Anaphylatoxin C5a-mediated reduction in cytokine and chemokine production depends on Sphk1.

<p>BMDMs from <i>Sphk1</i>^+/+ or <i>Sphk1^−/−</i> mice were stimulated with LPS (500 ng/ml), with LPS concomitant with C5a (1 nM), or without C5a. Tissue culture supernatants were harvested 2 h (TNF-α) or 8 h (IL-6 and KC) after the addition of stimuli. (<b>A</b>) TNF-α; (<b>B</b>); IL-6; (<b>C</b>) KC. Error bars represent s.d. *p<0.05 by Student's t-test; n = 5 for each genotype; representative for at least three independent experiments. (<b>D</b>) Reduced C5a-induced ERK1/2 and activation. BMDMs from <i>Sphk1</i>^+/+ or <i>Sphk1^−/−</i> mice were stimulated with C5a (10 nM), LPS (500 ng/ml), LPS and C5a, or saline for 5 min. Cell lysates were processed for immunoblotting with indicated antibodies. Representative of 3 independent experiments showing similar results. UD, undetected. (<b>E</b>) Model: Reduction of inflammatory cytokine production by phagocytes stimulated with C5a requires Sphk1. The model links Sphk1 activity to the cell surface expression of the anaphylatoxin receptor C5L2. LPS, C5a and inflammatory cytokines activate Sphk1 which is required to maintain S1P during inflammation. S1P regulates C5L2 cell surface expression on phagocytes. C5a, via C5L2 expressed on the cell surface, reduces neutrophil inflammation and inflammatory cytokine production by macrophages.</p

Sphingosine Kinase 1 Mediation of Expression of the Anaphylatoxin Receptor C5L2 Dampens the Inflammatory Response to Endotoxin

The complement anaphylatoxin C5a has a pathogenetic role in endotoxin-induced lung inflammatory injury by regulating phagocytic cell migration and activation. Endotoxin and C5a activate the enzyme sphingosine kinase (Sphk) 1 to generate the signaling lipid sphingosine-1-phosphate (S1P), a critical regulator of phagocyte function. We assessed the function of Sphk1 and S1P in experimental lung inflammatory injury and determined their roles in anaphylatoxin receptor signaling and on the expression of the two C5a receptors, C5aR (CD88) and C5L2, on phagocytes. We report that Sphk1 gene deficient (Sphk12/2) mice had augmented lung inflammatory response to endotoxin compared to wild type mice. Sphk1 was required for C5a-mediated reduction in cytokine and chemokine production by macrophages. Moreover, neutrophils from Sphk12/2 mice failed to upregulate the anaphylatoxin receptor C5L2 in response to LPS. Exogenous S1P restored C5L2 cell surface expression of Sphk12/2 mouse neutrophils to wild type levels but had no effect on cell surface expression of the other anaphylatoxin receptor, CD88. These results provide the first genetic evidence of the crucial role of Sphk1 in regulating the balance between expression of CD88 and C5L2 in phagocytes. S1P-mediated up-regulation of C5L2 is a novel therapeutic target for mitigating endotoxin-induced lung inflammatory injury

Sphingosine-1-phosphate (S1P) and anaphylatoxin C5a concentrations in plasma and lung tissue.

<p>S1P concentrations in plasma (<b>A</b>) or lung tissue lysate (<b>B</b>) or C5a concentrations in plasma (<b>C</b>) or lung tissue lysates (<b>D</b>) from <i>Sphk1</i>^+/+ or <i>Sphk1^−/−</i> mice were determined before or after i.p. LPS challenge (0.5 mg/kg), using the LC-MS/MS (46) or ELISA techniques. Error bars represent s.d. *p<0.005, **p<0.001, by Student's t-test. No significant differences in (<b>B, C, D</b>). n = 10 for each genotype and time point.</p

Genetic deletion of Sphk1 amplifies lung inflammation and lethality in mice.

<p>(<b>A</b>) Lung MPO activity. <i>Sphk1</i>^+/+ or <i>Sphk1^−/−</i> (n = 10 per time point of each genotype) mice were given LPS i.p. (0.5 mg/kg) and lungs were removed at the indicated times. Error bars represent s.d. *p<0.01 by Student's t-test. (<b>B, C</b>) Increased LPS-induced cytokine and chemokine production. TNF-α, IL-6, IL-1β, and KC were measured in plasma (<b>B</b>) and TNF-α, IL-6 and KC in lung tissue lysates (<b>C</b>) from <i>Sphk1</i>^+/+ or <i>Sphk1^−/−</i> mice, 1 h after i.p. LPS or saline injection. Error bars represent s.d. *p<0.05 by Student's t-test; n = 5 for each genotype. (<b>D</b>) Increased LPS-induced lethality. <i>Sphk1</i>^+/+ or <i>Sphk1^−/−</i> mice (n = 10/genotype, representative of three independent experiments) were given LPS i.p. (<b>E</b>) Increased LPS-induced lethality. <i>Sphk1</i>^+/+ or <i>Sphk1^−/−</i> mice (n = 10/genotype, representative of three independent experiments) were given LPS i.p. Differences in mortality were assessed by log-rank test (p<0.05). UD, undetected.</p

Sphk1 regulates cell surface expression of anaphylatoxin receptor C5L2.

