Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Age-associated Changes in Basal NF-ÎºB Function in Human CD4+ T Lymphocytes via Dysregulation of PI3 Kinase (dataset 1)

ABSTRACT: Immune impairment and high circulating level of pro-inflammatory cytokines are landmarks of human aging. However, the molecular basis of immune dysregulation and the source of inflammatory markers remain unclear. Here we demonstrate that in the absence of overt cell stimulation, gene expression mediated by the transcription factor NF-ÎºB is higher in purified and rested human CD4+ T lymphocytes from older compared to younger individuals. This increase of NF-ÎºB -associated transcription includes transcripts for pro-inflammatory cytokines such as IL-1 and chemokines such as CCL2 and CXCL10. We demonstrate that NF-ÎºB up-regulation is cell-intrinsic and mediated in part by phosphatidylinositol 3-kinase (PI3K) activity induced in response to metabolic activity, which can be moderated by rapamycin treatment. Our observations provide direct evidence that dysregulated basal NF-ÎºB activity may contribute to the mild pro-inflammatory state of aging. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood using Ficoll-Paque Plus density gradient centrifugation. CD4+ T cells were obtained by positive selection using anti-human CD4 microbeads. After a 4h incubation at 37ÂºC (5% CO2 incubator), cells were harvested and collected by centrifugation. Total cellular RNA was prepared using Qiagen RNeasy protocols. Labeling and amplification were done with the standard Illumina protocol, and the biotin-labeled cRNA was hybridized to Illumina's HumanRef-8 v2.0 Expression BeadChip. The arrays were washed, blocked and the biotin-labeled cRNA was detected by staining with streptavidin-Cy3. Samples were divided into 2 age groups, young and old, with the cut off at 65 years and above as old.

ORGANISM(S): Homo sapiens

SUBMITTER: Kevin Becker

PROVIDER: E-GEOD-62331 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

Age-associated changes in basal NF-κB function in human CD4+ T lymphocytes via dysregulation of PI3 kinase.

Bektas Arsun A Zhang Yongqing Y Lehmann Elin E Wood William H WH Becker Kevin G KG Madara Karen K Ferrucci Luigi L Sen Ranjan R

Aging 20141101 11

Immune impairment and high circulating level of pro-inflammatory cytokines are landmarks of human aging. However, the molecular basis of immune dys-regulation and the source of inflammatory markers remain unclear. Here we demonstrate that in the absence of overt cell stimulation gene expression mediated by the transcription factor NF-κB is higher in purified and rested human CD4+ T lymphocytes from older compared to younger individuals. This increase of NF-κB -associated transcription includes t ...[more]

PMID: 25553802

Similar Datasets

Project description:Obesity is associated with a chronic, low-grade, systemic inflammation that may contribute to the development of insulin resistance and type 2 diabetes. Resveratrol, a natural compound with anti-inflammatory properties, is shown to improve glucose tolerance and insulin sensitivity in obese mice and humans. Here we tested the effect of a 2-year resveratrol administration on the pro-inflammatory profile and insulin resistance caused by a high-fat, high-sugar (HFS) diet in white adipose tissue (WAT) from rhesus monkeys. Eighty mg/day of resveratrol for 12-month followed by 480 mg/day for the second year decreased adipocyte size, increased sirtuin 1 expression, decreased NF-kB activation and improved insulin sensitivity in visceral but not subcutaneous WAT from HFS-fed animals. These effects were reproduced in 3T3-L1 adipocytes cultured in media supplemented with serum from monkeys fed HFS +/- resveratrol diets. In conclusion, chronic administration of resveratrol exerts beneficial metabolic and inflammatory adaptations in visceral WAT from diet-induced obese monkeys. Twenty-four adult (7-13 years old) male rhesus monkeys (Macaca mulatta) were housed individually in standard nonhuman primate caging on a 12h light/12h dark cycle, room temperature (78 +/- 2 degrees F), and humidity at 60 +/- 20%. One pairing was maintained throughout the study; all other monkeys had extensive visual, auditory, and olfactory but limited tactile contact with monkeys housed in the same room. Monkeys received 2 meals per day at estimated ad libitum levels throughout the study. Water was always available ad libitum. Monkeys were monitored minimally 3 times daily by trained animal care staff. During baseline assessments, all monkeys were maintained on a commercially available closed formula monkey chow. After baseline assessment, four male rhesus monkeys remained on the healthy standard diet (SD), 10 male rhesus monkeys were begun on a high fat/high sucrose (HFS) diet and 10 male rhesus monkeys were begun on a high fat/high sucrose (HFS) diet plus Resveratrol, 80mg/day. After one year of dietary intervention, the amount of resveratrol was increased to 480mg/day for one additional year. Tissues were then harvested for the array experiments.

