The pro-apoptotic human BH3-only peptide harakiri is expressed in cryptococcus-infected perivascular macrophages in HIV-1 encephalitis patients

In the central nervous system (CNS), HIV-1 targets mainly microglia/macrophages. Like the CD4+ T cell depletion and neuronal loss in AIDS, apoptosis is thought to be involved in eliminating infected macrophages. In this study, we examined the expression of the pro-apoptotic BH3-peptide harakiri (Hrk) in brain tissues of AIDS patients. Immunoreactivity against Hrk was positive in perivascular macrophages infiltrated into some restricted lesions. Most of these immunopositive cells contained small inclusions positive for Grocott's methenamine silver staining. Confocal laser microscopy demonstrated that Hrk expression coincided with immunoreactivities against HIV-1 and Cryptococcus neoformans. Expression of Hrk mRNA was demonstrated in these cells by in situ hybridization, which indicated that Hrk is not phagocytosed material. Some pro-apoptotic bcl-family members, including Hrk, may contribute to the delayed hypersensitive reaction in AIDS, in macrophages eliminating opportunistic infection.     

Role of immune-derived diffusible mediators in AIDS-associated neurological disorders

Neurologic abnormalities are common in HIV-1 infected patients and often represent the dominant clinical manifestation of pediatric AIDS. Although the neurological dysfunction has been directly related to CNS invasion by HIV-1, the pathogenesis of neurologic disorders remains unclear. This review will first discuss the spectrum of potential interactions between HIV-1 and neural (neuronal and glial) cells, in the face of experimental data. Next, we will focus on the role of immune-derived cytokines and other soluble compounds which have been proposed to act as neurotoxic mediators and appear to play a role in the pathogenesis of AIDS-associated neurodegeneration.     

Human brain parenchymal microglia express CD14 and CD45 and are productively infected by HIV-1 in HIV-1 encephalitis

Microglia are endogenous brain macrophages that show distinct phenotypes such as expression of myeloid antigens, ramified morphology, and presence within the neural parenchyma. They play significant roles in a number of human CNS diseases including AIDS dementia. Together with monocyte-derived (perivascular) macrophages, microglia represent a major target of HIV-1 infection. However, a recent report challenged this notion based on findings in SIV encephalitis. This study concluded that perivascular macrophages can be distinguished from parenchymal microglial cells by their expression of CD14 and CD45, and that macrophages, but not microglia, are productively infected in SIV and HIV encephalitis. To address whether parenchymal microglia are productively infected in HIV encephalitis, we analyzed expression of CD14, CD45 and HIV-1 p24 in human brain. Microglia were identified based on their characteristic ramified morphology and location in the neural parenchyma. We found that parenchymal microglia are CD14+ (activated), CD45+ (resting and activated), and constitute approximately two thirds of the p24+ cells in HIV encephalitis cases. These results demonstrate that microglia are major targets of infection by HIV-1, and delineate possible differences between HIVE and SIVE. Because productively infected tissue macrophages serve as the major viral reservoir, these findings have important implications for AIDS.     

Immune privilege and HIV-1 persistence in the CNS

Summary:  Human immunodeficiency virus-1 (HIV-1) neuroinvasion occurs early (during period of initial viremia), leading to infection of a limited amount of susceptible cells with low CD4 expression. Protective cellular and humoral immunity eliminate and suppress viral replication relatively quickly due to peripheral immune responses and the low level of initial central nervous system (CNS) infection. Upregulation of the brain protective mechanisms against lymphocyte entry and survival (related to immune privilege) helps reduce viral load in the brain. The local immune compartment dictates local viral evolution as well as selection of cytotoxic lymphocytes and immunoglobulin G specificity. Such status can be sustained until peripheral immune anti-viral responses fail. Activation of microglia and astrocytes, due to local or peripheral triggers, increases chemokine production, enhances traffic of infected cells into the CNS, upregulates viral replication in resident brain macrophages, and significantly augments the spread of viral species. The combination of these factors leads to the development of HIV-1 encephalitis-associated neurocognitive decline and patient death. Understanding the immune-privileged state created by virus, the brain microenvironment, and the ability to enhance anti-viral immunity offer new therapeutic strategies for treatment of HIV-1 CNS infection.     

