Comparison of LSVs from MS spectra of <i>I</i>. <i>ricinus</i> ticks according to body part, origin and <i>Borrelia</i>-infectious status.

<p>Dashed line represent the threshold value for relevant identification (LSVs>1.8). LSV, log score value; NE, non-exposed; BF; <i>Borrelia</i>-free; BI; <i>Borrelia</i>-infeted.</p

Mass peak list distinguishing legs and idiosomes from laboratory reared and pathogen-free <i>I</i>. <i>ricinus</i> specimens.

<p>Mass peak list distinguishing legs and idiosomes from laboratory reared and pathogen-free <i>I</i>. <i>ricinus</i> specimens.</p

Comparison of MALDI-TOF MS spectra from legs, capitula and half-idiosomes of adult <i>I</i>. <i>ricinus</i> pathogen-free (i) or infected by <i>Borrelia afzelii</i> (ii).

<p>Representative MS spectra of legs (A, B), capitula (C, D) and half-idiosomes (E, F) from laboratory reared <i>I</i>. <i>ricinus</i> homogenized automatically using FastPrep-24 device with glass powder. a.u., arbitrary units; m/z, mass-to-charge ratio.</p

Assessment of <i>I</i>. <i>ricinus</i> MS spectra reproducibility according to tick body parts and <i>Borrelia</i> infectious status using composite correlation index (CCI).

<p>MS spectra from five specimens per body part and <i>B</i>. <i>afzelii</i> infectious status were analysed using the CCI tool. Body part and infectious status are indicated on the left side of the heat map. Levels of MS spectra reproducibility are indicated in red and blue revealing relatedness and incongruence between spectra, respectively. CCI matrix was calculated using MALDI-Biotyper v3.0. software with default settings (mass range 3.0–12.0 kDa; resolution 4; 8 intervals; auto-correction off). The values correspond to the mean coefficient correlation and respective standard deviations obtained for paired condition comparisons. CCI were expressed as mean ± standard deviation. BI, <i>Borrelia</i>-infected; PF, pathogen-free.</p

Principal component analysis (PCA) from MS spectra of idiosomes and legs of <i>I</i>. <i>ricinus</i> infected or not by <i>Borrelia sp</i>.

<p>PCA dimensional image from MS spectra of <i>I</i>. <i>ricinus</i> idiosomes (A) and legs (B) <i>Borrelia</i>-free (red dots, n = 10), infected by <i>B</i>. <i>afzelii</i> (green dots, n = 6), <i>B</i>. <i>burgdorferi</i> (blue dots, n = 3), <i>B</i>. <i>garinii</i> (yellow dots, n = 2), co-infected by <i>B</i>. <i>garinii</i> and <i>B</i>. <i>burgdorferi</i> (purple dots, n = 1). (C) PCA dimensional image from the same MS spectra of <i>I</i>. <i>ricinus</i> idiosomes (red dots, n = 22) and legs (green dots, n = 22). The contributions of PC1, PC2 and PC3 were 38.4%, 15.5% and 7.2%, respectively. Among the <i>Borrelia</i>-free specimens, five were laboratory reared and the other five came from field collection.</p

MSP dendrogram of MALDI-TOF MS spectra from legs, capitula and half-idiosomes of adult <i>I</i>. <i>ricinus</i> pathogen-free or infected by <i>Borrelia afzelii</i>.

<p>Five specimens per body part and <i>B</i>. <i>afzelii</i> infectious status were used to construct the dendrogram. The dendrogram was created using Biotyper v3.0 software and distance units correspond to the relative similarity of MS spectra. The specimens infected by <i>B</i>. <i>afzelii</i> were indicated by asterisks (*).</p

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

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

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

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