Figure 4

HA fragment induction of IFNβ is independent of MyD88, and TLR3 but dependent upon TRIF and TBK1. Thioglycollate elicited peritoneal macrophages from WT, MyD88 null, TLR3, TRIF null mice were stimulated with 200 ug/mL LMW HA fragments, LPS 10 ng/ml or p(I:C) 5 ug/ml for 3 hours; cells were collected in Trizol, RNA extracted, and cDNA was analyzed with real-time PCR (a) HA fragments require MyD88 to induce MIP1α but NOT IFNβ. (b) TLR3 is not necessary for HA induced IFNβ mRNA. (c) In the absence of TRIF, HA fragments induced significantly less IFNβ. (d,e) RAW macrophages were stimulated with 200 ug/mL HA fragments for 3 hours +/- the TBK1 inhibitor BX795; RNA was isolated and Real-time PCR performed. HA fragments and p(I:C) induced significantly less IFNβ in the presence of the TBK1 inhibitor but HA induced MIP1α was not significantly inhibited. Data reported as fold induction over WT unstimulated; data are mean of 3-7 experiments run in triplicate; * p < 0.05 HA vs US; # p < 0.05 HA vs HA + BX795; p < 0.05 p(I:C) vs p(I:C) + BX795.