Dysregulated interferon-gamma responses during lethal cytomegalovirus brain infection of IL-10-deficient mice.

Murine cytomegalovirus (MCMV) brain infection induces a transient increase in chemokine production, which precedes the infiltration of CD3(+) lymphocytes. In this study, we hypothesized that an absence of anti-inflammatory cytokines would result in sustained proinflammatory neuroimmune responses. Direct intracerebroventricular injection of MCMV into IL-10 knockout (KO) mice produced an unexpected result: while wild-type animals controlled MCMV, the infection was lethal in IL-10 KO animals. Identical infection of IL-4 KO animals did not produce lethal disease. To further characterize the role of IL-10, infected brain tissue from both wild-type and IL-10 KO animals was assessed for cytokine and chemokine levels, as well as viral gene expression. These data show vastly elevated levels of interferon (IFN)-gamma, and the IFN-gamma-inducible chemokines CXCL9 and CXCL10, as well as IL-6 in brain homogenates obtained from IL-10 KO animals. However, MCMV viral load, glycoprotein B mRNA levels, and titers of infectious virus were similar in both IL-10 KO and wild-type animals. Separation of cells isolated from murine brain tissue into distinct populations using FACS, along with subsequent quantitative RT real-time PCR, showed that brain-infiltrating CD45(hi)/CD11b(-) and CD45(hi)/CD11b(int) were the cellular source of IL-10 in the brain. Taken together, these data demonstrate that MCMV brain infection of IL-10-deficient mice causes lethal disease, which occurs in the presence of a dysregulated IFN-gamma-mediated neuroimmune response.

Cocaine-induced HIV-1 expression in microglia involves sigma-1 receptors and transforming growth factor-beta1.

The neuropharmacological properties of cocaine are known to be associated with the activation of sigma-1 receptors. Cocaine also has been shown to alter both cytokine production and HIV-1 expression in mononuclear phagocytes, including microglial cells. This study tested the hypothesis that sigma-1 receptors and transforming growth factor (TGF)-beta1 are involved in cocaine-induced up-regulation of HIV-1 expression in microglial cell cultures. Treatment of microglial cells with cocaine resulted in a concentration-dependent increase in viral expression assessed by measurement of p24 antigen levels in culture supernatants. This cocaine-mediated stimulation of HIV-1 expression was blocked by treatment of microglia with inhibitors of sigma-1 receptors (BD1047) and TGF-beta1 (SB-431542 and anti-TGF-beta1 antibodies). Microglia were also shown to constitutively express sigma-1 receptor mRNA. Thus, the results of this study support the notion that neuroimmunopharmacological properties of cocaine involve sigma-1 receptors and cytokines.

Anti-HIV-1 activity of propolis in CD4(+) lymphocyte and microglial cell cultures.

An urgent need for additional agents to treat human immunodeficiency virus type 1 (HIV-1) infection led us to assess the anti-HIV-1 activity of the natural product propolis in CD4(+) lymphocytes and microglial cell cultures. Propolis inhibited viral expression in a concentration-dependent manner (maximal suppression of 85 and 98% was observed at 66.6 microg/ml propolis in CD4(+) and microglial cell cultures, respectively). Similar anti-HIV-1 activity was observed with propolis samples from several geographic regions. The mechanism of propolis antiviral property in CD4(+) lymphocytes appeared to involve, in part, inhibition of viral entry. While propolis had an additive antiviral effect on the reverse transcriptase inhibitor zidovudine, it had no noticeable effect on the protease inhibitor indinavir. The results of this in vitro study support the need for clinical trials of propolis or one or more of its components in the treatment of HIV-1 infection.

Kappa-opioid receptor agonist inhibition of HIV-1 envelope glycoprotein-mediated membrane fusion and CXCR4 expression on CD4(+) lymphocytes.

