Histone H3 clipping is a novel signature of human neutrophil extracellular traps
- Department of Cellular Microbiology, Max Planck Institute for Infection Biology, Germany
- Microscopy Core Facility, Max Planck Institute for Infection Biology, Germany
- Protein Analysis Core Facility, Max Planck Institute for Infection Biology, Germany
- Institut für Pathologie, Charité - Universitätsmedizin Berlin, Germany
Figures
Figure 1 with 3 supplements
PMA-induced Histone H3 cleavage occurs in the N-terminal domain and is prevented by serine protease inhibition.
(A) Neutrophils were preincubated with the serine protease inhibitor AEBSF for 30 min in microcentrifuge tubes and then stimulated with PMA as indicated in the figure. Lysates were resolved by …
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Figure 1—source data 1
Time course of H3 cleavage and inhibition by AEBSF.
- https://cdn.elifesciences.org/articles/68283/elife-68283-fig1-data1-v2.zip
Figure 1—figure supplement 1
Cysteine protease inhibition by E64 does not inhibit histone H3 cleavage.
Neutrophils were preincubated with the cysteine protease inhibitor E64 (10 µM) or serine protease inhibitor AEBSF (150 µm) for 30 min and then stimulated with or without PMA 50 nM for 120 min. …
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Figure 1—figure supplement 1—source data 1
Cysteine protease inhibition does not inhibit Histone H3 cleavage.
- https://cdn.elifesciences.org/articles/68283/elife-68283-fig1-figsupp1-data1-v2.zip
Figure 1—figure supplement 2
AEBSF does not inhibit ROS production.
Neutrophils were preincubated with AEBSF at the specified concentrations for 30 min before stimulation as indicated. ROS production was assessed using a luminol-HRP assay. Representative trace of …
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Figure 1—figure supplement 2—source data 1
AEBSF does not inhibit the ROS burst.
- https://cdn.elifesciences.org/articles/68283/elife-68283-fig1-figsupp2-data1-v2.xlsx
Figure 1—figure supplement 3
AEBSF is not cytotoxic.
Neutrophils were incubated with AEBSF at the indicated concentration and cytotoxicity was measured by LDH (lactate dehydrogenase) release where 100% lysis was achieved by adding 0.1% (w/v) Triton …
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Figure 1—figure supplement 3—source data 1
AEBSF is not cytotoxic.
- https://cdn.elifesciences.org/articles/68283/elife-68283-fig1-figsupp3-data1-v2.xlsx
Figure 2 with 2 supplements
Identification of histone H3 cleavage sites in NET formation.
(A) Representative RP-HPLC chromatogram of acid extracted histones from NETs and corresponding 1D-SDS-PAGE and immunoblots to identify H3 and H4 containing fractions. Histone enriched supernatants …
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Figure 2—source data 1
Western blots and gels for histones H3 and H4 in RP-HPLC fractions.
- https://cdn.elifesciences.org/articles/68283/elife-68283-fig2-data1-v2.zip
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Figure 2—source data 2
RP-HPLC absorbance curve.
- https://cdn.elifesciences.org/articles/68283/elife-68283-fig2-data2-v2.xlsx
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Figure 2—source data 3
2D-E blots for Edman degradation.
- https://cdn.elifesciences.org/articles/68283/elife-68283-fig2-data3-v2.zip
Figure 2—figure supplement 1
Schematic summary of extraction and identification of histone H3 cleavage sites in NETs.
Neutrophils were stimulated with PMA (50 nM) to induce histone cleavage and the samples were collected in the presence of proteases inhibitors. The nuclear material was isolated by hypotonic lysis …
Figure 2—figure supplement 2
Separation of histone H3 by two dimensional electrophoresis.
Coomassie stained gel of pooled histone H3 containing fractions resolved by two dimensional electrophoresis (2-DE). All labelled spots were subsequently analysed by mass spectrometry to identify the …
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Figure 2—figure supplement 2—source data 1
2DE-gel showing spots for identification by mass spectrometry.
- https://cdn.elifesciences.org/articles/68283/elife-68283-fig2-figsupp2-data1-v2.zip
Figure 3 with 4 supplements
Screening and detection of cleaved H3 and NETs by 3D9.
(A) Immunoblots of lysates prepared from neutrophils stimulated with PMA (50 nM) for the times indicated in the figure. H3 C-terminal antibody was used as a control to detect all H3 forms while a …
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Figure 3—source data 1
Western blots with H3-terminal antibody and hybridoma clone 3D9.
