Research ArticleTransplantation

Donor pulmonary intravascular nonclassical monocytes recruit recipient neutrophils and mediate primary lung allograft dysfunction

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Science Translational Medicine  14 Jun 2017:
Vol. 9, Issue 394, eaal4508
DOI: 10.1126/scitranslmed.aal4508
  • Fig. 1. Donor intravenous clo-lip treatment ameliorates PGD after transplantation.

    (A) Experimental design. (B) Allograft histology. Representative histology of the heart-lung blocks of control PBS-lip and intravenous (iv) clo-lip–treated donors at 24 hours after reperfusion. Hematoxylin and eosin staining. Inset scale bars, 50 μm. (C) Allograft function measured by PaO2 on 100% FiO2 as a marker of lung injury at 24 hours after transplant. *P = 0.01, n = 3 to 6 per group. (D) Allograft edema measured by wet-to-dry ratio of allograft at 4 and 24 hours after reperfusion. *P < 0.001, n = 5 per group. (E) Allograft vascular permeability measured by Evans blue dye extravasation leak test of allograft at 4 and 24 hours after reperfusion. *P < 0.01, n = 5 per group. Unpaired Student’s t test was used to compare means.

  • Fig. 2. Intravenous clo-lip selectively depletes NCMs in donor lungs.

    (A) Experimental design. (B) Flow cytometry plots showing effects of monocyte depletion strategies on CMs, NCMs, and CD11b+ dendritic cells (DCs) in wild-type (WT) mice (full gating strategy is shown in fig. S2). (C) Effects of clo-lip and anti-CCR2 on the number of monocytes and relative composition of lung monocytes at 24 hours after treatment. *P < 0.01, n = 5 per group. (C) Data are representative of five experiments. Unpaired Student’s t test with Holm-Šidák correction for multiple comparisons was used to compare means.

  • Fig. 3. Influx of recipient neutrophils into the allograft is abrogated by depletion of donor NCMs.

    (A) Experimental design. (B to G) Intravital two-photon imaging at 2 hours, starting at t = 0 min through t = 30 min after reperfusion. Representative still images of control PBS-lip–treated donor allograft immediately after reperfusion (also refer to movies S1 and S2). Green, LysM+; red, Qdot655 blood vessels. Unpaired Student’s t test was used to compare means. Scale bar, 50 μm. (H) Experimental design and result of differential monocyte depletion strategies in donors and recipients. Combinations of donor and recipient treatments were used to selectively deplete the different monocyte populations. The allograft was harvested at 24 hours after transplantation, and neutrophil influx was determined using flow cytometry. *P = 0.001 compared to group I (all other comparisons to group I are not significant), n = 6 per group. Unpaired Student’s t test with Holm-Šidák correction for multiple comparisons was used to compare means.

  • Fig. 4. Pulmonary intravascular NCMs are dependent on CX3CR1 to recruit neutrophils, and reconstitution of depleted NCMs restores neutrophil influx in NR4A1-deficient mice.

    (A) Experimental design for CX3CR1 knockout transplants. (B) Representative flow plots and effects of CX3CR1 deletion on monocyte populations in donor lungs and posttransplant neutrophil infiltration of the allograft. *P = 0.01, n = 6 per group. Unpaired Student’s t test was used to compare means. (C) Experimental design for NR4A1 knockout transplants. (D) Representative flow plots and effects of NR4A1 deletion NCM reconstitution on monocyte populations in donor lungs and posttransplant neutrophil infiltration. *P = 0.01, n = 5 per group. Unpaired Student’s t test with Holm-Šidák correction for multiple comparisons was used to compare means.

  • Fig. 5. Compartmentalization of pulmonary NCMs and CMs and their response to intratracheal lipopolysaccharide.

