Lung Epithelial −Derived Microvesicles Regulate Migration via MicroRNA-17/221−Induced 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

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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 , 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 , 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 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 , 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, ; Macrophages are the first responders among all immunoregu- EV, ; Exo, ; 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 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 /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 Scientific, form leukocyte function Ag-1, Mac-1, gp150/95, and ad/b2 (32). Rockford, IL). Slides were air-dried and stained with PROTOCOL Hema 3 b fixative and solutions. Differential cell counts were evaluated under a light The 2 integrins serve as principal receptors that are recognized microscope. by complementary adhesion molecules on known as ICAM-1 and ICAM-2. The ICAM molecules are responsible for Macrophage culture allowing leukocytes to bind to endothelial cells at sites of in- Mouse bone marrow–derived macrophages (BMDMs) were isolated from

flammation (27, 33). Once the leukocytes have extravasated from mice as described previously (9). Isolated bone marrow cells were cul- Downloaded from the circulation, these differentiated /macrophages then tured with 30% L929-conditioned medium for 5–7 d to allow differen- tiation of the bone marrow monocyte/macrophage progenitors and interact with extracellular matrix (ECM) proteins through the further experimentation. Human THP1 monocytes were obtained from integrin b1 subfamily (27). Integrin b1 combines with at least nine the American Type Culture Collection and maintained in RPMI 1640 a integrins whereas the integrin b1 subfamily recognizes ECM with 10% FBS and 1% penicillin/streptomycin. Cells were cultured at proteins, including fibronectin, collagen, and laminins (34). It is 37˚C in a humidified atmosphere with 5% CO2 and 95% air. THP1 monocytes were differentiated into macrophages with 20 ng/ml PMA now generally accepted that integrin b is continuously internal- http://www.jimmunol.org/ 1 (Sigma-Aldrich) for 24 h and then further matured in fresh medium for ized and traffics to Rab11-positive recycling or lyso- an additional 24 h before use. somes to control cell migration (35, 36). Recent reports show that incorporation of integrin b1 into recycling endosomes is essential Lung epithelial cell isolation for macrophage migration (28). Primary alveolar epithelial cells were isolated from mice as described Collectively, our studies sought to explore the role of epithelial previously (41). Briefly, mouse lung tissue was washed with sterile PBS, MV-shuttling miRNAs in the development of lung inflammatory followed by infusion of 2 ml of dispase and 0.5 ml of 1% agarose. Lung responses after acid inhalation. We further describe the effects of tissue was then dissociated in DMEM with 25 mM HEPES and 200 U/ml DNase. Isolated cells were sequentially strained using 100-, 40-, and 20- MV-shuttling miRNAs on macrophage migration and the under- mm cell strainers, followed by incubation on plates precoated with CD45 by guest on September 26, 2021 lying mechanism of action involved in integrin b1 recycling. and CD16/32 Abs for 2 h. Suspended cells were further transferred to noncoated plates to remove fibroblasts. After another 2-h incubation, suspended epithelial cells were cultured in DMEM containing 10% FBS Materials and Methods prior to further experimentation. Materials Flow cytometry miRNA (miR)-17 mimics (HMI0264), miR-221 mimics (HMI0398), miR-320a mimics (HMI0470), miR-92a mimics (HMI0955), and HRP- Flow cytometric analysis of BALF MVs was performed as described conjugated anti–b-actin Ab (A3854) were purchased from Sigma- previously (40) with minor modifications. Isolated MVs were coupled to 10 ml of aldehyde/sulfate latex beads (Thermo Fisher Scientific) for 2 h, Aldrich (St. Louis, MO). Mouse anti-integrin b1 (sc-374430), mouse anti-PTEN (sc-7974), mouse anti–c-fos (sc-166904), and rabbit anti– and the MV-coated beads were then treated with 4% BSA for 1 h as a flotillin-1 (sc-25506) Abs were purchased from Santa Cruz