Toll-like receptor 2 signaling is a mediator of apoptosis in herpes simplex virus-infected microglia

BACKGROUND: Information regarding the response of brain cells to infection with herpes simplex virus (HSV)-1 is needed for a complete understanding of viral neuropathogenesis. We have recently demonstrated that microglial cells respond to HSV infection by producing a number of proinflammatory cytokines and chemokines through a mechanism involving Toll-like receptor 2 (TLR2). Following this cytokine burst, microglial cells rapidly undergo cell death by apoptosis. We hypothesized that TLR2 signaling might mediate the cell death process as well. METHODS: To test this hypothesis, we investigated HSV-induced cell death of microglia obtained from both wild-type and TLR2(-/- )mice. Cell death was studied by oligonucleosomal ELISA and TUNEL staining, and the mechanisms of apoptosis were further analyzed using murine apoptosis-specific microarrays. The data obtained from microarray analysis were then validated using quantitative real-time PCR assays. RESULTS: HSV infection induced apoptotic cell death in microglial cells from wild-type as well as TLR2 cells. However, the cell death at 24 h p.i. was markedly lower in knockout cells. Furthermore, microarray analyses clearly showed that the expression of pro-apoptotic genes was down-regulated at the time when wild-type cells were actively undergoing apoptosis, indicating a differential response to HSV in cells with or without TLR2. CONCLUSION: We demonstrate here that HSV induces an apoptotic response in microglial cells which is mediated through TLR2 signaling

Persistent Humoral Immune Responses in the CNS Limit Recovery of Reactivated Murine Cytomegalovirus

Background: Experimental infection of the mouse brain with murine CMV (MCMV) elicits neuroimmune responses that terminate acute infection while simultaneously preventing extensive bystander damage. Previous studies have determined that CD8 + T lymphocytes are required to restrict acute, productive MCMV infection within the central nervous system (CNS). In this study, we investigated the contribution of humoral immune responses in control of MCMV brain infection. Methodology/Principal Findings: Utilizing our MCMV brain infection model, we investigated B-lymphocyte-lineage cells and assessed their role in controlling the recovery of reactivated virus from latently infected brain tissue. Brain infiltrating leukocytes were first phenotyped using markers indicative of B-lymphocytes and plasma cells. Results obtained during these studies showed a steady increase in the recruitment of B-lymphocyte-lineage cells into the brain throughout the timecourse of viral infection. Further, MCMV-specific antibody secreting cells (ASC) were detected within the infiltrating leukocyte population using an ELISPOT assay. Immunohistochemical studies of brain sections revealed co-localization of CD138 + cells with either IgG or IgM. Additional immunohistochemical staining for MCMV early antigen 1 (E1, m112–113), a reported marker of viral latency in neurons, confirmed its expression in the brain during latent infection. Finally, using B-cell deficient (Jh 2/2) mice we demonstrated that B-lymphocytes control recovery of reactivated virus from latently-infected brain tissue. A significantly higher rate of reactivated virus was recovered from the brains of Jh 2/2 mice when compared t

Histoplasma capsulatum yeast phase-specific protein Yps3p induces Toll-like receptor 2 signaling

Histoplasma capsulatum is a common cause of fungal infection in certain geographic areas, and although most infections are asymptomatic, it is capable of causing histoplasmosis, a disseminated, life-threatening disease, especially in immunocompromised individuals. A deeper understanding of this host-pathogen interaction is needed to develop novel therapeutic strategies to counter lethal infection. Although several lines of evidence suggest that this fungus is neurotropic in HIV patients, little is known about the immunobiology of Histoplasma infection in the central nervous system [CNS]. The goal of the present study was to understand the innate neuroimmune mechanisms that recognize H. capsulatum during the initial stages of infection. Using a 293T stable cell line expressing murine Toll-like receptor 2 [TLR2], we show here that TLR2 recognizes H. capsulatum cell wall protein Yps3p and induces the activation of NF-κB. In further experiments, we tested the ability of Yps3p to induce signaling from TLR2 in primary microglial cells, the resident brain macrophages of the CNS. Our data show that H. capsulatum Yps3p induced TLR2 signaling in wild-type microglia, but not in microglia isolated from TLR2 KO mice, confirming that Yps3p is a ligand for TLR2. Furthermore, Yps3p-induced TLR2 signaling was suppressed by vaccinia virus-encoded TLR inhibitors. This is the first demonstration of a fungal protein serving as a TLR ligand and mediating signaling in primary brain cells

Herpes simplex virus induces neural oxidative damage via microglial cell Toll-like receptor-2

