Cytokine Expression In Microglia Research Articles

Inhibition of ANGPTL8 protects against diabetes-associated cognitive dysfunction by reducing synaptic loss via the PirB signaling pathway

BackgroundType 2 diabetes mellitus (T2D) is associated with an increased risk of cognitive dysfunction. Angiopoietin-like protein 8 (ANGPTL8) is an important regulator in T2D, but the role of ANGPTL8 in diabetes-associated cognitive dysfunction remains unknown. Here, we explored the role of ANGPTL8 in diabetes-associated cognitive dysfunction through its interaction with paired immunoglobulin-like receptor B (PirB) in the central nervous system.MethodsThe levels of ANGPTL8 in type 2 diabetic patients with cognitive dysfunction and control individuals were measured. Mouse models of diabetes-associated cognitive dysfunction were constructed to investigate the role of ANGPTL8 in cognitive function. The cognitive function of the mice was assessed by the Barnes Maze test and the novel object recognition test, and levels of ANGPTL8, synaptic and axonal markers, and pro-inflammatory cytokines were measured. Primary neurons and microglia were treated with recombinant ANGPTL8 protein (rA8), and subsequent changes were examined. In addition, the changes induced by ANGPTL8 were validated after blocking PirB and its downstream pathways. Finally, mice with central nervous system-specific knockout of Angptl8 and PirB−/− mice were generated, and relevant in vivo experiments were performed.ResultsHere, we demonstrated that in the diabetic brain, ANGPTL8 was secreted by neurons into the hippocampus, resulting in neuroinflammation and impairment of synaptic plasticity. Moreover, neuron-specific Angptl8 knockout prevented diabetes-associated cognitive dysfunction and neuroinflammation. Mechanistically, ANGPTL8 acted in parallel to neurons and microglia via its receptor PirB, manifesting as downregulation of synaptic and axonal markers in neurons and upregulation of proinflammatory cytokine expression in microglia. In vivo, PirB−/− mice exhibited resistance to ANGPTL8-induced neuroinflammation and synaptic damage.ConclusionTaken together, our findings reveal the role of ANGPTL8 in the pathogenesis of diabetes-associated cognitive dysfunction and identify the ANGPTL8-PirB signaling pathway as a potential target for the management of this condition.

Purpurogallin Reverses Neuronal Apoptosis and Enhances "M2" Polarization of Microglia Under Ischemia via Mediating the miR-124-3p/TRAF6/NF-κB Axis.

Purpurogallin (PPG) has been demonstrated to exert an anti-inflammatory function in neurological diseases. This study aimed at investigating the role of PPG on microglial polarization post ischemic stroke as well as the underlying mechanism. Mouse hippocampal neurons HT-22 and microglial BV2 cells were treated by oxygen and glucose deprivation to simulate an in-vitro ischemia model. qRT-PCR and ELISA examined expression of cytokines in microglia. CCK8 and flow cytometry measured HT-22 cell viability and apoptosis, respectively. The levels of miR-124-3p and TRAF6/NF-κB were determined. A mouse cerebral ischemia model was set up using middle cerebral artery occlusion (MCAO) method. After being dealt with PPG, the neurological functions, brain edema, neuronal apoptosis, and microglia activation of the mice were evaluated. As suggested by the results, PPG transformed "M1" to "M2" polarization of BV2 cells, and abated HT-22 cell apoptosis. PPG enhanced the neurological functions, alleviated brain edema, and decreased neuroinflammatory responses, and neuronal apoptosis in the brain lesions of MCAO mice. Furthermore, PPG enhanced miR-124-3p and repressed the TRAF6/NF-κB pathway. miR-124-3p suppressed the TRAF6/NF-κB pathway by targeting TRAF6. Collectively, PPG alleviates ischemia-induced neuronal damage and microglial inflammation by modulating the miR-124-3p/TRAF6/NF-κB pathway.

