Murine Microglial Cell Line BV-2 Research Articles

The citrus flavonoid, nobiletin inhibits neuronal inflammation by preventing the activation of NF-κB

Nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone) is one of the flavonoids found in shikuwasa, a popular citrus fruit in Okinawa, Japan. It exerts various pharmacological effects, such as anti-tumor, antioxidant, and anti-inflammatory activities. We herein investigated whether nobiletin attenuated lipopolysaccharide (LPS)-induced inflammatory responses in the murine microglial cell line BV-2 and neuroinflammation in mice induced by an intracerebral injection of LPS. In BV-2 cells, nobiletin significantly inhibited the LPS-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2) by preventing the mRNA expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively. Nobiletin also inhibited the LPS-induced mRNA expression of CCL2, CXCL1, IL-6, and TNFα. Nobiletin markedly attenuated the transcriptional activity of the NF-κB p65 subunit without affecting the degradation of IκBα or the nuclear localization of the NF-κB p65 subunit. Nobiletin also inhibited the LPS-induced activation of JNK, but not ERK or p38, in BV-2 cells. Furthermore, the administration of nobiletin significantly suppressed the accumulation of microglia and induction of the mRNA expression of CCL2, CXCL1, IL-6, and TNFα in the murine brain induced by injecting LPS into the striatum. Collectively, these results suggest the potential of nobiletin as a candidate anti-inflammatory drug for the prevention of neuroinflammation.

The anti-inflammatory and anti-oxidative effect of a classical hypnotic bromovalerylurea mediated by the activation of NRF2.

The Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2 (KEAP1-NRF2) system plays a central role in redox homeostasis and inflammation control. Oxidative stress or electrophilic compounds promote NRF2 stabilization and transcriptional activity by negatively regulating its inhibitor, KEAP1. We have previously reported that bromovalerylurea (BU), originally developed as a hypnotic, exerts anti-inflammatory effects in various inflammatory disease models. However, the molecular mechanism underlying its effect remains uncertain. Herein, we found that by real-time multicolor luciferase assay using stable luciferase red3 (SLR3) and green-emitting emerald luciferase (ELuc), BU potentiates NRF2-dependent transcription in the human hepatoblastoma cell line HepG2 cells, which lasted for more than 60h. Further analysis revealed that BU promotes NRF2 accumulation and the transcription of its downstream cytoprotective genes in the HepG2 and the murine microglial cell line BV2. Keap1 knockdown did not further enhance NRF2 activity, suggesting that BU upregulates NRF2 by targeting KEAP1. Knockdown of Nfe2l2 in BV2 cells diminished the suppressive effects of BU on the production of pro-inflammatory mediators, like nitric oxide (NO) and its synthase NOS2, indicating the involvement of NRF2 in the anti-inflammatory effects of BU. These data collectively suggest that BU could be repurposed as a novel NRF2 activator to control inflammation and oxidative stress.

CD74: a prospective marker for reactive microglia?

CD74: a prospective marker for reactive microglia?

The Role of Bromodomain and Extraterminal (BET) Proteins in Controlling the Phagocytic Activity of Microglia In Vitro: Relevance to Alzheimer’s Disease

The correct phagocytic activity of microglia is a prerequisite for maintaining homeostasis in the brain. In the analysis of mechanisms regulating microglial phagocytosis, we focused on the bromodomain and extraterminal domain (BET) proteins: Brd2, Brd3, and Brd4, the acetylation code readers that control gene expression in cooperation with transcription factors. We used pharmacological (JQ1) and genetic (siRNA) inhibition of BET proteins in murine microglial cell line BV2. Inhibition of BET proteins reduced the phagocytic activity of BV2, as determined by using a fluorescent microspheres-based assay and fluorescently labelled amyloid-beta peptides. Gene silencing experiments demonstrated that all brain-existing BET isoforms control phagocytosis in microglia. From a set of 84 phagocytosis-related genes, we have found the attenuation of the expression of 14: Siglec1, Sirpb1a, Cd36, Clec7a, Itgam, Tlr3, Fcgr1, Cd14, Marco, Pld1, Fcgr2b, Anxa1, Tnf, Nod1, upon BET inhibition. Further analysis of the mRNA level of other phagocytosis-related genes which were involved in the pathomechanism of Alzheimer's disease demonstrated that JQ1 significantly reduced the expression of Cd33, Trem2, and Zyx. Our results indicate the important role of BET proteins in controlling microglial phagocytosis; therefore, targeting BET may be the efficient method of modulating microglial activity.

