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Translational Stroke Research

, Volume 11, Issue 1, pp 108–121 | Cite as

Functional Dynamics of Neutrophils After Ischemic Stroke

  • Wei Cai
  • Sanxin Liu
  • Mengyan Hu
  • Feng Huang
  • Qiang Zhu
  • Wei Qiu
  • Xiaoming Hu
  • Jacob Colello
  • Song Guo ZhengEmail author
  • Zhengqi LuEmail author
Original Article

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 Neutrophils Extracellular traps Neuroprotection 

Notes

Availability of Data and Materials

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

Authors’ 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.

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).

Compliance with Ethical Standards

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.

Supplementary material

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ESM 1 (DOCX 20 kb)
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Fig. S1

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

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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)

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High Resolution Image (TIF 7167 kb)
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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)

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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)

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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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References

  1. 1.
    Dirnagl U, Iadecola C, Moskowitz MA. Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci. 1999;22:391–7.CrossRefGoogle Scholar
  2. 2.
    Shimamura N, Katagai T, Kakuta K, Matsuda N, Katayama K, Fujiwara N, et al. Rehabilitation and the neural network after stroke. Transl Stroke Res. 2017;8:507–14.CrossRefGoogle Scholar
  3. 3.
    Gelderblom M, Leypoldt F, Steinbach K, Behrens D, Choe CU, Siler DA, et al. Temporal and spatial dynamics of cerebral immune cell accumulation in stroke. Stroke. 2009;40:1849–57.CrossRefGoogle Scholar
  4. 4.
    Wu L, Walas S, Leung W, Sykes DB, Wu J, Lo EH, et al. Neuregulin1-beta decreases IL-1beta-induced neutrophil adhesion to human brain microvascular endothelial cells. Transl Stroke Res. 2015;6:116–24.CrossRefGoogle Scholar
  5. 5.
    Griffith JW, Sokol CL, Luster AD. Chemokines and chemokine receptors: positioning cells for host defense and immunity. Annu Rev Immunol. 2014;32:659–702.CrossRefGoogle Scholar
  6. 6.
    Cuartero MI, Ballesteros I, Moraga A, Nombela F, Vivancos J, Hamilton JA, et al. N2 neutrophils, novel players in brain inflammation after stroke: modulation by the PPARgamma agonist rosiglitazone. Stroke. 2013;44:3498–508.CrossRefGoogle Scholar
  7. 7.
    Brinkmann V. Neutrophil extracellular traps in the second decade. J Innate Immun. 2018;10:414–421.CrossRefGoogle Scholar
  8. 8.
    Lood C, Blanco LP, Purmalek MM, Carmona-Rivera C, de Ravin SS, Smith CK, et al. Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease. Nat Med. 2016;22:146–53.CrossRefGoogle Scholar
  9. 9.
    Jackson C, Sudlow C. Comparing risks of death and recurrent vascular events between lacunar and non-lacunar infarction. Brain. 2005;128:2507–17.CrossRefGoogle Scholar
  10. 10.
    Traylor M, Rutten-Jacobs LC, Thijs V, et al. Genetic associations with white matter hyperintensities confer risk of lacunar stroke. Stroke. 2016;47:1174–9.CrossRefGoogle Scholar
  11. 11.
    Norrving B. Long-term prognosis after lacunar infarction. Lancet Neurol. 2003;2:238–45.CrossRefGoogle Scholar
  12. 12.
    Lee CF, Venketasubramanian N, Wong KS, Chen CL. Comparison between the original and shortened versions of the National Institutes of Health Stroke Scale in ischemic stroke patients of intermediate severity. Stroke. 2016;47:236–9.CrossRefGoogle Scholar
  13. 13.
    Sucharew H, Khoury J, Moomaw CJ, Alwell K, Kissela BM, Belagaje S, et al. Profiles of the National Institutes of Health Stroke Scale items as a predictor of patient outcome. Stroke. 2013;44:2182–7.CrossRefGoogle Scholar
  14. 14.
    Fiebach JB, Stief JD, Ganeshan R, et al. Reliability of two diameters method in determining acute infarct size. Validation as new imaging biomarker. PLoS One. 2015;10:e140065.CrossRefGoogle Scholar
