Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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MiRNA profiling of sarcoidosis Treg cells

ABSTRACT: Sarcoidosis is characterized by exaggerated immune response to unknown agent and can affect different organs. One of the main players in the pathology of the disease are regulatory T cells (Tregs), however up to date there are no insides on the possible molecular alterations of this particular cell subset. Therefore, in the current study we looked for the global changes on miRNA and mRNA level of Tregs of patients with the most predominant form of the disease - pulmonary sarcoidosis (PS). For this purpose we sorted Treg cells from peripheral blood (PB) and bronchoalveolar lavage (BAL) and analyzed the global molecular changes. MiRNA analysis showed major changes in Tregs isolated from PB to the ones from BAL, pointing at the stronger impact of the microenvironment than the disease state. Additionally, several disease-related miRNAs of PB Tregs were identified like miR-155 and miR-223.

INSTRUMENT(S): Qubit, OpenArray NT cycler, PCR Applied Biosystems

ORGANISM(S): Homo sapiens

SUBMITTER: Neli Kachamakova-Trojanowska

PROVIDER: E-MTAB-6590 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Human milk (breastmilk) is much more than nutrition for the infant, containing an array of regulatory agents with immunoprotective and developmental functions. Amongst those, microRNAs (miRNAs) have recently been identified, with their properties, roles, origin and distribution in breastmilk as well as in the mammary gland being still undetermined. In this study, we examined the miRNA profile of different fractions of human milk (cells and lipids) using the OpenArray system (Applied Biosystems, 770 miRNA species measured per sample) and compared it with maternal peripheral blood mononuclear cells (PBMCs) and plasma. Although PBMCs were the richest group in miRNA species, plasma showed very low expression pattern. Thus, the human milk fractions (cells, lipid) and skim milk (not being investigated in this study) were found to conserve higher levels of miRNAs than blood in general. Specifically, human milk cell miRNA quantity was found relatively close to PBMCs, and higher than milk lipids. Correlation and clustering analyses indicated that miRNA expression and types of milk cells were highly similar to those in lipids. Milk miRNAs showed a slight correlation to PBMCs, so PBMCs potentially are not contributing to milk miRNAs. Plasma was different to all other three groups in miRNA content and expression pattern. Further, two infant formulae (a plant-based and a cow milk-based) were compared to human milk and found to contain significantly fewer miRNA species than human milk cells and lipids (p>0.001). Taken together with previous studies on miRNAs, our findings demonstrate that human milk is one of the richest sources of miRNAs among human body fluids. As a non-invasive and plentiful source of miRNAs, human milk could be used as a disease biomarker for the mammary gland, with potential in assessing lactation performance. Finally, gene target and pathways analyses identified several target mRNAs regulated by miRNAs found to be abundant in breastmilk. Given the recently identified stability and function of food-derived miRNAs in regulating mammalian genes, we propose that breastmilk is a rich source of miRNA ingested by the infant during the first months of life, and which potentially contribute to early infant development. 10 exclusively breastfeeding dyads were recruited. 10 whole milk and 10 whole blood samples were collected and fractionated to obtain 10 milk cells, 10 milk lipid, 10 mononeucleoted blood cells (PBMCs), and 10 plasma. In addition to the above 40 samples, 2 infant formula were profiled. 4 different extraction kits were used, miRNeasy mini Kit for human milk cell and PBMC samples. miRCURY RNA Isolation-Biofluids Kit for human milk lipid samples and both infant formulae. mirVana PARIS Kit for plasma samples. NanoDrop 2000 and Bioanalyzer 2100 were used to determine concentration and purity of the extracted miRNA from all samples (n=42). miRNA OpenArray panel system (Life Technologies, CA, USA) was used to profile 754 human mature miRNAs in samples. RNU48, RNU44 and U6 rRNA were used as housekeeping controls for normalisation. ath-miR159a was used as a negative control for human samples. GeneGO and Ingenuity Pathway Analysis were used to determine biological pathways. Please note that normalization of miRNAs was done in R but without generating deltaCT values, thus [1] only the list of normalized miRNA with Ct vlaue between 8 and 29 and that detected in at least 4 samples out of 10 analysed in each group is provided ('normalized_miRNAs_list.txt') [2] the sample data tables contain raw data.

Project description:Rifampicin and efavirenz are commonly used medicines to treat tuberculosis (rifampicin) and HIV/AIDS (efavirenz). Rifampicin and efavirenz are metabolized in the liver and exposure to these medicines can alter microRNA expression in hepatocytes. Changes in microRNA expression may affect metabolism of these medicines and potentially influence how patient's respond to therapy. It is important to understand changes in microRNA expression in a hepatic cell-based model. Differentiated HepaRG cells were used because mRNA expression and induction of drug metabolizing enzymes are comparable to that of primary human hepatocytes yet differentiated HepaRG cells have an extended lifespan. Differentiated HepaRG cells were obtained from Merck Millipore and cultured as an adherent cell line at a density of 315000 cells/well using collagen I coated 24-well plates. Clinically relevant concentrations of rifampicin and efavirenz were selected as 6.4uM efavirenz (dissolved in 100% dimethyl sulfoxide) and 24.4uM rifampicin (dissolved in 100% dimethyl sulfoxide), while 0.02% dimethyl sulfoxide was used as control to minimize toxicity. HepaRG serum-free induction medium was used to dilute efavirenz and rifampicin. On day 7 since thawing differentiated HepaRG cells, these cells were treated with 6.4uM efavirenz or 24.4uM rifampicin or 0.02% dimethyl sulfoxide for a period of 24 hours. Three biological replicates were available for each treatment condition. Total RNA was isolated from the cells, after treatment, using the Quick-RNATM MiniPrep Kit from Zymo Research Corporation. MicroRNA expression profiling was performed using the TaqMan® OpenArray® Human MicroRNA Panel and QuantStudioTM 12K Flex system. The R/Bioconductor package “Automated Analysis of High-Throughput qPCR Data” were used for data analyses. CT values were used for differential expression analysis and microRNAs with a CT value > 35 was considered undetected. MicroRNAs undetected in any of the replicate samples or microRNAs with AmpScore < 1.24 or CqConf < 0.8 were excluded. Quantile normalization was followed by limma analyses to identify differentially expressed microRNAs for efavirenz versus dimethyl sulfoxide and rifampicin versus dimethyl sulfoxide.

