We also tested whether transfected c-di-GMP is capable of activating NF-B. This was particularly important to establish in light of a previous report that exogenous (untransfected) c-di-GMP failed to activate NF-B (Karaolis et al., 2007a) . There have also been inconsistent reports in the literature as to whether cytosolic DNA induces NF-B Stetson and Medzhitov, 2006a; Sun et al., 2006) . We found that c-di-GMP treatment led to the activation of NF-B, as assessed by using an electromobility gel shift assay, with similar kinetics to that of transfected pI:C and pdA:dT ( Fig. 3 B) . The probe-bound complex was confirmed to contain NF-B p65 by supershifting with an anti-p65 antibody ( Fig. 3 B) . We confirmed these results by using a RAW role for Irf 7 signaling as part of a positive feedback loop involving signaling through the type I IFN receptor (Ifnar). Indeed, Ifnar / macrophages exhibited a diminished response to c-di-GMP (Fig. 2 E) . Irf5 and Irf1 did not seem to be required for responsiveness to c-di-GMP (Fig. 2 F) . In addition, macrophages deficient in Rip2, Nalp3, or doubly deficient in Nod1/2 responded normally to c-di-GMP (unpublished data).