Mutational analysis of the mouse 6C2 MAIT TCR in response to antigen presenting cells infected with <i>E. coli</i>.

<p>Response of 6C2 MAIT hybridoma expressing different mutations of the TCR α chain (A, C) or TCR β chain (B, D) to LM1.8 fibroblasts (A and B) or CH27 B cells (C and D) cocultured with <i>E. coli</i> (MOI = 50). Data represent the mean+s.e.m. of three independent experiments.</p

The MAIT ligand is resistant to digestion with proteinase K and is not part of the lipid component of <10 kD <i>E. coli</i> culture.

<p>The <10 kD fraction <i>E. coli</i> lysate was spiked with OVA protein and left untreated (solid line) or digested overnight with proteinase K (dashed line). MAIT ligand activity was tested with LM1.8 fibroblasts and the 6C2 hybridoma (A) while OVA antigenic activity was tested with A20 cells and the DO11.10 hybridoma (B). An overnight culture of <i>E. coli</i> was spun down, resuspended in chloroform/methanol, spiked with α-GalCer, sonicated and subjected to lipid extraction. Aqueous (triangles), interphase (circles), and organic (squares) phases were dried out and resuspended in complete media. MAIT ligand activity was assessed with the 6C2 hybridoma and LM1.8 fibroblasts at indicated dilutions (C) while α-GalCer activity was detected using the DN32.D3 hybridoma and mCD1d-transfected A20 cells at indicated dilutions (D). Data represent the mean+s.e.m. of two independent experiments.</p

Mutational analysis of the mouse 6C2 MAIT TCR in response to MR1-overexpressing antigen presenting cells.

<p>Response of 6C2 MAIT hybridoma expressing different mutations of the TCR α chain (A, C) or TCR β chain (B, D) to overexpression of mouse MR1 on LM1.8 fibroblasts (A and B) or CH27 B cells (C and D). Data represent the mean+s.e.m. of three independent experiments.</p

The MAIT hybridoma response to MR1 mutants.

<p>(A) The 6C2 MAIT hybridoma was stimulated with mouse MR1 mutants expressed on LM1.8 fibroblasts in the absence (white bars) or presence of bacteria (black bars) at an MOI of 100. The IL-2 response in both cases could be blocked with an anti-MR1 antibody (data not shown). (B) The effect of the MR1 mutations on both the autoreactive response and the bacterial response is mapped on Phyre-generated mouse MR1 model. Mutations with an effect on both the autoreactive and bacterial response are shown in red, while mutations that only affected the autoreactive response are shown in pink. The residue attributed to the inability of human MR1 to activate mouse MAIT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053789#pone.0053789-Huang3" target="_blank">[19]</a> cells is shown in orange.</p

Effect of CS exposure on colonic pro-inflammatory and anti-inflammatory cytokine expression induced by DSS.

<p>Cytokine expression in colon homogenates was determined by real time qPCR analysis and normalized by the β-actin expression. Graph represents the mean of the fold expression of each cytokine with the expression level measured to control animals (no CS exposure, no DSS) used as a reference and set to one. Data are pooled from two independent experiments with a total of 17–20 mice/group; error bars represent SEM. NS, Non significant; Number on the graph represents p value.</p

Effect of CS exposure on colonic Th1/Th2/Th17 cytokine expression induced by DSS.

<p>Cytokine expression in colon homogenates was determined by real time qPCR analysis and normalized by the β-actin expression. Graph represents the mean of the fold expression of each cytokine with the expression level measured to control animals (no CS exposure, no DSS) used as a reference and set to one. Data are pooled from two independent experiments with a total of 17–20 mice/group; error bars represent SEM. NS, Non significant; Number on the graph represents p value.</p

CS exposure did not modulate the development of colitis induced by DSS in CD1d^−/− mice.

<p>A. Mice body weight changes during induction of colitis. Body weight changes were calculated by dividing body weight on the specified day by the body weight of the starting day (day 0) and expressed in percent (3</p

Effect of CS exposure on NKT cell recruitment.

<p>A. Percentage of iNKT cells (TCRβ⁺ CD1d tetramer⁺) in CD45⁺ cells in the colonic mucosa of mice exposed or not to CS. Colonic tissues of several mice per group (3 to 5 animals) were pooled before cell extraction in order to obtain enough cell for cytometry analysis. On the left: a representative dot plot is shown. Numbers indicate iNKT cell percentage. On the right: Graph represents the mean value ± SEM of the iNKT cell percentage in the colonic mucosa according to CS exposure (n = 32 mice/condition from 3 independent experiments). Number on the graph represents p value. B. Vα14 mRNA expression in the colon as determined by real time qPCR analysis after normalization by the β-actin expression (17</p

Effect of CS exposure on colonic cytokine expression induced by DSS in Jα18^−/− mice.

<p>Cytokines expression in colon homogenates was determined by real time qPCR analysis and normalized by the β-actin expression. Graph represents the mean of the fold expression of each cytokine with the expression level measured to control animals (no CS exposure, no DSS) used as a reference and set to one. Data are pooled from two independent experiments (11</p

Effect of CS exposure on neutrophils recruitment and activation induced by DSS colitis.

<p>A. CXCL1/KC and CXCL2/MIP-2 mRNA expression in colon homogenates was determined by real time qPCR analysis and normalized by the β-actin expression. Data are pooled from two independent experiments with a total of 17–20 mice/group; error bars represent SEM. NS, Non significant; Number on the graph represents p value. B. Mean ± SD of neutrophil (CD11c− Ly6G+) percentages of CD45+ cells in the colonic <i>lamina propria</i> (n = 5). NS, non significant<b>.</b> C. Colonic MPO level determined by ELISA. The graph represents the mean ± SD of the final values of MPO level expressed in ng/mg of protein (n = 10). NS, non significant.</p