Morphine affects HIV-induced inflammatory response without influencing viral replication in human monocyte-derived macrophages

Opiate-abusing individuals are in the top three risk-factor groups for HIV infection. In fact, almost 30% of HIV-infected individuals in the USA are reported to abuse opiates, highlighting the intersection of drugs of abuse with HIV/AIDS. Opiate-abusers are cognitively impaired and suffer from neurological dysfunctions that may lead to high-risk sexual behavior, poor adherence to antiretroviral regimens, and hepatitis-C virus infection. Collectively, these factors may contribute to accelerated HIV central nervous system (CNS) disease progression. To understand the role of morphine in disease progression, we sought to determine whether morphine influences HIV-induced inflammation or viral replication in human monocyte-derived macrophages (h-mdms) and MAGI cells infected with HIV and exposed to morphine. Chronic morphine exposure of HIV-infected h-mdms led to significant alterations in the secretion of IL-6 and monocyte chemoattractant protein 2 (MCP-2). Morphine enhanced IL-6 secretion and blunted MCP-2 secretion from HIV-infected h-mdms. However, exposure of HIV-infected h-mdms to morphine had no effect on tumor necrosis factor alpha secretion. Morphine had no effect on later stages of viral replication in HIV-infected h-mdms. Morphine had a potentially additive effect on the HIV-induced production of IL-6 and delayed HIV-induced MCP-2 production. These results suggest that in HIV-infected opiate-abusers, enhanced CNS inflammation might result even when HIV disease is controlled.     

Development of a human neuronal cell model for human immunodeficiency virus (HIV)-infected macrophage-induced neurotoxicity: apoptosis induced by HIV type 1 primary isolates and evidence for involvement of the Bcl-2/Bcl-xL-sensitive intrinsic apoptosis pathway

Neuronal apoptosis within the central nervous system (CNS) is a characteristic feature of AIDS dementia, and it represents a common mechanism of neuronal death induced by neurotoxins (e.g., glutamate) released from human immunodeficiency virus (HIV)-infected macrophages (HIV/macrophage-induced neurotoxicity). Neuronal apoptosis may result from activation of the intrinsic (mitochondrial/bcl-2 regulated) or extrinsic (death receptor) pathways, although which pathway predominates in CNS HIV infection is unknown. Apoptosis initiated by the intrinsic pathway is typically blocked by antiapoptosis Bcl-2 family proteins, such as Bcl-2 and Bcl-xL, but whether these can block HIV/macrophage-induced neuronal apoptosis is unknown. To determine the potential role of the Bcl-2 family in HIV/macrophage-induced neuronal apoptosis, we developed a unique in vitro model, utilizing the NT2 neuronal cell line, primary astrocytes and macrophages, and primary CNS HIV type 1 (HIV-1) isolates. We validated our model by demonstrating that NT2.N neurons are protected against HIV-infected macrophages by N-methyl-D-aspartate (NMDA) glutamate receptor antagonists, similar to effects seen in primary neurons. We then established stable NT2.N neuronal lines that overexpress Bcl-2 or Bcl-xL (NT2.N/bcl-2 and NT2.N/bcl-xL, respectively) and determined their sensitivity to macrophages infected with primary R5, X4, and R5/X4 HIV-1 isolates. We found that NT2.N/bcl-2 and NT2.N/bcl-xL neurons were resistant to apoptosis induced by either R5, X4, or R5/X4 isolates and that resistance was abrogated by a Bcl-2 antagonist. Thus, the NMDA receptor/bcl-2-regulated apoptotic pathway contributes significantly to HIV/macrophage-induced neuronal apoptosis, and Bcl-2 family proteins protect neurons against the spectrum of primary HIV-1 isolates. Modulation of bcl-2 gene expression may therefore offer adjunctive neuroprotection against development of AIDS dementia.     

Purinergic receptors are required for HIV-1 infection of primary human macrophages

Macrophages play a significant role in HIV infection, viral rebound, and the development of AIDS. However, the function of host proteins in viral replication is incompletely characterized in macrophages. Purinergic receptors P2X and P2Y are major components of the macrophage immune response to pathogens, inflammation, and cellular damage. We demonstrate that these receptors are necessary for HIV infection of primary human macrophages. Inhibition of purinergic receptors results in a significant reduction in HIV replication in macrophages. This inhibition is independent of viral strain and is dose dependent. We also identify that P2X(1), P2X(7), and P2Y(1) receptors are involved in viral replication. We show that P2X(1), but not P2X(7) or P2Y(1), is necessary for HIV entry into macrophages. We demonstrate that interaction of the HIV surface protein gp120 with macrophages stimulates an increase in ATP release. Thus, we propose that HIV's binding to macrophages triggers a local release of ATP that stimulates purinergic receptors and facilitates HIV entry and subsequent stages of viral replication. Our data implicate a novel role for a family of host proteins in HIV replication in macrophages and suggest new therapeutic targets to reduce the devastating consequences of HIV infection and AIDS.     

