Microglial Nitric Oxide Production Research Articles

TRPM2 contributes to LPS/IFNγ-induced production of nitric oxide via the p38/JNK pathway in microglia

Microglia are immune cells that maintain brain homeostasis at a resting state by surveying the environment and engulfing debris. However, in some pathological conditions, microglia can produce neurotoxic factors such as pro-inflammatory cytokines and nitric oxide (NO) that lead to neuronal degeneration. Inflammation-induced calcium (Ca2+) signaling is thought to underlie this abnormal activation of microglia, but the mechanisms are still obscure. We previously showed that combined application of lipopolysaccharide and interferon γ (LPS/IFNγ) induced-production of NO in microglia from wild-type (WT) mice is significantly reduced in microglia from transient receptor potential melastatin 2 (TRPM2)-knockout (KO) mice. Here, we found that LPS/IFNγ produced a late-onset Ca2+ signaling in WT microglia, which was abolished by application of the NADPH oxidase inhibitor diphenylene iodonium (DPI) and ML-171. In addition, pharmacological blockade or gene deletion of TRPM2 channel in microglia did not show this Ca2+ signaling. Furthermore, pharmacological manipulation and Western blotting revealed that Ca2+ mobilization, the proline-rich tyrosine kinase 2 (Pyk2), p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH2-terminal kinase (JNK) contributed to TRPM2-mediated LPS/IFNγ-induced activation, while the extracellular signal-regulated protein kinase (ERK) did not. These results suggest that LPS/IFNγ activates TRPM2-mediated Ca2+ signaling, which in turn increases downstream p38 MAPK and JNK signaling and results in increased NO production in microglia.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces microglial nitric oxide production and subsequent rat primary cortical neuron apoptosis through p38/JNK MAPK pathway

It has been widely accepted that microglia, which are the innate immune cells in the brain, upon activation can cause neuronal damage. In the present study, we investigated the role of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in regulating microglial nitric oxide production and its role in causing neuronal damage. The study revealed that TCDD stimulates the expression of inducible nitric oxide synthase (iNOS) as well as the production of nitric oxide (NO) in a dose- and time-dependent manner. Further, a rapid activation of p38 and JNK MAPKs was found in HAPI microglia following TCDD treatment. Blockage of p38 and JNK kinases with their specific inhibitors, SB202190 and SP600125, significantly reduced TCDD-induced iNOS expression and NO production. In addition, it was demonstrated through treating rat primary cortical neurons with media conditioned with TCDD treated microglia that microglial iNOS activation mediates neuronal apoptosis. Lastly, it was also found that p38 and JNK MAPK inhibitors could attenuate the apoptosis of rat cortical neurons upon exposure to medium conditioned by TCDD-treated HAPI microglial cells. Based on these observations, we highlight that the p38/JNK MAPK pathways play an important role in TCDD-induced iNOS activation in rat HAPI microglia and in the subsequent induction of apoptosis in primary cortical neurons.

Phytoalexin protects dopaminergic neurons against neurotoxicity by modulating myeloperoxidase levels in glial cells (P4199)

Abstract Myeloperoxidase functions as a key molecular component of the host defense system against diverse pathogens. We have previously reported that increased myeloperoxidase levels and activity is a distinguishing feature of rotenone-exposed glial cells, and that either overactivation or deficiency of myeloperoxidase leads to pathological conditions in the brain. Here, we provide that modulation of myeloperoxidase levels in glia by resveratrol confers protective effects on rotenone-induced neurotoxicity. We show that resveratrol significantly reduced myeloperoxidase levels but did not trigger abnormal nitric oxide production in microglia and astrocytes. Resveratrol-induced down-regulation of myeloperoxidase markedly attenuated rotenone-triggered inflammatory responses including phagocytic activity and reactive oxygen species production in primary microglia and astrocytes. In addition, impaired responses of primary mixed glia from MPO knockout mice to rotenone were relieved by treatment with resveratrol. We further show that rotenone-induced neuronal injury, particularly dopaminergic cell death, was attenuated by resveratrol in neuron-glia co-cultures, but not in neurons cultured alone. Collectively, our findings provide that resveratrol influences glial responses to rotenone by regulating both myeloperoxidase and nitric oxide, and thus protects against rotenone-induced neuronal injury.

