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1 doi:.8/nature85 Supplementary Methods Plasmid construction Murine RIG-I, HMGB and Rab5 cdnas were obtained by polymerase chain reaction with reverse transcription (RT-PCR) on total RNA from, and then cloned into pcaggs-cfp, pcaggs-yfp and pcaggs-rfp vectors, respectively, at the XhoI and NotI sites. Fluorescence Imaging HeLa cells (5 x ) were cultured on glass-bottom, 5-mm tissue culture dishes (MATSUNAMI Glass). Confocal microscopy studies were performed as described previously with an Olympus IX8 confocal microscope (OLYMPUS). Dual- or triplecolour images were acquired using a sequential acquisition mode to avoid crossexcitation. RNA analysis Primer sequences for quantitative real-time RT-PCR (qrt-pcr) analysis are as follows: HMGB sense 5'-CCAAAGGGGAGACCAAAAAG-' HMGB antisense 5'-TCATAGGGCTGCTTGTCATCT-' HMGB sense 5'-TGCCTTCTTCCTGTTTTGCT-' HMGB antisense 5'-GGACCCTTCTTTCCTGCTTC-' HMGB sense 5'-GGAGATGAAAGATTATGGACCAG-' HMGB antisense 5'-CTTTGCTGCCTTGGTG-'

2 doi:.8/nature85 GBP sense 5'-CTCAGCAGCAGTGCAAAAGG-' GBP antisense 5'-GCTCCTGGAGGGTTTCTGTG-' IRF7 sense 5'-GCAAGGGTCACCACACTA-' IRF7 antisense 5'-CAAGCACAAGCCGAGACT-' IL-p sense 5'-GACACGCCTGAAGAAGATGAC-' IL-p antisense 5'-TAGTCCCTTTGGTCCAGTGTG-' Supplementary references. Honda, K. et al., Role of a transductional-transcriptional processor complex involving MyD88 and IRF-7 in Toll-like receptor signaling. Proc. Natl. Acad. Sci. USA, 56-5 ().. Negishi, H. et al., Evidence for licensing of IFN-γ-induced IFN regulatory factor transcription factor by MyD88 in Toll-like receptor-dependent gene induction program. Proc. Natl. Acad. Sci. USA, 56-5 (6).. Gitlin, L. et al., Essential role of mda-5 in type I IFN responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus. Proc. Natl. Acad. Sci. USA, (6).

3 doi:.8/nature85 a (kda) 75 DNase I + DNase I + HMGB HMGB 8 6 HMGB 8 5 DNase I HMGBs 7 b Competitor: pull-down poly(i:c) poly(u) HMGB HMGB Competitor: Calf thymus DNA Bacterial DNA R87 HMGB Competitor: CpG-B ODN PD PS HMGB c Competitor: poly(u) pull-down CpG-B ODN PS R87 HMGB d Competitor: HMGB pull-down poly(i:c) (μg/ml) Supplementary Figure. Identification of HMGBs and its binding to DNA and RNA. a, Identification of HMGBs. Cytosolic extracts from stimulated with B- DNA for h were subjected to pull-down assay using biotin-conjugated and streptavidin (SA)-conjugated magnetic beads. Bound proteins were eluted by DNase I treatment. Eluted proteins, visualized by SDS-PAGE followed by silver staining (left panel), were analyzed by mass spectrometry. Immunoblot analysis using antibodies against HMGB, and is shown in the right panel. b, Direct binding of HMGBs to DNA, RNA, and/or base-free phosphorothioate deoxyribose homopolymers (PS). In vitro pull-down assays, using recombinant HMGB or and biotin-conjugated, were performed in the presence of increasing amounts of unconjugated nucleic acids (,,, or µg/ml), R87 (,,, or µg/ml) (upper and middle panel) or base-free deoxyribose homopolymers (PD and PS;.,.,., or µg/ml) (lower

4 doi:.8/nature85 panel). In the lower panel, increasing amounts of unconjugated or CpG- B (.,., or µg/ml) was also used. Note that the half maximal inhibitory concentration (IC 5 ) values for CpG-B ODN and PS were 5- and -fold lower than that for non-conjugated, respectively. c, In vitro pull-down assay using recombinant HMGB and biotin-conjugated poly(u) in the presence of increasing amounts of unconjugated CpG-B, PS or R87 (., or µg/ml).

