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Human Immunodeficiency Virus (HIV) is widely acknowledged for its profound impact on the immune system. Although HIV primarily affects peripheral CD4 T cells, its influence on the central nervous system (CNS) cannot be overlooked. Within the brain, microglia and CNS-associated macrophages (CAMs) serve as the primary targets for HIV and the simian immunodeficiency virus (SIV) in nonhuman primates. This infection can lead to neurological effects and establish a viral reservoir. Given the gaps in our understanding of how these cells respond in vivo to acute CNS infection, we conducted single-cell RNA sequencing (scRNA-seq) on myeloid cells from the brains of three rhesus macaques 12 days after SIV infection, along with three uninfected controls. Our analysis revealed six distinct microglial clusters including homeostatic microglia, preactivated microglia, and activated microglia expressing high levels of inflammatory and disease-related molecules. In response to acute SIV infection, the homeostatic and preactivated microglia population decreased, while the activated and disease-related microglia increased. All microglial clusters exhibited upregulation of MHC class I molecules and interferon-related genes, indicating their crucial roles in defending against SIV during the acute phase. All microglia clusters also upregulated genes linked to cellular senescence. Additionally, we identified two distinct CAM populations: CD14lowCD16hi and CD14hiCD16low CAMs. Interestingly, during acute SIV infection, the dominant CAM population changed to one with an inflammatory phenotype. Specific upregulated genes within one microglia and one macrophage cluster were associated with neurodegenerative pathways, suggesting potential links to neurocognitive disorders. This research sheds light on the intricate interactions between viral infection, innate immune responses, and the CNS, providing valuable insights for future investigations., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Xu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Perivascular macrophages (PVMs) and, to a lesser degree, microglia are targets and reservoirs of HIV and simian immunodeficiency virus (SIV) in the brain. Previously, we demonstrated that colony-stimulating factor 1 receptor (CSF1R) in PVMs was upregulated and activated in chronically SIV-infected rhesus macaques with encephalitis, correlating with SIV infection of PVMs. Herein, we investigated the role of CSF1R in the brain during acute SIV infection using BLZ945, a brain-penetrant CSF1R kinase inhibitor. Apart from three uninfected historic controls, nine Indian rhesus macaques were infected acutely with SIVmac251 and divided into three groups (n = 3 each): an untreated control and two groups treated for 20-30 days with low- (10 mg/kg/day) or high- (30 mg/kg/day) dose BLZ945. With the high-dose BLZ945 treatment, there was a significant reduction in cells expressing CD163 and CD206 across all four brain areas examined, compared with the low-dose treatment and control groups. In 9 of 11 tested regions, tissue viral DNA (vDNA) loads were reduced by 95%-99% following at least one of the two doses, and even to undetectable levels in some instances. Decreased numbers of CD163+ and CD206+ cells correlated significantly with lower levels of vDNA in all four corresponding brain areas. In contrast, BLZ945 treatment did not significantly affect the number of microglia. Our results indicate that doses as low as 10 mg/kg/day of BLZ945 are sufficient to reduce the tissue vDNA loads in the brain with no apparent adverse effect. This study provides evidence that infected PVMs are highly sensitive to CSF1R inhibition, opening new possibilities to achieve viral clearance., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Background: The SARS-CoV-2 virus activates maternal and placental immune responses. Such activation in the setting of other infections during pregnancy is known to impact fetal brain development. The effects of maternal immune activation on neurodevelopment are mediated at least in part by fetal brain microglia. However, microglia are inaccessible for direct analysis, and there are no validated non-invasive surrogate models to evaluate in utero microglial priming and function. We have previously demonstrated shared transcriptional programs between microglia and Hofbauer cells (HBCs, or fetal placental macrophages) in mouse models., Methods and Results: We assessed the impact of maternal SARS-CoV-2 on HBCs isolated from 24 term placentas (N = 10 SARS-CoV-2 positive cases, 14 negative controls). Using single-cell RNA-sequencing, we demonstrated that HBC subpopulations exhibit distinct cellular programs, with specific subpopulations differentially impacted by SARS-CoV-2. Assessment of differentially expressed genes implied impaired phagocytosis, a key function of both HBCs and microglia, in some subclusters. Leveraging previously validated models of microglial synaptic pruning, we showed that HBCs isolated from placentas of SARS-CoV-2 positive pregnancies can be transdifferentiated into microglia-like cells (HBC-iMGs), with impaired synaptic pruning behavior compared to HBC models from negative controls., Conclusion: These findings suggest that HBCs isolated at birth can be used to create personalized cellular models of offspring microglial programming., (© 2024. The Author(s).)

HIV-1 infection within the central nervous system (CNS) includes evolution of the virus, damaging inflammatory cascades, and the involvement of multiple cell types; however, our understanding of how Env tropism and inflammation can influence CNS infectivity is incomplete. In this study, we utilize macrophage-tropic and T cell-tropic HIV-1 Env proteins to establish accurate infection profiles for multiple CNS cells under basal and interferon alpha (IFN-α) or lipopolysaccharide (LPS)-induced inflammatory states. We found that macrophage-tropic viruses confer entry advantages in primary myeloid cells, including monocyte-derived macrophage, microglia, and induced pluripotent stem cell (iPSC)-derived microglia. However, neither macrophage-tropic or T cell-tropic HIV-1 Env proteins could mediate infection of astrocytes or neurons, and infection was not potentiated by induction of an inflammatory state in these cells. Additionally, we found that IFN-α and LPS restricted replication in myeloid cells, and IFN-α treatment prior to infection with vesicular stomatitis virus G protein (VSV G) Envs resulted in a conserved antiviral response across all CNS cell types. Further, using RNA sequencing (RNA-seq), we found that only myeloid cells express HIV-1 entry receptor/coreceptor transcripts at a significant level and that these transcripts in select cell types responded only modestly to inflammatory signals. We profiled the transcriptional response of multiple CNS cells to inflammation and found 57 IFN-induced genes that were differentially expressed across all cell types. Taken together, these data focus attention on the cells in the CNS that are truly permissive to HIV-1, further highlight the role of HIV-1 Env evolution in mediating infection in the CNS, and point to limitations in using model cell types versus primary cells to explore features of virus-host interaction. IMPORTANCE The major feature of HIV-1 pathogenesis is the induction of an immunodeficient state in the face of an enhanced state of inflammation. However, for many of those infected, there can be an impact on the central nervous system (CNS) resulting in a wide range of neurocognitive defects. Here, we use a highly sensitive and quantitative assay for viral infectivity to explore primary and model cell types of the brain for their susceptibility to infection using viral entry proteins derived from the CNS. In addition, we examine the ability of an inflammatory state to alter infectivity of these cells. We find that myeloid cells are the only cell types in the CNS that can be infected and that induction of an inflammatory state negatively impacts viral infection across all cell types.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurotropic coronavirus that invades the host central nervous system (CNS) and causes neurological dysfunction. Microglia are key immune cells in the CNS, however, whether and how they response to PHEV infection remains unclear. Herein, microglial activation and proliferation were detected in the CNS of PHEV-infected mice, as along with the proinflammatory response. Moreover, the production of proinflammatory cytokines induced by moderately activated microglia limited viral replication in the early stage of infection. Microglial depletion assays showed that during late infection, excess activation of microglia aggravated neurological symptoms, BBB destruction, and peripheral monocyte/macrophage infiltration into the CNS. Using an in vitro brain slice model, PHEV was identified to specifically and moderately induce microglial activation in the absence of peripheral immune cells infiltration. Consistently, macrophage clearance from circulating blood indicated that peripheral monocytes/macrophages crossing the BBB of mice were responsible for excess activation of microglia and CNS damage in late PHEV infection. Overall, our findings provide evidence supporting a dual role for microglia in the host CNS in response to coronavirus PHEV invasion., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhang, Li, Lu, Shi, Gao, Ma, Lan, Guan, Zhao, Gao and He.)

