Fig. 3.

Hyaluronan (HA) acts through TLR2 to block OPC maturation. (A) OPCs were exposed to 25 μg/mL hyaluronan alone or with 10 μg/mL neutralizing antibodies to TLR2, CD44, or TLR4 with controls of nonimmune mouse IgG or nonimmune rat IgG. (B) After 2 d in culture with 10 ng/mL CNTF and 15 μM T3, cells were assessed for O1/olig2 staining. Compared with untreated cells, significant differences in the percent O1/olig2 mature oligodendrocytes were observed with hyaluronan with or without each antibody treatment except for TLR2-blocking antibodies. Mixed glia derived from strain-matched postnatal day 0 (P0) wild-type pups (C) or TLR2-null pups (D) were cultured for 14 d and then grown in oligodendrocyte medium for 4 d. Cells were stained for O1 (green), olig2 (red), and DAPI (blue). (E) The density of O1⁺ cells and olig2⁺ cells was measured, standardized by wild-type levels. (White bars, wild-type; black bars, TLR2-null.) (F) Wild-type mixed glia were stained by biotinylated hyaluronan-binding protein (HABP), FITC-linked streptavidin (green), and DAPI (blue) after culture for 14 d and then were grown in oligodendrocyte medium for 4 d. FITC-linked streptavidin (green) detected endogenous hyaluronan in wild-type mixed glia cultures. (G) After chondroitinase treatment to remove hyaluronan, mixed glia were washed, stained for hyaluronan, and found to exhibit virtually no hyaluronan staining. (Scale bars, 20 μm in A and 100 μm in C, D, F, and G.)