TRAIL-mediated apoptosis in HIV-1-infected macrophages is dependent on the inhibition of Akt-1 phosphorylation

HIV-1 uses mononuclear phagocytes (monocytes, tissue macrophages, and dendritic cells) as a vehicle for its own dissemination and as a reservoir for continuous viral replication. The mechanism by which the host immune system clears HIV-1-infected macrophages is not understood. TRAIL may play a role in this process. TRAIL is expressed on the cell membrane of peripheral immune cells and can be cleaved into a soluble, secreted form. The plasma level of TRAIL is increased in HIV-1-infected patients, particularly those with high viral loads. To study the effect of elevated TRAIL on mononuclear phagocytes, we used recombinant human (rh) TRAIL and human monocyte-derived macrophages (MDM) as an in vitro model. Our results demonstrated rhTRAIL-induced apoptosis in HIV-1-infected MDM and inhibited viral replication, while having a reduced effect on uninfected MDM. HIV-1 infection significantly decreased Akt-1 phosphorylation; rhTRAIL exposure further decreased Akt-1 phosphorylation. Infection with a dominant-negative Akt-1 adenovirus potentiated rhTRAIL-induced apoptosis, while constitutively active Akt-1 blocked rhTRAIL-induced apoptosis in HIV-1-infected MDM. From this data we conclude the death ligand TRAIL preferentially provokes apoptosis of HIV-1-infected MDM, and the mechanism is reliant upon the inhibition of Akt-1 phosphorylation. Understanding this mechanism may facilitate the elimination of HIV-1-infected macrophages and lead to new therapeutic avenues for treatment of HIV-1 infection.     

Human immunodeficiency virus type-1 vulnerates nascent neuronal cells

Macrophages or microglial cells are the major target cells for HIV-1 infection in the brain. The infected cells release neurotoxic factors that may cause severe neuronal cell damage, especially in the basal ganglia and hippocampus. In this study, we used rat OHC to examine the region-specific neuronal cell damage caused by HIV-1-infected macrophages. When OHC was cocultured with HIV-1-infected MDM, we found that neuronal cells at the GCL of the DG were preferentially killed via apoptosis, and that projection of MF from GCL to PCL of the CA3 region was severely disturbed. We marked precursor cells around the DG region by using an EGFP-expressing retrovirus vector and found that these cells lost the ability to differentiate into neurons when exposed to HIV-1-infected MDM. In the DG, new neurons are normally incorporated into GCL or PCL, while in the presence of HIV-1-infected MDM, mature neurons failed to be incorporated into those layers. These data indicate that the neurotoxic factor(s) released from HIV-1-infected macrophages impede(s) neuronal cell repair in brain tissue. This suggests that DG is the region of the hippocampus most vulnerable to neuronal damage caused by HIV-1 infection, and that its selective vulnerability is most likely due to the highly active neurogenesis that takes place in this region.     

HIV infection enhances TRAIL-induced cell death in macrophage by down-regulating decoy receptor expression and generation of reactive oxygen species

Background

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) could induce apoptosis of HIV-1-infected monocyte-derived macrophage (MDM), but the molecular mechanisms are not well understood.

Methodology/Principal Findings

By using an HIV-1 Env-pseudotyped virus (HIV-1 PV)-infected MDM cell model we demonstrate that HIV-1 PV infection down-regulates the expression of TRAIL decoy receptor 1 (DcR1) and 2 (DcR2), and cellular FLICE-inhibitory protein (c-FLIP), but dose not affect the expression of death receptor 4 and 5 (DR4, DR5), and Bcl-2 family members in MDM cells. Furthermore, recombinant soluble TRAIL and an agonistic anti-DR5 antibody, AD5-10, treatment stimulates reactive oxygen species (ROS) generation and JNK phosphorylation.