Both substance use disorder and HIV infection continue to affect many individuals. Both have untoward effects on the brain, and the two conditions often co-exist. In the brain, macrophages and microglia are infectable by HIV, and these cells are also targets for the effects of drugs of abuse, such as the psychostimulant methamphetamine. To determine the interaction of HIV and methamphetamine, we isolated microglia and brain macrophages from SIV-infected rhesus monkeys that were treated with or without methamphetamine. Cells were subjected to single-cell RNA sequencing and results were analyzed by statistical and bioinformatic analysis. In the animals treated with methamphetamine, a significantly increased proportion of the microglia and/or macrophages were infected by SIV. In addition, gene encoding functions in cell death pathways were increased, and the brain-derived neurotropic factor pathway was inhibited. The gene expression patterns in infected cells did not cluster separately from uninfected cells, but clusters comprised of microglia and/or macrophages from methamphetamine-treated animals differed in neuroinflammatory and metabolic pathways from those comprised of cells from untreated animals. Methamphetamine increases CNS infection by SIV and has adverse effects on both infected and uninfected microglia and brain macrophages, highlighting the dual and interacting harms of HIV infection and drug abuse on the brain.

Alpha/beta interferon (IFN-α/β) signaling through the IFN-α/β receptor (IFNAR) is essential to limit virus dissemination throughout the central nervous system (CNS) following many neurotropic virus infections. However, the distinct expression patterns of factors associated with the IFN-α/β pathway in different CNS resident cell populations implicate complex cooperative pathways in IFN-α/β induction and responsiveness. Here we show that mice devoid of IFNAR1 signaling in calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) expressing neurons (CaMKIIcre:IFNAR fl/fl mice) infected with a mildly pathogenic neurotropic coronavirus (mouse hepatitis virus A59 strain [MHV-A59]) developed severe encephalomyelitis with hind-limb paralysis and succumbed within 7 days. Increased virus spread in CaMKIIcre:IFNAR fl/fl mice compared to IFNAR fl/fl mice affected neurons not only in the forebrain but also in the mid-hind brain and spinal cords but excluded the cerebellum. Infection was also increased in glia. The lack of viral control in CaMKIIcre:IFNAR fl/fl relative to control mice coincided with sustained Cxcl1 and Ccl2 mRNAs but a decrease in mRNA levels of IFNα/β pathway genes as well as Il6 , Tnf , and Il1β between days 4 and 6 postinfection (p.i.). T cell accumulation and IFN-γ production, an essential component of virus control, were not altered. However, IFN-γ responsiveness was impaired in microglia/macrophages irrespective of similar pSTAT1 nuclear translocation as in infected controls. The results reveal how perturbation of IFN-α/β signaling in neurons can worsen disease course and disrupt complex interactions between the IFN-α/β and IFN-γ pathways in achieving optimal antiviral responses. IMPORTANCE IFN-α/β induction limits CNS viral spread by establishing an antiviral state, but also promotes blood brain barrier integrity, adaptive immunity, and activation of microglia/macrophages. However, the extent to which glial or neuronal signaling contributes to these diverse IFN-α/β functions is poorly understood. Using a neurotropic mouse hepatitis virus encephalomyelitis model, this study demonstrated an essential role of IFN-α/β receptor 1 (IFNAR1) specifically in neurons to control virus spread, regulate IFN-γ signaling, and prevent acute mortality. The results support the notion that effective neuronal IFNAR1 signaling compensates for their low basal expression of genes in the IFN-α/β pathway compared to glia. The data further highlight the importance of tightly regulated communication between the IFN-α/β and IFN-γ signaling pathways to optimize antiviral IFN-γ activity., (Copyright © 2020 American Society for Microbiology.)

HIV-associated neuroinflammation is primarily driven by CNS macrophages including microglia. Regulation of these immune responses, however, remains to be characterized in detail. Using the SIV/macaque model of HIV, we evaluated CNS expression of triggering receptor expressed on myeloid cells 2 (TREM2) which is constitutively expressed by microglia and contributes to cell survival, proliferation, and differentiation. Loss-of-function mutations in TREM2 are recognized risk factors for neurodegenerative diseases including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and Nasu-Hakola disease (NHD); recent reports have also indicated a role for TREM2 in HIV-associated neuroinflammation. Using in situ hybridization (ISH) and qRT-PCR, TREM2 mRNA levels were found to be significantly elevated in frontal cortex of macaques with SIV encephalitis compared with uninfected controls (P = 0.02). TREM2 protein levels were also elevated as measured by ELISA of frontal cortex tissue homogenates in these animals. Previously, we characterized the expression of CSF1R (colony-stimulating factor 1 receptor) in this model; the TREM2 and CSF1R promoters both contain a PU.1 binding site. While TREM2 and CSF1R mRNA levels in the frontal cortex were highly correlated (Spearman R = 0.79, P < 0.001), protein levels were not well correlated. In SIV-infected macaques released from ART to study viral rebound, neither TREM2 nor CSF1R mRNA increased with rebound viremia. However, CSF1R protein levels remained significantly elevated unlike TREM2 (P = 0.02). This differential expression suggests that TREM2 and CSF1R play unique, distinct roles in the pathogenesis of HIV CNS disease.

The first oncolytic virotherapy employing HSV-1 (oHSV-1) was approved recently by the FDA to treat cancer, but further improvements in efficacy are needed to eradicate challenging refractory tumors, such as glioblastomas (GBM). Microglia/macrophages comprising approximately 40% of a GBM tumor may limit virotherapeutic efficacy. Here, we show these cells suppress oHSV-1 growth in gliomas by internalizing the virus through phagocytosis. Internalized virus remained capable of expressing reporter genes while viral replication was blocked. Macrophage/microglia formed a nonpermissive OV barrier, preventing dissemination of oHSV-1 in the glioma mass. The deficiency in viral replication in microglial cells was associated with silencing of particular viral genes. Phosphorylation of STAT1/3 was determined to be responsible for suppressing oHSV-1 replication in macrophages/microglia. Treatment with the oxindole/imidazole derivative C16 rescued oHSV-1 replication in microglia/macrophages by inhibiting STAT1/3 activity. In the U87 xenograft model of GBM, C16 treatment overcame the microglia/macrophage barrier, thereby facilitating tumor regression without causing a spread of the virus to normal organs. Collectively, our results suggest a strategy to relieve a STAT1/3-dependent therapeutic barrier and enhance oHSV-1 oncolytic activity in GBM. Significance: These findings suggest a strategy to enhance the therapeutic efficacy of oncolytic virotherapy in glioblastoma. Cancer Res; 78(3); 718-30. ©2017 AACR ., (©2017 American Association for Cancer Research.)

