Evidence for a novel gene expression program in peripheral blood mononuclear cells from Mycobacterium avium subsp. paratuberculosis-infected cattle

Abstract

A bovine-specific cDNA microarray system was used to compare gene expression profiles of peripheral blood mononuclear cells (PBMCs) from control uninfected (n = 4) and Johne's disease-positive (n = 6) Holstein cows. Microarray experiments were designed so that for each animal, a direct comparison was made between PBMCs stimulated in vitro with Mycobacterium avium subsp. paratuberculosis and PBMCs stimulated with phosphate-buffered saline (nil-stimulated PBMCs). As expected, M. avium subsp. paratuberculosis stimulation of infected cow PBMCs enhanced expression of gamma interferon transcripts. In addition, expression of 15 other genes was significantly affected (>1.25-fold change; P < 0.05) by in vitro stimulation with M. avium subsp. paratuberculosis. Similar treatment of control cow PBMCs with M. avium subsp. paratuberculosis resulted in significant changes in expression of 13 genes, only 2 of which were also affected in PBMCs from the infected cow PBMCs. To compare gene expression patterns in the two cow infection groups (infected cows and uninfected cows), a mixed-model analysis was performed with the microarray data. This analysis indicated that there were major differences in the gene expression patterns between cells isolated from the two groups of cows, regardless of in vitro stimulation. A total of 86 genes were significantly differentially expressed (P < 0.01) in M. avium subsp. paratuberculosis-stimulated PBMCs from infected cows compared to expression in similarly treated PBMCs from control cows. Surprisingly, a larger number of genes (110 genes) were also found to be significantly differentially expressed (P < 0.01) in nil-stimulated cells from the two infection groups. The expression patterns of selected genes were substantiated by quantitative real-time reverse transcriptase PCR. Flow cytometric analysis indicated that there were no gross differences in the relative populations of major immune cell types in PBMCs from infected and control cows. Thus, data presented in this report indicate that the gene expression program of PBMCs from M. avium subsp. paratuberculosis-infected cows is inherently different from that of cells from control uninfected cows.

FIG. 1.

(A) Representative M-A plot of raw cDNA microarray data. In the analysis, data were derived from a cDNA microarray in which RNA from nil (Cy3)-stimulated PBMCs and RNA from M. avium subsp. paratuberculosis (Cy5)-stimulated PBMCs from a Johne's disease-positive cow were compared. Raw cDNA data in the form of relative fluorescence intensity were log transformed and used to calculate M (difference in log intensities) and A (average log intensity) for each spot on the BOTL-3 microarray. Test genes (black circles), blanks and negatives (pink squares), GAPDH (yellow dots), and synthetic lambda Q gene control spots (blue triangles) were plotted as separate series. The red line indicates an M value of zero. (B) M-A plot of LOESS-normalized data. LOESS-corrected values were used to calculate normalized M and A values essentially as described above for panel A. As in panel A, the sample, blank or negative, GAPDH, and lambda Q control genes were plotted as separate series, and the red line indicates an M value of zero. Note the effect of LOESS normalization on the sample M values clustering around the M-0 line (compare this plot with that in panel A). (C) Representative plot of log Cy3 intensity versus log Cy5 intensity before LOESS normalization. Data from panel A were used to calculate log-transformed intensity values for every spot on the BOTL-3 cDNA microarray. Cy3 log intensity values were plotted against Cy5 log intensity values by using a color scheme identical to that described above for panel A. A line of unity was inserted to demonstrate the relationship of sample and control gene log intensity values in an ideal situation, where most points would cluster around the line. (D) Representative plot of log Cy3 intensity versus log Cy5 intensity after LOESS normalization. LOESS-normalized log Cy3 and log Cy5 intensity values were plotted against each other, and a line of unity (log Cy3 intensity = log Cy5 intensity) was inserted to demonstrate the relationship of sample and control gene points in an ideal situation. The colors are as described above for panel A. Note the effect of LOESS normalization both on the clustering of log Cy3-versus-log Cy5 points around the unity line and on the tailing at lower fluorescence intensities observed in the nonnormalized data shown in panel C.

FIG. 2.

