Tina M. Calderon

CCL2/Monocyte Chemoattractant Protein-1 Mediates Enhanced Transmigration of Human Immunodeficiency Virus (HIV)-Infected Leukocytes across the Blood–Brain Barrier: A Potential Mechanism of HIV–CNS Invasion and NeuroAIDS

Encephalitis and dementia associated with acquired immunodeficiency syndrome (AIDS) are characterized by leukocyte infiltration into the CNS, microglia activation, aberrant chemokine expression, blood–brain barrier (BBB) disruption, and eventual loss of neurons. Little is known about whether human immunodeficiency virus 1 (HIV-1) infection of leukocytes affects their ability to transmigrate in response to chemokines and to alter BBB integrity. We now demonstrate that HIV infection of human leukocytes results in their increased transmigration across our tissue culture model of the human BBB in response to the chemokine CCL2, as well as in disruption of the BBB, as evidenced by enhanced permeability, reduction of tight junction proteins, and expression of matrix metalloproteinases (MMP)-2 and MMP-9. HIV-infected cells added to our model did not transmigrate in the absence of CCL2, nor did this condition alter BBB integrity. The chemokines CXCL10/interferon-gamma-inducible protein of 10 kDa, CCL3/macrophage inflammatory protein-1α, or CCL5/RANTES (regulated on activation normal T-cell expressed and secreted) did not enhance HIV-infected leukocyte transmigration or BBB permeability. The increased capacity of HIV-infected leukocytes to transmigrate in response to CCL2 correlated with their increased expression of CCR2, the chemokine receptor for CCL2. These data suggest that CCL2, but not other chemokines, plays a key role in infiltration of HIV-infected leukocytes into the CNS and the subsequent pathology characteristic of NeuroAIDS.

MCP‐1 (CCL2) protects human neurons and astrocytes from NMDA or HIV‐tat‐induced apoptosis

Acquired immunodeficiency syndrome (AIDS)‐associated dementia is often characterized by chronic inflammation, with infected macrophage infiltration of the CNS resulting in the production of human immunodeficiency virus type 1 (HIV‐1) products, including tat, and neurotoxins that contribute to neuronal loss. In addition to their established role in leukocyte recruitment and activation, we identified an additional role for chemokines in the CNS. Monocyte chemoattractant protein‐1 (MCP‐1 or CCL2) and regulated upon activation normal T cell expressed and secreted (RANTES) were found to protect mixed cultures of human neurons and astrocytes from tat or NMDA‐induced apoptosis. Neuronal and astrocytic apoptosis in these cultures was significantly inhibited by co‐treatment with MCP‐1 or RANTES but not IP‐10. The protective effect of RANTES was blocked by antibodies to MCP‐1, indicating that RANTES protection is mediated by the induction of MCP‐1. The NMDA blocker, MK801, also abolished the toxic effects of both tat and NMDA. Tat or NMDA treatment of mixed cultures for 24 h resulted in increased extracellular glutamate ([Glu]e) and NMDA receptor 1 (NMDAR1) expression, potential contributors to apoptosis. Co‐treatment with MCP‐1 inhibited tat and NMDA‐induced increases in [Glu]e and NMDAR1, and also reduced the levels and number of neurons containing intracellular tat. These data indicate that MCP‐1 may play a novel role as a protective agent against the toxic effects of glutamate and tat.

HIV-tat induces formation of an LRP-PSD-95-NMDAR-nNOS complex that promotes apoptosis in neurons and astrocytes

HIV infection of the central nervous system can result in neurologic dysfunction with devastating consequences in AIDS patients. NeuroAIDS is characterized by neuronal injury and loss, yet there is no evidence that HIV can infect neurons. Here we show that the HIV-encoded protein tat triggers formation of a macromolecular complex involving the low-density lipoprotein receptor-related protein (LRP), postsynaptic density protein-95 (PSD-95), N-methyl-d-aspartic acid (NMDA) receptors, and neuronal nitric oxide synthase (nNOS) at the neuronal plasma membrane, and that this complex leads to apoptosis in neurons negative as well as positive for NMDA receptors and also in astrocytes. Blockade of LRP-mediated tat uptake, NMDA receptor activation, or neuronal nitric oxide synthase significantly reduces ensuing neuronal apoptosis, suggesting that formation of this complex is an early step in tat toxicity. We also show that the inflammatory chemokine, CCL2, protects against tat toxicity and inhibits formation of the complex. These findings implicate the complex in HIV-induced neuronal apoptosis and suggest therapeutic targets for intervention in the pathogenesis of NeuroAIDS.

