R. Bryan Rock

Role of microglia in central nervous system infections.

SUMMARY 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.

Central Nervous System Tuberculosis: Pathogenesis and Clinical Aspects

SUMMARY Tuberculosis of the central nervous system (CNS) is a highly devastating form of tuberculosis, which, even in the setting of appropriate antitubercular therapy, leads to unacceptable levels of morbidity and mortality. Despite the development of promising molecular diagnostic techniques, diagnosis of CNS tuberculosis relies largely on microbiological methods that are insensitive, and as such, CNS tuberculosis remains a formidable diagnostic challenge. Insights into the basic neuropathogenesis of Mycobacterium tuberculosis and the development of an appropriate animal model are desperately needed. The optimal regimen and length of treatment are largely unknown, and with the rising incidence of multidrug-resistant strains of M. tuberculosis, the development of well-tolerated and effective antibiotics remains a continued need. While the most widely used vaccine in the world largely targets this manifestation of tuberculosis, the BCG vaccine has not fulfilled the promise of eliminating CNS tuberculosis. We put forth this review to highlight the current understanding of the neuropathogenesis of M. tuberculosis, to discuss certain epidemiological, clinical, diagnostic, and therapeutic aspects of CNS tuberculosis, and also to underscore the many unmet needs in this important field.

Microglia as a pharmacological target in infectious and inflammatory diseases of the brain.

Following an eclipse of scientific inquiry regarding the biology of microglia that lasted 50 years, recognition toward the end of the 20th century of their neuropathogenic role in HIV-associated dementia and in neuroinflammatory/neurodegenerative diseases fueled a renaissance of interest in these resident macrophages of the brain parenchyma. Results of a large number of in vitro studies, using isolated microglial cells or glial/neuronal cell cultures, and parallel findings emerging from animal models and clinical studies have demonstrated that activated microglia produce a myriad of inflammatory mediators that both serve important defense functions against invading neurotropic pathogens and have been implicated in brain damage in infectious as well as neuroinflammatory/neurodegenerative diseases, such as multiple sclerosis, Alzheimers disease, Parkinsons disease, and amyotrophic lateral sclerosis. This review provides a brief background regarding the physiological and pathophysiological roles of microglia and highlights current pharmacological approaches that target activated microglia with the goal of ameliorating infectious and neuroinflammatory/neurodegenerative diseases of the brain. Although this aspect of the field of neuroimmunopharmacology is in its infancy, it holds great promise for developing new treatments and prevention of diseases that are, in many cases, epidemic throughout the world.

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.

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

Cannabinoid receptors CB1 and CB2 are primarily expressed in cells of the nervous and immune systems, respectively. Recently, the synthetic CB1/CB2 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 CB2 receptors. By reverse transcription-polymerase chain reaction, microglia were found to express both CB1 and CB2 receptors. Using additional CB1/CB2 receptor agonists and selective antagonists, we found that CB2 receptors are involved in WIN55,212-2’s antiviral activity and surprisingly that the CB1 receptor-selective antagonist SR141716A behaved as an agonist in these brain macrophages.

Potentiation of HIV-1 Expression in Microglial Cells by Nicotine: Involvement of Transforming Growth Factor-β1

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-β1 is involved in the enhanced expression of HIV-1 by nicotine.

Morphine stimulates CCL2 production by human neurons

BackgroundSubstances of abuse, such as opiates, have a variety of immunomodulatory properties that may influence both neuroinflammatory and neurodegenerative disease processes. The chemokine CCL2, which plays a pivotal role in the recruitment of inflammatory cells in the nervous system, is one of only a few chemokines produced by neurons. We hypothesized that morphine may alter expression of CCL2 by human neurons.MethodsPrimary neuronal cell cultures and highly purified astrocyte and microglial cell cultures were prepared from human fetal brain tissue. Cell cultures were treated with morphine, and cells were examined by RNase protection assay for mRNA. Culture supernatants were assayed by ELISA for CCL2 protein. β-funaltrexamine (β-FNA) was used to block μ-opioid receptor (MOR)s.ResultsMorphine upregulated CCL2 mRNA and protein in neuronal cultures in a concentration- and time-dependent fashion, but had no effect on CCL2 production in astrocyte or microglial cell cultures. Immunocytochemical analysis also demonstrated CCL2 production in morphine-stimulated neuronal cultures. The stimulatory effect of morphine was abrogated by β-FNA, indicating an MOR-mediated mechanism.ConclusionMorphine stimulates CCL2 production by human neurons via a MOR-related mechanism. This finding suggests another mechanism whereby opiates could affect neuroinflammatory responses.

