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• W2214005756 abstract "Membrane-coated extracellular vesicles (EVs) released by cells can serve as vehicles for delivery of biological materials and signals. Recently, we demonstrated that alcohol-treated hepatocytes cross-talk with immune cells via exosomes containing microRNA (miRNAs). Here, we hypothesized that alcohol-exposed monocytes can communicate with naive monocytes via EVs. We observed increased numbers of EVs, mostly exosomes, secreted by primary human monocytes and THP-1 monocytic cells in the presence of alcohol in a concentration- and time-dependent manner. EVs derived from alcohol-treated monocytes stimulated naive monocytes to polarize into M2 macrophages as indicated by increased surface expression of CD68 (macrophage marker), M2 markers (CD206 (mannose receptor) and CD163 (scavenger receptor)), secretion of IL-10, and TGFβ and increased phagocytic activity. miRNA profiling of the EVs derived from alcohol-treated THP-1 monocytes revealed high expression of the M2-polarizing miRNA, miR-27a. Treatment of naive monocytes with control EVs overexpressing miR-27a reproduced the effect of EVs from alcohol-treated monocytes on naive monocytes and induced M2 polarization, suggesting that the effect of alcohol EVs was mediated by miR-27a. We found that miR-27a modulated the process of phagocytosis by targeting CD206 expression on monocytes. Importantly, analysis of circulating EVs from plasma of alcoholic hepatitis patients revealed increased numbers of EVs that contained high levels of miR-27a as compared with healthy controls. Our results demonstrate the following: first, alcohol increases EV production in monocytes; second, alcohol-exposed monocytes communicate with naive monocytes via EVs; and third, miR-27a cargo in monocyte-derived EVs can program naive monocytes to polarize into M2 macrophages. Membrane-coated extracellular vesicles (EVs) released by cells can serve as vehicles for delivery of biological materials and signals. Recently, we demonstrated that alcohol-treated hepatocytes cross-talk with immune cells via exosomes containing microRNA (miRNAs). Here, we hypothesized that alcohol-exposed monocytes can communicate with naive monocytes via EVs. We observed increased numbers of EVs, mostly exosomes, secreted by primary human monocytes and THP-1 monocytic cells in the presence of alcohol in a concentration- and time-dependent manner. EVs derived from alcohol-treated monocytes stimulated naive monocytes to polarize into M2 macrophages as indicated by increased surface expression of CD68 (macrophage marker), M2 markers (CD206 (mannose receptor) and CD163 (scavenger receptor)), secretion of IL-10, and TGFβ and increased phagocytic activity. miRNA profiling of the EVs derived from alcohol-treated THP-1 monocytes revealed high expression of the M2-polarizing miRNA, miR-27a. Treatment of naive monocytes with control EVs overexpressing miR-27a reproduced the effect of EVs from alcohol-treated monocytes on naive monocytes and induced M2 polarization, suggesting that the effect of alcohol EVs was mediated by miR-27a. We found that miR-27a modulated the process of phagocytosis by targeting CD206 expression on monocytes. Importantly, analysis of circulating EVs from plasma of alcoholic hepatitis patients revealed increased numbers of EVs that contained high levels of miR-27a as compared with healthy controls. Our results demonstrate the following: first, alcohol increases EV production in monocytes; second, alcohol-exposed monocytes communicate with naive monocytes via EVs; and third, miR-27a cargo in monocyte-derived EVs can program naive monocytes to polarize into M2 macrophages. Extracellular vesicles (EVs), 3The abbreviations used are: EV, extracellular vesicle; AH, alcoholic hepatitis; ALD, alcoholic liver disease; EtOH, ethanol; MFI, mean fluorescence intensity; MV, microvesicles; miRNA/miR, microRNA; NTA, nanoparticle tracking analysis; qPCR, quantitative PCR. including exosomes and microvesicles (MVs), are heterogeneous, membranous, and cell-derived vesicles that are released by almost every cell type into their microenvironment. EVs have a lipid composition similar to those present on the plasma membranes of the parent cells (1Ratajczak J. Wysoczynski M. Hayek F. Janowska-Wieczorek A. Ratajczak M.Z. Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication.Leukemia. 2006; 20: 1487-1495Crossref PubMed Scopus (1058) Google Scholar, 2Kalra H. Simpson R.J. Ji H. Aikawa E. Altevogt P. Askenase P. Bond V.C. Borràs F.E. Breakefield X. Budnik V. Buzas E. Camussi G. Clayton A. Cocucci E. Falcon-Perez J.M. et al.Vesiclepedia: a compendium for extracellular vesicles with continuous community annotation.PLos Biol. 2012; 10: e1001450Crossref PubMed Scopus (861) Google Scholar3Momen-Heravi F. Balaj L. Alian S. Mantel P.Y. Halleck A.E. Trachtenberg A.J. Soria C.E. Oquin S. Bonebreak C.M. Saracoglu E. Skog J. Kuo W.P. Current methods for the isolation of extracellular vesicles.Biol. Chem. 2013; 394: 1253-1262Crossref PubMed Scopus (401) Google Scholar). EVs act as signaling conveyors in cell-to-cell communication and contain bioactive molecules, including proteins, microRNAs (miRNAs), and messenger RNAs (mRNAs). Based on the mode of biogenesis, EVs can be categorized into two main classes as follows: shedding MVs (originating from plasma membrane) and exosomes (derived from multivesicular bodies) (2Kalra H. Simpson R.J. Ji H. Aikawa E. Altevogt P. Askenase P. Bond V.C. Borràs F.E. Breakefield X. Budnik V. Buzas E. Camussi G. Clayton A. Cocucci E. Falcon-Perez J.M. et al.Vesiclepedia: a compendium for extracellular vesicles with continuous community annotation.PLos Biol. 2012; 10: e1001450Crossref PubMed Scopus (861) Google Scholar). Exosomal markers are CD63, CD81, and CD9, whereas microvesicles are annexin V-positive (3Momen-Heravi F. Balaj L. Alian S. Mantel P.Y. Halleck A.E. Trachtenberg A.J. Soria C.E. Oquin S. Bonebreak C.M. Saracoglu E. Skog J. Kuo W.P. Current methods for the isolation of extracellular vesicles.Biol. Chem. 2013; 394: 1253-1262Crossref PubMed Scopus (401) Google Scholar). Based on size distribution and CD63 expression, exosomes are defined as 40–150 nm and MVs as 150–1000 nm (4Momen-Heravi F. Bala S. Bukong T. Szabo G. Exosome-mediated delivery of functionally active miRNA-155 inhibitor to macrophages.Nanomedicine. 2014; 10: 1517-1527Crossref PubMed Scopus (208) Google Scholar, 5Pap E. The role of microvesicles in malignancies.Adv. Exp. Med. Biol. 2011; 714: 183-199Crossref PubMed Scopus (27) Google Scholar). Recently, EVs, including exosomes, have emerged as important signaling organelles in mediating local and systemic communication between cells and in the pathogenesis of different diseases (6György B. Szabó T.G. Pásztói M. Pál Z. Misják P. Aradi B. László V. Pállinger E. Pap E. Kittel A. Nagy G. Falus A. Buzás E.I. Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles.Cell. Mol. Life Sci. 2011; 68: 2667-2688Crossref PubMed Scopus (1472) Google Scholar7El Gazzar M. McCall C.E. MicroRNAs regulatory networks in myeloid lineage development and differentiation: regulators of the regulators.Immunol. Cell Biol. 2012; 90: 587-593Crossref PubMed Scopus (41) Google Scholar, 8Taylor D.D. Gercel-Taylor C. The origin, function, and diagnostic potential of RNA within extracellular vesicles present in human biological fluids.Front. Genet. 