Transcriptional Changes in CD16+ Monocytes May Contribute to the Pathogenesis of COVID-19.

The COVID-19 pandemic has caused more than three million deaths globally. The severity of the disease is characterized, in part, by a dysregulated immune response. CD16+ monocytes are innate immune cells involved in inflammatory responses to viral infections, and tissue repair, among other functions. We characterized the transcriptional changes in CD16+ monocytes from PBMC of people with COVID-19, and from healthy individuals using publicly available single cell RNA sequencing data. CD16+ monocytes from people with COVID-19 compared to those from healthy individuals expressed transcriptional changes indicative of increased cell activation, and induction of a migratory phenotype. We also analyzed COVID-19 cases based on severity of the disease and found that mild cases were characterized by upregulation of interferon response and MHC class II related genes, whereas the severe cases had dysregulated expression of mitochondrial and antigen presentation genes, and upregulated inflammatory, cell movement, and apoptotic gene signatures. These results suggest that CD16+ monocytes in people with COVID-19 contribute to a dysregulated host response characterized by decreased antigen presentation, and an elevated inflammatory response with increased monocytic infiltration into tissues. Our results show that there are transcriptomic changes in CD16+ monocytes that may impact the functions of these cells, contributing to the pathogenesis and severity of COVID-19.

The impact of substance abuse on HIV-mediated neuropathogenesis in the current ART era.

Approximately 37 million people worldwide are infected with human immunodeficiency virus (HIV). One highly significant complication of HIV infection is the development of HIV-associated neurocognitive disorders (HAND) in 15-55% of people living with HIV (PLWH), that persists even in the antiretroviral therapy (ART) era. The entry of HIV into the central nervous system (CNS) occurs within 4-8 days after peripheral infection. This establishes viral reservoirs that may persist even in the presence of ART. Once in the CNS, HIV infects resident macrophages, microglia, and at low levels, astrocytes. In response to chronic infection and cell activation within the CNS, viral proteins, inflammatory mediators, and host and viral neurotoxic factors produced over extended periods of time result in neuronal injury and loss, cognitive deficits and HAND. Substance abuse is a common comorbidity in PLWH and has been shown to increase neuroinflammation and cognitive disorders. Additionally, it has been associated with poor ART adherence, and increased viral load in the cerebrospinal fluid (CSF), that may also contribute to increased neuroinflammation and neuronal injury. Studies have examined mechanisms that contribute to neuroinflammation and neuronal damage in PLWH, and how substances of abuse exacerbate these effects. This review will focus on how substances of abuse, with an emphasis on methamphetamine (meth), cocaine, and opioids, impact blood brain barrier (BBB) integrity and transmigration of HIV-infected and uninfected monocytes across the BBB, as well as their effects on monocytes/macrophages, microglia, and astrocytes within the CNS. We will also address how these substances of abuse may contribute to HIV-mediated neuropathogenesis in the context of suppressive ART. Additionally, we will review the effects of extracellular dopamine, a neurotransmitter that is increased in the CNS by substances of abuse, on HIV neuropathogenesis and how this may contribute to neuroinflammation, neuronal insult, and HAND in PLWH with active substance use. Lastly, we will discuss some potential therapies to limit CNS inflammation and damage in HIV-infected substance abusers.

CCR2 on Peripheral Blood CD14+CD16+ Monocytes Correlates with Neuronal Damage, HIV-Associated Neurocognitive Disorders, and Peripheral HIV DNA: reseeding of CNS reservoirs?

HIV-associated neurocognitive disorders (HAND) occur in ~50% of HIV infected individuals despite combined antiretroviral therapy. Transmigration into the CNS of CD14+CD16+ monocytes, particularly those that are HIV infected and express increased surface chemokine receptor CCR2, contributes to neuroinflammation and HAND. To examine whether in HIV infected individuals CCR2 on CD14+CD16+ monocytes serves as a potential peripheral blood biomarker of HAND, we examined a cohort of 45 HIV infected people. We correlated CCR2 on CD14+CD16+ monocytes with cognitive status, proton magnetic resonance spectroscopy (1H-MRS) measured neurometabolite levels, and peripheral blood mononuclear cell (PBMC) HIV DNA copies. We determined that CCR2 was increased specifically on CD14+CD16+ monocytes from people with HAND (median [interquartile range (IQR)]) (63.3 [51.6, 79.0]), compared to those who were not cognitively impaired (38.8 [26.7, 56.4]) or those with neuropsychological impairment due to causes other than HIV (39.8 [30.2, 46.5]). CCR2 was associated with neuronal damage, based on the inverse correlation of CCR2 on CD14+CD16+ monocytes with total N-Acetyl Aspartate (tNAA)/total Creatine (tCr) (r2 = 0.348, p = 0.01) and Glutamine-Glutamate (Glx)/tCr (r2 = 0.356, p = 0.01) in the right and left caudate nucleus, respectively. CCR2 on CD14+CD16+ monocytes also correlated with PBMC HIV DNA copies (ρ = 0.618, p = 0.02) that has previously been associated with HAND. These findings suggest that CCR2 on CD14+CD16+ monocytes may be a peripheral blood biomarker of HAND, indicative of increased HIV infected CD14+CD16+ monocyte entry into the CNS that possibly increases the macrophage viral reservoir and contributes to HAND.

