To validate our findings in vivo, we injected mice intraperitoneally with c-di-GMP (200 nmol per mouse) and measured the production of type I IFNs in the blood 18 h later. B6 mice injected with c-di-GMP produced an IFN response to c-di-GMP, and this response was abolished in Irf3/7 double-deficient mice (Fig. 6 A) . Interestingly, single-deficient Irf3 / mice responded to c-di-GMP in vivo (unpublished data), implying a role for IRF7 in responses to c-di-GMP in vivo. To determine whether cytokine induction by c-di-GMP affected cellular responses in vivo, we collected splenic NK cells from mice injected with c-di-GMP Figure 5 . c-di-GMP does not stimulate known cytosolic pathways for sensing nucleic acids. (A) Mavs / and littermate wild-type macrophages were transfected with 3.3 µg/ml c-di-GMP, poly dA:dT (pdA:dT, DNA), or poly I:C (pI:C, RNA), and transcription of the IFN- gene (Ifnb) was assessed by quantitative RT-PCR, with normalization to ribosomal protein rps17 message. n.d., not detectable. (B) Mda5 / and wild-type control macrophages were stimulated and analyzed as in A. (C) Zbp1 / (DAI knockout) and wild-type control macrophages were stimulated and analyzed as in A. SeV, Sendai virus. Results are representative of at least three independent experiments. Data are means ± SD (n = 3). *, P < 0.01; and **, P < 0.001 as compared with wild-type bone marrow macrophages (Student's t test) .