sphingosine-kinase and Encephalitis

Sphingosine kinase 1 (Sphk1), a key enzyme responsible for phosphorylating sphingosine into sphingosine1-phosphate (S1P), plays an important role in mediating post-stroke neuroinflammation. However, the pathway and mechanism of the Sphk1-mediated inflammatory response remains unknown. In this study, we found that suppression of Sphk1 decreased IL17 production and relieved neuronal damage induced by microglia in cerebral ischemia reperfusion (IR) or in an in vitro oxygen-glucose deprivation reperfusion (OGDR) system. Inhibition of Sphk1 with an inhibitor or siRNA decreased tumor necrosis factor receptor-associated factor 2 (TRAF2) and nuclear factor-kappa B (NF-κB) sequentially in microglia in response to IR or OGDR. Moreover, we also found that after suppression of TRAF2 or NF-κB by siRNA in microglia, reductions in the downstream molecules NF-κB and IL-17 and in neuronal apoptosis were observed in response to OGDR. Taken together, we hypothesize that Sphk1, TRAF2 and NF-κB form an axis that leads to increased IL-17 and neuronal apoptosis. This axis may be a potential therapeutic target to control neuroinflammation in brain IR.

Topics: Animals; Animals, Newborn; Cells, Cultured; Disease Models, Animal; Encephalitis; Glucose; Hypoxia; Infarction, Middle Cerebral Artery; Interleukin-17; Male; Methanol; Microglia; NF-kappa B; Phosphotransferases (Alcohol Group Acceptor); Pyrrolidines; Rats; Rats, Sprague-Dawley; Reperfusion; Signal Transduction; Sulfones; TNF Receptor-Associated Factor 2

Sphingosine kinases (Sphks) are the rate-limiting kinases in the generation of sphingosine-1-phosphate, which is a well-established intracellular pro-survival lipid mediator. Sphk2 has been reported to be protective following experimental stroke. We investigated the role of Sphk1 in cerebral ischemia using a mouse middle cerebral artery occlusion (MCAO) model and an in vitro glucose-oxygen deprivation (OGD) model. Sphk expression and activity were assessed in the ischemic brain with quantitative PCR (qPCR), Western blot, immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). Pharmacological and gene knockdown approaches were utilized to investigate the effects of Sphk1 on stroke outcomes. The expression of Sphk1 but not that of Sphk2 was rapidly induced in the cortical penumbra over 96h after MCAO, and the microglia were one of the major cellular sources of Sphk1 induction. Consistently, Sphk activity was enhanced in the cortical penumbra. In contrast to the protective role of Sphk2, pharmacological inhibition and cortical knockdown of Sphk1 reduced infarction at 24 and 96h after reperfusion. Additionally, the Sphk1 inhibitor improved the neurological deficits at 96h after reperfusion. Mechanistically, Sphk1 inhibition and knockdown significantly attenuated MCAO-induced expression of inflammatory mediators in the cortical penumbra. Moreover, using a conditioned medium transfer approach, we demonstrated that OGD-treated neurons induced the expression of Sphk1 and pro-inflammatory mediators in primary microglia, and the microglial induction of pro-inflammatory mediators by ischemic neurons was blunted by Sphk1 inhibition. Taken together, our results indicate that Sphk1 plays an essential role in mediating post-stroke neuroinflammation.

Topics: Animals; Animals, Newborn; Brain; Brain Infarction; Calcium-Binding Proteins; Cell Hypoxia; Cytokines; Disease Models, Animal; Encephalitis; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Glucose; Infarction, Middle Cerebral Artery; Male; Mice; Microfilament Proteins; Microglia; Neurons; Nitric Oxide Synthase Type II; Phosphopyruvate Hydratase; Phosphotransferases (Alcohol Group Acceptor); Time Factors

Microglial activation has been implicated as one of the causative factors for neuroinflammation in various neurodegenerative diseases. The sphingolipid metabolic pathway plays an important role in inflammation, cell proliferation, survival, chemotaxis, and immunity in peripheral macrophages. In this study, we demonstrate that sphingosine kinase1 (SphK1), a key enzyme of the sphingolipid metabolic pathway, and its receptors are expressed in the mouse BV2 microglial cells and SphK1 alters the expression and production of proinflammatory cytokines and nitric oxide in microglia treated with lipopolysaccharide (LPS). LPS treatment increased the SphK1 mRNA and protein expression in microglia as revealed by the RT-PCR, Western blot and immunofluorescence. Suppression of SphK1 by its inhibitor, N, N Dimethylsphingosine (DMS), or siRNA resulted in decreased mRNA expression of TNF-alpha, IL-1beta, and iNOS and release of TNF-alpha and nitric oxide (NO) in LPS-activated microglia. Moreover, addition of sphingosine 1 phosphate (S1P), a breakdown product of sphingolipid metabolism, increased the expression levels of TNF-alpha, IL-1beta and iNOS and production of TNF-alpha and NO in activated microglia. Hence to summarize, suppression of SphK1 in activated microglia inhibits the production of proinflammatory cytokines and NO and the addition of exogenous S1P to activated microglia enhances their inflammatory responses. Since the chronic proinflammatory cytokine production by microglia has been implicated in neuroinflammation, modulation of SphK1 and S1P in microglia could be looked upon as a future potential therapeutic method in the control of neuroinflammation in neurodegenerative diseases.

Topics: Animals; Cell Line; Cytokines; Encephalitis; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gliosis; Interleukin-1beta; Lipopolysaccharides; Lysophospholipids; Mice; Microglia; Nitric Oxide; Nitric Oxide Synthase Type II; Phosphotransferases (Alcohol Group Acceptor); RNA Interference; RNA, Messenger; Sphingosine; Tumor Necrosis Factor-alpha; Up-Regulation