Our previous studies have shown that the suppressive effect of kappa-opioid receptor (KOR) ligand treatment on HIV-1(AT) (a T-tropic strain) expression in acutely infected CD4(+) lymphocytes is time-dependent. This finding implied that the inhibition observed following treatment with KOR agonists such as U50,488 (trans-3,4-dichloro-N-methyl-N[2-(1-pyrolidinyl)cyclohexyl]benzeneaceamide methanesulfonate) occurs at an early step in the viral replication cycle, perhaps as early as viral entry. In the present study, we examined the hypothesis that U50,488 treatment of CD4(+) lymphocytes inhibits HIV-1 envelope (Env) glycoprotein-mediated membrane fusion. We used a vaccinia virus-based assay to measure the effects of U50,488 treatment of CD4(+) lymphocytes on HIV-1 IIIB Env glycoprotein-mediated fusogenic activity, based on the cytoplasmic activation of a reporter gene. The results show that U50,488 inhibited Env-mediated cell fusion in a bell-shaped concentration-response manner with suppression ranging between 31 and 98% at concentrations of 10(-8) and 10(-10)M (N=9 experiments). U50,488 was also found to inhibit cell fusion when monitored in situ with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining. Blockade of the inhibitory activity of U50,488 by the KOR antagonist nor-bialtorphimine (nor-BNI) suggested that U50,488 was acting via a KOR-related mechanism. Using flow cytometry, we demonstrated that the chemokine co-receptor CXCR4, but not CD4, is down-regulated as a consequence of KOR activation, with 44.2+/-3.5% suppression at 10(-10)M U50,488. These findings support the hypothesis that KOR-related activation of CD4(+) lymphocytes inhibits HIV-1 entry via down-regulation of CXCR4.

Potentiation of HIV-1 expression in microglial cells by nicotine: involvement of transforming growth factor-beta 1.

HIV-1 infection and nicotine addiction are global public health crises. In the central nervous system, HIV-1 causes a devastating neurodegenerative disease. It is well recognized that microglial cells play a pivotal role in the neuropathogenesis of HIV-1 and that drugs of abuse not only contribute to the spread of this agent but may facilitate viral expression in these brain macrophages. Nicotine has been shown to stimulate the production of HIV-1 by in vitro-infected alveolar macrophages, and the HIV-1 protein gp120 binds to nicotinic receptors. In this study, we demonstrated the constitutive expression of nicotinic acetylcholine receptor mRNA in primary human microglial cells and showed that the pretreatment of microglia with nicotine increased HIV-1 expression in a concentration-dependent manner, as measured by p24 antigen levels in culture supernatants. We also found that nicotine robustly altered the gene expression profile of HIV-1-infected microglia and that the transforming growth factor-beta1 is involved in the enhanced expression of HIV-1 by nicotine.

Human neural precursor cells express functional kappa-opioid receptors.

Neural stem cells (NSCs) play an important role in the developing as well as adult brain. NSCs have been shown to migrate toward sites of injury in the brain and to participate in the process of brain repair. Like NSCs, cultured human neural precursor cells (NPCs) are self-renewing, multipotent cells capable of differentiating into neurons, astrocytes, and oligodendrocytes and of migrating toward chemotactic stimuli. Cellular and environmental factors are important for NPC proliferation and migration. Expression of kappa-opioid receptors (KORs) and mu-opioid receptors (MORs) in murine embryonic stem cells and of MORs and delta-opioid receptors in rodent neuronal precursors, as well as hippocampal progenitors has been reported by other investigators. In this study, we demonstrated robust expression of KORs in highly enriched (>90% nestin-positive) human fetal brain-derived NPCs. We found that KOR ligands, dynorphin(1-17) and trans-3,4-dichloro-N-methyl-N[2-(1-pyrolidinyl)cyclohexyl] benzeneacetamide methanesulfonate (U50,488) but not dynorphin(2-17), stimulated proliferation and migration of NPCs in a concentration-dependent manner. NPC proliferation was maximally stimulated at 10(-14) M dynorphin(1-17) and 10(-12) M U50,488. The KOR selective antagonist, nor-binaltorphimine, partially blocked the migratory and proliferative effects of KOR agonists supporting, at least in part, the involvement of a KOR-related mechanism. As has been described for rodent P19 embryonal carcinoma stem cells, retinoic acid treatment markedly suppressed KOR mRNA expression in human NPCs. Taken together, the results of this study suggest that activation of KORs alters functional properties of NPCs/NSCs that are relevant to human brain development and repair.

WIN55,212-2-mediated inhibition of HIV-1 expression in microglial cells: involvement of cannabinoid receptors.

Cannabinoid receptors CB(1) and CB(2) are primarily expressed in cells of the nervous and immune systems, respectively. Recently, the synthetic CB(1)/CB(2) agonist WIN55,212-2 was found to suppress replication of HIV-1 in microglial cell cultures. The present study was undertaken to test the hypothesis that WIN55,212-2's antiviral effect is mediated via CB(2) receptors. By reverse transcription-polymerase chain reaction, microglia were found to express both CB(1) and CB(2) receptors. Using additional CB(1)/CB(2) receptor agonists and selective antagonists, we found that CB(2) receptors are involved in WIN55,212-2's antiviral activity and surprisingly that the CB(1) receptor-selective antagonist SR141716A behaved as an agonist in these brain macrophages.

Differential apoptotic signaling in primary glial cells infected with herpes simplex virus 1.

