Research ArticleTHROMBOSIS

Neutrophil macroaggregates promote widespread pulmonary thrombosis after gut ischemia

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Science Translational Medicine  27 Sep 2017:
Vol. 9, Issue 409, eaam5861
DOI: 10.1126/scitranslmed.aam5861
  • Fig. 1. Gut I/R injury induces the formation of occlusive neutrophil-rich thrombi in the pulmonary vasculature.

    Mice were subjected to gut I/R injury or sham operation. (A) Representative hematoxylin and eosin (H&E) staining comparing intravascular leukocyte aggregates and fibrin in lungs of I/R-injured and sham-operated mice. (B) Quantification of the number of pulmonary intravascular leukocyte aggregates, normalized for the surface area of lung sections (I/R, n = 14; sham, n = 8). (C and D) Lungs were flushed and perfused with Microfil to identify defective vascular perfusion. (C) Representative photographs of the arterial and venous vasculature of the left lung lobe or (D) phase contrast images of the indicated lung vessel branches depicting vascular perfusion (vessel branches are color-coded with corresponding vessel sizes indicated) (I/R, n = 7; sham, n = 3). (E and F) Mice were administered DyLight 647–anti–Gr-1 antibody, before gut I/R injury and lung Microfil perfusion, to identify colocalization of neutrophil aggregates (red) and defective Microfil perfusion (blue) in the pulmonary arteries and veins. Confocal images (E) are from one representative experiment (vessels, yellow dotted lines), and the percentage (%) of blocked vessels with associated neutrophil aggregates was quantified (F) (I/R-artery, n = 5; I/R-vein, n = 3). (G) Representative confocal image depicting neutrophil aggregates (red) in the pulmonary circulation (green, collagen autofluorescence) after gut I/R injury. (H and I) H&E staining (H) and quantification (I) of pulmonary intravascular fibrin formation in I/R-injured mice with or without preneutrophil depletion (anti–Gr-1 and RB6-8C5). Scale bars, 50 μm (A), 1000 μm (C), 100 μm (D), 20 μm (E), 10 μm (G), and 50 μm (H). Error bars represent means ± SEM. *P < 0.05.

  • Fig. 2. Neutrophil-rich mixed arterial-venous thrombi in the lungs of ARDS patients and splanchnic circulation of ischemic mice.

    (A to C) Postmortem lung specimens from patients with ARDS or acute pulmonary edema (APE) or from explanted lungs from emphysema (EMP) patients. (A) H&E and Carstair’s staining of ARDS and EMP lung specimens to detect intravascular neutrophil aggregates (right, H&E, arrows) and associated fibrin formation (left, Carstair’s). (B) The number of pulmonary vessels (%) containing neutrophil aggregates in ARDS, APE, or EMP patients was quantified (ARDS, n = 12; APE, n = 11; EMP, n = 10). (C) Carstair’s staining depicting neutrophil-rich thrombi in both arteries and veins of ARDS specimens. ns, not significant. RBCs, red blood cells. (D to G) Mice were administered phycoerythrin (PE)–Gr-1 antibody before gut I/R injury. (D) Fluorescence images depicting neutrophil aggregates (red) in mesenteric veins (dotted line) after I/R injury. (E) Fluorescence images depicting neutrophil aggregates (red) in the systemic arterial (left ventricle of the heart) and venous (IVC) blood after sham operation or gut I/R injury. (F) Number of aggregated versus single neutrophils in mesenteric veins 30 to 90 min after I/R injury (n = 6). (G) The impact of ischemia time on neutrophil aggregate formation in mesenteric veins (n = 3 to 4). *P < 0.05; **P < 0.01; ***P < 0.005.

  • Fig. 3. Neutrophil-rich thrombus formation is platelet-dependent.

    (A to C) C57Bl/6 mice or the indicated genotype (A) were administered PE–Gr-1 and DyLight 647–GPIbβ antibodies before gut I/R injury. (A) Quantification of the impact of platelet depletion (C57BL/6JPlt-depleted), P-selectin deficiency (P-sel−/−), and hematopoietic P-selectin deficiency (P-selPlt−/−) on neutrophil aggregate formation in mesenteric veins during I/R injury (n = 3). (B and C) Confocal images of heterotypic platelet-neutrophil aggregates in mesenteric veins (B) and pulmonary vasculature (C) after I/R injury. (D) Histological immunostaining of ARDS specimens depicting platelets (integrin αIIb, brown, yellow arrowheads) within intravascular neutrophil aggregates (blue) in the lung vasculature (marked with yellow dotted lines). Scale bars, 50 μm (B), 10 μm (C), 200 μm (D, left), 20 μm (D, right). Error bars represent means ± SEM. **P < 0.01.

  • Fig. 4. Neutrophil aggregation is selectively induced by PS+ platelets.

