Hans Rempel

Hiv-1 Tat inhibits neprilysin and elevates amyloid β

Objective:Aging is a risk factor for amyloid beta (Aβ) accumulation and dementia. Since highly active antiretroviral therapies have effectively lengthened the life expectancy of individuals infected with HIV-1, we investigated the affect of HIV-1 Tat, a viral transactivating transcription factor, on Aβ degradation in the brain by neprilysin (NEP), a neuronal endopeptidase. Design and methods:Using neural cell membrane fractions from human brain aggregates, Tat inhibition of NEP activity was assessed in a fluorescence assay. Following treatment with Tat, conditioned medium of human brain aggregate cultures was assayed for Aβ1–40 by ELISA. We evaluated the potential consequence of Tat inhibition of NEP by immunostaining cortex sections from postmortem human brain for Aβ. Results:In an in vitro assay, Tat inhibited NEP activity by 80%. The cysteine-rich domain of Tat was essential for NEP inhibition. Recombinant Tat added directly to brain cultures, resulted in a 125% increase in soluble Aβ. Postmortem human brain sections from patients with HIV-1 infection (n = 14; 31–58 years old) had a significant increase in Aβ, compared to controls (n = 5; 30–52 years old). Correlative analysis identified a statistically significant relationship between Aβ load and duration of HIV-1 seropositive status. Conclusion:We have shown that Tat, which is found in the brains of patients with HIV-1 infection, inhibits the Aβ-degrading enzyme, NEP. Aβ staining was significantly increased in human brain sections from individuals with HIV-1 infection compared to controls. These results have important implications for individuals living and aging with HIV-1 infection.

Sialoadhesin Expressed on IFN-Induced Monocytes Binds HIV-1 and Enhances Infectivity

Background HIV-1 infection dysregulates the immune system and alters gene expression in circulating monocytes. Differential gene expression analysis of CD14+ monocytes from subjects infected with HIV-1 revealed increased expression of sialoadhesin (Sn, CD169, Siglec 1), a cell adhesion molecule first described in a subset of macrophages activated in chronic inflammatory diseases. Methodology/Principal Findings We analyzed sialoadhesin expression on CD14+ monocytes by flow cytometry and found significantly higher expression in subjects with elevated viral loads compared to subjects with undetectable viral loads. In cultured CD14+ monocytes isolated from healthy individuals, sialoadhesin expression was induced by interferon-α and interferon-γ but not tumor necrosis factor-α. Using a stringent binding assay, sialoadhesin-expressing monocytes adsorbed HIV-1 through interaction with the sialic acid residues on the viral envelope glycoprotein gp120. Furthermore, monocytes expressing sialoadhesin facilitated HIV-1 trans infection of permissive cells, which occurred in the absence of monocyte self-infection. Conclusions/Significance Increased sialoadhesin expression on CD14+ monocytes occurred in response to HIV-1 infection with maximum expression associated with high viral load. We show that interferons induce sialoadhesin in primary CD14+ monocytes, which is consistent with an antiviral response during viremia. Our findings suggest that circulating sialoadhesin-expressing monocytes are capable of binding HIV-1 and effectively delivering virus to target cells thereby enhancing the distribution of HIV-1. Sialoadhesin could disseminate HIV-1 to viral reservoirs during monocyte immunosurveillance or migration to sites of inflammation and then facilitate HIV-1 infection of permissive cells.

Invasive chronic inflammatory monocyte phenotype in subjects with high HIV-1 viral load

Human immunodeficiency virus type 1 (HIV-1)-infected monocytes trafficking into the central nervous system are a risk factor for HIV-1-associated dementia. We performed global gene expression analysis on CD14+ monocytes isolated from HIV-1-infected individuals and controls to identify HIV-1-related changes in monocyte phenotype. Monocytes from subjects with high viral load (HVL) had a significant increase in monocytes expressing CD16, CCR5, and MCP-1. There was also an increase in sialoadhesin, a macrophage marker of chronic inflammation. Expression of proinflammatory cytokine genes IL-1, IL-6, and TNF-alpha was unchanged in individuals with HIV-1 compared to control CD14+ monocytes. Differential gene expression identified by DNA microarray analysis was confirmed with reverse transcription polymerase chain reaction (RT-PCR), while increased protein expression was characterized by immunofluorescence. We concluded that there is a circulating CD14+ macrophage hybrid phenotype in subjects with HVL.

