Fig 1.
Immunostaining of NETs derived from HL-60 cells or primary neutrophils demonstrates the presence of H3.1-nucleosomes.
Neutrophil-like DMSO-differentiated HL-60 cells (A-D) or primary neutrophils isolated from human whole blood (E-F) treated with PMA were stained with anti-MPO (A and B, purple; E, yellow), anti-histone H3.1 (A, D, E and F, green; C, purple), anti-nucleosome (B and C, green) or anti-H3Cit (D, purple; F, yellow) antibodies. DNA and membranes were stained with DAPI (blue) and Cell MaskTM Orange (orange-red), respectively. Scale bars, 20µm.
DMSO, dimethyl sulfoxide; PMA, phorbol 12-myristate 13-acetate; MPO, myeloperoxidase; NETs, neutrophil extracellular traps.
Fig 2.
Molecular characterization of NETs from differentiated HL-60 cells demonstrates the presence of H3.1-nucleosomes.
NET extracts from DMSO-differentiated HL-60 cells treated with PMA (DMSO-dHL-60 + PMA NETs) (A, lane 1), HeLa chromatin extracts (A, lane 2), and proteins pulled-down by using magnetic particles coated with anti-nucleosome (A, lane 3), anti-H3.1 (A, lane 4), anti-MPO (A, lane 5), IgG2a isotype (A, lane 6) and IgG rabbit isotype (A, lane 7) were analyzed using SDS-PAGE followed by anti-MPO, anti-H4 and anti-H3 Western blotting. The anti-MPO antibody detects both α and β sub-units of the MPO protein. The H3 antibody targets the C-terminal extremity of the histone H3. Cell-free DNA fragment size distribution of DNA immunoprecipitated (IP, blue) or left in the supernatant (post-IP, red) by anti-H3.1 (B), anti-nucleosome (C), anti-MPO (D), IgG2A isotype (E), and IgG rabbit isotype(F)-coated particles were analyzed using Agilent 2100 Bioanalyzer.
DMSO, dimethyl sulfoxide; Ig, immunoglobulin; MPO, myeloperoxidase; NETs, neutrophil extracellular traps; PMA, phorbol 12-myristate 13-acetate; SDS-PAGE, sodium dodecyl-sulfate polyacrylamide gel electrophoresis.
Fig 3.
H3.1-nucleosome immunoassay linearity and sensitivity.
Three K2EDTA plasma samples containing low concentrations of H3.1-nucleosomes were each mixed into three other K2EDTA plasma samples of higher H3.1 concentrations [(A) Mix 1, (B) Mix 2 or (C) Mix 3] to prepare 11 proportions and measured with the H3.1-nucleosomes immunoassay. Scatter plots show the linearity of the measurement, expressed in ng/mL, over the measuring intervals (expected value). The red line shows the linear fit. The linearity plots show the difference between the linear and nonlinear fit across the measuring interval. (D) Combined results from Mixes 1–3. (E) H3.1-nucleosomes assay limits of blank (LOB), limits of detection (LOB) and limits of quantification (LOQ) estimated for three independent lots of reagents. CI, confidence.
Table 1.
H3.1-nucleosome immunoassay precision.
Table 2.
H3.1-nucleosome immunoassay spike recovery.
Fig 4.
Comparative measures of H3.1-nucleosomes, H3R8Cit-nucleosomes, dsDNA and MPO-DNA complexes in NETs derived from HL-60 cells.
Jitter plot analysis representation of the H3.1-nucleosomes (A), the H3Cit-nucleosomes (B), the dsDNA (C), and the MPO-DNA (D) measured for three independent productions of NET extracts (n = 3) from DMSO-differentiated HL-60 cells treated with PMA (NETs) compared with the untreated cells (Controls). ns represent non-significant p-value, *, ** and *** represent p-values <0.05, < 0.01 and <0.001, respectively, calculated using the Welch’s test. Cit, citrullinated histone H3; dsDNA, double-stranded DNA; MPO, myeloperoxidase; NETs, neutrophil extracellular traps; OD, optical density.
Fig 5.
The H3.1-nucleosome assay significantly distinguishes NETs-associated diseases from healthy controls.
(A) Jitter plot analysis representation of the circulating H3.1-nucleosomes level (ng/mL). The whiskers represent the 25th–75th percentile with median. **** represent p-value <0.0001, calculated using the Mann–Whitney U test. (B) ROC curve analysis of circulating H3.1-nucleosomes for discrimination of NETs patient’s vs control samples. The area under the curve is 0.91 (95% CI: 0.89–0.94; p < 0.0001). CI, confidence interval; NETs, neutrophil extracellular traps; ROC, receiver operating characteristic.