Navneet K. Dhillon

Neuronal Apoptosis Is Mediated by CXCL10 Overexpression in Simian Human Immunodeficiency Virus Encephalitis

Inflammatory mediators play a crucial role in the pathophysiology of several neurodegenerative diseases including acquired immune deficiency syndrome dementia complex. In the present study we identified a link between CXCL10 overexpression in the brain and human immunodeficiency virus dementia and demonstrated the presence of the chemokine CXCL10 and its receptor, CXCR3, in the neurons in the brains of macaques with simian human immunodeficiency virus encephalitis. Using human fetal brain cultures, we showed that treatment of these cells with either SHIV89.6P or viral gp120 resulted in induction of CXCL10 in neurons. Cultured neurons treated with the chemokine developed increased membrane permeability followed by apoptosis via activation of caspase-3. We confirmed the relevance of these findings in sections of human and macaque brains with encephalopathy demonstrating that neurons expressing CXCL10 also expressed caspase-3.

CXCL10-induced cell death in neurons: role of calcium dysregulation

Chemokines play a key role in the regulation of central nervous system disease. CXCL10 over‐expression has been observed in several neurodegenerative diseases, including multiple sclerosis, Alzheimers disease and HIV‐associated dementia. More recent studies by others and us have shown that CXCL10 elicits apoptosis in fetal neurons. The mechanism of CXCL10‐mediated neurotoxicity, however, remains unclear. In this study, we provide evidence for the direct role of Ca2+ dysregulation in CXCL10‐mediated apoptosis. We demonstrate that treatment of fetal neuronal cultures with exogenous CXCL10 produced elevations in intracellular Ca2+ and that this effect was modulated via the binding of CXCL10 to its cognate receptor, CXCR3. We further explored the association of intracellular Ca2+ elevations with the caspases that are involved in CXC10‐induced neuronal apoptosis. Our data showed that increased Ca2+, which is available for uptake by the mitochondria, is associated with membrane permeabilization and cytochrome c release from this compartment. The released cytochrome c then activates the initiator active caspase‐9. This initiator caspase sequentially activates the effector caspase‐3, ultimately leading to apoptosis. This study identifies the temporal signaling cascade involved in CXCL10‐mediated neuronal apoptosis and provides putative targets for pharmaceutical intervention of neurological disorders associated with CXCL10 up‐regulation.

Involvement of TRPC Channels in CCL2-Mediated Neuroprotection against Tat Toxicity

Chemokine (C-C motif) ligand 2 (CCL2), also known as monocyte chemoattractant protein-1, plays a critical role in leukocyte recruitment and activation. In the present study, we identify an additional role for CCL2 that of neuroprotection against HIV-1 transactivator protein (Tat) toxicity in rat primary midbrain neurons. Furthermore, we report the involvement of transient receptor potential canonical (TRPC) channels in CCL2-mediated neuroprotection. TRPC are Ca2+-permeable, nonselective cation channels with a variety of physiological functions. Blockage of TRPC channels resulted in suppression of both CCL2-mediated neuroprotection and intracellular Ca2+ elevations. Parallel but distinct extracellular signal-regulated kinase (ERK)/cAMP response element-binding protein (CREB) and Akt/nuclear factor κB (NF-κB) pathways were involved in the CCL2-mediated neuroprotection. Blocking TRPC channels and specific downregulation of TRPC channels 1 and 5 resulted in suppression of CCL2-induced ERK/CREB activation but not Akt/NF-κB activation. In vivo relevance of these findings was further corroborated in wild-type and CCR2 knock-out mice. In the wild-type but not CCR2 knock-out mice, exogenous CCL2 exerted neuroprotection against intrastriatal injection of HIV-1 Tat. These findings clearly demonstrate a novel role of TRPC channels in the protection of neurons against Tat through the CCL2/CCR2 axis.

