The mucosal adjuvant cyclic di-GMP enhances antigen uptake and selectively activates pinocytosis-efficient cells in vivo.

Effective mucosal adjuvants enhance the magnitude and quality of the vaccine response. Cyclic di-GMP (CDG) is a promising mucosal vaccine adjuvant. However, its in vivo mechanisms are unclear. Here, we showed, in mice, that CDG elicits stronger Ab and TH responses than the mammalian 2'3'-cyclic GMP-AMP (cGAMP), and generated better protection against Streptococcus pneumoniae infection than 2'3'-cGAMP adjuvanted vaccine. We identified two in vivo mechanisms of CDG. First, intranasally administered CDG greatly enhances Ag uptake, including pinocytosis and receptor-mediated endocytosis in vivo. The enhancement depends on MPYS (STING, MITA) expression in CD11C(+) cells. Second, we found that CDG selectively activated pinocytosis-efficient-DCs, leading to T(H) polarizing cytokines IL-12p70, IFNγ, IL-5, IL-13, IL-23, and IL-6 production in vivo. Notably, CDG induces IFNλ, but not IFNß, in vivo. Our study revealed previously unrecognized in vivo functions of MPYS and advanced our understanding of CDG as a mucosal vaccine adjuvant.

Deletion of the cyclic di-AMP phosphodiesterase gene (cnpB) in Mycobacterium tuberculosis leads to reduced virulence in a mouse model of infection.

Tuberculosis (TB) remains a major cause of morbidity and mortality worldwide. The pathogenesis by the causative agent, Mycobacterium tuberculosis, is still not fully understood. We have previously reported that M. tuberculosis Rv3586 (disA) encodes a diadenylate cyclase, which converts ATP to cyclic di-AMP (c-di-AMP). In this study, we demonstrated that a protein encoded by Rv2837c (cnpB) possesses c-di-AMP phosphodiesterase activity and cleaves c-di-AMP exclusively to AMP. Our results showed that in M. tuberculosis, deletion of disA abolished bacterial c-di-AMP production, whereas deletion of cnpB significantly enhanced the bacterial c-di-AMP accumulation and secretion. The c-di-AMP levels in both mutants could be corrected by expressing the respective gene. We also found that macrophages infected with ΔcnpB secreted much higher levels of IFN-ß than those infected with the wild type (WT) or the complemented mutant. Interestingly, mice infected with M. tuberculosis ΔcnpB displayed significantly reduced inflammation, less bacterial burden in the lungs and spleens, and extended survival compared with those infected with the WT or the complemented mutant. These results indicate that deletion of cnpB results in attenuated virulence, which is correlated with elevated c-di-AMP levels.

MPYS/STING-mediated TNF-α, not type I IFN, is essential for the mucosal adjuvant activity of (3'-5')-cyclic-di-guanosine-monophosphate in vivo.

The bacterial second messenger (3'-5')-cyclic-di-guanosine-monophosphate (CDG) is a promising mucosal adjuvant candidate that activates balanced Th1/Th2/Th17 responses. We showed previously that CDG activates stimulator of IFN genes (STING)-dependent IFN-I production in vitro. However, it is unknown whether STING or IFN-I is required for the CDG adjuvant activity in vivo. In this study, we show that STING(-/-) mice (Tmem173()) do not produce Ag-specific Abs or Th1/Th2/Th17 cytokines during CDG/Ag immunization. Intranasal administration of CDG did not induce TNF-α, IL-1ß, IL-6, IL-12, or MCP-1 production in STING(-/-) mice. Surprisingly, we found that the cytokine and Ab responses were unaltered in CDG/Ag-immunized IFNAR(-/-) mice. Instead, we found that CDG activates STING-dependent, IFN-I-independent TNF-α production in vivo and in vitro. Furthermore, using a TNFR1(-/-) mouse, we demonstrate that TNF-α signaling is critical for CDG-induced Ag-specific Ab and Th1/Th2 cytokine production. This is distinct from STING-mediated DNA adjuvant activity, which requires IFN-I, but not TNF-α, production. Finally, we found that CDG activates STING-dependent, but IRF3 stimulation-independent, NF-κB signaling. Our results established an essential role for STING-mediated TNF-α production in the mucosal adjuvant activity of CDG in vivo and revealed a novel IFN-I stimulation-independent STING-NF-κB-TNF-α pathway.