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

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

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

Proteomic Modeling for HIV-1 Infected Microglia-Astrocyte Crosstalk

Background: HIV-1-infected and immune competent brain mononuclear phagocytes (MP; macrophages and microglia) secrete cellular and viral toxins that affect neuronal damage during advanced disease. In contrast, astrocytes can affect disease by modulating the nervous system’s microenvironment. Interestingly, little is known how astrocytes communicate with MP to influence disease. Methods and Findings: MP-astrocyte crosstalk was investigated by a proteomic platform analysis using vesicular stomatitis virus pseudotyped HIV infected murine microglia. The microglial-astrocyte dialogue was significant and affected microglial cytoskeleton by modulation of cell death and migratory pathways. These were mediated, in part, through F-actin polymerization and filament formation. Astrocyte secretions attenuated HIV-1 infected microglia neurotoxicity and viral growth linked to the regulation of reactive oxygen species. Conclusions: These observations provide unique insights into glial crosstalk during disease by supporting astrocytemediated regulation of microglial function and its influence on the onset and progression of neuroAIDS. The results open new insights into previously undisclosed pathogenic mechanisms and open the potential for biomarker discovery an