- https://cdn.elifesciences.org/articles/68283/elife-68283-fig3-data1-v2.zip
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Figure 3—source data 2
Direct ELISA for cleaved H3.
- https://cdn.elifesciences.org/articles/68283/elife-68283-fig3-data2-v2.xlsx
Figure 3—figure supplement 1
Outline of immunisation and screening strategy for antibody production.
After selection of animals for hybridoma production, clone supernatants were screened similarly. Positive selection (+), negative selection (-). *Performed in the hybridoma screen only. ** …
Figure 3—figure supplement 2
Peptide inhibition of 3D9 binding to NETs.
Immunofluorescent microscopy of neutrophils stimulated with PMA for 3 hr and stained with 3D9 (1 µg/ml) in the presence of competition and control peptides. Prior to staining, 3D9 was preincubated …
Figure 3—figure supplement 3
3D9 immunoprecipitation.
Neutrophils were seeded in 10 ml petri dishes and stimulated for 3 hr as indicated. Before total cell lysis and immunoprecipitation, NETs were digested with benzonase to liberate histones and …
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Figure 3—figure supplement 3—source data 1
3D9 immunoprecipitation.
- https://cdn.elifesciences.org/articles/68283/elife-68283-fig3-figsupp3-data1-v2.zip
Figure 3—figure supplement 4
Detection of 3D9 cross reacting proteins from plasma and serum.
(A) Diluted serum (1 donor) and plasma (2 donors) were separated by SDS-PAGE in reducing and non reducing conditions (sample plus/minus 2 mM DTT). NETs and Non-stimulated neutrophils (NS) were used …
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Figure 3—figure supplement 4—source data 1
Detection of 3D9 cross-reacting proteins from plasma and serum by western blot.
- https://cdn.elifesciences.org/articles/68283/elife-68283-fig3-figsupp4-data1-v2.zip
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Figure 3—figure supplement 4—source data 2
Detection of 3D9 cross-reacting proteins from plasma and serum by ELISA.
- https://cdn.elifesciences.org/articles/68283/elife-68283-fig3-figsupp4-data2-v2.xlsx
Figure 4 with 3 supplements
Visualisation of the 3D9 epitope in the nucleosome core complex.
Visualization of the putative core epitope for 3D9 mapped on to histone H3 ribbon structure. The observed core binding site of 3D9 to the peptide arrays was depicted on histone H3 (light brown) in …
Figure 4—figure supplement 1
Binding profiles recorded for 3D9 on the linear peptide array.
Antibodies 3D9 (red) and isotype control IgG,k (blue) were incubated on arrays with overlapping linear 8-mer (LIN8 – [i, ii]) and 15-mer (LIN15 – [iii, iv]) peptides and binding measured by ELISA. …
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Figure 4—figure supplement 1—source data 1
Linear and helical peptide epitope mapping.
- https://cdn.elifesciences.org/articles/68283/elife-68283-fig4-figsupp1-data1-v2.xlsx
Figure 4—figure supplement 2
Binding profiles recorded for 3D9 on helical peptide mimics arrays.
Antibodies 3D9 (red) and isotype control IgG,k (blue) were incubated on arrays with overlapping helical 9-mer (HEL9; [i, ii]) and 15-mer (HEL; [iii, iv]) peptides. Separate arrays were used with …
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Figure 4—figure supplement 2—source data 1
Linear and helical peptide epitope mapping.
- https://cdn.elifesciences.org/articles/68283/elife-68283-fig4-figsupp2-data1-v2.xlsx
Figure 4—figure supplement 3
Fine epitope mapping by replacement analysis.
Linear (i) peptides were generated bearing single amino acid substitutions at each position of the native peptide sequence REIRRYQKSTELLIRK of histone H3. In addition, helical peptide mimics were …
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Figure 4—figure supplement 3—source data 1
Fine epitope mapping by replacement analysis.
- https://cdn.elifesciences.org/articles/68283/elife-68283-fig4-figsupp3-data1-v2.xlsx
Figure 5 with 1 supplement
Comparison of NET quantification using an anti-chromatin antibody versus 3D9.
