Regulation of microglial cell function by corticosteroids and disruption by organotins

Microglia cells are the resident brain macrophages regulating in the initiation and maintenance of neuroinflammation. Chronic or exacerbated activation of microglia can contribute to neurodegenerative diseases. In the present thesis, mechanism of endogenous corticosteroids and xenobiotics on microglia function were investigated using the mouse BV-2 microlgia cell line. Corticosteroids are potent modulators of inflammation and mediate their effects by binding to mineralocorticoid receptors (MR) and glucocorticoid receptors (GR). MR and GR are suggested to regulate microglia activation and suppression, respectively. We showed that GR and MR differentially regulate on nuclear factor kappaB (NF-?B) activation and neuroinflammatory parameters, including interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a). 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), converting inactive 11-dehydrocorticosterone to active corticosterone, is involved in the action of GR and MR. Both 11-dehydrocorticosterone and corticosterone showed biphasic effects with low/moderate concentrations potentiating IL-6 and TNF-a expression and NF-?B activation through MR. At high concentrations, corticosteroids suppressed these mediators through GR. We also showed that the silane AB110873, indentified by a MR pharmacophore, stimulates mitochondrial reactive oxygen species (ROS) generation and the production of the IL-6 by activating MR. Metabotropic glutamate receptor 5 (mGluR5) has been documented to modulate microglia function. Microglial activation induced by (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), a mGluR5 agonist, has been shown to decrease microglia activation and release of associated pro-inflammatory factors. Additionally, oxidative stress and inflammatory response of microglia cells are associated with AMP-dependent protein kinase (AMPK) and calcium-mediated signaling. Here, we investigated the relationship between oxidative stress and inflammation and AMPK and calcium-mediated pathways in the antagonism of mGluR5 with 2-methyl-6-(phenylethynyl)-pyridine (MPEP). MPEP significantly increased oxidative stress parameters and inflammatory mediators in a concentration-dependent manner. MPEP reduced ATP production and changed the phosphorylation state of AMPK. MPEP increased the elevation of free intracellular Ca2+([Ca2+]i) from endoplasmic reticulum (ER) through IP3 receptor. ER stress markers were induced by MPEP and blocked by a chemical chaperone (4-phenyl butyric acid, 4-PBA) and a calcium chelator (BAPTA-AM). AMPK activation abolished and inhibition potentiated ER stress induced by MPEP. The effect of MPEP on phospholipase C (PLC)-associated pathways was also investigated. A PLC inhibitor (U73122), and a Gi protein inhibitor (pertussis toxin, PTX) blocked MPEP-induced increase of [Ca2+]i. MPEP also significantly increased PLC activity. Furthermore, AICAR, BAPTA-AM, U73122, and PTX prevented oxidative stress and inflammatory response induced by MPEP. Excessive and chronic exposure to organotin compounds has been associated with neurotoxicity and neuroinflammation. Dibutyltin (DBT) is an organotin widely used as a stabilizer in polyvinyl chloride plastics. In the present study, we found that DBT promotes mitochondrial oxidative stress and induces ATP depletion, leading to AMPK activation in a time-dependent manner. DBT induced concentration-dependent increases in IL-6, NADPH oxidase-2 (NOX-2), TNF-? expression. NOX-2 inhibitor, apocynin inhibited the action of DBT, not only on IL-6 up-regulation but also an intracellular ROS production. DBT induced the nuclear translocation of NF-?B and NF-?B inhibitor Cay10512 blocked IL-6 expression induced by DBT. Furthermore, we showed a role for phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (Akt), extracellular signal-regulated protein kinase1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK), c-Jun NH(2)-terminal kinase (JNK), protein kinase C (PKC), PLC, and [Ca2+]i in the DBT-mediated toxicity. Trimethyltin (TMT) is an organotin with potent neurotoxic effects, characterized by neuronal destruction and neuroinflammation, which involves microglia activation as a consequence of neuronal damaged. In the present study, we found that TMT induces the expression of IL-6 and inducible nitric oxide synthase (iNOS). Cay10512 blocked TMT-induced translocation of NF-?B into nucleus. PD98059 and SB20190, inhibitors of ERK1/2 and p38 MAPK, respectively, inhibited the ability of TMT to induce IL-6 and iNOS expression. The proinflammatory action of TMT was substantially enhanced by low/moderate corticosterone and 11-dehydrocorticosterone but suppressed by dexamethasone. Spironolactone suppressed the effects of TMT and the potentiation by corticosterone on proinflammatory mediator expression. Similarly, the potentiation of corticosterone was inhibited by PD98059 and SB20190. In conclusion, a tightly coordinated activity of GR and MR regulates the NF-?B pathway and the control of inflammatory mediators in microglia cells. The balance of GR and MR activity is locally modulated by the action of 11ß-HSD1, which is upregulated by pro-inflammatory mediators. This study highlights the role of mGluR5 antagonism in mediating oxidative stress, ER stress and inflammation in microglial cells. The calcium dependent pathways are mediated through Gi protein-coupled receptors, PLC, and PI3 receptors. AMPK also may play a role in the regulation of mGluR5 by disturbing the energy balance. TMT and corticosterone influence the same signaling pathways to exert inflammatory responses. TMT also directly disturbed the local corticosteroid balance

