<i>C.</i><i>difficile-</i>mediated effects on human monocyte-derived DC IL-10 and IL-1β production.

<p>Human mDCs from nine healthy donors were stimulated with <i>C. difficile</i> at an MOI of 10. IL-10 (A) and IL-1β (B) secretion measured 8h post-infection. **p<0.01 and ***p<0.001 represent significant difference from uninfected control cells. Data was analysed using Mann-Whitney U test.</p

Known and putative virulence factor genes differentially expressed <i>in vivo i</i>n the <i>fliC</i> mutant compared to strain R20291.

<p>ND: non determined.</p

Flagellar genes differentially expressed <i>in vivo</i> in the <i>fliC</i> mutant.

<p>*From F1 region (late-stage flagellar genes) and **F3 region (early-stage flagellar genes) of the flagellar operon from <i>C. difficile</i> 630 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096876#pone.0096876-Aubry1" target="_blank">[39]</a>.</p

Time-dependent effects of <i>C.</i><i>difficile</i> R20291 and 630 infection on BMDC cytokine mRNA expression.

<p>BMDCs were infected with bacterial cultures at an MOI of 10 and mRNA expression of IL-12 family members; p35 (A), p40 (B), p19 (C), p28 (D), EBI3 (E), also IL-10 (F) and IL-1β (G) was quantified by real-time PCR. Data is presented as fold increase compared to expression in uninfected control cells. Data represent mean ± SEM, n = 3. *p<0.05, **p<0.001, and ***p<0.001 represent significant inter-strain difference. <i>P</i> values were obtained using ANOVA with Bonferroni post-test analysis.</p

Splenocyte and Naïve CD4⁺ T cell proliferation in response to <i>C.</i><i>difficile</i>-stimulated BMDCs.

<p>WT BMDCs were infected with PFA-fixed <i>C. difficile</i> strains at an MOI of 50. 24 h post-infection, infected BMDCs were co-cultured with CFSE-labelled splenocytes in the presence of anti-CD3/CD28. Proliferation of CD4⁺ gated cells 96 h post stimulation (A). Quantification presented as percentage of proliferating cells (B). Infected BMDCs were co-cultured with CFSE-labelled naïve CD4⁺ T cells from OT-II transgenic mice in the presence of OVA_323–339 and T cell proliferation was analysed (C). 1 µg/ml LPS utilised as a reference stimulus. ***p<0.001 represent significant difference from uninfected control cells. Data was analysed using ANOVA with Bonferroni post-test.</p

Functional clusters of <i>in vivo</i> differentially expressed genes.

<p>Differentially expressed genes in the <i>fliC</i> mutant compared to wild-type R20291 from caeca of 14 h post-infection mice were classified in functional groups according to their involvement in the biological process categories. (<b>A</b>) The number of analyzed genes is represented in green bars and the number of significantly differentially expressed genes is shown in black bars. Percentages of differentially regulated genes are indicated at right. (<b>B</b>) The numbers of up- and down-regulated genes for each cluster are indicated in green and black bars, respectively.</p

<i>C.</i><i>difficile</i>-mediated effects on BMDC cytokine production.

<p>BMDCs were infected with <i>C. difficile</i> cultures at an MOI of 10 and IL-12 (A), IL-23 (B) IL-27 (C), IL-10 (D) and IL-1β (E) was measured 8 h post-infection. 1 µg/ml LPS stimulation served as positive control. Data represent mean ± SEM of duplicate samples and are representative of three individual experiments. */^∧p<0.05, ^∧∧p<0.01 and ***/^∧∧∧p<0.001 represent significant difference from uninfected cells and significant inter-strain difference. <i>P</i> values were obtained using ANOVA with Bonferroni post-test analysis.</p

Membrane transport genes highly differentially expressed <i>in vivo</i> in the <i>fliC</i> mutant compared to strain R20291.

<p>Membrane transport genes highly differentially expressed <i>in vivo</i> in the <i>fliC</i> mutant compared to strain R20291.</p

Ability of sporulation of the <i>fliC</i> mutant compared to wild-type R20291 <i>in vivo</i> and <i>in vitro</i>.

<p>(<b>A</b>) Groups of 6 axenic mice were infected by oral gavage route with 1×10⁸<i>C. difficile</i> CFU. The <i>C. difficile</i> faecal vegetative cells and spores were measured by determining the concentration of CFU in faeces at 14 h post-infection by homogenising and plating on appropriated agar medium after heat shock (for spores) or not (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096876#s4" target="_blank">Materials and Methods</a>). Data represent the ratio of spores per vegetative cells. (<b>B</b>) Cultures in BHIS broth in anaerobic conditions were prepared from 2 successive subcultures as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096876#s4" target="_blank">Materials and Methods</a>. After heat shock, spores were quantified (CFU/ml) by performing serial dilutions and spread plating on BHIS agar supplemented with 0.1% bile salt taurocholate to induce germination. Data represent the ratio of spores per total cells. The presented values are the mean of 3 different cultures. Statistically significant difference is indicated by * for p<0,05.</p

<i>C.</i><i>difficile</i> toxins trigger BMDC IL-1β release by activating an ASC-containing inflammasome.

<p>BMDCs from WT C57BL/6 (genetically Nlrp1 deficient) mice and mice deficient in Nlrp3 and ASC were infected with <i>C. difficile</i> strains at an MOI of 10. Pro- and active caspase-1 (45 & 10 kDa, respectively) and pro- and mature IL-1β (31 & 17 kDa, respectively) release 8h post-infection was quantified by western blotting. 5 mM ATP served as a positive control (A). Quantification of IL-1β secretion in infected WT, Nlrp3 and ASC KO BMDC 8h post-infection. Data is represented as mean ± SEM, n = 4. ***p<0.001 represents significant difference from uninfected cells, ^∧p<0.05 and ^∧∧∧/’p<0.001 represent significant WT and Nlrp3^−/− difference from ASC^−/−. <i>P</i> values were obtained using ANOVA with Bonferroni post-test analysis (B & C).</p