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A new neutrophil subset promotes CNS neuron survival and axon regeneration

Abstract

Transected axons typically fail to regenerate in the central nervous system (CNS), resulting in chronic neurological disability in individuals with traumatic brain or spinal cord injury, glaucoma and ischemia–reperfusion injury of the eye. Although neuroinflammation is often depicted as detrimental, there is growing evidence that alternatively activated, reparative leukocyte subsets and their products can be deployed to improve neurological outcomes. In the current study, we identify a unique granulocyte subset, with characteristics of an immature neutrophil, that had neuroprotective properties and drove CNS axon regeneration in vivo, in part via secretion of a cocktail of growth factors. This pro-regenerative neutrophil promoted repair in the optic nerve and spinal cord, demonstrating its relevance across CNS compartments and neuronal populations. Our findings could ultimately lead to the development of new immunotherapies that reverse CNS damage and restore lost neurological function across a spectrum of diseases.

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Fig. 1: Zymosan-driven regrowth of optic nerve axons is enhanced by blockade of eye-infiltrating CXCR2+ neutrophils.
Fig. 2: Treatment of mice with αCXCR2 antiserum, starting on the day of i.o. zymosan injection, skews eye-infiltrating neutrophils toward an immature phenotype.
Fig. 3: Neuroregenerative neutrophils have characteristics of alternatively activated cells.
Fig. 4: CD14+Ly6Glo cells, purified 3 d following i.p. zymosan injection, are neuroregenerative.
Fig. 5: CD14+Ly6Glo cells induce RGC axon outgrowth, in part, via secretion of growth factors.
Fig. 6: CD14+Ly6Glo cells drive the regeneration of spinal cord axons.
Fig. 7: Human cell line–derived immature neutrophils are neuroregenerative.

Data availability

Single-cell RNA-seq data are available in the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/) under accession no. GSE144637. Other data that support the findings of this study are available from the corresponding author on request.

References

  1. 1.

    Lucas, T. et al. Differential roles of macrophages in diverse phases of skin repair. J. Immunol. 184, 3964–3977 (2010).

    CAS  PubMed  Google Scholar 

  2. 2.

    Tourki, B. & Halade, G. Leukocyte diversity in resolving and nonresolving mechanisms of cardiac remodeling. FASEB J. 31, 4226–4239 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Chinetti-Gbaguidi, G., Colin, S. & Staels, B. Macrophage subsets in atherosclerosis. Nat. Rev. Cardiol. 12, 10–17 (2015).

    CAS  PubMed  Google Scholar 

  4. 4.

    Segal, B. M. CNS chemokines, cytokines, and dendritic cells in autoimmune demyelination. J. Neurol. Sci. 228, 210–214 (2005).

    CAS  PubMed  Google Scholar 

  5. 5.

    Akiyama, H. et al. Inflammation and Alzheimer’s disease. Neurobiol. Aging 21, 383–421 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Shi, K. et al. Global brain inflammation in stroke. Lancet Neurol. 18, 1058–1066 (2019).

    PubMed  Google Scholar 

  7. 7.

    Miron, V. E. et al. M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination. Nat. Neurosci. 16, 1211–1218 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Giles, D. A. et al. Myeloid cell plasticity in the evolution of central nervous system autoimmunity. Ann. Neurol. 83, 131–141 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Bollaerts, I., Van houcke, J., Andries, L., De Groef, L. & Moons, L. Neuroinflammation as fuel for axonal regeneration in the injured vertebrate central nervous system. Mediators Inflamm. 2017, 9478542 (2017).

    PubMed  PubMed Central  Google Scholar 

  10. 10.

    Jassam, Y. N., Izzy, S., Whalen, M., McGavern, D. B. & El Khoury, J. Neuroimmunology of traumatic brain injury: time for a paradigm shift. Neuron 95, 1246–1265 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Yin, Y. et al. Macrophage-derived factors stimulate optic nerve regeneration. J. Neurosci. 23, 2284–2293 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Baldwin, K. T., Carbajal, K. S., Segal, B. M. & Giger, R. J. Neuroinflammation triggered by β-glucan/dectin-1 signaling enables CNS axon regeneration. Proc. Natl Acad. Sci. USA 112, 2581–2586 (2015).

    CAS  PubMed  Google Scholar 

  13. 13.

