first characterized in a kinetic assay with crude glutaminase from rat brain revealing an uncompetitive mechanism of inhibition. The inhibitors were then tested in vitro for their ability to prevent glutamate generation by HIV-infected macrophages, their effect upon macrophage viability, and HIV infection. To validate these findings, glutaminase specific siRNA was tested for its ability to prevent glutamate increase during infection. Our results show that both glutaminase specific small molecule inhibitors and glutaminase specific siRNA were effective at preventing increases in glutamate by HIV-1 infected macrophage. These findings support glutaminase as a potential component of the HAD pathogenic process and identify a possible therapeutic avenue for the treatment of neuroinflammatory states such as HAD.

HIV-1 infected monocyte-derived macrophages affect the human brain microvascular endothelial cell proteome: New insights into blood–brain barrier dysfunction for HIV-1-associated dementia

Blood–brain barrier (BBB) compromise and transendothelial migration of HIV-infected leukocytes into the central nervous system (CNS) underlies the neuropathogenesis of HIV-1 infection. How this occurs is incompletely understood. We used a proteomic platform integrating difference gel electrophoresis and tandem mass spectrometry peptide sequencing to determine the effects that HIV-1-infected macrophages have on human brain microvascular endothelial cell (HBMEC) protein profiles. HIV-1 infected monocyte-derived macrophages (MDM) induced the upregulation of over 200 HBMEC proteins. These included metabolic, voltage-gated ion channels, heat shock, transport, cytoskeletal, regulatory, and calcium binding proteins. Results were validated by Western blot analysis. We conclude that HIV-1-infected MDM affect the HBMEC proteome and, in this way, affect BBB dysfunction and the development of HIV-1 CNS disease.

Neurokinin-1 Receptor Antagonist (Aprepitant) Inhibits Drug-Resistant HIV-1 Infection of Macrophages in vitro

Despite the success of antiretroviral therapy in controlling HIV replication, treatment failure may ultimately occur in more than 50% of the individuals on antiretroviral therapy. Cellular targets offer an attractive alternative, as it may be more difficult for HIV to develop resistance to alternative cellular inhibitory pathways. We have previously shown that CP-96,345, a neurokinin-1 receptor (NK-1R) antagonist, inhibits HIV-1 infection of macrophages in vitro by downregulating CCR5 expression (Lai JP, Ho WZ, Zhan GX, Yi Y, Collman RG, Douglas SD 2001). We have now investigated the effects of a Food and Drug Administration (FDA)-approved NK-1R antagonist, aprepitant (Emend), on HIV infection of macrophages in an in vitro system. Aprepitant is in clinical use for the prevention of nausea and vomiting associated with cancer chemotherapy or following surgical procedures. Monocytes isolated from healthy donors were cultured for 7 days and then treated with or without aprepitant (10(-6) M) for 2 h, followed by HIV infection with drug-resistant strains for 2 h. Untreated and HIV-infected macrophages were used as controls. Culture supernatants were harvested for p24 enzyme-linked immunosorbent assay (ELISA) or HIV reverse transcriptase (RT) activity at different time points after infection. R5X4 tropic and AZT-resistant strains (R5X4 tropic: A012 and A018) and RT inhibitor-resistant HIV strains (R5 tropic: TC60 and TC49) were used for infection. Aprepitant suppressed HIV Bal infection of macrophages. Treatment with aprepitant (10(-6) M) inhibited infection of macrophages with the AZT-resistant viruses (A018, A012) by 0.7 log(10). Aprepitant also suppressed infection of macrophages with RT inhibitor-resistant virus (TC 49 and TC 60) by 0.5 log(10). Furthermore, aprepitant significantly enhanced the anti-HIV activity of antiretrovirals (AZT, Efavirenz, and Indinavir) in HIV Bal-infected macrophages, and aprepitant inhibited CCR5 expression on macrophages, ranging from 50.5 to 29.6%. Donor heterogeneity was observed in antiviral activity and CCR5 receptor expression. Aprepitant is active against HIV drug-resistant isolates and enhances the anti-HIV activity of the antiretrovirals. Aprepitant downregulates CCR5 expression on macrophages. NK-1R antagonists merit further investigation as potential HIV therapeutic and immunomodulatory agents.