Microglial cells and astrocytes are glial cell types that perform distinct functions and generate innate immune responses to counter invading pathogens. Herpes simplex virus 1 (HSV-1) is a neurotropic virus that is capable of causing severe, necrotizing encephalitis. HSV-1 infects both of these glial cell types. Microglial cells undergo an abortive infection, yet respond to viral infection by inducing a burst of proinflammatory cytokine and chemokine production. Following this cytokine burst, they rapidly succumb to cell death. In contrast, astrocytes do permit productive viral replication, but do not generate these same innate immune mediators. Although apoptosis has been implicated in a number of acute and chronic neurological disorders, little is known about apoptosis during viral encephalitis. In the present study, the authors investigated the effect of HSV-1 infection on cell survival and studied the mechanisms of cell-death in virus-infected, primary murine glial cells. The authors report that although apoptosis occurred rapidly in microglia, it was delayed during productive infection of astrocytes. Furthermore, microarray studies revealed significant variations in the expression of apoptotic genes between these two types of glial cells, indicating crucial differences in signaling pathways. Intrinsic as well as extrinsic pathways of apoptosis were found to be activated in both glial cell types. Specifically, genes involved in the tumor necrosis factor (TNF) signaling pathway were predominantly up-regulated in microglia, whereas genes of the Fas pathway were induced during HSV infection of astrocytes.

T cell-mediated restriction of intracerebral murine cytomegalovirus infection displays dependence upon perforin but not interferon-gamma.

The authors have previously reported that adoptive transfer of splenocytes suppresses murine cytomegalovirus (MCMV) brain infection following intracerebroventricular injection of immunodeficient mice and that depletion of Thy 1.2+ T lymphocytes abolishes this suppressive effect. Here the authors report that splenocytes depleted of CD4+ T lymphocytes prior to adoptive transfer retained their ability to control viral expression in the brain. In sharp contrast, depletion of the CD8+ T-cell population prior to transfer abolished the suppressive effect, with sixfold greater expression in the brain than when undepleted splenocytes were used. The authors went on to examine the contributions of cytokine- and perforin-mediated mechanisms in controlling MCMV brain infection using splenocytes from major histocompatibility (MHC)-matched IFN-gamma -knockout (GKO), and perforin-knockout (PKO) mice. When used in adoptive transfer studies, splenocytes from GKO mice controlled viral expression; however, cells from PKO mice could not control reporter gene expression or viral DNA replication in brain tissues. The authors have previously reported that the levels of the T-cell chemoattractant CXCL10 are highly elevated in the brains of MCMV-infected mice. Here the authors found that the receptor for this ligand, CXCR3, was not essential in mediating the suppressive effects of adoptive transfer. These data indicate that peripheral CD8+ T cells control MCMV brain infection through a perforin-mediated mechanism and that neither IFN-gamma nor CXCR3 play a critical role in this neuroprotective response.

Mycobacterium tuberculosis-induced cytokine and chemokine expression by human microglia and astrocytes: effects of dexamethasone.

Although corticosteroids are recommended as adjunctive therapy for tuberculous meningitis, the mechanism underlying their beneficial effect is poorly understood. In this study, human microglia and astrocytes were infected with Mycobacterium tuberculosis H37Rv, and cytokine and chemokine expression was examined with and without dexamethasone treatment. Microglia were the principal cells infected by tubercle bacilli, which elicited robust amounts of several cytokines and chemokines. Treatment with dexamethasone markedly suppressed production of these mediators. The results of this study support the concept that microglia play an important role in neuropathogenesis of tuberculosis and that dexamethasone could operate via modulation of the production of proinflammatory cytokines and chemokines by these brain macrophages.

Glial cell responses to herpesvirus infections: role in defense and immunopathogenesis.

Glial cells can respond to herpesvirus infections through the production of cytokines and chemokines. Although specific interactions between resident glia and lymphocytes that infiltrate the infected brain remain to be defined, the presence of T cell chemotactic signals in microglial cell supernatants following infection with cytomegalovirus or herpes simplex virus has led to the concept that chemokines initiate a cascade of neuroimmune responses that result in defense of the brain against herpesviruses. While chemokines may play a defensive role by attracting T cells into the brain, aberrant accumulation of lymphocytes may also induce brain damage. Host defense mechanisms must balance control of herpesvirus spread with associated undesirable immunopathologic effects. A growing body of evidence suggests that through complex networks of chemokines and cytokines produced in response to herpesvirus infection, glial cells orchestrate a cascade of events that result in successful defense of or damage to the brain.

Role of microglia in central nervous system infections.

