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RNA-Seq profiling of blood and jejunal samples from subclinical Holstein cattle naturally infected with Mycobacterium avium subsp. paratuberculosis revealed host transcriptional adaptations to a latent MAP infection

ABSTRACT: Bovine paratuberculosis (PTB), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic granulomatous intestinal disease that leads to substantial economic losses in the global dairy industry. Current diagnostic tests have limited sensitivity, as they can reliably detect only animals that have advanced stages of disease characterized by diffuse lesions and with the presence of clinical signs, but fail to identify those in earlier or subclinical stages, with focal or multifocal lesions in gut tissues. Previous studies have suggested that multifocal granulomas prevent lesions progression, but the molecular mechanisms involved in the establishment and maintenance of a chronic MAP infection are not fully understood. This study aimed to compare the whole transcriptomic profiles of peripheral blood (PB), jejunum (JE), and jejunal lymph node (JELN) samples from Holstein cattle with multifocal lesions and those without lesions in gut tissues. Total RNA was extracted from samples from 11 cows with multifocal lesions and 4 control cows without lesions. RNA libraries were prepared using 250 ng of RNA with the Illumina NEBNext Ultra Directional RNA library preparation kit and sequenced on an Illumina NovaSeq sequencer. On average, 34.08, 33.85, and 36.92 million raw reads were sequenced from the PB, JE and JELN samples, respectively. In cows with multifocal lesions compared to controls, 1,272 differentially expressed (DE) genes were identified in PB, mainly linked to translation, RNA splicing, and mRNA processing. In JE, 7 genes were DE and related to metabolic processes, oxidative stress response, and granulocyte migration. In JELN, 18 genes were DE and associated with the mTOR signaling, response to reactive oxygen species and hypoxia, and regulation of autophagy. The protein-to-protein interaction (PPI) analysis revealed that several differentially expressed genes are highly interconnected and associated with pathways related to splicing and translation inhibition, as well as activation of a robust innate immune response in peripheral blood. Overall, this study demonstrates that MAP persistence with multifocal granulomas results from a complex interplay of host innate immune activation and regulation, autophagy suppression, translational reprogramming, and metabolic adaptation, providing new insights into MAP pathogenesis and identifying potential biomarkers or therapeutic targets.

ORGANISM(S): Bos taurus

PROVIDER: GSE313938 | GEO | 2026/02/18

REPOSITORIES: GEO

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Project description:Purpose: Since the peripheral blood (PB) is the most available physiological fluid for the detection of biomarkers, in the current we examine whether it recapitulates, at least in part, the transcriptome of the ileocecal valve (ICV), the primary site of MAP colonization. For this purpose, RNA-Seq was used to identify host genes differentially expressed (DE) in ICV and PB samples collected from PTB-infected animals with focal or diffuse lesions in gut tissues versus control animals. Methods: RNA-Seq libraries were single-end sequenced in a 1x75 format using an Illumina NextSeq 500 sequencer. The raw reads were filtered by their length (minimum size 60 bp long) and percentage of ambiguous base N less than 5 % using Prinseq-lite. Trimmed reads were subsequently mapped to the Bos Taurus reference genome (Bos_taurus.UMD3.1. version 87) with TopHat mapper. The resulting alignment files were provided to Cufflinks to generate a transcriptome assembly for each condition. These assemblies were then merged together using Cuffmerge, which is included in the Cufflinks package. This merged assembly provided a uniform basis for calculating gene and transcript expression levels in each condition. The reads and merged assembly were fed to Cuffdiff which calculates expression levels and tested the statistical significance of each observed change in expression. The fold change (in log2 scale), P-values and false discovery rates (FDR) for each gene were obtained. The genes with a FDR-adjusted threshold ˂ 0.05 were considered differentially expressed (DE) when compared to the control group. Results: Our results demonstrated both shared, and PB and ICV-specific gene expression in response to a natural Map infection. As expected, the number of differentially expressed (DE) genes was larger in the ICV than in the PB samples. Among the DE genes in the PB and ICV samples, there were some common genes irrespective of the type of lesion including the C-X-C motif chemokine ligand 8 (CXCL8/IL8), apolipoprotein L (APOLD1), and the interferon inducible protein 27 (IFI27). The biological processes (BP) enriched in the ICV gene expression profiles from the cows with focal lesions included the killing of cells of other organism, defense response, immune response and the regulation of neutrophil chemotaxis. Two of these BP, the defense and immune response, were also enriched in the ICV and PB from the cows with diffuse lesions. Metabolic analysis of the DE genes revealed that the N-glycan biosynthesis, bile secretion, one-carbon pool by folate and purine metabolism were significantly enriched in the ICV from the cows with focal lesions. In the ICV from cows with diffuse lesions; the valine, leucine and isoleucine degradation route, purine metabolism, vitamin digestion and absorption and the cholesterol routes were enriched Conclusions: This study is the first to simultaneously describe transcriptomic changes in PB and ICV samples of cattle naturally infected with MAP. Several important observations were made. First, by comparing the transcriptomic profiles of two MAP-targeted tissues we described both unique and overlapping changes in the transcriptome of the infected cows versus the control group. Second, our study highlighted the ability of the RNA-Seq technology to reveal roles for genes that have not been previously implicated in the host response to MAP infection. Third, our transcriptomic analysis provided potential biomarkers for the development of future diagnostic tools, vaccines and therapeutics.