Activation of locus coeruleus-spinal cord noradrenergic neurons alleviates neuropathic pain in mice via reducing neuroinflammation from astrocytes and microglia in spinal dorsal horn

BackgroundThe noradrenergic neurons of locus coeruleus (LC) project to the spinal dorsal horn (SDH), and release norepinephrine (NE) to inhibit pain transmission. However, its effect on pathological pain and the cellular mechanism in the SDH remains unclear. This study aimed to explore the analgesic effects and the anti-neuroinflammation mechanism of LC-spinal cord noradrenergic pathway (LC:SC) in neuropathic pain (NP) mice with sciatic chronic constriction injury.MethodsThe Designer Receptors Exclusively Activated by Designer Drugs (DREADD) was used to selectively activate LC:SC. Noradrenergic neuron-specific retro–adeno-associated virus was injected to the spinal cord. Pain threshold, LC and wide dynamic range (WDR) neuron firing, neuroinflammation (microglia and astrocyte activation, cytokine expression), and α2AR expression in SDH were evaluated.ResultsActivation of LC:SC with DREADD increased the mechanical and thermal nociceptive thresholds and reduced the WDR neuron firing. LC:SC activation (daily, 7 days) downregulated TNF-α and IL-1β expression, upregulated IL-4 and IL-10 expression in SDH, and inhibited microglia and astrocytes activation in NP mice. Immunofluorescence double staining confirmed that LC:SC activation decreased the expression of cytokines in microglia of the SDH. In addition, the effects of LC:SC activation could be reversed by intrathecal injection of yohimbine. Immunofluorescence of SDH showed that NE receptor α2B-AR was highly expressed in microglia in CCI mice.ConclusionThese findings indicate that selective activation of LC:SC alleviates NP in mice by increasing the release of NE and reducing neuroinflammation of astrocytes and microglia in SDH.

Inhibition of repulsive guidance molecule-a protects dopaminergic neurons in a mouse model of Parkinson\u2019s disease

Repulsive guidance molecule-a (RGMa), a glycosylphosphatidylinositol-anchored membrane protein, has diverse functions in axon guidance, cell patterning, and cell survival. Inhibition of RGMa attenuates pathological dysfunction in animal models of central nervous system (CNS) diseases including spinal cord injury, multiple sclerosis, and neuromyelitis optica. Here, we examined whether antibody-based inhibition of RGMa had therapeutic effects in a mouse model of Parkinson’s disease (PD). We treated mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and found increased RGMa expression in the substantia nigra (SN). Intraventricular, as well as intravenous, administration of anti-RGMa antibodies reduced the loss of tyrosine hydroxylase (TH)-positive neurons and accumulation of Iba1-positive microglia/macrophages in the SN of MPTP-treated mice. Selective expression of RGMa in TH-positive neurons in the SN-induced neuronal loss/degeneration and inflammation, resulting in a progressive movement disorder. The pathogenic effects of RGMa overexpression were attenuated by treatment with minocycline, which inhibits microglia and macrophage activation. Increased RGMa expression upregulated pro-inflammatory cytokine expression in microglia. Our observations suggest that the upregulation of RGMa is associated with the PD pathology; furthermore, inhibitory RGMa antibodies are a potential therapeutic option.

MicroRNA-210 downregulates TET2 and contributes to inflammatory response in neonatal hypoxic-ischemic brain injury

