Research ArticleFibrosis

Platelet microparticles sustain autophagy-associated activation of neutrophils in systemic sclerosis

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Science Translational Medicine  25 Jul 2018:
Vol. 10, Issue 451, eaao3089
DOI: 10.1126/scitranslmed.aao3089
  • Fig. 1 Platelet microparticles from patients with SSc express HMGB1.

    (A) Whole blood from healthy controls (left) or patients (middle and right) was examined by flow cytometry. CD61+ events were identified as microparticles based on low forward and side scatter. HMGB1 staining is shown in the lower row (red), with isotype staining in black. Images shown are representative of 53 healthy controls, 57 SSc patients, and 6 SLE patients. (B) (Top) Platelet-derived microparticles (PDμP) were identified in platelet-free plasma based on side scatter and CD61 expression. (Bottom) Concentration of platelet-derived CD61+ microparticles and of CD61+ microparticles expressing HMGB1. Each dot corresponds to a single subject, and lines indicate means ± SEM. ***P < 0.0001 by analysis of variance (ANOVA) and Bonferroni test. (C) (Top) Purified platelets from five healthy donors were stimulated with collagen or TRAP-6, and representative platelet P-selectin expression (red) is shown. Bars indicate means ± SEM (five different experiments carried out independently). (Bottom left) Expression of HMGB1 (red) on microparticles released by collagen-stimulated platelets (5 min). (Bottom middle) Fraction of HMGB1+ microparticles released by collagen- or TRAP-6–stimulated platelets at various times. (Bottom right) Expression of CD61, CD42, CD62P (P-selectin), and CD142 (tissue factor) on collagen-microparticles. Each dot represents the result of a single independent assessment, and lines indicate means ± SEM.

  • Fig. 2 Microparticles activate neutrophils via HMGB1.

    (A) Neutrophils from healthy donors were stimulated or not with collagen-microparticles (Coll-μP), and membrane MPO expression (mMPO) was assessed by confocal microscopy and flow cytometry (upper histogram: MPO fluorescence intensity; middle: fraction of neutrophils expressing mMPO). Representative of 10 experiments carried out independently. (Bottom) Fibrin proteolysis, assessed as actual area of proteolysis (mm2), with increasing concentrations of collagen-microparticles. The effect of a given concentration of microparticles (100,000/μl) of different origin (Coll-μP or microparticles derived from SSc or healthy controls) on the ability to digest fibrin is also shown. Recombinant HMGB1 was used as positive control. Each dot represents a single donor. Lines indicate means ± SEM. *P < 0.01 and ****P < 0.0001 by ANOVA and Bonferroni test. (B) (Left) Neutrophils were stimulated with collagen-, control-, and SSc-microparticles or with HMGB1 and analyzed by confocal microscopy for the Cyto-ID autophagy tracer (green) or for DNA (white). (Right) Cyto-ID was quantified by flow cytometry in untreated (gray) or microparticle-stimulated neutrophils with (open circles) or without the HMGB1 inhibitor BoxA (red). HMGB1 (blue) is the positive control. (C) The concentration of the NET by-products, MPO-DNA complexes, released by neutrophils was assessed by ELISA (left), and NETs were visualized by confocal microscopy (right). White, DNA; red, platelet antigen CD42; blue, citrullinated H4 histone; green, Cyto-ID. ****P < 0.0001 by ANOVA and Bonferroni test. OD, optical density.

  • Fig. 3 Neutrophil activation, autophagy, and NETs are associated in the blood from SSc patients.

    (A) Representative image of neutrophils from patients with SSc. Green, MPO; white, nuclei. (B) Neutrophil membrane MPO expression (mMPO) from SSc patients and matched healthy volunteers was quantified by flow cytometry. Each symbol refers to a single subject. (C and D) Neutrophils from healthy volunteers (C) and SSc patients (D) were examined for autophagosomes by confocal microscopy (green). DNA was counterstained with Hoechst (white). (E) Transmission electron microscopy of neutrophils purified from the blood of SSc patients, immediately fixed, and processed. (F) The fraction of Cyto-ID+ neutrophils in the blood of SSc patients and healthy controls was assessed by flow cytometry. Each symbol refers to a single subject. Bars indicate means ± SEM. ****P < 0.0001. (G and H) Concentration of by-products of NET generation/catabolism, including citrullinated H4 histone (G) and soluble DNA-MPO complexes (H), in the plasma of SSc patients and healthy volunteers. (I) Correlation of citrullinated H4 and MPO-DNA complexes in plasma. Symbols depict individual observations in each subject, and bars indicate means ± SEM. ****P < 0.0001 by ANOVA and Bonferroni test, significantly different from healthy subjects. MFI, mean fluorescence intensity.

