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Cytokine polarized, alternatively activated bone marrow neutrophils drive axon regeneration

Nature Immunology volume 25, pages 957–968 (2024)Cite this article

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Abstract

The adult central nervous system (CNS) possesses a limited capacity for self-repair. Severed CNS axons typically fail to regrow. There is an unmet need for treatments designed to enhance neuronal viability, facilitate axon regeneration and ultimately restore lost neurological functions to individuals affected by traumatic CNS injury, multiple sclerosis, stroke and other neurological disorders. Here we demonstrate that both mouse and human bone marrow neutrophils, when polarized with a combination of recombinant interleukin-4 (IL-4) and granulocyte colony-stimulating factor (G-CSF), upregulate alternative activation markers and produce an array of growth factors, thereby gaining the capacity to promote neurite outgrowth. Moreover, adoptive transfer of IL-4/G-CSF-polarized bone marrow neutrophils into experimental models of CNS injury triggered substantial axon regeneration within the optic nerve and spinal cord. These findings have far-reaching implications for the future development of autologous myeloid cell-based therapies that may bring us closer to effective solutions for reversing CNS damage.

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Fig. 1: IL-4/G-CSF-polarized mouse BMNs exhibit an immature, alternatively activated phenotype.
Fig. 2: IL-4/G-CSF-polarized BMNs enhance RGC survival and axon regrowth.
Fig. 3: IL-4/G-CSF-polarized neutrophils exhibit a transcriptome indicative of alternative activation and a pro-regenerative phenotype.
Fig. 4: IL-4/G-CSF-polarized BMNs enhance axon regeneration, in part, via the production of IGF-1 and HB-EGF.
Fig. 5: IL-4/G-CSF-polarized neutrophils drive the regeneration of spinal cord axons.
Fig. 6: IL-4/G-CSF-polarized CD34+ BM cells display a transcriptome indicative of alternatively activated, immature neutrophils.
Fig. 7: IL-4/G-CSF-polarized CD34+ BM cells contain a pro-regenerative neutrophil subset.

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Data availability

Bulk RNA-seq data have been deposited in NCBI’s Gene Expression Omnibus (GEO) under accession code GSE244934. Source data are provided with this paper.

Code availability

All scripts are publicly available at: https://github.com/lahammond/2024_Jerome_Axon_Regeneration.

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Acknowledgements

We thank A. Sepeda and S. Atkins for technical assistance. This study was supported by NIH-NEI grants R01EY029159 (B.M.S.), R01EY028350 (B.M.S.) and K08EY029362 (A.R.S.).

Author information

Author notes

  1. These authors contributed equally: Andrew D. Jerome, Andrew R. Sas.

Authors and Affiliations

  1. Department of Neurology, The Ohio State University Wexner Medical Center, Columbus, OH, USA

    Andrew D. Jerome, Andrew R. Sas, Yan Wang, Luke A. Hammond, Jeffrey R. Atkinson, Tom Liu & Benjamin M. Segal

  2. The Neuroscience Research Institute, The Ohio State University, Columbus, OH, USA

    Andrew D. Jerome, Andrew R. Sas, Yan Wang, Luke A. Hammond, Tom Liu & Benjamin M. Segal

  3. The James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA

    Jing Wen & Amy Webb

  4. Department of Biomedical Informatics, The Ohio State University, Columbus, OH, USA

    Amy Webb

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Contributions

B.M.S. designed the study and supervised the study, data interpretation and manuscript editing. A.D.J. and A.R.S. designed the study and contributed to data interpretation under the supervision of B.M.S. A.D.J. and A.R.S. performed optic nerve crush experiments, tracing of optic nerve neurons, tissue clearance, immunohistochemical staining, optic nerve imaging, quantification of regenerating axons and intraocular injections. A.D.J. performed RGC neurite outgrowth assays, retinal whole mounts, RGC imaging and quantification, cytospins and Giemsa–Wright staining. A.D.J. and J.R.A. performed flow cytometry and analysis. Y.W. performed dorsal spinal cord injury experiments, tracing of spinal cord neurons, tissue clearance, immunohistochemical staining, imaging and quantification of regenerating axons. Y.W. performed dorsal root ganglia neurite outgrowth assays under the supervision of A.D.J. L.A.H. conducted postimage acquisition processing and analyses of spinal cord, DRG and sciatic nerve specimens. T.L. isolated RNA and prepared DNA libraries. J.W. performed western blot assays. A.W. analyzed RNA-seq data. B.M.S. and A.D.J. wrote the manuscript.

Corresponding author

Correspondence to Benjamin M. Segal.

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Competing interests

B.M.S., A.R.S., T.L. and A.D.J. are inventors on a patent titled ‘Engineered cells and uses thereof’, licensed by the Ohio State University. The remaining authors declare no competing interests.