Project description:Several versions of SWI/SNF complexes, BAF and PBAF, have been described based on unique subunit composition. We find that T cell development in the thymus and lymphoid periphery is largely normal in the absence of the PBAF-specific component BAF180. However, BAF180-deficient Th2 cells express high levels of the immunoregulatory cytokine IL-10. BAF180 binds directly to regulatory elements in the Il-10 locus but is replaced by BAF250 BAF complexes in the absence of BAF180, resulting in increased histone acetylation and CBP recruitment to the IL-10 locus. These results demonstrate that BAF180 is a repressor of IL-10 transcription in Th2 cells and suggest that the differential recruitment of different SWI/SNF subtypes can have direct consequences on chromatin structure and gene transcription. mouse primary T helper lymphocytes, naïve and Th2 cells, resting and stimulated, comparison of wild-type and BAF180/Pbrm1 deficient cells RNA from T helper cells was compared in the presence (WT) and absence (KO) of the Pbrm1/BAF180 gene. Comparison was made in 2 T helper subsets (Naive and Th2), each under 2 condtions (resting and stimulated), in triplicate (A,B,C). Resting naïve CD4+ T cells (N) were purified to 95% purity from the spleens and lymph nodes of 4-6 week old Balb/c mice (WT, or BAF180 cKO) using CD4+CD62L+ T cell Purification Kit II per manufacturer’s instructions (Miltenyi). Stimulated Naive cells (S) were prepared from resting naive cells by stimulation for 24 hours on anti-CD3 (1ug/ml), anti-CD28 (2 ug/ml) coated plates at 1-2 x 106/ml. Resting Th2 cells (2U) were prepared from resting naive cells by plating onto anti-CD3 (1ug/ml), anti-CD28 (2 ug/ml) coated plates at 1-2 x 106/ml in the presence of 10ng/ml IL-4, 10ug/ml anti-IFN-gamma. IL-2 (100U/ml) was added 24 hours later. Cultures were expanded in IL-2 (100U/ml) three days after initial culture. Stimulated Th2 cells (2S) were prepared from resting Th2 cells, stimulated with PMA (50ng/ml) and Ionomycin (500ng/ml) for 1.5 hours. RNA was harvested from 3 replicates of the primary Th cells under each condition. RNA was isolated using RNAeasy Kit (Qiagen), Quality and quantity of the total RNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips. RNA was labeled using the standard Illumina protocol and Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) Biotin labeled cRNA was hybridized to Illumina’s Sentrix MouseRef-8,v2 Expression BeadChips.

Project description:The prevention or delay of the onset of age-related diseases prolongs survival and improves quality of life while reducing the burden on the health care system. Activation of sirtuin 1 (SIRT1), an NAD+ deacetylase, improves metabolism and confers protection against physiological and cognitive disturbances in old age. SRT1720 is a specific SIRT1 activator that has health and lifespan benefits in adult mice fed a highfat diet. We found extension in lifespan, delayed onset of age-related metabolic diseases, and improved general health in mice fed a standard diet after SRT1720 supplementation. Inhibition of pro-inflammatory gene expression both in the liver and muscle of SRT1720-treated animals was noted. SRT1720 lowered phosphorylation of NF-κB pathway regulators in vitro only when SIRT1 was functionally present. Combined with our previous work, the current study further supports the beneficial effects of SRT1720 on health across the lifespan in mice. Groups of 28 week old male C57BL/6J mice were maintained on ad libitum AIN-93G SD diet, or an ad libitum AIN-93G diet supplemented with SRT1720 for the rest of their lives. SRT1720 was added at a dose of 1.33 g drug per kg of chow, formulated to provide daily doses of approximately 100 mg drug per kg bodyweight to the mice. 5 mice from each group were selected and RNA was extracted from both muscle and liver tissue using 1.0mm glass beads in a Precellys 24 Tissue Homogenizer and Qiagen RNeasy Mini Kits for Fibrous Tissue according to manufacturer's specifications. Quality and quantity of the total RNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips. RNA samples were labeled using the Illumina TotalPrep RNA Amplification Kit. In short, 0.5ug of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP. Arrays were hybridized using a total of 0.75ug of biotin-labeled cRNA at 58 degrees C for 16 hours to Illumina's Sentrix MouseRef-8 v2 Expression BeadChips. Each BeadChip has ~24,000 well-annotated RefSeq transcripts with approximately 30-fold redundancy. The arrays were washed, blocked and the biotin labeled cRNA was detected by staining with streptavidin-Cy3. Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina and the data was extracted using the Illumina GenomeStudio software(v1.6.0). Any spots at or below the background were filtered out using an Illumina detection p value of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented below. Z-score = (raw value - avg)/std.

Dataset Information

Age-associated Changes in Basal NF-ÎºB Function in Human CD4+ T Lymphocytes via Dysregulation of PI3 Kinase (dataset 1)

Publications

Age-associated changes in basal NF-κB function in human CD4+ T lymphocytes via dysregulation of PI3 kinase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets