Lung Epithelial Cell−Derived Microvesicles Regulate Macrophage Migration via MicroRNA-17/221−Induced Integrin β1 Recycling This information is current as of September 26, 2021. Heedoo Lee, Duo Zhang, Jingxuan Wu, Leo E. Otterbein and Yang Jin J Immunol published online 3 July 2017 http://www.jimmunol.org/content/early/2017/07/01/jimmun ol.1700165 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2017/07/01/jimmunol.170016 Material 5.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 3, 2017, doi:10.4049/jimmunol.1700165 The Journal of Immunology Lung Epithelial Cell–Derived Microvesicles Regulate Macrophage Migration via MicroRNA-17/221–Induced Integrin b1 Recycling Heedoo Lee,* Duo Zhang,* Jingxuan Wu,* Leo E. Otterbein,† and Yang Jin* Robust lung inflammation is one of the prominent features in the pathogenesis of acute lung injury (ALI). Macrophage migration and recruitment are often seen at the early stage of lung inflammatory responses to noxious stimuli. Using an acid inhalation– induced lung injury model, we explored the mechanisms by which acid exposure initiates macrophage recruitment and migration during development of ALI. The lung epithelium comprises a large surface area and functions as a first-line defense against noxious insults. We found that acid exposure induced a remarkable microvesicle (MV) release from lung epithelium as detected in bronchoalveolar lavage fluid. Significantly elevated RNA, rather than protein, was found in these epithelium-derived MVs after Downloaded from acid and included several highly elevated microRNAs, including microRNA (miR)-17 and miR-221. Acid-induced epithelial MV release promoted macrophage migration in vitro and recruitment into the lung in vivo and required, in part, MV shuttling of miR- 17 and/or miR-221. Mechanistically, acid-induced epithelial MV miR-17/221 promoted b1 integrin recycling and presentation back onto the surface of macrophages, in part via a Rab11-mediated pathway. Integrin b1 is known to play an essential role in regulating macrophage migration. Taken together, acid-induced ALI results in epithelial MV shuttling of miR-17/221 that in turn modulates macrophage b integrin recycling, promoting macrophage recruitment and ultimately contributing to lung inflamma- 1 http://www.jimmunol.org/ tion. The Journal of Immunology, 2017, 199: 000–000. merging evidence has shown that extracellular vesicles that in addition to EV surface Ags, EVs can shuttle microRNAs (EVs) regulate diverse cellular and biological processes (miRNAs) that may play an essential role in mediating intercellular E related to human disease by facilitating cell–cell cross-talk communication (11–13). It is now generally accepted that single (1). EVs are classified into three major classes that include exo- miRNAs have diverse targets and can up- or downregulate hundreds somes (Exos), microvesicles (MVs), and apoptotic bodies (ABs) of genes (14), and MVs and Exos therefore act in part as messen- that are based on their size and mode of generation (2, 3). The gers in the coordination of innate immune responses to noxious smallest EVs (Exos) range in size from 50 to 150 nm and are stimuli (7, 9, 10). by guest on September 26, 2021 generated via an endocytic multivesicular body pathway (3). MVs Acute lung injury (ALI)/adult respiratory distress syndrome (200–1000 nm) and ABs (1000–5000 nm) are produced through (ARDS) is a complex syndrome with three overlapping phases direct budding of the plasma membrane or cellular rupture (4–6). characterized by the reduction of pulmonary compliance, re- Whereas the release of MVs is continuous and stimulated by a cruitment of inflammatory cells into the alveoli, and endothelial variety of cell stressors, ABs are only generated as a result of cell and epithelial damage (15, 16). Owing to the complexity of the death. In contrast, MVs and Exos are continuously released by live pathogenesis of ARDS, developing specific and effective therapies cells (7, 8) in either the absence or presence of noxious stimuli (9, has been exceedingly difficult. Common features of ALI/ARDS 10). Recent studies have shown that both proteins and nucleic acids include an intense inflammatory response in the lung parenchyma, are differentially sorted into the three types of EVs, reflecting their severe injury to the epithelial