Dibutyltin promotes oxidative stress and increases inflammatory mediators in BV-2 microglia cells

The organotin dibutyltin (DBT) is used as biocide and as stabilizer in the manufacture of silicones, polyvinyl chloride plastics, polyurethanes and polyester systems. Although the immuno- and neurotoxicity of DBT has been recognized, the underlying mechanisms remained unclear and the impact of DBT on microglia cells has not yet been established. We now used cultured mouse BV-2 cells as a model of activated microglia to investigate the impact of DBT on oxidative stress and pro-inflammatory cytokines. DBT, at subcytotoxic concentrations, increased intracellular reactive oxygen species (ROS), mitochondrial mass, mitochondrial ROS, and the mRNA expression of inducible nitric oxide synthase (iNOS) and NADPH-dependent oxidase-2 (NOX-2). ATP levels were decreased by DBT, followed by activation of AMP-activated protein kinase (AMPK). Moreover, DBT potentiated the expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Inhibition of NOX-2 diminished both ROS production and induction of IL-6 expression. The DBT-mediated increase in NF-κB activity and subsequent up regulation of IL-6 was abolished by co-treatment with a NF-κB inhibitor. Treatment of cells with pharmacological inhibitors indicated a role for mitogen-activated protein kinases (MAPKs), PI3K/Akt, protein kinase C (PKC) and phospholipase C (PLC) in the DBT-induced toxicity. Finally, the calcium chelator BAPTA-AM diminished oxidative stress and induction of IL-6 expression, indicating the involvement of increased intracellular calcium in the enhanced microglia activity upon exposure to DBT. Together, the results suggest that a potentiation of oxidative stress and pro-inflammatory cytokine expression in microglia cells contribute to the toxicity of DBT in the CNS

Inhibition of metabotropic glutamate receptor 5 induces cellular stress through pertussis toxin-sensitive G i -proteins in murine BV-2 microglia cells

BackgroundActivation of metabotropic glutamate receptor 5 (mGluR5) by (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) was shown to suppress microglia activation and decrease the release of associated pro-inflammatory mediators. In contrast, the consequences of mGluR5 inhibition are less well understood. Here, we used BV-2 cells, retaining key characteristics of primary mouse microglia, to examine whether mGluR5 inhibition by 2-methyl-6-(phenylethynyl)-pyridine (MPEP) enhances cellular stress and production of inflammatory mediators.MethodsBV-2 cells were treated with MPEP, followed by determination of cellular stress using fluorescent dyes and high-content imaging. The expression of inflammatory mediators, endoplasmic reticulum (ER)-stress markers and phosphorylated AMPK¿ was analyzed by quantitative PCR, ELISA and Western blotting. Additionally, phospholipase C (PLC) activity, cellular ATP content and changes in intracellular free Ca2+ ([Ca2+]i) were measured using luminescence and fluorescence assays.ResultsTreatment of BV-2 microglia with 100 ¿M MPEP increased intracellular reactive oxygen species (ROS), mitochondrial superoxide, mitochondrial mass as well as inducible nitric oxide synthase (iNOS) and IL-6 expression. Furthermore, MPEP reduced cellular ATP and induced AMPK¿ phosphorylation and the expression of the ER-stress markers CHOP, GRP78 and GRP96. The MPEP-dependent effects were preceded by a rapid concentration-dependent elevation of [Ca2+]i, following Ca2+ release from the ER, mainly via inositol triphosphate-induced receptors (IP3R). The MPEP-induced ER-stress could be blocked by pretreatment with the chemical chaperone 4-phenylbutyrate and the Ca2+ chelator BAPTA-AM. Pretreatment with the AMPK agonist AICAR partially abolished, whilst the inhibitor compound C potentiated, the MPEP-dependent ER-stress. Importantly, the PLC inhibitor U-73122 and the Gi-protein inhibitor pertussis toxin (PTX) blocked the MPEP-induced increase in [Ca2+]i. Moreover, pretreatment of microglia with AICAR, BAPTA-AM, U-73122 and PTX prevented the MPEP-induced generation of oxidative stress and inflammatory mediators, further supporting a role for Gi-protein-mediated activation of PLC.ConclusionsThe results emphasize the potential pathophysiological role of mGluR5 antagonism in mediating oxidative stress, ER-stress and inflammation through a Ca2+-dependent pathway in microglia. The induction of cellular stress and inflammatory mediators involves PTX-sensitive Gi-proteins and subsequent activation of PLC, IP3R and Ca2+ release from the ER

