It was previously reported that HEK293 cells stably transfected with various TLRs failed to respond to c-di-GMP (Karaolis et al., 2007a) , but the reason for this failure was unclear. For example, HEK293 cells might lack an essential accessory protein. To rule out a role for TLRs in sensing of c-di-GMP, we tested responses in Myd88 / Trif / double-knockout macrophages, which are deficient in all TLR signaling (Yamamoto et al., 2003) . Both wild-type and Myd88 / Trif / macrophages showed a robust induction of IFN- after c-di-GMP stimulation (Fig. 2 A) . As expected, the Myd88 / Trif / macrophages did not respond to LPS (Fig. 2 A) . These results indicated that TLR signaling is not required for responsiveness to c-di-GMP, and are consistent with c-di-GMP signaling via a cytosolic surveillance pathway.