Human immunodeficiency virus type 1 efficiently binds to human fetal astrocytes and induces neuroinflammatory responses independent of infection

<p>Abstract</p> <p>Background</p> <p>HIV-1 infects human astrocytes <it>in vitro </it>and <it>in vivo </it>but the frequency of infected cells is low and its biological significance is unknown. In studies <it>in vitro</it>, recombinant gp120 alone can induce profound effects on astrocyte biology, suggesting that HIV-1 interaction with astrocytes and its functional consequences extend beyond the limited levels of infection in these cells. Here we determined the relative efficiencies of HIV-1 binding and infection in human fetal astrocytes (HFA), mainly at the single cell level, using HIV-1 tagged with green fluorescence protein (GFP)-Vpr fusion proteins, termed HIV-GFP, to detect virus binding and HIV-1 expressing Rev and NefGFP fusion proteins to detect productive infection.</p> <p>Results</p> <p>Essentially all HFA in a population bound HIV-GFP specifically and independently of CCR5 and CXCR4. The dynamics of this binding at 37°C resembled binding of an HIV fusion mutant to CD4-positive cells, indicating that most of HIV-GFP arrested infection of HFA at the stage of virus-cell fusion. Despite extensive binding, only about 1% of HFA were detectably infected by HIV-RevGFP or HIV-NefGFP, but this proportion increased to the majority of HFA when the viruses were pseudotyped with vesicular stomatitis virus envelope glycoprotein G, confirming that HFA impose a restriction upon HIV-1 entry. Exposure of HFA to HIV-1 through its native proteins rapidly induced synthesis of interleukin-6 and interleukin-8 with increased mRNA detected within 3 h and increased protein detected within 18 h of exposure.</p> <p>Conclusion</p> <p>Our results indicate that HIV-1 binding to human astrocytes, although extensive, is not generally followed by virus entry and replication. Astrocytes respond to HIV-1 binding by rapidly increased cytokine production suggesting a role of this virus-brain cell interaction in HIV-1 neuropathogenesis.</p

HIV induces expression of complement component C3 in astrocytes by NF-κB-dependent activation of interleukin-6 synthesis

Background Abnormal activation of the complement system contributes to some central nervous system diseases but the role of complement in HIV-associated neurocognitive disorder (HAND) is unclear. Methods We used real-time PCR and immunohistochemistry to detect complement expression in postmortem brain tissue from HAND patients and controls. To further investigate the basis for viral induction of gene expression in the brain, we studied the effect of HIV on C3 expression by astrocytes, innate immune effector cells, and targets of HIV. Human fetal astrocytes (HFA) were infected with HIV in culture and cellular pathways and factors involved in signaling to C3 expression were elucidated using pharmacological pathway inhibitors, antisense RNA, promoter mutational analysis, and fluorescence microscopy. Results We found significantly increased expression of complement components including C3 in brain tissues from patients with HAND and C3 was identified by immunocytochemistry in astrocytes and neurons. Exposure of HFA to HIV in culture-induced C3 promoter activity, mRNA expression, and protein production. Use of pharmacological inhibitors indicated that induction of C3 expression by HIV requires NF-κB and protein kinase signaling. The relevance of NF-κB regulation to C3 induction was confirmed through detection of NF-κB translocation into nuclei and inhibition through overexpression of the physiological NF-κB inhibitor, I-κBα. C3 promoter mutation analysis revealed that the NF-κB and SP binding sites are dispensable for the induction by HIV, while the proximal IL-1β/IL-6 responsive element is essential. HIV-treated HFA secreted IL-6, exogenous IL-6 activated the C3 promoter, and anti-IL-6 antibodies blocked HIV activation of the C3 promoter. The activation of IL-6 transcription by HIV was dependent upon an NF-κB element within the IL-6 promoter. Conclusions These results suggest that HIV activates C3 expression in primary astrocytes indirectly, through NF-κB-dependent induction of IL-6, which in turn activates the C3 promoter. HIV induction of C3 and IL-6 in astrocytes may contribute to HIV-mediated inflammation in the brain and cognitive dysfunction

EcoHIV infection of mice establishes latent viral reservoirs in T cells and active viral reservoirs in macrophages that are sufficient for induction of neurocognitive impairment