Differential Expression of Microglial BDNF in Response to Acute Hyperglycemia and Hypoxia

Diabetes is one of the most common chronic disorders that affects the homeostasis of several systems including the central nervous system (CNS). Hyperglycemia, main feature of diabetes, is involved in pathogenesis of diabetic neuropathy. In addition, hyperglycemia increases mitochondrial oxygen consumption which leads to hypoxia and affects the neuronal survival, adversely. Brain‐derived neurotrophic (BDNF) is well known as neuroprotective factor which supports the survival of the neurons in the CNS. We hypothesize that microglial BDNF has a protective role against acute hyperglycemia and hypoxia. The objective of this study is to investigate the expression of BDNF in response to hyperglycemia and hypoxia. Murine microglial cell line BV‐2 cultured in Dulbecco's Modified Eagle Medium were used to investigate the effects of increased concentrations (30, 60, 90, 120 mM) at different durations (4, 8, 24 hours) on the viability of microglial cells. In addition, the effects of hypoxia on the survival rate of these cells was also assessed. Gene and protein expression of microglial BDNF was examined in response to increased concentrations of glucose and hypoxia as well as hypoxia inducible factor (HIF1‐alpha). Increased concentrations of glucose produced significant increase in gene expression of microglial BDNF and this response was concentration‐dependent (p < 0.001). On the other hand, gene expression of microglial BDNF was significantly decreased in response to hypoxia (4, 8, 24 hours) and this response was associated with reduced expression of HIF‐1 alpha (p < 0.05). In conclusion, the outcomes of this study demonstrated that expression of microglial BDNF was sensitive to increased concentration of glucose and decreased levels of oxygen. Further investigation will dissect the signaling pathways that are involved in this differential expression to gain a better understanding of the protective role of microglial BDNF against glucose and oxygen variability.

Proteomic dissimilarities of primary microglia and BV2 cells under stimuli.

Microglia are the static resident cells possessing the phagocytic properties in the central nervous system (CNS). In many relevant studies, the immortalized murine microglial cell line BV2 has been used as a tool in primary microglia (PM) relevant studies. Microglia participate in neuroinflammation by converting into M1- and M2-like phenotypes. In this study, we established M1- and M2-like phenotype response models by exposing PM and BV2 cells to lipopolysaccharides (LPS) and interlukin-4 (IL-4), respectively, and discovered the proteomic differences between the two types of microglia. It turned out that the BV2 cell responses to LPS and IL-4 were narrower and weaker than that of PM. In addition, irradiation, which has been shown to activate microglia and induces neuroinflammation, was also used as a treatment in this study. The results showed that BV2 cells have stronger capacity of DNA damage repair. Besides, irradiation had a negative effect on the regulation of KEGG pathways such as proteasome, ribosome, oxidative phosphorylation and TCA cycle in both cells. Furthermore, the KEGG pathways including cell cycle and DNA replication (significantly downregulated), and antigen processing and presentation and FC γ R mediated phagocytosis (significantly up-regulated) were only found in irradiated PM. These data demonstrate that PM is more fragile to irradiation. Results in this study indicate that BV2 cells only partially model PM, and thus, using BV2 in microglia related studies should be carefully considered.

Dopamine inhibits the expression of proinflammatory cytokines of microglial cells through the formation of dopamine quinone in the mouse striatum