Frontline Science: CXCR7 mediates CD14+CD16+ monocyte transmigration across the blood brain barrier: a potential therapeutic target for NeuroAIDS.

CD14+CD16+ monocytes transmigrate into the CNS of HIV-positive people in response to chemokines elevated in the brains of infected individuals, including CXCL12. Entry of these cells leads to viral reservoirs, neuroinflammation, and neuronal damage. These may eventually lead to HIV-associated neurocognitive disorders. Although antiretroviral therapy (ART) has significantly improved the lives of HIV-infected people, the prevalence of cognitive deficits remains unchanged despite ART, still affecting >50% of infected individuals. There are no therapies to reduce these deficits or to prevent CNS entry of CD14+CD16+ monocytes. The goal of this study was to determine whether CXCR7, a receptor for CXCL12, is expressed on CD14+CD16+ monocytes and whether a small molecule CXCR7 antagonist (CCX771) can prevent CD14+CD16+ monocyte transmigration into the CNS. We showed for the first time that CXCR7 is on CD14+CD16+ monocytes and that it may be a therapeutic target to reduce their entry into the brain. We demonstrated that CD14+CD16+ monocytes and not the more abundant CD14+CD16- monocytes or T cells transmigrate to low homeostatic levels of CXCL12. This may be a result of increased CXCR7 on CD14+CD16+ monocytes. We showed that CCX771 reduced transmigration of CD14+CD16+ monocytes but not of CD14+CD16- monocytes from uninfected and HIV-infected individuals and that it reduced CXCL12-mediated chemotaxis of CD14+CD16+ monocytes. We propose that CXCR7 is a therapeutic target on CD14+CD16+ monocytes to limit their CNS entry, thereby reducing neuroinflammation, neuronal damage, and HIV-associated neurocognitive disorders. Our data also suggest that CCX771 may reduce CD14+CD16+ monocyte-mediated inflammation in other disorders.

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.

Dopamine increases CD14+CD16+ monocyte migration and adhesion in the context of substance abuse and HIV neuropathogenesis.

Drug abuse is a major comorbidity of HIV infection and cognitive disorders are often more severe in the drug abusing HIV infected population. CD14+CD16+ monocytes, a mature subpopulation of peripheral blood monocytes, are key mediators of HIV neuropathogenesis. Infected CD14+CD16+ monocyte transmigration across the blood brain barrier mediates HIV entry into the brain and establishes a viral reservoir within the CNS. Despite successful antiretroviral therapy, continued influx of CD14+CD16+ monocytes, both infected and uninfected, contributes to chronic neuroinflammation and the development of HIV associated neurocognitive disorders (HAND). Drug abuse increases extracellular dopamine in the CNS. Once in the brain, CD14+CD16+ monocytes can be exposed to extracellular dopamine due to drug abuse. The direct effects of dopamine on CD14+CD16+ monocytes and their contribution to HIV neuropathogenesis are not known. In this study, we showed that CD14+CD16+ monocytes express mRNA for all five dopamine receptors by qRT-PCR and D1R, D5R and D4R surface protein by flow cytometry. Dopamine and the D1-like dopamine receptor agonist, SKF38393, increased CD14+CD16+ monocyte migration that was characterized as chemokinesis. To determine whether dopamine affected cell motility and adhesion, live cell imaging was used to monitor the accumulation of CD14+CD16+ monocytes on the surface of a tissue culture dish. Dopamine increased the number and the rate at which CD14+CD16+ monocytes in suspension settled to the dish surface. In a spreading assay, dopamine increased the area of CD14+CD16+ monocytes during the early stages of cell adhesion. In addition, adhesion assays showed that the overall total number of adherent CD14+CD16+ monocytes increased in the presence of dopamine. These data suggest that elevated extracellular dopamine in the CNS of HIV infected drug abusers contributes to HIV neuropathogenesis by increasing the accumulation of CD14+CD16+ monocytes in dopamine rich brain regions.

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.

Monocytes mediate HIV neuropathogenesis: mechanisms that contribute to HIV associated neurocognitive disorders.

HIV infected people are living longer due to the success of combined antiretroviral therapy (cART). However, greater than 40-70% of HIV infected individuals develop HIV associated neurocognitive disorders (HAND) that continues to be a major public health issue. While cART reduces peripheral virus, it does not limit the low level, chronic neuroinflammation that is ongoing during the neuropathogenesis of HIV. Monocyte transmigration across the blood brain barrier (BBB), specifically that of the mature CD14(+)CD16(+) population that is highly susceptible to HIV infection, is critical to the establishment of HAND as these cells bring virus into the brain and mediate the neuroinflammation that persists, even if at low levels, despite antiretroviral therapy. CD14(+)CD16(+) monocytes preferentially migrate into the CNS early during peripheral HIV infection in response to chemotactic signals, including those from CCL2 and CXCL12. Once within the brain, monocytes differentiate into macrophages and elaborate inflammatory mediators. Monocytes/macrophages constitute a viral reservoir within the CNS and these latently infected cells may perpetuate the neuropathogenesis of HIV. This review will discuss mechanisms that mediate transmigration of CD14(+)CD16(+) monocytes across the BBB in the context of HIV infection, the contribution of these cells to the neuropathogenesis of HIV, and potential monocyte/macrophage biomarkers to identify HAND and monitor its progression.