The nature of microglia fascinated many prominent researchers in the 19th and early 20th centuries, and in a classic treatise in 1932, Pio del Rio-Hortega formulated a number of concepts regarding the function of these resident macrophages of the brain parenchyma that remain relevant to this day. However, a renaissance of interest in microglia occurred toward the end of the 20th century, fueled by the recognition of their role in neuropathogenesis of infectious agents, such as human immunodeficiency virus type 1, and by what appears to be their participation in other neurodegenerative and neuroinflammatory disorders. During the same period, insights into the physiological and pathological properties of microglia were gained from in vivo and in vitro studies of neurotropic viruses, bacteria, fungi, parasites, and prions, which are reviewed in this article. New concepts that have emerged from these studies include the importance of cytokines and chemokines produced by activated microglia in neurodegenerative and neuroprotective processes and the elegant but astonishingly complex interactions between microglia, astrocytes, lymphocytes, and neurons that underlie these processes. It is proposed that an enhanced understanding of microglia will yield improved therapies of central nervous system infections, since such therapies are, by and large, sorely needed.

Intracerebral infection with murine cytomegalovirus induces CXCL10 and is restricted by adoptive transfer of splenocytes.

The brain's intrinsic immune system consists of glial cells that produce cytokines and chemokines in response to stimulation with cytomegalovirus (CMV). The present experiments were undertaken to determine whether this intrinsic glial cell response alone is sufficient to control CMV infection of the central nervous system (CNS) or whether effector cells from the somatic immune system are also required. Following stereotactic, intracerebroventricular (icv), injection of murine cytomegalovirus (MCMV) into immunocompetent (C.B-17) mice, viral spread in the brain was limited to the cells of the ventricular walls and the infection was resolved by 10 days post infection (p.i.). In contrast, icv infection of immunodeficient (C.B-17 SCID/bg) mice resulted in viral spread from the ventricles throughout the brain parenchyma and these mice succumbed to lethal disease. Adoptive transfer of total splenocytes from major histocompatibility complex (MHC)-matched, MCMV-primed animals restricted intracerebral viral infection to the periventricular cells and reduced levels of reporter gene expression from the viral genome. Peripheral immune cell transfer also protected immunodeficient animals from lethal disease. Depletion of Thy 1.2(+) cells from MCMV-primed splenocytes abolished the protective effect of adoptive transfer. Viral expression was found to be fourfold greater in the brains of animals given Thy 1.2-depleted splenocytes than from those receiving total undepleted cells. As MCMV infection proceeded in the brains of immunodeficient mice, levels of the T-cell chemoattractants CXCL10 and CCL2 remained elevated, whereas CXCL10 levels waned in the brains of animals receiving transferred splenocytes. Taken together, these results demonstrate the ability of T lymphocytes to restrict intracerebral viral spread and indicate that intrinsic glial cell responses alone are insufficient to control MCMV brain infection.

Kappa-opioid receptor ligands inhibit cocaine-induced HIV-1 expression in microglial cells.

Cocaine abuse has been implicated as a cofactor in human immunodeficiency virus (HIV)-1-associated dementia (HAD). In this study, we tested the hypothesis that exposure of microglial cells, the resident macrophages of the brain, to cocaine would potentiate HIV-1 expression. Because kappa-opioid receptor (KOR) agonists have been shown to suppress neurochemical and neurobehavioral responses to cocaine and to inhibit HIV-1 expression in microglial cell cultures, we also postulated that KOR ligands would inhibit cocaine-induced potentiation of HIV-1 expression. Human microglial cells were infected with HIV-1(SF162), an R5 isolate, and viral expression was quantified by measurement of p24 antigen in culture supernatants. Treatment of microglia with the KOR agonists trans-(+/-)-3,4-dichlor-N-methyl-N-(2[1-pyrrolidnyl])benzeneacetamide methanesulfonate and 8-carboxamidocyclazocine inhibited viral expression (maximal suppression of 42 and 48%, respectively). Consistent with the hypotheses, treatment of microglia with cocaine promoted HIV-1 expression (maximal enhancement of 54%), and pretreatment of microglia with these KOR agonists as well as with the KOR-selective antagonist nor-binaltorphimine abrogated cocaine-induced potentiation of viral expression. Results of flow cytometry studies suggested that the mechanism whereby KOR ligands inhibit cocaine's stimulatory effect on viral expression involves the suppression of cocaine-induced activation of extracellular signal-regulated kinase1/2, thereby blunting cocaine-enhanced up-regulation of the HIV-1 entry chemokine coreceptor CCR5. The findings of this study suggest that in addition to its neurotoxic effects, cocaine could foster development of HAD by potentiating viral expression in the brain and that this phenomenon is inhibited by KOR ligands.