Suppression of HIV replication by antiretroviral therapy (ART) or host immunity can prevent AIDS but not other HIV-associated conditions including neurocognitive impairment (HIV-NCI). Pathogenesis in HIV-suppressed individuals has been attributed to reservoirs of latent-inducible virus in resting CD4+ T cells. Macrophages are persistently infected with HIV but their role as HIV reservoirs in vivo has not been fully explored. Here we show that infection of conventional mice with chimeric HIV, EcoHIV, reproduces physiological conditions for development of disease in people on ART including immunocompetence, stable suppression of HIV replication, persistence of integrated, replication-competent HIV in T cells and macrophages, and manifestation of learning and memory deficits in behavioral tests, termed here murine HIV-NCI. EcoHIV established latent reservoirs in CD4+ T lymphocytes in chronically-infected mice but could be induced by epigenetic modulators ex vivo and in mice. In contrast, macrophages expressed EcoHIV constitutively in mice for up to 16 months; murine leukemia virus (MLV), the donor of gp80 envelope in EcoHIV, did not infect macrophages. Both EcoHIV and MLV were found in brain tissue of infected mice but only EcoHIV induced NCI. Murine HIV-NCI was prevented by antiretroviral prophylaxis but once established neither persistent EcoHIV infection in mice nor NCI could be reversed by long-acting antiretroviral therapy. EcoHIV-infected, athymic mice were more permissive to virus replication in macrophages than were wild-type mice, suffered cognitive dysfunction, as well as increased numbers of monocytes and macrophages infiltrating the brain. Our results suggest an important role of HIV expressing macrophages in HIV neuropathogenesis in hosts with suppressed HIV replication

A Common Path to Innate Immunity to HIV-1 Induced by Toll-Like Receptor Ligands in Primary Human Macrophages

Toll-like receptors (TLR) represent the best characterized receptor family transducing innate immune responses, the first line of defense against microbial invaders. This study was designed to investigate whether responses through TLR inhibit HIV-1 replication in its primary target cells. Primary human macrophages and lymphocytes from several different donors and HIV-1 infection in tissue culture were used exclusively in this work. We report that ligands of three different TLR: LPS, R848, and double stranded RNA, induce a common antiviral response in macrophages as assayed by measurement of HIV-1 p24 protein, gag DNA, and entry into cells. HIV-1 infection is arrested after efficient entry but prior to reverse transcription. TLR-ligand activated cells secrete antiviral factors that induce a similar restriction. HIV-1 infection of lymphocytes is not affected by exposure to TLR ligands or to antiviral factors secreted by activated macrophages. TBK1, but neither NF-κB nor JAK-STAT activity, is required in macrophages to mount this antiviral response; the combination of p38 MAPK and JNK are partially required for induction of antiviral activity. Based on transcriptional induction and inhibition, the TLR-linked antiviral activity is different from APOBEC3 A or G, interferon-β, NAMPT, or p21Cip1. The cell-type specificity, site of action, and requirement for signaling intermediates suggest that the TLR-linked antiviral activity is novel

Protective Efficacy of Serially Up-Ranked Subdominant CD8+ T Cell Epitopes against Virus Challenges

Immunodominance in T cell responses to complex antigens like viruses is still incompletely understood. Some data indicate that the dominant responses to viruses are not necessarily the most protective, while other data imply that dominant responses are the most important. The issue is of considerable importance to the rational design of vaccines, particularly against variable escaping viruses like human immunodeficiency virus type 1 and hepatitis C virus. Here, we showed that sequential inactivation of dominant epitopes up-ranks the remaining subdominant determinants. Importantly, we demonstrated that subdominant epitopes can induce robust responses and protect against whole viruses if they are allowed at least once in the vaccination regimen to locally or temporally dominate T cell induction. Therefore, refocusing T cell immune responses away from highly variable determinants recognized during natural virus infection towards subdominant, but conserved regions is possible and merits evaluation in humans

Significant Effects of Antiretroviral Therapy on Global Gene Expression in Brain Tissues of Patients with HIV-1-Associated Neurocognitive Disorders