(A) Confocal immunofluorescent microscopy of neutrophils stimulated with PMA (180 min) and stained with Hoechst and anti-chromatin antibody (PL2.3) or 3D9. Insets represent selected cells examined …
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Figure 5—source data 1
Characterisation of PL2.3 NET staining.
- https://cdn.elifesciences.org/articles/68283/elife-68283-fig5-data1-v2.csv
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Figure 5—source data 2
Characterisation of 3D9 NET staining.
- https://cdn.elifesciences.org/articles/68283/elife-68283-fig5-data2-v2.csv
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Figure 5—source data 3
Comparison of NET quantification using manual or automatic thresholding and segmentation procedures for chromatin antibody and 3D9.
- https://cdn.elifesciences.org/articles/68283/elife-68283-fig5-data3-v2.xlsx
Figure 5—figure supplement 1
Workflow of NET analysis methods.
Schematic of imaging, segmentation, thresholding and particle analysis for both automatic and semi-automatic NET quantification.
Figure 6
Detection by 3D9 of NETs from diverse stimuli.
Immunofluorescence microscopy of neutrophils left unstimulated (NS), stimulated with PMA (50 nM, 2.5 hr), nigericin (15 µM, 2.5 hr), TNF primed and then stimulated with heme (20 µM, 6 hr), and …
Figure 7
Detection of NETs in mixed cell fractions.
Immunofluorescence microscopy of non-purified peripheral blood mononuclear cell (PBMC) fractions treated with the NET stimuli, PMA (50 nM, 2.5 hr) or nigericin (15 µM, 2.5 hr), and then stained with …
Figure 8 with 2 supplements
Comparison of 3D9 detection in response to apoptotic, necroptotic, and necrotic cell death stimuli.
Confocal immunofluorescent microscopy of neutrophils stimulated with different cell death stimuli and subsequently stained with Hoechst, anti-neutrophil elastase (NE) and 3D9. NETs were induced with …
Figure 8—figure supplement 1
Comparison of PL2.3 detection in response to apoptotic, necroptotic, and necrotic cell death stimuli.
Confocal immunofluorescent microscopy of neutrophils stimulated with different cell death stimuli and subsequently stained with Hoechst, anti-neutrophil elastase (NE) and PL2.3. NETs were induced …
Figure 8—figure supplement 2
Staining of primary human neutrophils for markers of apoptosis and NETs.
Primary neutrophils were purified from whole blood and left unstimulated to allow spontaneous apoptosis. Samples were fixed and stained for DNA (Hoechst), cleaved caspase 3 (green) and cleaved …
Figure 9 with 1 supplement
Detection of clipped histone 3 and NETs in human tissues.
Paraffin embedded sections were stained with Hoechst, anti-NE and 3D9 or H3cit antibodies and examined by confocal microscopy at ×63 magnification (Plan Apochromat, glycerol, numerical aperture …
Figure 9—figure supplement 1
Hematoxylin and eosin (HE) stain of kidney section.
HE stained tissue overview of NETs represented in Figure 9B, C. Black box indicates area for further magnification in images (ii) and (iii). The red arrow indicates the location of the NET in the …
Figure 10 with 1 supplement
Comparison of Clipped H3, H3cit & H2B staining in the gallbladder from an appendicitis patient.
Paraffin embedded sections were stained with Hoechst, anti-NE and histone antibodies 3D9, H3cit and H2B and examined by confocal microscopy at ×63 magnification (Plan Apochromat, glycerol, numerical …
Figure 10—figure supplement 1
Hematoxylin & eosin (HE) stain of gallbladder and colocalization analysis.
(A) HE stained overview of gallbladder stained for NETs in Figure 10. Magnification increases from (i)-(iii) and boxes represent the field of view of the next image. Red box indicates the area …
Figure 11 with 2 supplements
Comparison of Clipped H3, H3cit, and H2B staining in the appendix of an appendicitis patient.
Paraffin embedded sections were stained with Hoechst, anti-NE and 3D9 or H3cit antibodies and examined by confocal microscopy at ×63 magnification (Plan Apochromat, glycerol, numerical aperture …
Figure 11—figure supplement 1
Hematoxylin and eosin stain of inflamed appendix and colocalization analysis.
(A) HE stained overview of appendix with appendicitis and peri-appendicitis stained for NETs in Figure 11. Magnification increases from (i)-(iii) and boxes represent the field of view of the next …
Figure 11—figure supplement 2
Time course of 3D9 and H3cit co-staining of primary human neutrophils stimulated with PMA.
Neutrophils were stimulated for 4 hr with PMA, fixed with paraformaldehyde and stained for cleaved H3R49 (3D9) and histone citrullination at R2, R8, and R17 (H3cit). Images were taken on an upright …
Author response image 1
Tables
Table 1
Mass spectrometry identification of proteins co-separating with histone H3 following RP-HPLC and 2-DE.
| Spot | score | accession no. | protein name | MW | pI | sequence coverage | Number of peptides |
|---|---|---|---|---|---|---|---|
| 1 | 99 | P84243 | Histone H3.3* | 15318 | 11.27 | 25.7 | 8 |
| 2 | 133 | P84243 | Histone H3.3 | 15318 | 11.27 | 25 | 7 |
| 2 | 78 | P0C0S5 | Histone H2A.Z | 13545 | 10.58 | 14.1 | 4 |
| 3 | 140 | P84243 | Histone H3.3 | 15318 | 11.27 | 22.1 | 7 |
| 4 | 193 | P84243 | Histone H3.3 | 15318 | 11.27 | 27.2 | 8 |
| 5 | 198 | P84243 | Histone H3.3 | 15318 | 11.27 | 27.2 | 9 |
| 6 | 317 | P06702 | Protein S100-A9 | 13234 | 5.71 | 70.2 | 10 |
| 7 | 486 | P06702 | Protein S100-A9 | 13234 | 5.71 | 81.6 | 15 |
| 8 | 483 | P06702 | Protein S100-A9 | 13234 | 5.71 | 82.5 | 14 |
| 9 | 57 | P31949 | Protein S100-A11 | 11733 | 6.56 | 9.5 | 2 |
| 10 | 344 | P31949 | Protein S100-A11 | 11733 | 6.56 | 45.7 | 8 |
| 11 | 343 | P05109 | Protein S100-A8 | 10828 | 6.51 | 37.6 | 9 |
| 12 | 140 | P05109 | Protein S100-A8 | 10828 | 6.51 | 25.8 | 5 |
| 13 | 136 | P25815 | Protein S100-P | 10393 | 4.75 | 35.8 | 6 |
| 14 | 48 | P06703 | Protein S100-A6 | 10173 | 5.33 | 16.7 | 3 |
Table 2
Table 3
List of immunisation, screening, and competition peptides.
| Immunisation | H - REIRRK(RRIER)C - NH2 KLH conjugated |
| Screening | (+) H - REIRRK(RRIER)C - NH2 (-) H - AARKSK(SKRAA)C - NH2* |
| Validation | H - REIRRK(RRIER) - NH2 competition peptide H - TGGVKK(KVGGT) - NH2 negative control |
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*
Alternative branched peptide used for screening.The N-terminal is represented by H (H2N) and the C-terminal is represented by NH2 (CONH2).
Table 4
Summary of identified 3D9 binding regions in the peptide array.
| Sample | Type | Peak | Epitope candidate |
|---|---|---|---|
| 3D9-1 | LIN8 | 47–58 | REIRRYQK |
| VALREIRR | |||
| EIRRYQKS | |||
| 60–68 | LLIRKLP | ||
| ELLIRKLP | |||
| LIN8 - Ac | 47–60 | VALREIRR | |
| REIRRYQK | |||
| RRYQKSTE | |||
| EIRRYQKS | |||
| LREIRRYQ | |||
| 61–68 | LLIRKLPF | ||
| LIN15 | 40–64 | HRYRPGTVALREIRR | |
| REIRRYQKSTELLIR | |||
| TVALREIRRYQKSTE | |||
| 1–33 | RYQKSTELLIRKLPF | ||
| LIN15 - Ac | 12–44 | HRYRPGTVALREIRR | |
| REIRRYQKSTELLIR | |||
| PGTVALREIRRYQKS | |||
| VALREIRRYQKSTEL | |||
| RPGTVALREIRRYQK | |||
| YRPGTVALREIRRYQ | |||
| RYRPGTVALREIRRY | |||
| TVALREIRRYQKSTE | |||
| ALREIRRYQKSTELL |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (human) | A549 human lung epithelial cells; A549 | American Type Cell Collection | ATCC:CCL-185 | |
| Genetic reagent (bovine) | Calf thymus DNA; purified DNA | Invitrogen | ||
| Antibody | Rabbit polyclonal anti-histone H3 C-terminal; anti-H3-C; αH3-C | Active Motif | Active Motif:61277 | Western blot (1:15000) |
| Antibody | Rabbit polyclonal Histone H4 C-terminal; αH4-C | Abcam | Abcam:ab10158 | Western blot (1:5000) |
