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Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that has been implicated in many biological processes, including cell migration, survival, proliferation, angiogenesis and immune and allergic responses. S1P levels inside cells are regulated tightly by the balance between its synthesis by sphingosine kinases and degradation by S1P lyases and S1P phosphatases. Activation of sphingosine kinase by any of a variety of agonists increases S1P levels, which in turn can function intracellularly as a second messenger or in an autocrine and/or paracrine fashion to activate and signal through S1P receptors present on the surface of the cell. This review summarizes recent findings on the roles of S1P as a mediator of the actions of cytokines, growth factors and hormones. [Copyright &y& Elsevier]

Background: Sphingosine-1-phosphate (S1P), which is produced by 2 sphingosine kinase (SphK) isoenzymes, SphK1 and SphK2, has been implicated in IgE-mediated mast cell responses. However, studies of allergic inflammation in isotype-specific SphK knockout mice have not clarified their contribution, and the role that S1P plays in vivo in a mast cell– and IgE-dependent murine model of allergic asthma has not yet been examined. Objective: We used an isoenzyme-specific SphK1 inhibitor, SK1-I, to investigate the contributions of S1P and SphK1 to mast cell–dependent airway hyperresponsiveness (AHR) and airway inflammation in mice. Methods: Allergic airway inflammation and AHR were examined in a mast cell–dependent murine model of ovalbumin (OVA)–induced asthma. C57BL/6 mice received intranasal delivery of SK1-I before sensitization and challenge with OVA or only before challenge. Results: SK1-I inhibited antigen-dependent activation of human and murine mast cells and suppressed activation of nuclear factor κB (NF-κB), a master transcription factor that regulates the expression of proinflammatory cytokines. SK1-I treatment of mice sensitized to OVA in the absence of adjuvant, in which mast cell–dependent allergic inflammation develops, significantly reduced OVA-induced AHR to methacholine; decreased numbers of eosinophils and levels of the cytokines IL-4, IL-5, IL-6, IL-13, IFN-γ, and TNF-α and the chemokines eotaxin and CCL2 in bronchoalveolar lavage fluid; and decreased pulmonary inflammation, as well as activation of NF-κB in the lungs. Conclusion: S1P and SphK1 play important roles in mast cell–dependent, OVA-induced allergic inflammation and AHR, in part by regulating the NF-κB pathway. [Copyright &y& Elsevier]

The nitrone spin trap 5,5-dimethyl-1-pyrroline N -oxide (DMPO) was originally synthesized for the study of free radicals. However we found that DMPO can also prevent inflammatory activation in RAW264.7 cells primed with lipopolysaccharide (LPS). Additionally, DMPO prevents lung damage, adipose tissue inflammation, systemic inflammation and insulin resistance in a mouse model of acute distress respiratory syndrome induced by intratracheal instillation with LPS. These effects of DMPO are more likely caused by changes in gene expression in innate immune cells. Thus herein we tested whether DMPO by itself can change the transcriptome and interactome in RAW 264.7 cells, as a model of macrophage. To accomplish our goal we incubated RAW 264.7 cells with (DMPO) or without (control) 50 mM DMPO for 6h. The transcriptome was analyzed using micro-array (Illumina) and corroborated by Nanostring (nCounter) technology. Bioinformatics analysis showed 79 differentially expressed genes (DEGs) in DMPO vs control comparison (ONE-way ANOVA; FDR = 0.05). DAVID databases for identifying enriched Gene Onthology terms and Ingenuity Pathway Analysis (IPA) for functional analysis showed that DMPO DEGs were consistent with a negative regulation of innate immune response. Functional analysis indicated that IRF7 and TLRs were related (predicted inhibitions) to the observed transcriptomic effects of DMPO. Functional data analyses are consistent with DMPO dampening LPS-induced inflammatory activation of RAW 264.7 cells by down regulating several genes related to the innate immune response. These effects were also supported by Nanostring data showing that DMPO downregulates the expression of inflammation markers (CCL2, IFNβ, COX-2 and iNOS). Remarkably, our data suggest that DMPO by itself shifts the transcriptomic profile of RAW264.7 cells towards a negative modulation of innate immune response. [ABSTRACT FROM AUTHOR]

