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Fig. 1. DNA-dependent generation of a heat-resistant small molecule activates the STING pathway. (A) Illustration of an activity assay for cellular factors that activate the STING pathway. (B) Cytosolic extracts from mock or ISD-transfected L929-shSTING cells were incubated with PFO-permeabilized THP1 cells together with 35 S-labeled IRF3. Dimerization of IRF3 was analyzed by native gel electrophoresis followed by autoradiography. (C) Similar to (B), except that in lanes 4 to 6, cytosolic extracts were heated at 95°C for 5 min to denature proteins and then the heat-resistant supernatant was incubated with PFO-permeabilized THP1 cells. (D) L929-shSTING cytosolic extracts were incubated with the indicated nucleic acids in the presence of ATP, and then the heat-resistant supernatant was assayed for its ability to stimulate IRF3 dimerization in permeabilized Raw264.7 cells. (E) THP1 cells stably expressing shRNA against GFP (control) or STING were permeabilized with PFO and then incubated with the heat-resistant supernatant from the reaction mixture containing DNA-supplemented L929 cytosolic extracts (lanes 2 and 5) or from DNA-transfected L929 cells (lanes 3 and 6). IRF3 activation was analyzed by native gel electrophoresis. (F) THP1 cells described in (E) were transfected with HT-DNA or poly(I:C) or infected with Sendai virus (SeV), followed by measurement of IRF3 dimerization. (G) Cytosolic extracts from the indicated cell lines were incubated with HT-DNA, and then heatresistant supernatants were assayed for their ability to stimulate IRF3 dimerization in permeabilized Raw264.7 cells. Unless noted otherwise, all results in this and other figures were representative of at least two independent experiments. Fig. 2. Purification and identification of the heatresistant STING activator. (A) Full scan nano-LC-MS spectra of active and inactive fractions from the C18 column. Arrows indicate an ion at +1 (675.11) and +2 (338.14) charge states present only in the active fraction. (B) Tandem mass (MS2) spectra after CID fragmentation of the ion with m/z =338.14(z =2) from the MS1 scan shown in (A). Arrows indicate the m/z values of the expected fragmentation patterns of cyclic GMP-AMP (cGAMP, bottom). Asterisk indicates an ion (m/z = 506) that resulted from a neutral loss of a water molecule (18) from the ion with m/z = 524. (C)Fractions(B7toB12)fromtheC18column were analyzed for the presence of cGAMP by selective reaction monitoring of the expected ions and for their ability to stimulate IRF3 dimerization. (D)Comparison of the CID MS2 spectra of the purified STING activator and chemically synthesized cGAMP. A Active Factor D (IRF3)2 F Extracts from DNAtransfected cells STING IRF3 ER Nucleus PFO Permeabilized Cells - HT-DNA+DNaseI poly(dA:dT) poly(dG:dC) ISD poly(I:C) ssRNA HT-DNA IRF3 Lane 1 2 3 4 5 6 7 8 IB: IRF3 IB: STING B C + - - - - + - + + + + - Mock Extract: DNA Extract: THP-1(perm.): (IRF3)2 IRF3 THP1: shGFP shSTING Mock HT-DNA poly(I:C) SeV Mock HT-DNA poly(I:C) SeV Lane 1 2 3 4 5 6 7 8 Lane 1 2 3 4 E Mock ISD - HEK293T - heat +heat HT-DNA Lane 1 2 3 4 5 6 THP1: shGFP shSTING In vitro reaction: Transfected Cells: + + + + (IRF3)2 IRF3 A 371.00 C Relative Abundance B Relative Abundance 100 80 60 40 20 0 100 80 60 40 20 0 30 20 338.14 313.11 371.00 675.11 391.00 444.89 518.89 664.01 775.71 738.83 300 400 500 600 700 800 m/z 10 136.05 0 136.05 MS1 (active fraction) 390.97 444.97 MS1 (background) 