Jianuo Liu

Neuromodulatory Activities of CD4+CD25+ Regulatory T Cells in a Murine Model of HIV-1-Associated Neurodegeneration

HIV-1-associated neurocognitive impairments are intrinsically linked to microglial immune activation, persistent viral infection, and inflammation. In the era of antiretroviral therapy, more subtle cognitive impairments occur without adaptive immune compromise. We posit that adaptive immunity is neuroprotective, serving in both the elimination of infected cells through CD8+ cytotoxic T cell activities and the regulation of neuroinflammatory responses of activated microglia. For the latter, little is known. Thus, we studied the neuromodulatory effects of CD4+ regulatory T cells (Treg; CD4+CD25+) or effector T cells in HIV-1-associated neurodegeneration. A newly developed HIV-1 encephalitis mouse model was used wherein murine bone marrow-derived macrophages are infected with a full-length HIV-1YU2/vesicular stomatitis viral pseudotype and injected into basal ganglia of syngeneic immunocompetent mice. Adoptive transfer of CD3-activated Treg attenuated astrogliosis and microglia inflammation with concomitant neuroprotection. Moreover, Treg-mediated anti-inflammatory activities and neuroprotection were associated with up-regulation of brain-derived neurotrophic factor and glial cell-derived neurotrophic factor expression and down-regulation of proinflammatory cytokines, oxidative stress, and viral replication. Effector T cells showed contrary effects. These results, taken together, demonstrate the importance of Treg in disease control and raise the possibility of their utility for therapeutic strategies.

Cytokine-Mediated Inhibition of Fibrillar Amyloid-β Peptide Degradation by Human Mononuclear Phagocytes

Vaccination therapy of AD animal models and patients strongly suggests an active role of brain mononuclear phagocytes in immune-mediated clearance of amyloid-β peptides (Aβ) in brain. Although Aβ uptake by macrophages can be regulated by pro- and anti-inflammatory cytokines, their effects on macrophage-mediated Aβ degradation are poorly understood. To better understand this mechanism of degradation, we examined whether pro- and anti-inflammatory cytokines affect the degradation of Aβ using primary cultured human monocyte-derived macrophages (MDM) and microglia using pulse-chase analysis of fibrillar and oligomer 125I-Aβ40 and Aβ42. Initial uptake of fibrillar Aβ40 and Aβ42 was 40% and its degradation was saturated by 120 h in both MDM and microglia, compared with an initial uptake of oligomeric Aβ less than 0.5% and saturation of degradation within 24 h. IFN-γ increased the intracellular retention of fibrillar Aβ40 and Aβ42 by inhibiting degradation, whereas IL-4, IL-10, and TGF-β1, but not IL-13 and IL-27, enhanced degradation. Fibrillar Aβ degradation in MDM is sensitive to lysosomal and insulin degrading enzyme inhibitors but insensitive to proteasomal and neprilysin inhibitors. IFN-γ and TNF-α directly reduced the expression of insulin degrading enzyme and chaperone molecules (heat shock protein 70 and heat shock cognate protein 70), which are involved in refolding of aggregated proteins. Coculture of MDM with activated, but not naive T cells, suppressed Aβ degradation in MDM, which was partially blocked by a combination of neutralizing Abs against proinflammatory cytokines. These data suggest that proinflammatory cytokines suppress Aβ degradation in MDM, whereas select anti-inflammatory and regulatory cytokines antagonize these effects.

T cell independent mechanism for copolymer‐1‐induced neuroprotection

Despite active investigation of copolymer‐1 (Cop‐1) for nearly 40 years the mechanisms underlying its neuroprotective properties remain contentious. Nonetheless, current dogma for Cop‐1 neuroprotective activities in autoimmune and neurodegenerative diseases include bystander suppression of autoimmune T cells and attenuation of microglial responses. In this report, we demonstrate that Cop‐1 interacts directly with primary human neurons and decreases neuronal cell death induced by staurosporine or oxidative stress. This neuroprotection is mediated through protein kinase Cα and brain‐derived neurotrophic factor. Dendritic cells (DC) uptake Cop‐1, deliver it to the injury site, and release it in an active form. Interactions between Cop‐1 and DC enhance DC blood brain barrier migration. In a rat model with optic nerve crush injury, Cop‐1‐primed DC induce T cell independent neuroprotection. These findings may facilitate the development of neuroprotective approaches using DC‐mediated Cop‐1 delivery to diseased nervous tissue.