We previously reported that dopamine (DA) attenuated lipopolysaccharide (LPS)-induced expression of proinflammatory cytokines through the formation of DA quinone (DAQ) in murine microglial cell line BV-2 and primary murine microglial cells. To reveal whether DA inhibits the expression of proinflammatory cytokines of microglial cells through the formation of DAQ in the central nervous system (CNS), in this study, we examined the effect of DAQ on LPS-induced mRNA expression of proinflammatory cytokines in C57BL/6 mouse brain under two experimental conditions: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration and l-dopa/carbidopa administration. Acute MPTP administration reduced the number of tyrosine hydroxylase-positive cells in the substantia nigra, and decreased the level of quinoprotein, an indicator of DAQ formation, in the striatum. Real-time RT-PCR analysis revealed that intraperitoneal administration of LPS increased the mRNA levels of proinflammatory cytokines, including tumor-necrosis factor-α and interleukin-1β, in the striatum. These increases were enhanced in MPTP-treated mice. On the other hand, l-dopa/carbidopa administration increased the level of quinoprotein, attenuated the LPS-induced mRNA expression of proinflammatory cytokines, and reduced the LPS-induced increase in the number of microglial cells in the striatum. These results suggest that DA attenuate the expression of proinflammatory cytokines in microglia through the formation of DAQ in the CNS.

Macrophage migration inhibitory factor meditates MPP+/MPTP-induced NLRP3 inflammasome activation in microglia cells

To explore the mechanisms of macrophage migration inhibitory factor (MIF)/nucleus factor-κB (NF-κB) in mediating 1-methyl-4-phenylpyridinium iodide (MPP +)/1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced activation of Nod-like receptor protein 3 (NLRP3) inflammasome in microglials and the its effects on neurons. Murine microglial cell line Bv-2 was infected with a lentivirus carrying MIF shRNA for MIF knockdown and then treated with MPP+. The total protein levels of NLRP3, caspase-1, p65 and p65 in the cell nuclei and cytoplasm were detected. ELISA was used to detect the levels of IL-1β and IL-18 in the culture supernatant, which served as the conditioned culture medium for MN9D cells, whose TH expression level was detected using Western blotting. The effect of stereotactic injection of an adeno-associated virus (AAV) carrying MIF shRNA on behaviors was assessed in a C57BL/6 mouse model of Parkinson disease (PD) induced by intraperitoneal MPTP injection. TH and Iba-1 immunohistochemistry was used to evaluate the number of substantia nigra neurons and the activation of microglia cells, and the protein expressions of MIF, NLRP3 and TH in the substantia nigra were detected with Western blotting. MPP+ significantly increased NLRP3 and MIF expressions in Bv-2 cells (P < 0.05). MIF knockdown in Bv-2 cells significantly lowered NLRP3 and caspase-1 protein expressions and IL-1β and IL-18 levels in the culture supernatant (P < 0.05) without affecting total protein level of p65. Bv-2 cells with MIF knockdown showed significantly lowered p65 protein expression in the nuclei but increased p65 expression in the cytoplasm (P < 0.05). The conditioned medium derived from Bv-2 cells with MIF knockdown, as compared with that from than MPP +-treated Bv-2 cells, significantly increased TH expression in MN9D cells (P=0.01). Compared with those in MPTP group, the mice receiving injections of AAV-MIF-shRNA had higher scores in pole test and open field test with lower scores in traction test, and showed increased TH-positive neurons, decreased Iba-1 microglia cell activation, reduced expressions of MIF and NLRP3, and increased expression of TH in he substantia nigra (all P < 0.05). Inhibition of MIF can reduce the expression of NLRP3 inflammasomes and inflammatory factor caused by MPP+ in microglia cells to relieve the damage of dopaminergic neurons and alleviate microglia cell activation, thus offering protection against neuroinflammation in Parkinson's disease.

Reversal of β-Amyloid-Induced Microglial Toxicity In Vitro by Activation of Fpr2/3.

