Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

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1 ARTICLE Reeived Jul Aepted Mr 7 Pulished My 7 DOI:./nomms7 Exosomes mintin ellulr homeostsis y exreting hrmful DNA from ells OPEN Akiko Tkhshi, Ryo Okd, Koji Ngo, Yuk Kwmt, Aki Hnyu, Shin Yoshimoto,, Mski Tksugi, Sugiko Wtne, Msto T. Knemki,, Chikshi Ouse & Eiji Hr,,7 Emerging evidene is reveling tht exosomes ontriute to mny spets of physiology nd disese through interellulr ommunition. However, the iologil roles of exosome seretion in exosome-sereting ells hve remined lrgely unexplored. Here we show tht exosome seretion plys ruil role in mintining ellulr homeostsis in exosome-sereting ells. The inhiition of exosome seretion results in the umultion of nuler DNA in the ytoplsm, therey using the tivtion of ytoplsmi DNA sensing mhinery. This event provokes the innte immune response, leding to retive oxygen speies (ROS)-dependent DNA dmge response nd thus indue senesene-like ell-yle rrest or poptosis in norml humn ells. These results, in onjuntion with oservtions tht exosomes ontin vrious lengths of hromosoml DNA frgments, indite tht exosome seretion mintins ellulr homeostsis y removing hrmful ytoplsmi DNA from ells. Together, these findings enhne our understnding of exosome iology, nd provide vlule new insights into the ontrol of ellulr homeostsis. The Cner Institute, Jpnese Foundtion for Cner Reserh (JFCR), Koto-ku, Tokyo -, Jpn. Grdute Shool of Life Siene, Hokkido University, Spporo, Hokkido -, Jpn. LSI Mediene Corportion, Chiyod-ku, Tokyo -7, Jpn. Deprtment of Moleulr Miroiology, Reserh Institute for Miroil Diseses (RIMD), Osk University, Suit, Osk -7, Jpn. Division of Moleulr Cell Engineering, Deprtment of Genetis, Ntionl Institute of Genetis, ROIS, SOKENDAI, Mishim, Shizuok -, Jpn. PRESTO, Jpn Siene nd Tehnology Ageny (JST), Kwguhi, Sitm -, Jpn. 7 CREST, Jpn Ageny for Medil Reserh nd Development (AMED), Chiyod-ku, Tokyo -, Jpn. Correspondene nd requests for mterils should e ddressed to A.T. (emil: kiko.tkhshi@jfr.or.jp) or to E.H. (emil: ehr@iken.osk-u..jp). NATURE COMMUNICATIONS :7 DOI:./nomms7

2 ARTICLE NATURE COMMUNICATIONS DOI:./nomms7 Higher eukryoti ells re equipped with vrious potent self-defene mehnisms to preserve ellulr homeostsis. One suh mehnism is ellulr senesene, whih loks the errnt prolifertion of ells t risk for neoplsti trnsformtion, nd is therefore elieved to t s n importnt tumour suppressive mehnism. Although irreversile ell-yle rrest is trditionlly onsidered s the mjor funtion of senesent ells, reent studies hve reveled some dditionl funtions of senesent ells. Most noteworthy, however, is the inresed seretion of vrious seretory proteins, suh s inflmmtory ytokines, hemokines, growth ftors nd mtrix metlloproteinses, into the surrounding extrellulr fluid 7. These newly reognised senesent phenotypes, termed the senesene-ssoited seretory phenotypes 9, reportedly ontriute to tumour suppression 7,, wound heling, emryoni development, nd even tumorigenesis promotion 9,. Thus, senesene-ssoited seretory phenotypes pper to e enefiil or deleterious, depending on the iologil ontext,. In ddition to seretory proteins, senesent ells lso inrese the seretion of lss of extrellulr vesiles lled exosomes 7. Exosomes re endosoml memrne vesiles with dimeters of B nm. They originte in the lte endosoml omprtment from the inwrd udding of endosoml memrnes, whih genertes intrellulr multi-vesiulr endosomes (MVEs),. Pools of exosomes re pked in the MVEs nd relesed into the extrellulr spe fter the fusion of MVEs with the plsm memrne,,. Emerging evidene hs indited tht exosomes ply importnt roles in interellulr ommunition, y serving s vehiles for trnsferring vrious ellulr onstituents, suh s proteins, lipids nd nulei ids, etween ells 7. However, very little is known out the iologil roles of exosome seretion in exosome-sereting ells. Erly hypotheses fvoured the notion