Proinflammatory Response In Microglia Research Articles

Lipopolysaccharide is a frequent and significant contaminant in microglia‐activating factors

Lipopolysaccharide (LPS/endotoxin) is a potent immunologic stimulant. Many commercial-grade reagents used in research are not screened for LPS contamination. LPS induces a wide spectrum of proinflammatory responses in microglia, the immune cells of the brain. Recent studies have demonstrated that a broad range of endogenous factors including plasma-derived proteins and bioactive phospholipids can also activate microglia. However, few of these studies have reported either the LPS levels found in the preparations used or the effect of LPS inhibitors such as polymyxin B (PMX) on factor-induced responses. Here, we used the Limulus amoebocyte lysate assay to screen a broad range of commercial- and pharmaceutical-grade proteins, peptides, lipids, and inhibitors commonly used in microglia research for contamination with LPS. We then characterized the ability of PMX to alter a representative set of factor-induced microglial activation parameters including surface antigen expression, metabolic activity/proliferation, and NO/cytokine/chemokine release in both the N9 microglial cell line and primary microglia. Significant levels of LPS contamination were detected in a number of commercial-grade plasma/serum- and nonplasma/serum-derived proteins, phospholipids, and synthetic peptide preparations, but not in pharmaceutical-grade recombinant proteins or pharmacological inhibitors. PMX had a significant inhibitory effect on the microglia-activating potential of a number of commercial-, but not pharmaceutical-grade, protein preparations. Novel PMX-resistant responses to alpha(2)-macroglobulin and albumin were incidentally observed. Our results indicate that LPS is a frequent and significant contaminant in commercial-grade preparations of previously reported microglia-activating factors. Careful attention to LPS levels and appropriate controls are necessary for future studies in the neuroinflammation field.

Resveratrol Inhibits Nitric Oxide and Prostaglandin E2Production by Lipopolysaccharide-Activated C6 Microglia

Resveratrol, a natural polyphenolic antioxidant found in red wine and grapes, has been reported to exert a variety of important pharmacological effects, including anti-inflammatory, cardioprotective, and cancer chemopreventive properties. In the present study, we investigated the effect of resveratrol on the production of nitric oxide (NO) and prostaglandin (PG) E2 by lipopolysaccharide (LPS)-activated C6 microglia. Exposure of cultured rat C6 astroglioma cells to LPS increased their release of NO and PGE2 and their inducible expression of NO synthase and cyclooxygenase-2, all of which were significantly inhibited by resveratrol pretreatment. Further studies revealed that resveratrol suppressed LPS-induced nuclear translocation and activation of nuclear factor kappaB (NF-kappaB). These results demonstrate a potent suppressive effect of resveratrol on pro-inflammatory responses of microglia by modulation of NF-kappaB activity, suggesting a therapeutic potential for this compound in neurodegenerative diseases accompanied by microglial activation.

Clearance of apoptotic neurons without inflammation by microglial triggering receptor expressed on myeloid cells-2

Elimination of apoptotic neurons without inflammation is crucial for brain tissue homeostasis, but the molecular mechanism has not been firmly established. Triggering receptor expressed on myeloid cells-2 (TREM2) is a recently identified innate immune receptor. Here, we show expression of TREM2 in microglia. TREM2 stimulation induced DAP12 phosphorylation, extracellular signal–regulated kinase phosphorylation, and cytoskeleton reorganization and increased phagocytosis. Knockdown of TREM2 in microglia inhibited phagocytosis of apoptotic neurons and increased gene transcription of tumor necrosis factor α and nitric oxide synthase-2, whereas overexpression of TREM2 increased phagocytosis and decreased microglial proinflammatory responses. Thus, TREM2 deficiency results in impaired clearance of apoptotic neurons and inflammation that might be responsible for the brain degeneration observed in patients with polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy/Nasu-Hakola disease.

