Dissecting Macrophage-Derived Microvesicles' Content and Function.

Abstract 3785 Microvesicles (MVs) are small membrane-bound vesicles released under normal homeostatic and stimulatory conditions by a wide variety of cell types. Microvesicles are collectively referred to as exosomes and microparticles which vary in size due to different cellular mechanisms responsible for their production. These microvesicles have a wide range of functions from facilitating communication to regulating cellular growth and differentiation. During their production; microvesicles become enriched in various molecules including proteins and nucleic acids. Previously, we have shown that plasma microvesicles derived from many cell lineages contain microRNAs (miRNAs). We also found that the majority of the peripheral blood microvesicles are derived from platelets while those originating from monocytic cells including macrophages represent the second largest population. Since microvesicles derived from mononuclear phagocytes are a large subpopulation in the plasma; we were interested in understanding their content and function. We hypothesized that the content and/or quantity of macrophage-derived microvesicles could induce the maturation of monocytes. To address our hypothesis, peripheral blood monocytes were treated in vitro for 4hr with GM-CSF; washed and cultured in media devoid of cytokines for 24 h then microvesicles were collected. Flow cytometry and electron)confocal microscropy were used to quantify and visualize microvesicles production. To examine the function of the microvesicles on macrophage maturation, the purified microvesicles were then cultured with freshly isolated monocytes. Macrophage differentiation was determined by cellular adherence using a crystal violet uptake assay and changes in surface antigen expression by flow cytometry. We also examined the genetic changes induced in monocytes incubated with the microvesicles compared to GM-CSF-treated cells. We found that freshly isolated monocytes treated with microvesicles from macrophages acquired phenotypic characteristics of a macrophage such as cellular adherence and surface antigen expression. We also found that treatment of naïve monocytes with the microvesicles induced molecular changes similar to GM-CSF treated monocytes. We found more than 7985 mRNAs that were similarly expressed between the two culture conditions. Notably, we observed the unique expression of 1324 and 1079 genes in the GM-CSF-treated compared to the microvesicle-treated cells, respectively. To begin dissecting the molecules contained in the microvesicles responsible for these changes, we performed mass-spectrometry and miRNA profiling. We observed the expression of miRs-223, -222,-191, -484, -016, -026a, and -155 in GM-CSF-derived microvesicles. Notably, these miRNAs were also expressed in the cells from which the microvesicles were released. We have begun bioinformatics analyses to predict whether the expression of the miRNAs may account for the decrease expression of specific genes in cells treated with the microvesicles that undergo differentiation. Many of the proteins found in the vesicles are important in facilitating protein:protein interactions and nucleic acid binding. Based on our observations; we postulate that microvesicles in areas of inflammation may contribute to the inflammatory response through the maturation of immune cells and activation of cells responsible for tissue repair. Thus, defining key components of this response may identify targets to regulate inflammation. Disclosures: No relevant conflicts of interest to declare.