Biotechnology blocking agent. The bead-bound MVs were then permeabilized and fixed (Santa Cruz, CA). Rabbit anti-Rab11 (ab128913), rabbit anti-TSG101 for 5 min with 0.2% Triton X-100 and 2% formaldehyde, followed by (ab30871), and PE-conjugated anti-pan cytokeratin Abs were purchased incubation with designated Abs. from Abcam (Cambridge, MA). Rabbit anti–caveolin-1 and anti–histone For analysis of BALF macrophages, cells were incubated with Ab b H3 Abs were purchased from Thermo Fisher Scientific (Waltham, MA) specific to integrin 1, followed by incubation with CD11b-FITC and a PE- and Technology (Danvers, MA), respectively. FITC-conjugated conjugated secondary Ab. Macrophages present in BALF were gated based anti-CD11b (11-0112-82) and allophycocyanin/Cy7-conjugated anti-CD11c on light scattering profiles, as described previously (42). FITC- and PE- (117323) Abs were purchased from eBioscience (San Diego, CA) and Bio- conjugated secondary Abs were used as negative controls. Flow cytometric Legend (San Diego, CA), respectively. analysis was performed using FACSCalibur instrument (BD Biosciences), and the data were analyzed using FlowJo software (Tree Star). Acid-induced ALI RNA preparation, reverse transcription, and quantitative Wild-type (WT) C57BL/6 mice (6–8 wk of age) were obtained from The real-time PCR Jackson Laboratory (Bar Harbor, ME). Hydrochloric acid (0.1 N, pH 1.5) was intratracheally instilled into the mouse lung. One day after instillation, Total RNAs were purified from the isolated BALF EVs or harvested mice were sacrificed and bronchoalveolar lavage fluid (BALF) was col- macrophages using miRNeasy mini kits (Qiagen). Purified RNA concen- lected, and lung tissue was harvested. All protocols and methods involving tration was measured using the NanoDrop Lite spectrophotometer (Thermo animals were approved by the Institutional Animal Care and Use Com- Fisher Scientific). A reverse transcription kit (Thermo Fisher Scientific) was mittee in accordance with approved guidelines. used to generate single-stranded cDNA from equal amounts of purified RNAs. SYBR Green–based real-time quantitative PCR (qPCR) was per- Categorizing the EVs from BALF formed for measuring miRNAs as previously described (43, 44). For relative b Previously reported protocols and techniques were applied to isolate three expression levels of mRNAs, tubulin was used as a reference housekeeping subpopulations of EVs from mouse BALF, including ABs, MVs, and Exos gene. The list of primers is shown in Table I. (9, 37, 38). BALF was centrifuged at 300 3 g for 5 min to remove the Direct miRNA transfection into MVs inflammatory cells. The supernatant was then collected and centrifuged at 2000 3 g for 10 min to pellet ABs (37, 38). To isolate MVs, the miRNA mimics or inhibitors (both at 10 pM) were directly transfected into AB-depleted supernatant was then centrifuged at 16,000 3 g for 40 min isolated BALF MVs with 0.1 M CaCl2, as previously described (45). The The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ FIGURE 1. Characterization of the EVs in the lung. (A) Three subpopulations of EVs were isolated from mouse BALF, including ABs, MVs, and Exos. The sizes of the isolated EVs were measured using dynamic light scattering (A, left panel) and NanoSight (A, right panel). (B) Representative Western blot of isolated EVs. (C) Pie graph indicates the average percentages of each type of EVs isolated from BALF (n = 8 mice per group). (D)Acid(0.1NHCl)wasintratracheally instilled into the lungs of mice. Twenty-four hours later BALF EVs were isolated and the amounts of EVs were determined. Data represent mean 6 SD (n =8mice per group). (E) Flow cytometry of isolated MVs to determine the proportion of cell type–specific markers. Dot graphs show the percentages of the indicated cell markers (E, right lower panels). For (A)and(B), data are representative of three independent experiments. *p , 0.05, **p , 0.01 between indicated groups. transfected MVs were washed with cold PBS followed by 16,000 3 g (46, 47). MV-containing miRNA levels were measured using qPCR . To confirm successful transfection, the transfected MVs (Supplemental Fig. 1A). The transfected MVs were used for further by guest on September 26, 2021 were treated with RNase, which rapidly degrades miRNAs outside of MVs experiments.