Microglial inflammatory activity is thought to be a major contributor to the pathology of neurodegenerative conditions such as Alzheimer's disease (AD), and strategies to restrain their behaviour are under active investigation. Classically, anti-inflammatory approaches are aimed at suppressing proinflammatory mediator production, but exploitation of inflammatory resolution, the endogenous process whereby an inflammatory reaction is terminated, has not been fully investigated as a therapeutic approach in AD. In this study, we sought to provide proof-of-principle that the major proresolving actor, formyl peptide receptor 2, Fpr2, could be targeted to reverse microglial activation induced by the AD-associated proinflammatory stimulus, oligomeric β-amyloid (oAβ). The immortalised murine microglial cell line BV2 was employed as a model system to investigate the proresolving effects of the Fpr2 ligand QC1 upon oAβ-induced inflammatory, oxidative, and metabolic behaviour. Cytotoxic behaviour of BV2 cells was assessed through the use of cocultures with retinoic acid-differentiated human SH-SY5Y cells. Stimulation of BV2 cells with oAβ at 100 nM did not induce classical inflammatory marker production but did stimulate production of reactive oxygen species (ROS), an effect that could be reversed by subsequent treatment with the Fpr2 ligand QC1. Further investigation revealed that oAβ-induced ROS production was associated with NADPH oxidase activation and a shift in BV2 cell metabolic phenotype, activating the pentose phosphate pathway and NADPH production, changes that were again reversed by QC1 treatment. Microglial oAβ-stimulated ROS production was sufficient to induce apoptosis of bystander SH-SY5Y cells, an effect that could be prevented by QC1 treatment. In this study, we provide proof-of-concept data that indicate exploitation of the proresolving receptor Fpr2 can reverse damaging oAβ-induced microglial activation. Future strategies that are aimed at restraining neuroinflammation in conditions such as AD should examine proresolving actors as a mechanism to harness the brain's endogenous healing pathways and limit neuroinflammatory damage.

Deacetyl Ganoderic Acid F Inhibits LPS-Induced Neural Inflammation via NF-κB Pathway Both In Vitro and In Vivo.

Microglia mediated neuronal inflammation has been widely reported to be responsible for neurodegenerative disease. Deacetyl ganoderic acid F (DeGA F) is a triterpenoid isolated from Ganoderma lucidum, which is a famous edible and medicinal mushroom used for treatment of dizziness and insomnia in traditional medicine for a long time. In this study the inhibitory effects and mechanisms of DeGA F against lipopolysaccharide (LPS)-induced inflammation both in vitro and in vivo were investigated. On murine microglial cell line BV-2 cells, DeGA F treatment inhibited LPS-triggered NO production and iNOS expression and affected the secretion and mRNA levels of relative inflammatory cytokines. DeGA F inhibited LPS-induced activation of the NF-κB pathway, as evidenced by decreased phosphorylation of IKK and IκB and the nuclear translocation of P65. In vivo, DeGA F treatment effectively inhibited NO production in zebrafish embryos. Moreover, DeGA F suppressed the serum levels of pro-inflammatory cytokines, including TNF-α and IL-6 in LPS-stimulated mice model. DeGA F reduced inflammatory response by suppressing microglia and astrocytes activation and also suppressed LPS-induced NF-κB activation in mice brains. Taken together, DeGA F exhibited remarkable anti-inflammatory effects and promising therapeutic potential for neural inflammation associated diseases.

Dopamine attenuates lipopolysaccharide-induced expression of proinflammatory cytokines by inhibiting the nuclear translocation of NF-κB p65 through the formation of dopamine quinone in microglia.

Many reports have indicated that dopamine has immunomodulatory effects on peripheral immune cells. The purpose of this study was to reveal the immunomodulatory effect of dopamine on the expression of proinflammatory cytokines in microglial cells, which are the immune cells of the central nervous system. In murine microglial cell line BV-2 cells, pretreatment with dopamine for 24 h attenuated the lipopolysaccharide (LPS)-induced expression of proinflammatory cytokines such as tumor-necrosis factor-α, interleukin-1β, and interleukin-6. Neither (5R)-8-chloro-3-methyl-5-phenyl-1,2,4,5-tetrahydro-3-benzazepin-7-ol; hydrochloride (SCH-23390) nor sulpiride, which are dopamine D1-like and D2-like receptor antagonists, respectively, affected the attenuation of LPS-induced expression of cytokines by dopamine. In addition, pretreatment with neither (−)-(6aR,12bR)-4,6,6a,7,8,12b-Hexahydro-7-methylindolo[4,3-a]phenanthridin (CY208-243) nor bromocriptine, dopamine D1-like and D2-like receptor agonists, respectively, was effective in doing so. However, N-acetylcysteine (NAC), which inhibits dopamine oxidation to dopamine quinone, did inhibit this attenuated expression. Dopamine increased the level of quinoproteins, and this increase was inhibited by NAC. Western blot and immunocytochemical analyses revealed that dopamine inhibited LPS-induced nuclear translocation of nuclear factor-kappa B (NF-κB) p65. Dopamine also attenuated the expression of cytokines and the nuclear translocation of NF-κB p65 induced by LPS in mouse microglial cells in primary culture. These results suggest that dopamine attenuated LPS-induced expression of cytokines by inhibiting the nuclear translocation of NF-κB p65 through the formation of dopamine quinone in microglial cells.