Modulation of experimental herpes encephalitis-associated neurotoxicity through sulforaphane treatment

Reactive oxygen species (ROS) produced by brain-infiltrating macrophages and neutrophils, as well as resident microglia, are pivotal to pathogen clearance during viral brain infection. However, unchecked free radical generation is also responsible for damage to and cytotoxicity of critical host tissue bystander to primary infection. These unwanted effects of excessive ROS are combated by local cellular production of antioxidant enzymes, including heme oxygenase-1 (HO-1) and glutathione peroxidase 1 (Gpx1). In this study, we showed that experimental murine herpes encephalitis triggered robust ROS production, as well as an opposing upregulation of the antioxidants HO-1 and Gpx1. This antioxidant response was insufficient to prevent tissue damage, neurotoxicity, and mortality associated with viral brain infection. Previous studies corroborate our data supporting astrocytes as the major antioxidant producer in brain cell cultures exposed to HSV-1 stimulated microglia. We hypothesized that stimulating opposing antioxidative responses in astrocytes, as well as neurons, would mitigate the effects of ROS-mediated neurotoxicity both in vitro and during viral brain infection in vivo. Here, we demonstrate that the addition of sulforaphane, a potent stimulator of antioxidant responses, enhanced HO-1 and Gpx1 expression in astrocytes through the activation of nuclear factor-E2-related factor 2 (Nrf2). Additionally, sulforaphane treatment was found to be effective in reducing neurotoxicity associated with HSV-stimulated microglial ROS production. Finally, intraperitoneal injections of sulforaphane into mice during active HSV infection reduced neuroinflammation via a decrease in brain-infiltrating leukocytes, macrophage- and neutrophil-produced ROS, and MHCII-positive, activated microglia. These data support a key role for astrocyte-produced antioxidants in modulating oxidative stress and neuronal damage in response to viral infection.     

Retrovirally mediated IFN-beta transduction of macrophages induces resistance to HIV, correlated with up-regulation of RANTES production and down-regulation of C-C chemokine receptor-5 expression

Constitutive expression of IFN-beta by HIV target cells may be an alternative or complementary therapeutic approach for the treatment of AIDS. We show that macrophages derived from CD34+ cells from umbilical cord blood can be efficiently transduced by a retroviral vector carrying the IFN-beta coding sequence. This results in resistance to infection by a macrophage-tropic HIV type 1, as shown by the drastic reduction in the HIV DNA copy number per cell and in p24 release. Moreover, IFN-beta transduction totally blocked secretion of proinflammatory cytokines after HIV infection. The constitutive IFN-beta production also resulted in an increased production of IL-12 and IFN-gamma Th1-type cytokines and of the beta-chemokines macrophage-inflammatory protein-1alpha, macrophage-inflammatory protein-1beta, and RANTES. RANTES was found to be involved in the HIV resistance observed, and this was correlated with a down-regulation of the CCR-5 HIV entry coreceptor. These results demonstrate the feasibility and the efficacy of such IFN-beta-mediated gene therapy. In addition to inhibiting HIV replication, IFN-beta transduction could have beneficial immune effects in HIV-infected patients by favoring cellular immune responses.     

Macrophages: a crucial reservoir for human immunodeficiency virus in the body.

The replication of Human Immunodeficiency Virus (HIV) in cells of macrophage lineage represents a key pathogenetic event of the neurological damages typically found during the course of this disease. Macrophages are persistently infected cells and thus not susceptible to the cytophatic effect typical of infected activated CD4-lymphocytes. The resistance of macrophages to HIV infection is at least in part mediated by the autocrine production of the nerve growth factor (NGF), a neurokine able to sustain the survival of some cells of bone marrow origin, including monocyte-derived macrophages. This anti-apoptotic effect of NGF in HIV-infected macrophages can be even more relevant at the central nervous system level, where many cells are able to physiologically produce NGF, thus further increasing the survival of macrophages infected by HIV, and enhancing the damages that these cells may induce upon bystander neurons. The proapoptotic effect of soluble factors released by HIV-infected macrophages may heavily affect the survival and functions also of astrocytes, that in turn become unable to sustain neuronal homeostasis. Taken together, this information supports the importance of therapeutic attempts aimed at attacking virus replication in infected macrophages and/or to selectively eliminate these chronically infected and persistently virus-producing cells.     