tht exosomes my funtion s ellulr grge gs tht expel unusle ellulr onstituents from ells,9.however,thishsnot een expliitly proven. Sine exosome seretion is reportedly inresed in some senesent ells 7, we exmined the effets of the inhiition of exosome seretion in senesent ells. Surprisingly, we disovered tht reduing exosome seretion provokes retive oxygen speies (ROS)-dependent DNA dmge response (DDR), in oth senesent nd non-senesent ells. Interestingly, the tivtion of ROS DDR is onsequene of the umultion of nuler DNA frgments in the ytoplsm, where they re reognised y STING,ytoplsmiDNAsensor.This response ws llevited y the overexpression of ytoplsmi DNse, the inhiition of STING tivity or the inhiition of ROS generted y the interferon (IFN) pthwy. These results, together with the oservtions tht exosomes ontin hromosoml DNA frgments, indited tht exosome seretion plys n importnt role in mintining ellulr homeostsis y removing hrmful ytoplsmi DNA from ells, t lest in ertin types of norml humn ells. Notly, the inhiition of exosome seretion in mouse liver, using hydrodynmis-sed RNA interferene (RNAi), reveled tht this pthwy lso funtions in this tissue, suggesting tht this mhinery my ontriute more rodly to tissue homeostsis in vivo. Finlly, we extended these findings to the ntivirl tivity of exosome seretion, whih expels infeted denovirl DNA from ells. Thus, lthough we nnot exlude the possiilities tht exosome seretion mintins ellulr homeostsis y expelling not only ytoplsmi DNA ut lso other hrmful ellulr onstituents from ells, our findings delinete novel mehnism tht links exosome seretion nd ellulr homeostsis. Results Exosome seretion mintins ellulr homeostsis. To enhne our understnding of exosome iology, we first exmined the effets of the inhiition of exosome seretion in senesent ells. Pre-senesent (erly pssge) norml humn diploid firolsts (HDFs) were rendered senesent y either seril pssge or etopi expression of onogeni Rs, the most estlished wys to indue ellulr senesene (Supplementry Fig. ), nd then exosomes were isolted y ultrentrifugtion. The isolted extrellulr vesiles were onfirmed to e exosomes, sed on nnoprtile trking nlysis (), immuno-gold lelling for CD, well known exosome-ssoited protein, followed y trnsmission eletron mirosopy, nd western lotting nlysis of nonil exosoml mrkers (Supplementry Fig. d f). Consistent with previous report 7, exosome seretion ws signifintly inresed in senesent ells, regrdless of how the ellulr senesene ws indued (Supplementry Fig. f). We thus tried to inhiit exosome seretion y knoking down or, whih re essentil omponents of exosome iogenesis nd seretion, respetively, using previously vlidted smll interfering RNAs (sirnas),7 in senesent ells. In greement with studies using severl humn ner ell lines, the depletion of either or sustntilly redued exosome seretion, s judged y nd western lotting nlyses of nonil exosoml mrkers (Fig.,). Interestingly, however, this ws ompnied y poptoti ell deth (Fig. f), showing tht there is n inverse orreltion etween the levels of exosome seretion nd the inidene of poptosis. To our surprise, moreover, similr ut less pronouned effet ws lso oserved in pre-senesent ells (Fig. ). These results re unlikely to e the off-trget effets of the sirna oligos, sine the introdution of sirna-resistnt or DNA into the knokdown ells ttenuted the effets of the sirna oligos (Fig. e,f). Furthermore, two struturlly unrelted hemil inhiitors of N-sphingomyelinse (nsmse), GW9 nd Spiroepoxide, whih re well known inhiitors of exosome prodution 9,, lso hd the sme effets in HDFs (Supplementry Fig. ) nd other types of norml humn ells (Supplementry Fig., e g). Colletively, these results strongly suggested tht exosome seretion plys ruil role in mintining ellulr homeostsis t lest in ertin types of norml humn ells, regrdless of whether the ells re senesent. Exosome seretion prevents the errnt tivtion of DDR.To sustntite this ide, we next sought to