RNA expression of reactivated Antigen-specific memory CD4 T cells previously exposed to tolerogenic or immunogenic stimulation
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ABSTRACT:
PROVIDER: PRJNA606741 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11088661_1.fastq.gz | Fastqsanger.gz | |||
| SRR11088661_2.fastq.gz | Fastqsanger.gz | |||
| SRR11088662_1.fastq.gz | Fastqsanger.gz | |||
| SRR11088662_2.fastq.gz | Fastqsanger.gz | |||
| SRR11088663_1.fastq.gz | Fastqsanger.gz |
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Project description:Activated and memory CD4 T cells play important roles in many autoimmune diseases often acting as upstream co-ordinators of inflammation and tissue destruction. A major therapeutic strategy is to turn off or tolerize these CD4 T cells thereby providing a cure. While much is understood about inducing tolerance in naïve CD4 T cells, little is known about whether or how to induce tolerance in previously activated or memory CD4 T cells. Here, we use RNA-sequencing to investigate the consequences of activating mouse CD4 memory T cells with antigen delivered in the absence of adjuvant, a classic tolerance-inducing strategy for naïve T cells. To examine the long term consequences of exposing memory CD4 T cells to tolerizing signals, we first reactivated them with antigen delivered with (control) or without (experimental) adjuvant, rested the cells and then reactivated them with antigen and adjuvant 5 days prior to isolating RNA for gene expression analysis.
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2014-07-01 | E-GEOD-58949 | biostudies-arrayexpress
Project description:Human monocyte-derived dendritic cells (moDCs) have been used as an in vitro model for studying tolerance and immunity. However, the underlying metabolic states of tolerogenic (dexamethasone and vitamin D3-treated), immature and immunogenic (mature, LPS-treated) moDCs have not been completely characterized. Through transcriptomic analyses, we determined that tolerogenic moDCs exhibit augmented catabolic pathways with respect to oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO) and glycolysis. Functionally, tolerogenic moDCs showed the highest mitochondrial membrane potential, production of reactive oxygen species and superoxide, and increased mitochondrial spare respiratory capacity. Tolerogenic and mature moDCs manifested differential FAO gene expression with FAO activity being significantly higher in tolerogenic and immature moDCs than in mature. In addition, tolerogenic and mature moDCs demonstrated similar levels of glycolytic rate, but not glycolytic capacity and reserve, which were more pronounced in tolerogenic and immature moDCs. Finally, tolerogenic and immature moDCs, but not mature moDCs, showed high plasticity to compensate the intracellular ATP content after inhibition of different energetic metabolic pathways. Overall, tolerogenic moDCs exhibit a metabolic signature of increased, stable OXPHOS programing and high plasticity for metabolic adaptation. These findings provide a framework for future research of metabolic properties of human DCs.
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Project description:Human monocyte-derived dendritic cells (moDCs) have been used as an in vitro model for studying tolerance and immunity. However, the underlying metabolic states of tolerogenic (dexamethasone and vitamin D3-treated), immature and immunogenic (mature, LPS-treated) moDCs have not been completely characterized. Through transcriptomic analyses, we determined that tolerogenic moDCs exhibit augmented catabolic pathways with respect to oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO) and glycolysis. Functionally, tolerogenic moDCs showed the highest mitochondrial membrane potential, production of reactive oxygen species and superoxide, and increased mitochondrial spare respiratory capacity. Tolerogenic and mature moDCs manifested differential FAO gene expression with FAO activity being significantly higher in tolerogenic and immature moDCs than in mature. In addition, tolerogenic and mature moDCs demonstrated similar levels of glycolytic rate, but not glycolytic capacity and reserve, which were more pronounced in tolerogenic and immature moDCs. Finally, tolerogenic and immature moDCs, but not mature moDCs, showed high plasticity to compensate the intracellular ATP content after inhibition of different energetic metabolic pathways. Overall, tolerogenic moDCs exhibit a metabolic signature of increased, stable OXPHOS programing and high plasticity for metabolic adaptation. These findings provide a framework for future research of metabolic properties of human DCs. Total RNA of sixteen samples from four moDC types (tolerogenic, LPS-tolerogenic, immature and mature) were extracted by Trizol (Invitrogen) followed by a clean-up procedure using RNeasy Micro Kit (Qiagen). All RNA samples had an integrity number ≥9.6 assessed by Agilent Bioanalyzer. Total RNA samples were amplified using TargetAmp™ and the biotinilated cRNA was prepared by Nano-g™ Biotin-aRNA Labeling Kit for the Illumina® System (Epicentre). After the hybridization to the Illumina Human HT-12 v4 Beadchips for 17 h at 58°C, the arrays were washed, stained (Illumina Wash Protocol) and then scanned using BeadArray Scanner 500GX. Array data were extracted at the probe level without background correction using Illumina GenomeStudio software. These raw data were quantile normalized and log2 transformed. Technical replicates were obtained from the hybridization in duplicate of three samples. Pearson correlation analysis showed high correlation between the technical replicates (r>0.99). Differentially expressed genes (DEGs) were identified using Limma16 with Benjamini-Hochberg multiple testing correction (p<0.05). DEGs were further clustered into different groups according to the patterns of expression change among the different moDC types using STEM software17. The analysis was performed in R v.2.12.2 (http://www.R-project.org) with Bioconductor 2.12 (http://www.bioconductor.org) and enabled by Pipeline Pilot (www.accelrys.com).
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Project description:F4/80+ macrophages treated with TGFb2 are potently tolerogenic. Our understanding of the molecular mechanisms mediating the development of these tolerogenic properties is incomplete. We used microarray analysis to identify molecules that are involved in the tolerogenic mechanisms in murine TGFb-treated macrophages.
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