As shown in Fig. 2A, LPS (1 μg/ml) challenge of AMΦ, which were isolated from WT mice, resulted in an increase in TLR3 protein expression at 4 h and a marked increase at 6 h. However, in AMΦ from TLR4−/− mice, LPS failed to induce TLR3 expression (Fig. 2A), indicating that TLR4 signaling mediates the LPS-induced up-regulation of TLR3. All TLRs, with the exception of TLR3, utilize MyD88, and TLR4/MyD88 signaling pathway is the primary signaling pathway used to induce the expression of pro-inflammatory cytokines17. However, TLR4 can signal through both MyD88-dependent and -independent pathways, and the MyD88-independent signaling for TLR4 results in the production of type I IFNs18. To address the role of MyD88 in mediating the LPS-TLR4-induced up-regulation of TLR3, LPS (1 μg/ml) was administered to AMΦ from MyD88−/− mice, and TLR3 expression was assessed. As shown in Fig. 2A, MyD88 deficiency prevented LPS-induced up-regulation of TLR3. The changes in the mRNA expression of TLR3 are shown in Fig. 2B, and these results corresponded to the observed changes in protein expression. As shown in Fig. 2C, the localization of TLR3 was further investigated in AMΦ by immunofluorescence. In the absence of LPS, TLR3 was found within small vesicles throughout the cytoplasm. TLR3-containing vesicles increased in a time-dependent manner in AMΦ from WT mice, but not in TLR4−/− or Myd88−/− mice, and similar results were observed using quantitative PCR (Fig. 2D).