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Nature Immunology thanks Richard Zigmond and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Laurie A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Kinetics of gene expression in BMN during polarization. Polarized and unpolarized Ly6G+ BMN express canonical markers of granulopoiesis.

a, RNA was extracted from Ly6G+ BM cells at serial time points during culture in the presence or absence of polarizing factors. Levels of transcripts encoding IL-4Ra (IL4r), arginase-1 (Arg1), mannose receptor (Mrc1), and F4/80 (Adgre1), were measured by RT-qPCR and normalized to Actb. Data are shown as fold increase over mean normalized levels in unpolarized BMN. Each symbol represents data derived from an individual mouse. Data are presented as mean values ± SEM. b, Normalized levels of myeloperoxidase (Mpo) and neutrophil elastase (Ela, also known as Elane) mRNA, extracted from Ly6G+ BM cells, following a 24 hour polarization under the indicated conditions (n = 5). Transcript levels were also measured in mature peripheral blood neutrophils, isolated from naïve mice, for an additional control. (n = 5). Each symbol represents an individual mouse. Data are presented as mean values ± SEM. c, Polarized and unpolarized BMN were analyzed by flow cytometry. Histograms showing MFI of MPO (left) and Elastase (right), gating on CD11b+Ly6G+ cells (Representative of n = 5 mice per group). a, Data shown from one experiment, representative of two independent experiments. b, c, Data are shown from one experiment, representative of four independent experiments. d, Flow cytometric gating strategy for assessing the purity of MACS purified Ly6G+ BM cells.

Source data

Extended Data Fig. 2 GFAP expression in retina from mice subjected to ONC injury and intraocular injection of unpolarized or polarized BMN versus PBS.

Retina were harvested on day 4 following ONC injury and intraocular injection of either IL-4/G-CSF polarized BMN, unpolarized BMN, or PBS. Each panel shows a representative retinal cross-section obtained from an individual mouse, stained with antibodies against GFAP (green). Scale bar, 100 μm. Data shown from one experiment, representative of two independent experiments.

Extended Data Fig. 3 IL-4/G-CSF-polarized BMNs produce HB-EGF protein.

Representative western blot of HB-EGF protein in BMN lysates Representative western blot, analyzing lysates from unpolarized BMN (left) and IL-4/G-CSF polarized BMN (right), displaying total protein (stain-free gel) and HB-EGF band on a PVDF membrane. Data shown from one experiment, representative of two independent experiments.

Extended Data Fig. 4 IL-4/G-CSF polarized Ly6G+ BMN accumulate in the sciatic nerve at 4 hours, and the spinal cord at 24 hours, following SCI and i.n. BMN injection.

a, Representative section of a sciatic nerve harvested 4 hours after SCI injury and i.n. injection of IL-4/G-CSF polarized BMN that were derived from tdTomato-reporter mice. Donor BMN are identified as tdTomato+ (red) and Ly6G+ (white) (scale bar, 50 μm). b, Representative flow cytometric pseudocolored dot plot of mononuclear cells isolated from sciatic nerves 4 hours after SCI injury and i.n. injection of either IL-4/G-CSF polarized tdTomato+ BMN (left) or PBS (right), gating on CD11b+Ly6G+ cells. c, Representative flow cytometric pseudocolored dot plot of spinal cord mononuclear cells isolated 24 hours following SCI injury and i.n. injection of either IL-4/G-CSF polarized tdT+ BMN (left) or PBS (right), gating on CD11b+Ly6G+ cells. a-c, Data shown from one experiment, representative of two independent experiments.

Extended Data Fig. 5 IL-4/G-CSF polarized CD34+ bone marrow cells, derived from individual donors, reproducibly induce neurite outgrowth of human cortical neurons.

a, Gating strategy for flow cytometric analysis and sorting of IL-4/G-CSF polarized CD34+ human BM cells. b, Length of the longest neurite grown by human primary cortical neurons following 24 hour culture with IL-4/G-CSF polarized, single cytokine polarized, or unpolarized human CD34+ BM cells. Some neurons were cultured in media alone (negative control) or with NGF (positive control). Each plot represents an independent experiment performed using BM cells derived from a unique BM donor. c, Length of the longest neurite grown by human primary cortical neurons following 24 hour culture with CD34+ or CD33+CD15+ cells, FACS sorted from IL-4/G-CSF polarized human BM stem cells. Some neurons were cultured in media alone (negative control) or with NGF (positive control). Each plot represents an independent experiment performed using BM cells derived from a unique BM donor. b, c, Data are presented as mean values ± SEM. Each symbol represents a single neuron with the n value listed under each experimental group. Statistical significance was determined by one-way ANOVA followed by Dunnett’s post hoc test.

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Jerome, A.D., Sas, A.R., Wang, Y. et al. Cytokine polarized, alternatively activated bone marrow neutrophils drive axon regeneration. Nat Immunol 25, 957–968 (2024). https://doi.org/10.1038/s41590-024-01836-7

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