Functional Dynamics of Neutrophils After Ischemic Stroke

Abstract

Neutrophils are forerunners to brain lesions after ischemic stroke and exert elaborate functions. However, temporal alterations of cell count, polarity, extracellular trap formation, and clearance of neutrophils remain poorly understood. The current study was aimed at providing basic information of neutrophil function throughout a time course following stroke onset in patients and animal subjects. We found that neutrophil constitution in peripheral blood increased soon after stroke onset of patients, and higher neutrophil count indicated detrimental stroke outcomes. Comparably, neutrophil count in peripheral blood of stroke mice peaked at 12 h after cerebral ischemia, followed by a 1-2-day spike in brain lesions. In stroke lesion, clearance of neutrophils peaked at 2 days after stroke and extracellular traps were mostly detected at 2-3 days after stroke. In neutrophil infiltrated into stroke lesion, expression of the N2 marker CD206 was relatively stable. We found that the N2 phenotype facilitated neutrophil clearance by macrophage and did not further induce neuronal death after ischemic injury compared with N0 or N1 neutrophils. Skewing neutrophil toward the N2 phenotype before stroke reduced infarct volumes at 1 day after tMCAO. Conditioned medium of ischemic neurons drove neutrophils away from the protective N2 phenotype and increased the formation of extracellular traps. Conclusively, neutrophil function has an important impact on stroke outcomes. Neutrophil frequency in the peripheral blood could be an early indicator of stroke outcomes. N2 neutrophils facilitate macrophage phagocytosis and are less harmful to ischemic neurons. Directing neutrophils toward the N2 phenotype could be a promising therapeutic approach for ischemic stroke.

Keywords: Brain ischemia; Extracellular traps; Neuroprotection; Neutrophils.

Fig. 1

Early elevation of neutrophil was correlated with detrimental outcomes of cerebral ischemic stroke. A total of 225 patients and 56 age- and gender-matched heathy controls (HC) were included in the study. a Comparison of neutrophil count and neutrophil to lymphocyte ratio (NLR) of AIS patients with or without infection and healthy controls (HC). **** P ≤ 0.001 compared with HC. b Linear regression analysis of neutrophil count or NLR with NIHSS scores at admission in AIS patients without infection (N = 167). c-d Correlation of neutrophil count or NLR with infarct diameter in patients without infection. Included AIS patients without infection were further divided into small infarction group (Infarct diameter ≤ 1.5cm, N = 93) c and large infarction group (Infarct diameter > 1.5cm, N = 74) d, linear regression analysis of infarct size and neutrophil of the two groups was performed respectively. e Linear regression analysis of neutrophil count or NLR with infarct volume of mice at 1d after tMCAO (N = 18).

Fig. 2

Temporal and spatial dynamics of neutrophil after ischemic stroke. Male C57/BL6 mice were subjected to 60min tMCAO and sacrificed at various time points. a Neutrophil constitution in bone marrow (BM) blood and spleen of mice as assessed with flow cytometry. N = 3-5. *P ≤ 0.05, **P ≤ 0.01, versus Sham in BM. ^##P ≤ 0.01, versus Sham in blood. ^{^^} P ≤ 0.01, ^{^^^} P ≤ 0.001, versus Sham in spleen. b Expression dynamics of neutrophil attracting chemokines (mRNA level) after ischemic stroke. N = 3. **P ≤ 0.01, *** P ≤ 0.001, versus Sham (Sh). c Temporal dynamics of infiltrated neutrophil after ischemic stroke as assessed with flow cytometry. Number in the representative flow panel = neutrophil per 10³ singlets in ischemic hemisphere. N = 3-5. *P ≤ 0.05, *** P ≤ 0.001, versus Sham (Sh). d Heat map showing expression dynamics of neutrophil related inflammatory mediators in ischemic hemisphere as analyzed with qPCR. N = 3. Statistic analysis was displayed in Fig. S2c. FSC, forward scatter; W, width; hi, high; NE, neutrophil elastase; MPO, myeloperoxidase; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; IFN, interferon; IL, interleukin; TLR, toll-like receptor; NLRP3, nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 3; PAD, peptidylarginine deiminase type; BAFF, B cell activating factor.