  • Fig. 4 Interaction with SSc-microparticles prompts neutrophil survival due to activation of autophagy.

    Human neutrophils from healthy donors were exposed (open triangles) or not (open squares) to SSc-microparticles and injected in NSG mice. (A) Human neutrophil counts in the mouse were assessed over time. Dependence of neutrophil survival on autophagy was assessed by treatment with the blocker of the autophagic flux, wortmannin, before the challenge with SSc-microparticles (black diamond) and the effect of pharmacological induction of autophagy by treatment with rapamycin (black circles). (B) The fraction of autophagic untreated and treated neutrophils, determined by the accumulation of Cyto-ID, was assessed at various times after injection. Symbols indicate the mean and bars indicate the SEM of three and four different experiments conducted independently. ****P < 0.0001, ***P < 0.005, and **P < 0.05, by ANOVA and Bonferroni test, significantly different from untreated neutrophils. Purified neutrophils were challenged (or not) with collagen microparticles from three different healthy donors. Independently purified neutrophils were injected in groups of three mice.

  • Fig. 5 Platelet microparticles trigger neutrophil and endothelial activation, lung inflammation, and fibrosis in NSG mice.

    (A) Human collagen-microparticles were injected into NSG mice, and their blood counts were assessed at various times based on the expression of the human CD61. (B) The fraction of murine neutrophils with adherent human microparticles, (C) the fraction of autophagic neutrophils identified based on the accumulation of Cyto-ID, (D) and the fraction of neutrophils that expressed MPO on the plasma membrane were assessed at various times after injection. (E to G) Lungs of NSG mice were harvested 24 hours after injection with phosphate-buffered saline (sham) (H to J) or with human collagen-microparticles. Sections were processed for confocal microscopy (E and H), and endothelial lining was identified by CD31 (blue), infiltrating neutrophils were identified by MPO (green), and nuclei were counterstained with Hoechst (white). Alternatively, sections were processed for histochemistry with hematoxylin and eosin (F and I) or Sirius red (G and J) and examined by immunofluorescence for infiltration of inflammatory cells. Scale bars, 300 μm. Concentrations of E-selectin (K) and of the NET by-product, citrullinated H4 histone, (L) assessed by ELISA in the plasma of mice injected with collagen-microparticles, to assess systemic endothelial activation and NET generation, respectively. Microparticles from the plasma of patients with SSc (μP) or of healthy volunteers (Control-μP) were injected into NSG mice in the presence or absence of the HMGB1 inhibitor BoxA. The fraction of murine neutrophils that expressed MPO on the plasma membrane (M) and the fraction of autophagic neutrophils (N) were assessed by flow cytometry, while the concentration in the plasma of low–molecular weight soluble DNA (O) or of citrullinated H4 histones (P) was assessed by ELISA. Symbols depict individual observations in each mouse injected with microparticles derived from a single donor. Lines represent the means ± SEM for each determination. ****P < 0.0001, significantly different from sham-treated mice (K and L) or from mice injected with control microparticles or with SSc-microparticles in the presence of BoxA (M to P) by ANOVA and Bonferroni test. LMW, low molecular weight.

  • Table 1 Microparticles from the blood of SSc patients trigger neutrophils and endothelial cell activation.
    Healthy
    controls
    SScSLE
    n53576
    Leukocytes/μl5793 ± 1066922 ± 458*5933.3 ± 113
    Neutrophils/μl3546 ± 834663 ± 5363500 ± 543
    Platelets (×103/μl)200 ± 5223 ± 17248 ± 5
    Whole blood
     CD66b+CD16
    neutrophils (%)
    1.0 ± 0.13.6 ± 1.221.6 ± 10.9*
     Cyto-ID+
    neutrophils (%)
    4.3 ± 178.9 ± 3.9*33.4 ± 6.4*
     P-selectin+
    platelets (%)
    6.2 ± 0.320.9 ± 1.6*20.1 ± 4.8*
     TF+ platelets (%)4.9 ± 0.622.3 ± 3.8*26.4 ± 6.6*
    Platelet-free plasma§
     MPO-DNA
    complexes (a.u.)
    0.2 ± 0.031.2 ± 0.08*1.1 ± 0.39*
     Platelet (CD61+)
    microparticles
    (×103/μl)
    19.1 ± 4.8184.4 ± 19.3*24.4 ± 11.4
     Platelet (CD61+)
    HMGB1+
    microparticles
    (×103/μl)
    2.9 ± 0.3133.4 ± 13.8*4.7 ± 1.0
    In vivo effects elicited by control-, SSC-, or SLE-microparticlesǁ
     Autophagic murine
    neutrophils (%)
    6.5 ± 0.662.3 ± 3.6*23.5 ± 6.5
     mMPO murine
    neutrophils (%)
    11.0 ± 0.677.8 ± 2.1*17.7 ± 3.9
     Citrullinated
    H4 (a.u.)
    0.06 ± 0.010.14 ± 0.01*0.07 ± 0.02
     Soluble E-selectin
    (ng/ml)
    10.7 ± 2.1223.2 ± 74.6*13.9 ± 1.2

