Lillie Lopez

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.

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.

Opposing Effects Mediated by the Chemokine Receptor CXCR2 on Myocardial Ischemia-Reperfusion Injury Recruitment of Potentially Damaging Neutrophils and Direct Myocardial Protection

Background—The timely reperfusion of ischemic myocardium limits infarction, but components of reperfusion, such as inflammation, may be injurious. The chemokine receptor CXCR2 mediates neutrophil chemotaxis. CXCR2 activation also inhibits hypoxia-induced death of isolated cardiac myocytes. This study assesses whether CXCR2 mediates protection in the intact heart and, if so, the magnitude of this protection relative to CXCR2-mediated chemotaxis of potentially damaging inflammatory cells. Methods and Results—After ischemia-reperfusion in vivo, CXCR2−/− mice exhibited infarcts that were 50.5% smaller (P <0.05) with 44.3% fewer inflammatory cells (P <0.05) than wild type mice. These data suggest that in this model, CXCR2-mediated chemotaxis may be important in myocardial cell death. To isolate the role of CXCR2 specifically on blood cells, adoptive transfer experiments were performed. After ischemia-reperfusion, infarcts were 53.4% smaller (P <0.05) and contained 65.0% fewer inflammatory cells (P <0.05) in lethally irradiated wild type mice reconstituted with CXCR2−/− compared with wild type bone marrow. Thus, CXCR2 on blood cells is important in myocardial damage, most likely because of CXCR2-mediated chemotaxis. To unmask whether CXCR2 mediates direct myocardial protection in the intact heart, wild type and CXCR2−/− hearts were studied in the absence of blood using Langendorff preparations. In this case, infarcts were 19.7% larger in CXCR2−/− than wild type hearts (P <0.05), revealing a novel CXCR2-mediated cardioprotective effect. Conclusions—CXCR2 exerts opposing effects on myocardial viability during ischemia-reperfusion with recruitment of damaging inflammatory cells predominant over direct tissue protection.

Mechanisms of HIV entry into the CNS: increased sensitivity of HIV infected CD14+CD16+ monocytes to CCL2 and key roles of CCR2, JAM-A, and ALCAM in diapedesis.

As HIV infected individuals live longer, the prevalence of HIV associated neurocognitive disorders is increasing, despite successful antiretroviral therapy. CD14+CD16+ monocytes are critical to the neuropathogenesis of HIV as they promote viral seeding of the brain and establish neuroinflammation. The mechanisms by which HIV infected and uninfected monocytes cross the blood brain barrier and enter the central nervous system are not fully understood. We determined that HIV infection of CD14+CD16+ monocytes resulted in their highly increased transmigration across the blood brain barrier in response to CCL2 as compared to uninfected cells, which did not occur in the absence of the chemokine. This exuberant transmigration of HIV infected monocytes was due, at least in part, to increased CCR2 and significantly heightened sensitivity to CCL2. The entry of HIV infected and uninfected CD14+CD16+ monocytes into the brain was facilitated by significantly increased surface JAM-A, ALCAM, CD99, and PECAM-1, as compared to CD14+ cells that are CD16 negative. Upon HIV infection, there was an additional increase in surface JAM-A and ALCAM on CD14+CD16+ monocytes isolated from some individuals. Antibodies to ALCAM and JAM-A inhibited the transmigration of both HIV infected and uninfected CD14+CD16+ monocytes across the BBB, demonstrating their importance in facilitating monocyte transmigration and entry into the brain parenchyma. Targeting CCR2, JAM-A, and ALCAM present on CD14+CD16+ monocytes that preferentially infiltrate the CNS represents a therapeutic strategy to reduce viral seeding of the brain as well as the ongoing neuroinflammation that occurs during HIV pathogenesis.

CCL2 disrupts the adherens junction: implications for neuroinflammation.