5 /- doi:.8/nature85 a.5 IFN-α mrna...5 : IFN-α mrna 5 6 RANTES mrna 8 poly(i:c):.5 RANTES mrna (h) (h) Hmgb +/+ Hmgb / b.8. : poly(i:c): LPS: (μg/ml) (μg/ml) (ng/ml) Hmgb +/+ Hmgb / c IFN-α mrna cdcs d, In vitro pull-down assay using recombinant HMGB and biotin-conjugated B- : (h).5 DNA in the IFN-β absence mrna or.5 presence IFN-α mrna of or.5 IL-6 µg/ml mrna unconjugated or... poly(i:c).... poly(i:c): (h) Hmgb +/+ Hmgb / Supplementary Figure. Essential roles of HMGBs in cytosolic nucleic acid receptor-mediated innate immune responses. a-c, Hypoinduction of various cytokine and chemokine genes in the absence of HMGB. Hmgb +/+ or Hmgb -/- (a) and cdcs (c) were lipofected with B- DNA or poly(i:c), and then mrna expression levels for the indicated genes measured by qrt-pcr., not detected. Asterisk, P <. as compared with Hmgb +/+ cells. For dose-response experiments, wild-type and Hmgb -/- littermate were stimulated with increasing amounts of (.,, 5, µg/ml) or poly(i:c) (.,, 5, µg/ml) for 6 h, or stimulated with LPS (, 5,, 5 ng/ml) for h. The mrna expression levels for the indicated cytokine genes measured by qrt-pcr (b). Data in all panels are mean ± s.d. (n = ). Note that the poly(i:c) response in cdcs may be mediated by both RLRs and TLR (ref. ). d, Induction of cytokine genes in the absence of HMGB. Wild-type and Hmgb - 5

6 doi:.8/nature85 d : (μg/ml)...5 poly(i:c): LPS: (μg/ml) 5 5 (ng/ml) Hmgb +/+ Hmgb / e : IFN-α mrna 6 (h) Hmgb +/+ + Hmgb / + Hmgb / + HMGB-si poly(i:c): IFN-α mrna 6 (h) f HMGB-si HMGB HMGB HMGB β-actin d, Induction of cytokine genes in the absence of HMGB. Wild-type and Hmgb - /- littermate were stimulated with increasing amounts of (.,, 5, µg/ml) or poly(i:c) (.,, 5, µg/ml) for 6 h, or stimulated with LPS (, 5,, 5 ng/ml) for h. The mrna expression levels for the indicated cytokine genes measured by qrt-pcr. Data in all panels are mean ± s.d. (n = ). e, Effect of HMGB knock down in Hmgb -/-. Hmgb -/- that were transduced with retrovirus expressing a sirna targeting HMGB (HMGB-si) or a control sirna () were stimulated with or poly(i:c), and mrna expression levels of the indicated genes measured by qrt-pcr. Hmgb +/+ expressing control sirna () were also analyzed for comparison. Data in all panels are mean ± s.d. (n = ). Asterisk, P <. as compared with -expressing cells. f, The effect of HMGB-targeting sirna. Wild-type were transduced with the indicated sirna retroviruses, and the expression of each HMGB protein was analyzed by immunoblot analysis. 6