P2RX7 INHIBITOR SUPPRESSES EXOSOME SECRETION, DELAYS PROTEOPATHIC TAU ACCUMULATION AND MEMORY IMPAIRMENT IN PS19 P301S TAU MICE

P2X7 receptor (P2XR7) is an ATP-gated cation channel, highly expressed in microglia, involved in Alzheimer's disease (AD) pathobiology. Series of studies also suggested that pharmacological inhibition or genetic deletion of P2XR7 could alter exosome secretion triggered by ATP stimulation. Recently, we showed that microglia could spread tau aggregates via secretion of extracellular vesicles throughout the brain. We hypothesize that P2XR7 inhibitor could inhibit exosome secretion by microglia and thus alleviate tauopathy in PS19 P301S tau transgenic mice. Murine microglial cell line BV-2 were treated with GSK1482160, a specific inhibitor of P2X7R, prior to ATP stimulation. Culture medium were collected and exosome number was measured using Nanoparticle Tracking Analysis (NTA) and CD9 ELISA. Three-months old P301S and control wild-type mice were treated with GSK1482160 (20mg/kg) or vehicle by oral gavage for 30 days. Hippocampal dependent memory capacity was assessed and hippocampal accumulation of tau aggregation and exosomal deposition was determined by immunohistochemistry. In addition, a separated cohort of animals was generated to quantify exosome and measure hippocampal p-Tau expression by ELISA. ATP stimulation of BV-2 cells significantly increased secretion of exosomes (30-150 nm), which was significantly inhibited by GSK1482160 pre-treatment in a dose-dependent manner as determined by NTA and CD9 ELISA. GSK1482160 also restored spontaneous alteration of PS19 mice in Y-maze test, and contextual and cued fear conditioning memory as compared to vehicle-treated PS19 mice. Interestingly, hippocampal accumulation of Alz50+ and MC1+ tau aggregation was significantly reduced in GSK1482160-treated group as compared to vehicle-treated group. GSK1482160 pre-treatment successfully suppresses exosome secretion by microglia in vitro. Its oral administration also significantly reduces spatial and fear conditional memory and tau accumulation in the hippocampal region of P301S Tau mouse model.

Triptolide Suppressed the Microglia Activation to Improve Spinal Cord Injury Through miR-96/IKKβ/NF-κB Pathway.

The effect of triptolide on spinal cord injury (SCI) and inflammatory response was observed by establishing SCI rat model. And in vitro experiments were conducted to determine the underlying mechanism of triptolide-mediated in murine microglial cell line BV2. To determine the underlying mechanism of triptolide in suppressing the microglia activation to improve SCI. Triptolide, as a major active ingredient of Chinese herb Tripterygium wilfordii, can promote spinal cord repair through inhibiting microglia activation, but the underlying mechanism is not clear. Locomotion recovery was accessed by Basso, Beattie, and Bresnahan score, the number of footfalls, stride length, and angle of rotation analysis. Expressions of microRNA 96 (miR-96), microglia activation marker Iba-1, and IκB kinase (IKKβ)/nuclear factor (NF)-κB-related proteins were detected by qRT-PCR or western blot. Inflammatory cytokines tumor necrosis factor-α and interleukin -1β were measured by enzyme-linked immuno sorbent assay. The regulation of miR-96 on IKKβ was confirmed by dual luciferase reporter assay. Triptolide promoted locomotion recovery of SCI rats, upregulated the expression of miR-96, decreased microglia activation marker Iba-1 and IKKβ/NF-κB-related proteins, and inhibited inflammatory cytokines tumor necrosis factor-α and interleukin-1β levels in spinal cord tissues and lipopolysaccharide -induced microglia. Triptolide suppressed the microglia activation and inflammatory cytokines secretion in BV2 cells through up-regulating miR-96. We confirmed the interaction between miR-96 and IKKβ, and IKKβ expression was negatively regulated by miR-96. Finally, we determined that triptolide suppressed the microglia activation and inflammatory cytokines secretion through miR-96/IKKβ pathway. Triptolide suppressed microglia activation after SCI through miR-96/IKKβ/NF-κB pathway. N/A.