    Fridlender, Z. G. et al. Polarization of tumor-associated neutrophil phenotype by TGF-β: ‘N1’ versus ‘N2’ TAN. Cancer Cell 16, 183–194 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Chen, F. et al. Neutrophils prime a long-lived effector macrophage phenotype that mediates accelerated helminth expulsion. Nat. Immunol. 15, 938–946 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Tsuda, Y. et al. Three different neutrophil subsets exhibited in mice with different susceptibilities to infection by methicillin-resistant Staphylococcus aureus. Immunity 21, 215–226 (2004).

    CAS  PubMed  Google Scholar 

  16. 16.

    Cuartero, M. I. et al. N2 neutrophils, novel players in brain inflammation after stroke modulation by the PPAR-γ agonist rosiglitazone. Stroke 44, 3498–3508 (2013).

    CAS  PubMed  Google Scholar 

  17. 17.

    Horckmans, M. et al. Neutrophils orchestrate post-myocardial infarction healing by polarizing macrophages towards a reparative phenotype. Eur. Heart J. 38, 187–197 (2017).

    CAS  PubMed  Google Scholar 

  18. 18.

    Yang, W. et al. Neutrophils promote the development of reparative macrophages mediated by ROS to orchestrate liver repair. Nat. Commun. 10, 1076 (2019).

    PubMed  PubMed Central  Google Scholar 

  19. 19.

    Neumann, S. & Woolf, C. J. Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury. Neuron 23, 83–91 (1999).

    CAS  PubMed  Google Scholar 

  20. 20.

    Newburger, P. E., Chovaniec, M. E., Greenberger, J. S. & Cohen, H. J. Functional changes in human leukemic cell line HL-60. A model for myeloid differentiation. J. Cell Biol. 82, 315–322 (1979).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Boss, M. A., Delia, D., Robinson, J. B. & Greaves, M. F. Differentiation-linked expression of cell surface markers on HL-60 leukemic cells. Blood 56, 910–916 (1980).

    CAS  PubMed  Google Scholar 

  22. 22.

    Olsson, I. & Olofsson, T. Induction of differentiation in a human promyelocytic leukemic cell line (HL-60). Production of granule proteins. Exp. Cell Res. 131, 225–230 (1981).

    CAS  PubMed  Google Scholar 

  23. 23.

    Ma, S. F. et al. Adoptive transfer of M2 macrophages promotes locomotor recovery in adult rats after spinal cord injury. Brain Behav. Immun. 45, 157–170 (2015).

    CAS  PubMed  Google Scholar 

  24. 24.

    Wynn, T. A. & Vannella, K. M. Macrophages in tissue repair, regeneration and fibrosis. Immunity 44, 450–462 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Schwartz, M. ‘Tissue-repairing’ blood-derived macrophages are essential for healing of the injured spinal cord: from skin-activated macrophages to infiltrating blood-derived cells? Brain Behav. Immun. 24, 1054–1057 (2010).

    CAS  PubMed  Google Scholar 

  26. 26.

    Gliem, M., Schwaninger, M. & Jander, S. Protective features of peripheral monocytes/macrophages in stroke. Biochim. Biophys. Acta 1862, 329–338 (2016).

    CAS  PubMed  Google Scholar 

  27. 27.

    von Leden, R. E., Parker, K. N., Bates, A. A., Noble-Haeusslein, L. J. & Donovan, M. H. The emerging role of neutrophils as modifiers of recovery after traumatic injury to the developing brain. Exp. Neurol. 317, 144–154 (2019).

    Google Scholar 

  28. 28.

    Semple, B. D., Bye, N., Ziebell, J. M. & Morganti-Kossmann, M. C. Deficiency of the chemokine receptor CXCR2 attenuates neutrophil infiltration and cortical damage following closed head injury. Neurobiol. Dis. 40, 394–403 (2010).

    CAS  PubMed  Google Scholar 

  29. 29.

    Herz, J. et al. Role of neutrophils in exacerbation of brain injury after focal cerebral ischemia in hyperlipidemic mice. Stroke 46, 2916–2925 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Brennan, F. H. et al. Complement receptor C3aR1 controls neutrophil mobilization following spinal cord injury through physiological antagonism of CXCR2. JCI insight 4, https://doi.org/10.1172/jci.insight.98254 (2019).

  31. 31.

