Lynn Pulliam

Unique monocyte subset in patients with AIDS dementia

BACKGROUND 15-30% of patients infected with HIV will develop a debilitating dementia. Whilst HIV enters the brain soon after infection, presumably within monocyte-derived macrophages, not all patients with HIV become demented. Blood monocytes probably cross the blood-brain barrier and give rise ultimately to parenchyma macrophages. We looked for a specific monocyte subset in AIDS patients with dementia. METHODS Peripheral blood monocytes from three groups were compared: AIDS patients with (n = 12) and without (n = 11) dementia, and ten HIV seronegative healthy controls. We used flow cytometry to analyse monocytes, and cell lysis and apoptosis assays to examine monocyte effects on human brain cells in vitro. FINDINGS We found a unique subset of monocytes in patients with AIDS dementia. These monocytes were more dense and granular and expressed CD14/CD16 and CD14/CD69. Means (SD) for CD14/CD16 in HIV-negative controls and in AIDS non-dementia and AIDS dementia patients were 6.5% (4), 16% (13), and 37% (21), respectively (p = 0.008 between the two groups of patients). The corresponding means for CD14/CD69 were 7% (6), 8% (10), and 69% (18) (p < 0.0001). INTERPRETATION CD69 is a member of the natural-killer-cell gene complex that is expressed after activation. Supernatants from cultures containing these dense cells can trigger apoptosis of human brain cells in vitro. The monocyte subset we found in patients with AIDS dementia might enter the brain and expose neural cells to toxic factors.

The HIV envelope protein gp120 is toxic to human brain-cell cultures through the induction of interleukin-6 and tumor necrosis factor-α

Objective: To investigate the induction of cytokines as a possible mechanism for the neurotoxicity of the HIV‐1 envelope protein gp120. Design: The gp120 protein was tested directly on primary human brain cultures to examine its ability to induce cytokines and its neurotoxicity on human neural cells because gp120 is known to be toxic to rodent ganglion cultures, and neural cells such as astrocytes and microglia produce cytokines when stimulated. Methods: Primary cultures of human brain cell aggregates, astrocytes and macrophages were exposed to HIV‐1 recombinant (r) gp120SF2. Induction of cytokines was assayed by enzyme‐linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT‐PCR); neurotoxicity of rgp120SF2 and interleukin (IL)‐6 on human brain cultures was examined by electron microscopy. Results: ELISA and RT‐PCR studies revealed that rgp120SF2 induced IL‐6 and tumor necrosis factor (TNF)‐&agr; in brain cultures; IL‐6 could also be induced by TNF‐&agr; added to brain cultures. Both IL‐6 and TNF‐&agr; were upregulated in astrocytes and macrophage cultures on rgp120SF2 treatment. Ultrastructural studies demonstrated that IL‐6 treatment for 72h induced large cytoplasmic vacuoles in neural cells with morphology consistent with neurons; rgp120SF2 treatment for 7 days resulted in chromatin condensation along the inner margins of nuclear envelopes of neural cells. Conclusions: Our results demonstrated that HIV‐1 rgp120SF2 can upregulate at least two known neurotoxic cytokines, IL‐6 and TNF‐&agr;, which may injure neural cells and contribute to the neuropathology observed in AIDS dementia patients.

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.

Exosomes as mediators of neuroinflammation

Exosomes are membrane-bound nanovesicles that are shed by cells of various lineages under normal as well as pathological conditions. Previously thought to be ‘extracellular debris’, exosomes have recently generated immense interest following their discovery as mediators of intercellular communication by delivering functional proteins, mRNA transcripts as well as miRNAs to recipient cells. Although suggested to primarily serve as signaling organelles which also remove unwanted cellular components in the brain, accumulating evidence suggests that exosomes can also significantly contribute to the development of several neuropathologies. Toxic forms of aggregated proteins such as α-synuclein, amyloid β and prions, that are responsible for the development of Parkinson’s disease, Alzheimer’s disease and Creutzfeldt-Jacob disease (CJD) respectively, have been shown to get effectively packaged into exosomes and spread from one cell to another, initiating an inflammatory cascade. In addition, exosomes secreted by resident brain cells in response to pathogenic stimuli such as viral proteins can also influence bystander cells by the transfer of dysregulated miRNAs that suppress the expression of essential genes in the recipient cells. Given the relevance of exosomes in brain communication and neuropathogenesis, novel therapeutic strategies are now being developed that exploit the biology of these vesicles to deliver anti-inflammatory molecules to the CNS. Exosomes may alter the way we think about brain disorders and their treatments.

Biomarkers of HIV-1 CNS infection and injury

While it is clear that HIV-1 can cause CNS dysfunction, current approaches to classification and diagnosis of this dysfunction rely on syndromic definitions or measures of abnormality on neuropsychological testing in the background context of HIV-1 infection. These definitions have been variably applied, offer only limited sensitivity or specificity, and do not easily distinguish active from static brain injury. Supplanting or augmenting these approaches with objective biologic measurements related to underlying disease processes would provide a major advance in classification, diagnosis, epidemiology, and treatment assessment. Two major avenues are now actively pursued to this end: 1) analysis of soluble molecular markers in CSF and, to a lesser degree, in blood, and 2) neuroimaging markers using anatomic, metabolic, and functional measurements. This review considers the rationale and prospects of these approaches. GLOSSARY: ADC = AIDS dementia complex; BBB = blood–brain barrier; DTI = diffusion tensor imaging; FA = fractional anisotropy; fMRI = functional MRI; HAART = highly active combination antiretroviral therapy; HIVE = HIV-1 encephalitis; MCP-1 (CCL2) = monocyte chemoattractant protein 1; MRS = MR spectroscopy; MTR = magnetization transfer imaging; NAA = N-acetylaspartate; NFL = neurofilament light chain protein.

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.