A loss of pyrin causes increased IL-1β protein levels in response to NLRP3 inflammasome stimuli.

<p>rpMΦs from <i>Mefv</i>^+/+ and <i>Mefv</i>^−/− littermate mice were exposed to LPS and the indicated stimuli as described in the method section. The concentration of IL-1β, <i>A</i>, and IL-6, <i>B</i>, cytokines in cell culture supernatants was determined by ELISA. <i>C</i>, LDH protein levels detected in cell supernatants. The total protein of cell lysates was measured to verify equivalent plating of cells. TiO2, titanium dioxide; MDP, muramyl dipeptide; Alum, aluminum hydroxide; CPPD, calcium pyrophosphate dihydrate; ATP, adenosine 5′ triphosphate. n = 6 mice per genotype. A student’s t test was used to calculate p values for WT versus <i>Mefv</i>^−/− cultures. #, p<0.05; *, p≤0.01 **, p≤0.001, ***, p<0.0001.</p

Inflammatory response in <i>Mefv</i>^+/+ and <i>Mefv</i>^−/− mice after LPS treatment.

<p>The body weight lost was measured between 0 h and 20 h post-LPS treatment, immune cells present in the peritoneal lavage fluid, and MPO activity as a measure of neutrophil presence in lavage samples were evaluated 20 h post-LPS treatment. For body temperature changes, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051105#pone-0051105-g002" target="_blank">Figure 2F, G</a>. <b>n.a</b>. not analyzed. n = 18. Differences between Mefv^+/+ and Mefv^−/− mice within each dose of LPS treatment were not statistically significant.</p

A loss of pyrin causes increased IL-1β release in response to NLRC4 and NLRP1b stimuli.

<p>rpMΦs from <i>Mefv</i>^+/+ and <i>Mefv</i>^−/− littermate mice were exposed to LPS and the indicated stimuli as indicated in the method section. The concentration of IL-1β, <i>A</i>, and IL-6, <i>B</i>, cytokines in cell culture supernatants was determined by ELISA. <i>C</i>, LDH protein levels detected in cell supernatants. The total protein of cell lysates was measured to verify equivalent plating of cells. LT, lethal toxin of <i>Bacillus anthracis</i>. n = 6 mice per genotype. Results are representative of at least 4 independent experiments. A student’s t test was used to calculate p values for WT versus <i>Mefv</i>^−/− cultures. #, p<0.05; *, p≤0.01.</p

Caspase-1 (CASP-1) activity is not elevated in <i>Mefv^−/−</i> macrophages.

<p><i>A</i>, rpMΦs from <i>Mefv</i>^+/+ and <i>Mefv</i>^−/− littermate mice were exposed to LPS and ATP as indicated in the method section. Levels of caspase-1 (upper panel) and IL-1β (middle panel) were detected by western blot in cell lysates (lanes 1 and 2) and cell culture supernatant (lanes 3 and 4). An equivalent amount of total protein was loaded in each lane. The amount of protein in the lysate and supernatant was verified by staining with Ponceau S, major protein band is shown (lower panel). <i>B</i> and <i>C</i>, <i>Mefv</i>^+/+, <i>Mefv</i>^−/−, and <i>Casp1^−/−</i> mice were treated with 10 µg of lethal toxin (LT) or PBS vehicle alone by intratracheal instillation and bronchoalveolar lavage (BAL) was collected at 2 h after treatment. Cells from the BAL were stained with fluorescently labeled CASP-1 inhibitor (FLICA), FAM-YVAD-FMK. Cells were analyzed via flow cytometry. <i>B</i>, Data is expressed as the mean of the fluorescence intensity (MFI). <i>C</i>, A representative plot of the fluorescence intensity of LT or vehicle-treated cell populations. <i>D</i>, rpMΦs from <i>Mefv</i>^+/+ and <i>Mefv</i>^−/− littermate mice were treated with LPS and the indicated stimuli as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051105#pone-0051105-g003" target="_blank">Figures 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051105#pone-0051105-g004" target="_blank">4</a>. The concentration of IL-18 in cell culture supernatants was determined by ELISA. A student’s t test was used to calculate p values for <i>Mefv</i>^+/+ versus <i>Mefv</i>^−/− cultures. #, p<0.05; *, p<0.01.</p

Analysis of blood collected from <i>Mefv</i>^+/+ and <i>Mefv</i>^−/− mice.

<p>Blood was collected by cardiac puncture. Whole blood containing 5 mM EDTA was used for analysis on the Heska Hematology Analyzer by the Animal Clinical Chemistry and Gene Expression Laboratory at UNC-Chapel Hill. n = 13 and 13 mice. <b>WBC</b>- white blood cells, <b>RBC</b>- red blood cells, <b>PLT</b>- platelets, <b>LYMPH</b> – lymphocytes, <b>GRAN</b> – granulocytes, <b>MONO</b> – monocytes, <b>HCT</b> - hematocrit, <b>HGB</b> hemoglobin. Differences between Mefv^+/+ and Mefv^−/− were not statistically significant.</p

NS1 variants and their characterization.