    Roth, T. L. et al. Transcranial amelioration of inflammation and cell death after brain injury. Nature 505, 223–228 (2014).

    CAS  PubMed  Google Scholar 

  32. 32.

    Kurimoto, T. et al. Neutrophils express oncomodulin and promote optic nerve regeneration. J. Neurosci. 33, 14816–14824 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Lorber, B., Berry, M., Douglas, M. R., Nakazawa, T. & Logan, A. Activated retinal glia promote neurite outgrowth of retinal ganglion cells via apolipoprotein E. J. Neurosci. Res. 87, 2645–2652 (2009).

    CAS  PubMed  Google Scholar 

  34. 34.

    Hou, Y. et al. N2 neutrophils may participate in spontaneous recovery after transient cerebral ischemia by inhibiting ischemic neuron injury in rats. Int. Immunopharmacol. 77, 105970 (2019).

    CAS  PubMed  Google Scholar 

  35. 35.

    Sagiv, J. Y. et al. Phenotypic diversity and plasticity in circulating neutrophil subpopulations in cancer. Cell Rep. 10, 562–573 (2015).

    CAS  Google Scholar 

  36. 36.

    Liu, C. Y. et al. Population alterations of l-arginase- and inducible nitric oxide synthase-expressed CD11b+/CD14/CD15+/CD33+ myeloid-derived suppressor cells and CD8+ T lymphocytes in patients with advanced-stage non-small cell lung cancer. J. Cancer Res. Clin. Oncol. 136, 35–45 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Rodriguez, P. C. et al. Arginase I–producing myeloid-derived suppressor cells in renal cell carcinoma are a subpopulation of activated granulocytes. Cancer Res. 69, 1553–1560 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Cloke, T., Munder, M., Taylor, G., Muller, I. & Kropf, P. Characterization of a novel population of low-density granulocytes associated with disease severity in HIV-1 infection. PLoS ONE 7, e48939 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Ssemaganda, A. et al. Characterization of neutrophil subsets in healthy human pregnancies. PLoS ONE 9, e85696 (2014).

    PubMed  PubMed Central  Google Scholar 

  40. 40.

    Mistry, P. et al. Transcriptomic, epigenetic, and functional analyses implicate neutrophil diversity in the pathogenesis of systemic lupus erythematosus. Proc. Natl Acad. Sci. USA 116, 25222–25228 (2019).

    CAS  PubMed  Google Scholar 

  41. 41.

    Benowitz, L. I., He, Z. & Goldberg, J. L. Reaching the brain: advances in optic nerve regeneration. Exp. Neurol. 287, 365–373 (2017).

    PubMed  Google Scholar 

  42. 42.

    Stoolman, J. S., Duncker, P. C., Huber, A. K. & Segal, B. M. Site-specific chemokine expression regulates central nervous system inflammation and determines clinical phenotype in autoimmune encephalomyelitis. J. Immunol. 193, 564–570 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Leon, S., Yin, Y., Nguyen, J., Irwin, N. & Benowitz, L. I. Lens injury stimulates axon regeneration in the mature rat optic nerve. J. Neurosci. 20, 4615–4626 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Yoon, C. et al. Low-density lipoprotein receptor-related protein 1 (LRP1)-dependent cell signaling promotes axonal regeneration. J. Biol. Chem. 288, 26557–26568 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Ruschel, J. et al. Axonal regeneration. Systemic administration of epothilone B promotes axon regeneration after spinal cord injury. Science 348, 347–352 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Giles, D. A., Duncker, P. C., Wilkinson, N. M., Washnock-Schmid, J. M. & Segal, B. M. CNS-resident classical DCs play a critical role in CNS autoimmune disease. J. Clin. Invest. 128, 5322–5334 (2018).

    PubMed  PubMed Central  Google Scholar 

  47. 47.

    Grifka-Walk, H. M., Giles, D. A. & Segal, B. M. IL-12-polarized Th1 cells produce GM-CSF and induce EAE independent of IL-23. Eur. J. Immunol. 45, 2780–2786 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Park, J. et al. Single-cell transcriptomics of the mouse kidney reveals potential cellular targets of kidney disease. Science 360, 758–763 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank S. Atkins for technical support. Financial support for this research was provided by the National Eye Institute (NEI), National Institutes of Health (R01EY029159 and R01EY028350 to B.M.S. and R.J.G; K08EY029362 to A.R.S.), the Wings of Life Foundation (C.Y.) and the Dr. Miriam and Sheldon G. Adelson Research Foundation (R.J.G.). B.M.S. holds the Stanley D. and Joan H. Ross chair in neuromodulation at the Ohio State University.

