Spread of neuroadapted Sindbis virus (NSV) to motor neurons (MN) of the spinal cord (SC) causes severe hind limb weakness in C57BL/6 mice and models the paralysis that can accompany alphavirus and flavivirus encephalomyelitis in humans. The fate of spinal MN dictates the severity of NSV-induced paralysis, and recent data suggest that MN damage can occur indirectly via the actions of activated microglial cells. Because the opioid receptor antagonist, naloxone (NAL), blocks microglial-mediated neurodegeneration in other models, we examined its effects during NSV infection. Drug treatment prevented paralysis and enhanced the survival of MN without altering NSV tropism, replication, or clearance from SC tissue. Further studies showed that NAL most effectively inhibited paralysis in a 72-hour window after NSV challenge, suggesting that the drug inhibits an early event in SC pathogenesis. Histochemical studies demonstrated that NAL blocked early microglial activation in SC tissue sections, and protein assays showed that the early induction of pathogenic IL-1β was blunted in SC homogenates. Finally, loss of glutamate transporter-1 (GLT-1) expression in SC, an astrocyte glutamate reuptake protein responsible for lowering toxic extracellular levels of glutamate and preventing MN damage, was reversed by NAL treatment. This GLT-1 loss proved to be highly IL-1β-dependent. Taken together, these data suggest that NAL is neuroprotective in the SC by inhibiting microglial activation that, in turn, maintains normal astrocyte glutamate homeostasis. We propose that drugs targeting such microglial responses may have therapeutic benefit in humans with related viral infections.
Neuroprotection; viral encephalitis; motor neurons; microglial activation; IL-1β; Glutamate transporter-1
Neuroadapted Sindbis virus (NSV) causes acute encephalitis and paralyzes and kills adult mice unless they are treated with primary immune serum after infection. To study the nature and specificity of curative antibodies, we gave mice 30 different monoclonal antibodies (MAbs) against Sindbis virus (SV) 24 h after lethal intracerebral inoculation of NSV. By the time of MAb treatment, NSV replication in the brain had been well established (7.5 X 10(7) PFU/g). Seventeen MAbs directed against multiple biological domains on the NSV E1 and E2 envelope glycoproteins prevented paralysis and death. Anticapsid MAbs failed to protect. Altogether, 15 of 17 curative MAbs either neutralized NSV infectivity or lysed NSV-infected cells with complement, but neither ability was necessary or sufficient to guarantee recovery. All 5 protective anti-E2 MAbs neutralized NSV infectivity; 6 of 10 protective anti-E1 MAbs neutralized NSV; 4 did not. Plaque assay or immunohistochemical staining showed that neutralizing and nonneutralizing curative MAbs decreased NSV in the brain, brainstem, and spinal cord. Despite high neutralization titers, hyperimmune anti-SV and anti-NSV mouse sera prevented only 6 and 30% of deaths, respectively, while primary immune sera prevented 50 (SV) and 90% (NSV) of deaths. Secondary intravenous immunization with a live virus apparently diminished, obscured, or failed to boost a class of protective antibodies. When separate mouse groups were given these 30 MAbs 24 h before lethal intracerebral inoculation of NSV, a slightly different set of 17 neutralizing or nonneutralizing anti-E1 and anti-E2 antibodies protected. Two nonneutralizing MAbs and hyperimmune anti-SV serum, which had failed to promote recovery, prophylactically protected 100% of the mice. The antibody requirements or mechanisms of prophylaxis and recovery may differ.