BackgroundNeonatal hypoxic-ischemic (HI) brain injury is a leading cause of acute mortality and chronic disability in newborns. Our previous studies demonstrated that HI insult significantly increased microRNA-210 (miR-210) in the brain of rat pups and inhibition of brain endogenous miR-210 by its inhibitor (LNA) provided neuroprotective effect in HI-induced brain injury. However, the molecular mechanisms underpinning this neuroprotection remain unclear.MethodsWe made a neonatal HI brain injury model in mouse pups of postnatal day 7 to uncover the mechanism of miR-210 in targeting the ten eleven translocation (TET) methylcytosine dioxygenase 2 that is a transcriptional suppressor of pro-inflammatory cytokine genes in the neonatal brain. TET2 silencing RNA was used to evaluate the role of TET2 in the neonatal HI-induced pro-inflammatory response and brain injury. MiR-210 mimic and inhibitor (LNA) were delivered into the brain of mouse pups to study the regulation of miR-210 on the expression of TET2. Luciferase reporter gene assay was performed to validate the direct binding of miR-210 to the 3′ untranslated region of the TET2 transcript. Furthermore, BV2 mouse microglia cell line was employed to confirm the role of miR-210-TET2 axis in regulating pro-inflammatory response in microglia. Post-assays included chromatin immunoprecipitation (ChIP) assay, co-immunoprecipitation, RT-PCR, brain infarct assay, and neurobehavioral test. Student’s t test or one-way ANOVA was used for statistical analysis.ResultsHI insult significantly upregulated miR-210, downregulated TET2 protein abundance, and increased NF-κB subunit p65 acetylation level and its DNA binding capacity to the interleukin 1 beta (IL-1β) promoter in the brain of mouse pups. Inhibition of miR-210 rescued TET2 protein level from HI insult and miR-210 mimic decreased TET2 protein level in the brain of mouse pups, suggesting that TET2 is a functional target of miR-210. The co-immunoprecipitation was performed to reveal the role of TET2 in HI-induced inflammatory response in the neonatal brain. The result showed that TET2 interacted with NF-κB subunit p65 and histone deacetylase 3 (HDAC3), a co-repressor of gene transcription. Furthermore, TET2 knockdown increased transcriptional activity of acetyl-p65 on IL-1β gene in the neonatal brain and enhanced HI-induced upregulation of acetyl-p65 level and pro-inflammatory cytokine expression. Of importance, TET2 knockdown exacerbated brain infarct size and neurological deficits and counteracted the neuroprotective effect of miR-210 inhibition. Finally, the in vitro results demonstrated that the miR-210-TET2 axis regulated pro-inflammatory response in BV2 mouse microglia cell line.ConclusionsThe miR-210-TET2 axis regulates pro-inflammatory cytokine expression in microglia, contributing to neonatal HI brain injury.

Butyrate and Dietary Soluble Fiber Improve Neuroinflammation Associated With Aging in Mice.

Aging results in chronic systemic inflammation that can alter neuroinflammation of the brain. Specifically, microglia shift to a pro-inflammatory phenotype predisposing them to hyperactivation upon stimulation by peripheral immune signals. It is proposed that certain nutrients can delay brain aging by preventing or reversing microglial hyperactivation. Butyrate, a short-chain fatty acid (SCFA) produced primarily by bacterial fermentation of fiber in the colon, has been extensively studied pharmacologically as a histone deacetylase inhibitor and serves as an attractive therapeutic candidate, as butyrate has also been shown to be anti-inflammatory and improve memory in animal models. In this study, we demonstrate that butyrate can attenuate pro-inflammatory cytokine expression in microglia in aged mice. It is still not fully understood, however, if an increase in butyrate-producing bacteria in the gut as a consequence of a diet high in soluble fiber could affect microglial activation during aging. Adult and aged mice were fed either a 1% cellulose (low fiber) or 5% inulin (high fiber) diet for 4 weeks. Findings indicate that mice fed inulin had an altered gut microbiome and increased butyrate, acetate, and total SCFA production. In addition, histological scoring of the distal colon demonstrated that aged animals on the low fiber diet had increased inflammatory infiltrate that was significantly reduced in animals consuming the high fiber diet. Furthermore, gene expression of inflammatory markers, epigenetic regulators, and the microglial sensory apparatus (i.e., the sensome) were altered by both diet and age, with aged animals exhibiting a more anti-inflammatory microglial profile on the high fiber diet. Taken together, high fiber supplementation in aging is a non-invasive strategy to increase butyrate levels, and these data suggest that an increase in butyrate through added soluble fiber such as inulin could counterbalance the age-related microbiota dysbiosis, potentially leading to neurological benefits.