<p>(<b>A</b>) <i>in vivo</i> cell surface expression of anaphylatoxin receptors CD88 and C5L2 in circulating Gr1⁺ neutrophils and F4/80⁺ peritoneal macrophages from <i>Sphk1</i>^+/+ or <i>Sphk1^−/−</i> mice was determined by flow cytometry. The percentage of C5L2⁺CD88⁺ cells is shown. There is a significant reduction of C5L2⁺ cells in <i>Sphk1^−/−</i> mice. (<b>B</b>) Bar graph depicts the mean fluorescence intensity (MFI ± s.d.) of C5l2 or CD88 cell surface expression of cells from five mice per genotype. (<b>C</b>) Total C5L2 expression (MFI) assessed by fixing and permeabilizing cells before staining with specific Ab to C5L2. Cell surface expression only, on non-permeabilized cells, is shown for comparison. Representative histograms of C5L2 expression by circulating neutrophils and peritoneal macrophages from <i>Sphk1</i>^+/+ or <i>Sphk1^−/−</i> mice are shown. (<b>D</b>) Percentage change of anaphylatoxin receptors CD88 and C5L2 neutrophil cell surface expression after <i>in vitro</i> stimulation with LPS (1 µg/ml) for 1 h compared to non-stimulated controls. (<b>E</b>) Exogenous S1P (250 nM) restores C5L2 cell surface expression of <i>Sphk1^−/−</i> PMNs to the level of <i>Sphk1</i>^+/+ PMNs. Error bars represent s.d. *p<0.05 by Student's t-test. Representative of at least 3 independent experiments with similar results. n.s., not significant.</p

Direct Evidence for Pitavastatin Induced Chromatin Structure Change in the <i>KLF4</i> Gene in Endothelial Cells

<div><p>Statins exert atheroprotective effects through the induction of specific transcriptional factors in multiple organs. In endothelial cells, statin-dependent atheroprotective gene up-regulation is mediated by Kruppel-like factor (<i>KLF</i>) family transcription factors. To dissect the mechanism of gene regulation, we sought to determine molecular targets by performing microarray analyses of human umbilical vein endothelial cells (HUVECs) treated with pitavastatin, and <i>KLF4</i> was determined to be the most highly induced gene. In addition, it was revealed that the atheroprotective genes induced with pitavastatin, such as nitric oxide synthase 3 (<i>NOS3</i>) and thrombomodulin (<i>THBD</i>), were suppressed by <i>KLF4</i> knockdown. Myocyte enhancer factor-2 (<i>MEF2</i>) family activation is reported to be involved in pitavastatin-dependent <i>KLF4</i> induction. We focused on <i>MEF2C</i> among the <i>MEF2</i> family members and identified a novel functional <i>MEF2C</i> binding site 148 kb upstream of the <i>KLF4</i> gene by chromatin immunoprecipitation along with deep sequencing (ChIP-seq) followed by luciferase assay. By applying whole genome and quantitative chromatin conformation analysis {chromatin interaction analysis with paired end tag sequencing (ChIA-PET), and real time chromosome conformation capture (3C) assay}, we observed that the <i>MEF2C</i>-bound enhancer and transcription start site (TSS) of <i>KLF4</i> came into closer spatial proximity by pitavastatin treatment. 3D-Fluorescence in situ hybridization (FISH) imaging supported the conformational change in individual cells. Taken together, dynamic chromatin conformation change was shown to mediate pitavastatin-responsive gene induction in endothelial cells.</p></div

The frequency of direct interaction between the kb −148 enhancer and promoter in the <i>KLF4</i> locus was affected by pitavastatin treatment.

<p>HUVECs were harvested and cultivated as described in the <i>Methods</i> section. (A) The localization of active Pol II obtained by ChIP-seq. The black arrow shows the <i>MEF2C</i> binding site identified by ChIP-seq. (B) A ChIA-PET library was constructed and sequenced. From the TSS of <i>KLF4</i>, 15 PETs originated and 13 of them interacted with a locus −148 kb upstream of the TSS, which result is identical with the <i>MEF2C</i> binding site observed by ChIP-seq and validated by luciferase assay in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096005#pone-0096005-g002" target="_blank">Figure 2</a>. The numbers in the middle indicate the location on chromosome 9 using the hg19 build program. (C) Quantitative 3C assay. HUVECs were incubated with 1 µM pitavastatin for 4 hours. Primers were designed for analyzing the crosslink frequency of the regions connected with the arches. The relative frequencies were compared between DMSO control (black arch) and statin treatment (red arch). The sequences of the primers are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096005#pone.0096005.s001" target="_blank">Table S2E in File S1</a>. The data (mean ± SD) is representative of three independent experiments with similar results. Note that the interaction between the TSS and kb −148 was increased by statin treatment.</p

3D-FISH confirms the proximity of <i>KLF4</i> and the <i>MEF2C</i> binding region detected by 3C.