2013; 4: 142Crossref PubMed Scopus (103) Google Scholar9de Vrij J. Maas S.L. Kwappenberg K.M. Schnoor R. Kleijn A. Dekker L. Luider T.M. de Witte L.D. Litjens M. van Strien M.E. Hol E.M. Kroonen J. Robe P.A. Lamfers M.L. Schilham M.W. Broekman M.L. Glioblastoma-derived extracellular vesicles modify the phenotype of monocytic cells.Int. J. Cancer. 2015; 137: 1630-1642Crossref PubMed Scopus (135) Google Scholar). The pathogenesis of alcohol-induced liver disease is complex and is associated with liver abnormalities ranging from steatosis and inflammation to cirrhosis and hepatocellular carcinoma (10.Mandrekar, P., and Ambade, A., (2012) in Cellular Signaling Pathways in Alcoholic Liver Disease, Trends in Alcoholic Liver Disease Research–Clinical and Scientific Aspects (Shimizu, I., ed) 10.5772/27412,Google Scholar, 11Gao B. Bataller R. Alcoholic liver disease: pathogenesis and new therapeutic targets.Gastroenterology. 2011; 141: 1572-1585Abstract Full Text Full Text PDF PubMed Scopus (1319) Google Scholar12Szabo G. Gut-liver axis in alcoholic liver disease.Gastroenterology. 2015; 148: 30-36Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar). The intercellular signaling during alcoholic liver disease (ALD) is key to its progression and pathogenesis (13Beier J.I. McClain C.J. Mechanisms and cell signaling in alcoholic liver disease.Biol. Chem. 2010; 391: 1249-1264Crossref PubMed Google Scholar, 14Louvet A. Mathurin P. Alcoholic liver disease: mechanisms of injury and targeted treatment.Nat. Rev. Gastroenterol. Hepatol. 2015; 12: 231-242Crossref PubMed Scopus (532) Google Scholar). Our previous studies explored the role of exosomes in various liver diseases and showed that circulating miRNAs in exosomes may serve as biomarkers to differentiate between different types of hepatocyte injury and inflammation (15Bala S. Petrasek J. Mundkur S. Catalano D. Levin I. Ward J. Alao H. Kodys K. Szabo G. Circulating microRNAs in exosomes indicate hepatocyte injury and inflammation in alcoholic, drug-induced, and inflammatory liver diseases.Hepatology. 2012; 56: 1946-1957Crossref PubMed Scopus (505) Google Scholar). Recent data showed a role for exosomes in communication between hepatocytes and monocytes/macrophages via horizontal transfer of hepatocyte-derived miR-122 to monocytes in the presence of alcohol (16Momen-Heravi F. Bala S. Kodys K. Szabo G. Exosomes derived from alcohol-treated hepatocytes horizontally transfer liver specific miRNA-122 and sensitize monocytes to LPS.Sci. Rep. 2015; 5: 9991Crossref PubMed Scopus (230) Google Scholar). Innate immune cells, including monocytes and macrophages, play an important role in the pathogenesis of ALD. However, the role of immune cell-derived EVs in intercellular signaling and communication has yet to be explored in alcohol-induced immunoregulation. Monocytes are the precursors of macrophages. In response to various signals, monocytes differentiate into macrophages with a polarized phenotype in the spectrum of M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes (17Mosser D.M. Edwards J.P. Exploring the full spectrum of macrophage activation.Nat. Rev. Immunol. 2008; 8: 958-969Crossref PubMed Scopus (6181) Google Scholar, 18Martinez F.O. Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment.F1000Prime Rep. 2014; 6: 13Crossref PubMed Scopus (2879) Google Scholar19Sica A. Mantovani A. Macrophage plasticity and polarization: in vivo veritas.J. Clin. Invest. 2012; 122: 787-795Crossref PubMed Scopus (3876) Google Scholar). Increasing evidence suggests that miRNAs, a class of non-coding RNAs, play an important role in macrophage polarization (7El Gazzar M. McCall C.E. MicroRNAs regulatory networks in myeloid lineage development and differentiation: regulators of the regulators.Immunol. Cell Biol. 2012; 90: 587-593Crossref PubMed Scopus (41) Google Scholar, 20Graff J.W. Dickson A.M. Clay G. McCaffrey A.P. Wilson M.E. Identifying functional microRNAs in macrophages with polarized phenotypes.J. Biol. Chem. 2012; 287: 21816-21825Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 21O'Connell R.M. Rao D.S. Chaudhuri A.A. Baltimore D. Physiological and pathological roles for microRNAs in the immune system.Nat. Rev. Immunol. 