O-sulphated HS induces intracellular IL-1β expression in vivo and in vitro.Figure 2Mice were systemically challenged via intraperitoneal (I.P.) injection with PBS, bovine kidney HS (2.5 mg/kg), heparin (20 U/kg), WT GAG (2.5 mg/kg), MPSIIIA GAG (2.5 mg/kg) or LPS (250 μg/kg). Animals were sacrificed 1 hour, 2 hours and 6 hours after I.P. challenge. The expression of Il1b was measured in whole brain using quantitative PCR 1 hour, 2 hours and 6 hours after I.P. challenge (n=6 mice per group). Data are expressed as mean ± STDEV and were tested by two-way ANOVA with Bonferroni's post-test; *P<0.05, **P<0.01, ***P<0.001. Symbols above bars are versus PBS at the relevant time-point. In vitro modelling. WT or MPSIIIA GAG (0.8 μg/ml HS) was applied to WT mixed glial cultures for 24 hours before harvesting the media or cell extract and measuring levels of IL-1β. Quantification of intracellular production of IL-1β (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; ***P<0.001. Symbols above bars are versus PBS. Quantification of IL-1β secreted into the media (n=3 independent experiments each with 3 inter-experimental replicates). A horizontal dotted line represents the limit of detection of the ELISA. Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; **P<0.01, ***P<0.001. Symbols above bars are versus PBS. WT mixed glial cultures were co-treated with MPSIIIA GAG (4 μg/ml) and/or the TLR4 inhibitor CLI-095 (1 μg/ml) for 24 hours. The intracellular production of IL-1β was measured (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; ***P<0.001. Symbols above bars are versus MPSIIIA GAG + vehicle. Multi-heparinase (HepM) and/or chondroitinase ABC (cABC) digests were performed on MPSIIIA GAG, and the resulting oligosaccharides applied to a WT mixed glial culture for 24 hours alongside heparin, bovine HS (bHS), porcine DS (pDS) and WT GAG. The intracellular production of IL-1β was measured (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; *P<0.05, ***P<0.001. Symbols above bars are versus MPSIIIA GAG alone. MPSIIIA GAG was 2-O-desulphated (hashed bars) utilising 120 mM NaOH, and compositional HS disaccharide analysis performed via RP-HPLC against untreated MPSIIIA GAG (black bars) to validate complete 2-O-desulphation. Percentage contribution of each modification were determined (n=3 GAG samples). Data are expressed as mean ± STDEV and were tested by two-way ANOVA with Bonferroni's post-test; 2S MPSIIIA GAG vs. 2S 2-ODS MPSIIIA GAG P<0.0001. NS, N-sulphated glucosamine; 2S, 2-O-sulphate group; 6S, 6-O sulphate group. MPSIIIA GAG was 2-O-desulphated and the resulting oligosaccharides applied to a WT mixed glial culture for 24 hours. The intracellular production of IL-1β was measured (n=3 independent experiments each with 3 inter-experimental replicates). Data are expressed as mean ± STDEV and were tested by one-way ANOVA with Tukey's post-test; MPSIIIA GAG vs. 2ODS MPSIIIA GAG P<0.0001. 2ODS, 2-O-desulphated.