Antiretroviral therapy (ART) has reduced morbidity and mortality in HIV-1 infection; however HIV-1-associated neurocognitive disorders (HAND) persist despite treatment. The reasons for the limited efficacy of ART in the brain are unknown. Here we used functional genomics to determine ART effectiveness in the brain and to identify molecular signatures of HAND under ART. We performed genome-wide microarray analysis using Affymetrix U133 Plus 2.0 Arrays, real-time PCR, and immunohistochemistry in brain tissues from seven treated and eight untreated HAND patients and six uninfected controls. We also determined brain virus burdens by real-time PCR. Treated and untreated HAND brains had distinct gene expression profiles with ART transcriptomes clustering with HIV-1-negative controls. The molecular disease profile of untreated HAND showed dysregulated expression of 1470 genes at p<0.05, with activation of antiviral and immune responses and suppression of synaptic transmission and neurogenesis. The overall brain transcriptome changes in these patients were independent of histological manifestation of HIV-1 encephalitis and brain virus burdens. Depending on treatment compliance, brain transcriptomes from patients on ART had 83% to 93% fewer dysregulated genes and significantly lower dysregulation of biological pathways compared to untreated patients, with particular improvement indicated for nervous system functions. However a core of about 100 genes remained similarly dysregulated in both treated and untreated patient brain tissues. These genes participate in adaptive immune responses, and in interferon, cell cycle, and myelin pathways. Fluctuations of cellular gene expression in the brain correlated in Pearson's formula analysis with plasma but not brain virus burden. Our results define for the first time an aberrant genome-wide brain transcriptome of untreated HAND and they suggest that antiretroviral treatment can be broadly effective in reducing pathophysiological changes in the brain associated with HAND. Aberrantly expressed transcripts common to untreated and treated HAND may contribute to neurocognitive changes defying ART

Genomic Relationships, Novel Loci, and Pleiotropic Mechanisms across Eight Psychiatric Disorders

Genetic influences on psychiatric disorders transcend diagnostic boundaries, suggesting substantial pleiotropy of contributing loci. However, the nature and mechanisms of these pleiotropic effects remain unclear. We performed analyses of 232,964 cases and 494,162 controls from genome-wide studies of anorexia nervosa, attention-deficit/hyper-activity disorder, autism spectrum disorder, bipolar disorder, major depression, obsessive-compulsive disorder, schizophrenia, and Tourette syndrome. Genetic correlation analyses revealed a meaningful structure within the eight disorders, identifying three groups of inter-related disorders. Meta-analysis across these eight disorders detected 109 loci associated with at least two psychiatric disorders, including 23 loci with pleiotropic effects on four or more disorders and 11 loci with antagonistic effects on multiple disorders. The pleiotropic loci are located within genes that show heightened expression in the brain throughout the lifespan, beginning prenatally in the second trimester, and play prominent roles in neurodevelopmental processes. These findings have important implications for psychiatric nosology, drug development, and risk prediction.Peer reviewe

Dissecting the Shared Genetic Architecture of Suicide Attempt, Psychiatric Disorders, and Known Risk Factors

Background Suicide is a leading cause of death worldwide, and nonfatal suicide attempts, which occur far more frequently, are a major source of disability and social and economic burden. Both have substantial genetic etiology, which is partially shared and partially distinct from that of related psychiatric disorders. Methods We conducted a genome-wide association study (GWAS) of 29,782 suicide attempt (SA) cases and 519,961 controls in the International Suicide Genetics Consortium (ISGC). The GWAS of SA was conditioned on psychiatric disorders using GWAS summary statistics via multitrait-based conditional and joint analysis, to remove genetic effects on SA mediated by psychiatric disorders. We investigated the shared and divergent genetic architectures of SA, psychiatric disorders, and other known risk factors. Results Two loci reached genome-wide significance for SA: the major histocompatibility complex and an intergenic locus on chromosome 7, the latter of which remained associated with SA after conditioning on psychiatric disorders and replicated in an independent cohort from the Million Veteran Program. This locus has been implicated in risk-taking behavior, smoking, and insomnia. SA showed strong genetic correlation with psychiatric disorders, particularly major depression, and also with smoking, pain, risk-taking behavior, sleep disturbances, lower educational attainment, reproductive traits, lower socioeconomic status, and poorer general health. After conditioning on psychiatric disorders, the genetic correlations between SA and psychiatric disorders decreased, whereas those with nonpsychiatric traits remained largely unchanged. Conclusions Our results identify a risk locus that contributes more strongly to SA than other phenotypes and suggest a shared underlying biology between SA and known risk factors that is not mediated by psychiatric disorders.Peer reviewe

Activation of NF-κB by R5 and X4 Human Immunodeficiency Virus Type 1 Induces Macrophage Inflammatory Protein 1α and Tumor Necrosis Factor Alpha in Macrophages

NF-κB is a transcriptional activator that often regulates inflammatory responses. We demonstrate that human immunodeficiency virus type 1 activates nuclear localization of NF-κB in macrophages in a manner dependent upon virus strain but independent of virus replication. Through the use of an inhibitor, NF-κB activation was found to be responsible for the cytokine and chemokine induction that we recently reported