HIV-1 infection and neurocognitive impairment in the current era

Brain HIV-1-infection may result in a syndrome of profound cognitive, behavioral and motor impairment known as AIDS dementia complex (ADC) in adults and HIV-related encephalopathy in children. Although the introduction of highly active antiretroviral therapy (HAART) has prolonged and improved the lives of infected individuals, it is clear that HAART does not provide complete protection against neurological damage in HIV/AIDS. HIV-1 associated dementia is a complex phenomenon, which could be the result of several mechanisms caused by those players using different intracellular signaling pathways. Understanding the causes of neurodegeneration during HIV-1 infection and the factors which certain individuals develop disease can provide researches on new therapeutic targets to positively affect disease outcomes. Controlling CNS viral replication with HAART is an essential primary approach, but it should be complemented with adjunctive CNS-directed therapeutics. Understanding the nature of HIV-1 infection within the CNS as well as inflammatory responses will ultimately lead to the elimination of HIV-associated neurocognitive disorders.     

Evidence of productively infected CD8+ T cells in patients with AIDS: implications for HIV-1 pathogenesis

CD8+ T lymphocytes play an important protective role against HIV infection. The onset of AIDS is associated with a decline in both the number of CD8+ T lymphocytes and anti-HIV cytotoxic activity in CD8+ T cells. The reason for this progressive failure of CD8+ T cells in HIV-1 infection remains unknown. Earlier reports have shown presence of viral DNA in CD8+ cells of HIV-1-infected patients; under some conditions, CD8+ T cells have been shown to express CD4 in vitro and can be susceptible to infection with HIV-1. However, whether CD8+ lymphocytes in vivo can be productively infected with HIV-1 remains unclear. In this study, we generated multiple CD8+ T-cell clones from two patients with AIDS. These clones were CD8+/CD3+ but did not express CD4. Several of these CD8+ clones from both patients were found to be endogenously infected with HIV-1 and spontaneously produced these viruses. CD8+ cell-produced HIV-1 was biologically competent because viruses produced by most of these clones could efficiently infect and replicate in peripheral blood lymphocytes from HIV-negative donors. In addition, some of these viruses were able to form syncytia in MT-2 cells indicating syncytium-inducing phenotype. Comparison of the sequences in V3 loop areas among different viruses showed changes in some of the clones from both patients. For the first time, this report provides direct evidence that mature CD8+ T cells can be productively infected with HIV-1 in patients with AIDS. Direct infection of CD8+ T lymphocytes may play a role in the eventual failure of these cells in HIV-1 infection.     

Viral participation in cellular and microenvironmental transformation and amplification towards malignancy in AIDS patients

HIV-1 infection would initially predispose to neoplastic transformation in terms of a progressive lymphocytic proliferation followed by the onset of an immunodeficiency state. Both virion genomic integration and also active host cell proliferation would perhaps participate in the establishment of an often multifocal primary CNS lymphoma of AIDS type. Repeated opportunistic infections in AIDS patients tend to especially involve the central nervous system to also carry an increased risk of neoplastic transformation of the reactive B lymphocytes reaching the brain. A microenvironmental set of circumstances in patients with AIDS would predispose to non-Hodgkin's lymphoma largely in terms of an HIV-1 infection that progresses concurrently with evolving cell replication, immunodeficiency, and repeated opportunistic infections as caused by several different potential pathogens. Epstein-Barr virus infection in particular appears closely related to Hodgkin's disease that develops in some AIDS patients. A viral role in the development of lymphomas and of Kaposi sarcoma in HIV-infected individuals would account for neoplastic aggressiveness and for a particular predilection for primary CNS lymphoma. Such a role perhaps implicates viral integration within the genome of host cells that are actively proliferating or else infected by multiple viral pathogens such as EBV, HIV-1, CMV, and Herpes virus.     

