Expression data of wild type and miR-155 knockout bone marrow derived dendritic cells treated with lipopolysaccharide (LPS)
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ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Aadel Chaudhuri
PROVIDER: E-GEOD-23641 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:In response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation (maturation) that exhibits specific mechanisms to control immunity. Here, we show that in response to Lipopolysaccharides (LPS), several microRNAs (miRNAs) are regulated in human monocyte-derived dendritic cells. Among these miRNAs, miR-155 is highly up-regulated during maturation. Using LNA silencing combined to microarray technology, we have identified the Toll-like receptor / interleukin-1 (TLR/IL-1) inflammatory pathway as a general target of miR-155. We further demonstrate that miR-155 directly controls the level of important signal transduction molecules. Our observations suggest, therefore, that in mature human DCs, miR-155 is part of a negative feedback loop, which down-modulates inflammatory cytokine production in response to microbial stimuli. We devised a strategy to functionally inhibit the mature form of miR-155 with the aim of identifying the signaling pathways controlled by miR-155 during DC maturation. A LNA-modified oligonucleotide, specifically designed for miR-155 knockdown (anti-miR-155 LNA) was introduced in moDCs by nucleofection prior LPS stimulation. 24h after transfection, a reduction in miR-155 levels of 8- and 32-fold was observed by qPCR in immature and mature moDCs respectively. A comparative microarray analysis (Affymetrix U133 2.0 chip) was performed among miR-155 silenced (anti-miR-155 LNA) and control transfected (scramble LNA) moDCs, exposed or not to LPS. Thus, four samples were totally analyzed. As expected from the limited expression of miR-155 in non-activated DCs, little variation in mRNA expression (177 probe sets differentially expressed employing a cut-off of 1.5) was detected upon miR-155 silencing. Conversely, in LPS-activated cells many mRNAs (1324 probe sets, 770 up-regulated and 554 down-regulated) were affected by miR-155 inhibition.
2009-03-04 | E-GEOD-13296 | biostudies-arrayexpress
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Project description:MicroRNAs (miRNAs) are small RNAs that play important regulatory roles in many cellular pathways. MiRNAs associate with members of the Argonaute (Ago) protein family and bind to partially complementary sequences on mRNAs and induce translational repression or mRNA decay. MiRNA expression can be controlled by transcription factors and can therefore be cell type- or tissue-specific. Here we have analyzed miRNA expression profiles in murine monocyte-derived dendritic cells (DCs) and macrophages upon stimulation with LPS, LDL, eLDL and oxLDL to identify not only stimuli-specific miRNA, but also to identify a hierarchical miRNA system involving miR-155. For this, miR-155 knockout dendritic cells and macrophages were also sequenced using the same stimuli. Sequencing of murine monocyte-derived dendritic cells and macrophages (each wild type and miR-155 knock out cells) matured and stimulated, respectively, by LPS, oxLDL, eLDL or LDL.
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Project description:In response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation (maturation) that exhibits specific mechanisms to control immunity. Here, we show that in response to Lipopolysaccharides (LPS), several microRNAs (miRNAs) are regulated in human monocyte-derived dendritic cells. Among these miRNAs, miR-155 is highly up-regulated during maturation. Using LNA silencing combined to microarray technology, we have identified the Toll-like receptor / interleukin-1 (TLR/IL-1) inflammatory pathway as a general target of miR-155. We further demonstrate that miR-155 directly controls the level of important signal transduction molecules. Our observations suggest, therefore, that in mature human DCs, miR-155 is part of a negative feedback loop, which down-modulates inflammatory cytokine production in response to microbial stimuli.
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Project description:The pro-inflammatory microRNA-155 (miR-155) is highly expressed in the serum and CNS lesions of patients with multiple sclerosis (MS). The whole-body deletion of miR-155 in mice confers resistance to a mouse model of MS, EAE, by reducing the encephalogenic potential of CNS-infiltrating Th17 T cells. However, cell intrinsic roles for miR-155 during EAE have not been formally determined. Here we utilize single-cell RNA sequencing and cell-specific conditional miR-155 knockouts to determine the importance of miR-155 expression in distinct immune cell populations. Time course single-cell sequencing revealed reductions in T cells, macrophages, and dendritic cells in whole-body miR-155 knockout mice compared with wild-type controls at day 21. Deletion of miR-155 in T cells, driven by CD4 cre, reduced disease severity similar to whole-body miR-155 knockouts. CD11c cre-driven deletion of miR-155 in dendritic cells also significantly reduced EAE disease score, with both T cell and dendritic cell-specific knockouts showing a reduction in Th17 T cell infiltration into the CNS. Although miR-155 is highly expressed in infiltrating macrophages during EAE, deletion of miR-155 using LysM cre did not affect disease severity. Taken together, these data show that while miR-155 is highly expressed in most infiltrating immune cells, miR-155 has distinct roles and requirements depending on the cell type. This provides insights into which functionally relevant cell types should be targeted by the next generation of miRNA therapeutics.