Sindbis virus (SV) is an alphavirus that causes encephalitis in mice and can lead to the apoptotic death of infected cells. To determine the step in virus replication during which apoptosis is triggered, we used UV-inactivated SV, chemicals that block virus fusion or protein synthesis, and cells that do and do not express heparan sulfate, the initial binding molecule for SV infection of many cells. In initial experiments, UV-inactivated neuroadapted SV (NSV) induced apoptosis in Chinese hamster ovary (CHO) cells lacking heparan sulfate in the presence of cycloheximide. When fusion of prebound UV-inactivated NSV was rapidly induced at the plasma membrane by exposure to acidic pH, apoptosis was induced in CHO cells with or without heparan sulfate in the presence or absence of cycloheximide in a virus dose-dependent manner. In N18 neuroblastoma cells, the relative virulence of the virus strain was an important determinant of apoptosis induced by UV-inactivated SV. Treatment of N18 cells with monensin to prevent endosomal acidification an hour before, but not 2 h after, exposure to live NSV blocked the induction of cell death, as did treatment with NH4Cl or bafilomycin A1. These studies indicate that SV can induce apoptosis at the time of fusion with the cell membrane and that virus replication is not required.
Wild-type Sindbis virus strain AR339 (SV) and a neurovirulent mutant (NSV), derived by neonatal and weanling mouse brain passage, both cause acute fatal encephalitis in neonatal mice, but NSV alone kills adult mice. NSV cannot be distinguished from SV by immune sera or simple biochemical tests. To localize the molecular changes associated with neuroadaptation, we used a new array of 30 anti-SV monoclonal antibodies to probe for differences between SV and NSV in four tests: immunoprecipitation, enzyme-linked immunosorbent assay binding, neutralization, and hemagglutination inhibition. Seventeen monoclonal antibodies detected differences. Both E1 and E2 glycoprotein gene products were altered during neuroadaptation, but the preponderance of changes was clustered on E2. The capsid protein C was not measurably altered. Mapping of both viruses with these monoclonal antibodies showed that during neuroadaptation SV topography substantially shifted, masking and unmasking biologically important neutralization and hemagglutination inhibition sites. These conformational rearrangements, predominantly on E2, coincided with the acquisition of increased neurovirulence and new lethality for adult mice.
Recognition of Staphylococcus aureus and its cell-wall component peptidoglycan (PGN) by microglia is mediated, in part, by Toll-like receptor 2 (TLR2). However, the pattern recognition receptor (PRR) CD14 can also bind PGN and enhance TLR2-mediated signaling in macrophages, suggesting a similar phenomenon might occur in microglia. To assess the functional significance of CD14 on microglial activation, we evaluated the responses of primary microglia isolated from CD14 knockout (KO) and wild type (WT) mice. PGN-dependent microglial activation was partially CD14-dependent as demonstrated by the attenuated expression of TNF-α, macrophage inflammatory protein-2 (MIP-2/CXCL2), and the soluble PRR pentraxin-3 in CD14 KO microglia compared to WT cells. In contrast, microglial responses to intact S. aureus occurred primarily via a CD14-independent manner. Collectively, these findings reveal the complex nature of gram-positive bacterial recognition by microglia, which occurs, in part, via CD14.
CD14; Microglia; S. aureus; Peptidoglycan; Lipopolysaccharide; Central nervous system; Pentraxin-3
In response to invading pathogens, Toll-like receptors (TLR) play a critical role in the initiation of the innate immune response, which can be either beneficial or detrimental to the host. In the present study, we demonstrated that central nervous system (CNS) glial cells are activated by Lymphocytic Choriomeningitis Virus (LCMV) in a TLR2-MyD88/Mal-dependent manner. Specifically, in response to LCMV, both astrocytes and microglial cells isolated from wild type (WT) mice produced chemokines, such as MCP-1, RANTES and TNF-α. Similar responses occurred in TLR3 KO and TLR4 KO glial cells. In striking contrast, both astrocytes and microglial cells isolated from mice deficient in TLR2, MyD88, and Mal did not produce any of these chemokines. In addition, LCMV infection of glial cells induced up-regulation of TLR2, MHC-class-I and II, CD40, CD86 in a MyD88-dependent manner. These results define a functional role for TLR signaling in viral infection-induced activation of CNS glial cells as well as for the immunopathology in the CNS.