determine the underlying mehnisms y fousing on pre-senesent ells. Interestingly, we noted tht the inhiition of exosome seretion provoked not only poptosis ut lso senesene-like irreversile ell-yle rrest in pre-senesent ells (Fig., nd Supplementry Figs nd,f). Sine the umultion of DNA dmge is known to use poptosis or ellulr senesene, depending on the degree of DNA dmge,, we tested whether the inhiition of exosome seretion provokes DNA dmge in pre-senesent ells. Indeed, the redution of exosome seretion y sirnas or hemil inhiitors inresed the signs of the DDR in norml humn ells, s judged y ghax foi nd the phosphoryltion of the onsensus trget sequenes (S/TQ) of Atxi telngietsi mutted (ATM) nd Atxi telngietsi nd Rd relted protein (ATR), key omponents of the DDR pthwy (Fig. d, Supplementry Figs d nd d,h). Importntly, moreover, the simultneous knokdowns of ATM nd ATR using vlidted sirna oligos olished the onset of senesene-like ell-yle rrest nd poptoti ell deth in ells with or depletion (Fig.,). These results re lso onsistent with the oservtion tht the inhiition of exosome seretion filed to indue senesene-like ell-yle rrest nd poptoti ell deth in humn ner ell lines, in whih the DDR nd/or ell-yle hekpoint pthwys re disrupted (Supplementry Fig. ). Tken together, while dditionl mehnisms my prtiipte, NATURE COMMUNICATIONS :7 DOI:./nomms7

3 NATURE COMMUNICATIONS DOI:./nomms7 ARTICLE +H-RsV Exosome Lte pssge p P-p Ser CD CD Tsg Exosome H-Rs p P-p Ser CD CD Tsg Reltive mount of exosomes/ell. Reltive mount of exosomes/ell.... Reltive numer of ells.. Lte pssge Dys d... Reltive numer of ells.. +H-RsV Dys e Lte pssge f +H-RsV Reltive mounts of poptoti ells 9 Reltive mounts of poptoti ells 9 Figure Inhiition of exosome seretion in senesent HDFs. (,) Senesent TIG- ells indued y seril pssge () or onogeni Rs expression () were trnsfeted with vlidted sirna oligos indited t the top of the pnel for twie t dy intervls. These ells were then sujeted to western lotting using ntiodies shown right () or to exosome isoltion followed y western lotting using ntiodies ginst nonil exosome mrkers shown right (exosome) nd NnoSight nlysis () for quntittive mesurement of isolted exosome prtiles. The representtive dt from three independent experiments re shown. ws used s loding ontrol. ( f) Senesent TIG- ells desried in, were sujeted to ell prolifertion nlysis (,d) or to poptosis nlysis t dy (e,f). The representtive dt from three independent experiments re shown. For ll grphs, error rs indite men±s.d. of triplite mesurements. (Po., Po.; one-wy nlysis of vrine). these dt strongly suggested tht exosome seretion plys key role in the mintenne of ellulr homeostsis y preventing the errnt tivtion of DDR pthwys, t lest in ertin types of norml humn ells. Exosomes exrete hrmful ytoplsmi DNA from ells. To further explore this notion, we nlysed how exosome seretion prevents the errnt tivtion of DDR pthwys. In seeking n explntion, we noted tht exosomes relesed from HDFs hve the potentil to tivte the DDR pthwy in reipient pre-senesent HDFs, depending on the mount of dded exosome (Supplementry Fig. ). This result led us to propose tht exosome seretion my prevent the errnt tivtion of the DDR pthwy, y exreting hrmful ellulr onstituents from ells. Exosomes re known to ontin vrious ellulr omponents, suh s proteins, lipids, RNA nd DNA,. Among them, DNA is prtiulrly interesting, euse frgmented DNA is known to tivte the DDR in norml NATURE COMMUNICATIONS :7 DOI:./nomms7

