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. 2015 Oct 20;112(42):E5699-705.
doi: 10.1073/pnas.1516465112. Epub 2015 Sep 14.

Activation of cyclic GMP-AMP synthase by self-DNA causes autoimmune diseases

Daxing Gao  1 , Tuo Li  1 , Xiao-Dong Li  1 , Xiang Chen  2 , Quan-Zhen Li  3 , Mary Wight-Carter  4 , Zhijian J Chen  5
Affiliations

Activation of cyclic GMP-AMP synthase by self-DNA causes autoimmune diseases

Daxing Gao et al. Proc Natl Acad Sci U S A. .

Abstract

TREX1 is an exonuclease that digests DNA in the cytoplasm. Loss-of-function mutations of TREX1 are linked to Aicardi-Goutieres Syndrome (AGS) and systemic lupus erythematosus (SLE) in humans. Trex1(-/-) mice exhibit autoimmune and inflammatory phenotypes that are associated with elevated expression of interferon (IFN)-induced genes (ISGs). Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that activates the IFN pathway. Upon binding to DNA, cGAS is activated to catalyze the synthesis of cGAMP, which functions as a second messenger that binds and activates the adaptor protein STING to induce IFNs and other cytokines. Here we show that genetic ablation of cGas in Trex1(-/-) mice eliminated all detectable pathological and molecular phenotypes, including ISG induction, autoantibody production, aberrant T-cell activation, and lethality. Even deletion of just one allele of cGas largely rescued the phenotypes of Trex1(-/-) mice. Similarly, deletion of cGas in mice lacking DNaseII, a lysosomal enzyme that digests DNA, rescued the lethal autoimmune phenotypes of the DNaseII(-/-) mice. Through quantitative mass spectrometry, we found that cGAMP accumulated in mouse tissues deficient in Trex1 or DNaseII and that this accumulation was dependent on cGAS. These results demonstrate that cGAS activation causes the autoimmune diseases in Trex1(-/-) and DNaseII(-/-) mice and suggest that inhibition of cGAS may lead to prevention and treatment of some human autoimmune diseases caused by self-DNA.

