Project description:RNA modifications are essential for the establishment of cellular identity. Although increasing evidence indicates that RNA modifications control the innate immune response, their role in monocyte-to-macrophage differentiation and polarisation is unclear. We profile 5hmC epitranscriptomes of monocytes and macrophages at resting, pro- and anti-inflammatory states.
Project description:Epigenetic modifications, such as cytosine methylation and histone modification, have been shown involved in the pathology of ischemic brain injury. Recent works have implicated 5-hydroxymethylcytosine (5hmC), a DNA base derived from 5-methylcytosine (5mC) through the oxidation by Ten-Eleven Translocation (TET) enzymes, in DNA methylation-related plasticity. In this study we show that 5hmC abundance could be induced to increase by ischemia injury. Genome-wide profiling of 5hmC identified differentially hydroxymethylated regions (DhMRs) associated with ischemic injury and DhMRs were found enriched among the genes involved in cell junction, neuronal morphogenesis and neurodevelopment. These data together suggest that 5hmC modification could serve as a potential therapeutic target for the treatment of ischemic stroke. To determine the genome-wide 5hmC distribution in both ischemic injury (I/R) and control mice (C57BL/6), we employed a previously established chemical labeling and affinity purification method, coupled with high-throughput sequencing (Song et al, Nature Biotechnology, 2011). The ischemic or matched control brain tissues from three pairs of ischemic mice and control mice were used for the analyses.
Project description:RNA modifications are essential for the establishment of cellular identity. Although increasing evidence indicates that RNA modifications control the innate immune response, their role in monocyte-to-macrophage differentiation and polarisation is unclear. We profile m6A epitranscriptomes of monocytes and macrophages at resting, pro- and anti-inflammatory states.
Project description:Epigenetic modifications, such as cytosine methylation and histone modification, have been shown involved in the pathology of ischemic brain injury. Recent works have implicated 5-hydroxymethylcytosine (5hmC), a DNA base derived from 5-methylcytosine (5mC) through the oxidation by Ten-Eleven Translocation (TET) enzymes, in DNA methylation-related plasticity. In this study we show that 5hmC abundance could be induced to increase by ischemia injury. Genome-wide profiling of 5hmC identified differentially hydroxymethylated regions (DhMRs) associated with ischemic injury and DhMRs were found enriched among the genes involved in cell junction, neuronal morphogenesis and neurodevelopment. These data together suggest that 5hmC modification could serve as a potential therapeutic target for the treatment of ischemic stroke.
Project description:So far, although differentiation and activation of Ag presenting cells, including monocytes, dendritic cells (DCs) and macrophages are accompanied by programmed remodelling of cell surface glycosylated molecules with potentially biologically important consequences, no global analysis of the expression of genes involved in glycan biosynthesis (essentially glycosyltransferases (GTs) and modification (mainly sulfotransferases) has been reported in these cells. Our project aims to obtain global information relating to the expression of genes encoding GTs and sulfotransferases in human monocytes, DCs and macrophages isolated or differentiated from the same donor. We also aimed to compare the expression profile of these genes in immature versus mature (immunostimulatory phenotype) DCs and macrophages (LPS stimulation, 4 and 18 hrs). To this end we have performed three independent experiments (n = 7 x 3 samples) using the dedicated gene microarray (glycogene-chip v3). Although, globally the data are interesting(please see the summary below), they lack significance and are not yet exploitable. The relatively high variability between samples probably results from the human donor sources given their heterogeneous genetic makeup and natural history. Thus, to reach significance, we believe that additional donors need to be analyzed. Of note, the time point “4hrs” can be omitted since it does not appear to be indicative. Three x 5 samples would be thus necessary. Utilizing a focused gene microarray, we intended to compare the glycosyltransferase (GT) and sulfotransferase gene expression profile of human monocytes relative to immature dendritic cells (DCs) and to macrophages and to characterize variations in DCs and macrophages undergoing maturation. Micro-array analysis indicated that monocytes express transcripts for a full set of enzymes involved in the biosynthesis of N- and O-glycans potentially elongated by poly-LacNAc chains with type II terminal sequences. Monocytes also express, at a very good level, genes encoding enzymes involved in glycosaminoglycan biosynthesis, but seem to have a limited capacity for glycolipid biosynthesis. Immature DCs and macrophages exhibit similar pattern of GT and sulfotransferase expression with only a limited number of cell type specific gene expression. Approximately 15 % of GT and sulfotransferase transcripts varied in both DCs and macrophages, the majority of them being up-regulated compared to monocytes. Strikingly, stimulation of DCs and Ms with lipopolysaccharide caused a general alteration in gene expression, in particular in DCs, mainly affecting genes found to be positively modulated during the differentiation steps. LPS treatment also caused a relatively minor increase in gene expression in macrophages whereas in DCs, the expression levels of a significant number of genes were enhanced. Validation of this analysis was partially provided by quantitative RT-PCR and flow cytometry of cell surface glycan epitopes. Collectively, this preliminary study implies an important modification of the pattern of glycosylation in differentiated and activated APCs with potential biological consequences. Our project aims to obtain global information relating to the expression of genes encoding GTs and sulfotransferases in human monocytes, DCs and macrophages isolated or differentiated from the same donor. We also aimed to compare the expression profile of these genes in immature versus mature (immunostimulatory phenotype) DCs and macrophages (LPS stimulation, 4 and 18 hrs).
Project description:We developed a simplified flow cytometry strategy in order to discriminate monocytes and macrophages in the lung of C57BL/6 mice. Using this strategy, we identified autofluorescent F4/80+ CD11c+ alveolar macrophages, non-autofluorescent CD64+Ly-6C- interstitial macrophages and Ly-6Chi monocytes residing in the lung of WT mice. A fraction of these Ly-6Chi monocytes corresponded to classical blood monocytes associated with the lung vasculature, but another fraction did not depend on CCR2, the chemokine receptor required for monocytes to egress from the bone marrow, as a population of lung Ly-6Chi monocytes was also present in the lung of Ccr2-/- mice. A remaining question was whether lung monocytes represented a particular population of monocytes that could be distinguishable from the classical CCR2-dependent blood monocytes. To address this issue, we performed a transcriptomic comparison of Ly-6Chi monocytes recovered from flushed lung of WT mice (â60% of CCR2- dependent classical blood monocytes and â40% of lung monocytes) and Ccr2-/- mice (more than 95% of lung monocytes). In addition, we tested whether exposure to TLR ligands would affect interstitial macrophages, and we compared to transcriptome of IM at steady-state and IM 1 week after administration of 50 µg CpG-DNA intratracheally.
Project description:Maternal and fetal monocytes and tissue macrophages (decidual macrophages, Hofbauer cells) at the feto-maternal interface have different methylome. Paired and balanced design. We compared maternal blood monocytes (MB) vs. cord blood monocytes (CB), maternal blood monocytes (MB) vs. decidual macrophages (Deci), cord blood monocytes (CB) vs placental macrophages (villi) and decidual macrophages (Deci) vs. placental macrophages (villi).