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Neutrophil transfer of miR-223 to lung epithelial cells dampens acute lung injury in mice

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Science Translational Medicine  20 Sep 2017:
Vol. 9, Issue 408, eaah5360
DOI: 10.1126/scitranslmed.aah5360
  • Fig. 1. Transfer of miR-223 during neutrophil-epithelial cell interactions.

    (A) Coculture setup for human neutrophils (PMNs) and human pulmonary epithelial cells. (B) Expression of miRNA in human epithelial cells after coculture of HPAEpiC with activated human PMNs (means ± SEM; n = 4). (C) hsa–miR-223 expression after coculture of HPAEpiC with activated human PMNs (means ± SEM; n = 6). (D and E) Expression of miR-223 in HPAEpiC after exposure of HPAEpiC cells to (D) fMLP or (E) LPS [means ± SEM; n = 2 for control (Ctr) group or n = 3 for fMLP and LPS groups]. (F) Epithelial cell miR-223 target vector luciferase activity after coculture of activated human PMNs with transfected pulmonary epithelial cells (Calu-3); data are normalized to control vector activity and compared to no coculture (means ± SEM; n = 3 independent experiments). (G) Setup for murine coculture. (H) mmu–miR-223 expression in mouse pulmonary epithelial (MLE-12) cells after coculture with activated murine PMNs derived from wild-type (WT) or miR-223−/y mice (means ± SEM; n = 11 for the control group, n = 6 for mouse WT PMNs, and n = 7 for miR-223−/y PMNs). (I) mmu–miR-223 expression in MLE-12 cells or E-10 cells after coculture with activated murine PMNs (means ± SEM; n = 7 for MLE-12 control group or n = 8 for all other conditions) [*P < 0.05, Student’s t test or analysis of variance (ANOVA)]. n.s., not significant.

  • Fig. 2. PMN-dependent transfer of miR-223 to human pulmonary epithelial cells.

    (A) hsa–miR-223 release into human PMN–derived supernatants was compared to culture medium only (means ± SEM; n = 4 in the culture medium–only group and n = 6 in the PMN-derived supernatant group) after human PMN activation. (B) hsa–miR-223 in PMN supernatant–derived microvesicles compared to medium only (means ± SEM; n = 4). (C) Expression of miR-223 in HPAEpiC cells after incubation with PMN-derived or control supernatants containing microvesicles (+MV) or depleted of microvesicles (−MV) (means ± SEM; n = 7). (D) hsa–miR-223 expression after coculture of activated human PMNs with HPAEpiC cells in the presence or absence of the endocytosis inhibitor monodansylcadaverine (MDC) (means ± SEM; n = 8). (E) Pulmonary expression of mmu–miR-223 in a mouse model of VILI (means ± SEM; n = 6 mice per group). (F) mmu–miR-223 expression in isolated mouse AT-II cells after VILI (means ± SEM; n = 5 mice in the control group and n = 6 mice in the VILI group). (G) mmu–miR-223 expression in AT-II cells isolated from a mouse bone marrow (BM) chimera (WT→miR-223−/y and miR-223−/y→WT) after exposure to VILI (means ± SEM; n = 5 mice in the WT→miR-223−/y VILI group and n = 4 mice in all other groups). (H) mmu–miR-223 expression in murine lung tissue during VILI after antibody-mediated depletion of PMNs (means ± SEM; n = 5 mice in the control-treated group, n = 4 mice in the 1A8 antibody-treated group, and n = 7 mice in the control-treated VILI group). (I) Intravital microscopy of HL-60 cells, transfected with a dye-labeled miRNA mimic (red) and labeled with calcein dye (green), after intravenous injection into mice ventilated for 30 min (top) or after 90 min of high tidal ventilation to induce VILI (bottom) (two representative images; n = 3 mice). (J) Immunohistochemical (IHC) costaining of AT-II cells [fluorescein isothiocyanate (FITC)–pro–surfactant protein C (SPC); green] and Qtracker-labeled and intravenously injected HL-60 cells (Qtracker-655; red) in murine lung tissue after induction of VILI (one representative image; n = 3 mice). (K) hsa–miR-223 expression in BAL fluid derived from patients with ARDS or healthy controls (median and 25 to 75% percentiles; n = 8 controls and n = 55 ARDS patients; Table 1) (*P < 0.05, Student’s t test, ANOVA, or nonparametric analysis Mann-Whitney test).