Keywords: DNaseII; Trex1; autoimmune disease; cGAMP; cGAS.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
cGAS deletion rescues the lethality of Trex1−/− mice. Survival curves of Trex1−/−, Trex1−/−cGas+/−, and Trex1−/−cGas−/− mice are shown. All mice were on a C57BL/6 background. Statistical analysis was performed with a Mantel–Cox test.
Fig. 2.
Fig. 2.
cGAS mediates multiorgan inflammation in Trex1−/− mice. (A and C) Representative H&E-stained heart (A) and skeletal muscle (C) sections from 12-wk-old Trex1−/−, Trex1−/−cGas+/−, and Trex1−/−cGas−/− mice. Blue-stained cells indicate leukocytes that infiltrate the heart. (B and D) Blinded analysis of the indicated tissues of 12-wk-old Trex1−/−, Trex1−/−cGas+/−, and Trex1−/−cGas−/− mice as well as Trex1+/− mice. Histological scores were calculated as described in Materials and Methods. Statistical analysis was performed with a two-tailed, unpaired Student's t test. ***P < 0.001.
Fig. S1.
Fig. S1.
cGAS is responsible for the pathology and splenomegaly of Trex1−/− mice. (A) Representative Picro-Sirius Red (PSR; to measure fibrosis)-stained heart sections from mice of indicated genotypes. (B) Representative H&E-stained skin sections. (C) Blinded analysis of the skin tissues represented in B. (D) Representative H&E-stained (Upper; ×10) and Periodic Acid Schiff (PAS; to detect glycogen) stained (Lower; ×10) kidney sections. (E) Blinded analysis of the kidney tissues represented in D. (F) Spleens from 12-wk-old Trex1−/− and Trex1−/−cGas−/− mice. (G and H) Spleen cell numbers (G) and spleen weights (H) were measured from 12-wk-old Trex1−/− and Trex1−/−cGas−/− mice. Statistical analysis was performed with a two-tailed, unpaired Student's t test. *P < 0.05; ***P < 0.001.
Fig. 3.
Fig. 3.
cGAS is essential for the expression of ISGs and inflammatory cytokines as well as cGAMP overproduction in Trex1−/− mice. (A–E) qRT-PCR analysis of indicated ISGs (A–C), TNF-α (D), and IL12p40 (E) in hearts from 12-wk-old mice of indicated genotypes. Fold changes are relative to Trex1−/−cGas−/−mice. Error bars represent SD. *P < 0.05; **P < 0.01; ***P < 0.001. (F and G) cGAMP levels in mouse hearts of indicated genotypes were quantified by LC-MS. Upper shows zoomed chromatograms displaying relative intensities of endogenous cGAMP (red) and internal standard (green). Lower shows the calculated amounts of cGAMP (fmol) in each sample. Error bars represent SEM.
Fig. S2.
Fig. S2.
cGAS is essential for ISG expression in Trex1−/− mice. (A) qRT-PCR analysis of indicated ISGs, TNF-α, and IL12p40 in hearts from 12-wk-old mice of indicated genotypes. Fold changes are relative to Trex+/− mice. (B) qRT-PCR analysis of indicated ISGs, TNF-α, and IL12p40 in kidneys from 12-wk-old mice. Fold changes are relative to Trex1−/−cGas−/−mice. (C) qRT-PCR analysis of indicated ISGs in PBMCs from 5-wk-old mice. Fold changes are relative to Trex1−/−cGas−/−mice. (D) Measurement of IL-12 (total) in the plasma of 12-wk-old mice. Error bars represent SD. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. S3.
Fig. S3.
cGAS is required for ISG induction in different cells types from Trex1−/− mice. GM-CSF–derived BMDCs (A), BMDMs (B), and mouse embryonic fibroblasts (MEF; C) from indicated genotypes were analyzed by qRT-PCR for expression of ISGs. Fold changes are relative to Trex1−/−cGas−/−mice. Error bars represent SD of triplicate assays. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 4.
Fig. 4.
cGAS deletion mitigates autoimmunity in Trex1−/− mice. (A–C) Detection of autoantibodies, including ssDNA IgG (A), ssDNA IgG2b subtype (B), and ANA IgG (C) in the sera of 12-wk-old mice of indicated genotypes. (D and E) Flow-cytometric analysis of intracellular IFN-γ in response to PMA plus ionomycin in splenic CD4+ T cells (D) and CD8+ T cells (E) from 12-wk-old Trex1−/− and Trex1−/−cGas−/− mice. (F and G) Flow-cytometric analysis of CD44hiCD62lo cells in splenic CD4+ T cells (F) and CD8+ T cells (G) from 12-wk-old Trex1−/− and Trex1−/−cGas−/− mice. Error bars represent SD. **P < 0.01; ***P < 0.001.
Fig. S4.
Fig. S4.
cGAS is required for the generation of autoantibodies in Trex1−/− mice. Measurements of the indicated autoantibodies in the sera of 12-wk-old (A–C) and 5-mo-old (D–I) mice are shown. Error bars represent SD. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. S5.
