Mycobacterium bovis-BCG vaccination induces specific pulmonary transcriptome biosignatures in mice

Abstract

Background: In the present study, we applied microarray technology to define biosignatures by microarray transcriptome analysis in lung and spleen samples after BCG vaccination and M. bovis infection of BALB/c mice. The aims were two-fold, namely to define biosignatures that could predict vaccine success before challenge, and biomarker patterns that correlated with anamnestic protective responses following exposure to virulent M. bovis. Further, these biosignatures should be detectable without in vitro antigenic challenge.

Principal findings: After BCG vaccination, we defined a specific pulmonary gene expression signature related to the connective tissue development and function network that predicted vaccine success before M. bovis challenge. In addition, a Th17-related cytokine profile was found that correlated with vaccine-induced protective immunity following infection with virulent M. bovis in the lung as well as additional genes that were up-regulated in the spleens of vaccinated animals post-infection related to neutrophil biology and inflammation.

Conclusions: This study has therefore prioritized both biomarkers predicting vaccination success before challenge and bio-signatures that are potentially associated with protective immune responses that will be useful to evaluate future vaccine candidates.

Figure 1. Venn diagram showing the distribution of genes that define vaccine success after BCG vaccination but before M. bovis challenge in lung and spleen.

Comparisons of at least 1.7-times over- or under-expressed gene obtained for UU, VI and UI samples relative to VU, resulting in gene expression patterns that predict protection in lung (A) and in spleen (B). A list of these genes is provided in Table S1.

Figure 2. Pulmonary gene signature after BCG vaccination but before M. bovis challenge.

By using unsupervised hierarchical clustering of transcripts and samples with centroid linkage and Pearson centered measure, lung samples were segregated into two distinct groups based on the 164 genes identified previously (Fig.1A). All VU samples (red squares at the bottom) were clustered together with two UU samples. Bright red represents a 3.4-fold increase in expression above the mean and bright blue represents a 3.4-fold change decrease in the expression from the average to all UU mice. Yellow indicates the unchanged expression level of a partiuclar gene between vaccinated and uninfected control mice (VU and UU groups, respectively).

Figure 3. Connective tissue disorder network genes are differentially modulated after BCG vaccination but before M. bovis challenge in lungs.

The connective tissue disorder network was identified after Ingenuity Analysis (p-value 1.65E-4) based on a subset of 164 genes identified previously in lung (Fig.1A). The relationship between individual genes is depicted in this figure. Red: up-regulated; Blue: down-regulated; White: not found to be modulated.

Figure 4. Confirmation of the modulation of selected genes by quantitative RT-PCR.

To confirm the expression patterns of some genes identified in the microarray studies, real-time RT-PCR (QRT-PCR) was performed using total RNA collected from vaccinated and unvaccinated mice prior to infection form an experiment independent from the original experiment that provided samples for the microarray analysis. Data are expressed as log10 relative expression levels in vaccinated compared to naïve control mice.

Figure 5. Venn diagrams of genes defining protective host responses after M. bovis challenge.

Comparisons of genes that were over or under-expressed at least 1.7- times in the lungs and spleens of vaccinated compared to unvaccinated mice after M. bovis challenge are shown (UI samples relative to VI samples at day 3 and 14 post challenge). (A) Lung cell responses; (B) spleen cells responses (B). Genes involved are listed in Table S2).

Figure 6. Transcription profiles in lung and spleen cells of selected genes differentially modulated between vaccinated and unvaccinated mice post-infection.

Each line shows expression levels determined by microarray analysis in lung (A) and spleen cells (B) of individual transcripts between unvaccinated and vaccinated samples. (A) Pulmonary (lung cell) expression profiles of TH17 pathway related genes. (B) Spleen cell expression profiles of genes related to neutrophil-biology. For (B): N = genes related to neutrophil activity; Q =  genes related to neutrophil chemotaxis; IL8 =  genes related to interleukin-8 biosynthesis.