518.92 592.24 667.63 758.70 152.07 MS2 of 524.10 m/z=338.14 (z=2+) * 506.03 428.12 232.23 330.17 540.14 556.33 100 200 300 400 500 600 m/z 330.06 524.06 232.06 428.05 540.05 248.06 346.05 cGAMP (m/z=675.107, Z=1 + ) 152.05 Relative Abundance of cGAMP D Relative Abundance Relative Abundance Lane 1 2 3 4 5 6 G BMDM THP1 MEF(primary) Lane 1 2 3 4 5 6 100 0 100 0 100 0 100 0 100 0 100 30 20 10 0 30 20 10 0 0 0 5 10 15 Retention Time (min) Mock ISD HT-DNA L929 (IRF3)2 IRF3 (IRF3)2 IRF3 (IRF3)2 IRF3 IRF3 Assay IRF3 (IRF3)2 100 200 300 m/z 400 500 600 B7 B8 B9 B10 B11 B12 152.0 524.0 STING activator Purified from Cells 152.1 524.0 Chemically Synthesized cGAMP 428.1 506.0 136.1 153.2 232.1 330.1 428.0 525.1 540.2 541.2 100 200 300 400 500 600 m/z 525.2 136.3 153.3 330.1 232.3 410.2 506.1 540.2 REPORTS www.sciencemag.org SCIENCE VOL 339 15 FEBRUARY 2013 827 Downloaded from www.sciencemag.org on February 14, 2013
REPORTS 828 double-stranded DNA (G:C50), poly(deoxyinosinedeoxycytidine) [poly(dI:dC)], or herring testis DNA (HT-DNA; fig. S1C) activated IRF3 in permeabilized THP1 cells, indicating that this activity was independent of DNA sequence. To determine whether the STING activator is a protein, we incubated the cytoplasmic extracts at 95°C to denature most proteins and then incubated the “heat supernatant” with permeabilized THP1 cells. Surprisingly, the heat supernatant from the ISD-transfected or HT-DNA–transfected cells caused IRF3 dimerization (Fig. 1C). This activity was resistant to treatment with Benzonase (Novagen, fig. S1D), which degrades both DNA and RNA, or proteinase K (fig. S1D). Thus, the STING activator is probably not a protein, DNA, or RNA. To test whether DNA could stimulate the generation of the heat-resistant STING activator in vitro, we incubated HT-DNA with L929shSTING cytoplasmic extracts (S100) in the presence of ATP (fig. S1E). The reaction mixture was heated at 95°C to denature proteins. Remarkably, incubation of the supernatant with permeabilized Raw264.7 cells led to IRF3 di- Relative Abundance of cGAMP IFNβ RNA (fold) 800 600 400 200 0 0 2 4 6 8hr Retention time (min) merization (Fig. 1D, lane 2). This activity depended on the addition of DNA to the cytoplasmic extracts. Other DNAs, including poly(dA:dT), poly(deoxyguanosine-deoxycytidine), and ISD, also stimulated the generation of the STING activator in L929-shSTING cytoplasmic extracts, whereas poly(inosine-cytidine) [poly(I:C)] and single-stranded RNA had no activity (Fig. 1D). Similar results were obtained with permeabilized THP1 cells (fig. S1F). Knockdown of STING in the permeabilized THP1 cells abolished IRF3 activation by the heat-resistant factor generated by DNA transfected into L929 cells or DNA added to L929 cytosolic extracts (Fig. 1E). Control experiments showed that the knockdown of STING inhibited the activation of IRF3 and induction of IFN-b and tumor necrosis factor–a in THP1 cells by HT-DNA transfection (fig. S1, G and H), but IRF3 activation by poly(I:C) transfection or Sendai virus infection, which is known to activate the RIG-I pathway, was unaffected by the STING knockdown (Fig. 1F). We also tested cytoplasmic extracts from several cell lines for their ability to produce the heat-resistant STING activator (Fig. 1G). Incubation of HT-DNA with extracts from A B C D IFNβ (pg/ml) 200 150 100 50 0 0 2 4 6 8 hr cGAMP stimulation (time course) E 100 F 80 G 50 0 100 50 0 100 50 0 100 50 0 IFNβ RNA (fold) Mock HSV1 VSV DNA 10 20 30 40 600 400 200 0 0 3 10 