HIV-1 Gene Expression and Replication in Neuronal and Glial Cell Lines with Immature Phenotype: Effects of Nerve Growth Factor

Encephalopathy and neurological disorders are a major manifestation of pediatric AIDS. Although HIV-1 can replicate in cells of neuronal and glial origin, it is yet unclear whether immature neural cells, which are present during nervous system development, can support HIV-1 replication and whether neurotrophic factors can modulate HIV-1 gene expression. In this study we show that a glial cell line with a phenotype closely resembling immature glial cells is more permissive to HIV-1 infection and replication than a neuroblastic cell line. After HIV-1 infection or after transfection of these cells with the HIV-1 LTR-CAT reporter gene alone or in the presence of Tat, both HIV-1 replication and viral gene expression progressively decrease in the neuronal cell line, while they increase in the glial cell line. In both cell types viral gene expression and replication are augmented by the addition to the cells of nerve growth factor (NGF) at concentrations which induce neuronal differentiation. However, these effects are again more evident with the glial cell type, suggesting that immature glial cells may represent one of the major targets and reservoirs of HIV-1 in the developing nervous system. As NGF and Tat act synergistically in inducing HIV-1 gene expression, these data also suggest that during development the presence of high levels of neural trophic factors may activate viral replication and render the CNS more susceptible to the deleterious effects of HIV-1 infection.     

Macrophage/microglial accumulation and proliferating cell nuclear antigen expression in the central nervous system in human immunodeficiency virus encephalopathy

This study was performed to quantitate and characterize the mononuclear phagocytes (MPs) in human immunodeficiency virus encephalopathy (HIVE) by immunohistochemistry in an effort to gain insights into potential mechanisms of central nervous system (CNS) accumulation. Single- and double-labeled studies using antibodies against CD14, CD16, CD68, proliferating cell nuclear antigen (PCNA), Ki-67, von Willebrand factor, and HIV-1 p24 were performed using brain tissue from patients with HIVE, HIV-1 infection without encephalitis, and seronegative controls. A substantial increase in MPs was observed in CNS tissue from patients with HIVE, relative to seronegative controls and patients with acquired immune deficiency syndrome but without encephalitis, as determined by CD68 and CD16 immunohistochemistry. A large proportion of CD16+ MPs in HIVE CNS tissue were PCNA+, but do not appear to be proliferating, based on limited Ki-67 positivity. Although virtually all cells positive for HIV-1 p24 were PCNA+, there were many PCNA+ cells where HIV-1 p24 expression was not detected. PCNA positivity was also observed in some endothelial cells and ependymal cells in HIVE CNS. Our results would support a role for HIV-1-induced alterations in MP trafficking and homeostasis in the pathogenesis of HIVE.     

A system for the high efficiency replication of HIV-1 in neural cells and its application to anti-viral evaluation.

Stable transfection of H4 neuroglioma cells with the Epstein-Barr virus-based eucaryotic CD4 expression vector pKS286 generated the cell line, H4/CD4, in which greater than 90% of cells express surface CD4 receptors. Optimal conditions for infection of H4/CD4 cells with HIV-1 were determined; these included a cocultivation with growth-arrested, chronically infected T cells. Under these conditions, 3-days after infection up to 50% of H4/CD4 cells expressed HIV-1 antigens as detected by immunofluorescence assay, the number of intracellular HIV-1 RNA copies reached 10(3) molecules per cell as determined by liquid hybridization, and virus production ranged from 0.2 to 1.0 micrograms HIV-1 p24 core antigen per ml of culture supernatant, comparable to that measured under the same conditions in HIV-1 infected T cells. Giant cells and cytolysis were common. Inhibition of HIV-1 infection by nucleoside analogues in H4/CD4 cells was comparable to that in T cells, suggesting that the early stages of HIV-1 infection were similar in both cell systems. Infection in the presence of soluble CD4 reduced HIV-1 expression to the levels determined in CD4-negative H4 cells. This system may be useful for screening of drugs intended to block HIV-1 replication in the brain and for the evaluation of the HIV-1 life cycle in brain cells.     