7 doi:.8/nature85 g mrna HMGB HMGB HMGB h HMGB HMGB HMGB β-actin i... Stimulation:... Stimulation: HSV- DNA '-triphosphate RNA Bacterial DNA Vaccinia virus DNA Calf thymus DNA LPS ISD j...5 : (μg/ml) poly(i:c). 5 (μg/ml)..8. LPS.5 poly(i:c). LPS (ng/ml) Dose:. 5 (μg/ml). 5 (μg/ml) 5 5 (ng/ml) k IFN-β (pg/ml) IL-6 (pg/ml) 8 8 poly(i:c) expression levels of the indicated genes measured by qrt-pcr. Hmgb +/+ 6 6 expressing control sirna () were also analyzed for comparison. Data in all panels are mean ± s.d. (n = ). Asterisk, P <. as compared with :. 5 (μg/ml). 5 (μg/ml) -expressing cells. poly(i:c) LPS f, The effect of HMGB-targeting sirna. Wild-type were transduced with the indicated sirna retroviruses, and the expression of each HMGB protein Dose:. 5 (μg/ml). 5 (μg/ml) 5 5 (ng/ml) was analyzed by immunoblot analysis. g, h, The effect of sirna targeting of all HMGBs. Wild-type were transduced with retrovirus expressing sirna targeting all HMGB () or control sirna (), and then the expression of each HMGB measured by qrt-pcr (g) and immunoblot analysis (h). Asterisk, P <. as compared with -expressing cells. i, Impaired innate immune responses to cytosolic stimulation with various nucleic acids in HMGB-deficient cells. expressing sirna targeting all HMGBs () or control sirna () were stimulated with the indicated nucleic acids for 6 h or stimulated with LPS ( ng/ml) for h, and then mrna expression levels for the IL-6 gene measured by qrt-pcr. Data in all panels are mean ± s.d. (n = ). Asterisk, P <. as compared with -expressing cells. 7

8 doi:.8/nature85 expression levels for the IL-6 gene measured by qrt-pcr. Data in all panels are mean ± s.d. (n = ). Asterisk, P <. as compared with -expressing cells. j, k, Impairment of innate immune responses to various doses of nucleic acid ligands in HMGB-deficient cells. expressing sirna targeting all HMGBs () or control sirna () were stimulated with increasing amount of (.,, 5, µg/ml) or poly(i:c) (.,, 5, µg/ml) for 6 h, or stimulated with LPS (, 5,, 5 ng/ml) for h. The mrna expression levels for the indicated cytokine genes measured by qrt-pcr (j) or by ELISA (k). Data in all panels are mean ± s.d. (n = ). l... Stimulation: IRF7 mrna GBP mrna IκB-α mrna IFN-β IFN-γ TNF-α m IFN-γ: (units/ml) p-stat β-actin l, Response of HMGB-deficient cells to various cytokine stimuli. expressing sirna targeting all HMGBs () or control sirna () were stimulated with ( µg/ml) for 6 h, or stimulated with IFN-β (5 units/ml) for 6 h, or TNF-α ( ng/ml) or IFN-γ ( units/ml) for h. The mrna expression levels for the indicated target genes measured by qrt-pcr. Data in all panels are mean ± s.d. (n = ). Essentially the same results were obtained with different doses of these ligands. m, IFN-γ-induced activation of STAT in HMGB-deficient cells. expressing sirna targeting all HMGBs () or control sirna (), described above, were stimulated with IFN-γ ( or units/ml) for min. The phosphorylated STAT and β-actin was detected by using anti-phospho-stat (p-stat) and anti-β-actin antibody, respectively. 8

9 doi:.8/nature85 n. poly(i:c).8. Time (h): RAW6.7 o p.8. : Time (h):. 5 (μg/ml) 5 poly(i:c)... poly(i:c) (μg/ml) NIHT poly(i:c) LPS LPS 5 5 (ng/ml) Dose:. 5 (μg/ml). 5 (μg/ml) 5 5 (ng/ml) RAW levels for the indicated cytokine genes measured by qrt-pcr (j) or by ELISA.. (k). Data in all panels are mean ± s.d. (n = ). l, Response of HMGB-deficient cells to various cytokine stimuli. expressing sirna targeting all HMGBs () or control sirna () were stimulated with ( µg/ml) for 6 h, or stimulated with IFN-β (5 units/ml) for 6 h, or TNF-α ( ng/ml) or IFN-γ ( units/ml) for h. The mrna expression levels for the indicated target genes measured by qrt-pcr. Data q.8 poly(i:c) in all panels are mean ± s.d. (n = ). obtained with different doses of these ligands. Essentially the same results were NIHT.6 m, IFN-γ-induced activation of STAT in HMGB-deficient cells. :. 5 (μg/ml). 5 (μg/ml) expressing.6 sirna targeting all poly(i:c) HMGBs ().6 or LPS control sirna (), described above, were stimulated. with IFN-γ ( or. units/ml) for min. The.8 phosphorylated.5.. STAT and β-actin was detected by using anti-phospho-stat (p-stat) and anti-β-actin antibody, respectively. Dose:. 5 (μg/ml). 5 (μg/ml) 5 5 (ng/ml) n, o, Impairment of innate immune responses to cytosolic stimulation with nucleic acids in HMGB-deficient RAW6.7 cells. RAW6.7 cells expressing sirna targeting all HMGBs () or control sirna () were stimulated with or poly(i:c) for the indicated periods. The mrna expression levels for the IFN-β gene measured by qrt-pcr. Asterisk, P <. as compared with -expressing cells. (n). or expressing RAW6.7 cells were stimulated with increasing amounts of (.,, 5, µg/ml) or poly(i:c) (.,, 5, µg/ml) for 6 h, or stimulated with LPS (, 5,, 5 ng/ml) for h. The mrna expression levels for the indicated cytokine genes measured by qrt-pcr (o). Data in all panels are mean ± s.d. (n = ). p, q, Innate immune responses to cytosolic nucleic acids in HMGB-deficient NIHT cells. NIHT cells expressing sirna targeting all HMGBs () or control sirna () were stimulated with or poly(i:c) for the indicated periods. The mrna expression 6 levels for the IFN-β gene measured by qrt-pcr. Asterisk, P <. as compared with -expressing cells. (p). or expressing NIHT cells were stimulated with increasing amount of (.,, 5, µg/ml) or poly(i:c) (.,, 5, µg/ml) for 9 h, 9