FIGURE 2. Expression of miRNAs in BALF EVs after acid aspiration. (A and B)MVs(A) and Exos (B) were isolated from BALF after acid administration. Protein, RNA, and normalized RNA (RNA/protein) amounts are shown. (C and D) Expression of miRNAs in MVs (C) and Exos (D) by qPCR. Left panels show the fold change of miRNAs after normalization with EV RNAs. Right panels show the fold increase in miRNAs after normalization with EV protein. *p , 0.05 versus control group. 4 MV REGULATES MACROPHAGE MIGRATION

FIGURE 3. Acid-induced lung injury promotes macrophage recruitment and lung inflammation. (A and B) H&E staining of mouse lung tissue and BALF cells 24 h after acid aspiration. Data represent mean 6 SD (n = 4–6 mice per group). (C) Cytokine expression in BALF 24 h after acid. *p , 0.05, **p , 0.01 versus control or between indicated groups.

Vesicle uptake assay cell surface was stripped with 50 mM sodium 2-mercaptoethanesulfonate (MESNA; Sigma-Aldrich), followed by quenching of the residual MESNA Downloaded from Vesicle uptake determination was performed as previously described (48) with 20 mM iodoacetamide (Sigma-Aldrich). The cells were then har- with a minor modification. Isolated MVs were labeled with CSFE, fol- vested with lysis buffer containing 1% Triton X-100 and inhibi- lowed by washing with cold PBS. WT mice (intratracheal instillation) or tors. Biotinylated proteins were precipitated with streptavidin agarose BMDMs were then treated with the prepared MVs for 24 h, followed by (Thermo Fisher Scientific) and subjected to Western blot analysis using an collection of BALF cells or BMDMs. The collected cells were trypsinized Ab specific to integrin b1. The percentage internalization was determined and washed with acid buffer (pH 2.6) to remove surface-bounded MVs using the band intensity of internalized integrin b1 relative to total labeled (48), followed by cytospin preparation. Uptake of the MVs and the MV- integrin b1 (without MESNA). containing miRNAs was analyzed using confocal microscopy and qPCR, For the recycling assay, biotin-labeled surface integrins were incubated http://www.jimmunol.org/ respectively. for 30 min to allow internalization, and the remaining biotin on the cell surface was stripped as described above. The internalized integrins are able Integrin internalization, endocytic recycling, and to recycle back to the cell surface by incubating at 37˚C. The cell surface degradation assay was then stripped again to remove the recycled integrins. Biotinylated proteins were precipitated and analyzed by Western blot analysis. The Integrin trafficking assays were performed as previously described (49) with percentage of recycled integrins was determined by comparing the band a minor modification. For the internalization assay, macrophage-surface intensity of the remaining integrin b1 in the cells to the total internalized proteins were biotinylated with 0.5 mg/ml Sulfo-NHS-SS-Biotin (Thermo integrin b1. Fisher Scientific) for 20 min at 4˚C, followed by washing with TBS. In- Degradation of cell surface integrins was performed as previously de- ternalization of surface proteins was induced by incubating cells at 37˚C. scribed with a minor modification (40). Surface-biotinylated macrophages After internalization of the surface proteins, the remaining biotin on the were further cultured for the designated time points. Biotinylated proteins by guest on September 26, 2021

FIGURE 4. MVs and MV-containing miR-17/221 are actively transferred from lung epithelial cells to recipient macrophages. (A and B) pPCR analysis of lung epithelial cells (A) and BALF cells (B) 24 h after acid instillation. Data represent mean 6 SD (n = 4 per group). (C and D) Schematic illustration of MV uptake analysis in vivo (C). CFSE-labeled MVs (D) or acid-induced MVs. (E) MV uptake after intratracheally instillation into the lungs of mice. Data represent mean 6 SD (n = 3 per group). (F) Western blot analysis of THP1-differentiated macrophages transfected with the indicated miRNA mimics.

Graph shows expression levels of integrin b1 in the transfected macrophages. *p , 0.05 versus control group. The Journal of Immunology 5

were then precipitated and the remaining integrin b1 was analyzed by ment systems (R&D Systems), according to the manufacturer’s recom- Western blot analysis. The percentage of degraded integrins was calculated mendations. using the signal intensity of the remaining integrin b in the cells at the 1 Statistical analysis indicated time points relative to total labeled integrin b1. Ab-based integrin trafficking assay For all experiments, the exact n values and statistical significances are shown in the corresponding figure and figure legends. Representative data Ab-based trafficking assays were performed as previously described (40) of identical results are shown in the figures. Statistical analysis was per- with minor modifications. The surface integrins on cultured macrophages formed with an unpaired two-tailed Student t test. A p value ,0.05 was were labeled with anti–integrin b1 Ab (E-11) for 20 min at 4˚C, followed considered statistically significant (*p , 0.05, **p , 0.01). by internalization of the labeled integrin with incubation at 37˚C for 30 min. The remaining labeled integrins on the cell surface were then stripped with acid buffer (pH 2.5). The cells were then fixed and permeabilized with Results 100% cold methanol for 10 min at 4˚C. Recycling endosomes were stained Lung epithelial cell–derived MVs are robustly upregulated in with anti-Rab11 IgG. The internalized integrin b1 and Rab11-postive recy- BALF after acid inhalation cling endosomes were visualized using FITC-conjugated and Alexa Fluor 594–conjugated secondary Ab, respectively, using a Leica SP5 point-scanning EVs are classified into ABs, MVs, and Exos based on their size, confocal microscope (Leica Microsystems). Staining intensities and coeffi- protein markers, and modes of generation. As shown Fig. 1A, the cient values were analyzed using ImageJ 1.51h software (National Institutes size of MVs (172.8 6 3.2 nm) measured by both dynamic light of Health, Bethesda, MD). scattering and NanoSight were significantly larger than Exos 6 Transwell migration assay (122.6 1.7 nm). We also verified expression of selective protein makers for each type of EVs (Fig. 1B) and found that MVs were Macrophage migration analyses were performed using 6.5-mm-diameter the principal form of EVs in mouse BALF after acid inhalation. Downloaded from polycarbonate with 8.0-mm microporous membranes (CoStar, Cam- bridge, MA), as described previously (9). Macrophages (3 3 104) were Sixty-five percent of EVs fell into the size range of MVs (Fig. 1C). placed in the inner chamber coated with BD Matrigel basement membrane More interestingly, MVs showed the greatest induction compared matrix (BD Biosciences). EV-depleted 10% FBS was added in the outer with other types of EVs (Fig. 1D). To determine the source of the well with isolated BALF MVs transfected with miRNA mimics or inhib- BALF MVs, we analyzed surface markers using Abs against itors. After 24 h incubation, migrated cells were fixed with 4% formal- CD11b, CD11c (myeloid lineage markers), and pan cytokeratin dehyde and stained with hematoxylin. Migrated cells were counted using a . http://www.jimmunol.org/ light microscope. (epithelial cell marker). As shown in Fig. 1E, 70% of BALF MVs were derived from lung epithelial cells whereas a small Western blot analysis amount of MVs originated from myeloid cells. Furthermore, ep- Harvested cells were lysed with RIPA buffer containing 1% Triton X-100, ithelial cell–derived MVs were dramatically upregulated com- protease inhibitor, and phosphatase inhibitor. Western blotting analysis was pared with myeloid cell–derived MVs, which showed no change performed as described previously (39). Densitometric analyses were or a very slight induction following acid inhalation (Fig. 1E). performed using ImageJ software (National Institutes of Health), and the gene expression was normalized to b-actin. MV RNA is upregulated more robustly than MV protein in the ELISA presence of noxious stimuli by guest on September 26, 2021 Mouse BALF was collected 24 h after acid aspiration and centrifuged at We analyzed the composition of isolated MVs and isolated Exos. 300 3 g for 5 min to pellet the inflammatory cells. TNF, IL-1b, IL-6, In the presence of acid exposure, both MV RNA and MV protein IL-10, and IL-23 levels were analyzed using DuoSet ELISA develop- expression were enhanced. Interestingly, after normalization

