We also tested whether MAP kinase pathways are activated by c-di-GMP. A previous report found that overlay of c-di-GMP induced ERK but not p38 in human macrophages (Karaolis et al., 2007a) . In contrast, we found that cytosolic c-di-GMP stimulated p38, ERK1/2, and JNK phosphorylation in mouse bone marrow macrophages transfected with c-di-GMP (Fig. 3 D) . Although a difference between human and mouse macrophages may explain the Data are means ± SD (n = 3). *, P < 0.05 compared with unstimulated cells (Student's t test) . tion, and is downstream of two cytosolic viral RNA sensors, RIG-I and Mda5 (Sun et al., 2006) . To test if c-di-GMP signals through the cytosolic RNA-sensing pathway, we tested c-di-GMP responses in Mavs / and Mda5 / macrophages. Loss of Mavs or Mda5 did not affect the induction of IFN- by c-di-GMP (Fig. 5, A and B; and Fig. S1 C) , suggesting that c-di-GMP does not signal through the cytosolic RNA signaling apparatus (Gitlin et al., 2006; Kato et al., 2008; . DAI (encoded by the Zpb1 gene) is a recently identified molecule that has been proposed to function as a sensor of cytosolic DNA (Takaoka et al., 2007; Wang et al., 2008) . We found that bone marrow-derived macrophages from Zbp1deficient mice (Ishii et al., 2008) still responded to c-di-GMP (Fig. 5 C) . However, as previously reported (Ishii et al., 2008) , Zbp1-deficient macrophages also responded normally to DNA (Fig. 5 C) . These results imply that macrophages express at least one cytosolic DNA sensor that is distinct from In addition to bone marrow macrophages, we observed induction of IFN- by cytosolic c-di-GMP in a variety of other cell types, including peritoneal macrophages, conventional dendritic cells, L929 cells, and RAW 264.7 macrophages (Fig. 4, A-D) . These cells also responded to transfected DNA and RNA. Interestingly, however, we were unable to detect significant induction of type I IFNs by c-di-GMP in MEFs or in 293T cells, even though the cytosolic pathways for detecting DNA and RNA are intact in these cells (Fig. 4 , E and F). These results suggest that at least one component of the host signaling pathway responding to c-di-GMP is distinct from that used for responses to cytosolic RNA or DNA, and is differentially expressed in different cell types.