30 100 300 cGAMP Conc. (nM) IFNβ RNA (fold) 60 40 20 IFNβ (pg/ml) 120 80 40 0 cGAMP c-di-GMP 0 3 10 30 100 300 1000 Concentration (nM) 0 DNA: 0 1 2 3 4 6 (hr) Endogenous IRF3 cGAMP Activity Assay Fig. 3. DNA transfection and DNA virus infection induce IFN-b through cGAMP. (A) Chemically synthesized cGAMP (100 nM) was delivered to digitoninpermeabilized L929 cells for the indicated times, then IFN-b RNA and secreted protein were measured by qRT-PCR (inset) and ELISA, respectively. Unless noted otherwise, the error bars in this and all other panels denote SEM (n =3).(B) Similar to (A), except that different concentrations of cGAMP were delivered into L929 cells for 8 hours, followed by qRT-PCR analyses of IFN-b RNA. (C) Similar to (B), except that different concentrations of cGAMP and c-di-GMP were delivered into L929 cells, followed by ELISA assays for IFN-b.(D) L929 cells were infected with HSV-1D34.5 or VSV-DM51-GFP, transfected with HT-DNA, or mock-treated. An aliquot of the cell extracts was directly analyzed for IRF3 dimerization (top), whereas another aliquot was heated to denature Lane: 1 2 3 4 5 6 (IRF3) 2 IRF3 (IRF3) 2 IRF3 15 FEBRUARY 2013 VOL 339 SCIENCE www.sciencemag.org primary mouse embryo fibroblasts (MEFs), mouse bone marrow–derived macrophages (BMDMs), and L929 cells led to generation of the heatresistant factor that activated IRF3. Human cell extracts from THP1, but not human embryonic kidney (HEK) 293Tcells, were also able to produce this STING activator. These results are in agreement with our previous finding that primary MEFs, BMDMs, and L929 and THP1 cells, but not HEK293T cells, possessed the STING-dependent, RNA polymerase III–independent, pathway to induce type I interferons (3). We next used several chromatographic steps, including a STING-Flag affinity purification step, to purify the heat-resistant STING activator from L929 cell extracts (fig. S2, A and B). Previous research has shown that the bacterial molecules cyclic diadenylate monophosphate (c-di-AMP) and cyclic diguanylate monophosphate (c-di-GMP) bind to STING and induce type I interferons (8, 9). However, using nano–liquid chromatography– mass spectrometry (nano-LC-MS), we did not detect MS or MS/MS spectra consistent with those expected of c-di-GMP ([M+H] + = 691) or c-di-AMP ([M+H] + = 659). In-depth examination Endogenous IRF3 cGAMP Activity Assay THP1 Endogenous IRF3 Mock HSV1 VSV DNA Lane: 1 2 3 4 cGAMP Activity Assay Mock HSV1 VACV Lane: 1 2 3 (IRF3) 2 IRF3 (IRF3) 2 IRF3 (IRF3) 2 IRF3 (IRF3) 2 IRF3 proteins and the heat-resistant supernatant was assayed for its ability to stimulate IRF3 dimerization in permeabilized Raw264.7 cells (bottom). (E)The heat-resistant supernatant from (D) was fractionated by HPLC using a C18 column, and the presence of cGAMP in the fractions was measured by mass spectrometry using SRM. (F) L929 cells were transfected with HT-DNA (4 mg/ml) for the indicated time, then IFN-b RNA was measured by qRT-PCR and IRF3 dimerization was analyzed by native polyacrylamide gel electrophoresis (PAGE). AliquotsofthecellextractsweretestedforthepresenceofcGAMPonthebasis of its ability to induce IRF3 dimerization after delivery into Raw264.7 cells. (G) THP1 cells were infected with HSV-1D34.5 and vaccinia virus (VACV) for 6 hours, then the activation of endogenous IRF3 and generation of cGAMP activity were measured as described in (F). on February 14, 2013 www.sciencemag.org Downloaded from
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