Research ArticleCell death

The pseudokinase MLKL activates PAD4-dependent NET formation in necroptotic neutrophils

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Science Signaling  04 Sep 2018:
Vol. 11, Issue 546, eaao1716
DOI: 10.1126/scisignal.aao1716
  • Fig. 1 Substrate availability determines RIPK1 signaling by caspase-8–driven apoptosis or RIPK3/MLKL-dependent necroptosis.

    (A) Western blot analysis for the indicated proteins in lysates from wild-type (WT) and Ripk1D138N/D138N bone marrow neutrophils primed with interferon-γ (IFN-γ) for 1 hour and treated with combinations of birinapant, z-VAD-fmk, and Nec-1s for 6 hours. Extracellular signal–regulated kinase (ERK) [p44/42 mitogen-activated protein kinase (MAPK)] was used as a loading control. Blots are representative of two to four independent experiments using bone marrow from two to three mice per sample. For quantification of pooled experiments, see fig. S1. MW, molecular weight; WCL, whole-cell lysate. (B) Flow cytometric analysis of mouse bone marrow neutrophil viability assessed by PicoGreen DNA dye exclusion on cells from WT and Ripk1D138N/D138N mice treated for 6 hours as indicated after IFN-γ priming. Data are means ± SEM of six independent experiments. DMSO, dimethyl sulfoxide. (C) Flow cytometric analysis of mouse bone marrow neutrophil viability assessed by PicoGreen DNA dye exclusion on cells from WT, Ripk3−/−, Mlkl−/−, and Ripk3−/−Mlkl−/− mice treated with IFN-γ for 12 hours as indicated. Data are means ± SEM of six independent experiments. (D) Flow cytometric analysis of mouse bone marrow neutrophil viability assessed by PicoGreen DNA dye exclusion on cells from WT and Casp8−/−Ripk3−/− mice treated with IFN-γ for 14 hours as indicated. Data are means ± SEM of six independent experiments. (E) Western blot analysis for the indicated proteins in lysates from WT, Ripk3−/−, and Mlkl−/− bone marrow neutrophils primed for 1 hour with IFN-γ and treated for 4 hours as indicated. ERK (p44/42 MAPK) was used as a loading control. Blots are representative of four independent experiments. (F) In the presence of z-VAD-fmk, birinapant preferentially promotes RIPK3/MLKL-dependent necroptosis. In the absence of the necroptotic effectors RIPK3 and MLKL, RIPK1 promotes caspase-8–dependent apoptosis. *P < 0.05, **P < 0.01, and ***P < 0.005 by analysis of variance (ANOVA)/Tukey’s tests.

  • Fig. 2 Neutrophil necroptosis is morphologically similar to NETosis.

    (A) Transmission electron microscopy analysis of IFN-γ–primed mouse bone marrow neutrophils treated as indicated. Black-filled arrowheads highlight NET formation, and open arrowheads indicate NET absence. (B to F) Immunogold transmission electron microscopy analysis of IFN-γ–primed mouse bone marrow neutrophils treated as indicated. Sections were stained for dsDNA (B, E, and F), H3Cit (C), or neutrophil elastase (Neut elastase) (D). Black-filled arrowheads highlight immunogold staining of NETs, whereas open arrowheads highlight cytoplasmic or nuclear staining. Images are representative of 25 images taken over the course of three independent experiments. Scale bars, 1 μm. (G and H) Immunofluorescence microscopy analysis of dsDNA (green channel) and H3Cit (red channel) in IFN-γ–primed neutrophils treated as indicated. Images (G) are representative of three independent experiments performed in triplicate. Quantified data (H) are means ± SEM of all experiments. *P < 0.05 by ANOVA/Tukey’s tests. PMA, phorbol 12-myristate 13-acetate; CpB, compound B.

  • Fig. 3 Human and mouse necroptotic neutrophils release NETs that are sensitive to DNase I and inhibited by Nec-1s.

