TANK prevents IFN-dependent fatal diffuse alveolar hemorrhage by suppressing DNA-cGAS aggregation

(A) Survival rate of WT (n = 10, 6 males and 4 females), Tank^−/− (n = 19, 10 males and 9 females), Il6^−/−Tank^−/− (n = 10, 5 males and 5 females) and Ifnar2^−/−Tank^−/− (n = 15, 7 males and 8 females) mice observed daily following a single i.p. administration of pristane. ###P < 0.001, N.S., not significant (Log-rank test). (B) Prevalence of diffuse alveolar hemorrhage according to gross pathology in the lungs of WT (n = 9), Tank^−/− (n = 12), Il6^−/−Tank^−/− (n = 13), and Ifnar2^−/−Tank^−/− (n = 10) mice harvested 7 d after pristane treatment. (C, D) Serum anti–double-stranded DNA Ab levels (C) and total IgG1 or IgM levels (D) in WT (n = 10), Tank^−/− (n = 11), Il6^−/−Tank^−/− (n = 10), and Ifnar2^−/−Tank^−/− (n = 12) mice with or without 7 d pristane treatment. Results are representative of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, N.S., not significant (t test).

Source data are available for this figure.

(A) Total numbers of peritoneal exudate cells (PECs) after pristane treatment in WT (n = 3) and Tank^−/− (n = 3) mice. (B) Flow cytometry analysis of PECs in WT and Tank^−/− mice. The proportion of Ly6C^high inflammatory monocytes and Ly6G⁺Ly6C^int neutrophils among CD11b⁺ PECs is shown. (C) Numbers of Ly6C^high monocytes, Ly6G⁺Ly6C^int neutrophils, and CD11c⁺PDCA1⁺ pDCs in PECs from WT (n = 3) and Tank^−/− (n = 3) mice at indicated time point after pristane treatment. (D) Quantitative PCR analysis for the expression of Ifnb1, Isg15, and Cxcl10 in total PECs obtained from WT (n = 3) and Tank^−/− (n = 3) mice with or without pristane treatment. *P < 0.05, **P < 0.01, ***P < 0.001, N.S., not significant (t test).

Source data are available for this figure.

(A) HEK293T cells were transfected with the IFN-β-promoter luciferase reporter and Renilla control plasmids together with indicated expression plasmids with or without the TANK expression plasmid. The luciferase reporter activity was determined 48 h after transfection. (B) BM-plasmacytoid dendritic cell from WT and Tank^−/− mice were stimulated with 0.3 μM poly (U), 1 μM R-848, and 1 μM CpG-DNA for 6 h. Then, total RNA was prepared and the expression of Ifnb and Irf7 was determined by QPCR. Results are representative of at least three independent experiments. (C) Peritoneal exudate cells from WT and Tank^−/− mice were transfected with indicated amounts of double-stranded DNA and poly (I:C). Total RNA was prepared 6 h after transfection, and the expression of Ifnb1, Isg15, and Cxcl10 was examined by QPCR. *P < 0.05, **P < 0.01, ***P < 0.001 (t test).

Source data are available for this figure.

(A) Survival rate of WT (n = 10, 6 males and 4 females), Tank^−/− (n = 20, 11 males and 9 females), Tlr7^−/−Tank^−/− (n = 15, 8 males and 7 females), Myd88^−/−Tank^−/− (n = 13, 6 males and 7 females), Mavs^−/−Tank^−/− (n = 15, 7 males and 8 females) and Tmem173^gt/gtTank^−/− (n = 19, 10 males and 9 females) mice after a single i.p. administration of pristane. #P < 0.05, ##P < 0.01, ###P < 0.001, N.S., not significant (Log-rank test). (B) Flow cytometry analysis of peritoneal exudate cells (PECs) in WT, Tank^−/− and Tmem173^gt/gtTank^−/− mice. The proportion of Ly6C^high inflammatory monocytes and Ly6G⁺Ly6C^int neutrophils among CD11b⁺ PECs is shown. (C) Numbers of Ly6C^high monocytes, Ly6G⁺Ly6C^int neutrophils and CD11c⁺PDCA1⁺ plasmacytoid dendritic cells in PECs from WT (n = 3), Tank^−/− (n = 3) and Tmem173^gt/gtTank^−/− (n = 3) mice at indicated time point after pristane treatment. (D) Quantitative PCR analysis for the expression of Ifnb1, Isg15, and Cxcl10 in total PECs obtained from WT (n = 3), Tank^−/− (n = 3), and Tmem173^gt/gtTank^−/− (n = 3) mice with or without pristane treatment. *P < 0.05, **P < 0.01, ***P < 0.001, N.S., not significant (t test).

Source data are available for this figure.

(A) BMDCs from WT and Tank^−/− mice were infected with Newcastle disease virus and VACV for 24 h. Then total RNA was prepared, and the expression of Ifnb1, Isg15, and Cxcl10 was examined by QPCR. (B) BMDCs from WT and Tank^−/− mice were treated with ABT737 together with Z-VAD-Fmk for 6 h, and the expression of Ifnb was determined by QPCR. (C) Macrophages from WT and Tank^−/− mice were transfected with Herring testis DNA for indicated periods. Then cell lysates were prepared and subjected to the immunoblot analysis using anti-p-TBK1, p-IRF3, and β-actin Abs. p-TBK1 and p-IRF3 were quantified and normalized to β-actin. (D) Macrophages from WT and Tank^−/− mice were stimulated with cGAMP and double-stranded DNA for 8 h. Total RNA was prepared after stimulation, and the expression of Ifnb1, Isg15, and Cxcl10 was examined by QPCR. (E) Macrophages from WT and Tank^−/− mice were treated with DMXAA for 2 h. Total RNA was prepared 24 h after transfection, and the expression of Ifnb1 and Isg15 was examined by QPCR. *P < 0.05, **P < 0.01, ***P < 0.001 (t test).

Source data are available for this figure.

(A) Macrophages from WT and Tank^−/− mice were stimulated with dsDNA for 2 h. Then concentrations of cGAMP in the cell lysates were measured by ELISA. (B) Cell lysates from WT and Tank^−/− macrophages were subjected to the immunoblot analysis using anti-cGAS, TANK, and β-actin Abs. (C, D) Macrophages from WT and Tank^−/− mice stimulated with Cy3-dsDNA for indicated periods. Then immunostaining was performed using AF488-anti-cGAS Ab and DAPI. (C, D) The representative images are shown in (C), and the numbers of cGAS puncta were quantified by images obtained from three independent experiments (D). (E, F) Macrophages from WT and Tank^−/− mice were stimulated with dsDNA for 2 h. Then immunostaining was performed using FITC-anti-Ub (FK2) and AF568-cGAS Abs and DAPI. (E, F) The representative images are shown in (E), and the numbers of cGAS-Ub aggregates were quantified by images obtained from four (WT) and six (Tank^−/−) independent experiments (F). *P < 0.05, **P < 0.01 (t test).

Source data are available for this figure.