Conclusions/Significance

HIV infection facilitates TRIAL-induced cell death in MDM by down-regulating the expression of TRAIL decoy receptors and intracellular c-FLIP. Meanwhile, the agonistic anti-DR5 antibody, AD5-10, induces apoptosis synergistically with TRAIL in HIV-1-infected cells. ROS generation and JNK phosphorylation are involved in this process. These findings potentiate clinical usage of the combination of TRAIL and AD5-10 in eradication of HIV-infected macrophage and AIDS.     

Dysregulation of macrophage-secreted cathepsin B contributes to HIV-1-linked neuronal apoptosis

Chronic HIV infection leads to the development of cognitive impairments, designated as HIV-associated neurocognitive disorders (HAND). The secretion of soluble neurotoxic factors by HIV-infected macrophages plays a central role in the neuronal dysfunction and cell death associated with HAND. One potentially neurotoxic protein secreted by HIV-1 infected macrophages is cathepsin B. To explore the potential role of cathepsin B in neuronal cell death after HIV infection, we cultured HIV-1(ADA) infected human monocyte-derived macrophages (MDM) and assayed them for expression and activity of cathepsin B and its inhibitors, cystatins B and C. The neurotoxic activity of the secreted cathepsin B was determined by incubating cells from the neuronal cell line SK-N-SH with MDM conditioned media (MCM) from HIV-1 infected cultures. We found that HIV-1 infected MDM secreted significantly higher levels of cathepsin B than did uninfected cells. Moreover, the activity of secreted cathepsin B was significantly increased in HIV-infected MDM at the peak of viral production. Incubation of neuronal cells with supernatants from HIV-infected MDM resulted in a significant increase in the numbers of apoptotic neurons, and this increase was reversed by the addition of either the cathepsin B inhibitor CA-074 or a monoclonal antibody to cathepsin B. In situ proximity ligation assays indicated that the increased neurotoxic activity of the cathepsin B secreted by HIV-infected MDM resulted from decreased interactions between the enzyme and its inhibitors, cystatins B and C. Furthermore, preliminary in vivo studies of human post-mortem brain tissue suggested an upregulation of cathepsin B immunoreactivity in the hippocampus and basal ganglia in individuals with HAND. Our results demonstrate that HIV-1 infection upregulates cathepsin B in macrophages, increases cathepsin B activity, and reduces cystatin-cathepsin interactions, contributing to neuronal apoptosis. These findings provide new evidence for the role of cathepsin B in neuronal cell death induced by HIV-infected macrophages.     

Bcl-2 upregulation by HIV-1 tat during infection of primary human macrophages in culture

The ability of cells of the human monocyte/macrophage lineage to host HIV-1 replication while resisting cell death is believed to significantly contribute to their ability to serve as a reservoir for viral replication in the host. Although macrophages are generally resistant to apoptosis, interruption of anti-apoptotic pathways can render them susceptible to apoptosis. Here we report that HIV-1(BAL )infection of primary human monocyte-derived macrophages (MDM) upregulates the mRNA and protein levels of the anti-apoptic gene, Bcl-2. Furthermore, this upregulation can be quantitatively mimicked by treating MDM with soluble HIV-1 Tat-86 protein. These results suggest that in infecting cells of the monocyte/macrophage lineage, HIV-1 may be benefiting from additional protection against apoptosis caused by specific upregulation of cellular anti-apoptotic genes.     

The role of cytochrome P450 2E1 on ethanol-mediated oxidative stress and HIV replication in human monocyte-derived macrophages