Bradykinin decreases nitric oxide release from microglia via inhibition of cyclic adenosine monophosphate signaling

Bradykinin (BK) is a major potent inflammatory mediator outside the central nervous system. In Alzheimer's disease, BK release and BK receptor expression in brain tissues are upregulated relatively early during the course of the disease. Hence, BK was believed to promote neuroinflammation. However, BK was recently reported to possess anti-inflammatory and neuroprotective roles. Exposure of BV2 microglial cell line to BK lead to a decrease in NO release from unstimulated cells as well as a dose-dependent attenuation, mediated by both B1 and B2 receptors, in lipopolysaccharide (LPS)-induced NO production. In this study we examined whether cyclic adenosine monophosphate (cAMP) signaling is involved in BK-mediated effect in microglial nitric oxide (NO) production. A protein kinase A (PKA) inhibitor mimicked the effects of BK, while cAMP elevating agents antagonized BK-mediated NO decrease. Moreover, BK inhibited the activation of cAMP responsive element binding protein (CREB). In addition, BK protected microglial cells from death triggered by combinations of LPS and each of the cAMP elevating agents. Finally, the addition of Gαi protein inhibitor abrogated the effects of BK on NO release, and the expression of Gαi protein in the plasma membrane was induced by BK. These results suggest that BK-mediated reduction in microglial NO production depends on coupling to Gi protein and also involves inhibition of cAMP-PKA-CREB signaling.

Temperature- and time-dependent changes in TLR2-activated microglial NF-κB activity and concentrations of inflammatory and anti-inflammatory factors

Therapeutic hypothermia protects neurons following injury to the central nervous system (CNS). Microglia express toll-like receptors (TLRs) that play significant roles in pathological processes in sterile CNS injury. We have examined the effects of culture temperature on the TLR2-activated microglial production of cytokines and nitric oxide (NO), which are known to be associated with CNS damage, and the possible involvement of nuclear factor-κB (NF-κB) activation underlying such effects. Rat microglia were cultured with a selective TLR2 agonist, Pam(3)CSK(4), under hypothermic, normothermic, and hyperthermic conditions, and with Pam(3)CSK(4) in the presence of a NF-κB activation inhibitor at 37 °C. Cytokine and NO levels and NF-κB p65 activation were measured. The production of tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), and NO and the activation of NF-κB p65 were reduced by hypothermia, but augmented by hyperthermia at 3-6, 24-48, 48, and 0.5 h, post-treatment initiation, respectively. Pharmacological inhibition of NF-κB activation impaired the Pam(3)CSK(4)-induced TNF-α, IL-10, and NO production. In TLR2-activated microglia, hypothermia reduced, while hyperthermia increased, the early activation of NF-κB and the subsequent NF-κB-mediated production of TNF-α, IL-10, and NO in a time-dependent manner, suggesting that attenuation of these factors via suppression of NF-κB in microglia is one possible neuroprotective mechanism of therapeutic hypothermia. Moreover, temperature-dependent changes in microglial TNF-α production during the early phase and IL-10 and NO production during the late phase indicate that these factors might be useful as clinical markers to monitor hypothermia-related neuronal protection and hyperthermia-related neuronal injury.

Inhibitory role of kinins on microglial nitric oxide and tumor necrosis factor-α production

Brain inflammation is sustained by chronic activation of microglia and the over-production of pro-inflammatory cytokines and nitric oxide (NO), which in turn can be highly neurotoxic. Microglial activation can be regulated by neuropeptides such as bradykinin (BK) and other members of the kinin family. Kinins are well known inflammatory regulators outside the CNS. Although the kinin system is well distributed throughout the brain, the precise role of BK in the CNS is not yet clear. The aim of this study was to examine and characterize the effects of BK and related kinins on the production of NO and TNF-α in microglia. We found that BK and selective agonists for both B1 and B2 receptors, attenuated both NO and TNF-α levels in the media of BV2 microglial cells that had been stimulated with LPS. The effects of BK that were observed in BV2 cells were confirmed in primary neonatal rat microglial cells as well. In addition, all kinin agonists reduced the expression of iNOS and TNF-α protein and mRNA levels in LPS-stimulated BV2 cells. Also, while LPS activated the nuclear factor-κB (NF-κB) pathway, BK inhibited NF-κB activation by preventing degradation of the κB protein (IκB) inhibitor, abolishing translocation of p65 and p50 subunits to the nucleus and inhibiting NF-κB transcription activity. These results suggest a role for bradykinin in modulation of glial inflammation, as evidenced by attenuation of NO and TNF-α synthesis pathways in activated microglial cells.