Table I. Sequences of primers used in qPCR

Name Primer Sequence for Reverse Transcription (59→39) Primer Sequence for qPCR (59→39) Universal primer GGTGTCGTGGAGTCGGCAATTCAGTTGAG miR-221 CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGAAACCCA ACACTCCAGCTGGGAGCTACATTGTCTGCT miR-320a CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCGCCCTC ACACTCCAGCTGGGAAAAGCTGGGTTGAG miR-92a CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACAGGCCG ACACTCCAGCTGGGTATTGCACTTGTCCC miR-17 CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCTACCTGC ACACTCCAGCTGGGCAAAGTGCTTACAGT miR-185 CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCAGGAAC ACACTCCAGCTGGGTGGAGAGAAAGGCAGT miR-21 CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCAACATC ACACTCCAGCTGGGTAGCTTATCAGACTGA miR-20a CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCTACCTGC ACACTCCAGCTGGGTAAAGTGCTTATAGT miR-93 CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCTACCTGC ACACTCCAGCTGGGCAAAGTGCTGTTCGTG miR-181a CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACTCACCG ACACTCCAGCTGGGAACATTCAACGCTGTC let-7b CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAACCACAC ACACTCCAGCTGGGTGAGGTAGTAGGTTGT Rab11 Forward: TGGGAAAACAATAAAGGCACAGA Reverse: ATGTGAGATGCTTAGCAATGTCA

Integrin b1 Forward: ATGCCAAATCTTGCGGAGAAT Reverse: TTTGCTGCGATTGGTGACATT Rab4 Forward: GGAGCGGTTCAGGTCTGTG Reverse: AGTAAGCGCATTGTAGGTTTCTC b-Tubulin Forward: CTGGGAGGTGATCGGGGAA Reverse: GCACATACTTCTTACCGTAGGCT Pre–miR-17 Forward: GTCAGAATAATGTCAAAGTGCTTAC Reverse: GTCAGCATAATGCTACAAGTGCCC Pre–miR-221 Forward: ATCCAGGTCTGGGGCATGAA Reverse: TTCCAGGTAGCCTGAAACCC Pre–miR-92a Forward: CTTTCTACACAGGTTGGGATTTG Reverse: CCAAACTCAACAGGCCGGGA Pre–miR-320a Forward: GCCTCGCCGCCCTCC Reverse: CCCACATCCTTTTTCGCCCT 6 MV REGULATES MACROPHAGE MIGRATION with MV protein, MV RNA remained highly elevated (Fig. 2A). In the levels of mature miR-17/221 were highly elevated in BALF contrast, both Exo RNAs and Exo proteins, although elevated after macrophages (Fig. 4B). In the absence of any changes in pre- acid exposure, showed no significant upregulation when normal- miRNA expression, the increased mature forms of miR-17/221 izated with MV protein (Fig. 2B). We next analyzed several in macrophages suggest that these mature miRNAs are in part miRNAs in the BALF of acid-induced MVs and Exos generation. transferred from other cells, such as lung epithelial cells (Table I). miR-221, 320a, 92a, and 17 were significantly higher (.2-fold) in To directly examine MV uptake by macrophages in vivo, we MVs after acid exposure. No significant alteration of these miRNAs labeled MVs with CFSE. The labeled MVs were delivered intra- was observed in BALF Exos (Fig. 2C, 2D). tracheally to WT mice and BALF was collected 24 h later (Fig. 4C). More than 90% of cells in the BALF were macrophages. As Acid-induced lung injury promotes macrophage recruitment shown in Fig. 4D, we successfully visualized efficient uptake of and lung inflammation labeled MVs by BALF macrophages. Importantly, we confirmed Macrophage activation, recruitment, and migration play an es- that uptake of acid-induced MVs led to a significant increase in sential role in initiation of lung immune responses (9, 50–52). Acid miR-17 and 221, but not their precursor forms, in recipient BALF instillation induced a massive increase in cell infiltration in both macrophages (Fig. 4E). This observation indicated that MVs and lung tissue and BALF (Fig. 3A). Among all infiltrated cells, MV-containing miRNAs were actively delivered to macrophages macrophages were the most predominant cell type present in in vivo. We also noted that miR-17 and 221 significantly upreg- BALF up to 24 h after acid inhalation (Fig. 3B). influx ulated expression of integrin b1, an essential complex required for in the airways peaked 12 h after acid inhalation (Fig. 3B), whereas macrophage recruitment and migration (27, 32, 53) (Fig. 4F). eosinophils and were detected in low amounts in the Downloaded from BALF (data not shown), indicating a differential cellular response Acid-induced MV miRNAs modulate macrophage recruitment in the setting of acid exposure. We also confirmed that proin- and migration in the lung flammatory cytokines, including TNF, IL-1b, and IL-6, were To determine the effects of MVs and MV miRNAs on lung in- significantly increased 24 h after acid inhalation in the BALF flammatory responses and macrophage recruitment, we employed compared with sham controls (Fig. 3C). “loss of function” and “gain of function” approaches. Fig. 5A il-

lustrates our experimental design. We first isolated MVs from http://www.jimmunol.org/ Macrophages respond to epithelial cell–derived MVs and control mice or acid-treated mice. We then manipulated the MV miRNAs miRNA compositions in these isolated MVs using miRNA mimics To determine the target and recipient cells of the epithelial-derived or inhibitors. We inserted miR-17 and 221 mimics into the MVs MV-shuttling miRNAs in the BALF, we first examined expression obtained from naive mice to augment their expression. The suc- of miR-17, 320a, 92a, and 221 in the isolated lung epithelial cells cessful transfection of the miRNAs was confirmed using RNase and BALF inflammatory cells (70–90% of the BALF inflammatory (Supplemental Fig. 1A). In separate experiments, we inserted cells are macrophages; Fig. 3B). We found that levels of these miR-17 and 221 inhibitors into MVs harvested from acid-exposed mature miRNAs (mature miR-17, 320a, 92a, and 221), but not mice, given that these miRNAs were already upregulated in re- their precursor forms, were dramatically decreased in the lung sponse to acid. These modified MVs were delivered to WT mice by guest on September 26, 2021 epithelial cells in response to acid exposure (Fig. 4A). In contrast, via inhalation. After 24 h, BALF was harvested from the modified

FIGURE 5. MV-containing miR-17/221 facilitate macrophage recruitment into the lung. (A) Schematic illustration of the functional analysis of MV-containing miR- NAs on macrophage recruitment in vivo. MVs isolated from control mice (MV-con) and mice after acid aspiration (MV-acid) were then transfected with miR-17 and 221 mimics (MV-con + miR-17/221 mim) and inhibitors (MV-acid + miR-17/221 inh). Nonspecific RNA oligonucleotides were transfected into MVs as negative controls (MV-con + nc and MV-acid + nc). Twenty- four hours after instillation, BALF cells were isolated and stained with H&E (B, left panel). Dot graph shows the total cell count and macrophage count in the BALF (B, right panel). *p , 0.05 versus MV-con + nc group. The Journal of Immunology 7

MV-treated mice and we observed significantly elevated BALF MV-shuttling miR-17/221 upregulated integrin b1 in cell counts after inhalation of MVs enriched with miR-17/221 macrophages mimics. Treating WT mice with MVs obtained from acid-exposed We next evaluated the role of miR-17/221 in MV-induced up- mice resulted in significantly elevated BALF cell counts. Inter- regulation of integrin b1 expression in macrophages in vivo and estingly, after enrichment with miR-17/221 inhibitors, the MVs in vitro. To test this, isolated MVs were delivered to WT mice via obtained from acid-exposed mice failed to increase cell infiltration inhalation and BALF cells were isolated and analyzed by flow into the lung as measured in BALF (Fig. 5B). Moreover, the cytometry 24 h later. Overexpression of miR-17/221 induced ex- predominant inflammatory cells remained macrophages after MV- pression of integrin b whereas miR-17/221 inhibitors abolished mediated cell recruitment, indicating that macrophages are the 1 the effects of acid-induced MVs on integrin b expression in most likely target responding to MVs (Fig. 5B). 1 macrophages (Fig. 7A). This observation was further confirmed Acid-induced MVs upregulate expression of integrin b1in with BMDMs in vitro by Western blot (Fig. 7B, Supplemental macrophages Fig. 1A–C) and real-time PCR (Fig. 7C). We observed that Rab11, a Rab GTPase that associates with recycling of endocy- As described above, integrin b1 is crucial for macrophage migration into injured sites though specific interaction with ECM (27, 32, 53). tosed proteins, was also upregulated in macrophages in a similar b We showed that miR-17 and 221 significantly upregulated integrin manner to integrin 1 (Fig. 7B, 7C). Importantly, MV-shuttling b1 in macrophages (Fig. 4F). We confirmed that BALF macro- miR-17/221 had no effect on Rab4, an important regulator of phages expressed significantly more integrin b1 after exposure to vesicular transport (Fig. 7C). acid (Fig. 6A). We next studied the effects of MVs on integrin b Downloaded from 1 MV-containing miR-17 and 221 induce trafficking of integrin expression in macrophages in vitro. After confirming the efficient b into recycling endosomes uptake of MVs by cultured macrophages in vitro (Fig. 6B), we 1 b treated human macrophages with PBS (control), MVs obtained Surface-expressed integrin 1 is continuously internalized via from control mice, MVs obtained from acid-treated mice, Exos , and the internalized integrins are then trafficked to obtained from control mice, or Exos obtained from acid-treated Rab11-positive recycling endosomes or (35, 36). Given

mice. MVs obtained from acid-exposed mice stimulated remark- that MV-shuttling miR-17/221 significantly upregulated Rab11 http://www.jimmunol.org/ able expression of integrin b1 compared with all other treatment (Fig. 7B), we next examined the effect of MV-shuttling miR-17/ groups (Fig. 6C). Similar results were observed using mouse 221 on integrin trafficking in macrophages. THP1 macrophages macrophages (Fig. 6D). were transfected with miR-17/221 mimics, and upregulation of by guest on September 26, 2021

FIGURE 6. Acid-induced MVs upregulate expression of integrin b1 in macrophages. (A) BALF cells were isolated 24 h after acid aspiration. Based on the light-scattering profiles, BALF macrophages were gated (G, granulocyte; L, ; M, macrophages). Expression levels of CD11b (macrophage high high marker) and CD29 (integrin b1) in the macrophages were measured using flow cytometry. Dot graph shows the percentage of CD11b CD29 cells. Data represent mean 6 SD (n = 4 mice per group). (B) Mouse BMDMs were treated with CFSE-labeled MVs for 1 d, followed by MV uptake assay, as described in Materials and Methods.(C and D) Two types of EVs were isolated from control mice (MV-con and Exo-con) and mice after acid instillation (MV-acid and Exo-acid). (C and D) Human THP1-differentiated macrophages and BMDMs were treated with the isolated EVs for 1 d, followed by Western blot analysis. Left panel shows expression levels in treated macrophages of integrin b1 as dot graphs. Right panels show representative images of the Western blot analysis. Data represent mean 6 SD (n = 4 or 6 per group). *p , 0.05, **p , 0.01 between indicated groups. 8 MV REGULATES MACROPHAGE MIGRATION

