STING manifests self DNA-dependent inflammatory disease

Abstract

Inflammatory autoimmune diseases such as systemic lupus erythematosus (SLE) and polyarthritis are characterized by chronic cytokine overproduction, suggesting that the stimulation of host innate immune responses, speculatively by persistent infection or self nucleic acids, plays a role in the manifestation of these disorders. Mice lacking DNase II die during embryonic development through comparable inflammatory disease because phagocytosed DNA from apoptotic cells cannot be adequately digested and intracellular host DNA sensor pathways are engaged, resulting in the production of a variety of cytokines including type I IFN. The cellular sensor pathway(s) responsible for triggering DNA-mediated inflammation aggravated autoimmune disease remains to be determined. However, we report here that Stimulator of IFN Genes (STING) is responsible for inflammation-related embryonic death in DNase II defective mice initiated by self DNA. DNase II-dependent embryonic lethality was rescued by loss of STING function, and polyarthritis completely prevented because cytosolic DNA failed to robustly trigger cytokine production through STING-controlled signaling pathways. Our data provides significant molecular insight into the causes of DNA-mediated inflammatory disorders and affords a target that could plausibly be therapeutically controlled to help prevent such diseases.

Fig. 1.

Role of STING in facilitating cytokines production by apoptotic DNA. (A) DNA from thymocytes treated with or without Dex (10 μM, 5 h). (B) STING expression as analyzed by immunoblot and IFNβ ELISA of Sting^+/+ and Sting^−/− BMDM treated with dsDNA90 or aDNA. ND, nondetectable. (C) Confocal analysis of BMDMs transfected with aDNA and immunostained with anti-STING antibody. (Magnification: 1260×.) (D) Microarray of Sting^+/+ or Sting^−/− BMDM transfected with aDNA (6 h). (E) ELISA of IL-β, IL-6, and TNFα in BMDMs treated with or without aDNA. (F) Cxcl10 mRNA level levels in BMDMs treated with STING and/or DNase II siRNA after exposure for 6 h to apoptotic thymocytes (aCell). Cxcl10 mRNA level was normalized to the expression of GAPDH (Left). *P < 0.05, **P < 0.005 by Student’s t test.

Fig. 2.

STING-dependent gene regulation in E17 Sting^−/− DNase II^−/−embryos. (A) STING and DNase II expression by qPCR and Western blot from E17 fetal liver wild-type (eWT), Sting^−/− DNase II^+/+(eStingKO), Sting^+/− DNase II^−/− (eDNaseIIKO), and Sting^−/− DNase II^−/− (eDoubleKO) embryos. (B) Macroscopic view of E17 Sting^+/− DNase II^+/−(Left), eDNaseIIKO (Center), and eDoubleKO (Right) embryos. (C) H&E and TUNEL stains from mice livers same as A. Feulgen stain of BMDM after incubation with thymocytes (+) Dex. (Magnification: 200×.)

Fig. 3.

STING-dependent gene regulation in E17 Sting^−/− DNase II^−/− embryos. (A) Gene array analysis of fetal liver RNA isolated from E17 mice same as Fig. 2A. Data are from the mean of three independent embryos. (B) qPCR analysis of Ifnβ1, Irf7, Ifit3, and Ccl5 mRNA level from RNA same as (A). Error bars indicate SD. *P < 0.05, ***P < 0.0005 by Student’s t test. (C) Immunoblot analysis to determine the levels of p-Iκb and p-p65 in MEFs from Sting^+/+DNase II^+/+ (WT), Sting^−/− DNase II^+/+ (SKO), and Sting^−/− DNase II^−/− (DKO) treated with dsDNA90 and ssDNA90 for 3 h. M, mock. (D) Confocal analysis of MEFs isolated from WT and E17 DKO embryos transfected with dsDNA90 for 3 h and stained with anti–NF-κB and IRF-3 antibody. (Magnification: 1260×.)

Fig. 4.

Generation and characterization of Sting^−/− DNase II^−/− adult mice. (A) Mendelian analysis of the progeny DKO-deficient mice. Northern blotting with Sting and DNaseII cDNA as probe from liver of 8-wk-old Sting^+/+DNase II^+/+ (WT), and DKO mice. (B) Feulgen stain of peritoneal macrophages stained with Schiff reagent after incubation with thymocytes treated with or without Dex (10 μM, 5 h). (C) Macroscopic view of the normal fore and hind paws of DKO at the age of 12 mo. (D) H&E stain of joint sections from 6-mo-old WT and DKO mice. B, bone; C, cartilage; S; synovial. (Magnification: 200×.) (E) TNF-α, IL1-β, and IL-6 levels in serum from 8-mo-old DKO was measured by ELISA. Wild-type mice are shown as control for DKO. Data are shown with mean of five independent mice. Error bars indicate SD.