Common Transcriptional Signatures in Brain Tissue from Patients with HIV-Associated Neurocognitive Disorders, Alzheimer’s Disease, and Multiple Sclerosis

HIV-Associated Neurocognitive Disorders (HAND) is a common manifestation of HIV infection that afflicts about 50 % of HIV-positive individuals. As people with access to antiretroviral treatments live longer, HAND can be found in increasing segments of populations at risk for other chronic, neurodegenerative conditions such as Alzheimer’s disease (AD) and Multiple Sclerosis (MS). If brain diseases of diverse etiologies utilize similar biological pathways in the brain, they may coexist in a patient and possibly exacerbate neuropathogenesis and morbidity. To test this proposition, we conducted comparative meta-analysis of selected publicly available microarray datasets from brain tissues of patients with HAND, AD, and MS. In pair-wise and three-way analyses, we found a large number of dysregulated genes and biological processes common to either HAND and AD or HAND and MS, or to all three diseases. The common characteristic of all three diseases was up-regulation of broadly ranging immune responses in the brain. In addition, HAND and AD share down-modulation of processes involved, among others, in synaptic transmission and cell-cell signaling while HAND and MS share defective processes of neurogenesis and calcium/calmodulin-dependent protein kinase activity. Our approach could provide insight into the identification of common disease mechanisms and better intervention strategies for complex neurocognitive disorders. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11481-012-9409-5) contains supplementary material, which is available to authorized users

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

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

Prevention and treatment of HIV infection and cognitive disease in mice by innate immune responses

HIV associated neurocognitive impairment afflicts roughly half of infected individuals on antiretroviral therapy. This disease currently has no treatment. We have previously shown that type I interferon is induced by and partially controls infection and neuropathogenesis in mice infected by chimeric HIV, EcoHIV. Here we investigate the intentional ligation of the pattern recognition receptor Toll-like receptor 3 (TLR3) by polyinosinic-polycytidylic acid (poly I:C) for its ability to prevent or control infection and associated cognitive disease in EcoHIV infected mice. We tested topical, injection, and intranasal application of poly I:C in mice during primary infection through injection or sexual transmission or in established infection. We measured different forms of HIV DNA and RNA in tissues by real-time PCR and the development of HIV-associated cognitive disease by the radial arm water maze behavioral test. Our results indicate that poly I:C blocks primary EcoHIV infection of mice prior to reverse transcription and reduces established EcoHIV infection. Prevention or control of viral replication by poly I:C prevents or reverses HIV associated cognitive disease in mice. These findings indicate that poly I:C or other innate immune agonists may be useful in control of HIV cognitive disease

Infected mice lacking T cells develop cognitive impairment and have elevated number of monocytes/macrophages in the brain.

<p><b>A.</b> Control and EcoHIV-infected nude mice with or without ART were tested 24 h after fear conditioning training for contextual fear response and 24 h later for cued fear response. *P<0.05, **P<0.01, EcoHIV vs. EcoHIV+ART; #P<0.05, EcoHIV vs. PBS. <b>B.-D.</b> Flow cytometry analysis of macrophage or monocytes in brains of nude mice with EcoHIV, EcoHIV with cART or PBS showing representative dot plots. Cell populations were gated based on isotype control antibodies. <b>B.</b> Staining for CD45 and CD11b and negative for Ly-6G/C. <b>C.</b> Staining for CD45, CD11b and Ly-6C and negative for Ly-6G. <b>D.</b> As determined by flow cytometry using 5 mice per group, the number of cells in each population per total mouse brain are shown. <b>E.</b> Total and integrated vDNA was measured by QPCR and nested QPCR, respectively, in infiltrating leukocytes isolated from the brain of EcoHIV-infected mice with and without cART. *P<0.05, **P<0.01, ***P<0.001.</p

Features of EcoHIV-infected mice compared to HIV-infected people.

<p>Features of EcoHIV-infected mice compared to HIV-infected people.</p

LASER ART (L-ART) fails to reverse HIV-NCI in chronically-infected mice.

<p><b>A.</b> Two days prior to EcoHIV infection, mice were injected once with L-ART and their virus burdens in spleen and PC were evaluated seven days after infection. <b>B.</b> Design of the experiment. L-ART was administered twice, at 28 days and 56 days p.i. <b>C.</b> Results of RAWM conducted on chronically EcoHIV-infected L-ART-treated and untreated mice starting 82 days p.i.; L-ART treated, uninfected mice served as controls; left panel: errors; middle panel: latency; right panel: visible platform control. T represents the learning trial and RT the retention trial (*Mock vs. EcoHIV, <i>p</i><0.002; #Mock vs. EcoHIV+L-ART, <i>p</i><0.03). <b>D.</b> HIV burdens of treated and untreated EcoHIV-infected mice tested in spleen cells, peritoneal macrophages, and brain 94 days p.i.</p