Fig. 8: The TLR4 pathway is essential for the CSPG-activated inflammatory phenotype in M2 polarised macrophages.

a Experimental design to study the effect of CSPG treatment (5 μg/ml) on polarised BMDMs treated with or without TAK242 (100 μM), a pharmacological inhibitor of TLR4. Bar graphs showing the expression of inflammatory response genes in b M1 BMDMs and c M2 BMDMs. mRNA levels were determined by qPCR. Data were normalised with respect to control (no CSPGs). 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. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group, ^&p < 0.05, ^&&p < 0.01, ^&&&p < 0.001 vs. CSPG treated group, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. +CSPGs+TAK242 group, ^$p < 0.01, ^$$p < 0.001, ^$$$p < 0.001 vs. +TAK242 group. Data are presented as mean ± SEM (n = 13 in −CSPG and +CSPG groups; n = 7 in +CSPG +TAK242 group; n = 10 in −CSPG +TAK242 group). d Experimental design to compare the effect of CSPG treatment on BMDMs from C57/B6 WT and TLR4^−/− mice. e–h Bar graphs comparing immunomodulatory effects of CSPG treatment (4 h at 5ug/ml) between WT and TLR4^−/− polarised BMDMs. Differences in inflammatory gene expression by CSPG treatment were assessed in e M1− WT, (f) M1− TLR4^−/−, g M2-WT and h M2-TLR4^−/− BMDMs. mRNA levels of inflammatory response genes were determined by qPCR. Data was normalised by their respective controls (WT or TLR4^−/− BMDM without CSPGs), represented by dotted line. Results were assessed for normality using the Shapiro–Wilk test and analysed using a two-tailed unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001 vs. WT w/o CSPGs. Data are shown as mean ± SEM (n = 3 per group). Detailed statistics and exact p values are provided in Supplementary Table 8. Source data are provided as a Source Data file.