Cytokine polarized, alternatively activated bone marrow neutrophils drive axon regeneration

Extended Data 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 BMNΦ (n=4-5 mice per group). b, Normalized levels of myeloperoxidase (Mpo) and neutrophil elastase (Ela) mRNA, extracted from Ly6G+ BM cells, following a 24 hour polarization under the indicated conditions. Transcript levels were also measured in mature peripheral blood neutrophils, isolated from naïve mice, for an additional control (black symbols). (n=5 mice per group). c, Polarized and unpolarized BMNΦ were analyzed by flow cytometry. Histograms showing MFI of MPO (left) and Elastase (right), gating on CD11b+Ly6G+ cells (n=4-5 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.

Extended Data 2. GFAP expression in retina from mice subjected to ONC injury and i.o. injection of unpolarized or polarized BMN versus PBS. Retina were harvested on day 4 following ONC injury and i.o. 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

Extended Data 3. Representative western blot of HBEGF protein in BMNΦ lysates. Representative western blot, analyzing lysates from unpolarized BMNΦ (left) and IL-4/G-CSF polarized BMN (right), displaying total protein (stain-free gel) and HBEGF band on a PVDF membrane.

Extended Data 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 (tdT)-reporter mice. Donor BMN are identified as tdT+ (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 tdT+ 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+ BMNΦ (left) or PBS (right), gating on CD11b+Ly6G+ cells.

Extended Data 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, Mean neurite length of the longest neurite grown by primary cortical neurons following 24 hour culture with polarized or unpolarized CD34+ BM cells. Each plot represents an experiment performed using BM cells derived from a unique BM donor (n > 150 neurons per condition). c, Mean neurite length of the longest neurite grown by primary cortical neurons following 24 hour culture with CD34+ or CD33+CD15+ cells FACS sorted from IL-4/G-CSF polarized BM cells. Each plot represents an experiment performed using BM cells derived from a unique BM donor (n > 200 neurons per condition). b, c, Each symbot represents a single neuron. Statistical significance was determined by one-way ANOVA followed by Dunnett’s post hoc test.