West Nile virus (WNV) is a neurotropic flavivirus that can cause significant neurological disease. Mouse models of WNV infection demonstrate that a proinflammatory environment is induced within the central nervous system (CNS) after WNV infection, leading to entry of activated peripheral immune cells. We utilized ex vivo spinal cord slice cultures (SCSC) to demonstrate that anti-inflammatory mechanisms may also play a role in WNV-induced pathology and/or recovery. Microglia are a type of macrophage that function as resident CNS immune cells. Similar to mouse models, infection of SCSC with WNV induces the upregulation of proinflammatory genes and proteins that are associated with microglial activation, including the microglial activation marker Iba1 and CC motif chemokines CCL2, CCL3, and CCL5. This suggests that microglia assume a proinflammatory phenotype in response to WNV infection similar to the proinflammatory (M1) activation that can be displayed by other macrophages. We now show that the WNV-induced expression of these and other proinflammatory genes was significantly decreased in the presence of minocycline, which has antineuroinflammatory properties, including the ability to inhibit proinflammatory microglial responses. Minocycline also caused a significant increase in the expression of anti-inflammatory genes associated with alternative anti-inflammatory (M2) macrophage activation, including interleukin 4 (IL-4), IL-13, and FIZZ1. Minocycline-dependent alterations to M1/M2 gene expression were associated with a significant increase in survival of neurons, microglia, and astrocytes in WNV-infected slices and markedly decreased levels of inducible nitric oxide synthase (iNOS). These results demonstrate that an anti-inflammatory environment induced by minocycline reduces viral cytotoxicity during WNV infection in ex vivo CNS tissue. IMPORTANCE West Nile virus (WNV) causes substantial morbidity and mortality, with no specific therapeutic treatments available. Antiviral inflammatory responses are a crucial component of WNV pathology, and understanding how they are regulated is important for tailoring effective treatments. Proinflammatory responses during WNV infection have been extensively studied, but anti-inflammatory responses (and their potential protective and reparative capabilities) following WNV infection have not been investigated. Minocycline induced the expression of genes associated with the anti-inflammatory (M2) activation of CNS macrophages (microglia) in WNV-infected SCSC while inhibiting the expression of genes associated with proinflammatory (M1) macrophage activation and was protective for multiple CNS cell types, indicating its potential use as a therapeutic reagent. This ex vivo culture system can uniquely address the ability of CNS parenchymal cells (neurons, astrocytes, and microglia) to respond to minocycline and to modulate the inflammatory environment and cytotoxicity in response to WNV infection without peripheral immune cell involvement., (Copyright © 2017 American Society for Microbiology.)

While HIV kills most of the cells it infects, a small number of infected cells survive and become latent viral reservoirs, posing a significant barrier to HIV eradication. However, the mechanism by which immune cells resist HIV-induced apoptosis is still incompletely understood. Here, we demonstrate that while acute HIV infection of human microglia/macrophages results in massive apoptosis, a small population of HIV-infected cells survive infection, silence viral replication, and can reactivate viral production upon specific treatments. We also found that HIV fusion inhibitors intended for use as antiretroviral therapies extended the survival of HIV-infected macrophages. Analysis of the pro- and anti-apoptotic pathways indicated no significant changes in Bcl-2, Mcl-1, Bak, Bax or caspase activation, suggesting that HIV blocks a very early step of apoptosis. Interestingly, Bim, a highly pro-apoptotic negative regulator of Bcl-2, was upregulated and recruited into the mitochondria in latently HIV-infected macrophages both in vitro and in vivo. Together, these results demonstrate that macrophages/microglia act as HIV reservoirs and utilize a novel mechanism to prevent HIV-induced apoptosis. Furthermore, they also suggest that Bim recruitment to mitochondria could be used as a biomarker of viral reservoirs in vivo.

Zika virus (ZIKV) is an emerging virus causally linked to neurological disorders, including congenital microcephaly and Guillain-Barré syndrome. There are currently no targeted therapies for ZIKV infection. To identify novel antiviral targets and to elucidate the mechanisms by which ZIKV exploits the host cell machinery to support sustained replication, we analyzed the transcriptomic landscape of human microglia, fibroblast, embryonic kidney and monocyte-derived macrophage cell lines before and after ZIKV infection. The four cell types differed in their susceptibility to ZIKV infection, consistent with differences in their expression of viral response genes before infection. Clustering and network analyses of genes differentially expressed after ZIKV infection revealed changes related to the adaptive immune system, angiogenesis and host metabolic processes that are conducive to sustained viral production. Genes related to the adaptive immune response were downregulated in microglia cells, suggesting that ZIKV effectively evades the immune response after reaching the central nervous system. Like other viruses, ZIKV diverts host cell resources and reprograms the metabolic machinery to support RNA metabolism, ATP production and glycolysis. Consistent with these transcriptomic analyses, nucleoside metabolic inhibitors abrogated ZIKV replication in microglia cells.

Background: HIV-1-infected and/or immune-activated microglia and macrophages are pivotal in the pathogenesis of HIV-1-associated neurocognitive disorders (HAND). Glutaminase, a metabolic enzyme that facilitates glutamate generation, is upregulated and may play a pathogenic role in HAND. Our previous studies have demonstrated that glutaminase is released to the extracellular fluid during HIV-1 infection and neuroinflammation. However, key molecular mechanisms that regulate glutaminase release remain unknown. Recent advances in understanding intercellular trafficking have identified microvesicles (MVs) as a novel means of shedding cellular contents. We posit that during HIV-1 infection and immune activation, microvesicles may mediate glutaminase release, generating excessive and neurotoxic levels of glutamate., Results: MVs isolated through differential centrifugation from cell-free supernatants of monocyte-derived macrophages (MDM) and BV2 microglia cell lines were first confirmed in electron microscopy and immunoblotting. As expected, we found elevated number of MVs, glutaminase immunoreactivities, as well as glutaminase enzyme activity in the supernatants of HIV-1 infected MDM and lipopolysaccharide (LPS)-activated microglia when compared with controls. The elevated glutaminase was blocked by GW4869, a neutral sphingomyelinase inhibitor known to inhibit MVs release, suggesting a critical role of MVs in mediating glutaminase release. More importantly, MVs from HIV-1-infected MDM and LPS-activated microglia induced significant neuronal injury in rat cortical neuron cultures. The MV neurotoxicity was blocked by a glutaminase inhibitor or GW4869, suggesting that the neurotoxic potential of HIV-1-infected MDM and LPS-activated microglia is dependent on the glutaminase-containing MVs., Conclusions: These findings support MVs as a potential pathway/mechanism of excessive glutamate generation and neurotoxicity in HAND and therefore MVs may serve as a novel therapeutic target.

Microglia and macrophages play a central role for demyelination in Theiler's murine encephalomyelitis (TME) virus infection, a commonly used infectious model for chronic-progressive multiple sclerosis. In order to determine the dynamic changes of microglia/macrophage polarization in TME, the spinal cord of Swiss Jim Lambert (SJL) mice was investigated by gene expression profiling and immunofluorescence. Virus persistence and demyelinating leukomyelitis were confirmed by immunohistochemistry and histology. Electron microscopy revealed continuous myelin loss together with abortive myelin repair during the late chronic infection phase indicative of incomplete remyelination. A total of 59 genes out of 151 M1- and M2-related genes were differentially expressed in TME virus-infected mice over the study period. The onset of virus-induced demyelination was associated with a dominating M1 polarization, while mounting M2 polarization of macrophages/microglia together with sustained prominent M1-related gene expression was present during the chronic-progressive phase. Molecular results were confirmed by immunofluorescence, showing an increased spinal cord accumulation of CD16/32(+) M1-, arginase-1(+) M2- and Ym1(+) M2-type cells associated with progressive demyelination. The present study provides a comprehensive database of M1-/M2-related gene expression involved in the initiation and progression of demyelination supporting the hypothesis that perpetuating interaction between virus and macrophages/microglia induces a vicious circle with persistent inflammation and impaired myelin repair in TME., (© 2014 International Society of Neuropathology.)

HIV-1 replication in the central nervous system (CNS) is typically limited by the availability of target cells. HIV-1 variants that are transmitted and dominate the early stages of infection almost exclusively use the CCR5 coreceptor and are well adapted to entering, and thus infecting, cells expressing high CD4 densities similar to those found on CD4+ T cells. While the "immune privileged" CNS is largely devoid of CD4+ T cells, macrophage and microglia are abundant throughout the CNS. These cells likely express CD4 densities that are too low to facilitate efficient entry or allow sustained replication by most HIV-1 isolates. Examination of CNS viral populations reveals that late in disease the CNS of some individuals contains HIV-1 lineages that have evolved the ability to enter cells expressing low levels of CD4 and are well-adapted to entering macrophages. These macrophage-tropic (M-tropic) viruses are able to maintain sustained replication in the CNS for many generations, and their presence is associated with severe neurocognitive impairment. Whether conditions such as pleocytosis are necessary for macrophage-tropic viruses to emerge in the CNS is unknown, and extensive examinations of macrophage-tropic variants have not revealed a genetic signature of this phenotype. It is clear, however, that macrophage tropism is rare among HIV-1 isolates and is not transmitted, but is important due to its pathogenic effects on hosts. Prior to the evolution of macrophage-tropic variants, the viruses that are predominately infecting T cells (R5 T cell-tropic) may infect macrophages at a low level and inefficiently, but this could contribute to the reservoir.

