Global Gene Expression and Systems Biology Analysis of Bovine Monocyte-Derived Macrophages in Response to In Vitro Challenge with Mycobacterium bovis

Abstract

Background Mycobacterium bovis, the causative agent of bovine tuberculosis, is a major cause of mortality in global cattle populations. Macrophages are among the first cell types to encounter M. bovis following exposure and the response elicited by these cells is pivotal in determining the outcome of infection. Here, a functional genomics approach was undertaken to investigate global gene expression profiles in bovine monocyte-derived macrophages (MDM) purified from seven age-matched non-related females, in response to in vitro challenge with M. bovis (multiplicity of infection 2∶1). Total cellular RNA was extracted from non-challenged control and M. bovis-challenged MDM for all animals at intervals of 2 hours, 6 hours and 24 hours post-challenge and prepared for global gene expression analysis using the Affymetrix® GeneChip® Bovine Genome Array.ResultsComparison of M. bovis-challenged MDM gene expression profiles with those from the non-challenged MDM controls at each time point identified 3,064 differentially expressed genes 2 hours post-challenge, with 4,451 and 5,267 differentially expressed genes detected at the 6 hour and 24 hour time points, respectively (adjusted P-value threshold ≤0.05). Notably, the number of downregulated genes exceeded the number of upregulated genes in the M. bovis-challenged MDM across all time points; however, the fold-change in expression for the upregulated genes was markedly higher than that for the downregulated genes. Systems analysis revealed enrichment for genes involved in: (1) the inflammatory response; (2) cell signalling pathways, including Toll-like receptors and intracellular pathogen recognition receptors; and (3) apoptosis.ConclusionsThe increased number of downregulated genes is consistent with previous studies showing that M. bovis infection is associated with the repression of host gene expression. The results also support roles for MyD88-independent signalling and intracellular PRRs in mediating the host response to M. bovis.

Transcriptome changes upon in vitro challenge with Mycobacterium bovis in monocyte-derived macrophages from bovine tuberculosis-infected and healthy cows

“…Of the 16,793 total genes presented on the chips, 2269 genes have false discovery rate (FDR) ≤0.01, p < 0.01, and fold change >2, in which we found that 1103 (2184 probes) were upregulated and 1166 (2318 probes) were downregulated. With such stringent criteria, M. bovis challenge was found to cause significant fold changes in thousands of genes, which might be attributed to the fact that the MOI used in this study was higher than that used by other researchers (MOI, 10 vs. 2) (Magee et al, 2012). CSF3, CCL20, and CSF2 were the top upregulated genes (+11.47, +11.04, and +9.81 in log 2, respectively; Table 1), whereas killer cell lectin-like receptor-like (LOC618591), RAS guanyl releasing protein 3 (calcium and DAG-regulated) (RASGRP3), and solute carrier family 1 (glial high affinity glutamate transporter), member 3 (SLC1A3) were the top downregulated genes (−7.14,−6.03, and −6.01 in log 2, respectively; A recent study by Magee et al (2012) addressed the same question using M. bovis at an MOI of 2 to challenge bovine MDMs and studied transcription at 6 h post infection.…”

“…Pathway analysis using different databases produced significant redundancy and unrelated functions; although this may simply be due to the large number of possible pathways, it is more likely that M. bovis challenge may lead to changes that are much more complicated than inflammatory responses and apoptosis. For example, activation of the RIG-I-like receptor pathway (23th) by viral RNA was shown previously (Magee et al, 2012). Intracellular Legionella pneumophila interact with RIG-I-like receptors (Monroe et al, 2009), as well; thus, it is possible that M. bovis also activates this pathway.…”

“…Other research groups have used high-throughput microarrays for global transcriptome analysis of macrophages or peripheral blood mononuclear cells (PBMC) from healthy and infected cows to identify key genes and pathways activated by in vitro M. bovis challenge (Magee et al, 2012). Our previous work indicated that macrophages from BTB-positive and healthy cows had distinct immune responses to in vitro challenge (Wang et al, 2011).…”

Genomic scan for identifying candidate genes for paratuberculosis resistance in sheep

“…2); another important category (37%) included the genes involved in metabolic processes and ion transport. Also Meade et al (2007), Magee et al (2012) and Nalpas et al (2013) have performed gene expression screenings, with DNA microarrays containing immunity genes, on peripheral blood mononuclear cells from non-infected versus M. bovisinfected cattle. Meade et al (2007) reported the lower relative expression of key innate immune genes in the infected animals.…”

“…Meade et al (2007) reported the lower relative expression of key innate immune genes in the infected animals. Magee et al (2012) revealed a differential expression for genes involved in: (1) the inflammatory response; (2) cell signalling pathways, including TLR and intracellular pathogen recognition receptors; and (3) apoptosis. Nalpas et al (2013) demonstrated the differential expression of immune, apoptotic and cell signalling genes in the non-infected versus infected animals.…”