A role for CXCL12 (SDF-1α) in the pathogenesis of multiple sclerosis: Regulation of CXCL12 expression in astrocytes by soluble myelin basic protein

The pathogenic mechanisms that contribute to multiple sclerosis (MS) include leukocyte chemotaxis into the central nervous system (CNS) and the production of inflammatory mediators, resulting in oligodendrocyte damage, demyelination, and neuronal injury. Thus, factors that regulate leukocyte entry may contribute to early events in MS, as well as to later stages of lesion pathogenesis. CXCL12 (SDF-1alpha), a chemokine essential in CNS development and a chemoattractant for resting and activated T cells, as well as monocytes, is constitutively expressed at low levels in the CNS and has been implicated in T cell and monocyte baseline trafficking. To determine whether CXCL12 is increased in MS, immunohistochemical analyses of lesions of chronic active and chronic silent MS were performed. CXCL12 protein was detected on endothelial cells (EC) in blood vessels within normal human brain sections and on a small number of astrocytes within the brain parenchyma. In active MS lesions, CXCL12 levels were high on astrocytes throughout lesion areas and on some monocytes/macrophages within vessels and perivascular cuffs, with lesser staining on EC. In silent MS lesions, CXCL12 staining was less than that observed in active MS lesions, and also was detected on EC and astrocytes, particularly hypertrophic astrocytes near the lesion edge. Experiments in vitro demonstrated that IL-1beta and myelin basic protein (MBP) induced CXCL12 in astrocytes by signaling pathways involving ERK and PI3-K. Human umbilical vein EC did not produce CXCL12 after treatment with MBP or IL-1beta. However, these EC cultures expressed CXCR4, the receptor for CXCL12, suggesting that this chemokine may activate EC to produce other mediators involved in MS. In agreement, EC treatment with CXCL12 was found to upregulate CCL2 (MCP-1) and CXCL8 (IL-8) by PI3-K and p38-dependent mechanisms. Our findings suggest that increased CXCL12 may initiate and augment the inflammatory response during MS.

Monocyte maturation, HIV susceptibility, and transmigration across the blood brain barrier are critical in HIV neuropathogenesis

HIV continues to be a global health crisis with more than 34 million people infected worldwide (UNAIDS: Report on the Global AIDS Epidemic 2010, Geneva, World Health Organization). HIV enters the CNS within 2 weeks of infection and establishes a spectrum of HAND in a large percentage of infected individuals. These neurologic deficits greatly impact the quality of life of those infected with HIV. The establishment of HAND is largely attributed to monocyte transmigration, particularly that of a mature CD14+CD16+ monocyte population, which is more susceptible to HIV infection, across the BBB into the CNS parenchyma in response to chemotactic signals. To enter the CNS, junctional proteins on the monocytes must participate in homo‐ and heterotypic interactions with those present on BMVECs of the BBB as they transmigrate across the barrier. This transmigration is responsible for bringing virus into the brain and establishing chronic neuroinflammation. While there is baseline trafficking of monocytes into the CNS, the increased chemotactic signals present during HIV infection of the brain promote exuberant monocyte transmigration into the CNS. This review will discuss the mechanisms of monocyte differentiation/maturation, HIV infectivity, and transmigration into the CNS parenchyma that contribute to the establishment of cognitive impairment in HIV‐infected individuals. It will focus on markers of monocyte subpopulations, how differentiation/maturation alters HIV infectivity, and the mechanisms that promote their increased transmigration across the BBB into the CNS.

Human immunodeficiency virus (HIV) infection of human macrophages is increased by dopamine: a bridge between HIV-associated neurologic disorders and drug abuse.

The prevalence of human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) that result from HIV infection of the central nervous system is increasing. Macrophages, the primary target for HIV within the central nervous system, play a central role in HIV-induced neuropathogenesis. Drug abuse exacerbates HAND, but the mechanism(s) by which this increased neuropathology results in more severe forms of HAND in HIV-infected drug abusers is unclear. The addictive and reinforcing effects of many drugs of abuse, such as cocaine and methamphetamine, are mediated by increased extracellular dopamine in the brain. We propose a novel mechanism by which drugs of abuse intensify HIV neuropathogenesis through direct effects of the neurotransmitter dopamine on HIV infection of macrophages. We found that macrophages express dopamine receptors 1 and 2, and dopamine activates macrophages by increasing ERK 1 phosphorylation. Our results demonstrate for the first time that dopamine increases HIV replication in human macrophages and that the mechanism by which dopamine mediates this change is by increasing the total number of HIV-infected macrophages. This increase in HIV replication is mediated by activation of dopamine receptor 2. These findings suggest a common mechanism by which drugs of abuse enhance HIV replication in macrophages and indicate that the drug abuse-heightened levels of central nervous system dopamine could increase viral replication, thereby accelerating the development of HAND.

Characterization of monocyte maturation/differentiation that facilitates their transmigration across the blood–brain barrier and infection by HIV: Implications for NeuroAIDS

The prevalence of human immunodeficiency virus 1 (HIV) associated neurocognitive disorders resulting from infection of the central nervous system (CNS) by HIV continues to increase despite the success of combination antiretroviral therapy. Although monocytes are known to transport HIV across the blood-brain barrier (BBB) into the CNS, there are few specific markers that identify monocyte subpopulations susceptible to HIV infection and/or capable of infiltrating the CNS. We cultured human peripheral blood monocytes and characterized the expression of the phenotypic markers CD14, CD16, CD11b, Mac387, CD163, CD44v6 and CD166 during monocyte/macrophage (Mo/Mac) maturation/differentiation. We determined that a CD14(+)CD16(+)CD11b(+)Mac387(+) Mo/Mac subpopulation preferentially transmigrates across our in vitro BBB model in response to CCL2. Genes associated with Mo/Mac subpopulations that transmigrate across the BBB and/or are infected by HIV were identified by cDNA microarray analyses. Our findings contribute to the understanding of monocyte maturation, infection and transmigration into the brain during the pathogenesis of NeuroAIDS.