<p>Plasmids expressing the NS1 variants NS1-HK and NS1-WSN were transiently transfected into HEK293T cells (A and B). (A) Expression of NS1 variants in the cells was examined by western blot analysis using an anti-MYC antibody. Tubulin was used as a loading control. (B) HEK293T cells expressing NS1 variants were fixed at 24 hr post-transfection, immunostained with anti-MYC (red) for NS1 detection, and examined under a confocal laser scanning microscope. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (C) Transfection of HEK293T cells was performed in triplicate by using the reporter construct (IFN-β-luc) and the NS1 expression plasmids (300 and 500 ng); the β-galactosidase expression plasmid served as an internal control. At 24 hr post-transfection, 20 HA of SeV was infected. After 16 hr infection, IFN-β-luc reporter activities were measured and normalized to β-galactosidase activities. Statistical analysis was performed using Student’s <i>t</i>-test (*** denotes a <i>p</i>-value of <0.005.).</p

Downregulation of the transcription of proinflammatory cytokines by NS1 variants.

<p>NS1 variants-transduced THP-1 macrophage cells were differentiated with TPA and then treated with LPS (1 μg/mL) for 6 hr and subsequently with ATP (2.5 mM) for 15 min. (A-C) mRNA levels of pro-IL-1β (A), pro-IL-18 (B), and TNF-α (C) were analyzed by quantitative real-time PCR using gene-specific primers. (D) The supernatants were harvested and subjected to ELISA to quantify TNF-α. Data represent the mean and standard deviation. Statistical analysis was performed using Student’s <i>t</i>-test to analyze the differences between control and NS1-expressing samples (* denotes a <i>p</i>-value of <0.05; ** denotes a <i>p</i>-value of <0.01; *** denotes a <i>p</i>-value of <0.005).</p

Downregulation effect of NS1 variants on the NLRP3 inflammasome and their interaction with NLRP3.

<p>(A) Differentiated THP-1 cells expressing NS1 variants were treated with LPS (1 μg/mL) for 6 hr, followed by treatment with ATP (2.5 mM) for 15 min. The cell lysates were obtained and subjected to western blot analysis using the indicated antibodies. (B and C) NS1 variants interacted with NLRP3, not with ASC or pro-caspase-1. The MYC-NS1-HK (B) or MYC-NS1-WSN (C) construct was co-transfected with a FLAG-tagged NLRP3 inflammasome protein construct (NLRP3, ASC, or pro-caspase-1) into HEK293T cells. The cells were harvested at 48 hr post transfection, and lysates were immunoprecipitated with anti-FLAG-M2 antibody. Protein interaction with NS1 was identified by western blot analysis with an anti-MYC antibody. (D) Differentiated THP-1 cells expressing NS1 variants were treated with LPS (1 μg/mL) for 6 hr, and subsequently with ATP (2.5 mM) for 15 min. The cells were harvested and lysates were immunoprecipitated with anti-MYC antibody. Endogenous NLRP3 protein interaction with NS1 was identified by western blot analysis with anti-NLRP3.</p

Downregulation of NF-κB activation by NS1 variants.

<p>(A) Downregulation of p65-mediated 2xκB-luc activity by NS1 variants. HEK293T cells were transfected with reporter constructs (2xκB-luc) and NS1 expression plasmids (300 and 500 ng) in the presence of p65-expressing plasmid. Each transfection was performed in triplicate, with the β-galactosidase expression plasmid serving as an internal control. After 26 hr transfection, 2xκB-luc reporter gene activities were measured and normalized to β-galactosidase activities. Statistical analysis was performed using Student’s <i>t</i>-test (*** denotes a <i>p</i>-value of <0.005). (B) Inhibition of the NF-κB pathway by NS1 variants in THP-1 macrophage cells. Transduced THP-1 cells were differentiated with TPA treatment, and then treated with LPS (1 μg/mL) for 6 hr, followed by treatment with ATP (2.5 mM) for 15 min. Each indicated proteins were identified by western blot analysis.</p

Plexin-B2 and Plexin-D1 in Dendritic Cells: Expression and IL-12/IL-23p40 Production

<div><p>Plexins are a family of genes (A,B,C, and D) that are expressed in many organ systems. Plexins expressed in the immune system have been implicated in cell movement and cell-cell interaction during the course of an immune response. In this study, the expression pattern of Plexin-B2 and Plexin-D1 in dendritic cells (DCs), which are central in immune activation, was investigated. Plexin-B2 and Plexin-D1 are reciprocally expressed in myeloid and plasmacytoid DC populations. Plasmacytoid DCs have high Plexin-B2 but low Plexin-D1, while the opposite is true of myeloid DCs. Expression of Plexin-B2 and Plexin-D1 is modulated upon activation of DCs by TLR ligands, TNFα, and anti-CD40, again in a reciprocal fashion. Semaphorin3E, a ligand for Plexin-D1 and Plexin-B2, is expressed by T cells, and interestingly, is dramatically higher on Th2 cells and on DCs. The expression of Plexins and their ligands on DCs and T cells suggest functional relevance. To explore this, we utilized chimeric mice lacking <em>Plxnb2</em> or <em>Plxnd1.</em> Absence of Plexin-B2 and Plexin-D1 on DCs did not affect the ability of these cells to upregulate costimulatory molecules or the ability of these cells to activate antigen specific T cells. Additionally, Plexin-B2 and Plexin-D1 were dispensable for chemokine-directed <em>in-vitro</em> migration of DCs towards key DC chemokines, CXCL12 and CCL19. However, the absence of either Plexin-B2 or Plexin-D1 on DCs leads to constitutive expression of IL-12/IL-23p40. This is the first report to show an association between Plexin-B2 and Plexin-D1 with the negative regulation of IL-12/IL-23p40 in DCs. This work also shows the presence of Plexin-B2 and Plexin-D1 on mouse DC subpopulations, and indicates that these two proteins play a role in IL-12/IL-23p40 production that is likely to impact the immune response.</p> </div