MicroRNA-124 regulates the expression of p62/p38 and promotes autophagy in the inflammatory pathogenesis of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor and nonmotor symptoms due to the selective loss of midbrain dopaminergic neurons. The evidence for a chronic inflammatory reaction mediated by microglial cells in the brain is particularly strong in PD. In our previous study, we have shown that brain-specific microRNA-124 (miR-124) is significantly down-regulated in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD and that it can also inhibit neuroinflammation during the development of PD. However, further investigation is required to understand whether the abnormal expression of miR-124 regulates microglial activation. In this study, we found that the expression of sequestosome 1 (p62) and phospho-p38 mitogen-activated protein kinases (p-p38) showed a significant increase in LPS-treated immortalized murine microglial cell line BV2 cells in an MPTP-induced mouse model of PD. Knockdown of p62 could suppress the secretion of proinflammatory cytokines and p-p38 of microglia. Besides, inhibition of p38 suppressed the secretion of proinflammatory cytokines and promoted autophagy in BV2 cells. Moreover, our study is the first to identify a unique role of miR-124 in mediating the microglial inflammatory response by targeting p62 and p38 in PD. In the microglial culture supernatant transfer model, the knockdown of p62 in BV2 cells prevented apoptosis and death of human neuroblastoma cell lines SH-SY5Y (SH-SY5Y) cells following microglia activation. In addition, the exogenous delivery of miR-124 could suppress p62 and p-p38 expression and could also attenuate the activation of microglia in the substantia nigra par compacta of MPTP-treated mice. Taken together, our data suggest that miR-124 could inhibit neuroinflammation during the development of PD by targeting p62, p38, and autophagy, indicating that miR-124 could be a potential therapeutic target for regulating the inflammatory response in PD.-Yao, L., Zhu, Z., Wu, J., Zhang, Y., Zhang, H., Sun, X., Qian, C., Wang, B., Xie, L., Zhang, S., Lu, G. MicroRNA-124 regulates the expression of p62/p38 and promotes autophagy in the inflammatory pathogenesis of Parkinson's disease.

In vitro models to study insulin and glucocorticoids modulation of trimethyltin (TMT)-induced neuroinflammation and neurodegeneration, and in vivo validation in db/db mice.

Brain susceptibility to a neurotoxic insult may be increased in a compromised health status, such as metabolic syndrome. Both metabolic syndrome and exposure to trimethyltin (TMT) are known to promote neurodegeneration. In combination the two factors may elicit additive or compensatory/regulatory mechanisms. Combined effects of TMT exposure (0.5-1μM) and mimicked metabolic syndrome-through modulation of insulin and glucocorticoid (GC) levels-were investigated in three models: tridimensional rat brain cell cultures for neuron-glia effects; murine microglial cell line BV-2 for a mechanistic analysis of microglial reactivity; and db/db mice as an in vivo model of metabolic syndrome. In 3D cultures, low insulin condition significantly exacerbated TMT's effect on GABAergic neurons and promoted TMT-induced neuroinflammation, with increased expression of cytokines and of the regulator of intracellular GC activity, 11β-hydroxysteroid dehydrogenase 1 (11β-Hsd1). Microglial reactivity increased upon TMT exposure in medium combining low insulin and high GC. These results were corroborated in BV-2 microglial cells where lack of insulin exacerbated the TMT-induced increase in 11β-Hsd1 expression. Furthermore, TMT-induced microglial reactivity seems to depend on mineralocorticoid receptor activation. In diabetic BKS db mice, a discrete exacerbation of TMT neurotoxic effects on GABAergic neurons was observed, together with an increase of interleukin-6 (IL-6) and of basal 11β-Hsd1 expression as compared to controls. These results suggest only minor additive effects of the two brain insults, neurotoxicant TMT exposure and metabolic syndrome conditions, where 11β-Hsd1 appears to play a key role in the regulation of neuroinflammation and of its protective or neurodegenerative consequences.