Transcriptional Control of Monocyte Gene Expression in Post-Traumatic Stress Disorder

Post-traumatic stress disorder (PTSD) confers an increased risk for disorders with an inflammatory etiology. PTSD-related dysregulation of the sympathetic nervous system (SNS) and hypothalamic-pituitary adrenal (HPA) axis and associated alterations in inflammatory activity may contribute to this increased risk. However, little is known about convergent SNS, HPA and inflammatory signaling at the level of the immune cell transcriptome in PTSD. To explore such signaling, we examined the prevalence of specific transcription factor binding motifs in the promoter regions of differentially expressed genes in monocytes from individuals with PTSD and matched controls. Participants included 49 men (24 PTSD+ and 25 trauma-exposed controls) and 18 women (10 PTSD+ and 8 controls). Men with PTSD showed up-regulation of target genes for the NF-κB/Rel family of transcription factors, which convey inflammatory signals, up-regulation of target genes for CREB/ATF transcription factors, which convey adrenergic signals from the SNS, and down-regulation of target genes for the glucocorticoid receptor, which conveys glucocorticoid signals from the HPA axis. Women with PTSD also showed significant up-regulation of target genes for NF-κB and non-significant down-regulation of target genes for GR, but significant down-regulation of target genes for CREB/ATF. Altered transcriptional control of monocyte gene expression could contribute to exaggerated inflammatory activity in PTSD.

Interferon-α drives monocyte gene expression in chronic unsuppressed HIV-1 infection

Objectives:HIV-1 infection dysregulates the innate immune system and alters leukocyte-gene expression. The objectives were two fold: to characterize the impact of HIV-1 infection on peripheral monocyte gene expression and to identify the predominant factor(s) responsible for altered gene expression. Design and methods:In a cross-sectional study (n = 55), CD14+ monocytes were isolated from 11 HIV-1 seronegative controls, 22 HIV-1 seropositive individuals with low-viral loads (LVL) and 22 HIV-1 seropositive individuals with high-viral loads (HVL). Monocyte gene expression data were collected for control, LVL and HVL individuals using high-density microarrays. We evaluated three HIV-1 disease-related peripheral factors, interferon (IFN)-α, IFN-γ and lipopolysaccharide (LPS) as candidates causing monocyte dysregulation, by comparing gene expression profiles between study individuals and monocytes treated with these factors in vitro. Plasma from HIV-1 positive individuals was quantified for LPS and soluble CD14. Results:Monocytes from HIV-1-infected individuals with viral loads above 10 000 RNA copies/ml (HVL) displayed an activated phenotype. Characterization of gene expression revealed an ongoing immune response to viral infection including inflammation and chemotaxis. Gene expression analysis of in-vitro-treated HIV-1 seronegative monocytes with IFN-α, IFN-γ or LPS demonstrated that IFN-α most accurately recapitulated the HIV-1 HVL profile. No LPS-induced gene expression signature was detected even in HIV-1 individuals with the highest LPS and sCD14 levels. Conclusion:Monocyte gene expression in individuals with HIV-1 viremia is predominantly due to IFN-α, whereas individuals with LVL have a nonactivated phenotype. In monocytes, there was no discernible expression profile linked to LPS exposure.

