Homogeneous Inflammatory Gene Profiles Induced in Human Dermal Fibroblasts in Response to the Three Main Species of Borrelia burgdorferi sensu lato

Abstract

In Lyme borreliosis, the skin is the key site for bacterial inoculation by the infected tick and for cutaneous manifestations. We previously showed that different strains of Borrelia burgdorferi sensu stricto isolated from tick and from different clinical stages of the Lyme borreliosis (erythema migrans, and acrodermatitis chronica atrophicans) elicited a very similar transcriptional response in normal human dermal fibroblasts. In this study, using whole transcriptome microarray chips, we aimed to compare the transcriptional response of normal human dermal fibroblasts stimulated by 3 Borrelia burgdorferi sensu lato strains belonging to 3 main pathogenic species (B. afzelii, B. garinii and B. burgdorferi sensu stricto) in order to determine whether "species-related" inflammatory pathways could be identified. The three Borrelia strains tested exhibited similar transcriptional profiles, and no species-specific fingerprint of transcriptional changes in fibroblasts was observed. Conversely, a common core of chemokines/cytokines (CCL2, CXCL1, CXCL2, CXCL6, CXCL10, IL-6, IL-8) and interferon-related genes was stimulated by all the 3 strains. Dermal fibroblasts appear to play a key role in the cutaneous infection with Borrelia, inducing a homogeneous inflammatory response, whichever Borrelia species was involved.

Fig 1. Measurement of IL-8 secretion by 6 different batches of fibroblasts coincubated with strains of 3 different species of the B. burgdorferi sl group.

IL-8 secretion of fibroblasts stimulated by B. burgdorferi ss (Bb ss) IBS19, B. garinii IBS6, and B. afzelii IBS17 at MOI of 100:1 after 24h of stimulation. Each bar shows the mean ± SDs of triplicate values (technical replicates) obtained for each batch of fibroblasts—fibroblasts batch #1 to fibroblasts batch #6 (biological replicates). The results are representative of 2 independent experiments.

Fig 2. Kinetics and titration of IL-8 secretion by fibroblasts co-incubated with different species of the B. burgdorferi sl group.

(A-C) Kinetic studies of IL-8 secretion of fibroblasts stimulated by B. burgdorferi ss (Bb ss) IBS19, B. garinii IBS6, and B. afzelii IBS17 at MOI of 100:1. (D-F) Levels of IL-8 secretion by fibroblasts stimulated by increasing concentrations (MOI of 1:1 = 1B, MOI of 10:1 = 10B, 50:1 = 50B, and 100:1 = 100B) of the 3 Borrelia strains at 24 hours. NEG: unstimulated fibroblasts. (A-F) Each bar shows the mean ± SDs of duplicate values obtained for fibroblasts batch #1.

Fig 3. Gene expression profiles obtained from human fibroblasts stimulated with the three species of the B. burgdorferi sl group.

Number of genes differentially expressed during fibroblast stimulation with Borrelia. 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 1.2–2.0-fold, the gray hatched areas correspond to changes of 2.0–5.0-fold and black areas represent changes ≥5.0-fold.

Fig 4. Intensity of transcriptional modifications.

The distribution of regulated genes according to the level of regulation is expressed as fold-change in comparison to unstimulated cells. (A) Comparison of transcription levels between Borrelia strains. *** P <0.001, NS: not significant, tested by ANOVA using GraphPad Prism6 software (GraphPad, San Diego, CA). (B) Transcription levels of common (stimulated by the 3 strains) and specific (stimulated by only one of the strains) genes.

Fig 5. QRT-PCR analysis of mRNA levels induced in human dermal fibroblasts by different species of the B. burgdorferi sl group.

For each batch of fibroblasts,–fibroblasts batch #1 to fibroblasts batch #6 (biological replicates)–, the mRNA levels of IL-8, CXCL1, IL-6 and SOD2 stimulated during 24h at MOI 100:1 by B. burgdorferi ss IBS19 (white bars), B. garinii IBS6 (gray hatched bars) and B. afzelii IBS17 (black bars) were normalized to the β-actin (A) or RNA polymerase II (B) housekeeping gene levels and expressed as relative changes in gene transcription compared with untreated cells. Each bar shows the mean ± SDs of duplicate values (technical replicates).

Fig 6. QRT-PCR analysis of mRNA levels repressed in human dermal fibroblasts by different species of the B. burgdorferi sl group.

For each batch of fibroblasts,–fibroblasts batch #1 to fibroblasts batch #6 (biological replicates)–, the mRNA levels of UBE2C, KIF20A, TOP2A, CEP55 and CDC20 stimulated during 24h at MOI 100:1 by B. burgdorferi ss IBS19 (white bars), B. garinii IBS6 (gray hatched bars) and B. afzelii IBS17 (black bars) were normalized to the β-actin (A) or RNA polymerase II (B) housekeeping gene levels and expressed as relative changes in gene transcription compared with untreated cells. Each bar shows the mean ± SDs of duplicate values (technical replicates).