Morphine enhances Tat-induced activation in murine microglia

There is increasing evidence that opiates accelerate the pathogenesis and progression of acquired immunodeficiency syndrome (AIDS), as well as the incidence of human immunodeficiency virus (HIV) encephalitis (HIVE), a condition characterized by inflammation, leukocyte infiltration, and microglial activation. The mechanisms, by which the HIV-1 transactivating protein Tat and opioids exacerbate microglial activation, however, are not fully understood. In the current study, we explored the effects of morphine and HIV-1 Tat1–72 on the activation of mouse BV-2 microglial cells and primary mouse microglia. Both morphine and Tat exposure caused up-regulation of the chemokine receptor CCR5, an effect blocked by the opioid receptor antagonist naltrexone. Morphine in combination with Tat also induced morphological changes in the BV-2 microglia from a quiescent to an activated morphology, with a dramatic increase in the expression of the microglial activation marker CD11b, as compared with cells exposed to either agent alone. In addition, the mRNA expression of inducible nitric oxide synthase (iNOS), CD40 ligand, Interferon-gamma-inducible protein 10 (IP-10), and the proinflammatory cytokines tumor necrosis factor alpha (TNFα), interleukin (IL)-1β, and IL-6, which were elevated with Tat alone, were dramatically enhanced with Tat in the presence of morphine. In summary, these findings shed light on the cooperative effects of morphine and HIV-1 Tat on both microglial activation and HIV coreceptor up-regulation, effects that could result in exacerbated neuropathogenesis.

Proinflammatory cytokines and HIV-1 synergistically enhance CXCL10 expression in human astrocytes

HIV encephalitis (HIVE), the pathologic correlate of HIV‐associated dementia (HAD) is characterized by astrogliosis, cytokine/chemokine dysregulation, and neuronal degeneration. Increasing evidence suggests that inflammation is actively involved in the pathogenesis of HAD. In fact, the severity of HAD/HIVE correlates more closely with the presence of activated glial cells than with the presence and amount of HIV‐infected cells in the brain. Astrocytes, the most numerous cell type within the brain, provide an important reservoir for the generation of inflammatory mediators, including interferon‐γ inducible peptide‐10 (CXCL10), a neurotoxin and a chemoattractant, implicated in the pathophysiology of HAD. Additionally, the proinflammatory cytokines, IFN‐γ and TNF‐α, are also markedly increased in CNS tissues during HIV‐1 infection. In this study, we hypothesized that the interplay of host cytokines and HIV‐1 could lead to enhanced expression of the toxic chemokine, CXCL10. Our findings demonstrate a synergistic induction of CXCL10 mRNA and protein in human astrocytes exposed to HIV‐1 and the proinflammatory cytokines. Signaling molecules, including JAK, STATs, MAPK (via activation of Erk1/2, AKT, and p38), and NF‐κB were identified as instrumental in the synergistic induction of CXCL10. Understanding the mechanisms involved in HIV‐1 and cytokine‐mediated up‐regulation of CXCL10 could aid in the development of therapeutic modalities for HAD.

Cocaine-mediated enhancement of virus replication in macrophages: Implications for human immunodeficiency virus-associated dementia

Injection drug use has been recognized as a major risk factor for acquired immunodeficiency syndrome (AIDS) from the outset of the epidemic. Cocaine, one of the most widely abused drugs in the United States, can both impair the functions of macrophages and CD4+ lymphocytes and also activate human immunodeficiency virus (HIV)-1 expression in these cells. Because the brain is the target organ for both cocaine and HIV, the objective of the present study was to explore the effects of cocaine on virus replication in macrophages, the target cells for the virus in the central nervous system (CNS). Cocaine markedly enhanced virus production in simian human immunodeficiency virus (SHIV)-infected monocyte-derived macrophages (MDMs) and in U1 cells, a chronically infected promonocytic cell line as monitored by enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry. Cocaine treatment also resulted in the activation of nuclear factor (NF)-κB and transcriptional activation of the HIV-LTR (long terminal repeat) gag-GFP (green fluorescent protein). Analyses of chemokines in cocaine-treated macrophages by real-time reverse transcriptase—polymerase chain reaction (RT-PCR) and Luminex assays suggested increased expression of interleukin (IL)-10, a cytokine that is known to promote HIV replication in MDMs. In addition to enhancing IL-10 expression, cocaine also caused an up-regulation of the macrophage activation marker, human leukocyte antigen (HLA)-DR, in MDMs. The synergistic effect of cocaine on virus replication and its enhancement of host activation markers suggest that cocaine functions at multiple pathways to accelerate HIV-associated dementia (HAD).

Roles of MCP-1 in development of HIV-dementia.

