Others have implicated TLR4, or the combination of TLR2 and TLR4 in response to hyaluronan fragments of various sizes [8,18]. TLR4, TLR3, and TLR9 are well established as inducers of IFN�� expression. Although recently vaccinia has been reported to induce IFN�� via TLR2, in general TSA TLR2 signaling pathways do not induce IFN��. [19-24]. In some models TLR2 agonists actually inhibit IFN�� production [25]. Thus, we wanted to determine which, if any, of the TLR receptors was involved in mediating HA-induced IFN�� expression. Thiolglycollate-elicited primary macrophages were harvested from TLR2 null, TLR4 null, and wild type (WT) mice and stimulated in serum-free media with 200 ug/mL of LMW HA for 3 hours. RNA was extracted and analyzed by real time quantitative PCR.

Both TLR2 null and WT macrophages responded briskly to HA fragments, with a marked and significant increase in IFN�� mRNA. However, the TLR4 null macrophages failed to produce IFN�� after LMW HA stimulation (Figure 3a). In contrast, LMW HA-induced MIP1�� production was reduced in macrophages from TLR2 null mice but robustly induced by macrophages lacking TLR4. In addition, macrophages from both TLR2 and TLR4 null mice demonstrated robust expression of IFN�� in response to poly (I:C), a TLR3 agonist. Figure 3 HA fragment induction of IFN�� 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 … TLR2 signaling thus proved to be necessary for the chemokine expression, but not the IFN�� expression induced by HA fragments.

In contrast, TLR4 appeared responsible for HA-induced IFN�� expression but not chemokine expression. Thus, the ability of HA fragments to stimulate the C-C chemokine MIP1�� and IFN�� is by two distinct pathways: HA fragments induced chemokine expression via TLR2 while the same HA fragments induced type I interferon via TLR4. To confirm the dependence of HA fragment-induced IFN�� on TLR4 we employed the pharmacologic inhibitor CLI-095 [26]. This molecule blocks TLR4 activation without inhibiting other MyD88-dependent signaling. Macrophages were pre-incubated with either CLI-095 or DMSO as a control for 30 minutes, stimulated with HA fragments or LPS for 3 hours, and then evaluated for IFN�� expression by RT PCR.

Consistent with the observations in the TLR4 null macrophages, CLI-095 inhibited HA fragment induction of IFN�� mRNA by 80%. As expected, CLI-095 also inhibited TLR4-dependent LPS induction of IFN�� (Figure 3b). Drug_discovery These data further support the finding that HA fragments induce IFN�� via a TLR4-dependent pathway. HA fragments induce IFN�� independent of MyD88 via TRIF The ability of HA fragments to induce IFN�� in a TLR4 dependent fashion was somewhat surprising.