Gene expression profiling of peripheral blood mononuclear cells from cattle infected with Mycobacterium paratuberculosis

Abstract

A bovine-specific cDNA microarray system containing 721 unique leukocyte expressed sequence tags (ESTs) and amplicons representing known genes was used to compare gene expression profiles of peripheral blood mononuclear cells (PBMCs) from clinical and subclinical Johne's disease-positive Holstein cows (n = 2 per group). Stimulation of PBMCs from clinically infected cows with Mycobacterium paratuberculosis tended to decrease expression of 83 genes (fold change, >1.5). Of these 83 genes, 16 displayed significant down regulation across both clinical cows (P < 0.1), including genes encoding microspherule protein 1, fibroblast growth factor, and the Lyn B protein kinase. Only eight genes from PBMCs of clinically infected cows exhibited a modest up regulation following stimulation with M. paratuberculosis, including those encoding bovine CD40L, gamma interferon, interleukin-10 (IL-10), and tissue inhibitor of matrix metalloproteinases (TIMP) 4. In contrast, stimulation of PBMCs from subclinically infected cows with M. paratuberculosis tended to up regulate expression of 71 genes representing 68 unique transcripts. Of these, 11 genes showed significant up regulation (fold change, >1.5; P < 0.1) across both animals, including those encoding bovine CD40L, several matrix metalloproteinases, and SPARC (secreted protein, acidic and rich in cystine). Repression of gene expression was also observed in PBMCs from the subclinical cows, with 16 genes being significantly down regulated (fold change, >1.5; P < 0.1) across both animals, including those encoding the bovine orthologs of cytochrome oxidase subunit III, IL-1 receptor type I, and fibrinogen-like 2 protein. Only one clone, representing an unknown bovine EST, was similarly down regulated in PBMCs from both the clinical and subclinical cows. Thus, the most prominent change induced by exposure of PBMCs from clinical cows to M. paratuberculosis in vitro tended to be repression of gene expression, while changes in similarly treated PBMCs from subclinical cows was balanced between gene activation and repression. Comparison of gene expression profiles between PBMCs from clinical and uninfected (control) cows stimulated with the general mitogen concanavalin A were highly similar (overall r = 0.84), suggesting that M. paratuberculosis-induced gene repression in clinically infected cow PBMCs was not due to a general failure of the immune response in these animals.

FIG. 1.

Scatter plots of total sample Cy3 (PBS) and Cy5 (M. paratuberculosis) relative fluorescence intensities corrected for GAPDH. (A) Average gene expression intensities for control uninfected cows, with plot of average normalized intensity values for uninfected control animal (n = 3) PBMCs stimulated with PBS (Cy3; y axis) or M. paratuberculosis (Cy5; x axis). RNA isolation, probe labeling, and microarray hybridizations were conducted as described in Materials and Methods. Raw microarray intensity data for the Cy3 and Cy5 channels across three microarrays (one per animal) were normalized using GAPDH control genes, also as described in Materials and Methods. Resulting normalized intensity values were used to derive a mean relative fluorescence intensity value for each gene on the BOTL-2 microarray across all animals. The resulting mean normalized Cy3 (PBS) and Cy5 (M. paratuberculosis) intensity values were plotted, with Cy3 values on the y axis and Cy5 values on the x axis. The correlation (r²) of all spots to the resulting regression line is shown (0.9956). (B) Plot of average gene expression intensities comparing PBS versus M. paratuberculosis stimulation of PBMCs derived from two clinical-stage Johne's disease-positive cows. In this analysis, average gene expression intensities for each gene on the BOTL-2 cDNA microarray were derived as described for panel A using the normalized data from two separate microarrays (one per animal). In this case, the correlation coefficient (r²) derived by regression of average PBS intensities against average M. paratuberculosis intensities was 0.88, and the line was skewed from unity (y = 0.8394x), likely due to the number of genes showing repressed expression following stimulation with M. paratuberculosis relative to simulation with PBS.