Lymphocytic Choriomeningitis Virus (LCMV); Toll-like receptors; CNS glial cells
Many alphaviruses cause more severe disease in young animals than in older animals. The age-dependent resistance to severe disease is determined primarily by maturation of the host, but strains of virus can be selected that overcome the increased resistance of mature animals. Sindbis virus (SV) strain AR339 causes fatal encephalitis in newborn mice and nonfatal encephalitis in weanling mice, whereas NSV, a neuroadapted strain of SV, causes fatal encephalitis in weanling as well as newborn mice. We have previously shown that the E2 glycoprotein of NSV contained His-55, whereas AR339 E2 had Gln-55 (S. Lustig, A. C. Jackson, C. S. Hahn, D. E. Griffin, E. G. Strauss, and J. H. Strauss, J. Virol. 62:2329-2336, 1988) and that SV with E2 containing Gly-172 was more virulent for newborn mice than SV with E2 containing Arg-172 (P. C. Tucker and D. E. Griffin, J. Virol. 65:1551-1557, 1991). Here we tested the virulence for both newborn and older mice of SV containing a number of different amino acids at E2 position 55 (His, Gln, Lys, Arg, Glu, Gly) in combination with both Gly-172 and Arg-172. All the viruses were virulent for newborn mice, but the residues at both 55 and 172 influenced the virulence of the virus, and there were differences in virulence observed among the various viruses. However, only viruses with His-55 were fully virulent for 14-day-old mice, and this virulence was independent of the residue at position 172. Virus with Lys-55 was virulent for 7-day-old mice, although slightly attenuated relative to His-55. Viruses with His-55 grew more rapidly and to higher titer in the brains of 7- and 14-day-old mice, in N18 neuroblastoma cells, and in BHK cells. Our data suggest that His-55 is important for neurovirulence in older mice and acts by increasing the efficiency of virus replication.
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.
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.
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.
We demonstrate here that HSV induces an apoptotic response in microglial cells which is mediated through TLR2 signaling.
Neuroadapted Sindbis virus (NSV) is a neuronotropic virus that causes a fulminant encephalomyelitis in susceptible mice due to death of motor neurons in the brain and spinal cord. We and others have found that uninfected motor neurons die in response to NSV infection, at least in part due to disrupted astrocytic glutamate transport, resulting in excitotoxic motor neuron death. Here, we examined the mechanisms of astrocyte dysregulation associated with NSV infection. Treatment of organotypic slice cultures with NSV results in viral replication, cell death, altered astrocyte morphology, and the downregulation of the astrocytic glutamate transporter, GLT-1. We have found that TNF-α can mediate GLT-1 downregulation. Furthermore, TNF-α deficient mice infected with NSV exhibit neither GLT-1 downregulation nor neuronal death of brainstem and cervical spinal cord motor neurons and have markedly reduced mortality. These findings have implications for disease intervention and therapeutic development for the prevention of CNS damage associated with inflammatory responses.
Astrocyte; TNF-α; Motor neuron; GLT-1; Glutamate; Virus
Astrocytes remove glutamate from the synaptic cleft via specific transporters, and impaired glutamate reuptake may promote excitotoxic neuronal injury. In a model of viral encephalomyelitis caused by neuroadapted Sindbis virus (NSV), mice develop acute paralysis and spinal motor neuron degeneration inhibited by the AMPA receptor antagonist, NBQX. To investigate disrupted glutamate homeostasis in the spinal cord, expression of the main astroglial glutamate transporter, GLT-1, was examined. GLT-1 levels declined in the spinal cord during acute infection while GFAP expression was preserved. There was simultaneous production of inflammatory cytokines at this site, and susceptible animals treated with drugs that blocked IL-1β release also limited paralysis and prevented the loss of GLT-1 expression. Conversely, infection of resistant mice that develop mild paralysis following NSV challenge showed higher baseline GLT-1 levels as well as lower production of IL-1β and relatively preserved GLT-1 expression in the spinal cord compared to susceptible hosts. Finally, spinal cord GLT-1 expression was largely maintained following infection of IL-1β-deficient animals. Together, these data show that IL-1β inhibits astrocyte glutamate transport in the spinal cord during viral encephalomyelitis. They provide one of the strongest in vivo links between innate immune responses and the development of excitotoxicity demonstrated to date.
glutamate transporters; interleukin-1β; viral encephalomyelitis; motor neuron; excitotoxicity
Microglia are macrophage-like cells that constantly sense the microenvironment within the central nervous system (CNS). In the event of neuronal stress or injury, microglial cells rapidly react and change their phenotype. This response may lead to a deleterious type of microglial activation, which is often associated with neuroinflammation and neurotoxicity in several neuropathological conditions. We investigated the molecular mechanisms underlying triggering of microglial activation by necrotic neuronal damage.
Primary cultures of microglia were used to study the effect of necrotic neurons on microglial inflammatory responses and toxicity towards cerebellar granule neurons (CGN). The mouse hippocampal cell line, HT22, was used in this study as the main source of necrotic neurons to stimulate microglia. To identify the signal transduction pathways activated in microglia, primary microglial cultures were obtained from mice deficient in Toll-like receptor (TLR) -2, -4, or in the TLR adapter protein MyD88.
Necrotic neurons, but not other necrotic cell types, induced microglial activation which was characterized by up-regulation of: i) MHC class II; ii) co-stimulatory molecules, i.e. CD40 and CD24; iii) β2 integrin CD11b; iii) pro-inflammatory cytokines, i.e. interleukin 6 (IL-6), IL-12p40 and tumor-necrosis factor (TNF); iv) pro-inflammatory enzymes such as nitric oxide synthase (iNOS, type II NOS), indoleamine 2,3-dioxygenase (IDO) and cyclooxygenase-2 (COX-2) and increased microglial motility. Moreover, microglia-conditioned medium (MCM) obtained from cultures of activated microglia showed increased neurotoxicity mediated through the N-methyl-D-aspartate receptor (NMDAR). The activation of microglia by necrotic neurons was shown to be dependent on the TLR-associated adapter molecule myeloid differentiation primary response gene (MyD88). Furthermore, MyD88 mediated enhanced neurotoxicity by activated microglia through up-regulation of the expression and activity of glutaminase, an enzyme that produces glutamate, which is an NMDAR agonist.
These results show that necrotic neurons activate in microglia a MyD88-dependent pathway responsible for a pro-inflammatory response that also leads to increased neurotoxic activity through induction of glutaminase. This finding contributes to better understanding the mechanisms causing increased neuroinflammation and microglial neurotoxicity in a neurodegenerative environment.
Neuroadapted Sindbis virus (NSV), given intranasally, caused fatal encephalitis in 100% of adult C57BL/6 mice and 0% of BALB/cBy mice. Most C57BL/6 mice developed severe kyphoscoliosis followed by hind-limb paralysis, while BALB/cBy mice did not. In situ hybridization for detecting NSV RNA and immunohistochemistry for detecting NSV antigen indicated that virus delivered by this route infected neurons of the olfactory region and spread caudally without infection of ependymal cells. Virus antigen was more abundant and infectious virus increased more rapidly and reached higher levels in C57BL/6 mice than in BALB/cBy mice. Surprisingly, infectious virus was cleared faster in C57BL/6 mice, and this was associated with more rapid production of neutralizing antibody. However, viral RNA was cleared more slowly in C57BL/6 mice. In both mouse strains, more infectious virus was present in the lumbar spinal cord than in the cervical spinal cord. These data suggest that genetic susceptibility to fatal NSV encephalomyelitis is determined at least in part by the efficiency of viral replication and spread in the central nervous system. The differences identified in this study provide possible phenotypes for mapping genetic loci involved in susceptibility.
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.
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.
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.
These studies demonstrate the importance of microglial cell TLR2 in inducing oxidative stress and neuronal damage in response to viral infection.