<p>HUVECs were incubated with 1 µM pitavastatin for 4 hours. (A) Probe design for the two-color 3D-FISH analysis of the target region on human chromosome 9q31.2. The numbers in the middle indicate the location on chromosome 9 using the hg19 build program. (B) Visualization of two-color 3D-FISH on structurally preserved HUVEC nuclei and an image of the 3D distance. FISH with probes K (red) and M (green) showing the <i>KLF4</i> gene and <i>MEF2C</i> binding region, respectively. Nuclei were counterstained with TOPRO-3 (blue). 3D reconstruction was carried out on the captured image with Imaris software. The left panel shows the representative image of HUVECs with DMSO and the right panel shows the representative image of HUVECs with statin treatment. Magnified views of each probe sets are shown on top of the whole images. (C) The distance between the <i>KLF4</i> gene and <i>MEF2C</i> binding region for each condition. The distance was measured using the 3D image processing and analysis software CTMS (Chromosome Territory Measurement Software) (Cybernet Co. Ltd.). 70 chromosomes were analyzed and all of the data are shown in this figure. The average distances between the <i>KLF4</i> gene and <i>MEF2C</i> binding region are 0.45 µm with DMSO and 0.38 µm with the statin. <i>P</i><0.05 compared with pitavastatin (−), Wilcoxon rank-sum test.</p

Genes up- or down-regulated by pitavastatin treatment through <i>KLF4</i> in HUVECs.

<p>Transcriptome data were derived from the average of an array performed 5 times with 1 µM pitavastatin-treated HUVECs and the average of duplicate arrays using HUVECs transfected with <i>KLF4</i> siRNA or control (Ctl) siRNA, and treated with 1 µM pitavastatin for 4 hours. Fold induction is the representation of a log2 fold change to standardize the induction rate. Whole clustering analysis (A) using 384 selected genes that had significant changes in expression compared to control treatment were selected (See the details in <i>Methods</i>). The cluster shown in (B) contains the genes induced by pitavastatin and suppressed with si<i>KLF4</i>. Note that <i>NOS3</i> and <i>THBD</i> are included in addition to <i>KLF4</i>. These genes are indicated with red arrows. <i>KLF2</i> is shown by black arrow. The cluster shown in (C) includes the genes reduced pitavastatin treatment and induced with si<i>KLF4</i>. The sequences of the applied siRNA are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096005#pone.0096005.s001" target="_blank">Table S2A in File S1</a>.</p

Binding of <i>MEF2C</i> at kb −148 from the TSS of the <i>KLF4</i> gene is essential to pitavastatin-mediated <i>KLF4</i> induction.

<p>(A) HUVECs were incubated with 1 µM pitavastatin for 4 hours. As described in <i>Methods</i>, Chromatin immunoprecipitation was performed followed by deep sequencing. The localization and magnitude of <i>MEF2C</i> binding in the <i>KLF4</i> transcription regulation region are illustrated. Two <i>MEF2C</i> binding sites in the <i>KLF4</i> locus (−98 and −148 kb, relative to the TSS) were detected by ChIP-seq analysis. The localization of H3K27ac obtained by ChIP-seq is shown in the third lane. (B) Schematic structure of the transcriptional regulation region of the <i>KLF4</i> gene. The sequences of the primers used are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096005#pone.0096005.s001" target="_blank">Table S2D in File S1</a>. (C) HUVECs were transiently transfected with a KLF4-luc, (−98 kb)-KLF4-luc and (−148 kb)-KLF4-luc plasmid together with the <i>Renilla</i> luciferase plasmid, and were treated with 1 µM pitavastatin for 12 hours. Luciferase activity was measured as described in the <i>Methods</i> section. Error bars indicate the S.D. (<i>n</i> = 3), *<i>P</i><0.01 compared with pitavastatin (−), Student's t test. (D) HUVECs were transiently transfected with KLF4-luc, wild-type enhancer (−148 kb)-KLF4-luc and (enhancer −148 kb)-KLF4-luc containing a point mutation in the <i>MEF2</i> binding element. Pitavastatin-mediated induction of promoter activity was abolished by mutation of the <i>MEF2C</i> binding site. Error bars indicate the S.D. (<i>n</i> = 3), *<i>P</i><0.01 compared with pitavastatin (−), Student's t test. The <i>Firefly</i> luciferase activity value was normalized by <i>Renilla</i> luciferase activity.</p