Cyclic dinucleotides act as intracellular second messengers, modulating a variety of cellular activities including innate immune activation. Although phosphodiesterases (PDEs) hydrolyzing c-di-GMP and c-di-AMP have been identified, no PDEs for cGAMPs have been reported. Here we identified the first three cGAMP-specific PDEs in V. cholerae (herein designated as V-cGAP1/2/3). V-cGAPs are HD-GYP domain-containing proteins and specifically break 3′3′-cGAMP, but not other forms of cGAMP. 3′3′-cGAMP is first linearized by all three V-cGAPs to produce 5′-pApG, which is further hydrolyzed into 5′-ApG by V-cGAP1. In this two-step reaction, V-cGAP1 functions as both a PDE and a 5′-nucleotidase. In vivo experiments demonstrated that V-cGAPs play non-redundant roles in cGAMP degradation. The high specificity of V-cGAPs on 3′3′-cGAMP suggests the existence of specific PDEs for other cGAMPs, including 2′3′-cGAMP in mammalian cells. The absolute requirement of the GYP motif for 3′3′-cGAMP degradation suggests that HD domain-containing PDEs in eukaryotes are probably unable to hydrolyze cGAMPs. The fact that all V-cGAPs attack 3′3′-cGAMP on one specific phosphodiester bond suggests that PDEs for other cGAMPs would utilize a similar strategy. These results will provide valuable information for identification and characterization of mammalian 2′3′-cGAMP-specific PDEs in future studies.

Dysregulation of immune responses in the gut often associates with inflammatory bowel diseases (IBD). Mouse CD1d1, an ortholog of human CD1d mainly participating in lipid-antigen presentation to NKT cells, is able to generate intrinsic signals upon stimulation. Mice with macrophage-specific CD1d1 deficiency (LymCD1d1−/−) acquire resistance to dextran sodium sulfate (DSS)–induced colitis, attributing to the transcriptional inhibition of NLRP3 inflammasome components. The hyperactivation of NLRP3 inflammasome accounts for gut epithelial proliferation and intestine-blood barrier integrity. Mechanistically, occupancy by the natural ligand glycosphingolipid iGb3, CD1d1 responds with intracellular Ser330 dephosphorylation thus to reduce the Peroxiredoxin 1 (PRDX1)–associated AKT-STAT1 phosphorylation and subsequent NF-κB activation, eventually causing transcriptional down-regulation of Nlrp3 and its immediate substrates Il1b and Il18 in macrophages. Therefore, the counterbalancing role of CD1d1 in macrophages appears to determine severity of DSS-mediated colitis in mice. These findings propose new intervention strategies for treating IBD and other inflammatory disorders.