Suppressed monocyte gene expression profile in men versus women with PTSD

There have been several attempts to use gene microarrays from peripheral blood mononuclear cells to identify new biological pathways or targets for therapy in Posttraumatic Stress Disorder (PTSD). The few studies conducted to date have yielded an unclear pattern of findings, perhaps reflecting the use of heterogeneous samples of circulating immune cells for analysis. We used gene microarrays on a homogeneous sample of circulating monocytes to test the hypothesis that chronic PTSD would be associated with elevated inflammatory activity and to identify new pathways dysregulated in the disorder. Forty-nine men (24 PTSD+ and 25 age-matched trauma-exposed PTSD- controls) and 18 women (10 PTSD+ and 8 age-matched PTSD- controls) were recruited. Gene expression microarray analysis was performed on CD14+ monocytes, immune cells that initiate and respond to inflammatory signaling. Male subjects with PTSD had an overall pattern of under-expression of genes on monocytes (47 under-expressed versus 4 over-expressed genes). A rigorous correction for multiple comparisons and verification with qPCR showed that of only 3 genes that were differentially expressed, all were under-expressed. There was no transcriptional evidence of chronic inflammation in male PTSD+ subjects. In contrast, preliminary data from our pilot female PTSD+ subjects showed a relatively balanced pattern of increased and decreased expression of genes and an increase in activity of pathways related to immune activation. The results indicate differential patterns of monocyte gene expression in PTSD, and the preliminary data from our female pilot subjects are suggestive of gender dimorphism in biologic pathways activated in PTSD. Changes in immune cell gene expression may contribute to medical morbidity in PTSD.

Differential cognitive impairment in HCV coinfected men with controlled HIV compared to HCV monoinfection

Background:Individuals infected with both HIV and hepatitis C virus (HCV) have shown impaired performance on different neuropsychological (NP) tests; however, whether coinfected individuals with controlled HIV and minimal liver damage in the era of antiretroviral therapy have impairment is understudied. Methods:Nineteen HCV monoinfected, 17 HIV/HCV coinfected, and 17 control male participants were evaluated for depression, attention, executive function, information processing, fine motor speed, and verbal/visual learning/memory. Eleven controls and 14 HIV monoinfected participants with controlled viral load from a previous study were also included for comparison. At time of testing, participants were not using drugs or alcohol and did not have cirrhosis. A global deficit score (GDS) was calculated from 7 domains of NP tests and alterations in specific domains were determined. Results:HIV/HCV subjects had a higher depression score (11.1 ± 7.5) than controls (5.4 ± 4.1, P = 0.010) and a higher GDS score (0.77 ± 0.47) than HCV (0.46 ± 0.34, P = 0.036), HIV (0.45 ± 0.36, P = 0.008), and controls (0.30 ± 0.29, P = 0.001). Coinfection was associated with worse scores in attention working memory (P =0.007), executive function (P = 0.01), fine motor function (P = 0.011), verbal learning/memory (P < 0.001), and visual learning/memory (P < 0.001) compared to controls. Within the HCV group, viral load was associated with lower attention, executive function, and information processing speed and positively with GDS. Conclusions:Coinfection significantly increased the risk of cognitive impairment in subjects with controlled HIV viral loads. In HCV monoinfected but not coinfected subjects, HCV viral load correlated with worsening GDS, suggesting different pathways for NP impairment.

Peripheral biomarkers do not correlate with cognitive impairment in highly active antiretroviral therapy–treated subjects with human immunodeficiency virus type 1 infection