Inhibitory effects of chalcone glycosides isolated from Brassica rapa L. ‘hidabeni’ and their synthetic derivatives on LPS-induced NO production in microglia

Activation of microglia induces the production of various inflammatory mediators including nitric oxide (NO), leading to neurodegeneration in many central nervous system diseases. In this study, we examined the effects of chalcone glycosides isolated from Brassica rapa L. ‘hidabeni’ on lipopolysaccharide (LPS)-induced NO production using rat immortalized microglia HAPI cells. 4′-O-β-d-Glucopyranosyl-3′,4-dimethoxychalcone (A2) inhibited LPS-induced inducible NO synthase (iNOS) expression and NO production. However, A2 did not affect nuclear factor-κB and mitogen-activated protein kinase pathways. The signal transduction and activator of transcription 1 (STAT1), which is activated via production of IFN-β by LPS, is an important transcription factor responsible for LPS-induced iNOS expression. A2 suppressed LPS-induced phosphorylation and nuclear translocation of STAT1, although it had no effects on LPS-induced IFN-β expression. These results indicate that the inhibitory effect of A2 is due to the prevention of STAT signaling. Moreover, structure–activity relationship studies on newly synthesized ‘hidabeni’ chalcone derivatives showed that 4′-O-β-d-glucopyranosyl-3′-methoxychalcone (A11), which has no functional groups in the B-ring, inhibits LPS-induced NO production more potently than A2.

Glycine enhances microglial intracellular calcium signaling. A role for sodium-coupled neutral amino acid transporters

The inhibitory neurotransmitter glycine is known to enhance microglial nitric oxide production. However, up to now, the mechanism is undocumented. Since calcium is an important second messenger in both immune and glial cells, we studied the effects of glycine on intracellular calcium signaling. We found that millimolar concentrations of glycine enhance microglial intracellular calcium transients induced by 100μM ATP or by 500nM thapsigargin. This modulation was unaffected by the glycine receptor antagonist strychnine and could not be mimicked by glycine receptor agonists such as taurine or β-alanine, indicating glycine receptor independency. The modulation of calcium responses could be mimicked by several structurally related amino acids (e.g., serine, alanine, or glutamine) and was inhibited in the presence of the neutral amino acid transporter substrate α-aminoisobutyric acid (AIB). We correlated these findings to immunofluorescence glycine uptake experiments which showed a clear glycine uptake which was inhibited by AIB. Furthermore, all amino acids that were shown to modulate calcium responses also evoked AIB-sensitive inward currents, mainly carried by sodium, as demonstrated by patch clamp experiments. Based on these findings, we propose that sodium-coupled neutral amino acid transporters are responsible for the observed glycine modulation of intracellular calcium responses.

Neuropeptide Y Modulation of Interleukin-1β (IL-1β)-induced Nitric Oxide Production in Microglia

Given the modulatory role of neuropeptide Y (NPY) in the immune system, we investigated the effect of NPY on the production of NO and IL-1β in microglia. Upon LPS stimulation, NPY treatment inhibited NO production as well as the expression of inducible nitric-oxide synthase (iNOS). Pharmacological studies with a selective Y(1) receptor agonist and selective antagonists for Y(1), Y(2), and Y(5) receptors demonstrated that inhibition of NO production and iNOS expression was mediated exclusively through Y(1) receptor activation. Microglial cells stimulated with LPS and ATP responded with a massive release of IL-1β, as measured by ELISA. NPY inhibited this effect, suggesting that it can strongly impair the release of IL-1β. Furthermore, we observed that IL-1β stimulation induced NO production and that the use of a selective IL-1 receptor antagonist prevented NO production upon LPS stimulation. Moreover, NPY acting through Y(1) receptor inhibited LPS-stimulated release of IL-1β, inhibiting NO synthesis. IL-1β activation of NF-κB was inhibited by NPY treatment, as observed by confocal microscopy and Western blotting analysis of nuclear translocation of NF-κB p65 subunit, leading to the decrease of NO synthesis. Our results showed that upon LPS challenge, microglial cells release IL-1β, promoting the production of NO through a NF-κB-dependent pathway. Also, NPY was able to strongly inhibit NO synthesis through Y(1) receptor activation, which prevents IL-1β release and thus inhibits nuclear translocation of NF-κB. The role of NPY in key inflammatory events may contribute to unravel novel gateways to modulate inflammation associated with brain pathology.