    (A and B) Imaging flow cytometric analysis of membrane permeability, phosphatidylserine (PS) exposure, and NET formation assessed by annexin V (AnV), Sytox DNA dye, and H3Cit staining of murine WT bone marrow neutrophils treated as indicated. Black-filled arrowheads highlight NET formation, whereas open arrowheads highlight decondensed nuclei. Data are representative of four independent experiments. For quantification of pooled experiments, see fig. S2G. FasL, Fas ligand; BF, bright field. (C) Flow cytometric analysis of membrane permeability and PS exposure assessed by propidium iodide and annexin V staining of neutrophils stimulated for 12 hours as indicated. Data are representative of five independent experiments. G-CSF, granulocyte colony-stimulating factor. (D to H) Flow cytometric analysis of cellular viability and NET formation assessed by extracellular DNA and H3Cit staining of IFN-γ–primed mouse bone marrow neutrophils treated as indicated. Deoxyribonuclease I (DNase I) pretreatment eliminated the appearance of H3Cit+PicoGreen+ NET-producing cells (E), without effecting PicoGreen viable cells (F). Representative dot plots (D) were quantified (E and F), and data are means ± SEM of four independent experiments. (G and H) Flow cytometric analysis of cellular viability and NET formation of IFN-γ–primed mouse bone marrow neutrophils treated as indicated. NET formation (G) and viability (H) are means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.005 by Student’s t test or ANOVA/Tukey’s tests.

  • Fig. 4 RIPK1 activates PAD4-dependent NET formation.

    (A) Flow cytometric analysis of cellular viability and NET formation assessed by extracellular DNA and H3Cit staining of birinapant/z-VAD-fmk–treated WT ± Nec-1s, Ripk1D138N/D138N, and Pad4−/− neutrophils. Data are representative of six independent biological replicates from two independent experiments. (B) Flow cytometric analysis of NET formation by IFN-γ–primed WT and Ripk1D138N/D138N bone marrow neutrophils treated for 6 hours as indicated. Data are means ± SEM of three biological replicates representative from two independent experiments. (C) Western blot analysis of the indicated proteins in supernatants and WCLs from IFN-γ–primed WT, Ripk1D138N/D138N, and Pad4−/− bone marrow neutrophils treated with birinapant/z-VAD-fmk for the indicated times. Blots are representative of three independent experiments. For quantification of pooled experiments, see fig. S4A. (D and E) Flow cytometric analysis of NET formation (E) and cellular viability (F) from WT and Pad4−/− neutrophils treated for 6 hours as indicated. Data are means ± SEM of six independent biological replicates from two independent experiments. (F) Flow cytometric analysis of NET formation from IFN-γ–primed WT, Ripk3−/−, Mlkl−/−, and Tnfa−/− mouse bone marrow neutrophils treated for 6 hours as indicated. Data are means ± SEM of three independent biological replicates from three independent experiments. (G) Western blot analysis of the indicated proteins in supernatants and WCLs from IFN-γ–primed WT and Mlkl−/− bone marrow neutrophils treated with birinapant/z-VAD-fmk for the indicated times. Blots are representative of three independent experiments. For quantification of pooled experiments, see fig. S4B. (H) Western blot analysis of H3Cit in supernatants and WCLs of IFN-γ–primed WT and Ripk3−/− bone marrow neutrophils and treated with birinapant/z-VAD-fmk for the indicated times. Blots are representative of three independent experiments. For quantification of pooled experiments, see fig. S4C. *P < 0.05, **P < 0.01, and ***P < 0.005 by ANOVA/Tukey’s tests.

  • Fig. 5 Chromatin decondensation in Pad4−/− neutrophils.

    (A and B) Transmission electron microscopy analysis of immunogold staining for dsDNA or H3Cit (black spots) in bone marrow neutrophils from WT, Ripk1D138N/D138N, Ripk3−/−, Mlkl−/−, and Pad4−/− mice treated with IFN-γ/birinapant/z-VAD-fmk for 12 hours. Arrowheads highlight immunogold particles, black-filled arrowheads highlight NETs, and open arrowheads highlight cytoplasmic or nuclear staining. Images (A) are representative of two independent experiments. Quantified data (B) on the percentage of neutrophils with dsDNA-stained polymorphonuclear architecture versus decondensed nuclear morphology are means ± SEM of 45 to 64 neutrophils for each genotype from two independent experiments. (C) Necrotic stimuli may activate MLKL to trigger numerous cellular and biochemical changes, leading to PAD4-dependent H3Cit and NET formation. Scale bars, 500 nm. P < 0.0001 by χ2 test (B).

  • Fig. 6 Activated MLKL colocalizes to sites of DNA release in the plasma membrane.