Background: Integrase inhibitors are a promising class of antiretroviral drugs to treat chronic human immunodeficiency virus (HIV) infection. During HIV infection, macrophages can extravasate from the blood to the brain, while producing chemotaxic proteins and cytokines, which have detrimental effects on central nervous system cells. The main goal of this study was to understand the effects of raltegravir (RAL) on human brain macrophage production of immune-mediators when infected with HIV, but did not compare with other antiretroviral agents., Methods: Pro-inflammatory cytokines, IFN-γ, IL-10, IL-12-p70, IL-1, IL-8, TNF-α, and IL-6 were measured simultaneously in tissue culture supernatants from primary brain derived macrophages, microglia. We tested the effects of RAL on markers of astrocytosis and neurite integrity in primary human neuroglial cultures., Results: RAL administered at 20 nM effectively suppressed HIV infection in microglia over 9 days. Only IL-8, IL-10, and TNF-α were above the detection limit in the majority of samples and RAL significantly suppressed the rate of cytokine production in HIV-infected microglia. During RAL-alone, the rate of IL-8 secretion was higher., Conclusions: RAL did not affect neurite area but inhibited astrocyte growth in the neuroglial cultures. Exploring the effects of RAL on pro-inflammatory molecule production in brain macrophages may contribute to designing ARV neuroprotective strategies in chronic HIV infection.

Background: Chemokine CXCL8 is an important neutrophil chemoattractant implicated in various neurodegenerative disorders. Cytokine/chemokine imbalance, with an increase in proinflammatory cytokines like interleukin-1β and tumor necrosis factor-α within the central nervous system, is a hallmark of human immunodeficiency virus (HIV)-1 infection. We previously reported that HIV-1 infection is linked to upregulation of CXCL8 in brain tissues and human astrocytes. Chemokines play crucial roles in trafficking of leukocytes and trafficking of HIV-1-infected across the blood-brain barrier play an important role in HIV-1 central nervous system disease. In the post-antiretroviral therapy era, low level of productive replication of HIV-1 in brain is a critical component of neuropathogenesis regulation. The present study investigated the effect of CXCL8 on productive infection of HIV-1 in human monocytes-derived macrophages (MDM) and primary human microglia., Results: Human MDM and microglia were infected with the blood or brain derived HIV-1 isolates, HIV-1ADA or HIV-1JRFL. Treatment with CXCL8 significantly upregulated HIV-1p24 levels in supernatants of both HIV-1-infected MDM as well as microglia. In addition, the formation of 2-long terminal repeat (LTR) circles, a measure of viral genome integration, was significantly higher in CXCL8-treated, HIV-1-infected MDM and microglia. Transient transfection of U937 cells with HIV-1 LTR luciferase reporter construct resulted in increased promoter activity when treated with CXCL8. Moreover, increased nuclear translocation of nuclear factor-κB was seen in HIV-1-infected MDM following CXCL8 treatment. Blocking CXCL8 receptors CXCR1 and CXCR2 abrogated the CXCL8-mediated enhanced HIV-1 replication., Conclusion: Our results show that CXCL8 mediates productive infection of HIV-1 in MDM and microglia via receptors CXCR1 and CXCR2. These results demonstrate that CXCL8 exerts its downstream effects by increasing translocation of nuclear factor-κB into the nucleus, thereby promoting HIV-1 LTR activity.

HIV infection in the brain and the resultant encephalitis affect approximately one third of individuals infected with HIV, regardless of treatment with antiretroviral drugs. Microglia are the resident phagocytic cell type in the brain, serving as a "first responder" to neuroinvasion by pathogens. The early events of the microglial response to productively infected monocyte/macrophages entering the brain can best be investigated using in vitro techniques. We hypothesized that activation of microglia would be specific to the presence of simian immunodeficiency virus (SIV)-infected macrophages as opposed to responses to macrophages in general. Purified microglia were grown and stimulated with control or SIV-infected macrophages. After 6 h, aliquots of the supernatant were analyzed for 23 cytokines using Millipore nonhuman primate-specific kit. In parallel experiments, morphologic changes and cytokine expression by individual microglia were examined by immunofluorescence. Surprisingly, the presence of macrophages was more important to the microglial response rather than whether the macrophages were infected with SIV. None of the cytokines examined were unique to co-incubation with SIV-infected macrophages compared with control macrophages, or their supernatants. Media from SIV-infected macrophages, however, did induce secretion of higher levels of IL-6 and IL-8 than the other treatments. As resident macrophages in the brain, microglia would be expected to have a strong response to infiltrate innate immune cells such as monocyte/macrophages. This response is triggered by incubation with macrophages, irrespective of whether or not they are infected with SIV, indicating a rapid, generalized immune response when infiltrating macrophages entering the brain.

Despite the availability of effective vaccines, Japanese encephalitis virus (JEV) infections remain a leading cause of encephalitis in many Asian countries. The virus is transmitted to humans by Culex mosquitoes, and, while the majority of human infections are asymptomatic, up to 30% of JE cases admitted to hospital die and 50% of the survivors suffer from neurological sequelae. Microglia are brain-resident macrophages that play key roles in both the innate and adaptive immune responses in the CNS and are thus of importance in determining the pathology of encephalitis as a result of JEV infection.

It is estimated that half of HIV-infected adults and children will present at one time during their disease course with a neurologic disorder. The neurologic sequelae of HIV infection arise as a direct result of viral replication as well as from the subsequent neuroinflammatory processes. HIV enters the CNS early in infection and resides primarily in long-lived perivascular macrophages and microglia. CNS immunosurveillance is an integral part of normal brain function. Circulating lymphocytes play a vital role in support of brain plasticity under normal and traumatic circumstances. Malfunctions of this immunologic niche can impair brain homeostasis, resulting in neural impairment. Combination therapies that lower CNS viral load and improve immune homeostasis and neuroprotection will be required to address the neuropathogenesis of HIV infection.

Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS) can result in neurological dysfunction with devastating consequences in a significant proportion of individuals with acquired immune deficiency syndrome. HIV-1 does not infect neurons directly but induces damage indirectly through the accumulation of activated macrophage/microglia (M/M) cells, some of which are infected, that release neurotoxic mediators including both cellular activation products and viral proteins. One mechanism for the accumulation of activated M/M involves the development in infected individuals of an activated peripheral blood monocyte population that traffics through the blood-brain barrier, a process that also serves to carry virus into CNS and establish local infection. A second mechanism involves the release by infected and activated M/M in the CNS of chemotactic mediators that recruit additional monocytes from the periphery. These activated M/M, some of which are infected, release a number of cytokines and small molecule mediators as well as viral proteins that act on bystander cells and in turn activate them, thus amplifying the cascade. These viral proteins and cellular products have neurotoxic properties as well, both directly and through induction of astrocyte dysfunction, which ultimately lead to neuronal injury and death. In patients effectively treated with antiretroviral therapy, frank dementia is now uncommon and has been replaced by milder forms of neurocognitive impairment, with less frequent and more focal neuropathology. This review summarizes key findings that support the critical role and mechanisms of monocyte/macrophage activation and inflammation as a major component for HIV-1 encephalitis or HIV-1 associated dementia.