Stimulation of glucocorticoid‐induced tumor necrosis factor receptor family‐related protein ligand (GITRL) induces inflammatory activation of microglia in culture

Glucocorticoid-induced tumor necrosis factor receptor family-related protein ligand (GITRL) is a member of the tumor necrosis factor superfamily (TNFSF) and is known to act as a costimulator in the immune system by binding to GITR. GITRL is expressed in endothelial cells, dendritic cells, macrophages, and B cells, but it is not known whether GITRL is expressed in brain microglia cells. Here, we investigated the expression of GITR and GITRL and their potential role in microglia cells. Using BV-2 mouse microglia cells and mouse primary microglia cultures, we have demonstrated that 1) both GITR and GITRL are expressed in microglia cells; 2) stimulation of GITRL induces inflammatory activation of microglia on the basis of production of nitric oxide (NO) and expression of inducible nitric oxide synthase, cyclooxygenase-2, CD40, and matrix metalloproteinase-9; 3) GITRL-mediated microglial NO production partially depends on p38 MAPK, JNK, and nuclear factor-kappaB pathways; and 4) GITRL stimulation also induces microglia cell death. These results indicate that GITR and GITRL are functionally expressed on brain microglia and that the stimulation of GITRL can induce inflammatory activation of microglia. The GITR/GITRL system may play an important role in neuroinflammation.

Obovatol attenuates microglia‐mediated neuroinflammation by modulating redox regulation

Obovatol isolated from the medicinal herb Magnolia obovata exhibits a variety of biological activities. Here, the effect of obovatol and its mechanism of action on microglial activation, neuroinflammation and neurodegeneration were investigated. In microglial BV-2 cells stimulated with lipopolysaccharide (LPS), we measured nitric oxide (NO) and cytokine production, and activation of intracellular signalling pathways by reverse transcription-polymerase chain reaction and Western blots. Cell death was assayed in co-cultures of activated microglia (with bacterial LPS) and neurons and in LPS- induced neuroinflammation in mice in vivo. Obovatol inhibited microglial NO production with an IC50 value of 10 mM. Obovatol also inhibited microglial expression of proinflammatory cytokines and inducible nitric-oxide synthase, which was accompanied by the inhibition of multiple signalling pathways such as nuclear factor kappa B, signal transducers and activators of transcription 1, and mitogen-activated protein kinases. In addition, obovatol protected cultured neurons from microglial toxicity and inhibited neuroinflammation in mice in vivo. One molecular target of obovatol in microglia was peroxiredoxin 2 (Prx2), identified by affinity chromatography and mass spectrometry. Obovatol enhanced the reactive oxygen species (ROS)-scavenging activity of Prx2 in vitro, thereby suppressing proinflammatory signalling pathways of microglia where ROS plays an important role. Obovatol is not only a useful chemical tool that can be used to investigate microglial signalling, but also a promising drug candidate against neuroinflammatory diseases. Furthermore, our results indicate that Prx2 is a novel drug target that can be exploited for the therapeutic modulation of neuroinflammatory signalling.

Inhibitory effect of fucoidan on nitric oxide production in lipopolysaccharide‐activated primary microglia

1. Microglial activation plays an important role in the pathogenesis of neurodegenerative diseases by producing various pro-inflammatory cytokines. Microglia-derived nitric oxide (NO) is critical for the lipopolysaccharide (LPS)-induced selective loss of dopaminergic neurons. 2. Fucoidan is a sulphated polysaccharide extracted from brown seaweeds. It has a variety of biological actions, including anticoagulant, antiviral and anti-inflammatory effects. The aim of the present study was to investigate the effects of fucoidan on LPS-induced cellular activation in microglia and to evaluate the inhibitory mechanisms involved. 3. To investigate the effects of fucoidan on LPS-induced cellular activation in microglia, primary microglial cells were preincubated with fucoidan (31.25, 62.5 and 125 microg/mL) for 10 min, followed by stimulation with LPS (0.01 microg/mL). Then, cell shape and NO production were determined 24 h after LPS stimulation, whereas inducible nitric oxide synthase (iNOS) mRNA and protein expression were determined at 6 and 18 h after LPS stimulation, respectively. To evaluate the inhibitory mechanisms involved, mitogen-activated protein kinase (MAPK) activation was also evaluated. 4. Lipopolysaccharide transformed cells into an amoeboid shape, whereas 62.5 microg/mL fucoidan inhibited this activation. Moreover, 125 microg/mL fucoidan significantly inhibited microglial NO production to 75% of that in LPS-treated group and also significantly diminished the expression of iNOS mRNA and protein by nearly 50%. Fucoidan (125 microg/mL) also suppressed phosphorylation of p38 and extracellular signal-regulated kinase (ERK) by approximately 50%, but not that of c-Jun N-terminal kinase. 5. The results provide the first evidence that fucoidan has a potent inhibitory effect against LPS-induced NO production by microglia. The results also suggest that this inhibitory action of fucoidan involves suppression of p38 and ERK phosphorylation.