  • Fig. 3. PARP-1 is a miR-223 gene target in human pulmonary epithelial cells.

    (A) Baseline hsa–miR-223 expression in lentiviral-transduced, human Calu-3 (lenti-223) cells compared to control Calu-3 (lenti-ctr) cells (means ± SEM; n = 4). (B) Epithelial cell miR-223 target vector luciferase activity in transfected human Calu-3 cells compared to control vector activity (means ± SEM; n = 6). (C) Analysis of PARP-1 transcripts in lentiviral-transduced human Calu-3 cells compared to control Calu-3 cells using real-time reverse transcription polymerase chain reaction (RT-PCR) relative to the housekeeping gene β-actin (means ± SEM; n = 8). (D and E) Western blot analysis of PARP-1 protein in lentiviral-transduced human Calu-3 cells compared to control Calu-3 cells (one representative blot of three is shown; quantification by densitometry, n = 3). (F) Schematic of luciferase reporter plasmids showing the binding sequence between PARP-1 3′UTR and hsa–miR-223. (G and H) Luciferase activities of PARP-1 3′UTR WT plasmid or mutated (MUT) plasmid compared to control 3′UTR plasmid after transfection into lentiviral-transduced human Calu-3 cells (lenti-ctr or lenti-223) (means ± SEM; n = 12). (I) PARP-1 transcripts in the human Calu-3 cell line after coculture with human PMNs measured by real-time RT-PCR relative to the housekeeping gene β-actin (means ± SEM; n = 6) (*P < 0.05; Student’s t test).

  • Fig. 4. Acute lung injury in miR-223−/y mice.

    WT and miR-223−/y mice were exposed to VILI. (A) Albumin detected in mouse BAL fluid was measured in WT and miR-223−/y mice (means ± SEM; n = 5 mice in the WT control group and n = 7 mice in all others groups). (B) The neutrophil enzyme myeloperoxidase (MPO) was measured in BAL fluid from WT and miR-223−/y mice (means ± SEM; n = 5 mice in the WT VILI group and n = 6 mice in all others groups). (C and D) Interleukin-6 (IL-6) and Cxcl-1 protein concentrations in BAL fluid from WT and miR-223−/y mice were measured by enzyme-linked immunosorbent assay (ELISA) [means ± SEM; (C) n = 6 mice in the control group, n = 13 mice in the WT VILI group, and n = 12 mice in the miR-223−/y VILI group; (D) n = 6 mice in the control group, n = 8 mice in the WT VILI group, and n = 13 mice in the miR-223−/y VILI group]. (E) Representative slides of pulmonary histology for WT and miR-223−/y mice [hematoxylin and eosin (H&E) staining]. Scale bar, 100 μm. (F) Scoring of lung injury in slides from (E). (G and H) Pulmonary PARP-1 protein expression in WT or miR-223−/y mice at baseline (Ctr) or during acute lung injury (VILI) relative to the housekeeping gene β-actin (one representative blot of three is shown; quantification by densitometry, n = 3). (I) PARP-1 enzyme activity in WT or miR-223−/y mice (n = 5 mice in the miR-223−/y VILI group or n = 4 mice for all other groups). HPF, high-power field. (J) Representative immunohistochemical staining for PARP-1 activity in WT and miR-223−/y mice (*P < 0.05, ANOVA).

  • Fig. 5. Pulmonary bacterial infection in miR-223−/y mice.