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Project description:MYC regulates the expression of multiple microRNA (miRNA) genes and defines the Burkitt lymphoma (BL) miRNA signature. Here, we investigate the role of the MYC-regulated miRNAs by gain- and loss-of-function analysis. Overexpression of 5 miRNAs that were significantly downregulated by MYC resulted in strong (miR-150, miR-26a, miR-26b) and mild (miR-29a, let-7a) impaired cell growth. Overexpression of miR-155 increased proliferation of BL cells. By RNA immunoprecipitation of Argonaute 2 in BL cells with and without miR-155 we identified 54 miR-155 target genes. Using an shRNA approach we identified TBRG1 (NIAM1) as a miR-155 target gene that copied the miR-155-induced phenotype upon its inhibition. Analysis of TBRG1 protein expression and miR-155 levels in primary cases of B-cell lymphoma revealed that miR-155 levels are significantly lower in TBRG1 positive cases suggesting that TBRG1 is also regulated by miR-155 in primary B-cell lymphoma. Our data demonstrate that overexpression of individual MYC-repressed miRNAs has a strong suppressive effect on BL cell growth, whereas overexpression of miR-155 enhances B-cell lymphoma growth by targeting the tumor suppressor gene TBRG1. Gene expression profile was performed in ST486 Burkitt lymphoma cell line in 4 samples: ST486 EV (empty MXW-PGK-IRES-GFP vector) total cell lysate, ST486 EV Ago2-IP, ST486 miR-155 (ST486 with ectopic miR-155) total cell lysate, ST486 miR-155 Ago2-IP.
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Project description:Toll-like receptor (TLR)-induced maturation of dendritic cells (DCs) leads to the production of proinflammatory cytokines as well as the upregulation of various molecules involved in T cell activation. These are believed to be the critical events that account for the induction of the adaptive immune response. Here, we have examined the role of micro-RNA-155 (miR-155) in DC function and the induction of immunity. Using a model where the transfer of self-antigen-pulsed, TLR-matured DCs can induce a functional CD8 T cell response and autoimmunity, we find that DCs lacking miR-155 have an impaired ability to break immune tolerance. Importantly, transfer of self- antigen-pulsed DCs overexpressing miR-155 was sufficient to break tolerance in the absence of TLR stimuli. Although these unstimulated DCs induced T cell function in vivo, there was no evidence for the upregulation of costimulatory ligands or cytokine secretion. Further analysis showed that miR-155 influenced the level of the phosphatase SHIP1 in DCs, and that the lack of SHIP1 in DCs was sufficient to break T cell tolerance in vivo, again in the absence of TLR induced DC maturation. Our study demonstrates that the overexpression of miR-155 in DCs is a critical event that is alone sufficient to break self tolerance and promote a CD8-mediated autoimmune response in vivo. This process is independent of the induction of conventional DC maturation markers, indicating that miR-155 regulation of SHIP represents a unique axis that regulates DC function in vivo. Dendritic cell culture: Bone marrow was flushed from tibias and femurs of mice with HBSS. Bone marrow cells were cultured at 2x106 cells/ml in 10 ml non-tissue culture treated dishes in RPMI 1640 containing 10% LPS-free FBS, Penicillin:streptomycin glutamine 2-mercaptoethanol (cRPMI), with 40ng/ml murine GM-CSF (Peprotech). On day 3 of cultures 10ml of fresh media was added also containing 40ng/ml GM-CSF. On days 6 and 8, 10ml of media was removed and centrifuged to collect cells, which were resuspended in 10ml fresh media containing GM-CSF and were added back to dishes. Non-adherent cells were isolated on day 9. DCs were plated in 24-well plates at 2x106 cells/ml in 1ml of cRPMI with or without the class B CpG ODN1826 (ACGT DNA Technologies corporation, Toronto, ON, Canada) at 10?M final concentration overnight. Array analysis of mi-RNA expression: BMDCs from WT mice were either stimulated with CpG or left in media alone overnight. Whole RNA was isolated using mirVana miRNA isolation kit following the manufacturers instructions. RNA samples were labeled and hybridized to Agilent mouse miRNA 8x15K arrays (Agilent). Data was generated from 8 individual samples (4 unstimulated and 4 CpG-treated).
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