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Sphingosine-1-phosphate induces an antiinflammatory phenotype in macrophages.

ABSTRACT: Activated macrophages acquire a proinflammatory (classic) or antiinflammatory (alternative) phenotype that influences atherosclerosis. The present study investigated whether sphingosine-1-phosphate (S1P), with its known antiinflammatory effects, could regulate the inflammatory phenotype of lipopolysaccharide (LPS)-stimulated mouse macrophages. Activation of macrophages by LPS significantly increases proinflammatory cytokine secretion. Pretreatment of macrophages with 500 nmol/L S1P markedly reduced LPS-mediated secretion of tumor necrosis factor-alpha, monocyte chemoattractant protein-1, and interleukin-12. Such antiinflammatory actions were also evident in LPS-stimulated macrophages treated with the S1P1 receptor-specific agonist SEW2871. Pharmacological antagonism of the S1P1 receptor on macrophages using the S1P1-specific antagonist VPC44116 also blocked proinflammatory cytokine secretion in response to LPS. Studies using bone marrow-derived macrophages from S1P2-deficient mice revealed that the S1P2 receptor did not play a pivotal role in this process. Thus, activation of the S1P1 receptor in mouse macrophages limits the expression of proinflammatory cytokines. Furthermore, we demonstrated that S1P increased arginase I activity and inhibited LPS-induced inducible NO synthase activity in LPS-treated macrophages, again through S1P1 receptor activation on macrophages. Analysis of a 1.7-kb region of the murine inducible NO synthase promoter revealed the presence of putative nuclear factor kappaB, activator protein-1, and STAT-1 response elements. Using inducible NO synthase promoter-reporter constructs, we found that S1P significantly reduced the nuclear factor kappaB-mediated induction of inducible NO synthase. These findings demonstrate an important role for S1P in the regulation of macrophage phenotypic switching. Therefore, we conclude that S1P promotes the production of an alternative antiinflammatory macrophage phenotype through activation of the macrophage S1P1 receptor.

SUBMITTER: Hughes JE

PROVIDER: S-EPMC2875063 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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Sphingosine-1-phosphate induces an antiinflammatory phenotype in macrophages.

Hughes Jeniter E JE Srinivasan Suseela S Lynch Kevin R KR Proia Richard L RL Ferdek Pawel P Hedrick Catherine C CC

Circulation research 20080306 8

Activated macrophages acquire a proinflammatory (classic) or antiinflammatory (alternative) phenotype that influences atherosclerosis. The present study investigated whether sphingosine-1-phosphate (S1P), with its known antiinflammatory effects, could regulate the inflammatory phenotype of lipopolysaccharide (LPS)-stimulated mouse macrophages. Activation of macrophages by LPS significantly increases proinflammatory cytokine secretion. Pretreatment of macrophages with 500 nmol/L S1P markedly redu ...[more]

PMID: 18323526

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Project description:Apolipoprotein E (apoE) exerts potent antiinflammatory effects. Here, we investigated the effect of apoE on the functional phenotype of macrophages.Human apoE receptors very-low-density lipoprotein receptor (VLDL-R) and apoE receptor-2 (apoER2) were stably expressed in RAW264.7 mouse macrophages. In these cells, apoE downregulated markers of the proinflammatory M1 phenotype (inducible nitric oxide synthase, interleukin [IL]-12, macrophage inflammatory protein-1?) but upregulated markers of the antiinflammatory M2 phenotype (arginase I, SOCS3, IL-1 receptor antagonist [IL-1RA]). In addition, M1 macrophage responses (migration, generation of reactive oxygen species, antibody-dependent cell cytotoxicity, phagocytosis), as well as poly(I:C)- or interferon-?-induced production of proinflammatory cytokines; cyclooxygenase-2 expression; and activation of nuclear factor-?B, I?B, and STAT1, were suppressed in VLDL-R- or apoER2-expressing cells. Conversely, the suppression of the M2 phenotype and the enhanced response to poly(I:C) were observed in apoE-producing bone marrow macrophages derived from VLDL-R-deficient mice but not wild-type or low-density lipoprotein receptor-deficient mice. The modulatory effects of apoE on macrophage polarization were inhibited in apoE receptor-expressing RAW264.7 cells exposed to SB220025, a p38 mitogen-activated protein kinase inhibitor, and PP1, a tyrosine kinase inhibitor. Accordingly, apoE induced tyrosine kinase-dependent activation of p38 mitogen-activated protein kinase in VLDL-R- or apoER2-expressing macrophages. Under in vivo conditions, apoE-/- mice transplanted with apoE-producing wild-type bone marrow showed increased plasma IL-1RA levels, and peritoneal macrophages of transplanted animals were shifted to the M2 phenotype (increased IL-1RA production and CD206 expression).ApoE signaling via VLDL-R or apoER2 promotes macrophage conversion from the proinflammatory M1 to the antiinflammatory M2 phenotype. This effect may represent a novel antiinflammatory activity of apoE.

Project description:Background: The pathophysiology of non-alcoholic steatohepatitis involves hepatocyte lipotoxicity due to excess saturated free fatty acids and concomitant proinflammatory macrophage effector responses. These include the infiltration of macrophages into hepatic cords in response to incompletely understood stimuli. Stressed hepatocytes release an increased number of extracellular vesicles (EVs), which are known to participate in intercellular signaling and coordination of the behavior of immune cell populations via their cargo. We hypothesized that hepatocyte-derived lipotoxic EVs that are enriched in sphingosine 1-phosphate (S1P) are effectors of macrophage infiltration in the hepatic microenvironment. Methods: Lipotoxic EVs were isolated from palmitate treated immortalized mouse hepatocytes and characterized by nanoparticle tracking analysis. Lipotoxic EV sphingolipids were quantified using tandem mass spectrometry. Wildtype and S1P1 receptor knockout bone marrow-derived macrophages were exposed to lipotoxic EV gradients in a microfluidic gradient generator. Macrophage migration toward EV gradients was captured by time-lapse microscopy and analyzed to determine directional migration. Fluorescence-activated cell sorting along with quantitative PCR and immunohistochemistry were utilized to characterize the cell surface expression of S1P1 receptor on intrahepatic leukocytes and hepatic expression of S1P1 receptor, respectively. Results: Palmitate treatment induced the release of EVs. These EVs were enriched in S1P. Palmitate-induced S1P enriched EVs were chemoattractive to macrophages. EV S1P enrichment depended on the activity of sphingosine kinases 1 and 2, such that, pharmacological inhibition of sphingosine kinases 1 and 2 resulted in a significant reduction in EV S1P cargo without affecting the number of EVs released. When exposed to EVs derived from cells treated with palmitate in the presence of a pharmacologic inhibitor of sphingosine kinases 1 and 2, macrophages displayed diminished chemotactic behavior. To determine receptor-ligand specificity, we tested the migration responses of macrophages genetically deleted in the S1P1 receptor toward lipotoxic EVs. S1P1 receptor knockout macrophages displayed a marked reduction in their chemotactic responses toward lipotoxic palmitate-induced EVs. Conclusions:Palmitate-induced lipotoxic EVs are enriched in S1P through sphingosine kinases 1 and 2. S1P-enriched EVs activate persistent and directional macrophage chemotaxis mediated by the S1P1 receptor, a potential signaling axis for macrophage infiltration during hepatic lipotoxicity, and a potential therapeutic target for non-alcoholic steatohepatitis.

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Sphingosine-1-phosphate induces an antiinflammatory phenotype in macrophages.

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Sphingosine-1-phosphate induces an antiinflammatory phenotype in macrophages.

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