1. Volume 39, Issue 3, Pages 482-495 (September 2013)
Oxidative Damage of DNA Confers Resistance to Cytosolic Nuclease TREX1 Degradation and Potentiates STING-Dependent Immune Sensing 
Nadine Gehrke, Christina Mertens, Thomas Zillinger, Jörg Wenzel, Tobias Bald, Sabine Zahn, Thomas Tüting, Gunther Hartmann, Winfried Barchet 
Immunity 
Volume 39, Issue 3, Pages (September 2013)
DOI: /j.immuni
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2. Immunity 2013 39, 482-495DOI: (10.1016/j.immuni.2013.08.004)
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3. Figure 1
DNA from UV-Irradiated Cells Shows Enhanced Immune Stimulation
(A) Genomic DNA was isolated from RMA cells in which cell death was induced either through freeze-thaw cycles, shear stress from passage through a fine needle, or ultrasonication. In addition, DNA was isolated from apoptotic RMA cells 18 hr after either serum starvation, UV-C light (250 and 1,000 mJ/cm2) or γ irradiation (30 Gy), transfection with poly(I:C), or treatment with puromycin (1 μg/ml) or brefeldin A (5 μg/ml). Purified genomic DNA was transfected (1 μg/ml) into murine mDCs. Transfection with poly(I:C) was used as positive control. IFN-α was quantified after 18–24 hr in the supernatants of murine mDCs.
(B) Murine mDCs were transfected with genomic DNA isolated from RMA cells that were UV irradiated at doses of 250 or 1,000 mJ/cm2. Transfection with poly(I:C) was used as positive control. After 18–24 hr, IFN-α, IL-12p40, IL-6, and CXCL-10 was measured in the supernatants. Comparing differential stimulation by unmodified and UV-C-damaged (250 mJ/cm2) DNA, statistical significance (∗p < 0.05) was reached for all cytokines tested.
(C and D) Murine macrophages (C) and murine keratinocyte-derived SP1 cells (D) were stimulated with genomic DNA from untreated or UV-irradiated RMA cells. poly(dA:dT) and poly(I:C) were used as positive controls. After 18–24 hr, IFN-α was analyzed.
(E) Murine mDCs were stimulated with genomic DNA of untreated or UV-C-irradiated RMA cells with and without DNase I digestion. poly(dA:dT) was used as control. IFN-α was measured after 18–24 hr.
(F) Human CD14+ monocytes were transfected with genomic DNA from untreated or UV-irradiated human A549 cells or from murine RMA cells. Transfection with poly(dA:dT) was used as positive control. After 18–24 hr, IFN-α was measured.
Data are representative of at least three independent experiments each in triplicate and shown as means + SEM; ∗p < 0.05 (Student’s t test).
Immunity  , DOI: ( /j.immuni )
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4. Figure 2
Direct UV Irradiation of Cell-Free DNA Increases Its Type I IFN Induction
(A) Genomic DNA isolated from RMA cells was directly irradiated with UV-C light at the doses (mJ/cm2) indicated. Murine mDCs were subsequently transfected with the genomic DNA.
(B) Murine mDCs were transfected with genomic DNA that after isolation from RMA cells was directly irradiated with UV-A or UV-B light at the doses indicated.
IFN-α was measured by ELISA in supernatants after 18–24 hr. Data are representative of at least three independent experiments (means + SEM). ∗p < 0.05 (Student’s t test). See also Figure S1.
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5. Figure 3 ROS Damage of DNA Enhances Its Innate Immune Recognition
(A and B) RMA cells were incubated with ROS (A) and superoxide (B) detection reagent (ENZO Life Sciences) before UV irradiation. After 30 min, the mean fluorescence of the cells was determined by flow cytometry.
(C) The relative content of 8-OHdG in genomic DNA isolated from untreated and UV-irradiated RMA cells was measured with a competitive 8-hydroxy-2-deoxy-guanosine EIA Kit in which absorption at 405 nm is reversely correlated with 8-OHG content.