    (A) Two-photon imaging of Cx3cr1gfp/+ lungs. Green, Cx3cr1+; red, Qdot655 blood vessels; blue, second harmonic generation collagen; yellow, autofluorescent alveolar macrophages. Filled arrow, intravascular cell; open arrow, extravascular cell. (B) Morphology of the lung myeloid cell populations in the donor lung using immunogold electron microscopy in the Cx3cr1gfp/+ reporter mouse. Left: Immunoelectron microscopy of fixed Cx3cr1gfp/+ lung, with white circles highlighting gold nanoparticles staining for GFP. Right: Electron microscopy micrographs of postsort cells from WT B6 mouse lung. (C) Immunoelectron microscopy of Cx3cr1gfp/+ donor lungs at 4 hours after reperfusion, with white circles highlighting gold nanoparticles staining for GFP. Left and middle: NCMs bound to the endothelium with areas of exposed, thickened basement membrane and endothelial cell blebbing. Right: Neutrophil bound to the endothelium in the vicinity of a blebbing endothelial cell. (D) Representative compartmental staining of NCMs and alveolar macrophages. As negative control, FMO+1 (fluorescence minus one control + 1) staining is shown. it, intratracheal. (E) Experimental design and representative flow diagrams of intravenous and intratracheal anti-CD45 staining in LPS-treated and control mice along with neutrophil infiltration into the lungs with and without intravenous clo-lip pretreatment. Unpaired Student’s t test, not significant. n = 5 per group. LPS, lipopolysaccharide.

  • Fig. 6. Transcriptional profiling of murine posttransplant donor-derived NCMs.

    (A) Experimental plan to isolate and sort NCMs from donor-naïve, +2-hour, and +24-hour posttransplant lung. (B) Sorting strategy with representative flow plots from each time point. (C) Principle components (PC) analysis of samples. (D) Gene ontology process enrichment analysis using K-means clustering. (E) TLR and nuclear factor κB signaling pathway genes, which were differentially expressed by cutoff of adjusted P value of <0.05 by pairwise comparison between time points, with the normalized counts of genes of interest depicted. *P < 0.04 by one-way analysis of variance (ANOVA), n = 4 per group. Heat map scale bars represent log2 scale.

  • Fig. 7. NCMs are dependent on MyD88/TRIF signaling to produce CXCL2.

    (A) Experimental design. (B) Effects of reconstitution of NCM-depleted donor lungs with either WT or Myd88/Trif−/− NCMs. *P < 0.01, n = 5 per group. (C) Cxcl2 transcript expression level in donor-derived NCMs measured by quantitative polymerase chain reaction at 2 hours after transplant. As control, the baseline expression of Cxcl2 in WT pulmonary NCMs is shown. *P = 0.01. (D) Effect of NCM depletion on CXCL2 cytokine levels in allograft circulation when NCMs are depleted. As control, the CXCL2 levels in left pulmonary vein blood of WT mice are shown. *P < 0.01, n = 5 per group. Unpaired Student’s t test with Holm-Šidák correction for multiple comparisons was used to compare means. (E) Effect of CXCL2 blockade on neutrophil recruitment in the recipient at 24 hours after transplant. *P = 0.001, n = 5 per group. Unpaired Student’s t test was used to compare means.

  • Fig. 8. Myeloid cell populations in human donor lungs and immediate postreperfusion changes.

    (A) Representative gating strategy of human lungs flushed and used in clinical transplantation. After excluding doublets and dead cells, and including only CD45+ cells, neutrophils were identified as CD15+CD16+SSChigh. After gating out CD15+ events, an HLA-DR+CD11b+ gate was used to identify monocytes and macrophages. Alveolar macrophages (AMs) were identified as CD15HLA-DR+CD11b+CD169+CD206+. After gating out AMs, NCMs were identified as CD16++CD14dim, intermediate monocytes (IntMs) as CD16+CD14+, and CMs as CD14+CD16. (B) Changes in NCMs and neutrophils at 90 min after reperfusion. Data are expressed as cell count per AM to standardize across patients. Biopsies were taken serially from the same location in the lung. *P = 0.02 (by paired Student’s t test), n = 8. Immunofluoresence microscopy of prereperfusion (C) and postreperfusion (D) human lung samples depicting endothelial-bound intravascular CD16+CD14dim NCMs (filled white arrow) in contrast with CD16 CD14high CMs (open arrow) and CD16+CD14+ neutrophils (filled white chevron). Green, CD31; blue, DAPI (4′,6-diamidino-2-phenylindole); red, CD16; yellow, CD14. Scale bars, 10 μm.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/394/eaal4508/DC1

    Materials and Methods

    Fig. S1. Cytokine analysis of the lung allograft.

