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Modulation of reactive oxygen species and inflammatory cytokines by FoxO3a promotes host survival against Salmonella typhimurium

ABSTRACT: We employed microarray expression profiling to identify genes which are differentially regulated in the absence of FoxO3a, upon Salmonella typhimurium (ST) infection. ST induced gene expression changes were measured in Wild-type and FoxO3a-/- bone marrow derived macroophages at 6 hours post infection. The array showed an enrichment of MAPK signaling and alterations in inflammatory cytokines and ROS regulators in the absence of FoxO3a. Gene expression changes in bone marrow derived macrophages was measured after infection with 10 MOI of Salmonella typhimurium. Control samples were left untreated. Two mice from each genotype were used for the experiment.

ORGANISM(S): Mus musculus

PROVIDER: GSE82212 | GEO | 2016/08/11

SECONDARY ACCESSION(S): PRJNA324369

REPOSITORIES: GEO

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Project description:Persistence of memory CD4+ T cells in ECs was coupled with the inactivation of FOXO3a transcriptional activities, which we have previously identified as a critical regulator of TCM survival. Indeed, expression levels of transcriptional targets of FOXO3a, endowed with pro-apoptotic and anti-proliferative functions, were lower in TCM and TEM from ECs as compared to ST individuals. Silencing the transcriptionally active form of FOXO3a by siRNA rescued TCM and TEM of STs from Fas-mediated apoptosis. Moreover the expression of FOXO3a dominant negative form (FOXO3a Nt) rescued the long-term survival of TCM from STs as these cells persisted as long as those derived from ECs. Overall, these studies indicate that FOXO3a activation is an important mediator of the shortened survival and heighteined turnover of TCM and TEM in chronic HIV infection. Targeting this pathway may provide a strategy to preserve memory T cell numbers in HIV infection. Keywords: comparative gene profile, cell-type comparison Isolation of CD4+T cell sub-populations. Peripheral blood mononuclear cells (PBMCs) from healthy adult individuals were isolated by Ficoll-HyPaque (Pharmacia) density gradient. We first enriched for CD4+ T cells using negative immunomagnetic beads selection (Automacs, Myltenii), cells were then labeled with anti-CD4-APCcy7, anti-CD45RA-ECD, anti-CD27-FITC and anti-CCR7-PEcy7 and sorted into Naive cells described as CD4+, CD45RA+, CD27+ and CCR7+, Central Memory cells (TCM) described as CD4+, CD45RA-, CD27+ and CCR7+ cells and Effector Memory cells (TEM) described as CD4+, CD45RA-, CD27- and CCR7- cells. Sorting was performed using a BDAria (BD Pharmingen). Purity of the TCM and TEM sub-populations was ranging from 96 to 99%. All procedures were done at 4C to avoid any changes in cell phenotype or gene expression. Sample RNA was extracted using an RNA extraction kit (Quiagen), then amplified using the MessageAmp RNA kit (Ambion) as per the manufacturer’s instructions. The amplified RNA (aRNA) was then verified for quality and quantity using the Agilent Bioanalyser and measuring the OD. Universal human RNA (Stratagene) was also prepared in the same way. Sample probes were prepared by direct labelling with 3 µg of the aRNA Cy-5 (R values) fluorescent dye while the universal RNA probes were prepared by direct labelling of universal aRNA with Cy-3 (G values). All patient samples were hybridized against amplified universal RNA at 37 ºC for 18h on a custom human Immune array. Detailed information on the labeling and hybridization procedures can be obtained at the Microarray Centre web page (University Health Network).

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Modulation of reactive oxygen species and inflammatory cytokines by FoxO3a promotes host survival against Salmonella typhimurium

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