Microglia signaling as a target of donepezil

Donepezil is a reversible and noncompetitive cholinesterase inhibitor. The drug is considered as a first-line treatment in patients with mild to moderate Alzheimer's disease. Recently, anti-inflammatory and neuroprotective effects of the drug have been reported. "Cholinergic anti-inflammation pathway" has major implications in these effects. Here, we present evidence that donepezil at 5-20 microM directly acts on microglial cells to inhibit their inflammatory activation. Our conclusion is based on the measurement of nitric oxide and proinflammatory mediators using purified microglia cultures and microglia cell lines: donepezil attenuated microglial production of nitric oxide and tumor necrosis factor (TNF)-alpha, and suppressed the gene expression of inducible nitric oxide synthase, interleukin-1 beta, and TNF-alpha. Subsequent studies showed that donepezil inhibited a canonical inflammatory NF-kappaB signaling. Microglia/neuroblastoma coculture and animal experiments supported the anti-inflammatory effects of donepezil. Based on the studies using nicotinic acetylcholine receptor antagonists, the donepezil inhibition of microglial activation was independent of acetylcholine and its receptor. Thus, inflammatory activation signaling of microglia may be one of the direct targets of donepezil in the central nervous system. It should be noted, however, that there is a large gap between the therapeutic dose of the drug used clinically and the concentration of the drug that exerts the direct action on microglial cells.

Identification of KT-15073 as an Inhibitor of Lipopolysaccharide-Induced Microglial Activation

Neuroinflammation has recently been implicated as an important mechanism responsible for the progression of neurodegenerative diseases. Activated microglia produce various proinflammatory cytokines and nitric oxide (NO) that are toxic to neurons. Thus, inhibition of microglial activation may alleviate neuroinflammatory and neurodegenerative processes. Among several fluorovinyloxyacetamide derivatives that were screened by microglia cell-based assay, a novel synthetic compound KT-15073 was identified to strongly attenuate the microglial production of NO and tumor necrosis factor-alpha (TNF-alpha). This compound also suppressed the gene expression of interleukin-1beta, inducible nitric oxide synthase, and TNF-alpha. KT-15073 inhibited the nuclear translocation and DNA binding of nuclear factor-kappaB as well as phosphorylation of p38 mitogen-activated protein kinase. In addition, KT-15073 reduced the cytotoxicity of lipopolysaccharide (LPS)-stimulated microglia toward B35 neuroblastoma cells in the microglia/neuroblastoma coculture, suggesting that the compound might exhibit the neuroprotective activity. Thus, KT-15073 has an anti-inflammatory activity in microglia, and may have a therapeutic potential for the treatment of neuroinflammatory or neurodegenerative diseases.