Macrophages are tissue cells from the innate-immune system where they play a number of homeostatic and defense functions. Inside the tissues and under tissue-specific microenvironmental pressures monocytes are recruited and differentiated to specific phenotypes. This phenotype is a consequence of the expression of specific genes that are under the control of one or more transcription factors. In this context, inflammatory phenotype of adipose tissue (AT) macrophages (ATM-M1) is responsible for adipose tissue oxidative stress and inflammation mediators that reduce whole-body insulin sensitivity and cause a number of metabolic abnormalities-associated to obesity. Intratracheal instillation of the nitrone spin trap 5,5-dimethyl-1-pirroline N -oxide (DMPO) to diet-induced obese-mice reduced markers of AT oxidative stress and inflammation, reduced serum concentration of inflammatory cytokines and improved insulin sensitivity. Thus we hypothesized that DMPO may produce transcripcional effects in macrophages at the AT and maybe other tissues. To approach this hypothesis we determined the transcripcional effects of DMPO in RAW264.7 cells after 6h incubation and with or without lipopolysaccharide (LPS) to model transcriptional profile of ATM. Microarray data showed that LPS caused an M1-transcriptional pattern, whereas DMPO reduced these changes. Remarkable effects were observed in the expression of IRF-7 and PPAR-d, master regulators of genes that determine M1 and M2 macrophage phenotype. LPS induced IRF-7, but reduced PPARd expression; whereas DMPO reduced IRF-7, but induced PPAR-d expression. Taking together our data suggest that DMPO may serve as a structural platform for the design of novel compounds to reduce AT inflammation and, thus other inflammatory abnormalities-associated to obesity, such as insulin resistance and metabolic syndrome. Supported by PROICO 2-3214 & PICT-2014-3369 (to DCR), PROICO 10-0414 (To SEGM) and PIP2015-2017-112215-0100603CO (To DCR, SEGM & SEGM). [ABSTRACT FROM AUTHOR]

5,5-dimethyl-1-pirroline N -oxide (DMPO) is a nitrone spin trap originally synthesized as a nitrone spin trap to study free radicals by electron spin resonance spectroscopy and recently by immuno-spin trapping. Herein we envisioned at studying what are the mechanisms involved in these anti-inflammatory effects of this old drug with new properties. To accomplish this goal we used a well known model of macrophage-like cells (RAW264.6) primed with LPS; which induce a well known MAPK signaling cascade that ends in activation of NF-kB—the master regulator of inflammation, inducible nitric oxide (iNOS) expression and nitric oxide synthesis. DMPO blocked NO synthesis, iNOS induction and MAPK signaling; but it did not affect LPS binding to LPS to membrane receptors. Thus we hypothesized that DMPO, and likewise other nitrones, may somehow affect very early LPS triggered signaling downstream of LPS-receptor binding. In silico data showed that DMPO binds to a very narrow sequence of aminoacids inside the TIR domain of TLR-2. TIR domains are conserved throughout TLRs (TLR-4; 6; 10) and species, particularly in a region called BB-loop which is responsible for downstream signal transduction. Molecular dynamics data shows that DMPO binds almost exclusively to these residues located at the BB-loop. Taking together, our data indicate that DMPO anti-inflammatory effect, is at least in part due to its binding to specific residues in the cytoplasmic portion of TLRs, thus further signaling is damped. Supported by PROICO 2-3214 & PICT-2014-3369 (to DCR), PROICO 10-0414 (To SEGM) and PIP2015-2017-112215-0100603CO (To DCR, SEA & SEGM). [ABSTRACT FROM AUTHOR]