Chemokine CCL2 modulation of neuronal excitability and synaptic transmission in rat hippocampal slices.

J. Neurochem. (2011) 116, 406–414.

Acetyl-L-carnitine protects neuronal function from alcohol-induced oxidative damage in the brain.

The studies presented here demonstrate the protective effect of acetyl-L-carnitine (ALC) against alcohol-induced oxidative neuroinflammation, neuronal degeneration, and impaired neurotransmission. Our findings reveal the cellular and biochemical mechanisms of alcohol-induced oxidative damage in various types of brain cells. Chronic ethanol administration to mice caused an increase in inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine adduct formation in frontal cortical neurons but not in astrocytes from brains of these animals. Interestingly, alcohol administration caused a rather selective activation of NADPH oxidase (NOX), which, in turn, enhanced levels of reactive oxygen species (ROS) and 4-hydroxynonenal, but these were predominantly localized in astrocytes and microglia. Oxidative damage in glial cells was accompanied by their pronounced activation (astrogliosis) and coincident neuronal loss, suggesting that inflammation in glial cells caused neuronal degeneration. Immunohistochemistry studies indicated that alcohol consumption induced different oxidative mediators in different brain cell types. Thus, nitric oxide was mostly detected in iNOS-expressing neurons, whereas ROS were predominantly generated in NOX-expressing glial cells after alcohol ingestion. Assessment of neuronal activity in ex vivo frontal cortical brain tissue slices from ethanol-fed mice showed a reduction in long-term potentiation synaptic transmission compared with slices from controls. Coadministration of ALC with alcohol showed a significant reduction in oxidative damage and neuronal loss and a restoration of synaptic neurotransmission in this brain region, suggesting that ALC protects brain cells from ethanol-induced oxidative injury. These findings suggest the potential clinical utility of ALC as a neuroprotective agent that prevents alcohol-induced brain damage and development of neurological disorders.

Genomic and proteomic microglial profiling: pathways for neuroprotective inflammatory responses following nerve fragment clearance and activation

Microglia, a primary immune effector cell of the central nervous system (CNS) affects homeostatic, neuroprotective, regenerative and degenerative outcomes in health and disease. Despite these broad neuroimmune activities linked to specific environmental cues, a precise cellular genetic profile for microglia in the context of disease and repair has not been elucidated. To this end we used nucleic acid microarrays, proteomics, immunochemical and histochemical tests to profile microglia in neuroprotective immune responses. Optic and sciatic nerve (ON and SN) fragments were used to stimulate microglia in order to reflect immune consequences of nervous system injury. Lipopolysaccharide and latex beads‐induced microglial activation served as positive controls. Cytosolic and secreted proteins were profiled by surface enhanced laser desorption ionization‐time of flight (SELDI‐TOF) ProteinChip®, 1D and 2D difference gel electrophoresis. Proteins were identified by peptide sequencing with tandem mass spectrometry, ELISA and western blot tests. Temporal expression of pro‐inflammatory cytokines, antioxidants, neurotrophins, and lysosomal enzyme expression provided, for the first time, a unique profile of secreted microglia proteins with neuroregulatory functions. Most importantly, this molecular and biochemical signature supports a broad range of microglial functions for debris clearance and promotion of neural repair after injury.

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 systems 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 astrocyte-mediated 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 and therapeutics that may influence the course of HIV-1-mediated neurodegeneration.