BackgroundAlcohol consumption is considered to be a major health problem among people living with HIV/AIDS. Our previous reports have shown that ethanol reduced intracellular concentrations of antiretroviral drugs elvitegravir and darunavir in the HIV-1-infected U1 cell line. Ethanol also increased HIV-1 replication despite the presence of elvitegravir. Our previous finding has also shown that the levels of cytochrome P450 enzyme 2E1 (CYP2E1) and oxidative stress in blood monocytes were induced, while the concentration of alcohol in the plasma was reduced in HIV-1-infected alcohol users compared to uninfected alcohol users. However, the role of CYP2E1 in ethanol-enhanced oxidative stress and HIV-1 replication is still unclear.MethodsThis study examined the chronic effects (14 days) of ethanol on HIV viral load, oxidative DNA damage, expression of CYP2E1, expression of antioxidant enzymes (AOEs), expression of reactive oxygen species (ROS) in human monocyte-derived macrophages (MDM). Further, to evaluate the role of CYP2E1 in mediating ethanol-induced viral replication, CYP2E1 siRNA and CYP2E1 selective inhibitor were used in the HIV-1-infected U1 cell line following ethanol treatment.ResultsChronic ethanol exposure demonstrated an increase in oxidative DNA damage and CYP2E1 expression in both non-infected and HIV-1-infected MDM. Our results showed that ethanol chronic exposure increased HIV-1 replication by ~3-fold in HIV-1-infected MDM. This ethanol-enhanced HIV-1 replication was associated with an increased oxidative DNA damage, an increased expression of CYP2E1, and a decreased expression of antioxidant enzyme PRDX6. In HIV-1-infected U1 cell line, we observed a decreased viral replication (~30%) and a decreased DNA damage (~100%) after repression of CYP2E1 by siRNA, upon ethanol exposure. We also observed a decreased viral replication (~25%) after inhibition of CYP2E1 by using selective CYP2E1 inhibitor.ConclusionsThe data suggest that chronic ethanol exposure increases HIV-1 replication in MDM, at least in part, through CYP2E1-mediated oxidative stress. These results are clinically relevant to potentially find effective treatment strategies for HIV-1-infected alcohol users.     

Productive HIV-1 infection of macrophages restricted to the cell fraction with proliferative capacity.

Retroviruses establish productive infection only in proliferating cells. Macrophages are often considered to be non-proliferating in vitro yet are susceptible to HIV-1 infection. This has led to the conclusion that HIV-1 can establish infection independent of host cell proliferation. We here report that a small proportion of macrophages does have proliferative capacity. A comparable small fraction of monocyte derived macrophages (MDM) supported productive HIV-1 infection as demonstrated in limiting dilution culture. Fluorescence activated cell sorting on the basis of incorporation of BrdUrd, a thymidine analog, and subsequent PCR analysis revealed the presence of proviral DNA only in the BrdUrd positive cell fraction with DNA synthesizing activity. To identify which phase of cell cycle is required for establishment of productive infection, growth arrest in G1 or G1/S phase prior to inoculation was performed. gamma-Irradiation, which arrests primary cells in G1, prevented both cell proliferation and establishment of productive infection in MDM. Treatment of MDM with aphidicolin, a specific inhibitor of DNA polymerase alpha and delta which arrests cells in G1/S phase of the cell cycle, also inhibited DNA synthesis but did not prevent establishment of productive infection which is completely analogous to observations in T cells. Our data thus indicate that not cell division itself but cellular conditions that coincide with cell proliferation are apparently indispensable for establishment of productive infection.     

Neurovirulent simian immunodeficiency virus infection induces neuronal, endothelial, and glial apoptosis.

BACKGROUND: Studies of human immunodeficiency virus type 1 (HIV-1) associated dementia have shown neuronal loss in discrete areas. The presence and mechanism of neuronal death, however, has remained quite elusive. One mechanism of cell death, apoptosis, has been clearly demonstrated outside the central nervous system (CNS) in HIV-1 infection but has not been firmly established within the CNS. Therefore, we set out to ascertain whether neuronal cell loss in simian immunodeficiency virus (SIV) encephalitis, an animal model of HIV-1-associated dementia, is a result of apoptosis. MATERIALS AND METHODS: With the aid of an in situ technique for identifying the 3'-OH ends of newly fragmented DNA characteristic of apoptosis, in conjunction with specific detected morphological criteria via light microscopy, we have examined encephalitic and nonencephalitic brains of macaques infected with a neurovirulent, neuroendotheliotropic strain of SIV to see if virus is spatially associated with apoptosis of neurons and non-neuronal cell types. RESULTS: We demonstrate the presence of DNA damage, indicative of apoptosis, in neurons, endothelial cells, and glial cells of the CNS of SIV-infected macaques. Furthermore, we observe an association between the localization of cells with significant DNA fragmentation and perivascular inflammatory cell infiltrates containing SIV-infected macrophages and multinucleated giant cells. Quantitative analysis reveals significantly more cells with DNA fragmentation in the CNS of macaques infected with neurovirulent, neuroendotheliotropic SIV strains as compared with strictly lymphocyte-tropic SIV strains and SIV negative controls. CONCLUSIONS: Our findings of apoptosis in SIV-infected CNS may potentially lead to a better understanding of the AIDS dementia complex, ultimately providing a basis for better treatments.     

