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Macrophage Microvesicles Induce Macrophage Differentiation and miR-223 Transfer

ABSTRACT: Microvesicles (MV) are small membrane-bound particles comprised of exosomes and various sized extracellular vesicles. These are released by a number of cell types. Microvesicles have a variety of cellular functions from communication to mediating growth and differentiation. Microvesicles contain proteins and nucleic acids. Previously, we showed that plasma microvesicles contain microRNAs (miRNAs). Based on our previous report, the majority of peripheral blood microvesicles are derived from platelets while mononuclear phagocytes, including macrophages, are the second most abundant population. Here, we characterized macrophage-derived microvesicles and whether they influenced the differentiation of naïve monocytes. We also identified the miRNA content of the macrophage-derived microvesicles. We found that RNA molecules contained in the macrophage-derived microvesicles were transported to target cells, including monocytes, endothelial cells, epithelial cells and fibroblasts. Furthermore, we found that miR-223 was transported to target cells and was functionally active. Based on our observations, we hypothesize that microvesicles bind to and activate target cells. Furthermore, we find that microvesicles induce the differentiation of macrophages. Thus, defining key components of this response may identify novel targets to regulate host defense and inflammation. We used GeneChip microarrays to examine changes in gene expression induced by MV in primary monocyte-derived macrophages (MDM) and in THP1 cells, and compare this to cells treated with GM-CSF and PMA, respectively.

ORGANISM(S): Homo sapiens

PROVIDER: GSE41889 | GEO | 2013/03/08

SECONDARY ACCESSION(S): PRJNA178399

REPOSITORIES: GEO

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Project description:Microvesicles (MV) are small membrane-bound particles comprised of exosomes and various sized extracellular vesicles. These are released by a number of cell types. Microvesicles have a variety of cellular functions from communication to mediating growth and differentiation. Microvesicles contain proteins and nucleic acids. Previously, we showed that plasma microvesicles contain microRNAs (miRNAs). Based on our previous report, the majority of peripheral blood microvesicles are derived from platelets while mononuclear phagocytes, including macrophages, are the second most abundant population. Here, we characterized macrophage-derived microvesicles and whether they influenced the differentiation of naM-CM-/ve monocytes. We also identified the miRNA content of the macrophage-derived microvesicles. We found that RNA molecules contained in the macrophage-derived microvesicles were transported to target cells, including monocytes, endothelial cells, epithelial cells and fibroblasts. Furthermore, we found that miR-223 was transported to target cells and was functionally active. Based on our observations, we hypothesize that microvesicles bind to and activate target cells. Furthermore, we find that microvesicles induce the differentiation of macrophages. Thus, defining key components of this response may identify novel targets to regulate host defense and inflammation. We used GeneChip microarrays to examine changes in gene expression induced by MV in primary monocyte-derived macrophages (MDM) and in THP1 cells, and compare this to cells treated with GM-CSF and PMA, respectively. All experiments were done in triplicates. Primary monocytes were collected from buffy coats (BC). The freshly isolated monocytes from three donors (Mono1-3) were either treated with GM-CSF or subjected to RNA isolation. Following treatment, MVs were isolated from the GM-CSF-treated macrophage cultures. RNA was isolated from the remaining cells for profiling (GM1-3). The isolated MVs were then used to treat new BC monocytes for 24 h (BC-GMCSF-MV24). A fraction of the new BC monocytes was subjected to RNA extraction for profiling (BC1-3). For THP1 cells, they were treated with either DMSO or PMA to produce MVs. The MVs were collected and the remaining cells lyzed for RNA extraction and profiling (DMSO1-3 and PMA1-3). The collected MVs from the DMSO or PMA-treated THP1 cells were incubated with new THP1 for 24 h and designated DMSO-MV24 or PMA-MV24. We had a total of 24 samples.

Project description:Macrophages play a critical role in the pathogenesis of many diseases, including rheumatoid arthritis, inflammatory bowel disease and atherosclerosis. Monocytes recruited into tissues from peripheral blood differentiate into macrophages. There is limited data concerning the global changes in the expression of genes during monocyte to macrophage, and how the patterns of change identify the mechanism contributing to differentiation or macrophage function. Employing the microarray technology, we examined the transcriptional profile of in vitro adherence-induced differentiation of primary human monocytes into macrophages. We found the significant up regulation of genes contributing to the functions of macrophage, including signature patterns defining the induction of genes contributing to immunity and defense; lipid, fatty acid and steroid metabolism; cell adhesion and; carbohydrate metabolism; amino acid metabolism and endocytosis. In contrast, a variety of transcription factors were down regulated during monocyte to macrophage differentiation, suggesting that transcriptional repression may be important for the transition from monocytes to macrophages. However, a limited number of transcription factors were up regulated, among these was C/EBPA, which may contribute to differentiation by regulating down stream genes, which a characteristic of differentiated macrophages. These observations suggest that examination of the transcriptional profile in monocytes and macrophages in patients may identify relevant therapeutic targets in diseases such as rheumatoid arthritis and atherosclerosis. Experiment Overall Design: Total 9 samples were analyzed. Repeats of 3 total RNA samples from monocytes at 0 hour, 3 samples from monocytes at 16 hours, and other 3 samples of monocytes at 168 hours after differentiation were collected. Following the Affymetrix protocol, 5-10 mg of high quality RNA was used to make labeled cRNA. The size distribution of fragmented and unfragmented labeled cRNA were assessed by 2100 Bioanalyzer (Agilent Technologies). 20mg of fragmented labeled cRNA was hybridized to Human U133A GeneChip at 45C for 18 hours. Affymetrix fluidics station was used to perform the washing and staining of the chips. GeneChip 3000 scanner system was used to scan the chips. The data were normalized by dCHIP method.

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Macrophage Microvesicles Induce Macrophage Differentiation and miR-223 Transfer

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