Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Timecourse of Interferon-Gamma Treatment of Bone Marrow-Derived Macrophages (BMDMs)

ABSTRACT: The systematic temporal gene expression analysis of primary macrophages activated under immune (interferon-gamma (IFN-g)) and productive viral infection with murine cytomegalovirus (mCMV). The primary objective of the study is to define, in an unbiased manner, cause-and-effect relationships in the program of gene activation in this cellular system. The even spacing and time intervals (every 30 minutes) makes this study amenable to modelling of gene networks in the system. A kinetic analysis of IFN-g treatment on the gene expression of murine (BALB/c) bone marrow-derived macrophages (BMDMs) over the first 12 hours, sampling every 30 minutes. Agilent mouse genome arrays were used to determine the differences in gene expression between mock and IFN-g treated. The 25 samples collected for the mock IFN-g treatment were pooled and treated as a pooled control. Pooled control was labelled with Cy3 dye and hybridised with every other sample (labelled with Cy5 dye) = 25 dual-dye array hybridisations. The reference channel of the dual-channel hybridisations was only used to normalise the expression of the test channel, rather than used to calculate ratio data. Thus, the normalised data represent log2 single-channel data.

ORGANISM(S): Mus musculus

SUBMITTER: Nigel Binns

PROVIDER: E-GEOD-42504 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

The transcription factor STAT-1 couples macrophage synthesis of 25-hydroxycholesterol to the interferon antiviral response.

Blanc Mathieu M Hsieh Wei Yuan WY Robertson Kevin A KA Kropp Kai A KA Forster Thorsten T Shui Guanghou G Lacaze Paul P Watterson Steven S Griffiths Samantha J SJ Spann Nathanael J NJ Meljon Anna A Talbot Simon S Krishnan Kathiresan K Covey Douglas F DF Wenk Markus R MR Craigon Marie M Ruzsics Zsolts Z Haas Jürgen J Angulo Ana A Griffiths William J WJ Glass Christopher K CK Wang Yuqin Y Ghazal Peter P

Immunity 20121227 1

Recent studies suggest that the sterol metabolic network participates in the interferon (IFN) antiviral response. However, the molecular mechanisms linking IFN with the sterol network and the identity of sterol mediators remain unknown. Here we report a cellular antiviral role for macrophage production of 25-hydroxycholesterol (cholest-5-en-3β,25-diol, 25HC) as a component of the sterol metabolic network linked to the IFN response via Stat1. By utilizing quantitative metabolome profiling of all ...[more]

PMID: 23273843

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Project description:Intracellular pathogens including the apicomplexan and opportunistic parasite Toxoplasma gondii profoundly modify their host cells in order to establish infection. We have shown previously that intracellular T. gondii inhibit up-regulation of regulatory and effector functions in murine macrophages (MΦ) stimulated with interferon (IFN)-γ, which is the cytokine crucial for controlling the parasites’ replication. Using genome-wide transcriptome analysis we show herein that infection with T. gondii leads to global unresponsiveness of murine macrophages to IFN-γ. More than 61% and 89% of the transcripts, which were induced or repressed by IFN-γ in non-infected MΦ, respectively, were not altered after stimulation of T. gondii-infected cells with IFN-γ. These genes are involved in a variety of biological processes, which are mostly but not exclusively related to immune responses. Analyses of the underlying mechanisms revealed that IFN-γ-triggered nuclear translocation of STAT1 still occurred in Toxoplasma-infected MΦ. However, STAT1 bound aberrantly to oligonucleotides containing the IFN-γ-responsive gamma-activated site (GAS) consensus sequence. Conversely, IFN-γ did not induce formation of active GAS-STAT1 complexes in nuclear extracts from infected MΦ. Mass spectrometry of protein complexes bound to GAS oligonucleotides showed that T. gondii-infected MΦ are unable to recruit non-muscle actin to IFN-γ-responsive DNA sequences, which appeared to be independent of stimulation with IFN-γ and of STAT1 binding. IFN-γ-induced recruitment of BRG-1 and acetylation of core histones at the IFN-γ-regulated CIITA promoter IV, but not β-actin was diminished by >90% in Toxoplasma-infected MΦ as compared to non-infected control cells. Remarkably, treatment with histone deacetylase inhibitors restored the ability of infected macrophages to express the IFN-γ regulated genes H2-A/E and CIITA. Taken together, these results indicate that Toxoplasma-infected MΦ are unable to respond to IFN-γ due to disturbed chromatin remodelling, but can be rescued using histone deacetylase inhibitors. Comparison of 4 different RNA pools with a 2-Color-Loop Design including 10 microarrays: [1] T. gondii infected and IFN-gamma treated, [2] T. gondii infected and untreated, [3] Non-infected and IFN-gamma treated, and [4] Non-infected and untreated.

Project description:Analysis of Mtb infected murine macrophages derived from C57Bl/6 WT, TPL2KO, IFNARKO & TPL2IFNAR DKO mice [Set 1] Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of mortality and morbidity worldwide, causing approximately 1.4 million deaths per year. Key immune components for host protection during tuberculosis include the cytokines IL?12, IL?1 and TNF??, as well as IFN?? and CD4+ Th1 cells. However, immune factors determining whether individuals control infection or progress to active tuberculosis are incompletely understood. Excess amounts of type I interferon have been linked to exacerbated disease during tuberculosis in mouse models and to active disease in patients, suggesting tight regulation of this family of cytokines is critical to host resistance. In addition, the immunosuppressive cytokine IL?10 is known to inhibit the immune response to Mtb in murine models through the negative regulation of key pro-inflammatory cytokines and the subsequent Th1 response. We show here, using a combination of transcriptomic analysis, genetics and pharmacological inhibitors that the TPL-2-ERK1/2 signaling pathway is important in mediating host resistance to tuberculosis through negative regulation of type I interferon production. The TPL-2-ERK1/2 signalling pathway regulated production by macrophages of several cytokines important in the immune response to Mtb as well as regulating induction of a large number of additional genes, many in a type I IFN dependent manner. In the absence of TPL-2 in vivo, excess type I interferon promoted IL-10 production and exacerbated disease. These findings describe an important regulatory mechanism for controlling tuberculosis and reveal mechanisms by which type I interferon may promote susceptibility to this important disease. Macrophages were derived from C57Bl/6 bone marrow, plated and infected with Mtb H37Rv (or not) in duplicate wells. Samples were then harvested for RNA at time 0 (uninfected only), 15m, 30m, 1hr, 3hr, 6hr and 24hr.

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Dataset Information

Timecourse of Interferon-Gamma Treatment of Bone Marrow-Derived Macrophages (BMDMs)

Publications

The transcription factor STAT-1 couples macrophage synthesis of 25-hydroxycholesterol to the interferon antiviral response.

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