10 doi:.8/nature85 by qrt-pcr. Asterisk, P <. as compared with -expressing cells. (p). or expressing NIHT cells were stimulated with increasing amount of (.,, 5, µg/ml) or poly(i:c) (.,, 5, µg/ml) for 9 h, or stimulated with LPS (, 5,, 5 ng/ml) for h. The mrna expression levels for the indicated cytokine genes measured by qrt-pcr (q). Data in all panels are mean ± s.d. (n = ).

11 doi:.8/nature85 a pg/ml IL-β Macrophages Hmgb +/+ Hmgb -/- b pg/ml IL-β RAW6.7 : + : + c Viable cells (%) 8 6 RAW (μg/ml) Supplementary Figure. Involvement of HMGBs in the activation of the inflammasome pathway and cell death by stimulation. Hmgb +/+ or Hmgb -/- fetal liver haematopoietic progenitor-derived macrophages (a), and RAW6.7 cells expressing sirna targeting all HMGBs () or control sirna () (b) were lipofected with, and then the levels of secreted mature IL-β at h determined by ELISA. RAW6.7 cells were primed with 5 ng/ml LPS for 6 h to become competent for inflammasome activation. Data are mean ± s.d. (n = ). Asterisk, P <. as compared with wild-type cells or -expressing cells., not detected. (c) RAW6.7 cells expressing or were stimulated with increasing amounts of. The cells were collected after h stimulation and stained with trypan blue. The percentage of living cells compared with untreated cells was calculated. Note that RAW6.7 cells expressing are more resistant to the DNA-induced death. Data are mean ± s.d. (n = ).

12 doi:.8/nature85 a Virus titer (x 5 ) VSV HSV- b.8. IFN-α mrna.6.8. IFN-α mrna RAW6.7 VSV: (h).6.. HSV-: IFN-α mrna IFN-α mrna 6 (h) c Virus titer (x 5 ) 8 6 VSV 8 6 HSV- RAW6.7 Supplementary Figure. A requirement for HMGBs in anti-viral responses. a, expressing sirna targeting all HMGBs () or control sirna () were infected with VSV or HSV- for h, and then viral titers measured. Data are mean ± s.d. (n = ). Asterisk, P <. as compared with cells expressing. b, c, RAW6.7 cells expressing sirna targeting all HMGBs () or control sirna () were infected with VSV or HSV-. Type-I IFN mrna expression levels (b) and viral titers (c) measured. Asterisk, P <.; double asterisk, P <.5 as compared with cells expressing., not detected.