Immunomodulatory Role of Complement Proteins in the Neuropathology Associated with Opiate Abuse and HIV-1 Co-Morbidity

The complement system which is a critical mediator of innate immunity plays diverse roles in the neuropathogenesis of HIV-1 infection such as clearing HIV-1 and promoting productive HIV-1 replication. In the development of HIV-1 associated neurological disorders (HAND), there may be an imbalance between complement activation and regulation, which may contribute to the neuronal damage as a consequence of HIV-1 infection. It is well recognized that opiate abuse exacerbates HIV-1 neuropathology, however, little is known about the role of complement proteins in opiate induced neuromodulation, specifically in the presence of co-morbidity such as HIV-1 infection. Complement levels are significantly increased in the HIV-1-infected brain, thus HIV-induced complement synthesis may represent an important mechanism for the pathogenesis of AIDS in the brain, but remains underexplored. Anti-HIV-1 antibodies are able to initiate complement activation in HIV-1 infected CNS cells such as microglia and astrocytes during the course of disease progression; however, this complement activation fails to clear and eradicate HIV-1 from infected cells. In addition, the antiretroviral agents used for HIV therapy cause dysregulation of lipid metabolism, endothelial, and adipocyte cell function, and activation of pro-inflammatory cytokines. We speculate that both HIV-1 and opiates trigger a cytokine-mediated pro-inflammatory stimulus that modulates the complement cascade to exacerbate the virus-induced neurological damage. We examined the expression levels of C1q, SC5b-9, C5L2, C5aR, C3aR, and C9 key members of the complement cascade both in vivo in post mortem brain frontal cortex tissue from patients with HAND who used/did not use heroin, and in vitro using human microglial cultures treated with HIV tat and/or heroin. We observed significant expression of C1q and SC5b-9 by immunofluorescence staining in both the brain cortical and hippocampal region in HAND patients who abused heroin. Additionally, we observed increased gene expression of C5aR, C3aR, and C9 in the brain tissue of both HIV-1 infected patients with HAND who abused and did not abuse heroin, as compared to HIV negative controls. Our results show a significant increase in the expression of complement proteins C9, C5L2, C5aR, and C3aR in HIV transfected microglia and an additional increase in the levels of these complement proteins in heroin-treated HIV transfected microglia. This study highlights the a) potential roles of complement proteins in the pathogenesis of HIV-1-related neurodegenerative disorders; b) the combined effect of an opiate, like heroin, and HIV viral protein like HIV tat on complement proteins in normal human microglial cells and HIV transfected microglial cells. In the context of HAND, targeting selective steps in the complement cascade could help ameliorating the HIV burden in the CNS, thus investigations of complement-related therapeutic approaches for the treatment of HAND are warranted.     

Localization of HIV-1 co-receptors CCR5 and CXCR4 in the brain of children with AIDS.

The chemokine receptors CCR5 and CXCR4 are co-receptors together with CD4 for human immunodeficiency virus (HIV)-1 entry into target cells. Macrophage-tropic HIV-1 viruses use CCR5 as a co-receptor, whereas T-cell-line tropic viruses use CXCR4. HIV-1 infects the brain and causes a progressive encephalopathy in 20 to 30% of infected children and adults. Most of the HIV-1-infected cells in the brain are macrophages and microglia. We examined expression of CCR5 and CXCR4 in brain tissue from 20 pediatric acquired immune deficiency syndrome (AIDS) patients in relation to neuropathological consequences of HIV-1 infection. The overall frequency of CCR5-positive perivascular mononuclear cells and macrophages was increased in the brains of children with severe HIV-1 encephalitis (HIVE) compared with children with mild HIVE or non-AIDS controls, whereas the frequency of CXCR4-positive perivascular cells did not correlate with disease severity. CCR5- and CXCR4-positive macrophages and microglia were detected in inflammatory lesions in the brain of children with severe HIVE. In addition, CXCR4 was detected in a subpopulation of neurons in autopsy brain tissue and primary human brain cultures. Similar findings were demonstrated in the brain of adult AIDS patients and controls. These findings suggest that CCR5-positive mononuclear cells, macrophages, and microglia contribute to disease progression in the central nervous system of children and adults with AIDS by serving as targets for virus replication.     