    (A to C) Mice were administered indicated fluorescence probes (A and C) or Alexa 488–annexin V (PS+ platelets) and DyLight 647–anti-GPIbβ antibodies (platelets) (B) before needle puncture of mesenteric veins, followed by local microinjection of either ADP or Thr/CRP. (A) Representative confocal images depict neutrophil aggregate formation and detachment (red) from ADP or Thr/CRP thrombi (green) ~30 min after agonist injection. (B) Annexin V binding to thrombi (PS+ platelets) at the indicated times after agonist injection (n = 3). (C) Confocal images depicting PS+ platelets (green/yellow) within detaching neutrophil aggregates (red) from Thr/CRP-stimulated thrombi. (D and E) Confocal images depicting neutrophil aggregates (red) anchored by PS+ platelets (green/blue, cyan) in mesenteric veins, as demonstrated by channel overlays (white and cyan in magnified images, respectively) (D), and in pulmonary vasculature (dotted line) (E) after gut I/R injury. (F) Confocal images depicting PS+ platelet formation (green/blue, cyan) in intestinal microvasculature during gut I/R injury. (G and H) Percent (%) of intestinal vessels containing PS+ platelets (G) and the percent of platelets in a PS+ state in the intestinal microvasculature (H) after I/R injury [I/R, n = 4 (G); sham, n = 3 (G); I/R, n = 5 (H)]. (I) Confocal image depicting PS+ platelets [green/blue, cyan (arrows)] adherent to pulmonary vasculature (dotted line) after gut I/R injury. (J) Confocal images depict occlusive neutrophil aggregates (green) and colocalized fibrin (red) and platelets (blue) in the pulmonary vasculature 2 hours after gut I/R injury (n = 5). Scale bars, 50 μm (D), 20 μm (E), 100 μm (F), 10 μm (I), and 50 μm (J). Error bars represent means ± SEM. *P < 0.05; *P < 0.01; ***P < 0.005; ****P < 0.0001.

  • Fig. 5. Remnants of PS+ platelets induce neutrophil macroaggregation.

    (A) Representative differential interference contrast (DIC) images depicting spread human platelets pre-Thr/CRP stimulation (Thr/CRP 0′), remnant platelets post-Thr/CRP stimulation (Thr/CRP 9.3′) after adhesion to fibrinogen, and shed microparticles from Thr/CRP-stimulated platelets in suspension. Scale bar, 1 μm. (B and C) Representative phase contrast images depicting neutrophil macroaggregate formation on remnant PS+ platelets (B) or PS platelets (C) at the indicated shear rates (n = 3). (D) Quantification of the percentage of adherent neutrophils aggregated (left) and size of neutrophil aggregates formed (right) after neutrophil perfusion over remnant PS+ platelets, relative to microparticle coperfusion (1× or 10 to 100× MPs) over PS platelets. Error bars represent means ± SEM (n = 3). ****P < 0.001. (E) Representative confocal images depicting the extent of incorporation of large remnant PS+ platelet membrane fragments within aggregating neutrophils on remnant PS+ platelets or after microparticle coperfusion (1× MP) over PS platelets (n = 3 to 5). Scale bars, 10 μm.

  • Fig. 6. Rolling neutrophils extract membranes from fragile remnant PS+ platelets.

    (A) Fluorescence and DIC images depicting the level of filamentous actin (phalloidin) in remnant PS+ human platelets (annexin V). (B and C) DIC images demonstrate remnant platelet membrane deformation and detachment (B), quantification of platelet detachment, and calculated drag forces at the indicated shear rates (C) (means ± SEM) (n = 9). (D and E) Representative scanning electron microscopy (SEM) images depicting the size of membrane fragments (MFs) pulled by neutrophils from spread remnant platelets (D) and the integrity of PS+ and PS platelet membranes after neutrophil perfusion (E). (F and G) Representative fluorescence images depict the ripping and dragging of remnant PS+ platelets (green) by rolling neutrophils (*, red) from nonspread platelets (F) (ripped platelet, yellow dotted circles and white arrows; dragged platelet, yellow and white dotted circles and white arrows) over the indicated time frames and percent of total PS+ platelets being ripped or dragged (means ± SEM) (n = 3) (G). (H to M) Confocal and SEM images depicting the ripping of remnant PS+ platelet membranes by rolling neutrophils (*, red) from nonspread platelets (green) (H) over the indicated time frames (yellow and white circles and yellow arrows) and spread platelets (J to M), platelet membrane wrapping (I and J, white arrows), and bridging adjacent neutrophils (K and L, white arrows). (M) Representative fluorescence image depicting the extensive surface coating of aggregating neutrophils (hollow and unlabeled) by remnant PS+ platelet membranes (fluorescent) after perfusion over spread PS+ platelets. Scale bars, 3.8 μm (D, left), 1 μm (D, middle), 2 μm (D, right), 2.5 μm (E, left), 2 μm (E, right), 10 μm (F), 10 μm (H), 1 μm (J), 2 μm (K, left), 1 μm (K, right), and 10 μm (M). 2D, two-dimensional.

