Fig 2: TLR2 deficiency on astrocytes abolished the decreased CSPGs deposition and upregulated astrocytes induced by SCDEs treatment. a Representative images of immunofluorescent analysis of CS56 (red), GFAP (green), and DAPI (blue) in TLR2f/f sham group, TLR2f/f group, TLR2f/f with SCDEs treatment group and TLR2-/-group at 2 weeks after SCI. Scale bars = 200 µm. Right images are high-resolution versions of the boxed regions in the left images, scale bars = 50 µm. b–d Quantitative analysis of CSPGs area and the intensity value of CS56 and GFAP (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n = 6). e Representative images of immunofluorescent analysis of TLR2 (red), GFAP (green), and DAPI (blue) in TLR2f/f and TLR2-/- mice at 2 weeks after SCI. Scale bars = 20 µm. f Quantitative analysis of TLR2-positive astrocytes **P < 0.01, n = 6)

Fig 3: SMU-Z1 is a TLR1/2, but not TLR2/6 agonist. A) HEK-Blue hTLR2 cells were treated with SMU-Z1 and anti-hTLR1, anti-hTLR2, or anti-hTLR6 antibodies for 24 h. SMU-Z1 strongly activates QUANTI-Blue SEAP signaling at 30 × 10-9 m. Anti-hTLR1-IgG and anti-hTLR2-IgA antibodies inhibit SMU-Z1-triggered SEAP signaling in a dose-dependent manner, whereas anti-hTLR6-IgG has no such influence. B) The positive control Pam2CSK4, a TLR2/6 agonist, had different responses to TLR1, 2 and 6 specific antibodies compared to SMU-Z1. C) Flow cytometric analysis for 0 × 10-6, 1 × 10-6, and 5 × 10-6 m of SMU-Z1 and Pam3CSK4. Results demonstrate excellent NF-?B activation capacity of SMU-Z1 compared to the positive control Pam3CSK4. D–G) SMU-Z1 has stimulated D,E) TNF-a and F,G) IL-1ß production in Raw 264.7 macrophage cells and human PBMC, respectively. H) SMU-Z1 and Pam3CSK4 activate nitric oxide (NO) production in Raw cells in a dose-dependent manner. I) NO activation of SMU-Z1 can be inhibited by the TLR1/2 inhibitor, CU-CPT22, in Raw 264.7 cells. SMU-Z1 stimulates J) IL-6 and K) IL-8 mRNA production at different concentrations in human PBMC. Data are present as mean ± SD of triplicates and the results are representative of three independent experiments.

Fig 4: Structures of the hit compounds obtained from HTS, and bioactivity validation of the optimized compound SMU-Z1. A) Chemical structures of the hits from the HTS. B) The initial hit compounds obtained for TLR2 activation. HEK-Blue hTLR2 cells were incubated with indicated compounds for 24 h, and the cell culture supernatants were detected by QUANTI-Blue (Invivogen) to evaluate SEAP signaling at 620 nm (OD620). Pam3CSK4 was used as a positive control in the experiment. C) Comparison of SMU-Z1 with RF04289 and Pam3CSK4 for the SEAP activation in HEK-Blue hTLR2 cells. D) HEK-Blue human TLR3, TLR4, TLR5, TLR7, and TLR8 cells were incubated with SMU-Z1 (10 × 10-6 m) with the control of TLR-specific agonists separately for 24 h, and the activation was evaluated by QUANTI-Blue at OD620. As positive control, agonists that selectively activate a specific TLR were used: TLR3, Poly I:C (10 µg mL-1); TLR4, LPS (10 ng mL-1); TLR5, FLA-BS (1 µg mL-1); TLR7 and TLR8, R848 (5 µg mL-1). E) SMU-Z1 stabilized more TLR2 protein in HEK-Blue hTLR2 cells as the concentrations increase in the cultures. Cells were treated with SMU-Z1 (0–10 × 10-6 m) for 24 h, and the cell lysates were detected using Western blot and rabbit anti-TLR2 antibody. GAPDH served as the cell lysates input control. Data presented are mean ± SD and the figures shown are representative of three independent experiments.

Fig 5: General pathological changes such as shortening of colorectal length, number of tumors, and total volume of the tumor. NC: WT without DMH-DSS; KC: TLR2-/- without DMH-DSS; NT: WT with DMH-DSS; KT: TLR2-/- with DMH-DSS. *P < 0.05, KT vs NT group (n = 6); ***P < 0.001, KT vs NT group (n = 6)