    Pulmonary infection in miR-223−/y mice (filled circles) and WT mice (empty circles) was induced by intratracheal administration of S. aureus. (A) Albumin detected in the BAL fluid was measured [(means ± SEM; n = 4 mice in the control group, n = 8 mice in the WT group infected with S. aureus, and n = 6 mice in the miR-223−/y group infected with S. aureus]. (B) Myeloperoxidase was measured in BAL fluid from miR-223−/y mice infected with S. aureus. IL-6 and Cxcl-1 proteins were measured in BAL fluid using ELISA: (C) IL-6 and (D) Cxcl-1 [(B and C) means ± SEM; n = 8 mice in the control group, n = 13 mice in the WT group infected with S. aureus, and n = 11 mice in the miR-223−/y group infected with S. aureus; (D) means ± SEM; n = 8 mice in the control group, n = 12 mice in the WT mouse group infected with S. aureus, n = 11 mice in the miR-223−/y group infected with S. aureus]. Transcripts were analyzed in mouse lung tissue for (E) IL-6, (F) IL-1β, and (G) Cxcl-1 by real-time RT-PCR relative to the housekeeping gene β-actin. [(E) means ± SEM; n = 4 WT or miR-223−/y mice in the control groups and n = 7 WT or miR-223−/y mice in the S. aureus infection groups; (F and G) means ± SEM; n = 4 WT or miR-223−/y mice in the control groups, n = 8 mice in the WT S. aureus infection groups, and n = 7 in the miR-223−/y S. aureus infection group]. (H) Survival of WT and miR-223−/y mice after pulmonary infection with S. aureus bacteria (n = 9 mice per group). Number of bacterial colony-forming units (CFU) 4 hours after infection with intratracheal S. aureus (I) (means ± SEM; n = 8 mice in the WT group and n = 7 mice in the miR-223−/y group) and in BAL fluid when mice were moribund (J) (means ± SEM; n = 7 mice in the WT group and n = 9 mice in the miR-223−/y group) (*P < 0.05 and §P < 0.05 in the post hoc analysis between WT mice and miR-223−/y mice after S. aureus exposure).

  • Fig. 6. Genetic knockdown of PARP-1 with shRNA in miR-223−/y mice with acute lung injury.

    (A and B) Pulmonary PARP-1 protein knockdown in miR-223−/y mice using PARP-1 shRNA [one representative blot of three is shown; quantification by densitometry in (B), means ± SEM; n = 3 mice]. (C) Immunohistochemical staining for pulmonary PARP-1 activity in miR-223−/y mice treated with Parp-1 shRNA or control shRNA (n = 3 mice). (D) Quantification of immunohistochemical staining in (C) [means ± SEM; n = 6 mice in the nonventilated control shRNA group, n = 4 in the nonventilated Parp-1 shRNA group, and n = 10 or 5 mice in the control shRNA or Parp-1 shRNA ventilated (VILI) groups]. (E) Albumin detected in BAL fluid in miR-223−/y mice treated with control or Parp-1 shRNA and cytokine concentrations in BAL fluid measured by ELISA. (F) IL-6 and (G) Cxcl-1 protein. Pulmonary transcripts in miR-223−/y mice treated with control or Parp-1 shRNA for (H) IL-6, (I) IL-1β, (J) Cxcl-1, and (K) tumor necrosis factor–α (Tnfα), assessed by RT-PCR relative to the housekeeping β-actin gene [(F to K) means ± SEM; n = 4 mice in the control groups and n = 5 mice in the VILI groups] (*P < 0.05, ANOVA).

  • Fig. 7. Pulmonary overexpression of miR-223 during acute lung injury in WT mice.

