Fig. 10. Histones and G-CSF shorten mature neutrophil lifespan selectively.

a Representative neutrophil death time-courses of Ly6G^high (blue) and Ly6G^low (grey) BM neutrophils sorted from naïve WT mice and Ly6G^low cells (pink) from the BM of symptomatic WT mice infected with C. albicans in the absence or in the presence of 1 ng/mL G-CSF (Representative of 2 independent experiments. b Effect of recombinant histone H3 and G-CSF alone or in combination, on mature and immature neutrophil half-life curves for Ly6G^high (black) naive Ly6G^low (grey) and infected Ly6G^low (pink). Differences between curves are highlighted by coloured windows. Each point is the average half-life from three separate field measurements. c Fold increase over 4 healthy donors (HD) in histone H3 in the plasma of 36 human patients with bacterial sepsis, quantified from western immunoblots (left). DNA concentrations in the plasma of 4 HDs and 36 sepsis patients (right). d Half-life HD blood neutrophils incubated with plasmas from 22 HDs (blue) or 36 sepsis patients (pink). Representative of two independent experiments. e Representative cell death curves of HD neutrophils incubated with HD (lower) or sepsis patient plasma (upper) in the presence of a control or anti-histone H3 and anti-G-CSF antibodies. Average and SD for 4 FOV per condition. f Quantification of the half-life of HD neutrophils incubated with either HD (n = 5 donors) or sepsis patient (n = 5 donors) plasmas alone or in combination with anti-histone H3 or anti-G-CSF antibodies (3 FOV per donor). g Quantification of the mean fluorescence intensity (MFI) of surface markers in HD neutrophils incubated with HD or sepsis patient plasma alone or HD plasma supplemented with histone H3 or G-CSF, measured by flow cytometry. Each dot corresponds to a different plasma donor (n = 6). Panel: CD15-BV510, CD66b-FITC, PD-L1-PE, CD11b-PECy5, CD16-APC, CD10-BUV496, CD101-PECy7. Representative of two independent experiments. h Correlation between the fold-increase in sepsis patient plasma histone H3 (left plot) or G-CSF (right plot) and the relative abundance of S100A8 (red) and S100A9 (blue) measured by mass spectrometry. Boxes depict medium and high histone H3 containing plasmas. i Correlation between plasma G-CSF and histone H3 levels in sepsis patient plasmas. Dot sizes and colours are relative to plasma S100A8 levels. j Half-life of HD neutrophils incubated with plasmas from survivors (S) or deceased (D) sepsis patients (left), plasma G-CSF levels (middle) and neutrophil lifespan shortening activity in plasmas segregated into high (>200 pg/mL) and medium (dotted box, <200 pg/mL) G-CSF levels (right). k Survival curves of sepsis patients segregated into high (red), medium (yellow) and low (blue) neutrophil lifespan shortening activity (left), patients with high (red) and medium (blue) G-CSF levels and patients with medium G-CSF with low neutrophil lifespan shortening activity (blue) or high G-CSF and medium G-CSF with high neutrophil lifespan shortening activity (red) (right). Statistical analysis by simple linear regression, unpaired two-sided Mann–Whitney or parametric t-test and two-sided Log-rank (Mantel–Cox) test for survival analysis (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).