Project description:Purpose: The goal of this RNA-seq study was to profile changes in miRNA expression in bovine alveolar macrophages isolated from Holstein-Friesian and challenged with M. bovis over a 48 hour time course. Background: Mycobacterium bovis, the causative agent of bovine tuberculosis, is a major infectious disease problem for global agriculture, resulting in losses of billions of Euros annually. The bacterium spreads via an airborne route and is taken up by alveolar macrophages in the lung. MicroRNAs (miRNAs) — short non-coding RNAs that post-transcriptionally regulate gene expression — are increasingly being recognised as key regulators of the immune system. There is mounting evidence for the role of miRNAs in regulating the early innate immune response to mycobacterial infections; however, the underlying processes are not fully understood yet. Results: In total, 2.5 billion raw sequencing reads were generated. 93% of post-filtered reads aligned to known miRNAs. The expression of miRNAs varied over a wide range: miR-21 was expressed at approximately 430,000 reads per million (RPM), but the majority were more lowly expressed (1000-10,000 RPM). One, six, and 40 miRNAs were identified as significantly differentially expressed at 2, 24 and 48 h post-infection (hpi), respectively (FDR < 0.05); the differential expression at 48 hpi of three (out of four) of these miRNAs was confirmed by RT-qPCR. No miRNAs were detected to be differentially expressed 6 hpi. Many of the differentially expressed miRNAs have been shown to have a role in the host response to mycobacterial pathogens and other infections in other species. MicroRNA-155, for example, which we observed to be upregulated in response to M. bovis has recently been shown to control autophagy in murine macrophages infected with the human pathogen Mycobacterium tuberculosis. Pathway analysis suggests that upregulated miRNAs target endocytosis and lysosome trafficking, thus potentially enhancing bacterial survival. An independent gene silencing experiment with a bovine macrophage cell line (Bomac) has confirmed downregulation of IRAK1 by miR-146a and miR-146b. Moreover, downregulation of another predicted target, TGFRB2, by miR-146a was also confirmed. These findings support the target prediction and pathway analysis results. Our results suggest that several miRNAs have an important role in the alveolar macrophage response to pathogenic mycobacteria.

Project description:Background: Mycobacterium bovis, the causative agent of bovine tuberculosis, is a major cause of mortality in global cattle populations. Macrophages are among the first cells 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. Results: Comparison of M. bovis-challenged MDM gene expression profiles with the non-challenged MDM controls at each time point identified 3,529 differentially expressed genes after 2 hours post-challenge, with 5,211 and 6,150 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 larger 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 (PRRs)-receptors; and (3) apoptosis. Conclusions: The 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 important roles for MYD88-independent signalling and intracellular PRRs in mediating the host response to M. bovis, which to our knowledge have not been reported previously. Affymetrix GeneChip® Bovine Genome Arrays were used to examine gene expression of bovine monocyte-derived macrophages (MDM) after in vitro challenge with Mycobacterium bovis across a time series of 2 hr, 6 hr and 24 hr post-challenge. A 0 hr control treatment was also generated and seven different age-matched female Holstein-Friesian cattle were used for each time-point/treatment combination for a total of 49 microarrays.

Project description:Background: Mycobacterium bovis, the causative agent of bovine tuberculosis, is a major cause of mortality in global cattle populations. Macrophages are among the first cells 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. Results: Comparison of M. bovis-challenged MDM gene expression profiles with the non-challenged MDM controls at each time point identified 3,529 differentially expressed genes after 2 hours post-challenge, with 5,211 and 6,150 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 larger 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 (PRRs)-receptors; and (3) apoptosis. Conclusions: The 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 important roles for MYD88-independent signalling and intracellular PRRs in mediating the host response to M. bovis, which to our knowledge have not been reported previously.

Dataset Information

Early response of bovine alveolar macrophages to infection with live and heat-killed Mycobacterium bovis

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