24: STING, not type I IFN signaling, is required for the mucosal adjuvant activity of Cyclic-di-GMP in vivo

Abstract

Mucosal vaccination prevents the onset of disease, blocks early colonization, and reduces the risk of horizontal transmission. Currently, there is no vaccine formulation containing a mucosal adjuvant approved for human use. Cyclic-di-GMP (CDG) is a small molecule found only in bacteria, regulating bacterial growth and virulence. CDG exhibits strong immunostimulatory properties in mammalian cells. Karaolis et al. first discovered that CDG activates human immature Dendritic cells (DC), and enhancing DC’s ability to stimulate T cells. Importantly, intranasal co-immunization of CDG and antigens protects host from pathogen infections, including H5N1 influenza, and Streptococcus pneumonia infections in mice. Thus, CDG is a promising mucosal adjuvant candidate. The mechanism by which CDG acts as a mucosal adjuvant is unknown. STING (stimulator of interferon genes) has been identified as a ligand for CDG in mammalian cells activating TBK1-IRF3-type I IFN signaling. Here, using a STING-/- mouse (Tmem173), we investigated the in vivo role of STING in the mucosal adjuvant activity of CDG. We found that STING-/- mice fail to generate antigen-specific antibody response after intranasal immunization of an antigen and CDG. Furthermore, the productions of Th1/Th2/Th17 cytokines, proinflammatory cytokines and type I IFN are all missing in CDG immunized STING-/- mice. Surprisingly, we found that type I IFN signaling is not required for the mucosal adjuvant activity of CDG because IFNAR1-/- mice have the same antigen-specific antibody response as the wild-type mice. We further found that CDG activates STING-dependent but TBK1-type I IFN signaling-independent proinflammatory cytokine production in macrophages and DCs. This is distinct from STING-mediated DNA vaccine adjuvant activity, which requires type I IFN signaling. Thus, our results establish an essential role of STING in mediating the mucosal adjuvant activity of CDG in vivo and reveal novel TBK1-type I IFN stimulation-independent function of STING.