(D) Murine mDCs were stimulated with the genomic DNA from untreated or UV-irradiated RMA cells tested in (C). After 18–24 hr, secretion of IFN-α was measured.
(E) RMA cells were treated in culture with 0.1 or 1 mM H2O2 for 10 or 30 min, respectively, or were left untreated. Subsequently the genomic DNA was isolated and used to stimulate mDCs. poly(I:C) was used as positive control.
(F) Genomic DNA of RMA cells was directly incubated with 0.1, 10, or 100 μM H2O2 for 3 min. DNA was precipitated, washed, and resuspended to remove residual H2O2 and then used to transfect mDCs.
(G) dsDNA with oxidized guanosines (8-OHG) was generated as a 1.5 kB PCR product with 0%, 0.1%, or 10% 8-OH-dGTP in the dNTP mixture. After column purification, the quantity and integrity of the PCR products was determined on agarose gels, and the dose-dependent incorporation of 8-OHdG in the PCR products was verified with the 8-OHG EIA kit.
(H) Murine mDCs were stimulated with the PCR products or poly(I:C) as positive control.
Data are representative of at least three independent experiments (means + SEM). ∗p < 0.05 (Student’s t test). See also Figure S1.
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6. Figure 4
ROS Damage Enhances Cytosolic Recognition of Bacterial, Viral, and Self-DNA in Neutrophil Extracellular Traps
(A–C) Bacterial genomic DNA from E. coli (A) and viral genomic DNA from HSV-1 (B) or adenovirus (C) were incubated with hydrogen peroxide or hypochlorite, purified by precipitation, and then transfected into murine mDCs. After 18–24 hr, the IFN-α secretion of mDCs was measured in the supernatants.
(D and E) Human neutrophils were MACS purified from whole blood and were exposed to 40 nM PMA for 3 hr. The relative content of 8-OHG in human neutrophil and NET DNA was determined (D). Subsequently, isolated neutrophil and NET DNA was transfected into human monocytes. poly(dA:dT) served as positive control. After 18–24 hr IFN-α was measured in the supernatants (E).
(F) Genomic DNA isolated from hydrogen peroxide- or hypochlorite-treated RMA cells was added either naked uncomplexed together with LL-37 peptide to human monocytes. After 18–24 hr, the secretion of hIFN-α was quantified in the culture supernatants by ELISA.
Data shown are representative of three (A–D) and four (E, F) independent experiments (means + SEM). ∗p < 0.05 (Student’s t test).
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7. Figure 5 UV-Induced Oxidation of DNA in LE
(A) Genomic DNA of RMA cells was UV irradiated (250 mJ/cm2). DNA was complexed with Dotap, and 50 μg of complexed DNA was injected i.v. into C57BL/6J mice. IFN-α was measured in mouse serum 4 and 6 hr after injection. Results of the 6–11 individual mice per group are shown (means + SEM indicated). ∗∗p < 0.01 (Student’s t test).
(B and C) Genomic DNA of RMA cells was UV irradiated (250 mJ/cm2). 200 μg of naked uncomplexed genomic DNA was injected i.v. in C57BL/6J (B) and MRL/lpr (C) mice. After 4, 6, and 8 hr, serum IFN-α was quantified. Results of 12–17 individual mice per group are shown (means + SEM). ∗∗p < 0.01 (Student’s t test).
(D) Typical clinical presentation of sun-induced LE lesions are shown. Immunohistological expression pattern of 8-OHdG and MxA in sun-induced skin lesions in SCLE compared to sun-exposed skin of healthy controls (HC). DAB (8-OHdG) and Fast red (MxA) were used as chromogen. Magnification ×200.
(E) Clinical scoring of 8-OHdG and MxA staining intensities comparing expression of UV-induced SCLE skin lesions (n = 5) with sun-exposed nonlesional skin of healthy controls (HC) (n = 5). ∗∗p < 0.01 (Student’s t test).