Fig. 3

Polarization and clearance dynamics of neutrophil after ischemic stroke. a Polarization dynamics of neutrophils were assessed by CD206 expression with flow cytometry. Mean fluorescent intensity (MFI) of CD206 on neutrophils in Ipsilateral brain (Ip), blood (Bl) and spleen (S) was analyzed. N = 3-5. *** P ≤ 0.001, versus Sham in Ip brain. b Gating strategy of engulfed neutrophil (CD45^hiCD11b⁺Ly6G⁺F4/80⁺) and un-engulfed neutrophils by macrophage (CD45^hiCD11b⁺Ly6G⁺F4/80⁻). c Flow cytometry analysis of neutrophil clearance by macrophage at indicated time points. N = 3-5. d Immuno-staining of macrophages/microglia (Iba1⁺, red) phagocytosis of neutrophils (Ly6G⁺, green) in stroke lesions at indicated time points. White arrows show neutrophil engulfed by microglia/macrophage. N = 3-4. e-f Comparison of CD206 and MPO expression in engulfed and un-engulfed neutrophils by macrophages in ipsilateral brain as analyzed with flow cytometry. N = 3-5. *P ≤ 0.05. **P ≤ 0.01, ***P ≤ 0.001, compared with 6h.

Fig. 4

Protection of N2 neutrophil against ischemic stroke. Neutrophils were isolated from bone marrow of male C57/BL6 mice with magnetic sorting. a Time line of neutrophil induction and treatment to ischemic neuron. b Viability of ischemic neuron was assessed with immuno-staining of anti-NeuN antibodies (red). Data were summarized from 3 independent experiments. **P ≤ 0.01, ***P ≤ 0.001 versus OGD neuron without treatment. Scale bar = 20μm. c Time line of in vivo N2 induction with TGFβ treatment, tMCAO and blood (Bl) analysis. d Flow cytometric analysis of neutrophil phenotypic shift at 1d after TGFβ injection (before tMCAO). N = 9 in each group. *P ≤ 0.05, versus PBS treated group. e Infarct volume analysis at 1d after tMCAO with TTC staining. Dash-line outlined the infarct area. N = 9 in each group. **P ≤ 0.01, versus PBS treated group. NETs, neutrophil extracellular traps; OGD, oxygen glucose deprivation; CM, conditioned medium; Bl, blood; TGFβ, transforming growth factor beta; d, day.

Fig. 5

Neutrophil extracellular traps (NETs) formation time course after ischemic stroke. NETs formation was analyzed with immuno-staining of citrullinated histone 3 (CitH3) in coronal frozen brain slices at different time points after tMCAO. a Representative image showing NETs formation in striatum stroke lesion (co-localization of Ly6G (green) and CitH3 (red)) at 3d after ischemic stroke. b NETs formation dynamics in stroke lesion at striatum (STR) and cortex (CTX) as assessed with immuno-staining of CitH3 (red). NETs formation peaked at 2-3d after tMCAO. N = 3-4. **P ≤ 0.01, ***P ≤ 0.001 versus 6h. Scale bar = 20μm. c Presence of NETs formation was confirmed with immunol staining of chromatin dye Sytox green (green) and neutrophil elastase (NE) at 2d after tMCAO. Experiments were repeated for 3 times and representative images from one of the scanning were presented. d Presence of NETs formation was confirmed with scanning electronic microscopy (SEM) at 2d after tMCAO. N = 4. e Expression dynamics of MPO in neutrophil in ipsilateral brain (Ip), blood and spleen. N = 3-4. *P ≤ 0.05 versus Sham in ipsilateral brain; ^#P ≤ 0.05, ^###P ≤ 0.001 versus Sham in blood. CitH3, citrullinated histone 3; STR, striatum; CTX, cortex; d, day; Ip, ipsilateral brain; MFI, mean fluorescence intensity; Sh, Sham.

Fig. 6

Impact of neuronal ischemia on neutrophil polarity and NETs formation. a Time line of neuron conditioned medium after 6h oxygen glucose deprivation (OGD CM) collection and treatment of neutrophils. b Flow cytometric analysis of neutrophil polarity after neuron OGD CM treatment for 24h with CD206 expression. Data were summarized from 3 independent experiments. ***P ≤ 0.001 versus control (Ctrl). c Polarization of neutrophils after neuron OGD CM treatment for 24h was analyzed with mRNA expression of CD206, Arg1, IL-10 or TGFβ by qPCR. Data were summarized from 3 independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 versus conditioned medium of healthy neuron (CM). d MPO expression in neutrophils after neuron OGD CM treatment for 24h was analyzed with flow cytometry. Data were summarized from 3 independent experiments. e Neutrophils were treated with neuron OGD CM for 24h then subjected to PMA treatment (20nmol/L for 3h) to induce NETs formation. Neutrophils treated with neuron OGD CM were prone to form NETs. Scale bar = 20μm. Data were summarized from 3 independent experiments. **P ≤ 0.01 versus control (Ctrl). OGD, oxygen glucose deprivation; CM, conditioned medium; NETs, neutrophil extracellular traps; h, hour; MFI, mean fluorescence intensity; Ctrl, control; Arg1, arginase 1.