<p>Abstract</p> <p>Background</p> <p>Using a murine model of herpes simplex virus (HSV)-1 encephalitis, our laboratory has determined that induction of proinflammatory mediators in response to viral infection is largely mediated through a Toll-like receptor-2 (TLR2)-dependent mechanism. Published studies have shown that, like other inflammatory mediators, reactive oxygen species (ROS) are generated during viral brain infection. It is increasingly clear that ROS are responsible for facilitating secondary tissue damage during central nervous system infection and may contribute to neurotoxicity associated with herpes encephalitis.</p> <p>Methods</p> <p>Purified microglial cell and mixed neural cell cultures were prepared from C57B/6 and TLR2^-/-mice. Intracellular ROS production in cultured murine microglia was measured via 2', 7'-Dichlorofluorescin diacetate (DCFH-DA) oxidation. An assay for 8-isoprostane, a marker of lipid peroxidation, was utilized to measure free radical-associated cellular damage. Mixed neural cultures obtained from β-actin promoter-luciferase transgenic mice were used to detect neurotoxicity induced by HSV-infected microglia.</p> <p>Results</p> <p>Stimulation with HSV-1 elevated intracellular ROS in wild-type microglial cell cultures, while TLR2^-/-microglia displayed delayed and attenuated ROS production following viral infection. HSV-infected TLR2^-/-microglia produced less neuronal oxidative damage to mixed neural cell cultures in comparison to HSV-infected wild-type microglia. Further, HSV-infected TLR2^-/-microglia were found to be less cytotoxic to cultured neurons compared to HSV-infected wild-type microglia. These effects were associated with decreased activation of p38 MAPK and p42/p44 ERK in TLR2^-/-mice.</p> <p>Conclusions</p> <p>These studies demonstrate the importance of microglial cell TLR2 in inducing oxidative stress and neuronal damage in response to viral infection.</p

Reactive oxygen species drive herpes simplex virus (HSV)-1-induced proinflammatory cytokine production by murine microglia

Abstract Background Production of reactive oxygen species (ROS) and proinflammatory cytokines by microglial cells in response to viral brain infection contributes to both pathogen clearance and neuronal damage. In the present study, we examined the effect of herpes simplex virus (HSV)-1-induced, NADPH oxidase-derived ROS in activating mitogen-activated protein kinases (MAPKs) as well as driving cytokine and chemokine expression in primary murine microglia. Methods Oxidation of 2', 7'-dichlorodihydrofluorescin diacetate (H2DCFDA) was used to measure production of intracellular ROS in microglial cell cultures following viral infection. Virus-induced cytokine and chemokine mRNA and protein levels were assessed using real-time RT-PCR and ELISA, respectively. Virus-induced phosphorylation of microglial p38 and p44/42 (ERK1/2) MAPKs was visualized using Western Blot, and levels of phospho-p38 were quantified using Fast Activated Cell-based ELISA (FACE assay). Diphenyleneiodonium (DPI) and apocynin (APO), inhibitors of NADPH oxidases, were used to investigate the role of virus-induced ROS in MAPK activation and cytokine, as well as chemokine, production. Results Levels of intracellular ROS were found to be highly elevated in primary murine microglial cells following infection with HSV and the majority of this virus-induced ROS was blocked following DPI and APO treatment. Correspondingly, inhibition of NADPH oxidase also decreased virus-induced proinflammatory cytokine and chemokine production. In addition, microglial p38 and p44/42 MAPKs were found to be phosphorylated in response to viral infection and this activation was also blocked by inhibitors of NADPH oxidase. Finally, inhibition of either of these ROS-induced signaling pathways suppressed cytokine (TNF-α and IL-1β) production, while chemokine (CCL2 and CXCL10) induction pathways were sensitive to inhibition of p38, but not ERK1/2 MAPK. Conclusions Data presented herein demonstrate that HSV infection induces proinflammatory responses in microglia through NADPH oxidase-dependent ROS and the activation of MAPKs.</p

Human cytomegalovirus mediates APOBEC3B relocalization early during infection through a ribonucleotide reductase-independent mechanism

