CpG B and R848 induced gene expression in IRAK2 wildtype and deficient mouse macrophages
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Youzhong Wan
PROVIDER: E-GEOD-24264 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Compared to wildtype macrophages, IRAK2 deficient macrophages show higher induced gene expression in responsse to CpG B, but not R848 Manuscipt title: The dual function of IRAK2 in TLR9-mediated interfereon and proinflammatory cytokine production
2010-09-23 | GSE24264 | GEO
Project description:Exciting discoveries related to IL-1R/TLR signaling in development of atherosclerosis plaque have triggered intense interest in the molecular mechanisms by which innate immune signaling modulates the onset and development of atherosclerosis. Previous studies have clearly shown the definitive role of proinflammatory cytokine IL-1 in the development of atherosclerosis. Recent studies have provided direct evidence supporting a link between innate immunity and atherogenesis. While it is still controversial about whether infectious pathogens contribute to cardiovascular diseases, direct genetic evidence indicates the importance of IL-1R/TLR signaling in atherogenesis. In this study, we examined the role of IRAK4 kinase activity in modified LDL-mediated signaling using bone marrow-derived macrophage as well as in vivo model of atherosclerosis. First, we found that the IRAK4 kinase activity was required for modified LDL-induced NFκB activation and expression of a subset of proinflammatory genes, but not for the activation of MAPKs in bonemarrow-derived macrophage. IRAK4 kinase inactive knock-in (IRAK4KI) mice were bred onto ApoE-/- mice to generate IRAK4KI/ApoE-/- mice. Importantly, the aortic sinus lesion formation was impaired in IRAK4KI/ApoE-/- mice compared to that in ApoE-/- mice. Furthermore, proinflammatory cytokine production was reduced in the aortic sinus region of IRAK4KI/ApoE-/- mice compared to that in ApoE-/- mice. Taken together, our results indicate that the IRAK4 kinase plays an important role in modified LDL-mediated signaling and the development of atherosclerosis, suggesting that pharmacological inhibition of IRAK4 kinase activity might be a feasible approach in the development of anti-atherosclerosis drugs. To identify global changes in gene expression, we examined gene expression profiles of macrophages from wild-type and IRAK4 kinase-inactive knock-in mice in response to acLDL stimulation using the Illumina microarray with probes for 23,000 transcripts. Bone marrow-derived macrophages from wild-type and IRAK4 kinase-inactive knock-in mice were treated with acLDL for 24 hours.
2010-10-23 | E-GEOD-24894 | biostudies-arrayexpress
Project description:Murine dendritic cells were derived from bone-marrow of 4 mice using GM-CSF. The DC were treated with RNA from gram-positive bacteria Listeria monocytogenes packaged in DOTAP or TLR7 agonist R848. Negative controls were DOTAP with no RNA or mock.
2012-05-08 | E-MTAB-1139 | biostudies-arrayexpress
Project description:Bone marrow derived macrophages from wt and card9 KO mice were stimulated with CpG, Curdlan or TDB, an analogon to the mycobacterial cord factor TDM for 48h, respectively. Keywords: card9 knockout, macropphage, TDB wt or card9 KO macrophages stimulated for 48h
2009-01-31 | E-GEOD-10530 | biostudies-arrayexpress
Project description:Lipid A (a hexaacylated 1,4 bis-phosphate) is a potent immune stimulant for TLR4/MD-2. Upon lipid A ligation, the TLR4/MD-2 complex dimerizes and initiates signal transduction. Historically, studies also suggested the existence of TLR4/MD-2-independent LPS signaling. Here we define the role of TLR4 and MD-2 in LPS signaling by using genome wide expression profiling in TLR4- and MD-2-deficient macrophages after stimulations with peptidoglycan-free LPS and synthetic E.coli lipid A. Of the 1,396 genes found significantly induced or repressed by any one of the treatments in the wildtype macrophages, none was present in the TLR4- or MD-2-deficient macrophages, confirming that the TLR4/MD-2 complex is the only receptor for endotoxin, and are both absolutely required for responses to LPS. Using a molecular genetics approach, we investigated the mechanism of TLR4/MD-2 activation by combining the known crystal structure of TLR4/MD-2 with computer modeling. We used lipid IVa, a defined lipid A mimetic to model the activation of mouse TLR4/MD2. The two phosphates on lipid A were predicted to interact extensively with the two positively charged patches mouse TLR4 according to our dimeric murine TLR4/MD-2/lipid IVa model. These two patches are composed of K263, R337, and K360 (Positive Patch 1), and K367 and R434 (Positive Patch 2). When either Positive Patch was abolished by mutagenesis into Ala, the responses to LPS and lipid A were almost abrogated. Thus, ionic interactions between the two phosphates on lipid A and the two positively charged patches on murine TLR4 appear to be essential for LPS receptor activation. The gene expression profile of macrophages from C57BL/6 and MD-2-deficient mice following either 10 ng LPS /mL, 100 ng lipid A/mL or 10 nM Pam2 stimulation for 2 hours were compared to PBS-stimulated control cells . In vitro differentiated macrophages from two individual WT and MD-2-deficient mice were cultured and stimulated with agonists separately, comparing the gene expression to PBS-stimulated control cells from the same mouse. Comparisons of PBS-stimulated WT cells to PBS-stimulated MD-2-deficient cells were performed to directly compare basal gene expression in the two genotypes.
2011-07-30 | E-GEOD-31067 | biostudies-arrayexpress
Project description:Gene expression kinetics for BM-DM from C57BL/6 mice challenged by poly(I:C) , R848, poly(I:C)+R848 examined at 6 time points including 0.5, 1, 2, 4, 8, 12 h.
