Fig. 7: CSPG digestion reduces the inflammatory effects of CSPGs in M2 polarised BMDMs in vitro and in vivo. | Nature Communications

Fig. 7: CSPG digestion reduces the inflammatory effects of CSPGs in M2 polarised BMDMs in vitro and in vivo.

From: Chondroitin sulfate proteoglycans prevent immune cell phenotypic conversion and inflammation resolution via TLR4 in rodent models of spinal cord injury

Fig. 7

a Experimental design for evaluating the effects of ChABC conditioned media on polarised BMDMs after CSPG (5 μg/ml) activation. b mRNA level of ChABC gene expression produced by non-polarised (M0) BMDMs at different LV-ChABC titre transfection. Results were assessed for normality using the Shapiro–Wilk test and one-way ANOVA with Tukey post hoc test was used to analyse significant differences. ***p < 0.001 vs. control group (0 GC/ml), ###p < 0.001 vs. 2e7 GC/ml group, &&&p < 0.001 vs. 4e7 GC/ml group. Relative fold changes presented as mean ± SEM (n = 3 per group). The optimum titration was 1e8 GC/ml, which was used for further experiments. c Immunocytochemistry of BMDMs transfected with LV-GFP (green) to confirm (d) the percentage of transfection (68%) at 1e8 U/ml (n = 3 per group). Data are shown as mean ± SEM. Bar graphs of inflammatory gene expression by CSPG treatment with or without ChABC enriched medium in e M2 and f M1 BMDMs. mRNA levels of inflammatory response genes were determined by qPCR. **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control (no CSPG) group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. +CSPG+ ChABC group; &p < 0.05, &&p < 0.01, &&&p < 0.001, &&&&p < 0.0001 vs. +ChABC group; $p < 0.05, $$p < 0.01, $$$p < 0.001, $$$$p < 0.0001 vs. +CSPG group. Results were assessed for normality using the Shapiro–Wilk test and one-way ANOVA with Tukey post hoc test was used to analyse differences between conditions. Data are shown as mean ± SEM (n = 4 per group). g Experimental design for cytokine gene expression analysis in sorted cells from contused rat spinal cord at 7 dpi. h Bar graphs showing inflammatory cytokine gene expression measured by qPCR in sorted macrophages and microglial cells at 7 dpi, showing reduced pro inflammatory cytokine gene expression in response to CSPG digestion in both populations after SCI. *p < 0.05, ***p < 0.001 vs. 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 (CCL5, IL6 n = 3; Il1b, CCL3, CXCL10 n = 9, per treatment and cell population). Detailed statistics and exact p values are provided in Supplementary Table 8. Source data are provided as a Source Data file.

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