Cutting Edge: cGAS Is Required for Lethal Autoimmune Disease in the Trex1-Deficient Mouse Model of Aicardi-Goutières Syndrome

Abstract

Detection of intracellular DNA triggers activation of the stimulator of IFN genes-dependent IFN-stimulatory DNA (ISD) pathway, which is essential for antiviral immune responses. However, chronic activation of this pathway is implicated in autoimmunity. Mutations in TREX1, a 3' repair exonuclease that degrades cytosolic DNA, cause Aicardi-Goutières syndrome and chilblain lupus. Trex1 (-/-) mice develop lethal, IFN-driven autoimmune disease that is dependent on activation of the ISD pathway, but the DNA sensors that detect the endogenous DNA that accumulates in Trex1 (-/-) mice have not been defined. Multiple DNA sensors have been proposed to activate the ISD pathway, including cyclic GMP-AMP synthase (cGAS). In this study, we show that Trex1 (-/-) mice lacking cGAS are completely protected from lethality, exhibit dramatically reduced tissue inflammation, and fail to develop autoantibodies. These findings implicate cGAS as a key driver of autoimmune disease and suggest that cGAS inhibitors may be useful therapeutics for Aicardi-Goutières syndrome and related autoimmune diseases.

Figure 1. cGAS is required for lethal autoimmune disease in Trex1-deficient mice

Survival curves of cGAS^+/+Trex1^−/− (n=33), cGAS^+/−Trex1^−/− (n=44), and cGAS^−/−Trex1^−/− (n=29) mice. Both cGAS-deficient and Trex1-deficient mice were on a pure C57BL/6 background. Statistical analysis was performed with a Log-rank (Mantel-Cox) test. *p < 0.0001.

Figure 2. cGAS drives tissue inflammation in Trex1-deficient mice

(A) Representative H&E-stained heart, skin, and glandular stomach tissue sections from cGAS^+/+Trex1^−/− and cGAS^−/−Trex1^−/− mice (20X original magnification). (B) Histological scores of inflammation in the indicated tissues from cGAS^+/+Trex1^−/−, cGAS^+/−Trex1^−/−, cGAS^−/−Trex1^−/− and cGAS^−/−Trex1^+/+ mice. All histological analysis was performed in a blinded manner. Data are representative of at least four mice of each genotype. Statistical analysis was performed comparing cGAS^−/−Trex1^−/− to cGAS^+/+Trex1^−/− and cGAS^−/−Trex1^+/+ mice using a one-way ANOVA with Tukey’s multiple comparison posttest. *p < 0.05, **p < 0.005, ****p < 0.0001.

Figure 3. cGAS-dependent autoantibodies in Trex1-deficient mice

(A) Autoantibodies against heart antigens evaluated by blotting neat and 1:5 diluted heart extracts from Rag2^−/−Trex1^−/− mice with sera from mice of the indicated genotype. Immunoblots were prepared with sera from different mice harvested at the age indicated, rather than with sera harvested repeatedly from a single mouse. Data are representative of two independent experiments. (B) Antinuclear antibodies (ANA) in sera from littermate cGAS^+/+Trex1^−/−, cGAS^+/−Trex1^−/−, and cGAS^−/−Trex1^−/− mice (left panels) analyzed by immunofluorescence staining of HEp-2 cell-coated slides. ANA in serum from cGAS^−/−Trex1^+/− mice was analyzed as a control (right panel). Data are representative of three independent experiments with a total of at least three mice of each genotype.

Figure 4. cGAS promotes type I IFN production in Trex1-deficient mice

(A) Quantification by RT-PCR of a panel of IFN-stimulated gene transcripts in total peripheral blood cells from cGAS^+/+Trex1^−/− and cGAS^−/−Trex1^−/− mice relative Trex1^+/+ mice. Data are representative of one independent experiment with at least four mice of each genotype. (B) Analysis of cGAMP production in whole heart extracts from cGAS^+/+Trex1^−/−, cGAS^−/−Trex1^−/−, and Trex1^+/+ mice by LC-MS/MS. Data are plotted relative to the internal standard (cGAMP*). Data are representative of one independent experiment with at least five mice of each genotype. Statistical analysis was performed with a Mann-Whitney test. *p < 0.05.