Age-related changes following in vitro stimulation with Rhodococcus equi of peripheral blood leukocytes from neonatal foals

Abstract

Rhodococcus equi is an intracellular bacterium primarily known as an equine pathogen that infects young foals causing a pyogranulomatuous pneumonia. The molecular mechanisms mediating the immune response of foals to R. equi are not fully elucidated. Hence, global genomic high-throughput tools like gene expression microarrays might identify age-related gene expression signatures and molecular pathways that contribute to the immune mechanisms underlying the inherent susceptibility of foals to disease caused by R. equi. The objectives of this study were 2-fold: 1) to compare the expression profiles at specific ages of blood leukocytes from foals stimulated with virulent R. equi with those of unstimulated leukocytes; and, 2) to characterize the age-related changes in the gene expression profile associated with blood leukocytes in response to stimulation with virulent R. equi. Peripheral blood leukocytes were obtained from 6 foals within 24 hours (h) of birth (day 1) and 2, 4, and 8 weeks after birth. The samples were split, such that half were stimulated with live virulent R. equi, and the other half served as unstimulated control. RNA was extracted and the generated cDNA was labeled with fluorescent dyes for microarray hybridizations using an equine microarray. Our findings suggest that there is age-related differential expression of genes involved in host immune response and immunity. We found induction of genes critical for host immunity against pathogens (MHC class II) only at the later time-points (compared to birth). While it appears that foals up to 8-weeks of age are able to initiate a protective inflammatory response against the bacteria, relatively decreased expression of various other immune-related genes points toward inherent diminished immune responses closer to birth. These genes and pathways may contribute to disease susceptibility in foals if infected early in life, and might thus be targeted for developing preventative or therapeutic strategies.

Figure 1. Sample hybridization scheme for microarrays.

a. Pairwise hybridization of stimulated (with live, virulent R. equi) and unstimulated samples was done at each time-point; b. Common reference design to hybridize samples from W-2, W-4, and W-8, with D-1 acting as the reference.

Figure 2. Real-time PCR validation of microarray results.

The y-axis represents the fold-change of the selected genes for validation using real-time PCR (white bars) and microarray (black bars). The Y-axis represents fold-change of selected genes by real-time PCR (delta-delta Ct method) and by microarray (normalized log2 (Cy5/Cy3)). * represents significant (P<0.05) difference in fold-change of IFN-γ at W-8 compared to D-1. IFN-γ has no corresponding black bar representing microarray data, as this gene was not printed on the microarray. The error bars indicate standard error of the mean (SEM) which was calculated as SEM = SD/√n, where SD represents standard deviation and n is the sample size.

Figure 3. Venn diagram for pairwise comparison of the gene expression profile of stimulated versus unstimulated leukocytes.

The 4 time-points are designated in the diagram as 1: day 1, 2: week 2, 3: week 4, 4: week 8. The letters represent: a-genes found in 1 only; b-genes found in 2 only; c-genes found in 3 only; d-genes found in 4 only; e-genes in 1&2 only; f-genes in 1&3 only; g-genes in 2&3 only; h-genes in 1&4 only; i-genes in 2&4 only; j-genes in 3&4 only; k-genes in 1,2,3 only; l-genes in 1,2,4 only; m-genes in 1,3,4 only; n-genes in 2,3,4 only; and, o-genes in 1,2,3, and4. *Only genes with an annotated accession were used for generating this figure.

Figure 4. Venn diagram for temporal gene expression changes.

This Venn diagram depcts DE genes unique to each comparison: day1-week2 (C1); day1-week4 (C2); and, day1-week8 (C3). *Only genes with an annotated accession were used for generating this figure.