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Functional Dynamics of Neutrophils After Ischemic Stroke

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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.

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Availability of Data and Materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Funding

This work was supported by the Chinese Natural Science Foundation (NCSF) grants (81671178 to Z. L). Z. L is also supported by the Guangdong Natural Science Foundation grant (2017A030311013).

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Author notes

  1. Wei Cai, Sanxin Liu and Mengyan Hu contributed equally to this work.

Authors and Affiliations

  1. Department of Neurology, the Third Affiliated Hospital of Sun Yat-sen University, 600 Tianhe Road, Guangzhou, Guangdong, 510630, People’s Republic of China

    Wei Cai, Sanxin Liu, Mengyan Hu, Qiang Zhu, Wei Qiu & Zhengqi Lu

  2. Center of Clinical Immunology, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, People’s Republic of China

    Wei Cai & Feng Huang

  3. Pittsburgh Institute of Brain Disorders and Recovery, and Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA

    Xiaoming Hu

  4. Department of Internal Medicine, Ohio State University College of Medicine and Wexner Medical Center, Columbus, OH, 43201, USA

    Jacob Colello & Song Guo Zheng

Authors
  1. Wei Cai
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  2. Sanxin Liu
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Contributions

W.C. designed and performed the experiments, collected and analyzed data, and drafted the manuscript. S.L. carried out immunostaining, imaging, and quantification and drafted the manuscript. M.H. performed animal experiments and collected data. F.H. and Q.Z. contributed to the experimental design and the manuscript. W.Q. designed the experiment and critically revised the manuscript. X.H. contributed to the experimental design and revised the manuscript. S.G.Z and Z.L. designed and supervised the study and critically revised the manuscript. J.C. edited and revised the MS. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Song Guo Zheng or Zhengqi Lu.

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Conflict of Interest

The authors declare that they have no conflicts of interest.

Ethics Approval

All animal experiments were approved by the Third Affiliated Hospital of Sun Yat-sen University and performed following the Guide for the Care and Use of Laboratory Animals and Stroke Treatment. Clinic research was approved by the ethics committee of the Third Affiliated Hospital of Sun Yat-sen University.

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Electronic Supplementary Material

ESM 1

(DOCX 20 kb)

Fig. S1

Inclusion process of patients with acute ischemic stroke. (PNG 53 kb)

High Resolution Image (TIF 3034 kb)

Fig. S2

Neutrophil functional dynamics after ischemic stroke. a Alteration time course of neutrophil to lymphocyte ratio (NLR) in peripheral blood was assessed with flow cytometry. N = 3–4. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 versus Sham (Sh). b Representative images showing infiltrated neutrophil in ischemic lesion of striatum (STR) or cortex (CTX) in Sham brain and stroke lesion at 1-3d after tMCAO. N = 4. Scale bar = 20 μm. c Expression dynamics of neutrophil related inflammatory mediators after ischemic stroke as assessed with mRNA analyzed with qPCR. N = 3. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, versus Sham (Sh). Lym, lymphocyte; Neu, neutrophil; h, hour; d, day; STR, striatum; CTX, cortex; 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. (PNG 469 kb)

High Resolution Image (TIF 7167 kb)

Fig. S3

Dynamics of neutrophil polarity and clearance. a Representative flow cytometric panels showing mean fluorescence intensity (MFI) of CD206 on neutrophils in ischemic brain (Ip brain), blood and spleen at indicated time points. Statistic analysis was shown in Fig. 3. N = 3–5. b To confirm CD206 expression, brain infiltrated neutrophils were magnetic sorted and stained with anti-CD206 antibodies at 2d after stroke. Samples were collected from 4 ischemic hemispheres. c Representative flow cytometric panels showing clearance dynamics of neutrophil by macrophage at indicated time points. Gating strategy and statistic analysis were displayed in Fig. 3. N = 3–5. d Representative flow cytometric panels showing expression of CD206 and MPO of neutrophil engulfed (pink) or un-engulfed (blue) by macrophage at indicated time points. Statistic analysis were displayed in Fig. 3. N = 3–5. (PNG 519 kb)

High Resolution Image (TIF 7110 kb)

Fig. S4

Manufacturing neutrophil polarity in vitro and the impact of neutrophil phenotype on macrophage phagocytosis. Neutrophils were isolated from bone marrow of male C57/BL6 mice with magnetic sorting. N1 phenotype was induced with LPS (1 μg/ml) + IFNγ (40 ng/ml) for 3 h. N2 neutrophil was induced with TGFβ (20 ng/ml) for 3 h. NETs was induced with PMA (20 nmol/L) for 3 h. a Increased mRNA expression of IFNγ and TNFα after N1 induction. b Elevated mRNA RNA expression of CD206, Arginase 1 and IL-10 after N2 induction. Data were summarized from 3 independent experiments. *P ≤ 0.05, ***P ≤ 0.001 versus N0. c-d CD206 c and IL-10 d expression at 24 h after N2 induction. Data were summarized from 3 independent experiments. **P ≤ 0.01, ***P ≤ 0.001 versus N0. e Comparison of IL-10 expression by CD206− and CD206+ neutrophil before or after N2 induction. Upper number in representative flow panels showed the IL-10+ percentage in CD206− neutrophils while the lower number displayed the IL-10+ percentage in CD206+ cells. Data were summarized from 3 independent experiments. ***P ≤ 0.001 versus CD206− neutrophils. f Macrophages were pre-stained with lived-cell tracker (CMTPX, red) and then treated with various subsets of neutrophils (macrophages: neutrophils = 1: 10). N2 neutrophils facilitated phagocytosis by macrophages. White arrow emphasized neutrophil engulfed by macrophage. Scale bar = 10 μm. Data were summarized from 3 independent experiments. ***P ≤ 0.001 versus N0. IFN, interferon; TNF, tumor necrosis factor; Arg1, arginase 1; IL, interleukin; NETs, neutrophil extracellular traps. (PNG 309 kb)

High Resolution Image (TIF 5631 kb)

Fig. S5

Expression dynamics of MPO in neutrophil after ischemic stroke. Representative flow cytometric panels showing dynamics of the mean fluorescence intensity (MFI) of MPO on neutrophils in ischemic brain (Ip brain), blood and spleen at indicated time points. Statistic analysis was shown in Fig. 5. N = 3–5. (PNG 205 kb)

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Cai, W., Liu, S., Hu, M. et al. Functional Dynamics of Neutrophils After Ischemic Stroke. Transl. Stroke Res. 11, 108–121 (2020). https://doi.org/10.1007/s12975-019-00694-y

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