Inhibition of Neuroinflammation by AIBP: Spinal Effects upon Facilitated Pain States

Apolipoprotein A-I binding protein (AIBP) reduces lipid raft abundance by augmenting the removal of excess cholesterol from the plasma membrane. Here, we report that AIBP prevents and reverses processes associated with neuroinflammatory-mediated spinal nociceptive processing. The mechanism involves AIBP binding to Toll-like receptor-4 (TLR4) and increased binding of AIBP to activated microglia, which mediates selective regulation of lipid rafts in inflammatory cells. AIBP-mediated lipid raft reductions downregulate LPS-induced TLR4 dimerization, inflammatory signaling, and expression of cytokines in microglia. In mice, intrathecal injections of AIBP reduce spinal myeloid cell lipid rafts, TLR4 dimerization, neuroinflammation, and glial activation. Intrathecal AIBP reverses established allodynia in mice in which pain states were induced by the chemotherapeutic cisplatin, intraplantar formalin, or intrathecal LPS, all of which are pro-nociceptive interventions known to be regulated by TLR4 signaling. These findings demonstrate a mechanism by which AIBP regulates neuroinflammation and suggest the therapeutic potential of AIBP in treating preexisting pain states.

Inhibition of histone deacetylase 1 or 2 reduces induced cytokine expression in microglia through a protein synthesis independent mechanism

Histone deacetylase (HDAC) inhibitors prevent neural cell death in invivo models of cerebral ischaemia, brain injury and neurodegenerative disease. One mechanism by which HDAC inhibitors may do this is by suppressing the excessive inflammatory response of chronically activated microglia. However, the molecular mechanisms underlying this anti-inflammatory effect and the specific HDAC responsible are not fully understood. Recent data from invivo rodent studies have shown that inhibition of class I HDACs suppresses neuroinflammation and is neuroprotective. In our study, we have identified that selective HDAC inhibition with inhibitors apicidin, MS-275 or MI-192, or specific knockdown of HDAC1 or 2 using siRNA, suppresses the expression of cytokines interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α) in BV-2 murine microglia activated with lipopolysaccharide (LPS). Furthermore, we found that in the absence of HDAC1, HDAC2 is up-regulated and these increased levels are compensatory, suggesting that these two HDACs have redundancy in regulating the inflammatory response of microglia. Investigating the possible underlying anti-inflammatory mechanisms suggests an increase in protein expression is not important. Taken together, this study supports the idea that inhibitors selective towards HDAC1 or HDAC2, may be therapeutically useful for targeting neuroinflammation in brain injuries and neurodegenerative disease.

TREM2/DAP12 Signal Elicits Proinflammatory Response in Microglia and Exacerbates Neuropathic Pain.

Recent studies have revealed that activated microglia in the spinal dorsal horn exacerbate neuropathic pain, which has suggested that suppression of microglial activity should be considered as a therapeutic target. However, only a few molecules have been identified as regulators of microglial activity. In this study, we focused on a receptor complex of TREM2 and DAP12, both of which are expressed by microglia and have been implicated in the pathogenesis of Alzheimer's disease, and demonstrated that TREM2/DAP12 signaling promoted proinflammatory responses in microglia and exacerbates neuropathic pain. The present results revealed the functional significance of TREM2/DAP12 signaling in microglial activation after neuronal injury, and could help in the development of treatments for neuropathic pain and neurodegenerative diseases.

Sequential activation of microglia and astrocyte cytokine expression precedes increased Iba-1 or GFAP immunoreactivity following systemic immune challenge.

Activation of the peripheral immune system elicits a coordinated response from the central nervous system. Key to this immune to brain communication is that glia, microglia, and astrocytes, interpret and propagate inflammatory signals in the brain that influence physiological and behavioral responses. One issue in glial biology is that morphological analysis alone is used to report on glial activation state. Therefore, our objective was to compare behavioral responses after in vivo immune (lipopolysaccharide, LPS) challenge to glial specific mRNA and morphological profiles. Here, LPS challenge induced an immediate but transient sickness response with decreased locomotion and social interaction. Corresponding with active sickness behavior (2-12 h), inflammatory cytokine mRNA expression was elevated in enriched microglia and astrocytes. Although proinflammatory cytokine expression in microglia peaked 2-4 h after LPS, astrocyte cytokine, and chemokine induction was delayed and peaked at 12 h. Morphological alterations in microglia (Iba-1(+)) and astrocytes (GFAP(+)), however, were undetected during this 2-12 h timeframe. Increased Iba-1 immunoreactivity and de-ramified microglia were evident 24 and 48 h after LPS but corresponded to the resolution phase of activation. Morphological alterations in astrocytes were undetected after LPS. Additionally, glial cytokine expression did not correlate with morphology after four repeated LPS injections. In fact, repeated LPS challenge was associated with immune and behavioral tolerance and a less inflammatory microglial profile compared with acute LPS challenge. Overall, induction of glial cytokine expression was sequential, aligned with active sickness behavior, and preceded increased Iba-1 or GFAP immunoreactivity after LPS challenge.

