Fig. 3: CSPG digestion alters phenotypically distinct immune cell clusters during the resolution phase of inflammation after spinal cord injury.

a Bar graphs showing changes in the expression of classic M1 and M2 markers in the microglial cell population (cluster 1 in t-SNE analysis, Fig. 1f, g) at 7 dpi. Microglial cells exhibit significantly reduced expression of the pro-inflammatory (M1) marker MHC II in the LV-ChABC treated group, compared to LV-GFP treated animals. b FACS plot histogram of MHC-II expression in microglial cells at 7 dpi showing reduced expression in the LV-ChABC-treated group (blue) compared with control treatment (LV-GFP; green). Grey colour represents the isotype control. c Bar graphs showing changes in the expression of classic M1 and M2 markers in in the macrophage cell population (clusters 2 and 3 in t-SNE analysis, Fig. 1f, g), at 7 dpi. Macrophages exhibit significantly reduced expression of pro-inflammatory (M1) markers in LV-ChABC-treated animals compared to LV-GFP controls. d FACS plot histogram of M1 markers in the macrophage population at 7 dpi showing reduced expression in LV-ChABC treated group (blue) compared with control treatment (LV-GFP; green). Grey colour represents the isotype controls. e, g Graphs showing the changes in the expression of M1 and M2 markers in CD43^high and CD43^low macrophages (t-SNE cluster 3 and 2, respectively, Fig. 1f, g, i), at 7 dpi. Pro-inflammatory M1 marker reduction exerted by CSPG digestion is higher in the CD43^low population (G). f, h FACS plot histograms of pro-inflammatory marker expression IN CD43^high and CD43^low macrophages, respectively, at 7 dpi showing reduced expression in the LV-ChABC treated group (blue) compared with control treatment (LV-GFP; green). Grey colour represents the isotype controls. a, c, e, g *p < 0.05, **p < 0.01 versus control (LV-GFP) group. Results were assessed for normality using the Shapiro–Wilk test and analysed using a two-tailed unpaired t test. Data are shown as mean ± SEM (a and c: LV-GFP n = 9, LV-ChABC n = 11; e and g: n = 4 per treatment). MFI mean fluorescence intensity. i Experimental design for phenotype gene expression analysis in sorted cells at 7 dpi. j Expression levels of microglial (GPR34 and FcRls) and monocyte/macrophage (CCR2) enriched genes evaluated by qPCR in sorted cells. ***p < 0.001, ****p < 0.0001 versus sorted microglial gene expression. Results were assessed for normality using the Shapiro–Wilk test and analysed using a two-tailed unpaired t test. Data are shown as mean ± SEM (n = 13 per cell population). k gene expression of classic M1 and M2 phenotype markers measured by qPCR in sorted monocytes/macrophages and microglial cells at 7 dpi. LV-ChABC treatment redirects monocytes/macrophages and microglial cells toward a pro-repair (M2) phenotype after SCI. *p < 0.05, **p < 0.01 versus normalised control group (LV-GFP treatment). Results were assessed for normality using the Shapiro–Wilk test and analysed using a two-tailed unpaired t test. Data are shown as mean ± SEM (iNOS, MHC-II n = 3; CD68, Arg I, CD206 n = 10 per treatment for macrophages; MHC-II n = 3; iNOS, CD68, Arg I, CD206 n = 10 per treatment for microglial cells). Detailed statistics and exact p values are provided in Supplementary Table 8. Source data are provided as a Source Data file.