| Antibody | Rabbit monoclonal anti-histone H4 N terminal | Upstate Millipore, Sigma Aldrich | SigmaAldrich:05–858 | Western blot (1:30000) |
| Antibody | Rabbit monoclonal anti-GAPDH; GAPDH | Cell Signalling Technology | CST:2118 | Western blot (1:5000) |
| Antibody | Mouse monoclonal 3D9; 3D9: mouse anti-cleaved histone 3 3D9 | This paper | Custom synthesis by Genscript. Western blot (1 µg/ml), immunofluorescence (1–2 µg/ml) | |
| Antibody | Rat monoclonal anti-histone H3 N-terminal; anti H3-N; αH3N | Active Motif | Active motif:61647 | Western blot (1:15000) |
| Antibody | Mouse monoclonal anti-H2A-H2B-DNA; PL2.3; anti-chromatin | PMID:1371530 | Immunofluorescence (1 µg/ml) | |
| Antibody | Rabbit polyclonal anti neutrophil elastase; anti-NE; αNE | Calbiochem | Calbiochem 481001 | Immunofluorescence (1:500) |
| Antibody | Rabbit polyclonal anti-histone H3 (citrullinine R2+R8+R17); anti- H3cit | Abcam | Abcam:ab5103 | Immunofluorescence (1 µg/ml) |
| Antibody | Chicken polyclonal anti-histone H2B | abcam | Abcam:ab134211 | Immunofluorescence (1:400) |
| Antibody | Polyclonal Sheep anti-ELANE: anti-NE | LSBio | LSBio:LS-B4244-50 | Immunofluorescence (5 µg/ml) |
| Antibody | Rabbit polyclonal Anti-mouse Bridging antibody | Active Motif | Active motif:53017 | (50 µg) |
| Antibody | Mouse monoclonal (1H7A8) IgG1, k, isotype control | Genscript | Provided by Genscript as an isotype control (1 µg/ml) | |
| Peptide, recombinant protein | Recombinant human histone H3.1 | New England Biolabs | NEB:M2503S | |
| Peptide, recombinant protein | Peptide H-REIRRK(RREIR)C-NH2; immunising peptide REIRR | This paper | Custom synthesis by Eurogentec | |
| Peptide, recombinant protein | Peptide H-AARKSK(SKRAA)C-NH2; negative screening peptide | This paper | Custom synthesis by Eurogentec | |
| Peptide, recombinant protein | Peptide H-REIRRK (RRIER_NH2; competition peptide) | This paper | Custom synthesis by Eurogentec | |
| Peptide, recombinant protein | Peptide H-TGGVKK(KVGGT)-NH2; negative control peptide | This paper | Custom synthesis by Eurogentec | |
| Commercial assay or kit | BD OptEIA TMB substrate reagent set | BD Biosciences | RRID:AB_2869044 | |
| Commercial assay or kit | Picogreen Assay kit; Quant-iT PicoGreen dsDNA Assay Kits and dsDNA Reagents | Thermofisher Scientific | Thermofisher:P7589 | |
| Commercial assay or kit | Cytoxicity assay; CytoTox 96 Non-Radioactive Cytotoxicity assay | Promega | Promega:G1780 | |
| Chemical compound, drug | Hoechst DNA stain (33342) | Invitrogen Molecular Probes | Invitrogen:H1399 | Immunofluorescence 1 µg/ml |
| Chemical compound, drug | Neutrophil elastase inhibitor GW311616A; NEi | Biomol | Biomol:Cay27957-1 | |
| Chemical compound, drug | AEBSF hydrochloride; peflabloc | Millipore, Sigma Aldrich | Sigma Aldrich:124839 | |
| Chemical compound, drug | Cathepsin G inhibitor I; CGi | Calbiochem, Sigma Aldrich | Sigma Aldrich:219372 | |
| Software, algorithm | Fiji; Image J | https://doi.org/10.1038/nmeth.2019 | ||
| Software, algorithm | NETalyser | This paper | (Scripts deposited at https://github.com/tulduro/NETalyser) | |
| Software, algorithm | Volocity | Perkin Elmer |
