Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects

Abstract

Invasion of microbial DNA into the cytoplasm of animal cells triggers a cascade of host immune reactions that help clear the infection; however, self DNA in the cytoplasm can cause autoimmune diseases. Biochemical approaches led to the identification of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) as a cytosolic DNA sensor that triggers innate immune responses. Here, we show that cells from cGAS-deficient (cGas(-/-)) mice, including fibroblasts, macrophages, and dendritic cells, failed to produce type I interferons and other cytokines in response to DNA transfection or DNA virus infection. cGas(-/-) mice were more susceptible to lethal infection with herpes simplex virus 1 (HSV1) than wild-type mice. We also show that cGAMP is an adjuvant that boosts antigen-specific T cell activation and antibody production in mice.

Figure 1. cGAS is essential for IRF3 activation and IFN-β induction by DNA transfection and DNA virus infection in fibroblasts and macrophages

(A) Mouse lung fibroblasts were transfected with different forms of DNA or poly[I:C] (2 µg/ml) for 24 h followed by measurement of IFN-β protein by ELISA. Unless indicated otherwise, lipofectamine 2000 was used in all transfection experiments. (B) Lung fibroblasts were infected with the indicated viruses or stimulated with 2’3’cGAMP (200 nM) for 9 h followed by measurement of IFN-β RNA by q-RT-PCR. (C) Lung fibroblasts were transfected with HT-DNA or infected with HSV1 or Sendai virus for the indicated times. Cell extracts were analyzed for IRF3 dimerization by native gel electrophoresis. (D) BMDMs were transfected with HT-DNA, ISD or infected with the indicated virus for 20 h, then IFN-β levels were measured by ELISA. For the q-RT-PCR results in this and other figures, the error bars represent standard deviations of triplicate measurements. Unless otherwise indicated, the ELISA results in this and other figures represent variation ranges of duplicate measurements. ‘ND’ in this and other figures indicates ‘not detected’.

Figure 2. cGAS is essential for type-I interferon induction by DNA transfection and DNA virus infection in dendritic cells

(A) GM-CSF-induced dendritic cells (GM-CSF DCs) were transfected with HT-DNA or ISD or infected with Sendai virus for 20 h followed by measurement of IFN-α by ELISA. (B) GM-CSF DCs were transfected with the indicated DNA or viruses for 20 h, and then IFN-β was measured by ELISA. (C & D) Flt3L-induced dendritic cells (Flt3L-DCs) were incubated with the indicated DNA for 16 h in the absence or presence of lipofectamine 2000 (C), or infected with HSV1 or Sendai virus (D) for 16 h, followed by measurement of IFN-α by ELISA. Lipo: lipofectamine 2000.

Figure 3. cGAS is essential for immune defense against HSV1 infection in vivo

(A & B) WT and cGas^−/− mice (n=5 each) were infected i.v. with HSV1 at 1× 10⁷ pfu per mouse, and then sera were collected at different time points as indicated. The levels of IFNα and IFNβ were measured by ELISA. The error bars indicate standard deviations. (C) WT and cGas^−/− mice (n=5 each) were infected i.v. with HSV1 at 1×10⁶ pfu per mouse. The survival of each mouse was monitored for 7 days. (D) Brains of the HSV1 infected mice were excised on day 3 to prepare homogenates, which were used to measure viral titers by the plaque assay. The viral titer was recorded as pfu per gram brain (pfu/g). The experiments shown in C and D were repeated in an independent set of experiments, except that the HSV1 infectious dose was increased to 1×10⁷ pfu per mouse (fig. S5A and S5B).

Figure 4. cGAMP is an adjuvant that stimulates T cell activation and antibody production in a STING-dependent manner

(A) WT and Sting^gt/gt mice were injected intramuscularly with OVA alone or OVA + 2’3’cGAMP on day 1 and day 10. Sera were collected at the indicated time points to measure OVA-specific IgG1 by ELISA. The error bar indicates SEM. Data is representative of three independent experiments, each with 5 mice per group. (B & C) WT mice were immunized with OVA or OVA + 2’3’cGAMP as above. Spleen leukocytes were collected on day 7, and stimulated with OVA peptides known to complex with MHC class I (H-2K^b) or class II (I-A^b). Secretion of IFNγ (B) and IL-2 (C) by T cells was measured by ELISA. *P < 0.05, **P < 0.01, and ***P < 0.001; Student’s t-test. (D) Similar to (B), except that FACS analysis was performed to measure the percentage of CD8 T cells bearing the T cell receptor specific for an OVA-H-2K^b tetramer. *P < 0.05, Student’s t-test. Data shown in (B–D) are representative of two independent experiments, each with 3 mice per group.