Neuropathologies in transgenic mice expressing human immunodeficiency virus type 1 Tat protein under the regulation of the astrocyte-specific glial fibrillary acidic protein promoter and doxycycline

The human immunodeficiency virus type 1 (HIV-1) Tat protein is a key pathogenic factor in a variety of acquired immune deficiency syndrome (AIDS)-associated disorders. A number of studies have documented the neurotoxic property of Tat protein, and Tat has therefore been proposed to contribute to AIDS-associated neurological diseases. Nevertheless, the bulk of these studies are performed in in vitro neuronal cultures without taking into account the intricate cell-cell interaction in the brain, or by injection of recombinant Tat protein into the brain, which may cause secondary stress or damage to the brain. To gain a better understanding of the roles of Tat protein in HIV-1 neuropathogenesis, we attempted to establish a transgenic mouse model in which Tat expression was regulated by both the astrocyte-specific glial fibrillary acidic protein promoter and a doxycycline (Dox)-inducible promoter. In the present study, we characterized the phenotypic and neuropathogenic features of these mice. Both in vitro and in vivo assays confirmed that Tat expression occurred exclusively in astrocytes and was Dox-dependent. Tat expression in the brain caused failure to thrive, hunched gesture, tremor, ataxia, and slow cognitive and motor movement, seizures, and premature death. Neuropathologies of these mice were characterized by breakdown of cerebellum and cortex, brain edema, astrocytosis, degeneration of neuronal dendrites, neuronal apoptosis, and increased infiltration of activated monocytes and T lymphocytes. These results together demonstrate that Tat expression in the absence of HIV-1 infection is sufficient to cause neuropathologies similar to most of those noted in the brain of AIDS patients, and provide the first evidence in the context of a whole organism to support a critical role of Tat protein in HIV-1 neuropathogenesis. More importantly, our data suggest that the Dox inducible, brain-targeted Tat transgenic mice offer an in vivo model for delineating the molecular mechanisms of Tat neurotoxicity and for developing therapeutic strategies for treating HIV-associated neurological disorders.     

Mononuclear phagocyte immunity and the neuropathogenesis of HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1)-associated dementia is a neuroinflammatory brain disorder that is fueled by viral infection and immune activation of brain mononuclear phagocytes (MP; macrophages and microglia). MP serve as a reservoir for persistent viral infection, a vehicle for viral dissemination throughout the brain, and a major source of neurotoxic products that when produced in abundance, affect neuronal function. Such neurotoxic substances secreted by MP lead to clinical neurological impairment (cognitive, behavior, and motor abnormalities), which occurs usually years after the initial viral infection. How HIV-1 evades the immune function characteristic for MP as a first line of defense, including phagocytosis and intracellular killing, is not well understood despite more than two decades of study. In this report, we review the complex role(s) played by MP in the neuropathogenesis of HIV-1 infection. The clinical manifestations, pathology and pathogenesis, and treatment options are discussed in relationship to innate and adaptive immunity. Particular emphasis is given to the diversity of MP functions and how it may affect the disease process and manifestations. New insights into disease mechanisms are provided by advances in enhanced magnetic resonance imaging and proteomics to identify cell movement and genetic profiles of disease. New therapeutic strategies are discussed based on current knowledge of HIV-1-associated dementia pathogenesis.     

Characterization of HIV-1 Gag-specific T cell responses in chronically infected Indian population

India is at the epicentre of the global HIV/AIDS epidemic in South-east Asia, predominated by subtype C infections. It is important to characterize HIV-1-specific T cell responses in this particular population with the aim of identifying protective correlates of immunity to control HIV-1 infection. In this study, we performed a comprehensive analysis of the breadth and magnitude of T cell responses directed at HIV-1 subtype C Gag, one of the most conserved HIV-1 proteins. The study population consisted of antiretroviral naive, chronic HIV-1 subtype C-infected individuals at various stages of infection. We used recent advanced techniques such as enzyme-linked immunospot (ELISPOT) assay and intracellular cytokine staining to quantify the total CD4(+) and CD8(+) T cell response to HIV-1 gag at single peptide level, regardless of HLA haplotype of the infected individual. The p24-Gag was identified as the most frequently recognized subunit protein with the greatest magnitude of CD4(+) and CD8(+) T cell responses. Stronger and broader CD8 T cell responses were recognized, contrasting with the weaker and narrower CD4 T cell responses with regard to Gag protein subunits. The magnitude of the HIV-specific interferon (IFN)-gamma responses was observed to be higher than the corresponding interleukin (IL)-2 response, indicating the persistence of antigenic load in chronically infected Indian population due to the probable dysfunction of HIV-specific, IFN-gamma-secreting CD8 T cells in absence of IL-2 help.