Project description:To characterize the relationship between IRF5 and Lyn in innate immune responses at a genome-wide scale, we performed gene expression microarray analysis for wild-type (WT), Irf5-KO, Lyn-KO and Lyn/Irf5-DKO bone marrow-derived dendritic cells (BMDCs) with or without 6 h-stimulation with 150 nM CpG-B ODN. We extracted the transcripts that were upregulated by CpG-B ODN in WT BMDCs, further upregulated in Lyn-KO BMDCs and then downregulated in Lyn/Irf5-DKO BMDCs (fold change â?¥ 2, false discovery rate < 0.05). When these 79 transcripts were sorted by their expression levels in Lyn-KO BMDCs, most of the top 20 genes (13 out of 20) were those encoding type I interferons (IFNs), indicating that Lyn particularly suppresses IRF5 induction of type I IFNs. BMDCs from WT, Irf5-KO, Lyn-KO and Lyn/Irf5-DKO mice in a C57BL/6 background were unstimulated or stimulated with CpG-B ODN. Biological triplicate for each genotype were analyzed (24 samples in total).
Project description:To characterize the relationship between IRF5 and Lyn in innate immune responses at a genome-wide scale, we performed gene expression microarray analysis for wild-type (WT), Irf5-KO, Lyn-KO and Lyn/Irf5-DKO bone marrow-derived dendritic cells (BMDCs) with or without 6 h-stimulation with 150 nM CpG-B ODN. We extracted the transcripts that were upregulated by CpG-B ODN in WT BMDCs, further upregulated in Lyn-KO BMDCs and then downregulated in Lyn/Irf5-DKO BMDCs (fold change ≥ 2, false discovery rate < 0.05). When these 79 transcripts were sorted by their expression levels in Lyn-KO BMDCs, most of the top 20 genes (13 out of 20) were those encoding type I interferons (IFNs), indicating that Lyn particularly suppresses IRF5 induction of type I IFNs.
Project description:Synthetic oligonucleotides (ODNs) containing CpG motifs stimulate human plasmacytoid dendritic cells (pDCs) to produce type-1 interferons (IFN) and pro-inflammatory cytokines. Previous studies demonstrated that interferon regulatory factors (IRFs) played a central role in mediating CpG-induced pDC activation. This work explores the inverse effects of IRF-5 and IRF-8 on CpG dependent gene expression. Results from RNA interference and microarray studies indicate that IRF-5 up-regulates TLR9-driven gene expression whereas IRF-8 down-regulates the same genes. Several findings support the conclusion that IRF-8 inhibits TLR9 dependent gene expression by directly blocking the activity of IRF-5. First, the inhibitory activity of IRF-8 is only observed when IRF-5 is present. Second, proximity ligation analysis shows that IRF-8 and IRF-5 co-localize within the cytoplasm of resting human pDC and co-translocate to the nucleus after CpG stimulation. Taken together, these findings suggest that two transcription factors with opposing functions control TLR9 signaling in human pDCs. CAL-1 cells were transfected with siRNA targeting IRF-5 (IRF-5si) and left unstimulated (n=4, technical repeats) or stimulated with K-type CpG ODN (n=4, technical repeats). CAL-1 cells were transfected with siRNA targeting IRF-8 (IRF-8si) and left unstimulated (n=4, technical repeats) or stimulated with K-type CpG ODN (n=4, technical repeats). CAL-1 cells were transfected with control siRNA (Contsi) and left unstimulated (n=4, technical repeats) or stimulated with K-type CpG ODN (n=4, technical repeats).
Project description:mouse primary BMDCs were stimulated with tlr ligands and gene expression changes were profiled on Affymetrix arrays BMDC were stimulated with 5 tlr ligands (LPS, pIC ,PAM, CpG, GRD) across 9 time points (.5, 1, 2, 4, 6, 8, 12, 16, 24 hours). Unstimulated cells were used as controls.
Project description:Bioinformatic analysis of microarray data was used to identify the regulatory patterns underlying changes in gene expression induced when RAW 264.7 macrophages were stimulated via TLR9 by CpG oligonucleotides (ODN) and/or via TLR3 by poly (I:C). While the genes activated by each ligand mediated similar functions, poly (I:C) elicited a larger and more diverse change in gene expression. Co-stimulation with both ligands accelerated gene expression and synergistically activated genes primarily associated with immune function. This is the first work to compare global changes in gene regulation triggered by distinct TLR pathways and clarify their impact on gene expression. RAW 264.7 cells were cultured for 5 days in DMEM supplemented with 10% FCS, 50 U/ml penicillin, 50 ug/ml streptomycin, and 1mM sodium pyruvate at 37C in a 5% CO2 in air incubator. Cells were washed, transferred into new flasks, and rested for 16 hr before being stimulated with 3.2 ug/ml CpG ODN and/or 32 ug/ml poly(I:C) for 4 - 12 hr. All experiments were independently repeated three times, and data from all experiments combined for analysis. Control samples included RAW 264.7 cells that were unstimulated and collected at 4 and 12 hours. All arrays used reverse transcribed mRNA from stimulted and unstimulated RAW cells as well as Universal Mouse Reference RNA purchased from Stratagene, La Holla, CA.
Project description:Transcription profiling of unstimulated and stimulated mouse CD4+ T cells extracted from wild type (WT), hCD2-Egr2 transgenic (Egr2 Tg) and Egr2loxP/loxP hCD2-Cre Egr3-/- (Egr2/3 DKO) mice.
Project description:CD14+ purified bovine monoctye stimulation with CpG ODN 2007 vs. GpC ODN 2007, CpG 2007 vs. Control, GpC 2007 vs. Control and Media vs Control (Control is unstimluated CD14+ purified bovine monocytes at time zero). Before stimulation, the CD14+ purified bovine monocytes were rested for 20 h. Then the cells were stimulated for 4hrs.
Project description:We compared in vitro mTECs-high stimulated with TLR9 ligand CpG ODN (1826) or non stimulated from MyD88 -/- and MyD88 +/+ mice: Thymi were enzymaticaly digested, cells were MACS enriched for CD45- fraction, and FACS sorted using BD Influx sorter. mTECs-high were gated as EpCAM+CD11c-Ly51-MHCII+CD80+. Cell from MyD88 -/- and MyD88 +/+ mice were incubated in RPMI with or without CpG ODN (1826) for 24 hours. Total RNA was isolated using RNeasy Plus Micro Kit (Qiagen). 4 samples per condition were used.
Project description:We measured global mRNA expression in unstimulated and Bone Marrow Derived Murine Dendritic Cells (BMDCs) stimulated with CD40L in presence or absence of Neuronal precursor cell (NPC) condition medium at 6h and 18h.
Project description:Immunostimulatory CpG ODN trigger an innate immune response characterized by the rapid production of pro-inflammatory cytokines and chemokines, an effect that persists for days to weeks in vivo. Previous studies established that gene up-regulation is maximal 3 hr after CpG ODN treatment of normal mice {Klaschik et al. JLB 2009}. The immunostimulatory activity of CpG ODN is blocked by the addition of immunosuppressive ODN expressing multiple TTAGGG motifs. To clarify the mechanism underlying this suppression, changes in gene expresssion were evaluated by microarray in mice treated with 400 ìg of CpG ODN + 400 ìg of suppressive ODN.