A novel anti-neuroinflammatory pyridylimidazole compound KR-31360

Excessive microglial activation with overexpression of proinflammatory cytokines and oxidative stress products is linked to the progression of several neurodegenerative diseases; therefore, suppression of microglial activation is a potential therapeutic approach against these diseases. Since nitric oxide (NO) is one of the major inflammatory mediators that are produced by activated microglia, inhibitory effects of novel synthetic compounds on microglial NO production were investigated. From the mouse microglia cell-based assays, an imidazo [4,5-b] pyridine compound KR-31360 was identified as an inhibitor of microglial NO production with an IC 50 value of 2 μM. Structure–activity relationship study indicated that 5-position of imidazo [4,5-b] pyridine ring is critical for the activity. KR-31360 also inhibited lipopolysaccharide (LPS)-induced secretion of tumor necrosis factor alpha (TNF-α) and transcription of TNF-α, interleukin-1 beta, and inducible nitric oxide synthase as well as activation of nuclear factor kappa B and mitogen-activated protein kinases. KR-31360 was neuroprotective by suppressing microglial neurotoxicity in a microglia–neuron coculture. The neuroprotective activity of the compound was most effective when microglia were pretreated with the compound prior to LPS challenge. The inhibitory effect of KR-31360 on microglial activation was further demonstrated in a mouse neuroinflammation model in vivo: compared to vehicle-injected animals, KR-31360 injection attenuated LPS-induced microglial activation as evidenced by isolectin B4 staining and proinflammatory gene expression of brain sections. DNA microarray analysis supported that KR-31360 targeted Toll-like receptor 4 pathways. In addition to being a new drug candidate against neuroinflammatory diseases, the compound may be a powerful tool for the better understanding of microglia biology and neuroinflammation.

Delayed treatments for stroke influence neuronal death in rat organotypic slice cultures subjected to oxygen glucose deprivation

A major limitation of current stroke therapies is the need to treat candidate patients within 3 h of stroke onset. Human umbilical cord blood cell (HUCBC) and the sigma receptor agonist 1,3, di-o-tolylguanidine (DTG) administration both caused significant reductions in brain damage in the rat middle cerebral artery occlusion model of stroke when administered at delayed timepoints. In vivo, these treatments suppress the infiltration of peripheral lymphocytes into the brain in addition to decreasing neurodegeneration. An ex vivo organotypic slice culture (OTC) model was utilized to characterize the efficacy of these treatments in mitigating neurodegeneration in ischemic brain tissue in the absence of the peripheral immune system. Slice cultures subjected to oxygen glucose deprivation (OGD) had significantly elevated levels of degenerating neurons and microglial nitric oxide production when compared to their normoxic counterparts. In cultures subjected to OGD, HUCBC but not DTG treatment reduced the number of degenerating neurons and the production of microglial derived nitric oxide back to levels detected in normoxic controls. These data show that HUCBC treatment can mediate direct neuroprotection and suppress innate inflammation in ischemic brain tissue in the absence of the peripheral immune system, whereas DTG requires peripheral effects to mediate neuroprotection. These experiments yield insight into the mechanisms by which these neuroprotective treatments function at delayed timepoints following stroke.

Staphylococcus aureusInduces Microglial Inflammation via a Glycogen Synthase Kinase 3β-Regulated Pathway

A proinflammatory role for glycogen synthase kinase 3beta (GSK-3beta) has been demonstrated. Here, we addressed its roles on heat-inactivated Staphylococcus aureus-induced microglial inflammation. Heat-inactivated S. aureus induced tumor necrosis factor alpha (TNF-alpha) and nitric oxide (NO) production, at least in part, via a Toll-like receptor 2-regulated pathway. Neutralization of TNF-alpha largely blocked heat-inactivated S. aureus-induced NO. Heat-inactivated S. aureus activated GSK-3beta, and inhibiting GSK-3beta reduced TNF-alpha production as well as inducible NO synthase (iNOS)/NO biosynthesis. While activation of NF-kappaB was essential for heat-inactivated S. aureus-induced TNF-alpha and NO, inhibiting GSK-3beta blocked heat-inactivated S. aureus-induced NF-kappaB p65 nuclear translocation. Additionally, inhibiting GSK-3beta enhanced heat-inactivated S. aureus-induced interleukin-10 (IL-10) production (IL-10 is an anti-inflammatory cytokine which inhibits TNF-alpha production). Neutralization of IL-10 reduced TNF-alpha downregulation caused by GSK-3beta inhibition. These results suggest that GSK-3beta regulates heat-inactivated S. aureus-induced TNF-alpha and NO production in microglia mainly by activating NF-kappaB and probably by inhibiting IL-10.