CCR3 has been implicated as a co-receptor for human immunodeficiency virus type 1 (HIV-1), particularly in brain microglia cells. We sought to clarify the comparative roles of CCR3 and CCR5 in the central nervous system (CNS) HIV-1 infection and the potential utility of CCR3 as a target for manipulation via gene transfer. To target CCR3, we developed a single-chain antibody (SFv) and an interfering RNA (RNAi), R3-526. Coding sequences for both were cloned into Tag-deleted SV40-dervied vectors, as these vectors transduce brain microglia and monocyte-derived macrophages (MDM) highly efficiently. These anti-CCR3 transgenes were compared to SFv-CCR5, an SFv against CCR5, and RNAi-R5, an RNAi that targets CCR5, for the ability to protect primary human brain microglia and MDM from infection with peripheral and neurotropic strains of HIV-1. Downregulation of CCR3 and CCR5 by these transgenes was independent from one another. Confocal microscopy showed that CCR3 and CCR5 co-localized at the plasma membrane with each other and with CD4. Targeting either CCR5 or CCR3 largely protected both microglia and MDM from infection by many strains of HIV-1. That is, some HIV-1 strains, isolated from either the CNS or periphery, required both CCR3 and CCR5 for optimal productive infection of microglia and MDM. Some HIV-1 strains were relatively purely CCR5-tropic. None was purely CCR3-tropic. Thus, some CNS-tropic strains of HIV-1 utilize CCR5 as a co-receptor but do not need CCR3, while for other isolates both CCR3 and CCR5 may be required.

Neurokinin-1 receptor (NK-1R) antagonists suppress HIV-1 infection of macrophages in vitro. We have further investigated the anti-HIV-1 activity of aprepitant, a Food and Drug Administration-approved NK-1R antagonist, and its cytotoxic effect in the macrophage/microglia system. Aprepitant inhibited infection of macrophages with primary HIV-1 R5 strains (subtypes A, D, and H; UG275, BZ163, and BCF-KITA), while it had little effect on primary HIV-1 X4 strains (subtypes B and D, BZ167 and SE365). Aprepitant, when added to microglia cultures infected with CSF-derived HIV-1 strains (JAGO or JRFL), significantly inhibited viral replication. Aprepitant also enhanced the anti-HIV-1 activity of enfuvirtide (an HIV-1 fusion inhibitor) in HIV-1-infected macrophages. Over a concentration range of 10(-9) to 10(-5) M, aprepitant had little cytotoxic effect (less than 10%) on macrophages during the in vitro cultures. Autologous human serum (< or =20%) had little effect on the anti-HIV-1 activity of aprepitant in macrophages. These observations provide additional evidence to support the potential use of NK-1R antagonists as therapeutic and immunomodulatory agents for the treatment of HIV-1 infection.

The coronavirus mouse hepatitis virus (MHV) induces a minimal type I interferon (IFN) response in several cell types in vitro despite the fact that the type I IFN response is important in protecting the mouse from infection in vivo. When infected with MHV, mice deficient in IFN-associated receptor expression (IFNAR(-/-)) became moribund by 48 h postinfection. MHV also replicated to higher titers and exhibited a more broad tissue tropism in these mice, which lack a type I IFN response. Interestingly, MHV induced IFN-beta in the brains and livers, two main targets of MHV replication, of infected wild-type mice. MHV infection of primary cell cultures indicates that hepatocytes are not responsible for the IFN-beta production in the liver during MHV infection. Furthermore, macrophages and microglia, but not neurons or astrocytes, are responsible for IFN-beta production in the brain. To determine the pathway by which MHV is recognized in macrophages, IFN-beta mRNA expression was quantified following MHV infection of a panel of primary bone marrow-derived macrophages generated from mice lacking different pattern recognition receptors (PRRs). Interestingly, MDA5, a PRR thought to recognize primarily picornaviruses, was required for recognition of MHV. Thus, MHV induces type I IFN in macrophages and microglia in the brains of infected animals and is recognized by an MDA5-dependent pathway in macrophages. These findings suggest that secretion of IFN-beta by macrophages and microglia plays a role in protecting the host from MHV infection of the central nervous system.

Macrophages and microglia are critical in the acute inflammatory response and act as final effector cells of demyelination during chronic infection with the neutrotropic MHV-JHM strain of mouse hepatitis virus (MHV-JHM). Herein, we show that "immature" F4/80(+)Ly-6C(hi) monocytes are the first cells, along with neutrophils, to enter the MHV-JHM-infected central nervous system (CNS). As the infection progresses, macrophages in the CNS down-regulate expression of Ly-6C and CD62L, consistent with maturation, and a higher frequency express CD11c, a marker for dendritic cells (DCs). Microglia also express CD11c during this phase of the infection. CD11c(+) macrophages in the infected CNS exhibit variable properties of immature antigen-presenting cells (APCs), with modestly increased CD40 and MHC expression, and equivalent potent antigen uptake when compared with CD11c(-) macrophages. Furthermore, CDllc(+) and F4/80(+) macrophages and microglia are localized to areas of demyelination, in some instances directly associated with damaged axons. These results suggest that chronic CNS infection results in the appearance of CD11c-expressing macrophages from the blood that exhibit properties of immature APCs, are closely associated with areas of demyelination, and may act as final effectors of myelin destruction.

It is well known that infection by the human immunodeficiency virus (HIV) dysregulates cell physiology, but little information is available on the consequences of HIV infection in primary macrophages and microglia. The authors examined the relationship between cell proliferation and HIV infection in primary cultures of microglia and in human central nervous system (CNS). In cultures infected with HIV (ADA and BaL), granulocyte-macrophage colony-stimulating factor (GM-CSF)-mediated cell proliferation was reduced in productively infected (p24+) cells as compared to p24- cells. The reduction was observed with both Ki67 and BrdU labeling, suggesting a G1/S block. The reduction was insignificant when microglia were infected with a Vpr- mutant virus. In human CNS, proliferating (Ki67+) cells were rare but were increased in the HIV+ and HIV encephalitis (HIVE) groups compared to the HIV- group. A positive correlation between GM-CSF immunoreactivity and Ki67 counts, implicating GM-CSF as a growth factor in human CNS was found. The relationship between total macrophage (CD68+) proliferation and infected macrophage (p24+) proliferation was assessed in HIVE by double labeling. Whereas 1.2% of total CD68+ cells were Ki67+, only 0.5% of HIV p24+ cells were Ki67+ (P < .05). Furthermore, staining for CD45RB (as opposed to CD68) facilitated the identification of Ki67+ microglia, indicating that CD68 could underestimate proliferating microglia. The authors conclude that although there is increased expression of GM-CSF and increased cell proliferation in the CNS of HIV-seropositive individuals, cell proliferation in the productively infected population is actually suppressed. These data suggest that there might be a viral gain in the suppressed host cell proliferation.

HIV infects tissue macrophages and brain microglia, which express lower levels of CD4 and CCR5 than CD4+ T cells in peripheral blood. Mechanisms that enhance HIV tropism for macrophages in the CNS and other tissues are not well understood. Here, we identify an HIV envelope glycoprotein (Env) variant in the CD4-binding site of gp120, Asn 283 (N283), that is present at a high frequency in brain tissues from AIDS patients with HIV-associated dementia (HAD). N283 increases gp120 affinity for CD4 by decreasing the gp120-CD4 dissociation rate, enhancing the capacity of HIV Envs to use low levels of CD4 for virus entry and increasing viral replication in macrophages and microglia. Structural modeling suggests that the enhanced ability of Envs with N283 to use low levels of CD4 is due to a hydrogen bond formed with Gln 40 of CD4. N283 is significantly more frequent in brain-derived Envs from HAD patients (41%; n=330) compared with non-HAD patients (8%; n=151; P<0.001). These findings suggest that the macrophage-tropic HIV Env variant N283 is associated with brain infection and dementia in vivo, representing an example of a HIV variant associated with a specific AIDS-related complication.