Transfer of anti-Sindbis virus serum, obtained from peripherally inoculated donors, protected mice from an otherwise fatal intracerebral infection with neuroadapted Sindbis virus (NSV). F(ab)'2 preparations of serum were not protective, indicating that the Fc piece of immunoglobulin G was important. Complement-depleted animals were protected with anti-NSV serum, ruling out as essential the complement-fixing function of the Fc piece. The presence of protective antibody correlated with the ability of serum to inhibit T-cell cytotoxicity. However, experiments using athymic nude mice showed that T cells played no role in killing the mice since the 50% lethal dose was the same as that in normal BALB/c mice, and that T cells were not required for protection since athymic nude mice were protected with antibody alone. Cyclophosphamide treatment of NSV-infected mice ablated the protective capacity of anti-NSV serum. Therefore, a non-T cell, cyclophosphamide-sensitive cell was required for antibody-mediated protection.
Meningitis and meningoencephalitis caused by Escherichia coli are associated with high rates of mortality. When an infection occurs, Toll-like receptors (TLRs) expressed by microglial cells can recognize pathogen-associated molecular patterns and activate multiple steps in the inflammatory response that coordinate the brain's local defense, such as phagocytosis of invading pathogens. An upregulation of the phagocytic ability of reactive microglia could improve the host defense in immunocompromised patients against pathogens such as E. coli. Here, murine microglial cultures were stimulated with the TLR agonists Pam3CSK4 (TLR1/TLR2), lipopolysaccharide (TLR4), and CpG oligodeoxynucleotide (TLR9) for 24 h. Upon stimulation, levels of tumor necrosis factor alpha and the neutrophil chemoattractant CXCL1 were increased, indicating microglial activation. Phagocytic activity was studied after adding either E. coli DH5α or E. coli K1 strains. After 60 and 90 min of bacterial exposure, the number of ingested bacteria was significantly higher in cells prestimulated with TLR agonists than in unstimulated controls (P < 0.01). Addition of cytochalasin D, an inhibitor of actin polymerization, blocked >90% of phagocytosis. We also analyzed the ability of microglia to kill the ingested E. coli strains. Intracellularly surviving bacteria were quantified at different time points (90, 150, 240, and 360 min) after 90 min of phagocytosis. The number of bacteria killed intracellularly after 6 h was higher in cells primed with the different TLR agonists than in unstimulated microglia. Our data suggest that microglial stimulation by the TLR system can increase bacterial phagocytosis and killing. This approach could improve central nervous system resistance to infections in immunocompromised patients.
Microglia are crucial for the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Here, we show that the E3 ubiquitin ligase Peli1 is abundantly expressed in microglia and serves as a pivotal mediator of microglial activation during the course of EAE induction. Peli1 mediates the induction of chemokines and proinflammatory cytokines in microglia and, thereby, promotes recruitment of T cells into the central nervous system. Peli1-deficient mice are refractory to EAE induction despite their competent production of inflammatory T cells in the peripheral lymphoid organs. Notably, Peli1 regulates a novel signaling axis of the toll-like receptor pathway that mediates degradation of Traf3, a potent inhibitor of MAP kinase activation and gene induction. Ablation of Traf3 restores the microglial activation and EAE sensitivity of Peli1-deficient mice. These findings establish Peli1 as a microglia-specific mediator of autoimmune neuroinflammation and suggest a novel signaling mechanism of Peli1 function.
