Unknown,Transcriptomics,Genomics,Proteomics

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Global gene expression 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 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.

ORGANISM(S): Bos taurus

SUBMITTER: David MacHugh

PROVIDER: E-GEOD-33309 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Mycobacterium bovis (M. bovis) causes bovine tuberculosis (bTB). The challenges in controlling and eradicating this zoonotic disease are compounded by our incomplete understanding of the host immune response. In this study, we used high-throughput bulk RNA sequencing (RNA-seq) to characterise the response profiles of γδ T cells to antigenic stimulation using purified protein derivate from M. bovis (PPDb). γδ T cells are a subgroup of T cells that bridge innate and adaptive immunity and have known anti-mycobacterial response mechanisms. These cells are usually classified based on the expression of a pathogen-recognition receptor, Workshop Cluster 1 (WC1), into two main subsets: WC1.1+ and WC1.2+. Previous studies have identified a preferential transcriptomic response in WC1.1+ cells during natural bTB infection, suggesting a subset-specific response to mycobacterial antigens. This follow on study tested the hypothesis that a subset specific response would also be apparent from γδ T cells from infected cattle after repeat stimulation. Peripheral blood was collected from Holstein-Friesian cattle naturally infected with M. bovis, confirmed by a single intradermal comparative tuberculin test (SICTT) and IFN-γ ELISA and stimulated with 10 μg/ml PPDb for 6 hours. After whole blood stimulation, WC1.1+ and WC1.2+ γδ T cell subsets were isolated using magnetic cell sorting (n = 5 per group). High-quality RNA was extracted from each purified lymphocyte subset (WC1.1+ and WC1.2+) to generate transcriptomes using bulk RNA sequencing, resulting in 20 RNA-seq libraries. Transcriptomic analysis revealed 111 differentially expressed genes (DEGs) common to both WC1.1+ and WC1.2+ γδ T cell compartments, including upregulation of IL1A, IL1B, IL6, IL17A, IL17F, and IFNG genes (FDR-Padj. < 0.1). Interestingly, the WC1.2+ cells showed upregulation of IL10, CCL22, and GZMA (log2FC ≥ 1.5, and FDR-Padj. < 0.1). In conclusion, while WC1.1+ and WC1.2+ γδ T cells exhibit a conserved inflammatory response to PPDb, differences in anti-inflammatory and antimicrobial gene expression between these cell subsets provide new insights into their effector functions in response to mycobacterial antigens.

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.

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Global gene expression of bovine monocyte-derived macrophages in response to in vitro challenge with Mycobacterium bovis

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