Neurotoxic Activation of Microglia Is Promoted by a Nox1-Dependent NADPH Oxidase

Reactive oxygen species (ROS) modulate intracellular signaling but are also responsible for neuronal damage in pathological states. Microglia, the resident CNS macrophages, are prominent sources of ROS through expression of the phagocyte oxidase which catalytic subunit Nox2 generates superoxide ion (O2(.-)). Here we show that microglia also express Nox1 and other components of nonphagocyte NADPH oxidases, including p22(phox), NOXO1, NOXA1, and Rac1/2. The subcellular distribution and functions of Nox1 were determined by blocking Nox activity with diphenylene iodonium or apocynin, and by silencing the Nox1 gene in microglia purified from wild-type (WT) or Nox2-KO mice. [Nox1-p22(phox)] dimers localized in intracellular compartments are recruited to phagosome membranes during microglial phagocytosis of zymosan, and Nox1 produces O2(.-) in zymosan-loaded phagosomes. In microglia activated with lipopolysaccharide (LPS), Nox1 produces O2(.-), which enhances cell expression of inducible nitric oxide synthase and secretion of interleukin-1beta. Comparisons of microglia purified from WT, Nox2-KO, or Nox1-KO mice indicate that both Nox1 and Nox2 are required to optimize microglial production of nitric oxide. By injecting LPS in the striatum of WT and Nox1-KO mice, we show that Nox1 also enhances microglial production of cytotoxic nitrite species and promotes loss of presynaptic proteins in striatal neurons. These results demonstrate the functional expression of Nox1 in resident CNS phagocytes, which can promote production of neurotoxic compounds during neuroinflammation. Our study also shows that Nox1- and Nox2-dependent oxidases play distinct roles in microglial activation and that Nox1 is a possible target for the treatment of neuroinflammatory states.

Inhibition of nitric oxide production by the carbazole compound LCY-2-CHO via blockade of activator protein-1 and CCAAT/enhancer-binding protein activation in microglia

Excessive nitric oxide (NO) production by activated microglia plays a critical role in neurodegenerative disorders. In this study, we found that 9-(2-chlorobenyl)-9 H-carbazole-3-carbaldehyde (LCY-2-CHO) suppressed the NO production in lipopolysaccharide (LPS)/interferon-γ (IFNγ)-stimulated murine microglial N9 and BV-2 cells and in LPS-stimulated N9 cells and rat primary microglia. LCY-2-CHO had no cytotoxic effect on microglia. In activated N9 cells, LCY-2-CHO abolished the expression of inducible nitric oxide synthase (iNOS) protein and mRNA but failed to alter the stability of expressed iNOS mRNA and the enzymatic activity of expressed iNOS protein. LCY-2-CHO did not block DNA-binding activity of nuclear factor-κB (NF-κB) or cyclic AMP response element-binding protein (CREB), but abolished that of activator protein-1 (AP-1), CCAAT/enhancer-binding protein (C/EBP) and nuclear factor IL6 (NF-IL6). LCY-2-CHO attenuated the nuclear levels of c-Jun and C/EBPβ, but not those of p65, p50, C/EBPδ, signal transducer and activator of transcription-1 (STAT-1) or the nuclear expression of IFN regulatory factor-1 (IRF-1). LCY-2-CHO had no effect on the phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun NH 2-terminal kinase (JNK), MAPK-activated protein kinase-2 (MAPKAPK-2), STAT-1, CREB or c-Jun in LPS/IFNγ-stimulated N9 cells, whereas it attenuated the phosphorylation of C/EBPβ at Ser105 and Thr235 residues, which occurred concomitantly with LCY-2-CHO inhibition of C/EBPβ expression and phosphorylation. Taken together, these results suggest that LCY-2-CHO inhibits NO production in microglia through the blockade of AP-1 and C/EBP activation.

Expression of CXCL4 in microglia in vitro and in vivo and its possible signaling through CXCR3

Signaling through chemokine receptor CXCR3 in the brain has been implicated in various brain diseases, as CXCR3 and its ligands are found under these conditions. Recently, a new chemokine ligand for CXCR3 was reported. In humans, an alternatively spliced variant of CXCR3 expressed on microvascular endothelial cells, named CXCR3b, was shown to bind CXCL4. In the periphery, the cellular expression and functions of CXCL4 are well described but in the brain its expression and function are unknown. Here, we show that brain microglia are a cellular source of CXCL4 in vitro and in vivo under neurodegenerating conditions. Microglial migration induced by CXCL4 is absent in CXCR3-deficient microglia, indicating a role of CXCR3. CXCL4 furthermore attenuates lipopolysaccharide-induced microglial phagocytosis and nitric oxide production in microglia and BV-2 cells. Based on these findings, it is proposed that locally released CXCL4 may control microglia responses.