2010; 10: 111-122Crossref PubMed Scopus (1257) Google Scholar22Zhang Y. Zhang M. Zhong M. Suo Q. Lv K. Expression profiles of miRNAs in polarized macrophages.Int. J. Mol. Med. 2013; 31: 797-802Crossref PubMed Scopus (137) Google Scholar). We and others have shown that miRNAs can regulate cellular differentiation and functions in immune cells (21O'Connell R.M. Rao D.S. Chaudhuri A.A. Baltimore D. Physiological and pathological roles for microRNAs in the immune system.Nat. Rev. Immunol. 2010; 10: 111-122Crossref PubMed Scopus (1257) Google Scholar, 23Sharbati S. Sharbati J. Hoeke L. Bohmer M. Einspanier R. Quantification and accurate normalisation of small RNAs through new custom RT-qPCR arrays demonstrates Salmonella-induced microRNAs in human monocytes.BMC Genomics. 2012; 13: 23Crossref PubMed Scopus (34) Google Scholar, 24Saha B. Bruneau J.C. Kodys K. Szabo G. Alcohol-induced miR-27a regulates differentiation and M2 macrophage polarization of normal human monocytes.J. Immun. 2015; 194: 3079-3087Crossref PubMed Scopus (69) Google Scholar). miRNAs not only function within the cells of origin but can also be packaged into EVs and released into the extracellular environment, where they are transferred in an active form to other cells and exert functional effects (16Momen-Heravi F. Bala S. Kodys K. Szabo G. Exosomes derived from alcohol-treated hepatocytes horizontally transfer liver specific miRNA-122 and sensitize monocytes to LPS.Sci. Rep. 2015; 5: 9991Crossref PubMed Scopus (230) Google Scholar, 25Arroyo J.D. Chevillet J.R. Kroh E.M. Ruf I.K. Pritchard C.C. Gibson D.F. Mitchell P.S. Bennett C.F. Pogosova-Agadjanyan E.L. Stirewalt D.L. Tait J.F. Tewari M. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 5003-5008Crossref PubMed Scopus (2495) Google Scholar26Guay C. Menoud V. Rome S. Regazzi R. Horizontal transfer of exosomal microRNAs transduce apoptotic signals between pancreatic beta-cells.Cell Commun. Signal. 2015; 13: 17Crossref PubMed Scopus (106) Google Scholar, 27Landmann R. Ludwig C. Obrist R. Obrecht J.P. Effect of cytokines and lipopolysaccharide on CD14 antigen expression in human monocytes and macrophages.J. Cell. Biochem. 1991; 47: 317-329Crossref PubMed Scopus (65) Google Scholar28Bala S. Csak T. Momen-Heravi F. Lippai D. Kodys K. Catalano D. Satishchandran A. Ambros V. Szabo G. Biodistribution and function of extracellular miRNA-155 in mice.Sci. Rep. 2015; 5: 10721Crossref PubMed Scopus (103) Google Scholar). The possible contribution of circulating miRNAs transported via EVs to signal between monocytes and activate the process of monocyte differentiation and/or polarization has yet to be explored. In this study, we investigated the effect of alcohol on EV secretion by human monocytes and studied the role of alcohol-induced EVs as an alternative mode of cell-to-cell communication between monocytes. Our data demonstrate that the total number of EVs secreted from alcohol-treated monocytes is significantly increased as compared with untreated monocytes. EVs originating from alcohol-exposed monocytes induced differentiation and polarization of naive monocytes into M2 macrophages. We demonstrated the role for miR-27a in EVs from alcohol-treated monocytes in inducing activation, polarization, and increased phagocytic activity of naive monocytes. These in vitro observations were validated in vivo where we found an increased number of EVs in the circulation of