    *P < 0.001, significantly different from healthy controls.

    †Whole blood was analyzed by flow cytometry, as described in the Supplementary Materials. CD66b+CD16 refer to apoptotic neutrophils. Cyto-ID+ accumulation identifies autophagic cells. CD62P and CD142 are markers of platelet activation.

    P < 0.001, from patients with SSc.

    §MPO-DNA complexes were assessed by enzyme-linked immunosorbent assay (ELISA), and platelet-derived microparticles were assessed by cytometry in platelet-free plasma.

    ǁMicroparticles from controls and from patients with SSc or with SLE were injected in NSG mice. Autophagy and membrane MPO expression (mMPO) were determined by cytometry in the mouse blood, and citrullinated histone H4 and soluble E-selectin were determined by ELISA in platelet-free plasma.

    • Table 2 Cellular adhesion molecule ligand pairs potentially involved in microparticle-neutrophil interaction.

      Freshly purified healthy neutrophils were stimulated with autologous collagen-microparticles. Collagen-microparticles were treated with blocking monoclonal antibodies (mAbs) before incubation with neutrophils to identify the involvement of P-selectin (CD62P), glycoprotein Ib (CD42), or HMGB1. Neutrophils were treated with blocking mAbs before incubation with autologous collagen-microparticles to identify the involvement of PSGL1 (P-selectin receptor), activated Mac-1 (GPIb receptor), or RAGE (HMGB1 receptor). Cyto-ID+ was used to track autophagosomes by flow cytometry, and the soluble MPO-DNA complexes were determined by ELISA as bona fide NET-derived signals. Results are expressed as means ± SEM of six independent experiments carried out with different donors. N/A, not applicable; Ag, antigen.

      Blocking mAbs
      against
      Cyto-ID (%)MPO-DNA
      complexes
      (OD)
      Neutrophils
      alone
      N/A15.8 ± 1.70.35 ± 0.12
      Neutrophils +
      collagen-
      microparticles
      Irrelevant Ag81.3 ± 1.01.28 ± 0.46
      P-selectin79.3 ± 2.21.23 ± 0.48
      PSGL180.5 ± 1.01.37 ± 0.53
      CD4278.5 ± 1.51.24 ± 0.49
      Activated Mac-182.8 ± 4.01.21 ± 0.43
      HMGB115.0 ± 1.20.35 ± 0.15
      RAGE21.5 ± 1.40.41 ± 0.12

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/10/451/eaao3089/DC1

      Materials and Methods

      Fig. S1. Relative complexity of microparticles released from activated platelets.

      Fig. S2. Collagen-microparticles elicit neutrophil autophagy.

      Fig. S3. The kinetics of clearance of human neutrophils by splenic macrophages depends on their activation state.

      Fig. S4. Similar function of neutrophils from wild-type and NSG mice.

      Fig. S5. Viability is required for neutrophil response to platelet-derived microparticles.

      Table S1. SSc patients’ characteristics.

      Table S2. Primary data.

      References (6672)

    • This PDF file includes:

      • Materials and Methods
      • Fig. S1. Relative complexity of microparticles released from activated platelets.
      • Fig. S2. Collagen-microparticles elicit neutrophil autophagy.
      • Fig. S3. The kinetics of clearance of human neutrophils by splenic macrophages depends on their activation state.
      • Fig. S4. Similar function of neutrophils from wild-type and NSG mice.
      • Fig. S5. Viability is required for neutrophil response to platelet-derived microparticles.
      • Table S1. SSc patients’ characteristics.
      • Table S2. Primary data.
      • References (6672)

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