(F) MRL/lpr mice were injected i.c. into the earlobes every third day for 21 days with 10 μg of naked uncomplexed genomic DNA that was either untreated or UV irradiated. On day 21, mice were monitored for lupus-like skin lesions and 24 hr after the last injection ear thickness was determined with a caliper. Mean ear swelling in μm + SEM are shown (n = 6); ∗p < 0.05 (Student’s t test).
(G and H) To visualize immune cell infiltrates, histological sections of ear tissue were stained with hematoxylin and eosin (G) or anti-CD45 antibody (H). Arrowheads in the insert indicate sites of tissue destruction.
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8. Figure 6
Enhanced Immune Sensing of UV-Damaged DNA Is Independent of TLR9 but Requires STING Signaling
(A) TLR9-deficient mDCs were stimulated with genomic DNA isolated from RMA cells that were UV irradiated at doses indicated.
(B) Genomic DNA isolated from RMA cells was directly irradiated with UV-C light at doses (mJ/cm2) indicated and transfected into bone marrow cells of WT and TLR9-deficient mice. CpG ODN 1826 and M362 were used as control stimuli.
(C) WT and TLR9-deficient mice were injected twice into the earlobes with 10 μg genomic DNA that was either untreated or UV irradiated. 24 hr after the second injection, ear thickness was determined with a caliper. Mean ear swelling in μm + SEM are shown (n = 8).
(D) Immortalized murine macrophages were transduced with lentiviral shRNA vectors STING #1 or #2 and silencing of STING was verified by RT-PCR (not shown). Empty vector SHC001, as well as SHC002 with an unspecific shRNA sequence, were used as controls. The transduced cells were stimulated with genomic DNA of untreated or UV-C-irradiated RMA cells. The RIG-I ligand 3P-RNA was used as positive control.
(E) Murine WT and STING-deficient (Tmem173, Goldenticket mutation) mDCs were stimulated with genomic DNA isolated from RMA cells that were untreated or UV irradiated at doses indicated. poly(I:C) and 3P-RNA were used as controls.
(F) WT and STING-deficient mice were treated as described in (C) (n = 6).
Secretion of IFN-α was quantified in the supernatants after 18–24 hr by ELISA (A, B, D, E). Data are representative of at least three (A, B, D, E) or two (C and F) independent experiments (means + SEM). ∗p < 0.05 (Student’s t test). See also Figures S2–S4.
Immunity  , DOI: ( /j.immuni )
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9. Figure 7
Oxidized DNA Is Resistant to Cytosolic Exonuclease TREX1-Mediated Degradation
(A and B) Recombinant TREX1 protein was added to genomic DNA (A) or PCR products (B) that were either unmodified (UV 0) or UV damaged (UV 250), as well as to PCR product containing 8-OHG. DNA integrity was monitored over time by fluorophore intercalation. Data are representative of four (A) or two (B) independent experiments.
(C) Unmodified and UV-damaged PCR products of 0.7 kb and 0.5 kb were incubated for 30 min with increasing amounts of TREX1 protein and visualized on an agarose gel.
(D) Trex1 mRNA relative to β-actin in mDCs and in RAW cells with and without lentiviral TREX1 overexpression was determined by RT-PCR.
(E and F) RAW cells (E) and TREX1-overexpressing RAW cells (F) were stimulated with unmodified and UV-damaged genomic DNA or PCR product.
(G and H) WT and TREX1-deficient mDCs were stimulated with unmodified and oxidized genomic DNA (G) or PCR product (H).
Secretion of mouse IFN-β (E, F) or IFN-α (G, H) in the supernatants after 18–24 hr was quantified by ELISA. Data are representative of at least two independent experiments (means + SEM).
(I) WT and TREX1-deficient mice were injected twice into the earlobes with 10 μg genomic DNA that was either untreated or UV irradiated. At 24 hr after the second injection, ear thickness was determined with a caliper.
Mean ear swelling in μm + SEM are shown (n = 6), representative of two independent experiments. ∗p < 0.05 (Student’s t test). See also Figure S5.
Immunity  , DOI: ( /j.immuni )
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