patients with alcoholic hepatitis, which had increased levels of miR-27a in the EV cargo. Our results suggest that in the pathogenesis of alcoholic hepatitis, EVs play an important role in cell-cell communication between monocytes/macrophages. Confirmed cases of alcoholic hepatitis (n = 8) and healthy individuals (n = 8) were enrolled. The diagnosis of alcoholic hepatitis was performed by expert clinicians based on the patients' medical history, physical examination, and laboratory data and according to the guidelines of the American College of Gastroenterology (29O'Shea R.S. Dasarathy S. McCullough A.J. Practice Guideline Committee of the American Association for the Study of Liver Diseases, and Practice Parameters Committee of the American College of Gastroenterology Alcoholic liver disease.Hepatology. 2010; 51: 307-328Crossref PubMed Scopus (952) Google Scholar). Healthy controls were defined as being free of any systemic and non-systemic diseases based on patients' history and routine laboratory findings performed by primary care physicians and a history of “only social” alcohol consumption. To avoid selection bias, healthy controls and patients were enrolled consecutively. The study protocol was approved by the Institutional Review Board for the Protection of Human Subjects in Research at the University of Massachusetts Medical School (Worcester, MA), and written informed consents were obtained from all subjects. We obtained plasma samples from controls and patients for the study. RPMI 1640 cell culture media, antibiotics, and nonessential amino acids were purchased from Gibco. Exosome-depleted FBS (Exo-FBSTM) was purchased from System Biosciences (Mountain View, CA). CD14+ monocytes were isolated by MACS CD14 microbeads from Miltenyi Biotec (Auburn, CA). Human antibodies CD16 APC, CD14 FITC, and CD86 FITC were purchased from eBioscience (San Diego). Antibodies CD163 PE, CD68 PE, CD206 APC, and isotype control antibodies were purchased from Pharmingen. CD63 antibody was purchased from Abcam (Cambridge, MA). miR-27a mimic and scrambled control were purchased from Ambion Life Technologies, Inc. Dextran-FITC beads were purchased from Sigma. Primary human monocytes and THP-1 cells were cultured in RPMI 1640 cell culture media containing antibiotics, nonessential amino acids, and FBS. For EV isolation and quantification experiments, the cells were cultured in the presence of RPMI 1640 medium plus 10% exosome-depleted FBS (Exo-FBSTM) and 1% penicillin/streptomycin. For ethanol treatments, 25, 50, and 100 mm ethanol were added to the cells for various time points (24 and 48 h). At desired time points, culture media were harvested, and EVs were isolated or quantified using a NanoSight NS300 system (NanoSight, Amesbury, UK). For EV isolation from ethanol-treated and normal THP-1 cells, supernatants were centrifuged at 1500 × g for 10 min to remove cells followed by 10,000 × g for 20 min to deplete residual cellular debris, and then the supernatant was filtered through a 0.8-μm filter. The filtered supernatant was used to precipitate EVs with ExoQuick-TCTM, according to the manufacturer's guidelines (System Biosciences, Mountain View, CA). After isolation, EVs were resuspended in PBS. The concentration of EVs was determined by Bradford assay (30Momen-Heravi F. Saha B. Kodys K. Catalano D. Satishchandran A. Szabo G. Increased number of circulating exosomes and their microRNA cargos are potential novel biomarkers in alcoholic hepatitis.J. Transl. Med. 2015; 13: 261Crossref PubMed Scopus (215) Google Scholar). For co-culture experiments, EVs isolated from THP-1 cells or primary monocytes (ethanol-treated or non-treated) or from plasma of patients with alcoholic hepatitis or from healthy controls were added to normal