Figure 3

HA fragment induction of IFNβ is TLR-4 dependent. (a) Thioglycollate elicited peritoneal macrophages from C57BL/6 WT, TLR2 receptor null or TLR4 receptor null mice were stimulated with 200 ug/mL LMW HA fragments for 3 hours; cells were collected in Trizol, RNA extracted, and cDNA was analyzed with real-time PCR. HA fragment-induced MIP1α requires TLR2; HA induction of IFNβ was independent of TLR2 but required TLR4. Data demonstrate IFNβ induction compared to WT unstimulated levels; ^★ p < 0.05 compared to WT unstimulated; # = p <0.05 compared to genotype specific unstimulated). (b) Pharmacological inhibition of TLR4 significantly decreases HA fragment activation of IFNβ. RAW 264.7 macrophages were prestimulated with 1 ug/mL CLI-095 for thirty minutes, prior to HA fragments (200 ug/ml) or LPS (100 ng/ml) for 3 hours. Cells were collected in Trizol, RNA extracted, and cDNA was analyzed with real-time PCR. Blocking the TLR4 receptor with CLI-095 significantly inhibited both HA fragment and LPS induction of IFNβ. Values shown are mean fold induction over WT unstimulated; These figures are representative of at least 3-4 identical experiments done in triplicate ^★ p < 0.05 unstimulated stimulated.