integrin b1 and Rab11 was verified (Fig. 4F). To assess internal- integrin b1 was internalized after incubation at 37˚C. The labeled ization of integrin b1, cell surface proteins were labeled with integrin b1 that remained on the cell surface was then stripped. Sulfo-NHS-SS-Biotin at 4˚C. The remaining labeled proteins on As shown in Fig. 9C, robustly elevated colocalization was ob- the cell surface were then stripped using 50 mM MESNA, and the served between integrin b1 (green) and Rab11 (red) after ex- internalized proteins were precipitated and analyzed by Western posure of macrophages to acid-induced MVs and MV-shuttling blot. For the recycling assays, the internalized protein was recy- miR-17/221, suggesting that internalized integrin b1 traffics to cled back to the cell surface after another 37˚C incubation. The Rab11-positive recycling endosomes or lysosomes. Notably, recycled proteins were then stripped again and the labeled proteins acid-induced MVs enriched with the miR-17/221 inhibitor that remained in the endosomes were precipitated and analyzed by failed to induce colocalization of integrin b1 and Rab11, con- Western blot. Whereas the internalization of surface integrin b1 firming the functional significance of MV-containing miR-17/ was not significantly changed (Fig. 8A), the cell surface repre- 221 (Fig. 9C). sentation of intracellular integrin b1 was markedly increased after overexpression of miR-17/221 mimics in THP1 macrophages MV-containing miR-17/221 mediates macrophage migration via integrin b1 (Fig. 8B). Moreover, the degradation of the surface integrin b1 was significantly delayed in the presence of miR-17/221 over- We next examined the effects of MV-shuttling miR-17/221 on expression (Fig. 8C). Collectively, the surface integrin b1 was macrophage migration in vitro. As shown in Fig. 10A, we seeded highly augmented secondarily to enhanced recycling and delayed BMDMs in a Transwell insert coated with basement membrane degradation. Rab11 expression in miR-17/221–overexpressing matrix proteins and added the following agents into the Transwell macrophages was also upregulated in addition to integrin traf- lower compartment: 1) MVs obtained from control mice plus Downloaded from ficking processes (Fig. 8). control oligonucleotides, 2) acid-induced MVs plus control We further evaluated the effect of MV-shuttling miR-17/221 in miRNA oligonucleotides, 3) MVs obtained from control mice BMDMs and, as shown in Fig. 9A, acid-induced MVs and MV- enriched with miR-17/221 mimics, and 4) MVs obtained from shuttling miR-17/221 upregulated the recycling of integrin b1 in acid-exposed mice enriched with miR-17/221 inhibitors. To de- BMDMs. Significant colocalization between Rab11 and integrin termine the functional role of integrin b1 in MV-mediated mac- b1 in BMDMs was observed after treatment of acid-induced MVs rophage migration, we pretreated BMDMs with integrin b1 small http://www.jimmunol.org/ or MV-shuttling miR-17/221 (Fig. 9B). We next performed an Ab- interfering RNA (siRNA) (Fig. 10B). As expected, acid-induced based surface-labeling assay as described previously (40) target- MVs and MV-shuttling miR-17/221 significantly induced BMDM ing integrin b1. As illustrated in Fig. 9C, surface expression of migration compared with control MVs. The effect of acid-induced by guest on September 26, 2021

FIGURE 7. MV-containing miR-17/221 upregulate integrin b1 in macrophages. (A) BALF MVs were isolated from control mice (MV-con) and mice after acid instillation (MV-acid). miR-17 and 221 mimics (MV-acid + miR-17/221 mim) and inhibitors (MV-con + miR-17/221 inh) were directly transfected into MV-con and MV-acid, respectively. Nonspecific RNA oligonucleotides were transfected into MVs as negative controls (MV-con + nc and MV-acid + nc). The transfected MVs (20 mg/30 ml per mouse) were intratracheally instilled into mice. Twenty-four hours after instillation, BALF cells were isolated and gated by flow cytometry for CD11bhighCD29high. Data represent mean 6 SD (n = 4 or 5 mice per group). (B and C) BALF MVs were prepared as described in (A). BMDMs were treated with the MVs (5 mg/500 ml per well) for 24 h, followed by Western blot (B), and qPCR (C) for measuring protein and mRNA expression, respectively. Dot graphs show expression of the indicated genes from the treated macrophages. Data represent mean 6 SD (n =4or 6 per group). *p , 0.05 versus MV-con + nc or between indicated groups. The Journal of Immunology 9 Downloaded from

FIGURE 8. miR-17 and 221 promote endocytic recycling of integrin b1 in macrophages. (A–C) THP1-differentiated macrophages were transfected with miR-17 or 221 mimics. Surface proteins of the macrophages were labeled with EZ-Link Sulfo-NHS-SS-Biotin, followed by surface integrin internalization (A), endocytic recycling (B), and degradation assay (C). Biotinylated proteins were precipitated with streptavindin, followed by Western blot analysis. Data represent mean 6 SD (n = 3 per group). *p , 0.05 versus control group.

MVs was abolished after inhibition of miR-17/221 (Fig. 10C). We Discussion http://www.jimmunol.org/ further observed that depletion of integrin b1 decreased the EVs refer to a group of heterogeneous vesicles with different sizes, number of migrated cells (Fig. 10C). Additionally, acid-induced features, mechanisms of formation, and function (1–3, 5, 40, 54, MVs and MV-shuttling miR-17/221 failed to induce migration of 55). Depending on the cellular sources and stimuli, EVs are the integrin b1–deficient BMDMs, indicating that MV-shuttling generated via direct budding (MVs), secretion from cells via miR-17/221–mediated macrophage migration was, in part, integ- multivesicular bodies (Exos), or formation by plasma membrane rin b1–dependent (Fig. 10C). blebbing after undergoing apoptosis (ABs). Proteins derived from by guest on September 26, 2021