and endothelial cell barriers leading functional diversity (3, 9). Accumulating evidence further suggests to alveolar edema, decreased lung compliance, impaired gas ex- change, and hypoxemia (17, 18). This can occur in the presence of a broad range of agents, including oxidative stress, acid aspiration, *Division of Pulmonary and Critical Care Medicine, Department of Medicine, Bos- and infection (7, 10). The alveolar epithelium functions as a first- ton University, Boston, MA 02118; and †Department of Surgery, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, MA 02215 line defense against noxious insults and as such is critical in ORCIDs: 0000-0002-2168-3643 (H.L.); 0000-0002-3128-7601 (J.W.). maintaining the integrity and function of the lung during devel- opment of ALI (17–20). In addition to functioning as a barrier, Received for publication February 1, 2017. Accepted for publication June 11, 2017. recent evidence suggests that alveolar type I epithelial cells serve This work was supported by National Institutes of Health Grants R01 HL102076, R21 AI121644, and R01 GM111313 (all to Y.J.). important functions in innate immune responses and are under- Address correspondence and reprint requests to Dr. Yang Jin, Division of Pulmonary appreciated in lung cell–cell communication (21). An improved and Critical Care Medicine, Department of Medicine, Boston University, 72 East understanding of the mechanisms and significance of epithelium- Concord Street, Boston, MA 02118. E-mail address: [email protected] immune cell cross-talk in response to noxious stimuli may provide The online version of this article contains supplemental material. insight for new diagnostic/therapeutic approaches for treating Abbreviations used in this article: AB, apoptotic body; ALI, acute lung injury; ALI/ARDS. ARDS, adult respiratory distress syndrome; BAL, bronchoalveolar lavage; BALF, BAL fluid; BMDM, bone marrow–derived macrophage; ECM, extracellular matrix; Macrophages are the first responders among all immunoregu- EV, extracellular vesicle; Exo, exosome; HALI, hyperoxia-induced lung injury; MESNA, latory cells and are therefore involved in the initiation and de- sodium 2-mercaptoethanesulfonate; miR/miRNA, microRNA; MV, microvesicle; qPCR, velopment of lung inflammation (22–24). Macrophages present real-time quantitative PCR; siRNA, small interfering RNA; WT, wild-type. at barrier sites such as the lung are surveillance cells (25, 26). Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 How macrophages migrate and are recruited from the peripheral www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700165 2 MV REGULATES MACROPHAGE MIGRATION circulation into the lung in response to a noxious stumuli re- (4–6). Finally, the resulting supernatant was ultracentrifuged at 100,000 3 g mains largely unclear and is a complex process involving di- for1htopelletExos(39,40).Allvesicleswereresuspendedincold 2 verse cellular regulatory mechanisms (27–30). PBS and stored at 80˚C. Protein concentration was measured using a Bradford assay. Size and specific protein makers of the three types of Integrins are a family of heterodimeric adhesion receptors EVs were analyzed using dynamic light scattering instrumentation comprised of ∼17 a-chains and 8 b-chains integrins that pair (Brookhaven 90Plus nanoparticle size analyzer; Biomedical Engi- together in a specific pattern based on the cells in which they are neering Core, Boston University, Boston, MA) and Western blotting, expressed (31). These heterodimeric integrins have been classified respectively. into subfamilies according to the b-chain that they share (31). BAL and inflammatory cell counts Among the integrin subfamilies, integrins b and b serve critical 1 2 To obtain BALF cells from mice, bronchoalveolar lavage (BAL) was roles in monocyte/macrophage extravasation and migration into performed twice with 1 ml of cold PBS. Total inflammatory cell counts in sites of inflammation (27). Expression of the b2 subfamily is the BALF were determined using a hemocytometer as previously described limited to WBCs and is comprised of one of four heterodimeric (25). For cytospin preparations, the cell suspension was cytocentrifuged at isotypes depending on how they combine with the a-chains to 300 3 g for 5 min using a Shandon Cytospin 4 (Thermo Fisher