Involvement of Fc gamma RI (CD64) in the mechanism of HIV-1 inhibition by polyclonal IgG purified from infected patients in cultured monocyte-derived macrophages

The aim of this study was to investigate the mechanism of HIV-1 neutralization using monocyte-derived macrophages (MDM) in comparison to PBMC as target cells. For this purpose, we analyzed neutralizing activities of different human polyclonal IgG samples purified from sera of HIV-1-infected individuals using a single cycle infection assay. We found an increase of the neutralizing titer when macrophages vs PBMC were used as target cells. Moreover, polyclonal IgG from HIV-1-infected patients that are not able to neutralize virus when PBMC are used as target cells strongly inhibit MDM infection. Similar results were obtained with neutralizing mAbs. To explore the participation of FcgammaRs in HIV-1 inhibition, F(ab')(2) and Fab of these Igs were produced. Results indicated that both F(ab')(2) and Fab are less effective to inhibit virus replication in MDM. Moreover, competition experiments with Fc fragments of IgG from healthy donors or with purified monoclonal anti-human FcgammaRs Ab strengthen the participation of the FcgammaRs, and in particular of FcgammaRI (CD64) in HIV-1 inhibition on MDM. Mechanisms by which HIV-specific IgG inhibit virus replication in cultured macrophages are proposed and the benefit of inducing such Abs by vaccination is discussed.     

Human immunodeficiency virus type 1 inhibits DNA damage-triggered apoptosis by a Nef-independent mechanism

It is controversial whether the accessory human immunodeficiency virus type 1 (HIV-1) Nef protein inhibits or enhances apoptosis. To address this issue, we investigated the effect of Nef on programmed cell death with vectors or proviral HIV-1 constructs coexpressing Nef and green fluorescent protein from single bicistronic RNAs. This approach allows us to readily identify transfected or infected cells and to correlate cell death directly with Nef expression levels. We demonstrate that Nef does not significantly affect apoptosis in transfected or HIV-1-infected Jurkat T cells or primary human peripheral blood mononuclear cells. Unexpectedly, however, both nef⁺ and nef-defective HIV-1 infection blocked apoptosis in cells treated with UV light or etoposide but not cell death induced by CD95 antibody, TRAIL, Ly294002, or serum starvation. Our results show that HIV-1 infection inhibits DNA damage-induced but not death receptor-dependent cell death by a Nef-independent mechanism.     

vif-negative human immunodeficiency virus type 1 persistently replicates in primary macrophages, producing attenuated progeny virus.

The vif gene of human immunodeficiency virus type 1 (HIV-1) is required for efficient infection of primary T lymphocytes. In this study, we investigated in detail the role of vif in productive infection of primary monocyte-derived macrophages (MDM). Viruses carrying missense or deletion mutations in vif were constructed on the background of the monocytotropic recombinant NLHXADA-GP. Using MDM from multiple donors, we found that vif mutants produced in complementing or partially complementing cell lines were approximately 10% as infectious as wild-type virus when assayed for incomplete, complete, and circularized viral DNA molecules by quantitative PCR amplification or for viral core antigen p24 production by enzyme-linked immunosorbent assay. We then determined the structure and infectivity of vif mutant HIV-1 by using MDM exclusively both for virus production and as targets for infection. Biosynthetic labeling and immunoprecipitation analysis of sucrose cushion-purified vif-negative HIV-1 made in MDM revealed that the virus had reduced p24 content compared with wild-type HIV-1. Cell-free MDM-derived vif mutant HIV-1 was infectious in macrophages as determined by the synthesis and maintenance of full-length viral DNA and by the produc- tion of particle-associated viral RNA, but its infectivity was approximately 2,500-fold lower than that of wild-type virus whose titer was determined in parallel by measurement of the viral DNA burden. MDM infected with MDM-derived vif-negative HIV-1 were able to transmit the virus to uninfected MDM by cocultivation, confirming the infectiousness of this virus. We conclude that mutations in vif significantly reduce but do not eliminate the capacity of HIV-1 to replicate and produce infectious progeny virus in primary human macrophages.     