Sulforaphane Reduces Inflammatory Gene Expression in Microglia from Aged Mice

Increased oxidative stress resulting from heightened microglial activation is associated with age‐related cognitive impairment, neurodegenerative disease, and response to immune stimulus. The primary compensatory mechanism for neutralizing oxidative stress is through the nuclear factor E2‐related factor 2 (Nrf2) pathway which regulates transcription of antioxidant response element (ARE) genes. Here, BV2 cells and brain CD11b+ microglial cells isolated from young adult and aged mice were used to investigate the effects of sulforaphane (SFN), a potent Nrf2‐inducing bioactive, on ARE and inflammatory gene expression associated with increased age. We hypothesized that SFN would upregulate ARE genes in primary microglial cells and that this would attenuate elevated proinflammatory cytokine expression in microglia from aged mice. In support of our hypothesis, SFN upregulated antioxidant genes NAD(P)H dehydrogenase, quinone 1 (NQO1), heme oxygenase‐1 (HMOX1), and the modulatory subunit of glutamate‐cysteine ligase, modifier subunit (GCLM) in BV2 cells and primary microglia. Primary microglia from aged mice had higher expression of interleukin (IL‐)1β, IL‐6, and inducible nitric oxide synthase (iNOS) than adult controls, and SFN reduced IL‐1β and iNOS in microglia from aged mice. Taken together these data indicate that SFN is a potential therapeutic supplement that may be beneficial for reducing microglia‐mediated neuroinflammation and oxidative stress associated with aging.Supported by NIH RO1AG16710

Tyrphostin AG126 exerts neuroprotection in CNS inflammation by a dual mechanism.

The putative protein tyrosine kinase (PTK) inhibitor tyrphostin AG126 has proven beneficial in various models of inflammatory disease. Yet molecular targets and cellular mechanisms remained enigmatic. We demonstrate here that AG126 treatment has beneficial effects in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. AG126 alleviates the clinical symptoms, diminishes encephalitogenic Th17 differentiation, reduces inflammatory CNS infiltration as well as microglia activation and attenuates myelin damage. We show that AG126 directly inhibits Bruton's tyrosine kinase (BTK), a PTK associated with B cell receptor and Toll-like receptor (TLR) signaling. However, BTK inhibition cannot account for the entire activity spectrum. Effects on TLR-induced proinflammatory cytokine expression in microglia involve AG126 hydrolysis and conversion of its dinitrile side chain to malononitrile (MN). Notably, while liberated MN can subsequently mediate critical AG126 features, full protection in EAE still requires delivery of intact AG126. Its anti-inflammatory potential and especially interference with TLR signaling thus rely on a dual mechanism encompassing BTK and a novel MN-sensitive target. Both principles bear great potential for the therapeutic management of disturbed innate and adaptive immune functions.

Monomethyl fumarate inhibits the NFkB pathway and pro-inflammatory cytokine expression in microglia through HCA2 signaling via the AMPK/Sirt axis

Monomethyl fumarate inhibits the NFkB pathway and pro-inflammatory cytokine expression in microglia through HCA2 signaling via the AMPK/Sirt axis

PARP1 enhances inflammatory cytokine expression by alteration of promoter chromatin structure in microglia.