The encephalopathy caused by HIV, known clinically as HIV-associated dementia (HAD) and pathologically as HIV encephalitis (HIVE), results from intense infiltration of mononuclear cells, productive replication of the virus in monocyte-derived macrophages/microglia, abortive replication in astrocytes and activation of macrophages/microglia and astrocytes leading to neuronal degeneration in the brains of infected persons. Recent findings have suggested that development of HAD is based more on the activation process than on direct evidence of virus replication in the brain. Since HAD is based on the encephalitic process, major studies have been directed to the mechanisms regulating the inflammatory process. Monocyte chemoattractant protein 1, MCP-1, is a chemokine that is implicated in this process and also in the development of activation in the brain. In this review, we have attempted to identify mechanisms that induce expression of MCP-1 in the brain and the role that it plays in recruitment of mononuclear cells from blood to brain and in the activation processes of inflammatory and neural cells that lead to development of degenerative changes in the neuronal population.

Cocaine-mediated Alteration in Tight Junction Protein Expression and Modulation of CCL2/CCR2 Axis Across the Blood-Brain Barrier: Implications for HIV-Dementia

One of the hallmark features underlying the pathogenesis of HIV encephalitis is the disruption of blood–brain barrier (BBB). Cocaine, often abused by HIV-infected patients, has been suggested to worsen the HIV-associated dementia (HAD) via unknown mechanisms. The objective of the present study was to explore the effects of cocaine on BBB permeability using human brain microvascular endothelial cells (HBMECs). Additionally, because the chemokine CCL2 and its receptor CCR2 play a crucial role in the recruitment of inflammatory cells into the central nervous system in HAD brains, we tested for the effect of cocaine in modulating the CCL2/CCR2 axis. Our findings suggest that exposure of HBMECs to cocaine correlated with the breakdown of ZO-1 tight junction protein and reorganization of the cytoskeleton resulting in stress fiber formation. Furthermore, cocaine also modulated upregulation of the CCL2/CCR2 axis in monocytes. These findings conform to the multifaceted effects of cocaine leading to accelerated progression of HIV-1 neuropathogenesis.

Platelet-derived growth factor protects neurons against gp120-mediated toxicity

The human immunodeficiency virus (HIV)-1 envelope glycoprotein gp120 has been implicated in mediating neuronal apoptosis, a hallmark feature of HIV-associated dementia (HAD). Mitigation of the toxic effects of gp120 could thus be a potential mechanism for reducing HIV toxicity in the brain. In this study the authors hypothesized that neurotrophic factor, such as platelet-derived growth factor (PDGF), could protect the neurons against gp120-mediated apoptosis. SH-SY5Y cells treated with gp120 exhibited increased cell death when measured by lactate dehydrogenase (LDH) and deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay, with concomitant loss of neurites and increased cell rounding. Pretreatment with PDGF-BB, however, reduced gp120-associated neurotoxicity and rescued the neurite outgrowth. Additionally, gp120-mediated activation of caspase-3 was also significantly reduced in cells pretreated with PDGF-BB. Antiapoptotic effects of PDGF-BB were also confirmed by monitoring levels of anti- and proapoptotic genes, Bcl-xL and Bax, respectively. Furthermore, PDGF-mediated protection against gp120 involved the phosphoinositide (PI) 3-kinase/Akt pathway. Taken together these findings lead us to suggest that PDGF-BB could be considered as a therapeutic agent that can mitigate gp120-mediated neurotoxicity in HAD.

HIV-1 Tat Co-Operates with IFN-γ and TNF-α to Increase CXCL10 in Human Astrocytes

HIV-associated neurological disorders (HAND) are estimated to affect 60% of the HIV infected population. HIV-encephalitis (HIVE), the pathological correlate of the most severe form of HAND is often characterized by glial activation, cytokine/chemokine dysregulation, and neuronal damage and loss. However, the severity of HIVE correlates better with glial activation rather than viral load. One of the characteristic features of HIVE is the increased amount of the neurotoxic chemokine, CXCL10. This chemokine can be released from astroglia activated with the pro-inflammatory cytokines IFN-γ and TNF-α, in conjunction with HIV-1 Tat, all of which are elevated in HIVE. In an effort to understand the pathogenesis of HAND, this study was aimed at exploring the regulation of CXCL10 by cellular and viral factors during astrocyte activation. Specifically, the data herein demonstrate that the combined actions of HIV-1 Tat and the pro-inflammatory cytokines, IFN-γ and TNF-α, result in the induction of CXCL10 at both the RNA and protein level. Furthermore, CXCL10 induction was found to be regulated transcriptionally by the activation of the p38, Jnk, and Akt signaling pathways and their downstream transcription factors, NF-κB and STAT-1α. Since CXCL10 levels are linked to disease severity, understanding its regulation could aid in the development of therapeutic intervention strategies for HAND.