13 doi:.8/nature85 a TNF-α mrna RAW6.7 poly(i:c):... CpG-B ODN: TNF-α mrna 6 (h) (h) b Time (h): IFN-α mrna 6 6 RAW6.7 CpG-A ODN CpG-A ODN + DOTAP CpG-A ODN CpG-A ODN + DOTAP Supplementary Figure 5. A requirement for HMGBs in the nucleic acidmediated activation of TLRs. a, b, RAW6.7 cells expressing sirna targeting all HMGBs () or control sirna () were stimulated by the indicated TLR ligands, and then mrna expression levels for various cytokines measured by qrt-pcr. Data are mean ± s.d. (n = ). Asterisk, P <. as compared with -expressing cells., not detected.

14 doi:.8/nature85 a 6, IFN-β pg/ml,, : CpG-B ODN: (h) b IL-p mrna poly(i:c) R87 Tlr9 / cdcs Stimulation: PS + CpG-B ODN PS + CpG-B ODN Supplementary Figure 6. Inhibition of nucleic acid-mediated activation of innate immune responses by treatment with nucleotide analogues. a, were co-treated with µm CpG-B ODN at the indicated time points after lipofection, and then the induction of IFN-β measured by ELISA. Data are mean ± s.d. (n = ). Asterisk, P <. as compared with stimulation without CpG-B ODN. b, Bone marrow-derived Tlr9 -/- cdcs were stimulated by either 5 µg/ml poly(i:c) (without lipofection) or 5 µg/ml R87 for h in the absence of or following pretreatment with 5 µm PS or µm CpG-B ODN for min. IL-p mrna expression levels were measured by qrt-pcr. Data in all panels are mean ± s.d. (n = ). Asterisk, P <., pretreated cells versus non-pretreated cells., not detected.

15 doi:.8/nature85 a RIG-I HMGB Rab5 RIG-I+HMGB RIG-I+Rab5 HMGB+Rab5 RIG-I+HMGB+Rab5 b RIG-I HMGB mitotr RIG-I+HMGB RIG-I+mitoTR HMGB+mitoTR RIG-I+HMGB+mitoTR Supplementary Figure 7. Intracellular localization of HMGB and RIG-I. HeLa cells were transfected with CFP-tagged RIG-I (CFP-RIG-I) and YFPtagged HMGB (YFP-HMGB) expression vectors with or without a RFPtagged Rab5 (RFP-Rab5) expression vector. Sixteen hours after transfection, cells were stimulated with poly(i:c) for h, followed by fluorescence microscopic analysis using a laser-scanning confocal microscope. (a) Images of co-transfected (CFP-RIG-I, YFP-HMGB or RFP-Rab5) cells are shown. Upper and lower panels show single (RIG-I, HMGB or Rab5; from left to right panel) and merged (CFP-RIG-I+YFP-HMGB, CFP-RIG-I+RFP-Rab5, YFP- HMGB+RFP-Rab5 or CFP-RIG-I+YFP-HMGB+RFP-Rab5; from left to right panel) pictures, respectively. Scale bar, 5µm. A representative image of a pattern observed in many cells is shown. Moreover, the same observations were made in the absence of poly(i:c) stimulation, which we suspect is due to the activation of poly(i:c)-unstimulated cells upon DNA transfection of the vectors. Note that both RIG-I and HMGB merged partially with Rab5, indicating their recruitment on the endosomal membrane and possibly representing the occurrence of HMGB-instigated activation of RIG-I. (b) Cells 5

16 doi:.8/nature85 that were co-transfected with CFP-RIG-I and YFP-HMGB expression vectors were stained with MitoTracker Deep Red 6 (mitotr) after poly(i:c) stimulation. Upper and lower panels show single (CFP-RIG-I, YFP-HMGB or mitotr; from left to right panel) and merged (CFP-RIG-I+YFP-HMGB, CFP- RIG-I+mitoTR or YFP-HMGB+mitoTR; from left to right panel) pictures, respectively. Scale bar, 5µm. As shown here, a fraction of RIG-I merges with mitotr, while no merge is seen between HMGB and mitotr. Taken together with the data presented in (a), these data may be interpreted as follows: Following HMGB recognition of poly(i:c), RIG-I is activated and translocates to the mitochondria where it interacts with IPS-/MAVS. These observations may represent snapshots of the dynamic stages of nucleic acid-recognition and activation of the immune responses, wherein we observe some fraction of RIG-I interacts with HMGB, while another fraction has dissociated from HMGB and is bound with IPS-/MAVS. Although supported by our data, this model/interpretation requires more careful analysis. 6