  15. 15.
    Stetler RA, Cao G, Gao Y, Zhang F, Wang S, Weng Z, et al. Hsp27 protects against ischemic brain injury via attenuation of a novel stress-response cascade upstream of mitochondrial cell death signaling. J Neurosci. 2008;28:13038–55.CrossRefGoogle Scholar
  16. 16.
    Knight JS, Zhao W, Luo W, Subramanian V, O’Dell AA, Yalavarthi S, et al. Peptidylarginine deiminase inhibition is immunomodulatory and vasculoprotective in murine lupus. J Clin Invest. 2013;123:2981–93.CrossRefGoogle Scholar
  17. 17.
    Zhang X, Huang F, Li W, Dang JL, Yuan J, Wang J, et al. Human gingiva-derived mesenchymal stem cells modulate monocytes/macrophages and alleviate atherosclerosis. Front Immunol. 2018;9:878.CrossRefGoogle Scholar
  18. 18.
    Wang Y, Li M, Stadler S, Correll S, Li P, Wang D, et al. Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol. 2009;184:205–13.CrossRefGoogle Scholar
  19. 19.
    Garcia-Bonilla L, Benakis C, Moore J, Iadecola C, Anrather J. Immune mechanisms in cerebral ischemic tolerance. Front Neurosci. 2014;8:44.CrossRefGoogle Scholar
  20. 20.
    Zhao H, Garton T, Keep RF, Hua Y, Xi G. Microglia/macrophage polarization after experimental intracerebral hemorrhage. Transl Stroke Res. 2015;6:407–9.CrossRefGoogle Scholar
  21. 21.
    Wang J, Ye Q, Xu J, Benedek G, Zhang H, Yang Y, et al. DRalpha1-MOG-35-55 reduces permanent ischemic brain injury. Transl Stroke Res. 2017;8:284–93.CrossRefGoogle Scholar
  22. 22.
    Fan X, Jiang Y, Yu Z, Liu Q, Guo S, Sun X, et al. Annexin A2 plus low-dose tissue plasminogen activator combination attenuates cerebrovascular dysfunction after focal embolic stroke of rats. Transl Stroke Res. 2017;8:549–59.CrossRefGoogle Scholar
  23. 23.
    Li L, Tao Y, Tang J, Chen Q, Yang Y, Feng Z, et al. A cannabinoid receptor 2 agonist prevents thrombin-induced blood-brain barrier damage via the inhibition of microglial activation and matrix metalloproteinase expression in rats. Transl Stroke Res. 2015;6:467–77.CrossRefGoogle Scholar
  24. 24.
    Yu IC, Kuo PC, Yen JH, Paraiso HC, Curfman ET, Hong-Goka BC, et al. A combination of three repurposed drugs administered at reperfusion as a promising therapy for postischemic brain injury. Transl Stroke Res. 2017;8:560–77.CrossRefGoogle Scholar
  25. 25.
    Perez-de-Puig I, Miro-Mur F, Ferrer-Ferrer M, et al. Neutrophil recruitment to the brain in mouse and human ischemic stroke. Acta Neuropathol. 2015;129:239–57.CrossRefGoogle Scholar
  26. 26.
    Andzinski L, Kasnitz N, Stahnke S, Wu CF, Gereke M, von Köckritz-Blickwede M, et al. Type I IFNs induce anti-tumor polarization of tumor associated neutrophils in mice and human. Int J Cancer. 2016;138:1982–93.CrossRefGoogle Scholar
  27. 27.
    Ma Y, Yabluchanskiy A, Iyer RP, Cannon PL, Flynn ER, Jung M, et al. Temporal neutrophil polarization following myocardial infarction. Cardiovasc Res. 2016;110:51–61.CrossRefGoogle Scholar
  28. 28.
    Mangold A, Alias S, Scherz T, Hofbauer T, Jakowitsch J, Panzenböck A, et al. Coronary neutrophil extracellular trap burden and deoxyribonuclease activity in ST-elevation acute coronary syndrome are predictors of ST-segment resolution and infarct size. Circ Res. 2015;116:1182–92.CrossRefGoogle Scholar
  29. 29.
    Vajen T, Koenen RR, Werner I, Staudt M, Projahn D, Curaj A, et al. Blocking CCL5-CXCL4 heteromerization preserves heart function after myocardial infarction by attenuating leukocyte recruitment and NETosis. Sci Rep. 2018;8:10647.CrossRefGoogle Scholar
  30. 30.
    Bjornsdottir H, Welin A, Michaelsson E, et al. Neutrophil NET formation is regulated from the inside by myeloperoxidase-processed reactive oxygen species. Free Radic Biol Med. 2015;89:1024–35.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Neurologythe Third Affiliated Hospital of Sun Yat-sen UniversityGuangzhouPeople’s Republic of China
  2. 2.Center of Clinical Immunologythe Third Affiliated Hospital of Sun Yat-sen UniversityGuangzhouPeople’s Republic of China
  3. 3.Pittsburgh Institute of Brain Disorders and Recovery, and Department of NeurologyUniversity of Pittsburgh School of MedicinePittsburghUSA
  4. 4.Department of Internal MedicineOhio State University College of Medicine and Wexner Medical CenterColumbusUSA

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