  • Fig. 7. Rolling neutrophils rip and drag PS+ platelet membranes in vivo.

    (A to E) C57BL mice or mice of the indicated genotype were administered appropriate fluorescence probes and then subjected to needle puncture of mesenteric vein, followed by local Thr/CRP microinjection (A and D) or gut I/R injury (B, C, and E). (A and B) Representative confocal images depicting ripping (A, top) and dragging (A, bottom, and B) of PS+ platelets [P1–3, cyan-colored cells (yellow outline)] by rolling neutrophils [N1–3, red cells (white outline)] and PS+ platelets bridging adjacent neutrophils (A, bottom) on thrombi 30′ after agonist injection (A) or in intestinal vasculature after I/R injury (B) over the indicated time frames. (A) Top: Large platelet fragments ripped from P1 and P2 by N1. Bottom: P1 (tracked by yellow arrow) dragged by N1, P2 dragged by N2, and P3 dragged by N3, culminating in neutrophil macroaggregation by bridging of N1 and N2 via P2 and of N1 and N3 via P1 and P3. (B) P2 dragged by N1-P1 rolling complex (tracked by yellow arrow). (C) Representative confocal images showing neutrophils interacting with (left) and ripping (right) PS+ platelets from lung vasculature (dotted line) after gut I/R injury. (D) Left: The number of PS+ platelets/fragments ripped or dragged by single (gray) or aggregated (white) rolling neutrophils or detached independently of neutrophils (black) from mesenteric thrombi over 2′ and 30′ after agonist injection in P-sel+/+ and P-sel−/− mice (percent of total). (D) Right: The time remaining on thrombi for neutrophil-associated PS+ fragments in P-sel+/+ mice (white and gray) and neutrophil-free PS+ fragments in P-sel−/− mice (n = 3 and 9 thrombi). (E) Confocal images depicting large PS+ platelet membrane fragments (cyan) within neutrophil aggregates (red) in mesenteric vein after gut I/R injury. (F) All the plasma from an I/R-injured or naïve donor mouse (plasma) was administered to a naïve recipient mouse (recipient) via the portal vein and then subjected to confocal microscopy. The number of aggregated neutrophils in the mesenteric veins and pulmonary circulation of excited lungs was quantified and compared to gut I/R–injured mice (I/R) (means ± SEM; n = 3). Scale bars, 10 μm (A), 50 μm (B), 10 μm (C), and 50 μm (E).

  • Fig. 8. CypD deficiency reduces neutrophil aggregate formation and protects lung function after gut I/R injury.

    CypDPlt+/+ and CypDPlt−/− mice were subjected to gut I/R injury with or without the indicated fluorescent probes. (A) Representative images of confocal intravital microscopy examining PS+ platelet-neutrophil aggregates (green/blue-red) in CypDPlt−/− and CypDPlt+/+ mesenteric veins after gut I/R injury (n = 4). (B) The number of single and aggregated neutrophils was quantified in the mesenteric veins of CypDPlt+/+ and CypDPlt−/− mice 30 to 90 min after gut I/R injury (n = 4). (C) Representative H&E staining of lung sections detecting intravascular neutrophil aggregates in CypDPlt+/+ and CypDPlt−/− mice after gut I/R injury. (D) The number of aggregated neutrophils in the pulmonary vasculature of CypDPlt+/+ and CypDPlt−/− mice after gut I/R injury (without saline flush) was quantified and normalized for lung section surface area (CypDPlt+/+, n = 10; CypDPlt−/−, n = 8). (E and F) Representative confocal images assessing neutrophil aggregates (green, Gr-1 antibody) and colocalized fibrin (red) and platelets (blue) in CypDPlt+/+ and CypDPlt−/− pulmonary vasculature (E) and the number of fibrin-rich neutrophil aggregates in three lobes of each lung quantified (F) (n = 4). Scale bars, 50 μm (A and E) and 20 μm (C). Error bars represent means ± SEM. *P < 0.05. (G and H) Line graphs depict quantification of arterial blood oxygen levels (G) and survival rates (H) in CypDPlt−/− and CypDPlt+/+ mice after gut I/R injury [CypDPlt+/+, n = 9 (G); CypDPlt−/−, n = 7 (G); CypDPlt+/+, n = 12 (H); CypDPlt−/−, n = 8 (H)]. (I) Representative images of the arterial vasculature of the left lung lobe (top), or the indicated lobe vasculature (bottom), depicting the extent of Microfil perfusion in CypDPlt+/+ and CypDPlt−/− mice (left) (scale bars, 50 μm; CypDPlt+/+, n = 8; CypDPlt−/−, n = 6), and the number of nonperfused vessels with a diameter of ≥80 μm (percent of total vasculature for each genotype) (right) in the flushed lungs of gut I/R–injured mice [CypDPlt+/+, n = 8 (24 vessels/lobe, 3 lobes/mouse); CypDPlt−/−, n = 6 (24 vessels/lobe, 3 lobes/mouse)].