Neurotoxicity of macrophages infected by HIV1

Conclusion Neuronal astrocytes and perhaps oligodendrocytic lesions occur during the course of HIVinfection of CNS cells. Most of the results suggest that these lesions are indirectlyinduced by infected macrophages, probably monocytes, present in the brain. Twomechanisms of neurotoxicity have been studied to date, one testing soluble factors presentin supernatant of infected monocytes and the other the direct effect of adhering HIV-infected monocytes to neurons and astrocytes. These two mechanisms are not mutually exclusive. They both indicate a major role for monocytes in the induction of brain lesionsand the crucial importance of the neurotoxic approach in the study of HIV inducedencephalopathy.     

Macrophages exposed to lymphotropic and monocytotropic HIV induce similar CTL responses despite differences in productive infection

Macrophages are accessory cells that are vulnerable to infection by HIV-1. HTLV-IIIB, a lymphotropic strain of HIV, infects macrophages poorly resulting in either no or low levels of virus expression compared to high levels of productive infection after exposure of macrophages to the monocytotropic HIV strain Ada-M. Whether this results in an impaired ability of HTLV-IIIB-exposed macrophages to initiate protective cytotoxic T lymphocyte (CTL) immune responses against these strains is not well defined. We investigated the ability of monocyte-derived macrophages (MDM) exposed to lymphotropic and monocytotropic HIV strains to initiate primary CTL responses in vitro. MDM exposed to HTLV-IIIB induced a specific primary CTL response that was comparable to MDM exposed to the monocytotropic strain Ada-M despite marked differences in productive HIV infection in MDM between the two strains. CTL generated in this model were MHC-restricted, strain-specific, and CD8+. These data demonstrate that high levels of productive HIV infection in accessory cells are not a prerequisite for the generation of a primary CTL response, suggesting a novel immunologic interaction between MDM and lymphotropic HIV strains.     

HIV impairs TNF-alpha mediated macrophage apoptotic response to Mycobacterium tuberculosis

The factors that contribute to the exceptionally high incidence of Mycobacterium tuberculosis (MTb) disease in HIV(+) persons are poorly understood. Macrophage apoptosis represents a critical innate host cell response to control MTb infection and limit disease. In the current study, virulent live or irradiated MTb (iMTbRv) induced apoptosis of differentiated human U937 macrophages in vitro, in part dependent on TNF-alpha. In contrast, apoptosis of differentiated HIV(+) human U1 macrophages (HIV(+) U937 subclone) was markedly reduced in response to iMTbRv and associated with significantly reduced TNF-alpha release, whereas apoptosis and TNF-alpha release were intact to TLR-independent stimuli. Furthermore, reduced macrophage apoptosis and TNF-alpha release were independent of MTb phagocytosis. Whereas surface expression of macrophage TLR2 and TLR4 was preserved, IL-1 receptor associated kinase-1 phosphorylation and NF-kappaB nuclear translocation were reduced in HIV(+) U1 macrophages in response to iMTbRv. These findings were confirmed using clinically relevant human alveolar macrophages (AM) from healthy persons and asymptomatic HIV(+) persons at clinical risk for MTb infection. Furthermore, in vitro HIV infection of AM from healthy persons reduced both TNF-alpha release and AM apoptosis in response to iMTbRv. These data identify an intrinsic specific defect in a critical macrophage cellular response to MTb that may contribute to disease pathogenesis in HIV(+) persons.     

Cytokine-stimulated, but not HIV-infected, human monocyte-derived macrophages produce neurotoxic levels of l -cysteine

Approximately one-quarter of individuals with AIDS develop neuropathological symptoms that are attributable to infection of the brain with HIV. The cognitive manifestations have been termed HIV-associated dementia. The mechanisms underlying HIV-associated neuronal injury are incompletely understood, but various studies have confirmed the release of neurotoxins by macrophages/microglia infected with HIV-1 or stimulated by viral proteins, including the envelope glycoprotein gp120. In the present study, we investigated the possibility that l -cysteine, a neurotoxin acting at the N-methyl-d -aspartate subtype of glutamate receptor, could contribute to HIV-associated neuronal injury. Picomolar concentrations of gp120 were found to stimulate cysteine release from human monocyte-derived macrophages (hMDM) in amounts sufficient to injure cultured rat cerebrocortical neurons. TNF-alpha and IL-1beta, known to be increased in HIV-encephalitic brains, as well as a cellular product of cytokine stimulation, ceramide, were also shown to induce release of cysteine from hMDM in a dose-dependent manner. A TNF-alpha-neutralizing Ab and an IL-1betaR antagonist partially blocked gp120-induced cysteine release, suggesting that these cytokines may mediate the actions of gp120. Interestingly, hMDM infected with HIV-1 produced significantly less cysteine than uninfected cells following stimulation with TNF-alpha. Our findings imply that cysteine may play a role in the pathogenesis of neuronal injury in HIV-associated dementia due to its release from immune-activated macrophages but not virus-infected macrophages. Such uninfected cells comprise the vast majority of mononuclear phagocytes (macrophages and microglia) found in HIV-encephalitic brains.     

