James R. Lokensgard

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.

Morphine induces apoptosis of human microglia and neurons

Apoptosis plays a critical role in normal brain development and in a number of neurodegenerative diseases. Recently, opiates have been shown to promote apoptotic death of cells of the immune and nervous systems. In this study, we investigated the effect of morphine on apoptosis of primary human fetal microglial cell, astrocyte and neuronal cell cultures. Exposure of microglia and neurons to 10(-6) M morphine potently induced apoptosis of these brain cells (approximately fourfold increase above untreated control cells). In contrast to microglia and neurons, astrocytes were completely resistant to morphine-induced apoptosis. Concentration-response and time-course studies indicated that neurons were more sensitive than microglia to morphines effect on apoptosis. Naloxone blocked morphine-induced apoptosis suggesting involvement of an opiate receptor mechanism. Potent inhibition (>70%) of apoptosis by an inhibitor of caspase-3 as well as co-localization of active caspase-3 and DNA fragmentation in microglia or neurons treated with morphine indicated that caspase-3 is involved in the execution phase of morphine-induced apoptosis. The results of these in vitro studies have implications regarding the potential effect of opiates on fetal brain development and on the course of certain neurodegenerative diseases.

Neuropathogenesis of Congenital Cytomegalovirus Infection: Disease Mechanisms and Prospects for Intervention

SUMMARY Congenital cytomegalovirus (CMV) infection is the leading infectious cause of mental retardation and hearing loss in the developed world. In recent years, there has been an improved understanding of the epidemiology, pathogenesis, and long-term disabilities associated with CMV infection. In this review, current concepts regarding the pathogenesis of neurological injury caused by CMV infections acquired by the developing fetus are summarized. The pathogenesis of CMV-induced disabilities is considered in the context of the epidemiology of CMV infection in pregnant women and newborn infants, and the clinical manifestations of brain injury are reviewed. The prospects for intervention, including antiviral therapies and vaccines, are summarized. Priorities for future research are suggested to improve the understanding of this common and disabling illness of infancy.

Cutting Edge: TLR2-Mediated Proinflammatory Cytokine and Chemokine Production by Microglial Cells in Response to Herpes Simplex Virus

Recent studies indicate that TLRs are critical in generating innate immune responses during infection with HSV-1. In this study, we investigated the role of TLR2 signaling in regulating the production of neuroimmune mediators by examining cytokine and chemokine expression using primary microglial cells obtained from TLR2−/− as well as wild-type mice. Data presented here demonstrate that TLR2 signaling is required for the production of proinflammatory cytokines and chemokines: TNF-α, IL-1β, IL-6, IL-12, CCL7, CCL8, CCL9, CXCL1, CXCL2, CXCL4, and CXCL5. CXCL9 and CXCL10 were also induced by HSV, but their production was not dependent upon TLR2 signaling. Because TLR2−/− mice display significantly reduced mortality and diminished neuroinflammation in response to brain infection with HSV, the TLR2-dependent cytokines identified here might function as key players influencing viral neuropathogenesis.

Synthetic cannabinoid WIN55,212-2 inhibits generation of inflammatory mediators by IL-1β-stimulated human astrocytes

Activated glial cells have been implicated in the neuropathogenesis of many infectious and inflammatory diseases of the brain. A number of inflammatory mediators have been proposed to play a role in glial cell‐related brain damage; e.g., free radicals such as nitric oxide (NO), cytokines, and chemokines. Our laboratory has been interested in the effect of psychoactive drugs and their derivatives on the production of these mediators. Cannabinoids have been shown to possess immunomodulatory as well as psychoactive properties. We previously have shown that interleukin (IL)‐1β‐stimulated human astrocytes, but not microglia, produce NO. In this study, we investigated the effects of the synthetic cannabinoid WIN55,212‐2 on the production of several key inflammatory mediators by human fetal astrocytes activated by IL‐1β. Expression of the cannabinoid receptors CB1 and CB2 was detected on human astrocytes. WIN55,212‐2 (10−5 M) potently inhibited inducible NO synthase (iNOS) and corresponding NO production by IL‐1β‐stimulated astrocytes. The CB1 and CB2 receptor‐specific antagonists SR141716A and SR144528, respectively, partially blocked this suppressive effect. In addition, treatment of astrocytes with WIN55,212‐2 downregulated in a concentration‐dependent manner IL‐1β‐induced tumor necrosis factor (TNF)‐α release. Treatment with WIN55,212‐2 also inhibited production of the chemokines CXCL10, CCL2 and CCL5 by IL‐1β‐activated astrocytes. These findings indicate that WIN55,212‐2 inhibits the production of inflammatory mediators by IL‐1β‐stimulated human astrocytes and suggest that comparable agents may have therapeutic potential for the management of brain inflammation.