    Nanoparticle-mediated overexpression of mmu–miR-223 in C57BL/6J mice before acute lung injury induced by ventilation (VILI). (A) Parp-1 transcripts measured by real-time RT-PCR relative to the housekeeping gene β-actin in isolated murine AT-II cells before and after nanoparticle-mediated delivery of miR-223 mimic (either mimic-control or mimic-223) (means ± SEM; n = 5 mice per group). (B) Albumin detection in murine BAL fluid was assessed by ELISA in nanoparticle-treated WT mice (nanoparticles contained either miR-223 mimic or control mimic; means ± SEM; n = 7 mice in the control groups and n = 11 mice in the acute lung injury groups). (C) Lung wet–to–dry weight ratio of nanoparticle-treated murine lungs at baseline and after acute lung injury induced by ventilation (VILI) (means ± SEM; n = 4 mice per group). (D and E) IL-6 and Cxcl-1 protein concentrations in murine BAL fluid were assessed by ELISA [(D) means ± SEM; n = 5 mice in the control groups and n = 9 mice in the acute lung injury VILI groups; (E) means ± SEM; n = 4 mice in the control groups and n = 9 or 10 mice in the mimic-223– or mimic-control–treated VILI groups]. (F) Representative slides of mouse pulmonary histology before and after treatment with nanoparticles containing miR-223 mimic (mimic-223) or control mimic (H&E staining). Scale bar, 100 μm. (G) Scoring of lung injury in histological sections in (F). (H to J) Pulmonary transcripts were assessed by real-time RT-PCR relative to the housekeeping gene β-actin for (H) IL-6, (I) Cxcl1, and (J) Tnfα [means ± SEM; n = 4 mice in the mimic-control–treated groups and n = 5 mice in the mimic-223–treated groups for (H); n = 4 mice in the mimic-control–treated groups and n = 3 and 5 mice in the mimic-223–treated groups for (I); n = 4 and 5 mice in the mimic-control–treated groups and n = 3 and 6 mice in the mimic-223–treated groups for (J)] (*P < 0.05 and §P < 0.05 in the post hoc analysis between “mimic-ctr”–treated and “mimic-223”–treated mice after VILI, Student’s t test or ANOVA).

  • Fig. 8. Pulmonary bacterial infection in C57BL/6J mice overexpressing miR-223.

    After treating C57BL/6J mice with nanoparticles that contained either mimic-223 or mimic-control, mouse lungs were infected with S. aureus intratracheally. (A) Albumin detected in BAL fluid after S. aureus infection and nanoparticle treatment was measured by ELISA (means ± SEM; n = 5 mice per group). (B) Myeloperoxidase was measured in BAL fluid after S. aureus infection and nanoparticle treatment by ELISA (means ± SEM; n = 7 and 5 mice in the control groups and n = 10 mice in the S. aureus infection groups). Protein concentrations of (C) IL-6 and (D) Cxcl-1 were assessed by ELISA in control and S. aureus–infected C57BL/6J mice [(C and D) means ± SEM; n = 5 mice in the control groups and n = 10 mice in the S. aureus infection groups]. Mouse lung tissue was analyzed for (E) IL-6 and (F) IL-1β transcripts by real-time RT-PCR relative to the housekeeping gene β-actin [means ± SEM; (E) n = 9 mice in the mimic-control–treated group and n = 10 mice in the mimic-223–treated group; (F) n = 10 mice per group]. (G) Representative slides showing pulmonary histology for S. aureus–infected WT mice overexpressing miR-223 (mimic-223) or control mimic (H&E staining). Scale bar, 100 μm (means ± SEM; n = 5). (H) Mouse survival after pulmonary infection with S. aureus with or without treatment with nanoparticles containing miR-223 mimic or control mimic (n = 15 mice per group). (I) Lung bacterial colony-forming units 4 hours after infection of mice with intratracheal S. aureus and treated with nanoparticles containing miR-223 mimic or control mimic (means ± SEM; n = 5) (*P < 0.05, Student’s t test, ANOVA, or Mantel-Cox test).

  • Table 1. Characteristics of patients providing BAL fluid samples.

    hsa–miR-223 analysis was assessed in BAL fluid collected from 55 patients with ARDS within the first 7 days after diagnosis. Demographic data and APACHE II (Acute Physiology and Chronic Health Evaluation) clinical scores are shown. BAL samples from eight healthy nonsmokers (five males and three females) served as controls. n/a, not applicable.

