To examine the role of IRF3 in c-di-GMP induction of IFN-, we tested the ability of c-di-GMP to induce signaling in macrophages lacking Irf3. We observed that Irf3 / macrophages failed to induce IFN- in response to c-di-GMP ( Fig. 2 D) . We also tested Irf7 / macrophages and observed a partial requirement for IRF7 by the c-di-GMP pathway (Fig. 2 D) . Loss of Irf7 is likely compensated for by Irf3 in macrophages. The partial requirement for Irf7 may reflect a c-di-GMP responses in Tbk1 / macrophages (Fig. 2 B) . We observed very little IFN- induction by c-di-GMP in cells lacking Tbk1, suggesting that TBK1 is required for c-di-GMP signaling. We also observed that Tbk1 is required for the induction of IFN- by pdA:dT ( Fig. 2 B) , in agreement with a recently published report (Miyahira et al., 2009) . Although the data indicate a key role for Tbk1 in the response to c-di-GMP, it is formally possible, albeit unlikely, that Tbk1 protein rather than Tbk1 kinase activity is required for the response. We also tested the requirement for the TBK1related kinase Ikbke (also called IKK or IKK-i), which plays (Jiang et al., 2005) using recombinant IFN- as a standard. *, P < 0.01 as compared with transfected c-di-GMP (Student's t test). (B) Bone marrow macrophages were transfected with 3.3 µg/ml of the indicated molecules (or overlaid with 100 ng/ml LPS), and type I IFN was assessed after 6 h by bioassay as in A. *, P < 0.001 as compared with unstimulated cells (Student's t test). (C) Bone marrow macrophages were transfected with 3.3 µg/ml of the indicated molecules (or overlaid with 100 ng/ml LPS), and transcription of the IFN- gene (Ifnb) was assessed by quantitative RT-PCR, with normalization to ribosomal protein rps17 message. n.d., not detectably induced above background (lipofectamine alone). *, P < 0.001 as compared with unstimulated cells (Student's t test). (D) As in C, but transcription of the IFN-5 gene was assessed. n.d., not detectably induced above background (lipofectamine alone). *, P < 0.01 as compared with unstimulated cells (Student's t test). (E) c-di-GMP, treated or untreated with snake venom phosphodiesterase, was transfected at 3.3 µg/ml into B6 bone marrow macrophages and analyzed after 6 h for IFN- production by bioassay, as in A. *, P < 0.02 as compared with c-di-GMP treatment alone (Student's t test). Results in A-E are representative of at least three independent experiments. Data are means ± SD (n = 3). (F) Bone marrow macrophages were transfected with poly dA:dT (DNA) or with c-di-GMP. Total RNA was isolated after 6 h of stimulation. Probes were amplified and hybridized to whole-transcriptome spotted MEEBO microarrays. Each dot represents a single gene, and its position is determined by its induction in response to DNA (x axis) or c-di-GMP (y axis). Most genes lie on the diagonal, indicative of similar induction ratios by both treatments. Selected highly induced genes are labeled. Two independent microarray experiments gave similar results.