Maxim C.-J. Cheeran

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.

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.

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.

CXCL10 production from cytomegalovirus-stimulated microglia is regulated by both human and viral interleukin-10.

ABSTRACT Glial cells orchestrate immunocyte recruitment to focal areas of viral infection within the brain and synchronize immune cell functions through a regulated network of cytokines and chemokines. Since recruitment of T lymphocytes plays a critical role in resolving cytomegalovirus (CMV) infection, we investigated the production of a T-cell chemoattractant, CXCL10 (gamma interferon-inducible protein 10) in response to viral infection of human glial cells. Infection with CMV was found to elicit the production of CXCL10 from primary microglial cells but not from astrocytes. This CXCL10 expression was not dependent on secondary protein synthesis but did require the phosphorylation of p38 mitogen-activated protein (MAP) kinase. In addition, migration of activated lymphocytes toward supernatants from CMV-stimulated microglial cells was partially suppressed by anti-CXCL10 antibodies. Since regulation of central nervous system inflammation is essential to allow viral clearance without immunopathology, microglial cells were then treated with anti-inflammatory cytokines. CMV-induced CXCL10 production from microglial cells was suppressed following treatment with interleukin-10 (IL-10) and IL-4 but not following treatment with transforming growth factor β. The IL-10-mediated inhibition of CXCL10 production was associated with decreased CMV-induced NF-κB activation but not decreased p38 MAP kinase phosphorylation. Finally, CMV infection of fully permissive astrocytes resulted in mRNA expression for the viral homologue to human IL-10 (i.e., cmvIL-10 [UL111a]) in its spliced form and conditioned medium from CMV-infected astrocytes inhibited virus-induced CXCL10 production from microglial cells through the IL-10 receptor. These findings present yet another mechanism through which CMV may subvert host immune responses.

Neural precursor cell susceptibility to human cytomegalovirus diverges along glial or neuronal differentiation pathways

Cytomegalovirus (CMV) is a major cause of congenital brain disease, and its neuropathogenesis may be related to viral infection of rapidly dividing, susceptible neural precursor cells (NPCs). In the present study, we evaluated the susceptibility of human fetal brain‐derived NPCs (nestin+, A2B5+, CD133+) to infection with CMV. Data derived from these studies demonstrated that undifferentiated NPCs supported productive viral replication. After differentiation in the presence of serum, a treatment that promotes development of an astroglial cell phenotype (GFAP+, nestin−, A2B5−), viral expression was retained. However, differentiation of NPCs in medium containing platelet‐derived growth factor and brain‐derived neurotropic factor, conditions that support the development of neurons (Tuj‐1+, nestin−, A2B5−), resulted in reduced viral expression, with corresponding decreased CMV major immediate‐early promoter (MIEP) activity relative to undifferentiated cells. Further experiments showed that cellular differentiation into a neuronal phenotype was associated with elevated levels of various CCAAT/enhancer binding protein beta (C/EBP)‐β isoforms, which suppressed MIEP activity in cotransfected NPCs. Taken together, these data demonstrate that the susceptibility of primary human NPCs to CMV is retained concomitantly with differentiation into glial cells but is actively repressed following differentiation into neurons.

Interleukin-10 attenuates production of HSV-induced inflammatory mediators by human microglia

Infection of the central nervous system (CNS) with herpes simplex virus (HSV)‐1 initiates a rapidly progressive, necrotizing, and fatal encephalitis in humans. Even with the advent of antiviral therapy, effective treatments for HSV‐1 brain infection are limited because the cause of the resulting neuropathogenesis is not completely understood. We previously reported that human microglial cells, while nonproductively infected, respond to HSV‐1 by producing robust amounts of pro‐inflammatory mediators, such as tumor necrosis factor(TNF), interleukin (IL)‐1β, CCL5 (RANTES), and CXCL10 (IP‐10). Although initiation of immune responses by glial cells is an important protective mechanism in the CNS, unrestrained inflammatory responses may result in irreparable brain damage. To elucidate the potential immunomodulatory role of anti‐inflammatory cytokines, we investigated the effects of IL‐4, IL‐10, and transforming growth factor (TGF)‐β on microglial cell cytokine and chemokine production in response to HSV‐1. Results from these studies demonstrated a consistent IL‐10‐mediated suppression of TNF‐α (60% ± 2%), IL‐1β (68% ± 3%), CCL5 (62 ± 4%), but not CXCL10 production by HSV‐1‐infected microglial cells. This inhibition was associated with decreased HSV‐1‐induced activation of NF‐κB. These results suggest that IL‐10 has the ability to regulate microglial cell production of immune mediators and thereby, dampen the pro‐inflammatory response to HSV‐1.

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.