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A.R.S., K.S.C., A.D.J., C.Y. and A.L.K. performed experiments and data analysis. R.M. oversaw RNA-seq analysis. B.M.S. wrote the manuscript and coedited it with the help of the other authors. B.M.S., R.J.G. and A.R.S. directed the studies.

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Correspondence to Benjamin M. Segal.

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Extended data

Extended Data Fig. 1 Zymosan-induced RGC axon regeneration is independent of mature T and B cells.

a, Gating scheme for analysis of intraocular infiltrates by flow cytometry. b, C57BL/6 WT or RAG1 deficient mice were injected i.o. with zymosan or PBS on the day of ONC injury. Optic nerves were harvested 14 days later. Longitudinal sections were stained with fluorochrome-conjugated anti-GAP-43 antibodies to enumerate the density of regenerating axons at serial distances from the crush site (n = 6 nerves/ group). Data are shown as mean± sem. One of two independent experiments with similar results is shown. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test (P < 0.05, **P < 0.01, ***P < 0.001, compared with the PBS →WT group). c, Optic nerves were harvested on day 28 following i.o. injection of either PBS or zymosan. Mice received i.p. injections of either αCXCR2 antisera or control sera every other day from the day of ONC onward. The density of GAP-43+ regenerating axons was measured in optic nerve longitudinal sections at serial distances from the crush site (n = 10 nerves per group). Data are shown as mean± sem; statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test (*P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the i.o. PBS/ i.p. NRS group; #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the i.o. zymosan/ i.p. NRS group).

Extended Data Fig. 2 Immature neutrophils are mobilized into the circulation following treatment with i.o. zymosan and i.p. αCXCR2.

Mice received an i.o. injection of zymosan on day 0, and i.p. injections of NRS (blue) or αCXCR2 (red) on days 0, 2 and 4, post ONC injury. Peripheral blood cells were obtained on day 5 and analyzed by flow cytometry. a, Cell surface expression of Ly6G, CD14 and CD101. Upper panels, representative histograms. Lower panels, geometric Mean Fluorescence Intensity on gated Ly6G+ cells and percentage of CD101+ neutrophils. Each symbol represents data from an individual mouse (n = 3 mice/ group). Data are shown as mean± sem. One experiment representative of 3 with similar results is shown. Statistical significance was determined by two tailed unpaired Student’s t-test. b, Representative dot plots.

Extended Data Fig. 3 A population of alternatively activated, immature neutrophils is expanded in intraocular infiltrates following treatment with i.o. zymosan and i.p. αCXCR2.

Single-cell analysis using 10X Genomics of intraocular Ly6G+ cells from the NRS (left panels) or αCXCR2 (right panels) treatment groups, as in Fig. 3. a, Violin plots showing the cells expressing Arg1, Mrc, Hexb, Sgrn and Fpr1 in clusters 1 and 3 of the NRS and αCXCR2 treatment groups. b, Featureplots showing cluster-specific expression of Mrc (CD206, alternative activation marker), CXCR2 and S100a8 (maturation markers).

Extended Data Fig. 4 Adoptively transferred CD14+Ly6Glow cells induce RGC axon regeneration independent of TLR2 and dectin-1 or CCR2 signaling.

