Stroke-derived neutrophils demonstrate higher formation potential and impaired resolution of CD66b + driven neutrophil extracellular traps

Abstract

Background: Recent evidence suggests a merging role of immunothrombosis in the formation of arterial thrombosis. Our study aims to investigate its relevance in stroke patients.

Methods: We compared the peripheral immunological profile of stroke patients vs. healthy controls. Serum samples were functionally analyzed for their formation and clearance of Neutrophil-Extracellular-Traps. The composition of retrieved thrombi has been immunologically analyzed.

Results: Peripheral blood of stroke patients showed significantly elevated levels of DNAse-I (p < 0.001), LDG (p = 0.003), CD4 (p = 0.005) as well as the pro-inflammatory cytokines IL-17 (p < 0.001), INF-γ (p < 0.001) and IL-22 (p < 0.001) compared to controls, reflecting a T_H1/T_H17 response. Increased counts of DNAse-I in sera (p = 0.045) and Neutrophil-Extracellular-Traps in thrombi (p = 0.032) have been observed in patients with onset time of symptoms longer than 4,5 h. Lower values of CD66b in thrombi were independently associated with greater improvement of NIHSS after mechanical thrombectomy (p = 0.045). Stroke-derived neutrophils show higher potential for Neutrophil-Extracellular-Traps formation after stimulation and worse resolution under DNAse-I treatment compared to neutrophils derived from healthy individuals.

Conclusions: Our data provide new insight in the role of activated neutrophils and Neutrophil-Extracellular-Traps in ischemic stroke. Future larger studies are warranted to further investigate the role of immunothrombosis in the cascades of stroke.

Trial registration: DRKS, DRKS00013278, Registered 15 November 2017, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00013278.

Keywords: CD66b +; Immunothrombosis; Neutrophil-Extracellular-Traps; Stroke; Thrombus.

Fig. 1

Stroke patients show increased numbers of neutrophils with an accumulation of Low Density Granulocytes. A Representative scatter plot of isolated neutrophils from a patient with an ischemic occlusion showing a mean purity of 98.9% with B equal frequencies of CD66b⁺ neutrophils in stroke patients (n = 35) compared to control patients (n = 16; Healthy). Total numbers of isolated granulocytes are significantly increased in patients with stroke when compared to their controls. C Representative scatter dot plots of flow cytometric assessed surface expression of CD14, CD16 and CD66b on PBMCs showing D significantly increased low density granulocytes (LDGs), defined as CD14⁻ CD16⁺ CD66b⁺ lymphocytes, in stroke patients, despite comparable frequencies of CD66b expression in both groups. Data are representative of independent experiments with 35 stroke patients and 16 control patients (Healthy) and presented as mean ± SD. Mann-Whitney-U Test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Abbreviations: LDG, low density granulocytes; PBMCs, peripheral blood mononuclear cells

Fig. 2

Stroke patients accumulate pro-inflammatory cytokines and show increased NET formation within the retrieved thrombi. A CD4⁺ T cells of patients with ischemic stroke and control patients were assessed for their intracellular expression of the pro-inflammatory cytokines IL-17, IFN-y and IL-22 and show significantly higher absolute numbers of these T_H1 and T_H17 cytokines in peripheral blood. Data are representative for 35 stroke patients and 16 control patients (Healthy) and presented as mean ± SD. Mann-Whitney-U Test, * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. B Retrieved thrombi of 35 ischemic patients were analyzed via fluorescence microscopy for the expression of intact but activated CD66b⁺ neutrophils within the thrombus and apoptotic neutrophils in form of NET (Sytox Green positive). The MFI for CD66b expression and NET formation within the thrombi were determined. Correlation between these two parameters shows a significant slope for such. C Representative fluorescence images of a thrombus stained for NET (green; left image) and CD66b (red; middle image) as well as an overly of both (merged; right image)

Fig. 3

Neutrophils of stroke patients show elevated NET formation with worse resolution of already formed NET. A Representative images of neutrophils with or without NET formation for control patients (Healthy; upper row; n = 16) and stroke patients (Stroke; lower row; n = 35) Neutrophils are isolated from peripheral blood via ficoll density gradient and plated. NET formation is triggered by adding of 100 mM PMA (positive control) or DMSO (negative control) for 3 h. For the DNase-I control, samples are treated with 100 mM PMA for 3 h and afterwards with DNase-I for 30 min. Sytox Green is added to all samples 10 min before fixation with 4% PFA. NET formation is determined directly after fixation. B Quantification of the MFI (mean fluorescence intensity) of the Sytox Green positive images show significantly higher NET formation for patients with ischemic stroke for the positive control, but also already in resting neutrophils (negative control) and a worse resolution of NET upon DNAse-I treatment. C Neutrophils treated with serum of patients with ischemic stroke (3rd bar; n = 35) as well as serum of controls (2nd bar; n = 16) show significantly higher NET formation determined via MFI in comparison to the positive control (100 ng PMA) and the negative control (DMSO). D Represantative images of PMA stimulated neutrophils, showing nuclear expression (DAPI) of neutrophil elastase (NE) in green (anti-NE FITC) upon NETosis. E Serum of patients with ischemic stroke (3rd bar; n = 35) as well as serum of controls (2nd bar; n = 16) is analyzed for DNase-I levels and reveals higher levels of the enzyme in sera derived from stroke patients (p < 0.001). Data are representative of independent experiments with 35 stroke patients and 16 control patients (Healthy) and presented as mean ± SEM. Abbreviations: NET, neutrophil extracellular traps; MFI, mean fluorescence intensity. Mann-Whitney-U Test, * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001