Robust expression of TNF-α, IL-1β, RANTES, and IP-10 by human microglial cells during nonproductive infection with herpes simplex virus

Cytokine (TNF-α/β, IL-1β, IL-6, IL-18, IL-10, and IFN-α/β/γ) and chemokine (IL-8, IP-10, MCP-1, MIP-1α/β, and RANTES) production during herpes simplex virus (HSV) 1 infection of human brain cells was examined. Primary astrocytes as well as neurons were found to support HSV replication, but neither of these fully permissive cell types produced cytokines or chemokines in response to HSV. In contrast, microglia did not support extensive viral replication; however, ICP4 was detected by immunochemical staining, demonstrating these cells were infected. Late viral protein (nucleocapsid antigen) was detected in <10% of infected microglial cells. Microglia responded to nonpermissive viral infection by producing considerable amounts of TNF-α, IL-1β, IP-10, and RANTES, together with smaller amounts of IL-6, IL-8, and MlP-1α as detected by RPA and ELISA. Surprisingly, no interferons (α,β, or γ) were detected in response to viral infection. Pretreatment of fully permissive astrocytes with TNF-α prior to infection with HSV was found to dramatically inhibit replication, resulting in a 14-fold reduction of viral titer. In contrast, pretreatment of astrocytes with IL-1β had little effect on viral replication. When added to neuronal cultures, exogenous TNF-α or IL-1β did not suppress subsequent HSV replication. Exogenously added IP-10 inhibited HSV replication in neurons (with a 32-fold reduction in viral titer), however, similar IP-10 treatment did not affect viral replication in astrocytes. These results suggest that IP-10 possesses direct antiviral activity in neurons and support a role for microglia in both antiviral defense of the brain as well as amplification of immune responses during neuroinflammation.

Prolonged Microglial Cell Activation and Lymphocyte Infiltration following Experimental Herpes Encephalitis

Experimental murine herpes simplex virus (HSV)-1 brain infection stimulates microglial cell-driven proinflammatory chemokine production which precedes the presence of brain-infiltrating systemic immune cells. In the present study, we investigated the phenotypes and infiltration kinetics of leukocyte trafficking into HSV-infected murine brains. Using real-time bioluminescence imaging, the infiltration of luciferase-positive splenocytes, transferred via tail vein injection into the brains of HSV-infected animals, was followed over an 18-day time course. Flow cytometric analysis of brain-infiltrating leukocytes at 5, 8, 14, and 30 days postinfection (d.p.i.), was performed to assess their phenotype. A predominantly macrophage (CD45highCD11b+Ly6Chigh) and neutrophil (CD45highCD11b+Ly6G+) infiltration was seen early during infection, with elevated levels of TNF-α mRNA expression. By 14 d.p.i., the phenotypic profile shifted to a predominantly lymphocytic (CD45highCD3+) infiltrate. This lymphocyte infiltrate was detected until 30 d.p.i., when infectious virus could not be recovered, with CD8+ and CD4+ T cells present at a 3:1 ratio, respectively. This T lymphocyte infiltration paralleled increased IFN-γ mRNA expression in the brain. Activation of resident microglia (CD45intCD11b+) was also detected until 30 d.p.i., as assessed by MHC class II expression. Activated microglial cells were further identified as the predominant source of IL-1β. In addition, infected mice given primed immunocytes at 4 d.p.i. showed a significant increase in mortality. Taken together, these results demonstrate that intranasal infection results in early macrophage and neutrophil infiltration into the brain followed by prolonged microglial activation and T lymphocyte retention. Similar prolonged neuroimmune activation may contribute to the neuropathological sequelae observed in herpes encephalitis patients.