primary human monocytes in the concentration of 25–50 μg/ml (50 μl). This concentration was comparable with EVs secreted by primary monocytes in the presence of ethanol. The monocytes were stimulated with EVs (25 μl) on day 1 and day 3 and cultured for 5 days. The concentration and diameter of EVs derived from culture supernatant from primary monocytes, THP-1 cells, and patient plasma samples were identified by a NanoSight equipped with a fast video capture and NTA software. Before using the samples, the instrument was calibrated with 100 nm polystyrene beads (Thermo Scientific, Fremont, CA). The samples were captured for 90 s at room temperature. NTA software was used to measure particle concentration (particles/ml) and size distribution (in nanometers). For each sample, three measurements were taken, and the average value was determined. THP-1 human monocytes were plated into 12-well plates and were left untreated or treated with 50 mm ethanol for 24 h. After 24 h, ethanol-treated THP-1 cells and untreated cells were fixed with 2.5% glutaraldehyde in Sorenson Phosphate buffer (0.1 m, pH 7.4) for 2 h. The cells were then given three 5-min washes with PBS and fixed with 1% osmium tetroxide (OsO4) in 0.1 m PBS for 1 h. Next, the samples were rinsed and put through an alcohol dehydration series. The samples were mounted on aluminum, and the mounted specimens were coated with gold/palladium using a gold sputter coater. Then the samples were analyzed and examined with a MKII FEI Quanta 200 FEG MKII scanning electron microscope (FEI, Netherlands). EVs were isolated from THP-1 cells, and Western blot analysis for the exosomal marker CD63 was performed. EVs were resuspended in RIPA buffer, and the proteins were extracted. The proteins were run on a 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and blocked for 1 h in TBS containing 5% nonfat dry milk and 0.1% Tween 20 at room temperature (TBST). The blot was then incubated overnight with primary CD63 antibody at 4 °C on a rocker. The following day, the blot was washed three times with TBST and then incubated with the HRP-conjugated secondary antibody (goat anti-mouse IgG-HRP antibody, Santa Cruz Biotechnology, Dallas, TX) for 1 h at a dilution of 1:10,000. ClarityTM Western ECL substrate kit (Bio-Rad) was used to develop the blot, and the CD63 protein band was visualized with a Fujifilm LAS-4000 luminescent image analyzer. Cell surface marker analysis on monocytes/macrophages was performed by flow cytometry as described previously (31Zhang S. Kodys K. Li K. Szabo G. Human type 2 myeloid dendritic cells produce interferon-λ and amplify interferon-α in response to hepatitis C virus infection.Gastroenterology. 2013; 144: 414-425Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). FcR blocking reagent (BD Biosciences) was used to inhibit nonspecific binding of antibodies. The cells were incubated with appropriate antibody or isotype control for 30 min at 4 °C. Cells were washed with FACS buffer (PBS + 2% FBS) and acquired on a BD-LSR II (BD Biosciences). Data analysis was performed on FlowJo software (Tree Star, Ashland, OR). For CD14+ monocytes, we gated on the various markers and plotted the percentage of positive cells. The mean fluorescence intensity (MFI) is shown by histogram plots. Protein levels of TNFα, IL-10, IL-12, and TGFβ were measured in cell-free culture supernatant by ELISA. Whole blood cells isolated from human subjects were incubated overnight at 37 °C, and the levels of IL-10 and TGF-β secretion were determined by ELISA. Levels of TNFα, IL-12 (BD Biosciences), IL-10, and TGFβ (eBioscience, San Diego) were measured based on the manufacturers' recommendations and quantified using an ELISA reader. Mannose receptor-mediated endocytosis was measured as the cellular uptake of FITC-dextran and quantified by flow cytometry (32Op den Brouw M.L. Binda R.S. Geijtenbeek T.B. Janssen H.L. Woltman A.M. The mannose receptor acts as hepatitis B virus surface antigen receptor mediating interaction with intrahepatic dendritic cells.Virology. 