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/409/eaam5861/DC1

    Materials and Methods

    Fig. S1. Formation of leukocyte aggregates and fibrin in the pulmonary vasculature of mice after gut I/R injury.

    Fig. S2. Neutrophil aggregate and fibrin formation in the lung vasculature of I/R-injured mice and ARDS patients.

    Fig. S3. Neutrophil aggregate formation in the splanchnic circulation of the mouse after gut I/R injury.

    Fig. S4. P-selectin–expressing platelets on gut vasculature and within neutrophil aggregates after gut I/R injury.

    Fig. S5. Neutrophil aggregation requires potent platelet activation.

    Fig. S6. PS+ platelets selectively support neutrophil aggregation in vivo.

    Fig. S7. Occlusive neutrophil aggregate formation is associated with fibrin formation.

    Fig. S8. Rolling neutrophils pull membrane fragments from PS+ platelets.

    Fig. S9. Characterization of platelets deficient in CypD or Bak/Bax.

    Fig. S10. Conventional antiplatelet therapies do not prevent neutrophil aggregate formation after gut I/R injury.

    Fig. S11. Schematic illustration of the proposed mechanism linking gut I/R injury to pulmonary thrombosis.

    Table S1. ARDS: Patient details.

    Table S2. APE: Patient details.

    Table S3. Primary data (Excel file).

    Movie S1. Neutrophil aggregate formation on PS+ thrombi at sites of vascular injury.

    Movie S2. PS+ platelet-neutrophil aggregate formation in the mesenteric veins after gut I/R injury.

    Movie S3. Neutrophil ripping and dragging PS+ platelet membranes by rolling neutrophils in vitro (low magnification).

    Movie S4. Neutrophil ripping PS+ platelets in vivo and in vitro.

    Movie S5. Neutrophil extract PS+ platelets in the intestinal vasculature after gut I/R injury.

    References (6773)

  • Supplementary Material for:

    Neutrophil macroaggregates promote widespread pulmonary thrombosis after gut ischemia

    Yuping Yuan, Imala Alwis, Mike C. L. Wu, Zane Kaplan, Katrina Ashworth, David Bark Jr., Alan Pham, James Mcfadyen, Simone M. Schoenwaelder, Emma C. Josefsson, Benjamin T. Kile, Shaun P. Jackson*

    *Corresponding author. Email: shaun.jackson{at}sydney.edu.au

    Published 27 September 2017, Sci. Transl. Med. 9, eaam5861 (2017)
    DOI: 10.1126/scitranslmed.aam5861

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Formation of leukocyte aggregates and fibrin in the pulmonary vasculature of mice after gut I/R injury.
    • Fig. S2. Neutrophil aggregate and fibrin formation in the lung vasculature of I/R-injured mice and ARDS patients.
    • Fig. S3. Neutrophil aggregate formation in the splanchnic circulation of the mouse after gut I/R injury.
    • Fig. S4. P-selectin–expressing platelets on gut vasculature and within neutrophil aggregates after gut I/R injury.
    • Fig. S5. Neutrophil aggregation requires potent platelet activation.
    • Fig. S6. PS+ platelets selectively support neutrophil aggregation in vivo.
    • Fig. S7. Occlusive neutrophil aggregate formation is associated with fibrin formation.
    • Fig. S8. Rolling neutrophils pull membrane fragments from PS+ platelets.
    • Fig. S9. Characterization of platelets deficient in CypD or Bak/Bax.
    • Fig. S10. Conventional antiplatelet therapies do not prevent neutrophil aggregate formation after gut I/R injury.
    • Fig. S11. Schematic illustration of the proposed mechanism linking gut I/R injury to pulmonary thrombosis.
    • Table S1. ARDS: Patient details.
    • Table S2. APE: Patient details.
    • Legends for movies S1 to S5
    • References (6773)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S3. Primary data (Excel file).
    • Movie S1 (.mov format). Neutrophil aggregate formation on PS+ thrombi at sites of vascular injury.
    • Movie S2 (.mov format). PS+ platelet-neutrophil aggregate formation in the mesenteric veins after gut I/R injury.
    • Movie S3 (.mov format). Neutrophil ripping and dragging PS+ platelet membranes by rolling neutrophils in vitro (low magnification).
    • Movie S4 (.mov format). Neutrophil ripping PS+ platelets in vivo and in vitro.
    • Movie S5 (.mov format). Neutrophil extract PS+ platelets in the intestinal vasculature after gut I/R injury.

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