Gene expression profiles obtained from dermal fibroblasts stimulated with different strains of <i>B. burgdorferi</i> ss.

<p>(A) Venn diagram of genes significantly up-regulated (↑) or down-regulated (↓) after fibroblast stimulation with <i>Borrelia</i>, and compared with unstimulated fibroblasts. (B) Number of genes differentially expressed during fibroblast stimulation with <i>Borrelia</i>. The bars reflect the number of up-regulated genes (+) and down-regulated genes (-) for each strain. The light dotted areas correspond to gene expression changes of 1.7–5.0-fold, the grey hatched areas correspond to changes of 5.0–20.0-fold and black areas to changes ≥20.0-fold.</p

Role of OspC, <i>I. ricinus</i> salivary gland extracts (SGE) and Salp15 in <i>Borrelia</i>-induced fibroblast inflammation

<p>(A) IL-8 synthesis induced by wild-type strain 297 (wt), OspC-deficient (OspC −/−), and OspC-deficient strain 297 complemented with a plasmid carrying the ospC gene (OspC cp) in fibroblasts. (B) IL-8 synthesis in fibroblasts induced by <i>B. burgdorferi</i> ss N40 (Bb) in absence or in presence of human anti-TLR2 antibody (Ab aTLR2) or isotype control antibody (Ab isotype control). (C) IL-8 synthesis in fibroblasts coincubated with 20 µg/ml SGE alone, 30 µg/ml Salp15 alone, <i>B. burgdorferi</i> ss N40 (Bb) alone, with the combination of <i>Borrelia</i> and SGE at 20 µg/ml (Bb + SGE), 5 µg/ml (Bb + SGE 1∶4), 1 µg/ml (Bb + SGE 1∶20), and 0.2 µg/ml (Bb + SGE 1∶100), with the combination of <i>Borrelia</i> and Salp15 (Bb + Salp15), or with the combination of <i>Borrelia</i> and 20 µg/ml SGE heat-denaturated at 56°C for 1 hour (Bb + SGE 56°C), or at 98°C for 3 minutes (Bb + SGE 98°C). For (A), (B) and (C) fibroblasts were incubated with <i>Borrelia</i> at MOI of 100∶1 for 24 hours. The negative control was unstimulated cells (NEG). Each bar shows the mean ± SDs of triplicate values (expressed as % stimulation of IL-8 synthesis induced by <i>Borrelia</i> alone) and is representative of three independent experiments. ***P<0.001; and *P<0.05 compared with the corresponding stimulation induced by <i>Borrelia</i> alone. (D) Images of fibroblast cell cultures stimulated with SGE, showing SGE-induced cytotoxic effect at 6 and 24 hours (h). Images were taken at 100x (I, II and III) or at 200x magnification (IV, V and VI).</p

Measure of IL-8 secretion by fibroblasts co-incubated with different strains of <i>B. burgdorferi</i> ss.

<p>(A-C) IL-8 secretion of fibroblasts stimulated by different concentrations of <i>Borrelia</i> N40, Pbre, 1408 at MOI of 100∶1 (100B), 10∶1 (10B), and 1∶1 (1B) at 24 hours. (D-F) Kinetic studies of IL-8 secretions in the three strains. NEG: unstimulated fibroblasts. (A-F) Each bar shows the mean ± SDs of triplicate values and is representative of three independent experiments. ***P<0.001; **P<0.01; and *P<0.05 compared between stimulated and unstimulated cells.</p

QRT-PCR analysis of mRNA expression induced by <i>Borrelia</i> in kinetic experiments with fibroblasts.

<p>The mRNA levels of IL-8, IL-6, CXCL1, SOD2 and MMP-12 were normalized to the β-actin housekeeping gene level and expressed as relative changes in gene expression compared with untreated cells (NEG). Each bar shows the mean ± SDs of triplicate values and are representative of three independent experiments. **P<0.01; and *P<0.05 compared between stimulated and unstimulated cells.</p

Up-regulated genes in fibroblasts stimulated with <i>B. burgdorferi</i> in comparison to unstimulated fibroblasts.

1<p>For each strain, values shown correspond to the mean ratio of the duplicate measurement determined between normalized gene intensity values obtained after 24 hours of fibroblast stimulation with <i>B. burgdorferi</i> (MOI 100∶1) compared with gene intensity values from unstimulated cells.</p

Bacterial loads in the skin at the inoculation site was measured by quantitative PCR for the <i>B</i>. <i>burgdorferi flaB</i> gene and normalized to copies of mouse <i>gapdh</i>.

<p>Values between strains were not statistically different (two-way ANOVA test using the Sidak-Bonferroni method). # values close to the detection limit.</p

Bacterial loads in the skin.

<p>The spirochetal burden in the skin at the inoculation site was measured by quantitative PCR for the <i>B</i>. <i>burgdorferi flaB</i> gene and normalized to copies of mouse <i>gapdh</i>. Values represent relative expression + SD of three independent experiments. Values between strains were not statistically different (two-way ANOVA test using the Sidak-Bonferroni method). (d: days).</p

Inflammatory profiles of <i>B</i>. <i>burgdorferi</i> ss, parental strain and its clone 297/4.

<p>Levels of transcripts were measured by RT-qPCR from the skin at the inoculation site. The values were calculated using the 2-delta delta Ct method after normalization with <i>gapdh</i>. (h: hours, d: days). Two-way ANOVA was used to analyze the data. At least, three mice were analyzed for each time point.</p

Proteomic analyses of <i>B</i>. <i>burgdorferi</i> ss, 297 parental strain and its clone 297/4.

<p>(A) The Venn diagram shows the protein overlap and the proteins specifically identified in the parental strain and the clone 297/4. (B) Graphic representation of the breakdown of the proteins specifically identified in the clone. Categorization was based upon JCVI annotation. The percentages represent the fraction of that category within the proteins. (C) Gene location of the specific proteins identified in the clone 297/4.</p

Bacterial loads in the skin at the inoculation site was measured by quantitative PCR for the <i>B</i>. <i>burgdorferi flaB</i> gene and normalized to copies of mouse <i>gapdh</i>.

<p>Values between strains were not statistically different (two-way ANOVA test using the Sidak-Bonferroni method). # values close to the detection limit.</p