Glycine Induces Migration of Microglial BV-2 Cells via SNAT-Mediated Cell Swelling

Background/Aims: The neutral, non-essential amino acid glycine has manifold functions and effects under physiological and pathophysiological conditions. Besides its function as a neurotransmitter in the central nervous system, glycine also exerts immunomodulatory effects and as an osmolyte it participates in cell volume regulation. During phagocytosis, glycine contributes to (local) cell volume-dependent processes like lamellipodium formation. Similar to the expansion of the lamellipodium we assume that glycine also affects the migration of microglial cells in a cell volume-dependent manner. Methods: Mean cell volume (MCV) and cell migration were determined using flow cytometry and trans-well migration assays, respectively. Electrophysiological recordings of the cell membrane potential (V_mem) and swelling-dependent chloride (Cl^-) currents (ICl_swell, VSOR, VRAC) were performed using the whole-cell patch clamp technique. Results: In the murine microglial cell line BV-2, flow cytometry analysis revealed that glycine (5 mM) increases the MCV by ∼9%. The glycine-dependent increase in MCV was suppressed by the partial sodium-dependent neutral amino acid transporter (SNAT) antagonist MeAIB and augmented by the Cl^- current blocker DCPIB. Electrophysiological recordings showed that addition of glycine activates a Cl^- current under isotonic conditions resembling features of the swelling-activated Cl^- current (ICl_swell). The cell membrane potential (V_mem) displayed a distinctive time course after glycine application; initially, glycine evoked a rapid depolarization mediated by Na⁺-coupled glycine uptake via SNAT, followed by a further gradual depolarization, which was fully suppressed by DCPIB. Interestingly, glycine significantly increased migration of BV-2 cells, which was suppressed by MeAIB, suggesting that SNAT is involved in the migration process of microglial cells. Conclusion: We conclude that glycine acts as a chemoattractant for microglial cells presumably by a cell volume-dependent mechanism involving SNAT-mediated cell swelling.

Neuroprotective Effects of Annexin A1 Tripeptide after Deep Hypothermic Circulatory Arrest in Rats.

Resolution agonists, including lipid mediators and peptides such as annexin A1 (ANXA1), are providing novel approaches to treat inflammatory conditions. Surgical trauma exerts a significant burden on the immune system that can affect and impair multiple organs. Perioperative cerebral injury after cardiac surgery is associated with significant adverse neurological outcomes such as delirium and postoperative cognitive dysfunction. Using a clinically relevant rat model of cardiopulmonary bypass (CPB) with deep hypothermic circulatory arrest (DHCA), we tested the pro-resolving effects of a novel bioactive ANXA1 tripeptide (ANXA1sp) on neuroinflammation and cognition. Male rats underwent 2 h CPB with 1 h DHCA at 18°C, and received vehicle or ANXA1sp followed by timed reperfusion up to postoperative day 7. Immortalized murine microglial cell line BV2 were treated with vehicle or ANXA1sp and subjected to 2 h oxygen–glucose deprivation followed by timed reoxygenation. Microglial activation, cell death, neuroinflammation, and NF-κB activation were assessed in tissue samples and cell cultures. Rats exposed to CPB and DHCA had evident neuroinflammation in various brain areas. However, in ANXA1sp-treated rats, microglial activation and cell death (apoptosis and necrosis) were reduced at 24 h and 7 days after surgery. This was associated with a reduction in key pro-inflammatory cytokines due to inhibition of NF-κB activation in the brain and systemically. Treated rats also had improved neurologic scores and shorter latency in the Morris water maze. In BV2 cells treated with ANXA1sp, similar protective effects were observed including decreased pro-inflammatory cytokines and cell death. Notably, we also found increased expression of ANXA1, which binds to NF-κB p65 and thereby inhibits its transcriptional activity. Our findings provide evidence that treatment with a novel pro-resolving ANXA1 tripeptide is neuroprotective after cardiac surgery in rats by attenuating neuroinflammation and may prevent postoperative neurologic complications.