    ARDS patientGenderAgePrimary etiologyAPACHE II scoreARDS patientGenderAgePrimary etiologyAPACHE II score
    1Male27Aspiration2931Male63Sepsisn/a
    2Male77Sepsis4032Female37Pneumonia16
    3Male37Pneumonian/a33Female37Sepsis21
    4Male37Pneumonia1534Male57Aspiration21
    5Female20Trauman/a35Male23Aspiration15
    6Female59Pancreatitis3336Female28Aspiration18
    7Male27Sepsisn/a37Female57Aspiration13
    8Male62Sepsisn/a38Male53Sepsis33
    9Female52Pneumonia1539Female42Sepsis8
    10Male48Sepsis3040Female50Aspiration20
    11Male59Sepsisn/a41Male38Pneumonia12
    12Male37Pneumonia1542Female36Pneumonia13
    13Female44Unknown2143Male56Sepsis18
    14Male33Aspiration1344Female47Sepsis9
    15Female71Pneumonia2245Male50Sepsis22
    16Male52Sepsis1046Female54Sepsis24
    17Female53Pneumonia747Male58Pneumonia23
    18Male74Pneumonia3448Male53Pancreatitis19
    19Female45Pneumonia1449Male48Sepsis13
    20Female59Pancreatitis2050Female49Pneumonia25
    21Male37Inhalation1951Male69Pancreatitis19
    22Female60Aspiration1452Male57Pneumonia8
    23Male60Pneumonia2353Female43Aspiration5
    24Male64Aspiration4354Male50Pneumonia24
    25Male43Unknown2255Male42Aspiration15
    26Female47Pneumonia24
    27Male30Trauma20
    28Male53Aspiration27
    29Male39Sepsis13
    30Male45Aspiration30

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/408/eaah5360/DC1

    Fig. S1. miR-223 expression after coculture and baseline levels of miR-223 (Ct values).

    Fig. S2. NETosis inhibition and neutrophil-epithelial miRNA transfer detection.

    Fig. S3. PMN-derived microvesicles in murine BAL fluid and miR-223 in isolated murine pulmonary endothelial cells.

    Fig. S4. Bone marrow chimera and neutrophil depletion.

    Fig. S5. miR-223 in macrophages.

    Fig. S6. Wet–to–dry weight ratios in miR-223−/y mice.

    Fig. S7. Genetic knockdown of PARP-1 in miR-223−/y mice.

    Fig. S8. Epithelial cell death in miR-223–overexpressing C57BL/6J mice.

    Table S1. Significantly down-regulated genes in miR-223–overexpressing epithelial cells.

  • Supplementary Material for:

    Neutrophil transfer of miR-223 to lung epithelial cells dampens acute lung injury in mice

    Viola Neudecker,* Kelley S. Brodsky, Eric T. Clambey, Eric P. Schmidt, Thomas A. Packard, Bennett Davenport, Theodore J. Standiford, Tingting Weng, Ashley A. Fletcher, Lea Barthel, Joanne C. Masterson, Glenn T. Furuta, Chunyan Cai, Michael R. Blackburn, Adit A. Ginde, Michael W. Graner, William J. Janssen, Rachel L. Zemans, Christopher M. Evans, Ellen L. Burnham, Dirk Homann, Marc Moss, Simone Kreth, Kai Zacharowski, Peter M. Henson, Holger K. Eltzschig

    *Corresponding author. Email: viola.neudecker{at}med.uni-muenchen.de

    Published 20 September 2017, Sci. Transl. Med. 9, eaah5360 (2017)
    DOI: 10.1126/scitranslmed.aah5360

    This PDF file includes:

    • Fig. S1. miR-223 expression after coculture and baseline levels of miR-223 (Ct values).
    • Fig. S2. NETosis inhibition and neutrophil-epithelial miRNA transfer detection.
    • Fig. S3. PMN-derived microvesicles in murine BAL fluid and miR-223 in isolated murine pulmonary endothelial cells.
    • Fig. S4. Bone marrow chimera and neutrophil depletion.
    • Fig. S5. miR-223 in macrophages.
    • Fig. S6. Wet–to–dry weight ratios in miR-223−/y mice.
    • Fig. S7. Genetic knockdown of PARP-1 in miR-223−/y mice.
    • Fig. S8. Epithelial cell death in miR-223–overexpressing C57BL/6J mice.
    • Table S1. Significantly down-regulated genes in miR-223–overexpressing epithelial cells.

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