2009; 393: 84-90Crossref PubMed Scopus (39) Google Scholar33Piemonti L. Monti P. Allavena P. Leone B.E. Caputo A. Di Carlo V. Glucocorticoids increase the endocytic activity of human dendritic cells.Int. Immunol. 1999; 11: 1519-1526Crossref PubMed Scopus (78) Google Scholar, 34Malathi K. Dong B. Gale Jr., M. Silverman R.H. Small self-RNA generated by RNase L amplifies antiviral innate immunity.Nature. 2007; 448: 816-819Crossref PubMed Scopus (466) Google Scholar35Stuart L.M. Ezekowitz R.A. Phagocytosis: elegant complexity.Immunity. 2005; 22: 539-550Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar). For the phagocytosis assay, ∼2 × 105 cells per sample were incubated in medium containing 1 mg/ml FITC-dextran beads for 120 min at 4 or 37 °C. After incubation, cells were washed with PBS containing 2% FBS. Phagocytic assays performed at 4 °C were used as negative controls (i.e. a reduced phagocytosis condition) and accounted for the particles bound to the cells' extracellular surface. The cells were acquired in the LSR II (BD Biosciences) instrument to analyze the uptake of FITC-dextran beads by the cells. The phagocytosis was calculated by normalizing with the phagocytosis at 4 °C. The phagocytosis index was calculated as described previously (36Wang Z. Fayngerts S. Wang P. Sun H. Johnson D.S. Ruan Q. Guo W. Chen Y.H. TIPE2 protein serves as a negative regulator of phagocytosis and oxidative burst during infection.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 15413-15418Crossref PubMed Scopus (74) Google Scholar). We loaded THP-1-derived EVs with miR-27a mimic and scrambled mimic according to our previously optimized protocol (4Momen-Heravi F. Bala S. Bukong T. Szabo G. Exosome-mediated delivery of functionally active miRNA-155 inhibitor to macrophages.Nanomedicine. 2014; 10: 1517-1527Crossref PubMed Scopus (208) Google Scholar). Briefly, EVs were diluted in Gene Pulser® electroporation buffer (Bio-Rad) at a ratio of 1:1. miR-27a mimic or scrambled mimic at a final amount of 400 pmol was added to EV samples containing 1 μg/μl EV protein. For the dose-dependent experiments, 300, 100, and 40 pmol were loaded into the EVs. The mixtures (200 μl) were transferred into cold 0.2-cm electroporation cuvettes and electroporated at 150 kV and 100 microfarads with a Gene Pulser II System (Bio-Rad). The EVs were treated with 1 unit of RNase H to eliminate free-floating miR-27a mimic or scrambled mimic outside the EVs and were re-isolated using ExoQuick-TCTM. Based on the loading efficiency that we established earlier (4Momen-Heravi F. Bala S. Bukong T. Szabo G. Exosome-mediated delivery of functionally active miRNA-155 inhibitor to macrophages.Nanomedicine. 2014; 10: 1517-1527Crossref PubMed Scopus (208) Google Scholar) and dilution of EVs, a final concentration of 10, 25, and 75 pmol of miR-27a mimic was used in the co-culture experiments with primary human monocytes. EVs were lysed in QIAzol lysis reagent (Qiagen), and total RNA was isolated using Direct-zolTM RNA MiniPrep isolation kit (Zymo Research Corp, Irvine, CA). Optical density (260/280 and 260/230 ratios) was measured to check RNA quality and quantity. 