Microglial cells are central to brain immunity and intervene in many human neurological diseases. The aim of this study was to develop a convenient cellular model for human microglial cells, suitable for HIV studies. Microglia derive from the hematogenous myelomonocytic lineage, possibly as a distinct subpopulation but in any case able to invade the CNS, proliferate, and differentiate into ameboid and then ramified microglia in the adult life. We thus attempted to derive microglia-like cells from human monocytes. When cultured with astrocyte-conditioned medium (ACM), monocytes acquired a ramified morphology, typical of microglia. They overexpressed substance P and the calcium binding protein Iba-1 and dimly expressed class II MHC, three characteristics of microglial cells. Moreover, they also expressed a potassium inward rectifier current, another microglia-specific feature. These monocyte-derived microglia-like cells (MDMi) were CD4(+)/CD14(+), evocative of an activated microglia phenotype. When treated with lipopolysaccharide (LPS), MDMi lost their overexpression of substance P, which returned to untreated monocyte-derived macrophage (MDM) level. Compared with MDM, MDMi expressed higher CD4 but lower CCR5 levels; they could be infected by HIV-1(BaL), but produced less virus progeny than MDM did. This model of human microglia may be an interesting alternative to primary microglia for large scale in vitro HIV studies and may help to better understand HIV-associated microgliosis and chronic inflammation in the brain.

Cognitive, behavioral, and motor impairments, during progressive human immunodeficiency virus type 1 (HIV-1) infection, are linked to activation of brain mononuclear phagocytes (MP; perivascular macrophages and microglia). Activated MPs effect a giant cell encephalitis and neuroinflammatory responses that are mirrored in severe combined immunodeficient (SCID) mice injected with human monocyte-derived macrophages (MDM). Whether activated human MDMs positioned in the basal ganglia affect hippocampal neuronal plasticity, the brain subregion involved in learning and memory, is unknown. Thus, immunohistochemical techniques were used for detection of newborn neurons (polysialylated neuronal cell adhesion molecule [PSA-NCAM]) and cell proliferation (Ki-67) to assay MDM effects on neuronal development in mouse models of HIV-1 encephalitis. Immunodeficient (C.B.-17/SCID and nonobese diabetic/SCID, NOD/SCID) and immune competent (C.B.-17) mice were injected with uninfected or HIV-1-infected MDM. Sham-operated or unmanipulated mice served as controls. Neuronal plasticity was evaluated in the hippocampal dentate gyrus (DG) at days 7 and 28. By day 7, increased numbers of Ki-67+ cells, PSA-NCAM+ cells and dendrites in DG were observed in sham-operated animals. In contrast, significant reductions in neuronal precursors and altered neuronal morphology paralleled increased microglial activation in both HIV-1-infected and uninfected MDM-injected animals. DG cellular composition was restored at day 28. We posit that activated MDM induce inflammation and diminish DG neuronal plasticity. These data provide novel explanations for the cognitive impairments manifested during advanced HIV-1 infection., (Copyright 2005 Wiley-Liss, Inc.)

Complement activation in the brain contributes to the pathology of neuroinflammatory and neurodegenerative diseases such as neuro-AIDS. Using semiquantitative in situ hybridization and immunohistochemistry, we observed an early and sustained increase in the expression of C1q, the initial recognition subcomponent of the classical complement cascade, in the CNS during simian immunodeficiency virus (SIV) infection of rhesus macaques. Cells of the microglial/macrophage lineage were the sources for C1q protein and transcripts. C1q expression was observed in proliferating and infiltrating cells in SIV-encephalitic brains. All SIV-positive cells were also C1q-positive. Treatment with the CNS-permeant antiretroviral agent 6-chloro-2',3'-dideoxyguanosine decreased C1q synthesis along with SIV burden and focal inflammatory reactions in the brains of AIDS-symptomatic monkeys. Thus, activation of the classical complement arm of innate immunity is an early event in neuro-AIDS and a possible target for intervention.

Macrophages and microglia are the predominant cells infected with HIV-1 in the brain, yet the effects of productive HIV infection on the fate of these cells are poorly understood. In this study, we tested the hypothesis that HIV-1 expression influences cell death in infected macrophages and microglial cells. We detected apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) in the cerebral white matter of control and HIV encephalitis (HIVE) brains, and quantitatively analysed apoptotic cells with respect to their location (vessel-associated vs. parenchymal), CD68 expression, and HIV-1 p24 expression. There were more vessel-associated, but not more parenchymal, TUNEL+ cells in HIVE cases as compared to controls. Vessel-associated TUNEL+ cells were primarily endothelial cells (von Willebrand factor+) or macrophages (CD68+). TUNEL+/CD68+ cells were present in both control and HIVE cases in similar frequencies (2.1 +/- 0.7% vs. 1.9 +/- 0.7% of total CD68+ populations, respectively). In HIVE, TUNEL+/p24+ cells were 0.4 +/- 0.2% of the total p24+ cell population, which was lower than the frequency of TUNEL+/CD68+ cells (1.9 +/- 0.7%) in the total CD68+ macrophage population. These results suggest that HIV-1-infected macrophages and microglia are resistant to apoptosis, and may contribute to the formation of a central nervous system viral reservoir.

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.

The presence of specific neuroinvasive strains and necessity for brain viral replication for disease progression remain controversial issues in neuro-AIDS research. To investigate these questions, the authors injected human monocyte-derived macrophages (MDMs) infected with diverse viral strains were injected into the caudate and putamen of severe combined immunodeficient (SCID) mice. Independent of viral strain, infected MDMs became immunologically activated and elicited profound inflammatory reactions in brain areas most affected in humans. The intensity of neuropathologic changes, including microglial reactions, paralleled levels of viral infection and numbers of infected MDMs. The data suggest that HIV-1-associated neurological disease is related to the level of productive viral infection in activated macrophages. Virus infection, per se, may affect the ability of macrophages to respond to immune stimuli by overproduction of proinflammatory factors and neurotoxins, leading to neuronal dysfunction.

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.

Objectives: HIV-2 can use a broader range of co-receptors than HIV-1 in vitro, and is less dependent on CD4 for infection. The aim of this study was to detect productive HIV-2 infection in the brain and investigate whether HIV-2 has an expanded tropism for brain cells in vivo, in comparison with HIV-1, which productively infects macrophages/microglia., Design: Brain samples taken at autopsy from eight patients who died from AIDS, six HIV-2 and two HIV-1/HIV-2 dually seropositive, with HIV encephalitis (HIVE), collected in Abidjan, Côte d'Ivoire in 1991, were examined for the presence and localization of productive HIV-2 infection., Methods: Using immunohistochemistry, the presence of HIV-2 p26 in formalin-fixed, wax-embedded brain tissue sections was investigated. Double-staining with glial fibrillary acidic protein (GFAP), CD45- and CD68-specific antibodies was performed to identify infected cell types., Results: HIV-2 p26 was detected in brain tissue from four of the HIV-2 cases and one of the dually infected individuals. The productively infected cells were either microglia or infiltrating macrophages., Conclusions: The productively infected cells in the brains of HIV-2 infected individuals are macrophages/microglia. No evidence was found for productive infection of astrocytes, neurons or oligodendrocytes. Thus, the broader in vitro cell tropism, promiscuous coreceptor usage and relative independence of CD4 by HIV-2 compared to HIV-1 does not broaden its range of target cells in the brain.

The etiology of the central nervous system (CNS) alterations after human immunodeficiency virus (HIV) infection, such as dementia and encephalitis, remains unknown. We have used microarray analysis in a monkey model of neuroAIDS to identify 98 genes, many previously unrecognized in lentiviral CNS pathogenesis, whose expression is significantly up-regulated in the frontal lobe of simian immunodeficiency virus-infected brains. Further, through immunohistochemical illumination, distinct classes of genes were found whose protein products localized to infiltrating macrophages, endothelial cells and resident glia, such as CD163, Glut5, and ISG15. In addition we found proteins induced in cortical neurons (ie, cyclin D3, tissue transglutaminase, alpha1-antichymotrypsin, and STAT1), which have not previously been described as participating in simian immunodeficiency virus or HIV-related CNS pathology. This molecular phenotyping in the infected brains revealed pathways promoting entry of macrophages into the brain and their subsequent detrimental effects on neurons. These data support the hypothesis that in HIV-induced CNS disease products of activated macrophages and astrocytes lead to CNS dysfunction by directly damaging neurons, as well as by induction of altered gene and protein expression profiles in neurons themselves which are deleterious to their function.