Q-RT-PCR validation of cDNA microarray results for PBMCs from infected cows. Genes encoding IFN-γ and MMP 9 were selected for validation of cDNA microarray results by Q-RT-PCR. The IFN-γ gene was selected because enhanced expression of this cytokine is a well-documented effect following M. avium subsp. paratuberculosis stimulation of immune cells from Johne's disease-positive cows. The MMP 9 gene was selected as a representative of genes that exhibit repressed expression in M. avium subsp. paratuberculosis-stimulated PBMCs compared to the expression in nil-stimulated cells and because repression of MMP 9 gene expression was observed in previous studies. Q-RT-PCR was conducted as described in Materials and Methods by using gene-specific primers. Data were analyzed by using the 2^−ΔΔCt method essentially as described previously (21) with β-actin as the control gene and nil stimulation within animal as the calibrator. The data are the means ± standard errors of the means for independent results from four infected cows. MPTb, M. avium subsp. paratuberculosis-stimulated PBMCs.

FIG. 3.

Q-RT-PCR validation of cDNA microarray results for PBMCs from control cows. Genes encoding Sentrin-(SUMO-1), MMP 1, and MMP 23 were selected for Q-RT-PCR validation from among the genes that exhibit differential expression in nil-stimulated and M. avium subsp. paratuberculosis-stimulated PBMCs from control uninfected cows. The gene encoding Sentrin-(SUMO-1) was selected as a representative of genes that exhibit up-regulation in control cow PBMCs stimulated with M. avium subsp. paratuberculosis compared to expression in nil-stimulated cells. The genes encoding MMP 1 and MMP 23 were selected because each was apparently down-regulated by M. avium subsp. paratuberculosis stimulation of control cow PBMCs on cDNA microarrays and because of previous data suggesting that MMP gene regulation is a major and consistent effect of M. avium subsp. paratuberculosis on PBMCs from infected cows (13). For these reasons, an analysis of nil-stimulated and M. avium subsp. paratuberculosis-stimulated PBMCs from infected cows was also included in this study. Q-RT-PCR was conducted as described in Materials and Methods by using gene-specific primers. Data were analyzed by using the 2^−ΔΔCt method essentially as described previously (21) with β-actin as the control gene and nil stimulation within animal as the calibrator. The data are the means ± standard errors of the means for independent results from four infected cows and three control cows. MPTb, M. avium subsp. paratuberculosis.

FIG. 4.

Identification of numerous gene expression changes in both nil-stimulated and M. avium subsp. paratuberculosis-stimulated PBMCs when expression levels were compared across infection groups by using a mixed-model analysis. Data from microarray analysis of PBMCs from six infected cows and four control cows were combined and analyzed as described in Materials and Methods by using a two-stage mixed model in SAS. The resulting least square (LS) means were used to construct interaction tables containing relative expression information and confidence intervals for each gene on the BOTL-3 cDNA microarray. Data were imported into Excel, and the Data Filter command was used to select genes with various expression differences (fold changes) and significance values (P < 0.05, P < 0.01, and P < 0.001). The number of genes in each category was tabulated and used to construct plots. MPTb, M. avium subsp. paratuberculosis.

FIG. 5.

Q-RT-PCR validation of gene expression changes observed following microarray analysis of PBMCs from control and infected cows. Genes to be validated were selected from a list of the genes whose expression was most significantly different following nil stimulation of PBMCs from infected and control cows. Q-RT-PCR was performed as described in Materials and Methods, and data were analyzed by using the 2^−ΔΔCt method with β-actin as the control gene. Mean values for control cow PBMCs with nil stimulation or M. avium subsp. paratuberculosis (MPTb) stimulation were used as calibrators for calculation of all 2^−ΔΔCt values, so that the values for control cows always bracketed 1.0. For Q-RT-PCR analysis, samples were arranged in 96-well PCR plates so that comparisons could be made between PBMCs from infected cows and PBMCs from control cows, each stimulated with M. avium subsp. paratuberculosis or PBS (nil stimulation), on the same plate. The data are means ± standard errors of the means for 2^−ΔΔCt values for three or four infected cows and three or four control cows for each gene.

FIG. 6.

Flow cytometric analysis of major immune cell types in PBMCs from infected and control cows. Aliquots of PBMCs from infected and control cows used in a cDNA microarray analysis were immunostained as described in Materials and Methods to label specific immune cell types, and the relative percentages of each cell type were determined by flow cytometry. The percentage of each immune cell population was determined from density dot plots with side scatter as the y axis and FL-2 (fluorescence of PE) as the x axis. Quadrants were established based on similar plots of cells stained with irrelevant isotype control antibodies to determine the background fluorescence of the cells. The percentage of PE-positive cells was then determined as the percentage of all PBMCs in the upper or lower right quadrants. The data are the means ± standard errors of the means for six infected cow PBMC preparations and four control cow preparations for each cell type.