BackgroundPoly(ADP-ribose) polymerase 1 (PARP1) is a chromatin-associated enzyme that participates in processes such as transcription and DNA repair through the regulation of chromatin structure. Accumulating evidence suggests an important role for PARP1 enzymatic activity in promoting CNS inflammation by facilitating the expression of inflammatory cytokines in glial cells. However, the molecular mechanisms by which PARP1 enzymatic activity mediates this process are not well understood. In this report we sought to determine the molecular mechanisms by which PARP1 enzymatic activity facilitates the expression of Il1β and TNF in LPS-stimulated BV2 cells.MethodsPARP1 enzymatic activity and histone ADP-ribosylation were measured in LPS-stimulated BV2 cells by radioactive labelling with 32P-NAD+. To assess the effect of histone ADP-ribosylation on nucleosome structure, in vitro nucleosome remodeling, nuclease accessibility and binding assays were performed. These studies were complemented by chromatin immunoprecipitation assays in resting and LPS-stimulated BV2 cells in order to determine the occupancy of PARP1, nucleosomes and the RelA subunit of NF-κB, as well as ADP-ribosylation, at the Il1β and Tnf promoters. Finally, we determined the effect of pharmacological inhibition of PARP1 enzymatic activity on the LPS stimulation-dependent induction of Il1β and Tnf mRNA.ResultsOur results indicate that LPS stimulation induces PARP1 enzymatic activity and histone ADP-ribosylation in the chromatin compartment of BV2 cells. In vitro studies show that nucleosome-bound PARP1 disrupts nucleosome structure histone ADP-ribosylation, increasing the accessibility of nucleosomal DNA. Consistent with this PARP1 is constitutively associated with at the Il1β and Tnf promoters in resting BV2 cells. Upon stimulation with LPS, ADP-ribosylation is observed at these promoters, and this is correlated with increased recruitment of the transcription factor NF-κB, resulting in robust transcription of these inflammatory cytokines. Accordingly, pharmacological inhibition of PARP1 enzymatic activity reduces NF-κB recruitment, and Il1β and Tnf expression in LPS-stimulated microglia.ConclusionsCollectively, our data suggest that PARP1 facilitates inflammatory cytokine expression in microglia by increasing the accessibility of promoter DNA via histone ADP-riboyslation.

Peptidoglycan induces interleukin‐6 expression through the TLR2 receptor, JNK, c‐Jun, and AP‐1 pathways in microglia

We recently reported that peptidoglycan (PGN), a cell wall component of the Gram-positive bacterium, induces NF-κB activation and microglia activation. However, PGN-regulated AP-1 activation and cytokine expression in microglia remains unclear. This study investigated how PGN influences the signaling pathway involved in IL-6 production in microglia. IL-6 mRNA and protein level up-regulation were increased by PGN in a concentration- and time-dependent manner. In addition, PGN increased toll-like receptor-2 (TLR2) expression, but not TLR4 receptor up-regulation. Administration of TLR2 siRNA or TLR2 neutralized antibody effectively inhibited PGN-induced IL-6 expression. In contrast, PGN-induced IL-6 mRNA and protein up-regulation were attenuated by the SAPK/JNK (c-Jun N-terminal kinases) inhibitor SP600125. Treatment of microglia with PGN increased levels of JNK phosphorylation and c-Jun phosphorylation, and up-regulated of JNK kinase activity. Treatment of microglia with AP-1 inhibitors (Tanshinone IIA and curcumin) effectively reduced PGN-induced IL-6 expression. PGN also significantly increased c-Fos and phospho-c-Jun translocation to nucleus. In line with this, PGN also increased AP-1-DNA complexes formation, as determined by the electrophoretic mobility shift assay. Furthermore, PGN also increased IL-6 transcription activity determined by transfection with IL-6 promoter construct plasmid. Co-transfection with dominant negative mutant of JNK (DN-JNK), or treatment with SP600125, curcumin, or Tanshinone IIA effectively antagonized PGN-increased IL-6 transcription activity. Our data demonstrate that PGN-induced IL-6 expression is mediated by AP-1 activation through the TLR2 and JNK/c-Jun pathways in microglia.