Synergistic Antibacterial and Anti-inflammatory Activities of <i>Ocimum tenuiflorum</i> Ethanolic Extract against Major Bacterial Mastitis Pathogens

Mastitis is the most prevalent global illness affecting dairy cows. This bacterial infection damages and inflames the udder tissues. Several plant extracts have demonstrated synergistic antibacterial activities with standard drugs in mastitis treatment. Scant information exists on Ocimum tenuiflorum L. This study evaluated the antibacterial activity of O. tenuiflorum extract and its interaction with antibacterial drugs against common mastitis pathogens including Staphylococcus aureus, coagulase-negative Staphylococci (CNS), Streptococcus agalactiae, and Escherichia coli. Anti-inflammatory activities in LPS-stimulated RAW264.7 macrophage cells were also studied. The O. tenuiflorum extract exhibited antibacterial activities against S. aureus, CNS, and S. agalactiae with minimum inhibitory concentration (MIC) ranging from 3.9 to 31.2 µg/mL and minimum bactericidal concentration (MBC) ranging from 15.6 to 500 µg/mL. Combinations of O. tenuiflorum with penicillin or amoxicillin-clavulanic acid showed synergistic effects against all tested strains but an additive effect with cefazolin and gentamicin. Pretreatment of the extract significantly decreased the expression of inflammatory molecules (IL-6, TNF-α, IL-1β, iNOS, COX-2, and PGE2) generated by LPS in macrophages. Results suggested O. tenuiflorum effectiveness against various Gram-positive mastitis bacteria, with the potential to reduce antibacterial doses and combat inflammation

Development of Chrysin Loaded Oil-in-Water Nanoemulsion for Improving Bioaccessibility

Chrysin (5,7-dihydroxyflavone) is a remarkable flavonoid exhibiting many health-promoting activities, such as antioxidant, anti-inflammatory, and anti-Alzheimer’s disease (AD). Nevertheless, chrysin has been addressed regarding its limited applications, due to low bioaccessibility. Therefore, to improve chrysin bioaccessibility, a colloidal delivery system involving nanoemulsion was developed as chrysin nanoemulsion (chrysin-NE) using an oil-in-water system. Our results show that chrysin can be loaded by approximately 174.21 µg/g nanoemulsion (100.29 ± 0.53% w/w) when medium chain triglyceride (MCT) oil was used as an oil phase. The nanocolloidal size, polydispersity index, and surface charge of chrysin-NE were approximately 161 nm, 0.21, and −32 mV, respectively. These properties were stable for at least five weeks at room temperature. Furthermore, in vitro chrysin bioactivities regarding antioxidant and anti-AD were maintained as pure chrysin, suggesting that multistep formulation could not affect chrysin properties. Interestingly, the developed chrysin-NE was more tolerant of gastrointestinal digestion and significantly absorbed by the human intestinal cells (Caco-2) than pure chrysin. These findings demonstrate that the encapsulation of chrysin using oil-in-water nanoemulsion could enhance the bioaccessibility of chrysin, which might be subsequently applied to food and nutraceutical industries