Resveratrol inhibits nitric oxide and TNF-α production by lipopolysaccharide-activated microglia

Upon activation, brain macrophages, the microglia, release proinflammatory mediators that play important roles in eliciting neuroinflammatory responses associated with neurodegenerative diseases. As resveratrol, an antioxidant component of grape, has been reported to exert anti-inflammatory activities on macrophages, we investigated its effects on the production of TNF-alpha (TNF-α) and nitric oxide (NO) by lipopolysaccharide (LPS)-activated microglia. Exposure of cultured rat cortical microglia and a mouse microglial cell line N9 to LPS increased their release of TNF-α and NO, which was significantly inhibited by resveratrol. Further studies revealed that resveratrol suppressed LPS-induced degradation of IκBα, expression of iNOS and phosphorylation of p38 mitogen-activated protein kinases (MAPKs) in N9 microglial cells. These results demonstrate a potent suppressive effect of resveratrol on proinflammatory responses of microglia, suggesting a therapeutic potential for this compound in neurodegenerative diseases accompanied by microglial activation.

Expression of macrophage colony-stimulating factor and its receptor in microglia activation is linked to teratogen-induced neuronal damage

Prenatal exposure to teratogen agents is linked to the pathogenesis of neurodevelopment disorders, but the mechanisms leading to the neurodevelopmental disturbance are poorly understood. To elucidate this, an in vitro model of microglial activation induced by neuronal injury has been characterized. In this connection, exposure of primary microglial cells to the conditioned medium from the neuronal damage induced by teratogen, cyclophosphamide, is accompanied by a reactive microgliosis as assessed by reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, lectin histochemistry, double labeling immunohistochemistry and in situ hybridization. Our results showed that reactive microglia were capable of releasing various cytokines such as tumor necrosis factor-α, interleukin-1, interleukin-6, transforming growth factor-β and nitric oxide. Also, we have shown that macrophage colony-stimulating factor (M-CSF) was in fact produced by the reactive microglia. Concomitant to this was the increased expression of M-CSF receptor in these cells following the teratogen-induced neuronal injury. The up-regulation of M-CSF receptor suggests that the cells are capable of responding to self-derived M-CSF in an autocrine fashion. Results with antibody neutralization further suggest that microglial proinflammatory response, as manifested by cytokine expression in culture, is mediated by M-CSF, which acts as a molecular signal that initiates a microglial reaction. We therefore suggest that microglial activation following cyclophosphamide treatment is not only a response to the neuronal damage, but is also a cause of the damage during pathogenesis of neurodevelopment disorders. To this end, the increased expression of M-CSF and its receptor on microglia would be directly linked to the active cell proliferation and proinflammatory response in the teratogen-induced injury.

Inhibition of Alzheimer's β-Amyloid Induced Vasoactivity and Proinflammatory Response in Microglia by a cGMP-Dependent Mechanism

β-amyloid (Aβ) peptides are the major protein components of senile plaques in Alzheimer's disease (AD) brains. Vascular damage and reactive gliosis are found colocalized with amyloid deposits in AD brains, suggesting that the vasculature may be a clinically significant site of AD pathology. Our results show that freshly solubilized Aβ1–40enhances the vasoconstriction induced by endothelin-1 (ET-1) and increases resistance to relaxation triggered by nitric oxide (NO), suggesting that Aβ may oppose the NO/cGMP pathway. Using specific inhibitors and activators of the NO/cGMP pathway, we show that Aβ vasoactivity is not due to a modulation of nitric oxide synthase (NOS) or soluble guanylyl cyclase (sGC). However, we find that a selective cGMP phosphodiesterase (cGMP-PDE) inhibitor (dipyridamole) is able to interactively block the enhanced vasoconstriction as well as the opposition to relaxation induced by Aβ, suggesting that Aβ could effect the activity of this enzyme. Cyclic GMP levels, but not cAMP concentrations, are reduced after Aβ treatment of rat aortic rings, further substantiating this hypothesis. Moreover, in examination of this pathway in another cell type pertinent to AD, we find that Aβ induces a proinflammatory response in microglia as evidenced by increased leukotriene B4 release. We show that both dipyridamole and compounds which increase cGMP levels prevent Aβ-induced microglial inflammation. Our results suggest that therapeutic intervention aimed at reduction of microglial-mediated inflammation via inhibition of cGMP-PDE or elevation of cGMP may be beneficial in the treatment of AD.