Unknown,Transcriptomics,Genomics,Proteomics

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MiR expression profile in M1-polarized bone marrow derived macrophage cells compared with normal mouse aortic tissue.

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Andreas Schober

PROVIDER: E-GEOD-26556 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Macrophages rapidly engulf apoptotic cells to limit the release of noxious cellular contents and to restrict autoimmune responses against self antigens. Although factors participating in recognition and engulfment of apoptotic cells have been identified, the transcriptional basis for the sensing and silently disposing of apoptotic cells is unknown. Here we show that peroxisome proliferator activated receptor delta (PPARdelta) is induced when macrophages engulf apoptotic cells and functions as a transcriptional sensor of dying cells. Genetic deletion of PPARdelta decreases expression of opsonins, such as C1qb, resulting in impairment of apoptotic cell clearance and reduction in anti-inflammatory cytokine production. This increases autoantibody production and predisposes global and macrophage-specific PPARdelta deficient mice to autoimmune kidney disease, a phenotype resembling the human disease systemic lupus erythematosus. Thus, PPARdelta plays a pivotal role in orchestrating the timely disposal of apoptotic cells by macrophages, ensuring that tolerance to self is maintained. HoxA9 immortalized ES cell derived myeloid precursors were created and differentiated as in Odegaard et al, 2007. After expansion, macrophages were differentiated for 8 days in the presence of MCSF in 20% L929 conditioned media with 2.5% bovine serum albumin in low glucose media. Macrophages were harvested for RNA using Qiagen's RNeasy Kit. A common reference was generated using e17.5 C57Bl/6 embryos. 30ug of either reference or macrophage RNA was labeled with Cy3 and Cy5 (Amersham) and hybridized to Stanford Functional Genomics Facility mouse cDNA microarrays for 16 hours. The arrays were washed and scanned on an Agilent G2505A ChipScan v. A.6.3.1. Florescence values were extracted using GenePix Pro v. 5.0.0.51. The data were normalized using regression correlation. Subsequently, missing values were imputed and significantly differentially expressed genes were found with Significance Analysis of Microarrays. For the Nature Medicine manuscript, significantly differentially expressed genes including genes involved in the process of uptake of apoptotic cells were extracted and clustered by complete linkage using Cluster and visualized using TreeView. Key to genotypes: "J1 untreated" are WT differentiated macrophages; "18 Untreated" are PPARd-/- differentiated macrophages Genotype: Wild type (WT) or PPAR delta minus (PPARd -/-) macrophages genetic_modification_design

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MiR expression profile in M1-polarized bone marrow derived macrophage cells compared with normal mouse aortic tissue.

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