    (A to F) Transmission electron microscopy analysis of immunogold staining for MLKL (A to C) and pMLKL (D to F) in mouse bone marrow neutrophils treated with IFN-γ/birinapant/z-VAD-fmk (A and D), IFN-γ, (B and E), or IFN-γ/birinapant/z-VAD-fmk/Nec-1s (C and F). pMLKL was visualized in neutrophils treated with IFN-γ/birinapant/z-VAD-fmk (D), IFN-γ (E), or IFN-γ/birinapant/z-VAD-fmk/Nec-1s (F). Arrows highlight immunogold particles, black-filled arrowheads highlight NETs, and open arrowheads highlight cytoplasmic or nuclear staining. Images are representative of two independent experiments. For quantification of pooled experiments, see fig. S5K. Scale bars, 500 nm.

  • Fig. 7 Necroptosis leads to bacteriostatic NET formation in human and mouse neutrophils.

    (A and B) Growth of MRSA bacteria measured by colony formation assay after coculture with IFN-γ–primed human (A) or mouse (B) neutrophils treated as indicated. Data are means ± SEM pooled from three to six independent experiments performed in triplicate. (C) Growth of MRSA bacteria measured by colony formation assay after coculture with WT or Mlkl−/− mouse bone marrow neutrophils treated as indicated. Data are means ± SEM representative of three independent experiments performed in triplicate. (D) Flow cytometric analysis of cellular viability in WT or Mlkl−/− mouse bone marrow neutrophils stimulated with irradiated MRSA for 18 hours in the presence of IFN-γ (100 ng/ml) or G-CSF (100 ng/ml). Data are means ± SEM pooled from three independent experiments. (E) Body weight of WT and Mlkl−/− mice infected retro-orbitally with 107 colony-forming unit (CFU) of MRSA at the indicated times after infection. (F and G) Bacterial growth in WT and Mlkl−/− mice infected retro-orbitally with 106 CFU of MRSA as assessed by colony formation assay on the blood at day 7 (F) and kidney at day 14 (G) after infection. Data from at least 11 mice per group are pooled from two independent experiments. (H) Flow cytometric analysis of peripheral blood neutrophil number in WT and Mlkl−/− mice at 24 hours after retro-orbital infection with the indicated dose of MRSA. Data are means ± SEM of two independent experiments. (I) Bacterial growth in the blood of WT and Mlkl−/− mice at 24 hours after peritoneal infection with 107 CFU of MRSA. Data from at least 17 mice per group are pooled from two independent experiments. (J) Bacterial growth in the spleen, bone marrow, and blood of WT and neutrophil-specific Casp8Δ/Δ (Casp8fl/fl × S100a8-Cre) mice at 24 hours after retro-orbital infection with 107 CFU of MRSA. Data from at least 10 mice per group are pooled from two independent experiments. Dashed lines indicate the limit of detection (LOD) of colony formation assays. *P < 0.05, **P < 0.01, and ***P < 0.005 by Mann-Whitney test or Student’s t test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/546/eaao1716/DC1

    Fig. S1. RIPK1 kinase activity coordinates apoptosis and necroptosis.

    Fig. S2. Characterization of NETs by electron microscopy and imaging flow cytometry.

    Fig. S3. NET formation and cell death of human neutrophils triggered by necroptotic stimuli.

    Fig. S4. RIPK1 kinase activity and necroptotic signaling can activate PAD4.

    Fig. S5. Role of ROS, IFN-γ, and TNF in neutrophil necroptosis and NET formation.

    Fig. S6. MRSA dissemination in Mlkl−/− mice and Cre activity in S100a8-Cre transgenic mice.

  • This PDF file includes:

    • Fig. S1. RIPK1 kinase activity coordinates apoptosis and necroptosis.
    • Fig. S2. Characterization of NETs by electron microscopy and imaging flow cytometry.
    • Fig. S3. NET formation and cell death of human neutrophils triggered by necroptotic stimuli.
    • Fig. S4. RIPK1 kinase activity and necroptotic signaling can activate PAD4.
    • Fig. S5. Role of ROS, IFN-γ, and TNF in neutrophil necroptosis and NET formation.
    • Fig. S6. MRSA dissemination in Mlkl−/− mice and Cre activity in S100a8-Cre transgenic mice.

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