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.

Drug induced increases in CNS dopamine alter monocyte, macrophage and T cell functions: implications for HAND.

Central nervous system (CNS) complications resulting from HIV infection remain a major public health problem as individuals live longer due to the success of combined antiretroviral therapy (cART). As many as 70 % of HIV infected people have HIV associated neurocognitive disorders (HAND). Many HIV infected individuals abuse drugs, such as cocaine, heroin or methamphetamine, that may be important cofactors in the development of HIV CNS disease. Despite different mechanisms of action, all drugs of abuse increase extracellular dopamine in the CNS. The effects of dopamine on HIV neuropathogenesis are not well understood, and drug induced increases in CNS dopamine may be a common mechanism by which different types of drugs of abuse impact the development of HAND. Monocytes and macrophages are central to HIV infection of the CNS and to HAND. While T cells have not been shown to be a major factor in HIV-associated neuropathogenesis, studies indicate that T cells may play a larger role in the development of HAND in HIV infected drug abusers. Drug induced increases in CNS dopamine may dysregulate functions of, or increase HIV infection in, monocytes, macrophages and T cells in the brain. Thus, characterizing the effects of dopamine on these cells is important for understanding the mechanisms that mediate the development of HAND in drug abusers.

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.

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.

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.

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-1alpha) 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.

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-1alpha, 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.

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.

MCP-1/CCL2 protects cardiac myocytes from hypoxia-induced apoptosis by a G(alphai)-independent pathway.

Chemokines, in addition to their chemotactic properties, act upon resident cells within a tissue and mediate other cellular functions. In a previous study, we demonstrated that CCL2 protects cultured mouse neonatal cardiac myocytes from hypoxia-induced cell death. Leukocyte chemotaxis has been shown to contribute to ischemic injury. While the chemoattractant properties of CCL2 have been established, the protective effects of this chemokine suggest a novel role for CCL2 in myocardial ischemia/reperfusion injury. The present study examined the cellular signaling pathways that promote this protection. Treatment of cardiac myocyte cultures with CCL2 protected them from hypoxia-induced apoptosis. This protection was not mediated through the activation of G(alphai) signaling that mediates monocyte chemotaxis. Inhibition of the ERK1/2 signaling pathway abrogated CCL2 protection. Caspase 3 activation and JNK/SAPK phosphorylation were decreased in hypoxic myocytes co-treated with CCL2 as compared to hypoxia only-treated cultures. Expression of the Bcl-2 family proteins, Bcl-xL and Bag-1, was increased in CCL2-treated myocytes subjected to hypoxia. There was also downregulation of Bax protein levels as a result of CCL2 co-treatment. These data suggest that CCL2 cytoprotection and chemotaxis may occur through distinct signaling mechanisms.

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.

Inhibitory effect of brachytherapy on intimal hyperplasia in arteriovenous fistula.

PURPOSE: To determine whether endovascular radiation can inhibit intimal hyperplasia in a swine model of hemodialysis access. MATERIALS AND METHODS: Polytetrafluoroethylene arteriovenous grafts (6 mm in diameter) were placed between the common carotid artery and the external jugular vein bilaterally in 8 pigs. Two days after the surgery, fistulography was performed. Gamma radiation (12 Gy) was delivered endovascularly to one of the grafts at the venous anastomosis by using an iridium(192) source. Thus, the other graft in each pig served as an untreated control. Fistulas were evaluated with fistulography or venography 6 week after radiation. All grafts were then harvested for histological and immunohistochemical examination. RESULTS: Seven grafts on the treated side and 5 grafts on the control side remained patent for at least 6 weeks. Angiography demonstrated that the percentage stenosis at the venous anastomosis was significantly lower for the treated group (15.9 +/- 14.1 versus 32.6 +/- 16.7%, P = 0.045). Histopathologic analyses revealed that the mean intimal area and maximal intimal thickness were significantly lower with reduced smooth muscle cell proliferation at the venous anastomosis on the treated side compared to the control side (0.68 +/- 0.30 versus 1.06 +/- 0.29 mm(2), P = 0.017, and 0.18 +/- 0.08 versus 0.26 +/- 0.07 mm, P = 0.004, respectively). The residual lumen was significantly greater for the treated group (1.59 +/- 0.42 versus 1.06 +/- 0.37 mm(2), P = 0.031). No significant differences were found in the area, nor maximal thickness in the vein either proximal or distal to the anastomosis between the two groups. CONCLUSIONS: In an animal model of hemodialysis access, brachytherapy with iridium(192) delivered 2 days after graft implantation reduces intimal hyperplasia and stenosis at the venous anastomosis. The reduced smooth-muscle cells found in the radiated veins suggests that brachytherapy may exert its effect on neointimal formation by inhibition of smooth muscle cell proliferation.