Differential responses of human brain cells to West Nile virus infection

In recent years, West Nile virus (WNV) has emerged as a major cause of encephalitis in the United States. However, the neuropathogenesis of this flavivirus is poorly understood. In the present study, the authors used primary human brain cell cultures to investigate two neuropathogenic features: viral replication and induction of cytokines. Although neurons and astrocytes were found to support productive WNV infection, viral growth was poorly permissive in microglial cells. Compared to neuronal cultures that sustained viral growth for at least 2 weeks, replication peaked in astrocytes by 72 h post infection. In response to viral infection, astrocytes produced chemokines (CXCL10 and CCL5), but none of the cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-1β, IL-6, interferon α or γ) tested could be detected. Although microglial cells failed to support viral replication, WNV induced production of the proinflammatory cytokines IL-6 and TNF-α. Microglial cells also released robust amounts of the chemokines CXCL10 and CCL2, as well as lower levels of CCL5, in response to WNV infection. WNV-induced chemokine and cytokine production by microglia was coupled with activation of mitogen-activated protein kinase (MAPK) intracellular signaling pathways. Inhibition of p38 MAPK decreased chemokine production in response to WNV. Taken together, these findings suggest that microglial cell responses may influence the neuropathogenesis of WNV infection.

Cytomegalovirus induces cytokine and chemokine production differentially in microglia and astrocytes: Antiviral implications

Glial cells function as sensors for infection within the brain and produce cytokines to limit viral replication and spread. We examined both cytokine (TNF-α, IL-1β, and IL-6) and chemokine (MCP-1, MlP-1α, RANTES, and IL-8) production by primary human glial cells in response to cytomegalovirus (CMV). Although CMV-infected astrocytes did not produce antiviral cytokines, they generated significant quantities of the chemokines MCP-1 and IL-8 in response to viral infection. On the other hand, supernatants from CMV-stimulated purified microglial cell cultures showed a marked increase in the production of TNF-α and IL-6, as well as chemokines. Supernatants from CMV-infected astrocyte cultures induced the migration of microglia towards chemotactic signals generated from infected astrocytes. Antibodies to MCP-1, but not to MlP-1α, RANTES, or IL-8, inhibited this migratory activity. These findings suggest that infected astrocytes may use MCP-1 to recruit antiviral cytokine-producing microglial cells to foci of infection. To test this hypothesis, cocultures of astrocytes and microglial cells were infected with CMV. Viral gene expression in these cocultures was 60% lower than in CMV infected purified astrocyte cultures lacking microglia. These results support the hypothesis that microglia play an important antiviral role in defense of the brain against CMV. The host defense function of microglial cells may be directed in part by chemokines, such as MCP-1, produced by infected astrocytes.

TNF‐α‐induced chemokine production and apoptosis in human neural precursor cells

Recent studies have shown that proinflammatory cytokines damage rodent neural precursor cells (NPCs), a source of self‐renewing, multipotent cells that play an important role in the developing as well as adult brain. In this study, the effects of tumor necrosis factor α (TNF‐α) on cytokine and chemokine production by human NPCs (>98% nestin‐ and >90% A2B5‐positive), obtained from 6‐ to 8‐week‐old fetal brain specimens, were evaluated. NPCs stimulated with this proinflammatory cytokine were found to produce abundant amounts of the chemokines monocyte chemoattractant protein 1 (MCP‐1)/CC chemokine ligand 2 (CCL2) and interferon‐inducible protein 10 (IP‐10)/CXC chemokine ligand 10 (CXCL10) in a time‐ and concentration‐dependent manner. TNF‐α treatment also induced NPC apoptosis. Receptors for TNF [TNFRI (p55) and TNFRII (p75)] mRNA were constitutively expressed on NPCs. However, only TNFRI was involved in TNF‐α‐induced chemokine production and apoptosis by NPCs, as anti‐TNFRI but not anti‐TNFRII antibodies blocked the stimulatory effect. TNF‐α treatment induced p38 mitogen‐activated protein kinase (MAPK) phosphorylation in NPCs, and SB202190, an inhibitor of p38 MAPK, blocked TNF‐α‐induced chemokine production. Thus, this study demonstrated that NPCs constitutively express receptors for TNF‐α, which when activated, trigger via a p38 MAPK signaling pathway production of two chemokines, MCP‐1/CCL2 and IP‐10/CXCL10, which are involved in infectious and inflammatory diseases of the brain.