Preferential sensitivity of human dopaminergic neurons to gp120-induced oxidative damage

The dopamine (DA)-rich midbrain is known to be a key target of human immunodeficiency virus (HIV)-1. Studies of simian immunodeficiency virus (SIV)-induced neuropathogenesis recently established that there is a major disruption within the nigrostriatal dopaminergic system characterized by marked depletion of dopaminergic neurons, microglial cell activation, and reactive astrocytes. Using a human mesencephalic neuronal/glial culture model, which contains dopaminergic neurons, microglia, and astrocytes, experiments were performed to characterize the damage to dopaminergic neurons induced by HIV-1 gp120. Functional impairment was assessed by DA uptake, and neurotoxicity was measured by apoptosis and oxidative damage. Through the use of this mesencephalic neuronal/glial culture model, we were able to identify the relative sensitivity of dopaminergic neurons to gp120-induced damage, manifested as reduced function (decreased DA uptake), morphological changes, and reduced viability. We also showed that gp120-induced oxidative damage is involved in this neuropathogenic process.

CB2 Receptor Agonists Protect Human Dopaminergic Neurons against Damage from HIV-1 gp120

Despite the therapeutic impact of anti-retroviral therapy, HIV-1-associated neurocognitive disorder (HAND) remains a serious threat to AIDS patients, and there currently remains no specific therapy for the neurological manifestations of HIV-1. Recent work suggests that the nigrostriatal dopaminergic area is a critical brain region for the neuronal dysfunction and death seen in HAND and that human dopaminergic neurons have a particular sensitivity to gp120-induced damage, manifested as reduced function (decreased dopamine uptake), morphological changes, and reduced viability. Synthetic cannabinoids inhibit HIV-1 expression in human microglia, suppress production of inflammatory mediators in human astrocytes, and there is substantial literature demonstrating the neuroprotective properties of cannabinoids in other neuropathogenic processes. Based on these data, experiments were designed to test the hypothesis that synthetic cannabinoids will protect dopaminergic neurons against the toxic effects of the HIV-1 protein gp120. Using a human mesencephalic neuronal/glial culture model, which contains dopaminergic neurons, microglia, and astrocytes, we were able to show that the CB1/CB2 agonist WIN55,212-2 blunts gp120-induced neuronal damage as measured by dopamine transporter function, apoptosis and lipid peroxidation; these actions were mediated principally by the CB2 receptor. Adding supplementary human microglia to our cultures enhances gp120-induced damage; WIN55,212-2 is able to alleviate this enhanced damage. Additionally, WIN55,212-2 inhibits gp120-induced superoxide production by purified human microglial cells, inhibits migration of human microglia towards supernatants generated from gp120-stimulated human mesencephalic neuronal/glial cultures and reduces chemokine and cytokine production from the human mesencephalic neuronal/glial cultures. These data suggest that synthetic cannabinoids are capable of protecting human dopaminergic neurons from gp120 in a variety of ways, acting principally through the CB2 receptors and microglia.

Hemin inhibits NO production by IL-1β-stimulated human astrocytes through induction of heme oxygenase-1 and reduction of p38 MAPK activation

BackgroundHeme oxygenase (HO)-1 has been shown to attenuate oxidative injury and reduce apoptosis. HO-1 can be induced by various stimuli released during cellular injury, such as heme. Deleterious free heme is degraded by HO-1 to carbon monoxide, iron and biliverdin, which have potent anti-oxidant and anti-inflammatory properties. In this study, we tested the hypothesis that upregulation of HO-1 would inhibit production of the free radical (NO) by interlukin (IL)-1β-activated human astrocytes.MethodsTo measure NO production, inducible NO synthase (iNOS), HO-1 expression and mitogen-activated protein (MAP) kinase activation we used hemin as an HO-1 inducer and tin protoporphyrin (SnPP) IX as an inhibitor of HO-1 activity in human astrocyte cultures prior to IL-1β exposure. Transfection of astrocyte cultures was performed using a pLEX expression vector carrying the human HO-1 sequence prior to IL-1β treatment. Supernatants of astrocyte cultures pretreated with inhibitors of p38 MAPK or MEK1/2 prior to IL-1β exposure were collected for NO assay.ResultsIL-1β treatment of astrocytes alone induced undetectable amounts of HO-1 protein by western blot. However, HO-1 mRNA expression was modestly up-regulated in response to IL-1β stimulation. Pretreatment with hemin alone substantially induced both HO-1 mRNA and protein expression, and HO-1 mRNA expression was further enhanced when hemin was combined with IL-1β treatment. In contrast, IL-1β-induced iNOS mRNA expression and NO production were markedly inhibited by hemin treatment. When pretreated with SnPP, the inhibitory effect of hemin on IL-1β-induced NO production and iNOS expression was reversed, suggesting the involvement of HO-1. IL-1β-induced p38 MAPK activation, which is known to be required for NO production, was also down-regulated by hemin.ConclusionThese findings support the hypothesis that up-regulation of HO-1 in astrocytes is associated with down-regulation of iNOS expression and thereby NO production, an effect that involves the p38 MAPK signaling pathway, which suggests that this glial cell response could play an important protective role against oxidative stress in the brain.