Peli1; Ubiquitination; CNS inflammation; EAE; Traf3; c-IAP
Microglia, the innate immune effector cells of the CNS parenchyma, express TLR that recognize conserved motifs of microorganisms referred to as pathogen-associated molecular patterns (PAMP). All TLRs identified to date, with the exception of TLR3, use a common adaptor protein, MyD88, to transduce activation signals. Recently, we reported that microglial activation in response to the Gram-positive bacterium Staphylococcus aureus was not completely attenuated following TLR2 ablation, suggesting the involvement of additional receptors. To assess the functional role of alternative TLRs in microglial responses to S. aureus and its cell wall product peptidoglycan as well as the Gram-negative PAMP LPS, we evaluated primary microglia from MyD88 knockout (KO) and wild-type mice. The induction of TNF-α, IL-12 p40, and MIP-2 (CXCL2) expression by S. aureus- and peptidoglycan-stimulated microglia was MyD88 dependent, as revealed by the complete inhibition of cytokine production in MyD88 KO cells. In addition, the expression of additional pattern recognition receptors, including TLR9, pentraxin-3, and lectin-like oxidized LDL receptor-1, was regulated, in part, via a MyD88-dependent manner as demonstrated by the attenuated expression of these receptors in MyD88 KO microglia. Microglial activation was only partially inhibited in LPS-stimulated MyD88 KO cells, suggesting the involvement of MyD88-independent pathways. Collectively, these findings reveal the complex mechanisms for microglia to respond to diverse bacterial pathogens, which occur via both MyD88-dependent and -independent pathways.
We examined a variety of strains of Sindbis virus for the genetic changes responsible for differences in neurovirulence in mice. SV1A (a low passage of the AR339 strain of Sindbis virus), a neuroadapted Sindbis virus (NSV), and two laboratory strains of Sindbis virus (HRSP and Toto1101) were examined. NSV causes severe encephalomyelitis with hind-limb paralysis and high mortality after intracerebral inoculation in weanling mice. In contrast, SV1A causes only mild, nonfatal disease in weanling mice; however, in suckling mice, SV1A causes a fatal encephalomyelitis after either intracerebral or subcutaneous inoculation. The two laboratory strains used have a greatly reduced neurovirulence for suckling mice and are avirulent for weanling mice. The nucleotide sequences and encoded amino acid sequences of the structural glycoproteins of these four strains were compared. Hybrid genomes were constructed by replacing restriction fragments in a full-length cDNA clone of Sindbis virus, from which infectious RNA can be transcribed in vitro, with fragments from cDNA clones of the various strains. These recombinant viruses allowed us to test the importance of each amino acid difference between the various strains for neurovirulence in weanling and suckling mice. Glycoproteins E2 and E1 were of paramount importance for neurovirulence in adult mice. Recombinant viruses containing the nonstructural protein region and the capsid protein region from an avirulent strain and the E1 and E2 glycoprotein regions from NSV were virulent, although they were less virulent than NSV. Furthermore, changes in either E2 (His-55 in NSV to Gln in SV1A) or E1 (Ala-72 in NSV to Val in SV1A and Asp-313 in NSV to Gly in SV1A) reduced virulence. For virulence in suckling mice, we found that a number of changes in E2 and E1 can lead to decreased virulence and that in fact, a gradient of virulence exists.
Sindbis virus (SV) is an alphavirus that causes acute encephalomyelitis in mice. The outcome is determined by the strain of virus and by the age and genetic background of the host. The mortality rates after infection with NSV, a neurovirulent strain of SV, were as follows v: 81% (17 of 21) in BALB/cJ mice; 20% (4 of 20) in BALB/cByJ mice (P < 0.001); 100% in A/J, C57BL/6J, SJL, and DBA mice; and 79% (11 of 14) in immunodeficient scid/CB17 mice. Treatment with Nomega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthetase (NOS) inhibitor, increased mortality to 100% (P < 0.05) in NSV-infected BALB/cJ mice, to 95% (P < 0.001) in BALB/cByJ mice, and to 100% in scid/CB17 mice. BALB/cJ and BALB/cByJ mice had similar levels of inducible NOS mRNA in their brains, which were not affected by L-NAME or NSV infection. Brain NOS activity was similar in BALB/cJ and BALB/cByJ mice before and after infection and was markedly inhibited by L-NAME. NSV replication in the brains of BALB/cJ mice, BALB/cByJ mice, and mice treated with L-NAME was similar. Treatment of N18 neuroblastoma cells with NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside in vitro before infection increased cell viability at 42 to 48 h compared with untreated NSV-infected N18 cells with little effect on virus replication. These data suggest that NO protects mice from fatal encephalitis by a mechanism that does not directly involve the immune response or inhibition of virus growth but rather may enhance survival of the infected neuron until the immune response can control virus replication.