HIV-1 down-modulates gamma signaling chain of Fc gamma R in human macrophages: a possible mechanism for inhibition of phagocytosis

HIV-1 infection impairs a number of macrophage effector functions, thereby contributing to development of opportunistic infections and the pathogenesis of AIDS. FcgammaR-mediated phagocytosis by human monocyte-derived macrophages (MDM) is inhibited by HIV-1 infection in vitro, and the underlying mechanism was investigated in this study. Inhibition of phagocytosis directly correlated with the multiplicity of HIV-1 infection. Expression of surface FcgammaRs was unaffected by HIV-1 infection, suggesting that inhibition of phagocytosis occurred during or after receptor binding. HIV-1 infection of MDM markedly inhibited tyrosine phosphorylation of the cellular proteins, which occurs following engagement of FcgammaRs, suggesting a defect downstream of initial receptor activation. FcgammaR-mediated phagocytosis in HIV-infected MDM was associated with inhibition of phosphorylation of tyrosine kinases from two different families, Hck and Syk, defective formation of Syk complexes with other tyrosine-phosphorylated proteins, and inhibition of paxillin activation. Down-modulation of protein expression but not mRNA of the gamma signaling subunit of FcgammaR (a docking site for Syk) was observed in HIV-infected MDM. Infection of MDM with a construct of HIV-1 in which nef was replaced with the gene for the gamma signaling subunit augmented FcgammaR-mediated phagocytosis, suggesting that down-modulation of gamma-chain protein expression in HIV-infected MDM caused the defective FcgammaR-mediated signaling and impairment of phagocytosis. This study is the first to demonstrate a specific alteration in phagocytosis signal transduction pathway, which provides a mechanism for the observed impaired FcgammaR-mediated phagocytosis in HIV-infected macrophages and contributes to the understanding of how HIV-1 impairs cell-mediated immunity leading to HIV-1 disease progression.     

Generation of cytotoxic T cells against virus-infected human brain macrophages in a murine model of HIV-1 encephalitis

HIV-1 encephalitis (HIVE) and its associated dementia can occur in up to 20% of infected individuals, usually when productive viral replication in brain mononuclear phagocytes (macrophages and microglia) and depletion of CD4(+) T lymphocytes are most significant. T cells control viral replication through much of HIV-1 disease, but how this occurs remains incompletely understood. With this in mind, we studied HIV-1-specific CTL responses in a nonobese diabetic (NOD)-SCID mouse model of HIVE. HIV-1-infected monocyte-derived macrophages (MDM) were injected into the basal ganglia after syngeneic immune reconstitution by HLA-A*0201-positive human PBL to generate a human PBL-NOD-SCID HIVE mouse. Engrafted T lymphocytes produced HIV-1gag- and HIV-1pol-specific CTL against virus-infected brain MDM within 7 days. This was demonstrated by tetramer staining of human PBL in mouse spleens and by IFN-gamma ELISPOT. CD8, granzyme B, HLA-DR, and CD45R0 Ag-reactive T cells and CD79alpha-positive B cells migrated to and were in contact with human MDM in brain areas where infected macrophages were abundant. The numbers of productively infected MDM were markedly reduced (>85%) during 2 wk of observation. The human PBL-NOD-SCID HIVE mouse provides a new tool for studies of cellular immune responses against HIV-1-infected brain mononuclear phagocytes during natural disease and after vaccination.