FIGURE 9. MV-containing miR-17/221 induce the trafficking of integrin b1 into recycling endosomes. (A and B) MVs were isolated from control mice (MV-con) and mice after acid instillation (MV-acid). miR-17 and 221 mimics were directly transfected into MV-con (MV-con + miR-17/221 mim), as described in Materials and Methods. Nonspecific RNA oligonucleotides were transfected into MVs as negative controls (MV-con + nc and MV-acid + nc). BMDMs were treated with the transfected MVs for 1 d, followed by recycling assays (A) and double immunostaining (B) using the indicated Abs. Data represent mean 6 SD (n = 3 or 6 per group). (C) BMDMs were treated with the transfected MVs as described in (A), followed by labeling of surface integrin b1. Data are representative of three independent experiments. Scale bars, 50 mm. *p , 0.05 versus MV-con + nc group. 10 MV REGULATES MACROPHAGE MIGRATION Downloaded from http://www.jimmunol.org/

FIGURE 10. Integrin b1 is required for MV miR-17/221–induced macrophage migration. (A–C) Schematic illustration of the functional analysis of MV- containing miRNAs on macrophage migration (A). BMDMs were transfected with control siRNAs or integrin b1 siRNAs (50 nM), and expression levels of integrin b1 in the transfected BMDMs were assessed by Western blot (B). BALF MVs were prepared as described in Fig. 3D, and Transwell inserts were coated with basement membrane matrix. Migration assays of the transfected BMDMs were performed as described in Materials and Methods (C, left panel). Dot graph shows the migrated cell counts in each field (C, right panel). Data represent mean 6 SD (n = 6 per group). *p , 0.05 versus MV + nc group. by guest on September 26, 2021 parent cells are often detectable in EVs such as surfactant proteins been well documented (26, 56, 57). In this study, we show that that can be found in EVs released from lung epithelial cells (26). epithelial MVs and MV-shuttling miRNAs modulate, in part, We previously reported that development of hyperoxia-induced macrophage recruitment and migration. Unlike chemokines, MV- lung injury (HALI) is influenced by BALF EVs from lung epi- shuttling miRNAs directly regulate the cellular machinery neces- thelium (9, 26). In this study, we confirmed that epithelial cells sary for macrophage migration and include cell surface–expressed are the likely source of BALF EVs during the development of integrin b1 expression. ALI, regardless of the noxious stimuli. We put forth that lung The integrin b1 chain and distinct a-chains can form at least epithelium-derived EVs are dominant participants primarily in nine heterodimer combinations (27, 34) that permit a wide variety “sterile” stimuli-induced ALI. In the setting of bacterial infection– of ab combinations while sharing a common b1 chain. These associated ALI, such as bacterial pneumonia, a large amount of adhesion molecules interact with a diverse array of ECM proteins. BALF EVs is derived from macrophages, other immunomodula- We investigated the effects of MV-shuttling miR-17/221 on tory cells, and bacteria themselves (unpublished data). Although expression of integrin b1 in monocytes and macrophages. It has the pathogenesis of acid-induced lung injury is significantly dif- been reported that the integrin b1/Scr/PI3K-mediated signaling, ferent when compared with HALI, we found that MVs remained but not integrin b1/focal adhesion kinase–mediated signaling, the principal type of EVs present in BALF and that MV RNAs regulates macrophage migration (58, 59). Moreover, the in- were robustly altered after acid exposure. This is consistent with corporation of integrin b1–containing recycling endosomes our previous reports with HALI (9). Whether these observations onto their leading edge is critical for macrophage migration remain the same in the setting of pathogen-induced lung injury (28). requires further investigation. Collectively, our results suggest that epithelial MVs and MV- Our previous reports using the HALI model suggest that epi- shuttling miR-17/221 upregulate b1 integrin expression in mac- thelial MVs mediate the cross-talk between lung epithelial cells and rophages. Furthermore, our findings indicate that epithelial MVs alveolar macrophages via MV-shuttling miRNAs (9). However, a and MV-shuttling miR-17/221 promote b1 integrin recycling in different MV miRNA repertoire is involved in acid-induced lung part through an Rab11-mediated pathway. Rab11 belongs to the injury, compared with those involved in HALI. In the present Rab family of small GTPases and regulates membrane trafficking study, we demonstrated how epithelial MVs promote macrophage (36). Rab11 in particular regulates recycling of endocytosed recruitment and migration into the lung. In the absence of infec- proteins and is used as a recycling endosomal marker (36, 40). tious stimuli, the lung epithelium plays an essential role in releasing Colocalization of Rab11 and integrin b1 suggests that integrin b1 chemoattractants that target circulating macrophages. The role of is likely recycled with recycling endosomes and regulated by alveolar epithelial cell–mediated chemokine/cytokine release has Rab11. The Journal of Immunology 11

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