Genes activating <i>cxcl2</i>-pLuc expression in Raw 264.7 cells.

<p>Genes activating <i>cxcl2</i>-pLuc expression in Raw 264.7 cells.</p

Flow chart of the functional screen.

<p><b>A</b>) shows the workflow of the screen. <b>B</b>) shows an example of the analysis performed on a plate comprising 96 pools of 12. The hypothesis that individuals pools differ from the plate mean is tested using a linear model. Three pools (P1, P2, P3) give signal over the −logp>4 cut off used. Breakdown of pool P2 is shown in <b>C</b>) one constituent clone (#8) gives high-level induction. Dots represent results of four independent replicates, performed for each clone.</p

Functionally linked groups of proteins uncovered in the screen.

<p>Functionally linked groups of proteins uncovered in the screen.</p

Activation of proinflammatory promoters by library hits and their impact on LPS induced <i>cxcl2</i> activation in Raw 264.7 cells.

<p>Induction of three proinflammatory promoters (<i>ifnb</i>, <i>cxcl2</i> and <i>Lcn2</i>) by plasmids recovered from the library screen and positive controls are shown. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042388#s2" target="_blank">Results</a> are derived from 2 (for <i>ifnb</i> and <i>lcn2</i>) and between 3 and 13 independent experiments (for <i>cxcl2</i>), all performed in triplicate in each experiment. <b>A</b>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042388#s2" target="_blank">Results</a> are expressed as log fold increase of normalised promoter activity over background and 95% confidence intervals. <b>B</b>) and <b>C</b>) show bivariate plots illustrating activity of the various molecules on the <i>cxcl2</i>, <i>lcn2</i> and <i>ifnb</i> reporters. Effect estimates for molecules derived from the screen are marked as circles, and known components of signalling system as triangles. <b>D–F</b>) Raw 264.7 cells were transfected with the <i>cxcl2</i>-pLuc and EF1-rLuc reporters, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042388#s4" target="_blank">methods</a>, as well as with 60 ng/well expression plasmid, encoding known (TRAF6) (<b>D</b>) or novel proinflammatory molecules (<b>E</b> and <b>F</b>). The impact of over-expressed proinflammatory mediators on LPS induced <i>cxcl2</i> expression was tested. The activity profile of the cDNAs tested were classified according to three distinct patterns, as exemplified in D–F and shown in <b>G</b>). Genes highlighted in bold encode known components of inflammatory signal transduction (controls).</p

Selected screen hits are required for TLR4 but not for TLR7/8 induced cxcl2 induction.

<p>Raw 264.7 cells were transfected with the <i>cxcl2</i>-pLuc and EF1-rLuc reporters, as described in the methods, as well as with 10 pmol siRNA (ON-TARGET <i>plus</i> SMART pool, Dharmacon) aginst selected hits that are endogenously expressed in these cells. The impact of siRNA knockdown on <b>A</b>) LPS (20 ng/ml) or <b>B</b>) CL075 (2 µg/ml) induced <i>cxcl2</i> reporter activation was measured after 6 hrs stimulation. White bars: vehicle, grey bars: agonist added. Data was analysed by two-way ANOVA modelling effect of agonist (LPS or CL075) and siRNA (four categories). Significance was determined from contrast estimates from the model (***p<0.001, ****p<0.0001). A representative experiment of three, with similar results, is shown.</p

Screen hits interact with the canonical proinflammatory signalling network via distinct molecular mechanisms.

<p><b>A</b>) Raw 264.7 cells were transfected with the <i>cxcl2</i>-pLuc and EF1-rLuc reporters, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042388#s4" target="_blank">methods</a>, as well as with 30 ng/well expression plasmid, encoding for dominant negative (DN) mutants of known proinflammatory molecules (MyD88, TRAM, TIRAP, TRIF, IRAK1, TRAF6 and Ras, respectively). LPS (100 ng/ml) was used as a positive control (6 hrs stimulation) to test for the inhibitory activity of the DN constructs used. Data are derived from three independent experiments, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042388#s4" target="_blank">Methods</a>. *p<0.05 , **p<0.01, ***p<0.001 <b>B</b>) summarises the interactions observed in <b>A</b>.</p

A simulation of various sectioning strategies was performed using the data obtained from MRI scanning.

<p>A comparison of the total abscess volume calculated from each kidney and the estimation achieved by an average of abscess areas in sections is depicted for each slicing strategy. (A): 1 slice estimation, (B) 3 slice estimation, (C) 5 slice estimation, (D) 10 slice estimation.</p

Immunogenicity of Sortase A vaccine regimes.

<p>Mice were immunised with either a control regime comprising AdHu5 (Human adenovirus 5) expressing no transgene (Regime 1), or with AdHu5 expressing SrtA (Regime 2), or with the same vector followed 8 weeks later by a SrtA protein boost (Regime 3) (A). (B) Serological assay was performed on serum taken from immunised mice and specific antibody concentration to SrtA is presented in arbitrary luciferase units, calculated as log₁₀(Luciferase units- Background) in the LIPS assay used (C) An ELISPOT was performed from blood taken from immunized mice seven days on day 63 post adenoviral priming, or day 7 post protein boost. The difference between groups was not statistically significant. 15 days after infection, (D) bacterial recovery from the left kidney, and renal abscess formation (E,F) assessed by the MR technique were determined.</p

Quantification of abscess growth using MRI.

<p>Female Balb/c mice were injected intravenously with 10⁷ CFU <i>S</i>. <i>aureus</i> Newman. Kidneys were recovered at varying days post infection (DPI). A: The right kidney was fixed in paraformaldehyde, and processed for subsequent MRI analysis. Individual abscess in each kidney (A) and the total volume of abscess present per kidney (B) were calculated. Both the median volume of abscesses detected (A), and the total volume of abscesses (B) increase significantly over time, but (C) bacterial recovery in the left kidney changed little between 3 and 10 days post infection (C,D). Where no abscesses were detected, abscess volume was considered to be 1mm³ (Panel B), and when no bacteria were isolated, 1 CFU/g is plotted (Panel C). ** denotes p<0.01, * denotes p<0.05 by Mann-Whitney test.</p

Scanning fixed organs after agar embedding.

<p>Up to thirty-two paraformaldehyde-fixed kidneys can be placed in eight layers of four kidneys, and embedded in agarose containing the contrast agent. The tube is then subjected to MR imaging.</p