Toll-like receptor 2 (TLR2) is a pattern recognition receptor that plays an important role in enabling cells of the innate immune system to recognize conserved structural motifs on a wide array of pathogens including gram-positive bacteria. Although microglia have recently been shown to express TLR2, the functional significance of this receptor in mediating microglial activation remains unknown. To ascertain the importance of TLR2 in microglial responses to S. aureus and its cell wall product peptidoglycan (PGN), we evaluated primary microglia from TLR2 knockout (KO) and wild-type (WT) mice. TLR2 was found to play a pivotal role in PGN recognition and subsequent activation in primary microglia, as demonstrated by the attenuated expression of TNF-α, IL-12 p40, MIP-2, and MCP-1 in PGN-treated TLR2 KO microglia compared with WT cells. In contrast, the responses of TLR2 KO and WT microglia to S. aureus were qualitatively similar, indicating that alternative receptors are responsible for recognizing intact bacteria. Microarray analysis confirmed that TLR2 plays a central role in PGN recognition by primary microglia. The expression of MyD88, a central adapter molecule in TLR-dependent signaling, was similar in both TLR2 KO and WT microglia, suggesting that the defect in PGN recognition by the former is not due to alterations in this key signaling intermediate. These findings reveal the complex nature of gram-positive bacterial recognition by microglia, which occurs, in part, through engagement of TLR2.
Toll-like receptor 2; microglia; S. aureus; PGN
Japanese Encephalitis virus (JEV) is a common cause of acute and epidemic viral encephalitis. JEV infection is associated with microglial activation resulting in the production of pro-inflammatory cytokines including Interleukin-1 β (IL-1β) and Interleukin-18 (IL-18). The Pattern Recognition Receptors (PRRs) and the underlying mechanism by which microglia identify the viral particle leading to the production of these cytokines is unknown.
For our studies, we have used murine model of JEV infection as well as BV-2 mouse microglia cell line. In this study, we have identified a signalling pathway which leads to the activation of caspase-1 as the key enzyme responsible for the maturation of both IL-1β and IL-18 in NACHT, LRR and PYD domains-containing protein-3 (NLRP3) dependent manner. Depletion of NLRP3 results in the reduction of caspase-1 activity and subsequent production of these cytokines.
Our results identify a mechanism mediated by Reactive Oxygen Species (ROS) production and potassium efflux as the two danger signals that link JEV infection to caspase-1 activation resulting in subsequent IL-1β and IL-18 maturation.
Microglial cells respond to herpes simplex virus (HSV)-1 by producing proinflammatory cytokines and chemokines. Following this inflammatory burst, these cells undergo apoptotic cell death. We have recently demonstrated that both virus-induced immune mediator production and apoptosis were induced through Toll-like receptor (TLR) 2 signaling. Based upon these findings, we hypothesized that inhibition of TLR2 signaling may serve as a means to alleviate excessive neuroinflammation. In the present study, we cloned four vaccinia virus (VV) proteins which have been reported to disrupt either TLR signaling or NF-κB activation, and overexpressed them in HEK293T cells stably expressing murine TLR2, as well as in primary murine microglia. Using an NF-κB-driven luciferase reporter gene assay, we show that upon stimulation with HSV and Listeria monocytogenes, all four vaccinia proteins inhibited TLR2 signaling, with different levels of inhibition in the TLR2-expressing cell line and primary microglia. We found similar results when microglial cells were stimulated with the TLR4 ligand LPS and the TLR9 ligand CpG ODN. Taken together, these data provide evidence that these VV proteins can function as inhibitors of TLR signaling in primary microglial cells.