    Fig. S2. Representative gating strategy to evaluate the myeloid cell populations in the lungs.

    Fig. S3. Perfusion of the heart-lung block.

    Fig. S4. Effects of monocyte depletion strategies on the donor.

    Fig. S5. Effects of donor clo-lip treatment on the 24-hour posttransplant allograft.

    Fig. S6. Donor clo-lip treatment abrogates neutrophil influx after syngeneic lung transplant.

    Fig. S7. Effects of CX3CR1 deletion on nonmonocyte cell populations in the lung and blood and on the posttransplant allograft.

    Fig. S8. Effects of NR4A1 deletion on non-monocyte cell populations in the lung and blood and on the posttransplant allograft.

    Fig. S9. All neutrophils were of recipient origin after 24 hours of reperfusion in the allograft.

    Fig. S10. Representative gating strategy of GFP-expressing cells in the lungs of Cx3cr1gfp/+ and Cx3cr1gfp/gfp mice.

    Fig. S11. NCMs do not differentiate upon lipopolysaccharide challenge.

    Fig. S12. Immunofluorescence microscopy of human lung biopsy from a patient experiencing PGD.

    Fig. S13. Schematic to illustrate the role of pulmonary intravascular NCMs in mediating neutrophil infiltration and lung allograft injury.

    Table S1. Primary data.

    Movie S1. PBS-lip–treated control mice reveal profound neutrophil infiltration into the lung allograft after reperfusion.

    Movie S2. Intravenous clo-lip treatment of the donors abrogates neutrophil infiltration into the allograft after reperfusion.

    References (5257)

  • Supplementary Material for:

    Donor pulmonary intravascular nonclassical monocytes recruit recipient neutrophils and mediate primary lung allograft dysfunction

    Zhikun Zheng, Stephen Chiu, Mahzad Akbarpour, Haiying Sun, Paul A. Reyfman, Kishore R. Anekalla, Hiam Abdala-Valencia, Daphne Edgren, Wenjun Li, Daniel Kreisel, Farida V. Korobova, Ramiro Fernandez, Alexandra McQuattie-Pimentel, Zheng J. Zhang, Harris Perlman, Alexander V. Misharin, G. R. Scott Budinger, Ankit Bharat*

    *Corresponding author. Email: abharat{at}nm.org

    Published 14 June 2017, Sci. Transl. Med. 9, eaal4508 (2017)
    DOI: 10.1126/scitranslmed.aal4508

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Cytokine analysis of the lung allograft.
    • Fig. S2. Representative gating strategy to evaluate the myeloid cell populations in the lungs.
    • Fig. S3. Perfusion of the heart-lung block.
    • Fig. S4. Effects of monocyte depletion strategies on the donor.
    • Fig. S5. Effects of donor clo-lip treatment on the 24-hour posttransplant allograft.
    • Fig. S6. Donor clo-lip treatment abrogates neutrophil influx after syngeneic lung transplant.
    • Fig. S7. Effects of CX3CR1 deletion on nonmonocyte cell populations in the lung and blood and on the posttransplant allograft.
    • Fig. S8. Effects of NR4A1 deletion on non-monocyte cell populations in the lung and blood and on the posttransplant allograft.
    • Fig. S9. All neutrophils were of recipient origin after 24 hours of reperfusion in the allograft.
    • Fig. S10. Representative gating strategy of GFP-expressing cells in the lungs of Cx3cr1gfp/+ and Cx3cr1gfp/gfp mice.
    • Fig. S11. NCMs do not differentiate upon lipopolysaccharide challenge.
    • Fig. S12. Immunofluorescence microscopy of human lung biopsy from a patient experiencing PGD.
    • Fig. S13. Schematic to illustrate the role of pulmonary intravascular NCMs in mediating neutrophil infiltration and lung allograft injury.
    • Legend for table S1
    • Legends for movies S1 and S2
    • References (5257)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Primary data.
    • Movie S1 (.mov format). PBS-lip–treated control mice reveal profound neutrophil infiltration into the lung allograft after reperfusion.
    • Movie S2 (.mov format). Intravenous clo-lip treatment of the donors abrogates neutrophil infiltration into the allograft after reperfusion.

    [Download Table S1]

    [Download Movies S1 and S2]