A new neutrophil subset promotes CNS neuron survival and axon regeneration (original) (raw)

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.

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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.

Author information

Author notes

  1. These authors contributed equally: Andrew R. Sas, Kevin S. Carbajal.

Authors and Affiliations

  1. Department of Neurology, The Ohio State University Wexner Medical Center, Columbus, OH, USA
    Andrew R. Sas, Andrew D. Jerome & Benjamin M. Segal
  2. The Neuroscience Research Institute, The Ohio State University, Columbus, OH, USA
    Andrew R. Sas, Andrew D. Jerome & Benjamin M. Segal
  3. Department of Neurology, University of Michigan, Ann Arbor, MI, USA
    Andrew R. Sas, Kevin S. Carbajal & Benjamin M. Segal
  4. Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA
    Rajasree Menon
  5. Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA
    Choya Yoon, Ashley L. Kalinski & Roman J. Giger
  6. Neuroscience Graduate Program, University of Michigan, Ann Arbor, MI, USA
    Roman J. Giger

Authors

  1. Andrew R. Sas
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  2. Kevin S. Carbajal
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  3. Andrew D. Jerome
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  4. Rajasree Menon
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  5. Choya Yoon
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  6. Ashley L. Kalinski
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  7. Roman J. Giger
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  8. Benjamin M. Segal
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Contributions

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 toBenjamin 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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