Alterations to blood–brain barrier (BBB) adhesion molecules and junctional integrity during neuroinflammation can promote central nervous system (CNS) pathology. The chemokine CCL2 is elevated during CNS inflammation and is associated with endothelial dysfunction. The effects of CCL2 on endothelial adherens junctions (AJs) have not been defined. We demonstrate that CCL2 transiently induces Src-dependent disruption of human brain microvascular endothelial AJ. β-Catenin is phosphorylated and traffics from the AJ to PECAM-1 (platelet endothelial cell adhesion molecule-1), where it is sequestered at the membrane. PECAM-1 is also tyrosine-phosphorylated, an event associated with recruitment of the phosphatase SHP-2 (Src homology 2 domain-containing protein phosphatase) to PECAM-1, β-catenin release from PECAM-1, and reassociation of β-catenin with the AJ. Surface localization of PECAM-1 is increased in response to CCL2. This may enable the endothelium to sustain CCL2-induced alterations in AJ and facilitate recruitment of leukocytes into the CNS. Our novel findings provide a mechanism for CCL2-mediated disruption of endothelial junctions that may contribute to BBB dysfunction and increased leukocyte recruitment in neuroinflammatory diseases.

Microglia from Mice Transgenic for a Provirus Encoding a Monocyte-Tropic HIV Type 1 Isolate Produce Infectious Virus and Display in Vitro and in Vivo Upregulation of Lipopolysaccharide-Induced Chemokine Gene Expression

A large body of evidence has indicated that microglia are the predominant cellular location for HIV-1 in the brains of HIV-1-infected individuals and play a direct role in the development of HIV-1-associated dementia (HAD). Therefore, investigation of the mechanism by which HIV-1-infected microglia contribute to the development of HIV-associated dementia should be facilitated by the creation of a mouse model wherein microglia carry replication-competent HIV-1. To circumvent the inability of HIV-1 to infect mouse cells, we developed a mouse line that is transgenic for a full-length proviral clone of a monocyte-tropic HIV-1 isolate, HIV-1(JR-CSF) (JR-CSF mice), whose T cells and monocytes produce infectious HIV-1. We detected expression of the long terminal repeat-regulated proviral transgene in the microglia of these transgenic mice and demonstrated that it was increased by in vitro and in vivo stimulation with lipopolysaccharide. Furthermore, microglia isolated from JR-CSF mouse brains produced HIV-1 that was infectious in vitro and in vivo. We examined the effect that carriage of the HIV-1 provirus had on chemokine gene regulation in the brains of these mice and demonstrated that MCP-1 gene expression by JR-CSF mouse microglia and brains was more responsive to in vitro and in vivo stimulation with lipopolysaccharide than were microglia and brains from control mice. Thus, this study indicates that the JR-CSF mice may represent a new mouse model to study the effect of HIV-1 replication on microglia function and its contribution to HIV-1-associated neurological disease.

Lipid metabolites of the phospholipase A2 pathway and inflammatory cytokines are associated with brain volume in paediatric cerebral malaria.

BackgroundCerebral malaria (CM) remains a significant cause of morbidity and mortality in children in sub-Saharan Africa. CM mortality has been associated with increased brain volume, seen on neuroimaging studies.MethodsTo examine the potential role of blood metabolites and inflammatory mediators in increased brain volume in Malawian children with CM, an association study was performed between plasma metabolites, cytokine levels and phospholipase A2 (PLA2) activity with brain volume.ResultsThe metabolomics analysis demonstrated arachidonic acid and other lysophospholipids to be positively associated with brain swelling. These lipids are products of the PLA2 enzyme and an association of plasma PLA2 enzymatic activity with brain swelling was confirmed. TNFα, which can upregulate PLA2 activity, was associated with brain volume. In addition, CCL2 and IL-8 were also associated with brain volume. Some of these cytokines can alter endothelial cell tight junction proteins and increase blood brain barrier permeability.ConclusionsTaken together, paediatric CM brain volume was associated with products of the PLA2 pathway and inflammatory cytokines. Their role in causality is unknown. These molecules will need to undergo testing in vitro and in animal models to understand their role in processes of increased brain volume. These observations provide novel data on host physiology associated with paediatric CM brain swelling, and may both inform pathogenesis models and suggest adjunct therapies that could improve the morbidity and mortality associated with paediatric CM.