50 μl of EV suspension from the supernatant of ethanol-treated cells or normal cells was mixed with 500 μl of QIAzol lysis buffer, and the mixture was processed according to the manufacturer's protocol. The extracted RNA was eluted with 25 μl of RNase-free water. For miRNA analysis of exosomes, synthetic Caenorhabditis elegans (cel)-miR-39 was added during the total RNA isolation process and was used to normalize the qPCR data. TaqMan® miRNA assays (Applied Biosystems, Foster City, CA) were used for detection of miR-27a, miR-146a, and miR-9 expression according to the manufacturer's protocol, as described previously (4Momen-Heravi F. Bala S. Bukong T. Szabo G. Exosome-mediated delivery of functionally active miRNA-155 inhibitor to macrophages.Nanomedicine. 2014; 10: 1517-1527Crossref PubMed Scopus (208) Google Scholar). The miRNA levels were normalized, and the relative expression of the specific miRNAs was presented by 2−ΔΔCt. All the data are expressed as mean ± S.E. of the mean (S.E.), which was obtained from three or more independent experiments. For pairwise comparisons, non-parametric Mann-Whitney test or parametric Student's t test was used based on underlying distribution. Comparison between groups was performed with the Kruskal-Wallis non-parametric test or the analysis of variance parametric test. p value of less than 0.05 was considered statistically significant. GraphPad Prism version 6.05 (GraphPad Software Inc., La Jolla, CA) was used for statistical analysis. Monocytes become activated in the presence of alcohol and are characterized by changes in the expression of cell surface molecules and the secretion of cytokines and chemokines (24Saha B. Bruneau J.C. Kodys K. Szabo G. Alcohol-induced miR-27a regulates differentiation and M2 macrophage polarization of normal human monocytes.J. Immun. 2015; 194: 3079-3087Crossref PubMed Scopus (69) Google Scholar, 37McClain C.J. Hill D.B. Song Z. Deaciuc I. Barve S. Monocyte activation in alcoholic liver disease.Alcohol. 2002; 27: 53-61Crossref PubMed Scopus (101) Google Scholar, 38Szabo G. Petrasek J. Bala S. Innate immunity and alcoholic liver disease.Dig. Dis. 2012; 30: 55-60Crossref PubMed Scopus (85) Google Scholar). Recently, we have reported that alcohol-treated hepatocytes secrete increased numbers of exosomes (16Momen-Heravi F. Bala S. Kodys K. Szabo G. Exosomes derived from alcohol-treated hepatocytes horizontally transfer liver specific miRNA-122 and sensitize monocytes to LPS.Sci. Rep. 2015; 5: 9991Crossref PubMed Scopus (230) Google Scholar). Thus, we hypothesized that ethanol treatment can induce EV secretion by monocytes. To evaluate the EV secretion pattern of monocytes, we treated human blood monocytes with different concentrations of ethanol and measured the EVs using a Nanosight instrument coupled with NTA software. We observed that ethanol treatment (25–100 mm) resulted in a dose-dependent increase in the total number of EVs produced by monocytes (Fig. 1A). We found a significant increase in the number of secreted exosomes with the increasing concentrations of ethanol from 25 to 100 mm (Fig. 1A). These ethanol concentrations are relevant to human alcoholics because the 25 mm in vitro dose mimics binge drinking (∼0.1 g/dl) blood alcohol content and 100 mm approximates 0.4 g/dl blood alcohol content seen in chronic alcoholics (39Bala S. Marcos M. Gattu A. Catalano D. Szabo G. Acute binge drinking increases serum endotoxin and bacterial DNA levels in healthy individuals.PLoS ONE. 2014; 9: e96864Crossref PubMed Scopus (216) Google Scholar, 40Szabo G. Catalano D. Bellerose G. Mandrekar P. Interferon α and alcohol augment nuclear regulatory factor-κB acti" @default.
• W2214005756 created "2016-06-24" @default.
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• W2214005756 date "2016-01-01" @default.
• W2214005756 modified "2023-10-13" @default.
• W2214005756 title "MicroRNA Cargo of Extracellular Vesicles from Alcohol-exposed Monocytes Signals Naive Monocytes to Differentiate into M2 Macrophages" @default.
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