Transducing macrophages and other phagocytic cells has been problematic because these cells are largely nondividing and can phagocytose and degrade viral gene delivery vectors. Because of their carriage of the CCR5 chemokine receptor that functions as a coreceptor for most clinical strains of HIV, these cells are also key targets in early HIV infection and dissemination. We describe here a strategy to transduce these phagocytes, reduce cell membrane CCR5, and protect from infection with R5-tropic HIV. Recombinant Tag-deleted SV40 vectors were used to transduce unselected CCR5-bearing cell lines and primary cells with >98% efficiency. rSV40s were designed to express two different anti-CCR5 small interfering RNAs (siRNAs), driven by the adenoviral VA1 polymerase III (pol III) promoter, which localizes the transcripts in the cytoplasm. Transduction with both siRNAs substantially reduced CCR5 mRNA, which in turn decreased detectable cell membrane CCR5. Both CCR5+ cell lines and primary cells were used: SupT1/CCR5 cells, monocyte-derived macrophages (MDM), and primary human brain microglia. In addition, one siRNA, siRNA R5 #5, was designed to recognize conserved sequences in both murine and human CCR5 mRNA and effectively reduced CCR5 transcript in cells of both species. These siRNAs largely protected CCR5+ cell lines and primary human macrophages and brain microglia from challenge with R5-tropic HIV. Therefore, strategies to target CCR5 using rSV40-delivered, VA promoter-driven siRNAs may be useful therapeutic options for treating HIV infection.

Within the brain, glial cells are target cells for human cytomegalovirus (HCMV) and HIV. We infected cultures of unstimulated human microglial cells and astrocytes of embryonic origin and of monocyte-derived macrophages (MDM) with HCMV strain AD169 and observed down-regulation of the plasma membrane expression of CCR5 in the three cell types, and of CXCR4 and CD4 in microglial cells only. Cells were then coinfected simultaneously or at a 24-h interval with both AD169 and two different HIV-1 monocytotropic strains. HCMV late antigens and HIV-1 tat protein colocalized in the cytoplasm of 5-10% of microglia and MDM. p24 antigen levels decreased 10- to 40-fold in supernatants of MDM and the reduction was greater when HCMV infection was performed 24 h before HIV-1 infection. These data suggest that HCMV-induced reduction in the cell-surface expression of the primary co-receptor of HIV-1 monocytotropic strains may impair the ability of HIV to infect these cells.

DA strain of Theiler's murine encephalomyelitis virus (TMEV) persists in the mouse central nervous system (CNS) and induces demyelination while GDVII strain fails to persist or demyelinate. L* protein, which is synthesized only in DA but not GDVII, is believed important in virus persistence and demyelination. Because a major reservoir for DA persistence is infiltrated macrophages or microglia, a resident macrophage of the CNS, we investigated TMEV infection of Ra2 cells, a murine microglial cell line. We found that DA strain grew well in Ra2 cells, but not GDVII strain or DAL*-1 virus (which fails to synthesize L* protein), suggesting that L* protein plays an important role in virus growth in microglia. Interestingly, in contrast to virus growth, most Ra2 cells infected with DA strain survived with no evidence of virus-induced apoptosis. These results may be important in clarifying the pathogenesis of DA-induced demyelinating disease.

This review focuses on the role of the extended macrophage/monocyte family in the central nervous system during HIV or SIV infection. The accumulated data, buttressed by recent experimental results, suggest that these cells play a central, pathogenic role in retroviral-associated CNS disease. While the immune system is able to combat the underlying retroviral infection, the accumulation and widespread activation of macrophages, microglia, and perivascular cells in the CNS are held in check. However, with the collapse of the immune system and the disappearance of the CD4(+) T cell population, productive infection reemerges, especially in CNS macrophages. These cells, as well as noninfected macrophages, are stimulated to high levels of activation. When members of this cell group become highly activated, they elaborate a wide spectrum of deleterious substances into the neural parenchyma. In the final phases of HIV or SIV infection, this chronic, widespread, and dramatic level of macrophage/monocyte/microglial activation constitutes a self-sustaining state of macrophage dysregulation, which results in pathological alterations and the emergence of various neurological problems.

Opiate abuse has been postulated to be a cofactor in the immunopathogenesis of acquired immunodeficiency syndrome (AIDS). This study evaluated whether methadone, a drug widely prescribed for the treatment of drug abusers with opioid dependence, affects human immunodeficiency virus (HIV) infection of human immune cells. When added to human fetal microglia and blood monocyte-derived macrophage cultures, methadone significantly enhanced HIV infection of these cells. This enhancement was associated with the up-regulation of expression of CCR5, a primary coreceptor for macrophage-tropic HIV entry into macrophages. Most importantly, the addition of methadone to the cultures of latently infected peripheral blood mononuclear cells from HIV-infected patients enhanced viral activation and replication. Although the in vivo relevance of these findings remains to be determined, the data underscore the necessity of further studies to define the role of opioids, including methadone, in the immunopathogenesis of HIV infection and AIDS.

Infection of microglia and other cells of the macrophage/monocyte lineage in the central nervous system (CNS) by human immunodeficiency virus type I (HIV-1) underlies the development of giant cell encephalitis (GCE). It is currently unknown whether GCE depends on the emergence of virus populations specifically adapted to replicate in cells of the monocyte/macrophage lineage and whether this also leads to the specific targeting of macrophages in other nonlymphoid tissues. Autopsy samples from lymph node, brain (frontal region), lung, and full-thickness colon sections were obtained from nine study subjects with GCE and from nine without. The two groups showed no significant differences in CD4 counts, disease progression, or treatment history before death. Genetic relatedness between variants recovered from lymph node and nonlymphoid tissues was assessed by sequence comparison of V3 and p17(gag) regions using a newly developed method that scores the sample composition at successive nodes in a neighbor-joining tree. The association index enabled objective, numerical comparisons on the degree of tissue compartmentalization to be made. High proviral loads and p24 antigen expression in the brain were confined to the nine individuals with GCE. GCE was also associated with significantly higher proviral loads in colon samples (median of the GCE(+) group: 1,010 copies/10(6) cells; median of GCE(-) group, 10/10(6) cells; P = 0.006). In contrast, there were no significant differences in proviral load between the GCE(+) and GCE(-) groups in lymph node or lung samples, where HIV infection was manifested predominantly by infiltrates of lymphoid cells. V3 sequences from brain samples of individuals with GCE showed the greatest compartmentalization from those of lymph node, although samples from other tissues, particularly the colon, frequently contained variants phylogenetically related to those found in brain. The existence of shared, distinct populations of HIV specifically distributed in cells of the monocyte/macrophage lineage was further indicated by immunocytochemical detection of CD68(+), multinucleated giant cells expressing p24 antigen in samples of lung and colon in two individuals with GCE. This study provides the basis for future investigation of possible phenotypic similarities that underline the shared distributions of HIV variants infecting microglia and tissue macrophages outside the CNS.

