Transcriptional profiling of disease-induced host responses in bovine tuberculosis and the identification of potential diagnostic biomarkers

Abstract

Bovine tuberculosis (bTb) remains a major and economically important disease of livestock. Improved ante-mortem diagnostic tools would help to underpin novel control strategies. The definition of biomarkers correlating with disease progression could have impact on the rational design of novel diagnostic approaches for bTb. We have used a murine bTb model to identify promising candidates in the host transcriptome post-infection. RNA from in vitro-stimulated splenocytes and lung cells from BALB/c mice infected aerogenically with Mycobacterium bovis were probed with high-density microarrays to identify possible biomarkers of disease. In antigen-stimulated splenocytes we found statistically significant differential regulation of 1109 genes early (3 days) after infection and 1134 at a later time-point post-infection (14 days). 618 of these genes were modulated at both time points. In lung cells, 282 genes were significantly modulated post-infection. Amongst the most strongly up-regulated genes were: granzyme A, granzyme B, cxcl9, interleukin-22, and ccr6. The expression of 14 out of the most up-regulated genes identified in the murine studies was evaluated using in vitro with antigen-stimulated PBMC from uninfected and naturally infected cattle. We show that the expression of cxcl9, cxcl10, granzyme A and interleukin-22 was significantly increased in PBMC from infected cattle compared to naïve animals following PPD stimulation in vitro. Thus, murine transcriptome analysis can be used to predict immunological responses in cattle allowing the prioritisation of CXCLl9, CXCL10, Granzyme A and IL-22 as potential additional readout systems for the ante-mortem diagnosis of bovine tuberculosis.

Figure 1. Spleen gene signature after 3 and 14 days after infection with M. bovis.

The global transcriptional response in spleen cells of mice infected with M. bovis was compared to responses in uninfected mice. Genes were considered significantly modulated when their corrected p-values were below 0.05 with more than 2-fold change of expression at both time points. After 3 and 14 days post-infection (blue square and red squares, respectively at the bottom of graph), the mice were euthanized and their splenocytes were stimulated in vitro for 3 days with M7 protein pool (see Materials and Methods ). Black squares: Naïve control mice. Unsupervised hierarchical cluster was performed using a centroid linkage with a Person centered measure showing that 618 of these genes were modulated at both time points (see table S1 for list of these genes, with genes significantly modulated at both time points highlighted in bold).

Figure 2. Pulmonary gene signature after 14 days after infection with M. bovis.

The global transcriptional response in the lung of mice infected with M. bovis was compared the response of uninfected mice. Genes were considered significant when their correct p-value were below 0.05 with more than 2-fold change of expression. After 14 days post-infection the mice were euthanized and their lung cells were stimulated in vitro for 3 days with M7 protein pool (see Materials and Methods ). Unsupervised hierarchical cluster was performed using a centroid linkage with a Person centered measure showing that 282 of these genes were modulated (see table S2 for list of these genes).

Figure 3. Functional networks (A) and canonical pathways (B) most significantly modulated in lung cells 14 days after M. bovis infection.

Visualization of the trend and significance in the regulation of each network and pathway. Dark blue: all genes represented in a network. Light blue: genes that were up-regulated in a network. Cyan: gene those were down-regulated in a network. Fisher's exact test threshold value of p≤0.05.

Figure 4. Functional networks (A) and canonical pathways (B) most significantly modulated in spleen cells 3 and 14 days after M. bovis infection.

Visualization of the trend and significance in the regulation of each network and pathway are shown. Specific networks and pathways after 3 (dark blue bars) or 14 days p.i. (light blue bars) are indicated. Fisher's exact test threshold value of p≤0.05.

Figure 5. Induction of the inflammatory response network in lung (A) and spleen (B) cells at 14 days p.i.

Dark blue: all genes enriched in this network. Light blue: genes that were up-regulated in this network. Cyan: gene those were down-regulated in this network.

Figure 6. Phenotypic analysis of bovine T cell subsets that expressed the genes encoding for IL22, IL17a and GzmA.

Highly purified CD4⁺, CD8⁺, and CD8⁻/TCRδ⁺ T cell populations were isolated by FACS sorting and the mRNA expression of these genes was determined after stimulation with PPD-B. Results are presented as mean fold increase compared to media control values ± SEM.