Project description:Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne’s disease (JD), which is also known as paratuberculosis, in ruminants. The mechanisms of JD pathogenesis are still unclear, but it is known that MAP subverts the host immune system for years by using macrophages as its primary reservoir. The effect of MAP infection on macrophages is often studied in healthy or experimentally infected cows, but reports on primary macrophages from naturally infected cows are lacking. In our study, the transcriptome of primary monocyte-derived macrophages challenged with live MAP was analyzed using next-generation sequencing (RNA‑seq). The gene expression signatures of macrophages from cows diagnosed as negative or positive for JD [JD(–) or JD(+), respectively] revealed differential host–pathogen interplay, highlighting long-term mechanisms established during mycobacterial disease. In JD(–) cows, a considerable number of genes (1,436) were differentially expressed at 8 h post-infection (hpi). Interestingly, while expected inflammatory immune response pathways were among the significant pathways, others related to Hepatic fibrosis/hepatic stellate cell activation, lipid homeostasis, such as the LXR/RXR [liver X receptor/retinoid X receptor] activation, and autoimmune diseases such as atherosclerosis and rheumatoid arthritis were consistently among the top significant pathways. Unexpectedly, the macrophages from JD(+) cows did not present a clear response pattern to ex vivo MAP infection, neither during the early period nor at 24 hpi. Analyzing of the transcriptomic profile of the uninfected macrophages, revealed 868 genes that were differentially expressed in JD(+) versus JD(–) cows. The down-regulated genes particularly modified the general cell metabolism by down-regulating amino acid synthesis, cholesterol biosynthesis, and other energy production pathways while introducing a pro-fibrotic pattern associated with foam cells. These modifications show the apparent importance of the metabolic pathways hijacked by MAP to subvert the immune cells. Our findings support the hypothesis that MAP could induce a tolerant-like state in circulating peripheral blood monocytes when they are differentiating into macrophages, which could further promote the persistence. Similar to other mycobacteria and pathogen-associated molecular patterns, MAP could influence macrophage behavior in the long term. This report contributes to a better understanding of MAP control of immune cells and its mechanisms of survival.

Project description:An evaluation of multifocal lesions from patients with in-transit extremity melanoma to determine if all lesions from a patient harbor homogeneous patterns of gene expression; Gene expression profiling studies can help guide treatment for cancer patients by providing tools in the form of gene-expression signatures to characterize a tumor in terms of underlying biology, predicted response to therapy, metastatic progression and/or recurrence. The utility of gene signatures for defining therapeutic strategies in the treatment of extremity in-transit melanoma will be dependent on the genetic relationship between the multifocal lesions typically present in this disease and the extent to which a single lesion is representative of residual tumor burden. Using microarray-based gene expression profiling we examined 43 in-transit melanoma lesions across 17 patients with multifocal disease to determine whether one lesion could accurately characterize the underlying biology and genetic profile of a patient's tumor. Principal component analysis, unsupervised hierarchical clustering, one-way analysis of variance (ANOVA) and gene signatures predictive of chemosensitivity and oncogenic pathway activation showed gene expression patterns to be highly similar (p-values: <0.006; average r = 0.979) between lesions from a single patient but to be significantly different across patients (p<0.05). These findings demonstrate that individual melanoma tumor nodules in patients with multifocal disease are genetically similar and a single lesion can be used to predict response to chemotherapy, evaluate the activation status of oncogenic signaling pathways and characterize other aspects of the biology of an individual patient's disease. These results will facilitate the utilization of gene expression profiling in clinical trials of targeted therapy in melanoma allowing for more rational identification of candidates for specific therapies. Experiment Overall Design: Gene expression profiles were obtained from 43 lesions across 17 patients and evaluated using several different algorithms to determine the degree of correlation across multifocal lesions and to evaluate patterns of gene expression that are different across patients

Project description:Giant cell granulomas of the jaws often occur sporadically as single central or peripheral lesions. They are characterized by KRAS, FGFR1, or TRPV4 somatic mutations, the latter occurring exclusively in the central form. Less commonly, multiple giant cell lesions can develop in the context of syndromes such as cherubism, which is an autosomal dominant bone disease. Morphologically, giant cell granulomas can closely resemble other giant cell-rich lesions such as non-ossifying fibroma and aneurysmal bone cyst, and to a minor extent giant cell tumour of bone and chondroblastoma. The epigenetic basis of these giant cell-rich tumours is unclear and, recently, DNA methylation profile has been shown to be clinically useful for the diagnosis of other tumour types, including brain tumours as well as bone and soft tissue sarcomas. Therefore, we aimed to assess the DNA methylation profile of central and peripheral sporadic giant cell granulomas of the jaws and cherubism to test whether DNA methylation patterns can help to distinguish these entities. Additionally, we further compared the DNA methylation profile of these lesions with those of other giant cell-rich mimics to investigate if the microscopic similarities extend to the epigenetic level. Our results showed that central and peripheral sporadic giant cell granulomas of the jaws and cherubism share a related DNA methylation pattern with that of peripheral sporadic giant cell granulomas and cherubism appearing slightly distinct, while central sporadic giant cell granulomas show overlap with both of the former. Non-ossifying fibroma, aneurysmal bone cyst, giant cell tumour of bone, and chondroblastoma, on the other hand, have distinct methylation patterns. Therefore, DNA methylation profiling is currently not capable of clearly distinguishing sporadic and cherubism-associated giant cell lesions of the jaws. Conversely, it could discriminate sporadic giant cell granulomas from their giant cell-rich mimics.

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RNA-Seq profiling of blood and jejunal samples from subclinical Holstein cattle naturally infected with Mycobacterium avium subsp. paratuberculosis revealed host transcriptional adaptations to a latent MAP infection

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