HIV-1-Infected Astrocytes and the Microglial Proteome

The human immunodeficiency virus (HIV) invades the central nervous system early after viral exposure but causes progressive cognitive, behavior, and motor impairments years later with the onset of immune deficiency. Although in the brain, HIV preferentially replicates productively in cells of mononuclear phagocyte (MP; blood borne macrophage and microglia), astrocytes also can be infected, at low and variable frequency, particularly in patients with encephalitis. Among their many functions, astrocytes network with microglia to provide the first line of defense against microbial infection; however, very little is known about astrocytes’ consequences on MP. Here, we addressed this question using co-culture systems of HIV-infected mouse astrocytes and microglia. Pseudotyped vesicular stomatis virus/HIV was used to circumvent the absence of viral receptors and ensure cell genotypic uniformity for studies of intercellular communication. The study demonstrated that infected astrocytes show modest changes in protein elements compared to uninfected cells. In contrast, infected astrocytes induce robust changes in the proteome of HIV-1-infected microglia. Accelerated cell death and redox proteins, among others, were produced in abundance. The observations confirmed the potential of astrocytes to influence the neuropathogenesis of HIV-1 infection by specifically altering the neurotoxic potential of infected microglia and regulating viral maturation.

Monocyte Chemotactic Protein-1 Regulates Voltage-Gated K+ Channels and Macrophage Transmigration

Progressive human immunodeficiency virus (HIV)-1 infection and virus-induced neuroinflammatory responses effectuate monocyte-macrophage transmigration across the blood–brain barrier (BBB). A key factor in mediating these events is monocyte chemotactic protein-1 (MCP-1). Upregulated glial-derived MCP-1 in HIV-1-infected brain tissues generates a gradient for monocyte recruitment into the nervous system. We posit that the inter-relationships between MCP-1, voltage-gated ion channels, cell shape and volume, and cell mobility underlie monocyte transmigration across the BBB. In this regard, MCP-1 serves both as a chemoattractant and an inducer of monocyte-macrophage ion flux affecting cell shape and mobility. To address this hypothesis, MCP-1-treated bone marrow-derived macrophages (BMM) were analyzed for gene and protein expression, electrophysiology, and capacity to migrate across a laboratory constructed BBB. MCP-1 enhanced K+ channel gene (KCNA3) and channel protein expression. Electrophysiological studies revealed that MCP-1 increased outward K+ currents in a dose-dependent manner. In vitro studies demonstrated that MCP-1 increased BMM migration across an artificial BBB, and the MCP-1-induced BMM migration was blocked by tetraethylammonium, a voltage-gated K+ channel blocker. Together these data demonstrated that MCP-1 affects macrophage migratory movement through regulation of voltage-gated K+ channels and, as such, provides a novel therapeutic strategy for neuroAIDS.

HIV-1gp120 Induces Neuronal Apoptosis through Enhancement of 4-Aminopyridine-Senstive Outward K+ Currents

Human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) usually occurs late in the course of HIV-1 infection and the mechanisms underlying HAD pathogenesis are not well understood. Accumulating evidence indicates that neuronal voltage-gated potassium (Kv) channels play an important role in memory processes and acquired neuronal channelopathies in HAD. To examine whether Kv channels are involved in HIV-1-associated neuronal injury, we studied the effects of HIV-1 glycoprotein 120 (gp120) on outward K+ currents in rat cortical neuronal cultures using whole-cell patch techniques. Exposure of cortical neurons to gp120 produced a dose-dependent enhancement of A-type transient outward K+ currents (IA). The gp120-induced increase of IA was attenuated by T140, a specific antagonist for chemokine receptor CXCR4, suggesting gp120 enhancement of neuronal IA via CXCR4. Pretreatment of neuronal cultures with a protein kinase C (PKC) inhibitor, GF109203X, inhibited the gp120-induced increase of IA. Biological significance of gp120 enhancement of IA was demonstrated by experimental results showing that gp120-induced neuronal apoptosis, as detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and caspase-3 staining, was attenuated by either an IA blocker 4-aminopyridine or a specific CXCR4 antagonist T140. Taken together, these results suggest that gp120 may induce caspase-3 dependent neuronal apoptosis by enhancing IA via CXCR4-PKC signaling.