a, Mice were subjected to ONC injury on day 0 and received i.o. injections of either PBS, 4 h NΦ, or 3d NΦ, on days 0 and 3. Retina were harvested on day 14. The frequency of viable BRN3a+ RGC neurons in whole mounts, normalized to healthy retina (n = 10 retina per group). One experiment representative of 2 is shown. Statistical significance determined by one-way ANOVA followed by Tukey’s post hoc test. b, Peritoneal Ly6G+ cells were purified 3 days after i.p. zymosan injection (3d NΦ), and adoptively transferred into the eyes of naïve C57BL/6 WT or TLR2-/-dectin-1-/- double knock-out (dko) mice on days 0 and 3 post ONC injury. For negative controls, additional groups were injected i.o. with PBS. Optic nerves were harvested 14 days later and analyzed by GAP-43 immunohistochemistry. The figure shows the density of regenerating axons, at serial distances from the crush site (n = 8 nerves per group). One of 2 independent experiments is shown. Statistical significance determined by one-way ANOVA followed by Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with PBS/WT; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with PBS/dKO). c, GFAP (green) and IBA1 (red) IHC of retinal cross-sections obtained 7 or 14 days following ONC and i.o injection of either 3d NΦ or PBS. Representative images shown (n = 3 mice, 1 of 3 independent experiments, scale bar 80 μm). d, eGFP labeled 3d NΦ were injected i.o. on the day of ONC injury. Representative microscopic image of retinal cross-section prepared 3 days later (n = 3 mice, 1 of 2 independent experiments scale bar 200 μm). e, Representative flow cytometric analysis of intraocular infiltrates harvested from WT or Ccr2–/– mice on day 3 post ONC injury and i.o. injection of 3d NΦ (n = 5 mice per group). f, 3d NΦ were adoptively transferred into the eyes of C57BL/6 WT or Ccr2–/– mice on days 0 and 3 post ONC injury. Axonal densities at serial distances from the crush site, on day 14 post ONC injury (n = 6 nerves, 1 of 2 independent experiments is shown). Statistical significance determined by one-way ANOVA followed by Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, compared with PBS/WT; #P < 0.05, ##P < 0.01, ###P < 0.001 ####P < 0.0001, compared with PBS/ Ccr2–/–). a, b, f Data are shown as mean± sem.

Extended Data Fig. 5 Pro-regenerative neutrophils retain therapeutic efficacy when administered following CNS injury.

a, 3d NΦ were adoptively transferred into the eyes of mice on the day of ONC injury, or after a delay of 6, 12, or 24 hrs. NΦ adoptive transfer was repeated 3 days later. A control group was injected i.o. with PBS alone on days 0 and 3. Optic nerves were harvested on day 14 for quantification of axonal densities by GAP-43 IHC (n = 8 nerves per group). (*P < 0.05; **P < 0.01 *** P < 0.001 compared with PBS). b, 4 h or 3d NΦ were added to primary RGC cultures 4hrs after RGC plating. In other wells, RGC were cultured in media alone (No Tx), or in the presence of recombinant CNTF, as negative and positive controls, respectively. Neurite outgrowth was measured 24 hours later (n = 2000 RGCs per condition, one of two independent experiments shown). Statistical significance determined by one-way ANOVA followed by Tukey’s post hoc test. c, 4 h or 3d NΦ were added to primary DRG cultures 8hrs after DRG plating. In other wells, DRG were cultured in media alone (No Tx), or in the presence of recombinant NGF, for negative and positive controls, respectively. Neurite outgrowth was measured 24 hours later (n = 300 DRGs per condition, one of two independent experiments shown). Statistical significance determined by one-way ANOVA followed by Tukey’s post hoc test. a-c, Data shown as mean± sem.

Extended Data Fig. 6 NGF and IGF-1 drive RGC axon regeneration in a collaborative manner.

a, Quantification of a panel growth factors in unconditioned media (circles) and NCM (squares) by multiplexed antibody array. b, Primary RGC were cultured in the absence or presence of recombinant mouse CNTF, IGF-1, NGF, or a combination of IGF-1 and NGF. Neurite length was measured 24 hours later. Each symbol represents the mean of 200 RGCs in one independent experiment (n = 6 independent experiments shown). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test (**P < 0.01, ***P < 0.001 compared with No Tx; #P < 0.05 compared with NGF; ++P < 0.01, compared with IGF-1). c, Recombinant IGF-1 (blue bars), NGF (green), a combination of NGF and IGF1 (white), or PBS alone (black) was injected into the vitreous on days 0 and 3 post ONC injury. Optic nerves were harvested 14 days later. Density of regenerating axons in optic nerve sections, at serial distances from the crush site (n = 8 nerves per group). One experiment representative of 2 with similar results is shown. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with PBS; #P < 0.05 compared with NGF; +P < 0.05, ++P < 0.01, compared with IGF-1). b,c, Data shown as mean± sem.

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Sas, A.R., Carbajal, K.S., Jerome, A.D. et al. A new neutrophil subset promotes CNS neuron survival and axon regeneration. Nat Immunol 21, 1496–1505 (2020). https://doi.org/10.1038/s41590-020-00813-0

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