Fig. S5.
cGAS is required for the generation of hyperreactive T cells in Trex1−/− mice. (A–C) Flow-cytometric analysis of intracellular IFN-γ in response to PMA plus ionomycin in splenic CD4+ T cells or CD8+ T cells from 12-wk-old mice of indicated genotypes. (D) Similar to C, except that CD8 T cells in PBMC were analyzed. (E and F) Flow-cytometric analysis of T-bet in splenic CD4 (E) and CD8 (F) T cells from 12-wk-old mice. Error bars represent SD. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. S6.
Fig. S6.
cGAS is essential for infiltration of autoreactive immune cells in the hearts of Trex1−/− mice. qRT-PCR analysis of IFN-γ (A and B) or T-bet (C and D) RNA in hearts from 12-wk-old mice of indicated genotypes is shown. Fold changes are relative to Trex1+/− (A and C) or Trex1−/−cGas−/− (B and D). Error bars represent SD. **P < 0.01; ***P < 0.001.
Fig. S7.
Fig. S7.
Trex1−/− mice have higher percentages of activated and memory T cells. (A and B) Flow-cytometric analysis of surface expression of CD62L and CD44 in splenic CD4+ T cells (A) and CD8+ T cells (B) from 12-wk-old Trex1+/− and Trex1−/− mice. (C–E) Flow-cytometric analysis of CD69 (C) and Ly6c (D) in splenic CD8+ T cells and CD69 (E) in splenic CD4+ T cells from 12-wk-old mice. Error bars represent SD. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. S8.
Fig. S8.
cGAS is responsible for higher percentages of activated and memory T cells in Trex1−/− mice. (A and B) Representative flow-cytometric analysis of CD62L and CD44 expression in splenic CD4+ (A) and CD8+ T cells (B) from 12-wk-old mice of indicated genotypes. (C–E) Flow-cytometric analysis of expression of CD69 (C) and Ly6c (D) in splenic CD8+ T cells and CD69 (E) in splenic CD4+ T cells. Error bars represent SD. **P < 0.01; ***P < 0.001.
Fig. 5.
Fig. 5.
cGAS activation causes polyarthritis in DNaseII−/−Ifnar1−/− mice. (A and B) Representative foot pad pictures (A) and arthritis scores (B) of 7-mo-old WT, DNaseII−/−Ifnar1−/−, and DNaseII−/−cGAS−/− mice. Arthritis scores were calculated as described in Materials and Methods. (C) Detection of serum autoantibodies against dsDNA. Statistical analysis was performed with a two-tailed, unpaired Student's t test. Error bars represent SD. *P < 0.05; ***P < 0.001.
Fig. S9.
Fig. S9.
cGAS is responsible for embryonic lethality and ISG expression in DNaseII−/− mice. (A) Representative E17.5 embryos of indicated genotypes. (B and C) qRT-PCR analysis of indicated ISGs in E15.5 embryo limbs (B) and embryonic fibroblasts (C). Error bars represent SD *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 6.
Fig. 6.
cGAS is essential for ISG up-regulation and cGAMP production in fetal livers of DNaseII−/− mice. (A) qRT-PCR analysis of indicated ISGs in fetal liver from E15.5 mouse embryos of indicated genotypes (n = 3). (B) cGAMP levels in mouse fetal livers of indicated genotypes were quantified by LC-MS. Upper shows zoomed chromatograms displaying relative intensities of endogenous cGAMP (red) and internal standard (green). Lower shows the calculated amounts of cGAMP in each sample. Error bars represent SD (A) or SEM (B). *P < 0.05; **P < 0.01.
See this image and copyright information in PMC

Comment in

  • cGMP-AMP synthase paves the way to autoimmunity.
    Rodero MP, Crow YJ. Rodero MP, et al. Proc Natl Acad Sci U S A. 2015 Oct 20;112(42):12903-4. doi: 10.1073/pnas.1517578112. Epub 2015 Oct 8. Proc Natl Acad Sci U S A. 2015. PMID: 26450878 Free PMC article. No abstract available.

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References

    1. Pandey S, Kawai T, Akira S. Microbial sensing by Toll-like receptors and intracellular nucleic acid sensors. Cold Spring Harb Perspect Biol. 2015;7(1):a016246. - PMC - PubMed
    1. Wu J, Chen ZJ. Innate immune sensing and signaling of cytosolic nucleic acids. Annu Rev Immunol. 2014;32:461–488. - PubMed
    1. Goubau D, Deddouche S, Reis e Sousa C. Cytosolic sensing of viruses. Immunity. 2013;38(5):855–869. - PMC - PubMed
    1. Sun L, Wu J, Du F, Chen X, Chen ZJ. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science. 2013;339(6121):786–791. - PMC - PubMed
    1. Yoneyama M, et al. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol. 2004;5(7):730–737. - PubMed

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