Comment[ArrayExpressAccession] E-GEOD-44034 MAGE-TAB Version 1.1 Public Release Date 2013-12-08 Investigation Title The anti-inflammatory nature of HDL in macrophages is mediated by ATF3 Comment[Submitted Name] The anti-inflammatory nature of HDL in macrophages is mediated by ATF3 Experiment 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. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Schultze De Nardo Labzin Kono Seki Schmidt Kraut Kerkseik Bode Zimmer Kneilling Röcken Lütjohann Wright Schultze Latz Person First Name Joachim Dominic Larisa Hajime Reiko Susanne Michael Anja Niklas Sebastian Manfred Martin Dieter Sam Joachim Eicke Person Mid Initials V Person Email j.schultze@uni-bonn.de Person Affiliation LIMES (Life and Medical Sciences Center Genomics and Immunoregulation) Person Address Genomics and Immunoregulation, LIMES (Life and Medical Sciences Center Genomics and Immunoregulation), Carl-Troll-Strasse 31, Bonn, NRW, Germany Person Roles submitter Protocol Name P-GSE44034-1 P-GSE44034-5 P-GSE44034-6 P-GSE44034-2 P-GSE44034-3 P-GSE44034-4 P-GSE44034-7 Protocol Description The data were normalised using quantile normalisation with Partek Genomic Suite. ID_REF = VALUE = quantile normalized Detection Pval = please see Material and Methods section Standard Illumina hybridization protocol 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. 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). please see Material and Methods section Standard Illumina scanning protocol Protocol Type normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name TREATMENT STIMULATION PERIOD GENOTYPE CONDITION Experimental Factor Type treatment stimulation period genotype condition Comment[SecondaryAccession] GSE44034 Comment[GEOReleaseDate] 2013-12-08 Comment[ArrayExpressSubmissionDate] 2013-02-04 Comment[GEOLastUpdateDate] 2014-01-09 Comment[AEExperimentType] transcription profiling by array Comment[AdditionalFile:Data1] GSE44034_ATF3KO_non-normalized.txt Comment[AdditionalFile:Data2] GSE44034_carotid_artery_non-normalized.txt Comment[AdditionalFile:Data3] GSE44034_wt_non-normalized.txt SDRF File E-GEOD-44034.sdrf.txt