Upregulation of Fas ligand expression by human immunodeficiency virus in human macrophages mediates apoptosis of uninfected T lymphocytes.

Apoptosis has been proposed to mediate CD4+ T-cell depletion in human immunodeficiency virus (HIV)-infected individuals. Interaction of Fas ligand (FasL) with Fas (CD95) results in lymphocyte apoptosis, and increased susceptibility to Fas-mediated apoptosis has been demonstrated in lymphocytes from HIV-infected individuals. Cells undergoing apoptosis in lymph nodes from HIV-infected individuals do not harbor virus, and therefore a bystander effect has been postulated to mediate apoptosis of uninfected cells. These data raise the possibility that antigen-presenting cells are a source of FasL and that HIV infection of cells such as macrophages may induce or increase FasL expression. In this report, we demonstrate that HIV infection of monocytic cells not only increases the surface expression of Fas but also results in the de novo expression of FasL. Interference with the FasL-Fas interaction by anti-Fas blocking antibodies abrogates HIV-induced apoptosis of monocytic cells. Human monocyte-derived macrophages from healthy donors contain detectable FasL mRNA, which is further upregulated following HIV infection with monocytotropic strains. HIV-infected human macrophages result in the apoptotic death of Jurkat T cells and peripheral blood T lymphocytes. Interruption of the FasL-Fas interaction abrogates the HIV-infected macrophage-dependent death of T lymphocytes. These results provide evidence that human macrophages can provide a source of FasL, especially following HIV infection, and can thus participate in lymphocyte depletion in HIV-infected individuals.     

Functional synergy between CD40 ligand and HIV-1 Tat contributes to inflammation: implications in HIV type 1 dementia

HIV type 1 (HIV-1)-associated dementia (HAD) is believed to occur due to aberrant activation of monocyte-derived macrophages and brain-resident microglial cells by viral proteins as well as by the proinflammatory mediators released by infected cells. To investigate the inflammatory aspects of the disease, we examined the levels of soluble CD40L (sCD40L) in paired samples of plasma and cerebrospinal fluid obtained from 25 HIV-infected individuals. A significantly higher level of sCD40L was detected in both cerebrospinal fluid and plasma from HIV-infected patients with cognitive impairment, compared with their nonimpaired counterparts. The contribution of sCD40L to the pathogenesis of HAD was then examined by in vitro experiments. rCD40L synergized with HIV-1 Tat to increase TNF-alpha release from primary human monocytes and microglia, in an NF-kappaB-dependent manner. The mechanistic basis for this synergism was attributed to a Tat-mediated up-regulation of CD40 in monocytes and microglia. Finally, the CD40L-mediated increase in TNF-alpha production by monocytes was shown to be biologically important; immunodepletion experiments revealed that TNF-alpha was essential for the neurotoxic effects of conditioned medium recovered from Tat/CD40L-treated monocytes. Taken together, our results show that CD40 signaling in microglia and monocytes can synergize with the effects of Tat, further amplifying inflammatory processes within the CNS and influencing neuronal survival.     

Structure-activity relationships in platelet-activating factor. Part 13: synthesis and biological evaluation of piperazine derivatives with dual anti-PAF and anti-HIV-1 or pure antiretroviral activity

HIV-1 infection of the brain and PAF neurotoxicity are implicated in AIDS dementia complex. We previously reported that a trisubstituted piperazine derivative is able to diminish both HIV-1 replication in monocyte-derived macrophages and PAF-induced platelet aggregation. We report in this work new compounds obtained by modifying its piperazine substituents. The structure-activity relationship study shows that a better dual activity or even pure antiretroviral compounds can be obtained in this series.     

Macrophage response to Mycobacterium tuberculosis during HIV infection: relationships between macrophage activation and apoptosis

Human macrophages represent the first line of defense for the containment of Mycobacterium tuberculosis infection. After phagocytosis, macrophages express activation surface markers and produce proinflammatory cytokines and chemokines whose main role is to control pathogen spreading by recruiting peripheral lymphocytes and monocytes at the site of inflammation. However, in the case of a concomitant human immunodeficiency virus (HIV) infection, these signals strongly enhance the susceptibility to viral infection both at the viral entry and replication levels. Under these conditions, viral expansion extends beyond tissue macrophages to T cells and vice-versa, according to the emerging viral phenotype. In absence of an efficient immune response, Mycobacterium tuberculosis can replicate in macrophages in an uncontrolled fashion culminating in macrophage death by apoptosis. As a consequence, a more severe form of immunedepression, involving both innate and specific immune responses, could be responsible for both ematogenous mycobacterial dissemination and extrapulmonary form of tuberculosis in HIV-infected patients.