Dengue virus infection increases microglial cell migration

Activated microglial cells are present in dengue virus (DENV)-infected brains; however, the possible effects of DENV on microglia remain unclear. Here, we demonstrated DENV caused infection, including viral entry, RNA replication, viral protein expression, and virus release, in the murine microglial cell line BV2. DENV infection caused an increase in the formation of the multipolar phenotype in vitro and in vivo without affecting cell growth and cytotoxicity. DENV infection considerably increased cell motility and disrupting either actin filaments or clathrin retarded such effect. Increase in cell migration was only occurred by DENV infection following a clathrin-regulated endocytosis of DENV entry. Ultraviolet-inactivated DENV did not affect cell migration, and pharmacologically blocking toll-like receptor (TLR) 3 and TLR3-related signaling pathways reduced the DENV-induced increase in cell migration. These results demonstrate an advanced effect of DENV infection on microglial migration via a mechanism involving viral entry, RNA release, and TLR3 signal activation.

Down-Regulation of miRNA-128 Contributes to Neuropathic Pain Following Spinal Cord Injury via Activation of P38.

BackgroundNeuropathic pain (NPP) arises from a lesion or dysfunction of the somatosensory nervous system. Recent studies have demonstrated multiple microRNAs (miRNAs) play key roles in NPP development. This study aimed to investigate the effects of miR-128 on microglial cells.Material/MethodsWe established a compressive spinal cord injury (SCI) model and collected the spinal cord segment-derived conditioned medium (CM). We then measured the expression of miR-128 in the murine microglial cell line BV2 treated with CM-SCI or CM obtained from control (CM-NC). Furthermore, lentivirus production of miR-128 and scrambled control were transfected into BV2 cells, which were first treated with CM-SCI or CM-NC. Moreover, the effects of miR-128 on cell viability, M1/M2 microglial gene expression, inflammatory cytokines concentration, and the protein expression of P38 and phosphorylated P38 (P-P38) were investigated.ResultsThe expression of miR-128 was downregulated in murine microglial BV2 cells treated with CM-SCI. Overexpression of miR-128 markedly promoted the viability of murine microglial cells. In addition, miR-128 overexpression significantly decreased the expression levels of microglial M1 phenotypic markers CD86 and CD32, and increased the expression levels of M2 phenotypic markers Arg1 and CD206. Furthermore, miR-128 overexpression obviously decreased the concentration of TNF-α, IL-1β, and IL-6. We found that miR-128 overexpression significantly downregulated the expression levels of P38 andP-P38.ConclusionsOur findings indicate that down-regulation of miR-128 in murine microglial cells may contribute to the development of NPP following SCI via activation of P38. MiR-128 may be a potential intervention target for NPP.

Novel Insights into the Multiple Sclerosis Risk Gene ANKRD55.

An intronic variant in ANKRD55, rs6859219, is a genetic risk factor for multiple sclerosis, but the biological reasons underlying this association are unknown. We characterized the expression of ANKRD55 in human PBMCs and cell lines. Three ANKRD55 transcript variants (Ensembl isoforms 001, 005, and 007) could be detected in PBMCs and CD4(+) T cells but were virtually absent in CD8(+), CD14(+), CD19(+), and CD56(+) cells. Rs6859219 was significantly associated with ANKRD55 transcript levels in PBMCs and CD4(+) T cells and, thus, coincides with a cis-expression quantitative trait locus. The processed noncoding transcript 007 was the most highly expressed variant in CD4(+) T cells, followed by 001 and 005, respectively, but it was not detected in Jurkat, U937, and SH-SY5Y cell lines. Homozygotes for the risk allele produced more than four times more transcript copies than did those for the protective allele. ANKRD55 protein isoforms 005 and 001 were predominantly located in the nucleus of CD4(+) T cells and Jurkat and U937 cells. ANKRD55 was produced by primary cultures of murine hippocampal neurons and microglia, as well as by the murine microglial cell line BV2, and it was induced by inflammatory stimuli. ANKRD55 protein was increased in the murine mouse model of experimental autoimmune encephalomyelitis. Flow cytometric analysis of CNS-infiltrating mononuclear cells showed that CD4(+) T cells and monocytes expressed ANKRD55 in experimental autoimmune encephalomyelitis mice, with the higher fluorescence intensity found in CD4(+) cells. A low percentage of microglia also expressed ANKRD55. Together, these data support an important role for ANKRD55 in multiple sclerosis and neuroinflammation.