Xenon Enhances LPS-Induced IL-1β Expression in Microglia via the Extracellular Signal-Regulated Kinase 1/2 Pathway

The extracellular signal-regulated kinase (ERK) is involved in the cytokine production of immune cells. In this study, we show the influence of xenon on phosphatase activity, ERK 1/2 signalling and cytokine expression in microglia. The murine microglia cell line BV-2 was treated with 50ng/ml lipopolysaccharide (LPS) and 74% xenon in 21% O(2) and 5% CO(2). Cytokine levels were examined by gene expression analysis, Western blot and enzyme-linked immunosorbent assay. Phosphatase inhibition was assessed with p-nitrophenylphosphate and phosphorylation of ERK 1/2 via Western blot. Xenon significantly enhanced LPS-mediated IL-1β expression. ERK 1/2 phosphorylation was observed after xenon or LPS treatment which was inhibited by the use of the MEK inhibitor U0126. Xenon and LPS in combination superimposed individual effects on ERK 1/2 activation. Xenon decreased cellular phosphatase activity in microglia by 20% and inhibited dephosphorylation of ERK 1/2 up to 1h. The blocking of ERK 1/2 reduced IL-1β expression in xenon and LPS-treated cells to a level obtained by LPS alone. In conclusion, xenon enhanced LPS-induced IL-1β expression in microglia by activation of ERK 1/2 signalling. Xenon's interference with phosphatases may be a key feature to affect multiple intracellular signalling pathways.

Luteolin triggers global changes in the microglial transcriptome leading to a unique anti-inflammatory and neuroprotective phenotype

BackgroundLuteolin, a plant derived flavonoid, exerts a variety of pharmacological activities and anti-oxidant properties associated with its capacity to scavenge oxygen and nitrogen species. Luteolin also shows potent anti-inflammatory activities by inhibiting nuclear factor kappa B (NFkB) signaling in immune cells. To better understand the immuno-modulatory effects of this important flavonoid, we performed a genome-wide expression analysis in pro-inflammatory challenged microglia treated with luteolin and conducted a phenotypic and functional characterization.MethodsResting and LPS-activated BV-2 microglia were treated with luteolin in various concentrations and mRNA levels of pro-inflammatory markers were determined. DNA microarray experiments and bioinformatic data mining were performed to capture global transcriptomic changes following luteolin stimulation of microglia. Extensive qRT-PCR analyses were carried out for an independent confirmation of newly identified luteolin-regulated transcripts. The activation state of luteolin-treated microglia was assessed by morphological characterization. Microglia-mediated neurotoxicity was assessed by quantifying secreted nitric oxide levels and apoptosis of 661W photoreceptors cultured in microglia-conditioned medium.ResultsLuteolin dose-dependently suppressed pro-inflammatory marker expression in LPS-activated microglia and triggered global changes in the microglial transcriptome with more than 50 differentially expressed transcripts. Pro-inflammatory and pro-apoptotic gene expression was effectively blocked by luteolin. In contrast, mRNA levels of genes related to anti-oxidant metabolism, phagocytic uptake, ramification, and chemotaxis were significantly induced. Luteolin treatment had a major effect on microglial morphology leading to ramification of formerly amoeboid cells associated with the formation of long filopodia. When co-incubated with luteolin, LPS-activated microglia showed strongly reduced NO secretion and significantly decreased neurotoxicity on 661W photoreceptor cultures.ConclusionsOur findings confirm the inhibitory effects of luteolin on pro-inflammatory cytokine expression in microglia. Moreover, our transcriptomic data suggest that this flavonoid is a potent modulator of microglial activation and affects several signaling pathways leading to a unique phenotype with anti-inflammatory, anti-oxidative, and neuroprotective characteristics. With the identification of several novel luteolin-regulated genes, our findings provide a molecular basis to understand the versatile effects of luteolin on microglial homeostasis. The data also suggest that luteolin could be a promising candidate to develop immuno-modulatory and neuroprotective therapies for the treatment of neurodegenerative disorders.