The viral determinants that underlie human immunodeficiency virus type 1 (HIV-1) neurotropism are unknown, due in part to limited studies on viruses isolated from brain. Previous studies suggest that brain-derived viruses are macrophage tropic (M-tropic) and principally use CCR5 for virus entry. To better understand HIV-1 neurotropism, we isolated primary viruses from autopsy brain, cerebral spinal fluid, blood, spleen, and lymph node samples from AIDS patients with dementia and HIV-1 encephalitis. Isolates were characterized to determine coreceptor usage and replication capacity in peripheral blood mononuclear cells (PBMC), monocyte-derived macrophages (MDM), and microglia. Env V1/V2 and V3 heteroduplex tracking assay and sequence analyses were performed to characterize distinct variants in viral quasispecies. Viruses isolated from brain, which consisted of variants that were distinct from those in lymphoid tissues, used CCR5 (R5), CXCR4 (X4), or both coreceptors (R5X4). Minor usage of CCR2b, CCR3, CCR8, and Apj was also observed. Primary brain and lymphoid isolates that replicated to high levels in MDM showed a similar capacity to replicate in microglia. Six of 11 R5 isolates that replicated efficiently in PBMC could not replicate in MDM or microglia due to a block in virus entry. CD4 overexpression in microglia transduced with retroviral vectors had no effect on the restricted replication of these virus strains. Furthermore, infection of transfected cells expressing different amounts of CD4 or CCR5 with M-tropic and non-M-tropic R5 isolates revealed a similar dependence on CD4 and CCR5 levels for entry, suggesting that the entry block was not due to low levels of either receptor. Studies using TAK-779 and AMD3100 showed that two highly M-tropic isolates entered microglia primarily via CXCR4. These results suggest that HIV-1 tropism for macrophages and microglia is restricted at the entry level by a mechanism independent of coreceptor specificity. These findings provide evidence that M-tropism rather than CCR5 usage predicts HIV-1 neurotropism.

Progress in developing a small animal model of human immunodeficiency virus type 1 (HIV-1) disease would greatly facilitate studies of transmission, pathogenesis, host immune responses, and antiviral strategies. In this study, we have explored the potential of rats as a susceptible host. In a single replication cycle, rat cell lines Rat2 and Nb2 produced infectious virus at levels 10- to 60-fold lower than those produced by human cells. Rat-derived cells supported substantial levels of early HIV-1 gene expression, which was further enhanced by overexpression of human cyclin T1. Rat cells displayed quantitative, qualitative, and cell-type-specific limitations in the late phase of the HIV-1 replication cycle including relative expression levels of HIV-1 Gag proteins, intracellular Gag processing, and viral egress. Nb2 cells were rendered permissive to HIV-1 R5 viruses by coexpression of human CD4 and CCR5, indicating that the major restriction on HIV-1 replication was at the level of cellular entry. We also found that primary rat lymphocytes, macrophages, and microglia expressed considerable levels of early HIV-1 gene products following infection with pseudotyped HIV-1. Importantly, primary rat macrophages and microglia, but not lymphocytes, also expressed substantial levels of HIV-1 p24 CA and produced infectious virions. Collectively, these results identify the rat as a promising candidate for a transgenic small animal model of HIV-1 infection and highlight pertinent cell-type-specific restrictions that are features of this species.

The macrophage is well established as a target of HIV and simian immunodeficiency virus (SIV) infection and a major contributor to the neuropathogenesis of AIDS. However, the identification of distinct subpopulations of monocyte/macrophages that carry virus to the brain and that sustain infection within the central nervous system (CNS) has not been examined. We demonstrate that the perivascular macrophage and not the parenchymal microglia is the primary cell productively infected by SIV. We further demonstrate that although productive viral infection of the CNS occurs early, thereafter it is not easily detectable until terminal AIDS. The biology of perivascular macrophages, including their rate of turnover and replacement by peripheral blood monocytes, may explain the timing of neuroinvasion, disappearance, and reappearance of virus in the CNS, and questions the ability of the brain to function as a reservoir for productive infection by HIV/SIV.

Viral and cellular products from HIV-1-infected and/or immune competent mononuclear phagocytes (MP) (brain macrophages and microglia) affect neuronal function during HIV-1-associated dementia (HAD). Neurotoxic MP factors include, but are not limited to, pro-inflammatory cytokines, chemokines, platelet activating factor, arachidonic acid and its metabolites, nitric oxide, progeny virions and viral structural and regulatory proteins. The mechanisms for immune-mediated neural injury in HAD, only now, are being unraveled. In this regard, we reviewed the current knowledge of how postmitotic neurons, which can neither divide nor be replaced, are damaged by MP secretory activities. Linking neuronal function with brain MP activation was made possible by placing viral and/or immune products onto neurons and measuring cell signaling events or through ex vivo electrophysiological tests on MP-treated brain slices. Such linkages are shown, in this report, by select demonstrations of MP factors which cause neuronal dysfunction in HAD.

Microglial cells and astrocytes isolated from human embryonic proencephalon were compared to monocyte-derived macrophages (MDM) for their ability to replicate human cytomegalovirus (HCMV) in vitro. A specific cytopathic effect was observed in microglial cells and astrocytes, but not in MDM. A high percentage of glial cells but a low percentage of MDM expressed immediate-early and late viral antigens. The ability of HCMV-infected microglial cells and astrocytes to release viral particles in their supernatants was significantly higher than that of infected MDM. Human microglial cells and astrocytes at an early stage of development are highly susceptible to HCMV infection.

HIV-1 infection of the central nervous system can cause severe neurologic disease although only microglial cells and brain macrophages are susceptible to productive viral infection. Substances secreted by infected cells are thought to cause disease indirectly. Tumor necrosis factor alpha (TNF-alpha) is one candidate neurotoxin and is upregulated during HIV-1 infection of the brain, likely via activation of the transcription factor NF-kappaB. We used the proteasome inhibitors, MG132 and ALLN (N-acetyl-Leu-Leu-Norleucinal), to inhibit NF-kappaB activation in primary human fetal microglia (PHFM) and primary monocyte derived-macrophages, and showed that they could block TNF-alpha release stimulated by lipopolysaccharide (LPS) or TNF-alpha. In addition, we performed electrophoretic mobility shift analysis and determined that in microglia, the p50/p65 heterodimer of NF-kappaB is activated by LPS stimulation, and is inhibited by MG132. Thus, blockade of NF-kappaB activation in microglia in vitro can decrease production of TNF-alpha and may prove to be a novel strategy for treating HIV-1 dementia.

To investigate the pathogenetic role of human T lymphocyte virus type I (HTLV-I) in central nervous system disease, a rat model for HTLV-I-associated myelopathy/tropical spastic paraparesis, designated as HAM rat disease, has been established. Wistar-King-Aptekman-Hokudai strain rats with induced HTLV-I infection develop a chronic progressive myeloneuropathy with paraparesis of hind limbs after an incubation period of 15 months. In the affected spinal cord in these rats, white matter degeneration, demyelination and vacuolar change with microglia/macrophage infiltration are present as are the provirus DNA and the virus mRNA. To identify infected cells in the affected lesions, we carried out in situ hybridization of amplified fragments of the provirus DNA by polymerase chain reaction on thin sections, plus immunohistochemistry on the same sections. The provirus DNA was localized in some microglia/macrophages in the spinal cord lesion. In addition, the HTLV-I provirus was clearly evident not only in ED-1-negative lymphoid cells but also in ED-1-positive macrophages from lymph nodes. These observations suggest that cells of microglia/macrophage lineage may be one of dominant viral reservoirs in the spinal cords and lymph nodes in HAM rat disease. These infected microglia/macrophages may relate to cause the myeloneuropathy through neurotoxic cytokine synthesis.

HIV-1 is a hematogenously spread virus that most likely gains entry into the brain within blood-derived macrophages. Indeed, productive viral replication selectively occurs within perivascular and parenchymal blood-derived macrophages and microglia and HIV-infected macrophages have increased potential to bind and transmigrate through the blood-brain barrier. Once inside the brain, HIV-infected macrophages secrete a variety of pro-inflammatory mediators that display neuromodulatory and neurotoxic activities in several in vitro models for HIV-1 encephalitis. The final outcome regarding neuronal function and cell loss is regulated through intercellular interactions between these virus-infected cells and astrocytes. In this regard, both HIV-induced intracellular events in macrophages and interactions between HIV-infected macrophages and brain cells are reviewed as factors that might lead to neuronal injury in in vitro model systems for HIV-1 encephalitis.