Microglia; Toll-like receptors; vaccinia virus; NF-κB; HSV
Intestinal epithelial cells (IECs) form a barrier between invading microorganisms and the underlying host tissues. IECs express Toll-like receptors (TLRs) that recognize specific molecular signatures on microbes which activate intracellular signaling pathways leading to production of proinflammatory cytokines and chemokines. Stress hormones play an important role in modulation of proinflammatory cytokines and downregulation of immune responses. Here we demonstrated that expression levels of TLR-2, TLR-4, TLR-9 and TLR-11 were significantly increased in mouse IECs following infection with Toxoplasma gondii on day 8 post infection. In contrast, expression of TLRs was significantly decreased in infected mice subjected to cold water stress (CWS+INF). Expression of TLR-9 and TLR-11 in the mouse MODE-K cell line was significantly increased after infection. Expression of TLR-9 and TLR-11 in cells exposed to norepinephrine (NE) and parasites was significantly decreased when compared to cells exposed to parasites only. A significant increase was observed in SIGIRR, a negative regulator of TLRs in the CWS+INF group when compared to the INF group. Stress components were able to decrease expression levels of TLRs in IECs, decrease parasite load, and increase expression of a negative regulator thereby ameliorating intestinal inflammatory responses commonly observed during per oral T. gondii infection in C57BL/6 mice.
Toxoplasma gondii; Intestinal epithelial cells; Toll like receptors; stress hormones; innate immunity
Interferon (IFN)-gamma is an important component of the immune response to viral infections that can have a role both in controlling virus replication and inducing inflammatory damage. To determine the role of IFN-gamma in fatal alphavirus encephalitis, we have compared the responses of wild type C57BL/6 (WTB6) mice with mice deficient in either IFN-gamma (GKO) or the alpha-chain of the IFN-gamma receptor (GRKO) after intranasal infection with a neuroadapted strain of sindbis virus. Mortalities of GKO and GRKO mice were similar to WTB6 mice. Both GKO and GRKO mice had delayed virus clearance from the brain and spinal cord, more infiltrating perforin+ cells and lower levels of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 mRNAs than WTB6 mice. However, inflammation was more intense in GRKO mice than WTB6 or GKO mice with more infiltrating CD3+ T cells, greater expression of major histocompatibility complex-II and higher levels of interleukin-17A mRNA. Fibroblasts from GRKO embryos did not develop an antiviral response after treatment with IFN-gamma, but showed increases in TNF-alpha, IL-6, CXCL9 and CXCL10 mRNAs although these increases developed more slowly and were less intense than those of WTB6 fibroblasts. These data indicate that both GKO and GRKO mice fail to develop an IFN-gamma-mediated antiviral response, but differ in regulation of the inflammatory response to infection. Therefore, GKO and GRKO cannot be considered equivalent when assessing the role of IFN-gamma in CNS viral infections.
Microglia are the brain's tissue macrophages and are found in an activated state surrounding β-amyloid plaques in the Alzheimer's disease brain. Microglia interact with fibrillar β-amyloid (fAβ) through an ensemble of surface receptors composed of the α6β1 integrin, CD36, CD47, and the class A scavenger receptor. These receptors act in concert to initiate intracellular signaling cascades and phenotypic activation of these cells. However, it is unclear how engagement of this receptor complex is linked to the induction of an activated microglial phenotype. We report that the response of microglial cells to fibrillar forms of Aβ requires the participation of Toll like receptors (TLRs) and the co-receptor CD14. The response of microglia to fAβ is reliant upon CD14, which act together with TLR4 and TLR2 to bind fAβ and to activate intracellular signaling. We find that cells lacking these receptors could not initiate a Src-Vav-Rac signaling cascade leading to reactive oxygen species production and phagocytosis. The fAβ-mediated activation of p38 MAPK also required CD14, TLR4, and TLR2. Inhibition of p38 abrogated fAβ-induced reactive oxygen species production and attenuated the induction of phagocytosis. Microglia lacking CD14, TLR4, and TLR2 showed no induction of phosphorylated IκBα following fAβ. These data indicate these innate immune receptors function as members of the microglial fAβ receptor complex and identify the signaling mechanisms whereby they contribute to microglial activation.
microglia; fibrillar Aβ; Alzheimer's disease; CD14; TLR4; TLR2