Neuropsychological (NP) impairments in human immunodeficiency virus (HIV)-infected individuals remain high despite the introduction of highly active antiretroviral therapy (HAART). We sought to determine whether or not a monocyte gene expression profile along with other peripheral factors would correlate with neuropsychological impairment among HIV-infected individuals. Forty-four HIV-1-seropositive subjects (HIV+) on HAART and 11 HIV-1-seronegative controls (HIV−) had NP testing and blood drawn for monocyte gene expression analysis. All HIV+ subjects were assessed for CD4 counts, apolipoprotein E (ApoE) genotype, viral load, and plasma lipopolysaccharide (LPS) and soluble CD14 (sCD14). NP scores were normalized to age, gender, and education. Twenty-five percent of HIV+ individuals showed abnormal NP testing results (>1.5 SD below normal in two domains). HIV+ individuals had deficits in attention/working memory, verbal learning, and information processing speed compared to HIV− controls. There was no correlation between overall NP impairment and plasma viral load, level of education, age, ethnic diversity, sCD14, plasma LPS, CD4 cell count, ApoE genotype, or years of infection. However, greater years of infection had worse visual learning performance. sCD14 and CD4 nadir positively correlated with information processing speed and fine motor skills, respectively. LPS correlated with viral load but not cognitive impairment. Monocyte gene expression confirmed a chronic inflammatory profile that correlated with viral load but not cognition. No blood index or profile was associated with overall NP impairment.

Elevation of CD69+ monocyte/macrophages in patients with Alzheimer's disease.

In this report, we examined the presence of the activation marker, CD69, on monocytes derived from patients with Alzheimers disease (AD). We have previously shown that patients with AIDS dementia had an elevated percentage of a CD14+/CD69+ subset and that conditioned media from these M/M phi cultures were toxic to neural cultures. We therefore postulated that patients with AD might likewise have a higher monocyte subset and that this would be associated with neural toxicity. Flow analysis showed that AD patients (n = 13) had a higher percentage of CD69+ M/M phi over age matched controls (n = 14); this trend was statistically significant (p = 0.006). Side scatter (SSC), a measure of cellular granularity was also elevated in AD patients (p = 0.02). The elevated expression of human leukocyte antigen (HLA-DR) was not found to be significant between age-matched controls and AD patients. When conditioned media from M/M phi from five AD and two control patients were evaluated for neurotoxicity, three of the five culture supernatants from AD patients induced apoptosis in neural cell aggregate cultures. Electrophoretic mobility shift assays revealed that these three supernatants also triggered NF-kappaB translocation to the nucleus. Surprisingly, in vitro neurotoxicity was induced by M/M phi supernatants having a lower percentage of CD14+/CD69+ cells. Elevation of the CD14+/CD69+ subset in AD patients may therefore represent a manifestation in the peripheral blood of the pathological events occurring in the brain but may not be directly involved in neural cell toxicity.

A peripheral monocyte interferon phenotype in HIV infection correlates with a decrease in magnetic resonance spectroscopy metabolite concentrations

Objective:In spite of effective antiretroviral therapy (ART), cognition is impaired in upwards of 35% of the HIV-infected population. We investigated a possible link between peripheral immune activation and brain metabolite concentrations. Design and methods:Thirty-five HIV-seropositive (HIV+) and eight HIV-seronegative adults were recruited to this cross-sectional study. All HIV-positive patients were on ART or a treatment interruption. Participants were evaluated for monocyte gene expression, cognitive status, and brain metabolite concentrations using 4-Tesla short echo-time proton magnetic resonance spectroscopy. Absolute concentrations of brain metabolites in the frontal white matter (FWM), anterior cingulate cortex (ACC), and basal ganglia were derived and related to monocyte gene expression and global deficit scores. Results:Analysis of monocyte gene arrays revealed an interferon (IFN)-&agr;-induced activation phenotype. Fourteen genes having the greatest fold increase in response to HIV were IFN genes. Monocyte activation as measured by gene expression profiles strongly correlated with lower N-acetylaspartate (NAA) in FWM. The IFN response gene Interferon-gamma inducible protein-10 (IP-10) was activated in monocytes from HIV individuals and strongly correlated with plasma protein levels. Plasma IP-10 correlated significantly and inversely with ACC NAA, which was lower in HIV-positive patients with mild compared to no cognitive impairment. Conclusion:Chronic peripheral immune activation driven by a type 1 IFN correlates with neuronal injury in FWM and ACC and cognitive dysfunction. Easily measured IFN-induced blood markers may be clinically significant in following early neural cell damage.