Harmful neutrophil subsets in patients with ischemic stroke

(A–D) Polymorphonuclear neutrophil (PMN) death was measured immediately after sampling by staining with annexin V and 7-AAD. Results are expressed as the percentages of apoptotic (A and B) and necrotic (C and D) PMNs. (A and C) Values of PMN death in patients with IS at inclusion. (B and D) Follow-up of PMN death during the first week after IS symptoms. (E and F) Circulating NETs were quantified by MPO-DNA complex ELISA; results are expressed as ng/μL DNA. Values of circulating NETs in patients with IS at inclusion (E). Follow-up of intravascular NETosis during the first week after IS symptoms (F). All samples came from age-matched healthy controls (HCs, n = 22) and patients with IS at day 0 (n = 27), day 2 (n = 14), and day 7 (n = 14). Values are mean ± SEM. *Significantly different from controls p < 0.05, **p < 0.01, ***p < 0.001, adjusted for age. AAD = amino-actinomycin D; D0 = day 0; D2 = day 2; D7 = day 7; HC = healthy control; IS = ischemic stroke; MPO = myeloperoxidase; NET = neutrophil extracellular trap; SEM = standard error of mean.

(A–E) Analysis of senescent and immunosuppressive polymorphonuclear neutrophil (PMN) subsets in patients with IS at inclusion. Whole-blood samples were incubated for 45 minutes at 4°C with Pe-Cy7-anti-human CXCR4, PE-anti-human CD11b, and APC-antihuman CD62L (A and B) or with FITC-anti-human CD16, PE-anti-human CD11c, Pe-Cy7-antihuman CD11b, and APC-anti-human CD62L (C and D) antibodies. (A) Representative dot plots of the PMN phenotype according to CXCR4 and CD62L expression in a HC (left) and a patient with IS (right). (B) Representative dot plots of the PMN phenotype according to CD16 and CD62L expression in an HC (left) and a patient with IS (right). (C) Percentages of the CXCR4^bright/CD62L^dim senescent PMN subset. (D) Percentages of the CD16^bright/CD62L^dim immunosuppressive PMN subset. (E) Ratio between the senescent and the immunosuppressive PMN subsets. (F–H) Follow-up of senescent PMN subset (F), immunosuppressive PMN subset (G), and ratio between the senescent and the immunosuppressive PMN subsets (H) during the first week after IS symptoms. All samples came from age-matched HCs, (n = 22) and patients with IS at day 0 (n = 27), day 2 (n = 14), and day 7 (n = 14). Values are mean ± SEM. *Significantly different from controls p < 0.05, **p < 0.01, ***p < 0.001, adjusted for age. D0 = day 0; D2 = day 2; D7 = day 7; HC = healthy control; IS = ischemic stroke; SEM = standard error of mean.

(A and B) sJAM-C was quantified by ELISA; results are expressed as pg/mL. (A) Values of sJAM-C in age-matched HCs, (n = 22) and patients with IS at inclusion (n = 27) (patients included in set 1). (B) Follow-up of sJAM-C levels during the first week after IS symptoms in 10 patients. (C) Correlation between the levels of sJAM-C and of circulating PMN elastase. (D and E) Quantification of rTEM PMNs in patients with IS included in set 2. Whole-blood samples were incubated for 45 minutes at 4°C with FITCanti-human CD181 and PE-anti-human CD54 antibodies. (D) Representative dot plots of the PMN phenotype according to CXCR4 and CD62L expression in an HC (left) and a patient with IS (right). (E) Percentages of the rTEM PMN subset (CD54^high, CXCR1^low) in age-matched HCs (n = 17) and patients with IS at inclusion (n = 14). (F) Correlation between the percentage of rTEM PMN subset and the level of sJAM-C in patients with IS included in the set 2. *Significantly different from controls p < 0.05, **p < 0.01, ***p < 0.001, adjusted for age. D0 = day 0; D2 = day 2; D7 = day 7; HC = healthy control; IS = ischemic stroke; PMN = polymorphonuclear neutrophil; rTEM = reverse transendothelial migration; sJAM-C = soluble JAM-C.

(A and B) Expression of CD62L (A) and CD11b (B) at the surface of resting polymorphonuclear neutrophils (PMNs). Results are expressed as MFI. (C) Circulating levels of neutrophil elastase were quantified by ELISA. (D) ROS production by unstimulated PMNs; results are expressed in MFI. (E) Percentages of the CXCR4^bright/CD62L^dim senescent PMN subset. (F) Percentages of the CD16^bright/CD62L^dim immunosuppressive PMN subset. (G) sJAM-C was quantified by ELISA; results are expressed as pg/mL (H) PMN apoptosis was measured immediately after sampling by staining with annexin V and 7-AAD. Results are expressed as percentages of apoptotic PMNs. All measurements came from patients with minor-to-moderate stroke (IS NHISS ≤ 12, n = 17) and patients with severe stroke (IS NHISS > 12, n = 10) at the inclusion. Values are mean ± SEM. Statistical significance as determined by the nonparametric Mann-Whitney test is indicated. *Significantly different p < 0.05, **p < 0.01, ***p < 0.001. Correlation between the NIHSS score at inclusion and CD62L expression at the PMN surface (I), ROS production by unstimulated PMNs (J), ROS production by LPS-stimulated PMNs (K), percentage of the CXCR4^bright/CD62L^dim senescent PMN subset (L), sJAM-C level (M), and percentage of rTEM PMNs (N). AAD = amino-actinomycin D; D0 = day 0; IS = ischemic stroke; LPS = lipopolysaccharide; MFi = mean fluorescence intensity; NIHSS = NIH Stroke Scale; PMN = polymorphonuclear neutrophil; ROS = reactive oxygen species; SEM = standard error of mean; sJAM-C = soluble JAM-C.