Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Extensive Differential DNA Methylation in Bovine Tuberculosis Skin Test Positive and Skin Test Negative Cattle

ABSTRACT: Bovine tuberculosis (bTB), caused by Mycobacterium bovis, represents a significant issue for the global agriculture industry as well as for human health. Epigenetic modifications can alter the course of the immune response and differentially methylated regions (DMRs) could contribute to the failure of current generation tests to detect all TB-infected cattle. Whole Genome Bisulphite Sequencing (WGBS) was used to profile DNA methylation levels from peripheral blood of cattle naturally infected with M. bovis (positive for the single intradermal comparative tuberculin test (SICTT) and/or the interferon-γ release assay (IGRA) compared to negative controls [n=8/group, total of 16 WGBS libraries]. Although overall methylation profiles were similar across the genome, 224 DMRs and 159 Differentially Methylated Promoter Gene (DMPGs) were identified between groups with an excess of hypermethylated sites in bTB+ infected cattle (threshold >15% differential methylation). Genes located within these DMRs included the Interleukin 1 receptor (IL1R1) and MHC related genes (BOLA and BOLA-DQB) which may influence effective immunity. KEGG pathway analysis identified enrichment of genes involved in Calcium and MAPK signalling, as well as metabolism pathways. Analysis of DMRs in a subset of SICTT- cattle (n=4 group) which were IGRA+, and thereby potentially represent a risk for disease recurrence on farms, showed differential methylation of genes including Interleukin 10 Receptor, alpha (IL10RA), Interleukin 17F (IL17F) and host defence peptides (DEFB and BDEF109). This study has identified a number of immune gene loci at which differential methylation could impact on the host immune response and the ability of cattle to clear infection. Differential methylation of immune gene loci is an informative avenue to improve our understanding of the regulation of host immunity as well as the role of methylation on diagnostic test performance.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Bos taurus

SUBMITTER: Alia Parveen

PROVIDER: E-MTAB-13487 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Abstract: Bovine tuberculosis (bTB), caused by infection with Mycobacterium bovis, continues to cause significant issues for the global agriculture industry as well as for human health. An incomplete understanding of the host immune response contributes to the challenges of control and eradication of this zoonotic disease. In this study, high-throughput bulk RNA sequencing (RNA-seq) was used to characterize differential gene expression in γδ T cells – a subgroup of T cells which bridge innate and adaptive immunity and have specific anti-mycobacterial response mechanisms. γδ T cell subsets are classified on the basis of expression of a pathogen-recognition receptor known as Workshop Cluster 1 (WC1) and we hypothesised that bTB infection may alter the phenotype and function of specific γδ T cell subsets. Peripheral blood was collected from naturally M. bovis-infected (positive for single intradermal comparative tuberculin test (SICTT) and IFN-γ ELISA) and age- and sex-matched, non-infected control Holstein-Friesian cattle. γδ T subsets were isolated using fluorescence activated cell sorting (n = 10-12 per group) and high-quality RNA extracted from each purified lymphocyte subset (WC1.1+, WC1.2+, WC1- and γδ-) was used to generate transcriptomes using bulk RNA-seq (n = 6 per group, representing a total of 48 RNA-seq libraries). Relatively low numbers of differentially expressed genes (DEGs) were observed between most cell subsets; however, 163 genes were significantly differentially expressed in the M. bovis-infected compared to control groups for the WC1.1+ γδ T cell compartment (Log 2 FC ≥ 1.5 and FDR P adj ≤ 0.1). The majority of these DEGs (146) were significantly increased in expression in cells from the bTB+ cattle and included genes encoding transcription factor (TBX21 and EOMES), chemokine receptors (CCR5 and CCR7), granzymes (GZMA, GZMM and GZMH) and multiple killer cell immunoglobulin-like receptors (KIR) indicating cytotoxic functions. Biological pathway overrepresentation analysis revealed enrichment of genes with multiple immune functions including cell activation, proliferation, chemotaxis and cytotoxicity of lymphocytes. In conclusion, WC1.1+ γδ T cells have been proposed as major regulatory cell subset in cattle, and we provide evidence for preferential differential activation of this specific subset in cattle naturally infected with M. bovis. Understanding the role of these critical immune cells during mycobacterial infection contributes to our understanding of host immunity to bTB and identifies multiple novel cytotoxic functions of WC1.1+ γδ T cells.

Project description:Background: Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), a pathological infection with significant economic impact. Recent studies have highlighted the role of functional genomics to better understand the molecular mechanisms governing the host immune response to M. bovis infection. Furthermore, these studies may enable the identification of novel transcriptional markers of BTB that can augment current diagnostic tests and surveillance programmes. In the present study, we have analysed the transcriptome of peripheral blood leukocytes (PBL) from eight M. bovis-infected and eight control non-infected age-matched and sex-matched Holstein-Friesian cattle using the Affymetrix® GeneChip® Bovine Genome Array with features representing more than 23,000 gene transcripts and over 19,000 gene probe sets. Results: Control and infected animals had similar mean white blood cell counts. However, the mean number of lymphocytes was significantly increased in the infected group relative to the control group (P = 0.001), while the mean number of monocytes was significantly decreased in the BTB group (P = 0.002). Hierarchical clustering analysis using gene expression data from all 5,388 detectable mRNA transcripts unambiguously partitioned the animals according to their disease status. In total, 2,960 gene transcripts were differentially expressed (DE) between the infected and control animal groups (adjusted P-value threshold ≤ 0.05); with the number of genes showing decreased relative expression (1,563) exceeding those displaying increased relative expression (1,397). Systems analysis using the Ingenuity Systems Pathway Analysis (IPA) Knowledge Base revealed an over-representation of DE genes involved in the immune response functional category. More specifically, 64.5% of genes in the affects immune response subcategory displayed decreased relative expression levels in the infected animals compared to the control group. Conclusions: This study demonstrates that genome-wide transcriptional profiling of PBL can distinguish active M. bovis-infected animals from control non-infected animals. Furthermore, the results obtained support previous investigations demonstrating that mycobacterial infection is associated with host transcriptional suppression. These data support the use of transcriptomic technologies to enable the identification of robust, reliable transcriptional markers of active M. bovis infection Affymetrix GeneChip® Bovine Genome Arrays were used to examine gene expression of peripheral blood leukocytes from cattle infected with Mycobacterium bovis

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.

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Extensive Differential DNA Methylation in Bovine Tuberculosis Skin Test Positive and Skin Test Negative Cattle

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