Virtual screening as a strategy for the identification of xenobiotics disrupting corticosteroid action

Impaired corticosteroid action caused by genetic and environmental influence, including exposure to hazardous xenobiotics, contributes to the development and progression of metabolic diseases, cardiovascular complications and immune disorders. Novel strategies are thus needed for identifying xenobiotics that interfere with corticosteroid homeostasis. 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) and mineralocorticoid receptors (MR) are major regulators of corticosteroid action. 11β-HSD2 converts the active glucocorticoid cortisol to the inactive cortisone and protects MR from activation by glucocorticoids. 11β-HSD2 has also an essential role in the placenta to protect the fetus from high maternal cortisol concentrations.; We employed a previously constructed 3D-structural library of chemicals with proven and suspected endocrine disrupting effects for virtual screening using a chemical feature-based 11β-HSD pharmacophore. We tested several in silico predicted chemicals in a 11β-HSD2 bioassay. The identified antibiotic lasalocid and the silane-coupling agent AB110873 were found to concentration-dependently inhibit 11β-HSD2. Moreover, the silane AB110873 was shown to activate MR and stimulate mitochondrial ROS generation and the production of the proinflammatory cytokine interleukin-6 (IL-6). Finally, we constructed a MR pharmacophore, which successfully identified the silane AB110873.; Screening of virtual chemical structure libraries can facilitate the identification of xenobiotics inhibiting 11β-HSD2 and/or activating MR. Lasalocid and AB110873 belong to new classes of 11β-HSD2 inhibitors. The silane AB110873 represents to the best of our knowledge the first industrial chemical shown to activate MR. Furthermore, the MR pharmacophore can now be used for future screening purposes

11β-HSD inhibitors pharmacophore model.

<p>The hydrogen bond acceptor features are represented in green, and the hydrophobic features in blue. The shape of carbenoxolone, as a steric constraint to prevent too large molecules from fitting, is shown as a grey volume.</p

Effect of the silane AB110873 on the activation of mineralocorticoid (MR) and glucocorticoid receptors (GR).

<p>The impact of silane AB110873 on MR and GR transactivation was measured in HEK-293 cells transfected with MMTV-LacZ reporter, MR (<i>A,B</i>) or GR (<i>C</i>) and luciferase transfection control. Cells were incubated with the receptor ligands for 24 h, followed by analysis of reporter activity as given in the Methods section. <i>A</i>, Concentration-dependent activation of the human MR by the silane AB110873. Maximal activation at 30 µM was set as 100%. <i>B</i>, effect of the antagonist spironolactone (1 µM) on MR activation by aldosterone (5 nM) or by the silane AB110873 (20 µM); <i>C</i>, activation of human GR by cortisol (100 nM) and effect of AB110873 (20 µM) and antagonist RU-486 (1 µM) (MR and GR activity of the vehicle control was set as 1, data represent fold activation). Data were obtained from four independent experiments, each performed in triplicates (mean ± SD).</p

Binding of aldosterone and the silane AB110873 to the MR.

<p><i>A</i>, MR pharmacophore model with the cocrystallized ligand aldosterone. <i>B,</i> the silane AB110873 fitted to the MR ligand binding site with the pharmacophore model. Hydrogen bond acceptors are shown as red arrows, hydrophobic areas as yellow spheres. Exclusion volumes are not shown for clarity. Receptor binding pocket is colored by aggregated lipophilicity (grey)/hydrophilicity (blue).</p

Inhibition of 11β-HSD1 and 11β-HSD2 by compounds predicted by virtual screening.

<p>11β-HSD1-dependent conversion of cortisone (200 nM cortisone, 500 µM NADPH) to cortisol was determined by incubating lysates of HEK-293 cells stably expressing the human enzyme in the presence of vehicle or 20 µM of the respective compound for 10 min at 37°C. Similarly, 11β-HSD2-dependent conversion of cortisol (50 nM cortisol, 500 µM NAD⁺) to cortisone was measured. Data represent mean ± SD from at least three independent experiments.</p><p>T0504 and fenofibrate were used as synthetic positive controls, glycyrrhetinic acid as natural product positive control. N.D., not detectable (complete inhibition).</p