Project description:Introduction: Sarcoidosis is a multisystem immuno-inflammatory disorder of unknown etiology that most commonly involves the lungs. We hypothesized that an unbiased approach to identify pathways activated in alveolar macrophagesâa key immuno-inflammatory cell in the lungâcan shed light on the pathogenesis of this complex disease. Methods: We recruited 15 patients with various stages of sarcoidosis and 12 healthy controls. All subjects underwent bronchoscopy with lavage. For each subject, total RNA was extracted from bronchoalveolar (BAL) cells and hybridized to an Affymetrix GeneChip Human Genome U133A 2.0 microarray. Rigorous statistical methods were applied to identify differential gene expression between subjects with sarcoidosis vs. controls. To better elucidate pathways differentially activated between these groups, we applied gene set enrichment analysis (GSEA) to the transcriptional profiles of BAL cells. We used false discovery rate (FDR) < 0.01 to designate significant enrichment. Results: Sarcoid patients were either non-smokers or ex-smokers, all had lung involvement and only 2 were on systemic prednisone. Healthy controls were all non-smokers. Comparison of BAL cell gene expression between sarcoidosis and healthy subjects revealed over 1200 differentially expressed genes at an FDR cutoff < 0.01. Several previously described immune mediators, such as interferon gamma, were up-regulated in the sarcoidosis subjects. Since genes often exert their influence through functionally coherent modules, we performed GSEA based on global expression profiles of alveolar macrophages between the subject groups. We identified more than 200 gene sets enriched in patients with sarcoidosis whereas very few pathways were over-represented in the healthy controls. Many of the sarcoidosis-associated pathways mapped to inflammatory and immune-related processes including T-cell signaling, graft vs. host disease, IL-12, IL-23, and IL-17 pathways, and oxidative phosphorylation. However, we also found and confirmed significant alteration in Proteasome-related pathways. Conclusions: BAL cells in sarcoidosis are characterized by enrichment of distinct transcriptional networks involved in immuno-inflammatory and proteasomal processes. Our findings add to the growing evidence implicating airspace resident cells in the pathogenesis of sarcoidoisis and identify specific pathways whose activation may modulate disease progression. Total RNA from BAL cells of 15 subjects with sarcoidosis and 12 healthy controls was hybridized to 27 Affymetrix Genechip Human U133A 2.0 microarrays

Project description:Introduction: Sarcoidosis is a multisystem immuno-inflammatory disorder of unknown etiology that most commonly involves the lungs. We hypothesized that an unbiased approach to identify pathways activated in alveolar macrophages—a key immuno-inflammatory cell in the lung—can shed light on the pathogenesis of this complex disease. Methods: We recruited 15 patients with various stages of sarcoidosis and 12 healthy controls. All subjects underwent bronchoscopy with lavage. For each subject, total RNA was extracted from bronchoalveolar (BAL) cells and hybridized to an Affymetrix GeneChip Human Genome U133A 2.0 microarray. Rigorous statistical methods were applied to identify differential gene expression between subjects with sarcoidosis vs. controls. To better elucidate pathways differentially activated between these groups, we applied gene set enrichment analysis (GSEA) to the transcriptional profiles of BAL cells. We used false discovery rate (FDR) < 0.01 to designate significant enrichment. Results: Sarcoid patients were either non-smokers or ex-smokers, all had lung involvement and only 2 were on systemic prednisone. Healthy controls were all non-smokers. Comparison of BAL cell gene expression between sarcoidosis and healthy subjects revealed over 1200 differentially expressed genes at an FDR cutoff < 0.01. Several previously described immune mediators, such as interferon gamma, were up-regulated in the sarcoidosis subjects. Since genes often exert their influence through functionally coherent modules, we performed GSEA based on global expression profiles of alveolar macrophages between the subject groups. We identified more than 200 gene sets enriched in patients with sarcoidosis whereas very few pathways were over-represented in the healthy controls. Many of the sarcoidosis-associated pathways mapped to inflammatory and immune-related processes including T-cell signaling, graft vs. host disease, IL-12, IL-23, and IL-17 pathways, and oxidative phosphorylation. However, we also found and confirmed significant alteration in Proteasome-related pathways. Conclusions: BAL cells in sarcoidosis are characterized by enrichment of distinct transcriptional networks involved in immuno-inflammatory and proteasomal processes. Our findings add to the growing evidence implicating airspace resident cells in the pathogenesis of sarcoidoisis and identify specific pathways whose activation may modulate disease progression.

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MiRNA profiling of sarcoidosis Treg cells

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