Hepatocyte growth factor is a survival factor for endothelial cells and is expressed in human atherosclerotic plaques

Hepatocyte growth factor (HGF) has multiple effects on target cells upon activation of its receptor, c-Met. In endothelial cells, HGF induces migration, proliferation, and angiogenesis. HGF can also act as an anti-apoptotic factor for several cell types. The signal transduction pathways involved in mediating its anti-apoptotic effects have not been fully clarified. We demonstrated that HGF is anti-apoptotic for human endothelial cells, and identified the signaling pathways by which it mediates its effects. Human umbilical vein endothelial cells (HUVEC) exhibited significant levels of apoptosis after serum deprivation. HGF inhibited apoptosis in a dose dependent manner in serum-deprived cultures. HGF induced the phosphorylation of Akt and Erk1/2, cell survival factors, in a time dependent manner in serum deprived HUVEC. Inhibition of Akt and Erk1/2 activation abolished the anti-apoptotic effects of HGF. The transcription factor, NF-kappaB, can also play a role in promoting cell survival. However, NF-kappaB does not appear to contribute to the anti-apoptotic properties of HGF, as nuclear translocation of NF-kappaB was not detected in HGF-treated cultures. Endothelial cell migration, proliferation, and apoptosis contribute to the pathogenesis of atherosclerosis, and HGF may play a role in the development and progression of vascular lesions. Immunohistochemical analysis of human carotid artery sections demonstrated HGF protein localization within atherosclerotic lesions but not in normal vessels, suggesting that HGF may participate in atherogenesis.

HIV‐1 tat protein induces a migratory phenotype in human fetal microglia by a CCL2 (MCP‐1)‐dependent mechanism: Possible role in NeuroAIDS

Acquired immune deficiency syndrome (AIDS) encephalitis and dementia are characterized by neuronal loss, astrogliosis, and microglia activation and migration that contribute to the formation of multinucleated giant cells. Despite extensive evidence of pathological changes in the brain of infected individuals, the mechanisms of human immune deficiency virus type 1 (HIV‐1) entry, microglia migration, and viral propagation within the brain are still not completely understood. In this study, we report that the induction of a migratory phenotype in human fetal microglia by the HIV‐1 transactivator protein, tat, is mediated by the chemokine, CCL2. CCL2 or tat treatment alone induced rearrangement of actin and the formation of microglial processes. The time course of cell membrane ruffling induced by CCL2 was faster (5–30 min) than that elicited by tat treatment (2–3 h). Our previous data in human fetal microglia showed that tat induces CCL2 expression. Thus, we examined whether tat‐induced microglia membrane ruffling and process formation, critical components in cell migration, are mediated by the secretion of CCL2 by these cells. To test this hypothesis, we treated microglia with tat protein in the presence of neutralizing CCL2 antibodies. Co‐treatment with neutralizing CCL2 antibodies resulted in the loss of tat‐induced membrane ruffling. Tat treatment of microglia induced polarization of CCR2, the receptor for CCL2, to the leading edge of processes, further suggesting a CCL2‐dependent mechanism of tat‐induced microglia migration. Our data indicate that tat facilitates microglia migration by inducing autocrine CCL2 release. Our results suggest that tat induced CCL2 secretion may be one of the early signals during NeuroAIDS.

Neuroimmunity and the blood-brain barrier: molecular regulation of leukocyte transmigration and viral entry into the nervous system with a focus on neuroAIDS.

HIV infection of the central nervous system (CNS) can result in neurologic dysfunction with devastating consequences in a significant number of individuals with AIDS. Two main CNS complications in individuals with HIV are encephalitis and dementia, which are characterized by leukocyte infiltration into the CNS, microglia activation, aberrant chemokine expression, blood–brain barrier (BBB) disruption, and eventual damage and/or loss of neurons. One of the major mediators of NeuroAIDS is the transmigration of HIV-infected leukocytes across the BBB into the CNS. This review summarizes new key findings that support a critical role of the BBB in regulating leukocyte transmigration. In addition, we discuss studies on communication among cells of the immune system, BBB, and the CNS parenchyma, and suggest how these interactions contribute to the pathogenesis of NeuroAIDS. We also describe some of the animal models that have been used to study and characterize important mechanisms that have been proposed to be involved in HIV-induced CNS dysfunction. Finally, we review the pharmacologic interventions that address neuroinflammation, and the effect of substance abuse on HIV-1 related neuroimmunity.