Buprenorphine Decreases the CCL2-Mediated Chemotactic Response of Monocytes

Despite successful combined antiretroviral therapy, ∼60% of HIV-infected people exhibit HIV-associated neurocognitive disorders (HAND). CCL2 is elevated in the CNS of infected people with HAND and mediates monocyte influx into the CNS, which is critical in neuroAIDS. Many HIV-infected opiate abusers have increased neuroinflammation that may augment HAND. Buprenorphine is used to treat opiate addiction. However, there are few studies that examine its impact on HIV neuropathogenesis. We show that buprenorphine reduces the chemotactic phenotype of monocytes. Buprenorphine decreases the formation of membrane projections in response to CCL2. It also decreases CCL2-induced chemotaxis and mediates a delay in reinsertion of the CCL2 receptor, CCR2, into the cell membrane after CCL2-mediated receptor internalization, suggesting a mechanism of action of buprenorphine. Signaling pathways in CCL2-induced migration include increased phosphorylation of p38 MAPK and of the junctional protein JAM-A. We show that buprenorphine decreases these phosphorylations in CCL2-treated monocytes. Using DAMGO, CTAP, and Nor-BNI, we demonstrate that the effect of buprenorphine on CCL2 signaling is opioid receptor mediated. To identify additional potential mechanisms by which buprenorphine inhibits CCL2-induced monocyte migration, we performed proteomic analyses to characterize additional proteins in monocytes whose phosphorylation after CCL2 treatment was inhibited by buprenorphine. Leukosialin and S100A9 were identified and had not been shown previously to be involved in monocyte migration. We propose that buprenorphine limits CCL2-mediated monocyte transmigration into the CNS, thereby reducing neuroinflammation characteristic of HAND. Our findings underscore the use of buprenorphine as a therapeutic for neuroinflammation as well as for addiction.

Dopamine Increases CD14(+)CD16(+) Monocyte Transmigration across the Blood Brain Barrier: Implications for Substance Abuse and HIV Neuropathogenesis.

In human immunodeficiency virus-1 (HIV) infected individuals, substance abuse may accelerate the development and/or increase the severity of HIV associated neurocognitive disorders (HAND). It is proposed that CD14+CD16+ monocytes mediate HIV entry into the central nervous system (CNS) and that uninfected and infected CD14+CD16+ monocyte transmigration across the blood brain barrier (BBB) contributes to the establishment and propagation of CNS HIV viral reservoirs and chronic neuroinflammation, important factors in the development of HAND. The effects of substance abuse on the frequency of CD14+CD16+ monocytes in the peripheral circulation and on the entry of these cells into the CNS during HIV neuropathogenesis are not known. PBMC from HIV infected individuals were analyzed by flow cytometry and we demonstrate that the frequency of peripheral blood CD14+CD16+ monocytes in HIV infected substance abusers is increased when compared to those without active substance use. Since drug use elevates extracellular dopamine concentrations in the CNS, we examined the effects of dopamine on CD14+CD16+ monocyte transmigration across our in vitro model of the human BBB. The transmigration of this monocyte subpopulation is increased by dopamine and the dopamine receptor agonist, SKF 38393, implicating D1-like dopamine receptors in the increase in transmigration elicited by this neurotransmitter. Thus, elevated extracellular CNS dopamine may be a novel common mechanism by which active substance use increases uninfected and HIV infected CD14+CD16+ monocyte transmigration across the BBB. The influx of these cells into the CNS may increase viral seeding and neuroinflammation, contributing to the development of HIV associated neurocognitive impairments.