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Project description:DNA damage response kinase ATM regulates the genetic program of lymphocytes with phsiologically induced DNA DSBs. In bone marrow-derived macrophages, related kinase DNAPKcs is also responsible for activating DNA damage responses after infection with Listeria monocytogenes. Here we show that both ATM and DNA-PKcs regulate the genetic program of Listeria monocytogenes-infected macrophages. Two independent bone marrow-derived macrophage cultures for each genotype (LysMcre/+ and Scid: Atmc/c: LysMcre/+) were infected with Listeria monocytogenes for 24 hrs at an MOI of 5. RNA was isolated using RNeasy (Qiagen). Gene expression profiling was performed using Illumina MouseRef-8 expression microarrays.
2015-07-03 | E-GEOD-70467 | biostudies-arrayexpress
Project description:The global change of the miR expression profile during atherosclerosis is due to the infiltration of different types of leukocytes into the arterail vessel wall in addition to disease-specific regulation in vascular cells. Monocyte-derived macrophage accumulation in the subintimal region is critical in the formation of atherosclerotic plaques. It is currently unknown which miRs are involved in the atherogenic macrophage response. The comparison of the miR expression profile in LPS/Interferon-gamma activated mouse macrophages with the miR expression in the normal aortic vessel wall was performed to detect macrophage-enriched miRs. This screening may help to identify macrophage-enriched miRs in atherosclerotic vessels that may play a role in the macrophage function during atherogenesis. Bone marrow cells were harvested from femura of 6-8 week old female C57BL/6 mice, re-suspended in DMEM-F12/10% FCS/10% L929-conditioned medium, and cultured for 7 days to differentiate into primary macrophages. F4/80 and CD11b expression was determined by flow cytometry to confirm the macrophage phenotype. Macrophages were stimulated with LPS (100ng/ml, 14 hours) and INF-g (10ng/ml, 6 hours) and the M1 polarization was verified by quantification of mannose receptor C type 1 (MRC1), arginase II (ArgII), inducible nitric oxide synthase (iNOS), and arginase I (ArgI) by qRT-PCR. Total RNA (M1-type macrophages and aorta tissue) was isolated using mirVana microRNA Isolation Kit.
2014-01-01 | E-GEOD-26556 | biostudies-arrayexpress
Project description:We report the genomic regions enriched in Histone Deacetylase 3 (HDAC3) in mouse bone marrow derived macrophages. Furthermore, we also report the genomic acetylation pattern on Histone 3, Lysine 9 (H3K9) in macrophages with and without HDAC3 and/or treated with Th2 cytokine IL-4. HDAC3 enriched genomic regions in mouse bone marrow dervied macrophages and H3K9Ac enriched genomic regions in wild-type macrophages and macrophages treated with IL-4 and/or deficient in HDAC3.
2011-11-10 | E-GEOD-33596 | biostudies-arrayexpress
Project description:Elevated plasma levels of High Density Lipoprotein (HDL) are associated with decreased risk of cardiovascular disease (CVD). The protective role of HDL in atherosclerosis has been attributed primarily to its ability to remove excess cholesterol from lipid-laden macrophages (foam cells) within the arterial walls. However, clinical trials that raise HDL cholesterol levels have failed to show a benefit casting doubts on our basic understanding of HDL function. Atherosclerosis is a chronic inflammatory condition underlying CVD and driven in part by the recognition of metabolic danger signals by innate immune receptors on macrophages. A potential feature that could contribute to HDL’s protective effects in CVD could be HDL's anti-inflammatory nature, such as its ability to reduce endothelial cell activation. However, the molecular mechanisms by which HDL reduces inflammatory macrophage responses remain poorly understood and difficult to separate from its cholesterol depleting activity. Here we show that HDL protects against Toll like receptor (TLR)-induced inflammation both in vivo and in vitro under normocholesteremic conditions by suppressing the transcription of inflammatory cytokines in a manner independent of its ability to remove cellular cholesterol. We identify Activating Transcription Factor 3 (ATF3), a transcriptional repressor of the CREB family of basic leucine zipper transcription factors, as a HDL-inducible regulator of macrophage activation. HDL’s ability to down modulate TLR responses was severely compromised in ATF3-deficient cells demonstrating that ATF3 mediates HDL's anti-inflammatory effects and may explain the broad anti-inflammatory functions of HDL. Bone marrow-derived macrophages (BMDMs) were obtained by culturing bone marrow cells from 6 to 8 week old wildtype C57BL/6 mice in DMEM supplemented with 10% FCS, 10 mg ml-1 Ciprobay-500 and 40 ng ml-1 M-CSF (R & D Systems). BMDMs of wt mice were pretreated for 6 h with HDL (2 mg ml-1 ) then stimulated with CpG (100 nM) for 4 h. Further wild type or Atf3-deficient BMDMs were pretreated with 2 mg ml-1 HDL for 6 h and subsequently stimulated with CpG (100 nM) or P3C (50 ng ml-1) for 4 h. For carotid artery injury approximately 12-week old male WT and Atf3-deficient mice were anesthetized with i.p. injection of 150 mg/kg ketaminehydrochloride (Ketanest, Pharmacia) and 0.1 mg/kg xylazinehydrochloride (Rompun 2%, Bayer). A small incision from the cranial apex of the sternum to just below the mandible was made. After careful preparation of an approximately 6 mm long segment proximal of the bifurcation, the common carotid artery was electrically denuded. A 4 mm long lesion was made by applying two serial 5 second bursts of 2 Watt using 2 mm wide forceps. The skin was then sutured and the mice allowed to recover in individual cages before returning to their littermates. Three hours later the mice received a single 200 ?l i.v. injection of 20 mg/kg HDL or PBS.
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