Sinomenine inhibits activation of rat retinal microglia induced by advanced glycation end products

Diabetic retinopathy involves an inflammatory response in the retina characterized by an increase in inflammatory cytokines and activation of microglia. The degree of microglia activation may influence the extent of retina injury following an inflammatory stimulus. Cytokines, released by activated microglia, regulate the influx of inflammatory cells to the damaged area. Thus, therapeutic strategy to reduce cytokine expression in microglia would be neuroprotective. Sinomenine, an alkaloid isolated from the stem and root of Sinomenium acutum, has long been recognized as an anti-inflammatory drug for rheumatoid arthritis and also inhibits macrophage activation. In this study, we activated retinal microglia in culture with advanced glycation end products (AGEs) treatment and attempted to determine whether sinomenine could reduce the production of cytokines from the activated microglia at both gene and protein levels. Changes in inflammatory cytokines, TNF alpha, IL-1 beta and IL-6, were measured by semi-quantitative RT-PCR and enzyme-linked immunosorbent assay (ELISA) both in the presence and absence of AGEs. The effect of sinomenine on levels of reactive oxygen species (ROS) and the nuclear translocation of NF-kB p65 were studied with a laser confocal scanning microscope. AGEs treatment induced a significant release of TNF alpha, IL-1beta, and IL-6 from retinal microglia. Sinomenine could inhibit release of these cytokines. Sinomenine attenuated ROS production in a dose-dependent fashion and reduced the nuclear translocation of NF-kB p65 in AGEs-activated retinal microglia in culture.

TNFα and TNF receptor 1 expression in the mixed neuronal–glial cultures of hippocampal dentate gyrus exposed to glutamate or trimethyltin

We examined the expression and cellular localization of tumor necrosis factor alpha (TNFα) and its type 1 receptor (TNFR1) in mixed neuronal–glial cultures of rat hippocampal dentate gyrus exposed to glutamate (GLU) or trimethyltin (TMT). Our previous studies demonstrated that both pathogenic factors evoked neuronal apoptosis, however, TMT was more potent and caused cell death in almost 90% of neurons. Observed neurodegeneration was accompanied by morphological changes of microglia. In the current study, using RT-PCR and Western blotting analysis, we found that GLU and TMT induced increase in TNFα mRNA and protein levels. The induction of transcription was stronger following GLU treatment, however the protein production was much more intensive after TMT exposure. Double fluorescent labeling for TNFα, TNFR1 and cellular markers revealed cytokine expression in microglia and some neurons. On the other hand, majority of neuronal cells displayed TNFR1 immunoreactivity, in control and in treated cultures. Moreover, TMT led to a strong increase in TNFR1 expression in astrocytes, which displayed remarkable, granular staining for the cytokine receptor. Western blotting for TNFR1 revealed enhanced protein expression only in cultures treated with TMT. This is the first report demonstrating the changes of expression of TNFα and TNFR1 in hippocampal dentate gyrus cultures treated with GLU or TMT. Our results indicate that TNFα may be involved in the mechanism of neurotoxic effects evoked by both pathogenic factors and suggest that astrocytes via TNFR1 may enhance TMT-induced injury.

Role of microglia in the pathogenesis of osmotic-induced demyelination

Osmotic demyelination is a serious disease caused by rapid correction of hyponatremia. In humans, demyelinative lesions occur preferentially in the central pons, and thus are termed central pontine myelinolysis. Although accumulation of microglia has been reported in such demyelinative lesions, their role in the pathogenesis of osmotic demyelination remains unclear. We examined the expression of cytokines in microglia that accumulated in the demyelinative lesions in a rat model of osmotic demyelination. Hyponatremia was induced in rats by a combination of dDAVP infusion and liquid diet feeding. After 7 days, serum sodium levels were rapidly corrected by hypertonic saline injection. The rats developed severe motor deficits, and marked demyelinative lesions were found in the midbrain and cerebral cortex. In the area of the demyelinative lesions, massive accumulations of microglia were observed that expressed the proinflammatory cytokines TNF-α and IFN-γ as well as iNOS. In contrast, in hyponatremia corrected rats treated with lovastatin, which is known to inhibit microglial infiltration in various animal models of CNS disease, neurological impairments and the degree of demyelination were significantly ameliorated. Lovastatin also reduced the accumulation of microglia and decreased the expression of TNF-α in the demyelinative lesions. These results indicate that microglia play a detrimental role in the pathogenesis of osmotic demyelination by producing proinflammatory cytokines, and further suggest that lovastatin may be useful in repressing the demyelination.