Human cytomegalovirus (HCMV) infections can range from asymptomatic to severe, particularly in neonates and immunocompromised patients. HCMV has evolved strategies to overcome host-encoded antiviral defenses to achieve lytic viral DNA replication and dissemination and, under some conditions, latency and long-term persistence. Here, we show that HCMV infection causes the antiviral factor, APOBEC3B, to relocalize from the nuclear compartment to the cytoplasm. This overall strategy resembles that used by related herpesviruses. However, the HCMV relocalization mechanism utilizes a different viral factor(s) and available evidence suggests the involvement of at least one protein expressed at the early stages of infection. This knowledge is important because a greater understanding of this mechanism could lead to novel antiviral strategies that enable APOBEC3B to naturally restrict HCMV infection.The APOBEC3 family of DNA cytosine deaminases comprises an important arm of the innate antiviral defense system. The gamma-herpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus and the alpha-herpesviruses herpes simplex virus (HSV)-1 and HSV-2 have evolved an efficient mechanism to avoid APOBEC3 restriction by directly binding to APOBEC3B and facilitating its exclusion from the nuclear compartment. The only viral protein required for APOBEC3B relocalization is the large subunit of the ribonucleotide reductase (RNR). Here, we ask whether this APOBEC3B relocalization mechanism is conserved with the beta-herpesvirus human cytomegalovirus (HCMV). Although HCMV infection causes APOBEC3B relocalization from the nucleus to the cytoplasm in multiple cell types, the viral RNR (UL45) is not required. APOBEC3B relocalization occurs rapidly following infection suggesting the involvement of an immediate early or early (IE/E) viral protein. In support of this possibility, genetic (IE1 mutant) and pharmacologic (cycloheximide) strategies that prevent the expression of IE/E viral proteins also block APOBEC3B relocalization. In comparison, the treatment of infected cells with phosphonoacetic acid, which interferes with viral late protein expression, still permits A3B relocalization. These results combine to indicate that the beta-herpesvirus HCMV uses an RNR-independent, yet phenotypically similar, molecular mechanism to antagonize APOBEC3B. IMPORTANCEHuman cytomegalovirus (HCMV) infections can range from asymptomatic to severe, particularly in neonates and immunocompromised patients. HCMV has evolved strategies to overcome host-encoded antiviral defenses to achieve lytic viral DNA replication and dissemination and, under some conditions, latency and long-term persistence. Here, we show that HCMV infection causes the antiviral factor, APOBEC3B, to relocalize from the nuclear compartment to the cytoplasm. This overall strategy resembles that used by related herpesviruses. However, the HCMV relocalization mechanism utilizes a different viral factor(s) and available evidence suggests the involvement of at least one protein expressed at the early stages of infection. This knowledge is important because a greater understanding of this mechanism could lead to novel antiviral strategies that enable APOBEC3B to naturally restrict HCMV infection

Modulation of Experimental Herpes Encephalitis-Associated Neurotoxicity through Sulforaphane Treatment

Reactive oxygen species (ROS) produced by brain-infiltrating macrophages and neutrophils, as well as resident microglia, are pivotal to pathogen clearance during viral brain infection. However, unchecked free radical generation is also responsible for damage to and cytotoxicity of critical host tissue bystander to primary infection. These unwanted effects of excessive ROS are combated by local cellular production of antioxidant enzymes, including heme oxygenase-1 (HO-1) and glutathione peroxidase 1 (Gpx1). In this study, we showed that experimental murine herpes encephalitis triggered robust ROS production, as well as an opposing upregulation of the antioxidants HO-1 and Gpx1. This antioxidant response was insufficient to prevent tissue damage, neurotoxicity, and mortality associated with viral brain infection. Previous studies corroborate our data supporting astrocytes as the major antioxidant producer in brain cell cultures exposed to HSV-1 stimulated microglia. We hypothesized that stimulating opposing antioxidative responses in astrocytes, as well as neurons, would mitigate the effects of ROS-mediated neurotoxicity both in vitro and during viral brain infection in vivo. Here, we demonstrate that the addition of sulforaphane, a potent stimulator of antioxidant responses, enhanced HO-1 and Gpx1 expression in astrocytes through the activation of nuclear factor-E2-related factor 2 (Nrf2). Additionally, sulforaphane treatment was found to be effective in reducing neurotoxicity associated with HSV-stimulated microglial ROS production. Finally, intraperitoneal injections of sulforaphane into mice during active HSV infection reduced neuroinflammation via a decrease in brain-infiltrating leukocytes, macrophage- and neutrophil-produced ROS, and MHCII-positive, activated microglia. These data support a key role for astrocyte-produced antioxidants in modulating oxidative stress and neuronal damage in response to viral infection

Murine Cytomegalovirus Infection of Neural Stem Cells Alters Neurogenesis in the Developing Brain

Congenital cytomegalovirus (CMV) brain infection causes serious neuro-developmental sequelae including: mental retardation, cerebral palsy, and sensorineural hearing loss. But, the mechanisms of injury and pathogenesis to the fetal brain are not completely understood. The present study addresses potential pathogenic mechanisms by which this virus injures the CNS using a neonatal mouse model that mirrors congenital brain infection. This investigation focused on, analysis of cell types infected with mouse cytomegalovirus (MCMV) and the pattern of injury to the developing brain.We used our MCMV infection model and a multi-color flow cytometry approach to quantify the effect of viral infection on the developing brain, identifying specific target cells and the consequent effect on neurogenesis. In this study, we show that neural stem cells (NSCs) and neuronal precursor cells are the principal target cells for MCMV in the developing brain. In addition, viral infection was demonstrated to cause a loss of NSCs expressing CD133 and nestin. We also showed that infection of neonates leads to subsequent abnormal brain development as indicated by loss of CD24(hi) cells that incorporated BrdU. This neonatal brain infection was also associated with altered expression of Oct4, a multipotency marker; as well as down regulation of the neurotrophins BDNF and NT3, which are essential to regulate the birth and differentiation of neurons during normal brain development. Finally, we report decreased expression of doublecortin, a marker to identify young neurons, following viral brain infection.MCMV brain infection of newborn mice causes significant loss of NSCs, decreased proliferation of neuronal precursor cells, and marked loss of young neurons