4 ARTICLE NATURE COMMUNICATIONS DOI:./nomms7 Exosome Reltive mount of exosomes/ell e. Empty vetor si-res. DNA Erly pssge p P-p Ser CD CD Tsg d... Reltive numer of ells.. Dys Reltive mounts of poptoti ells μm μm μm f Empty vetor si-res. DNA γhax pst/q DAPI DNA dmge foi positive ells (%) Erly pssge Reltive mount of exosomes/ell.. Reltive mount of exosomes/ell.. Reltive mounts of poptoti ells DNA dmge foi positive ells (%) Reltive mounts of poptoti ells DNA dmge foi positive ells (%) Figure Inhiition of exosome seretion in pre-senesent HDFs. () Pre-senesent TIG- ells were sujeted to trnsfetion with indited sirna oligos twie (t dy intervls). These ells were then sujeted to western lotting using ntiodies shown right () or to exosome isoltion followed y western lotting using ntiodies ginst nonil exosome mrkers shown right (exosome) nd NnoSight nlysis () for quntittive mesurement of isolted exosome prtiles. The representtive dt from three independent experiments re shown. ws used s loding ontrol. ( d) Pre-senesent TIG- ells ultured under the onditions desried in were sujeted to ell prolifertion nlysis (), poptosis nlysis t dy () or to immunofluoresene stining for mrkers of DNA dmge (g-hax [red], phosphor-ser/thr ATM/ATR (pst/q) sustrte [green] nd,-dimidino--phenylindole [lue]) (d). The representtive dt from three independent experiments re shown. The histogrms indite the perentge of nulei tht ontin more thn foi positive for oth g-hax nd pst/q stining (d). At lest ells were sored per group (d). (e,f) Pre-senesent TIG- ells were infeted with retrovirus enoding flg-tgged wild-type or protein ontining mutted sirna levge site (lnes nd ) or empty vetor (lnes nd ). After seletion with puromyin, ells were trnsfeted with indited sirna oligos nd then sujeted to western lotting using ntiodies shown right, NnoSight nlysis for quntittive mesurement of isolted exosome prtiles, poptosis nlysis t dy or to immunofluoresene stining for mrkers of DNA dmge. ws used s loding ontrol. The representtive dt from three independent experiments re shown. For ll grphs, error rs indite men±s.d. of triplite mesurements. (Po.. Po.; one-wy nlysis of vrine). ells. Indeed, immuno-gold lelling of doule-strnded DNA (dsdna) followed y trnsmission eletron mirosopy reveled the presene of DNA in ertin proportion of the exosomes in MVEs (Fig. ). Moreover, eletrophoresis nd DNA sequening nlyses indited tht vrious lengths of dsdna frgments spnning ll hromosomes, ut not from mitohondri, re present in the exosomes relesed from HDFs (Fig.,). These results re onsistent with reent reports showing tht exosomes sereted from humn ner ell lines ontin dsdna from ll hromosomes,. Moreover, surose NATURE COMMUNICATIONS :7 DOI:./nomms7

5 NATURE COMMUNICATIONS DOI:./nomms7 ARTICLE sirna(): sirna(): sirna(): sirna(): ATM/ATR ATM ATM/ATR ATR Reltive numer of ells Dys Figure Exosomes seretion prevents ATM/ATR-dependent DDR. Pre-senesent TIG- ells were trnsfeted with two different sets of vlidted sirna oligos indited t the top of the pnel for twie t dy intervls. These ells were then sujeted to western lotting using ntiodies shown right () or to ell prolifertion nlysis (). ws used s loding ontrol (). The representtive dt from three independent experiments re shown. Error rs indite men±s.d. of triplite mesurements. grdient seprtion of exosomes prepred from pre-senesent HDFs reveled tht hromosoml DNA frgments were indeed present in the sme frtion ontining nonil exosome mrkers (Fig. d), further inditing tht the exosomes sereted from HDFs ontin hromosoml DNA frgments. Note tht lthough exosomes re formed in the ytoplsm, dmged nuler DNA is known to move into the ytoplsm,,7. Moreover, the mount of DNA in the exosomes (exosoml DNA) ws sustntilly inresed when pre-senesent HDFs were treted with DNA-dmging gents or rendered senesent, ompnied y the umultion of nuler DNA in the ytoplsm (Supplementry Fig. ). It is therefore most likely tht dmged nuler DNA is the soure of the exosoml DNA. Notly, the inhiition of exosome seretion lso indued the ytoplsmi umultion of nuler DNA in pre-senesent HDFs (Supplementry Fig. 7), inditing tht exosome seretion prevents the ytoplsmi umultion of nuler DNA. Consistent with this ide, the overexpression of Dnse, lysosoml DNA endonulese tht trgets dsdna in the ytoplsm,, diminished the ytoplsmi umultion of nuler DNA nd llevited the tivtion of the DDR pthwy in ells with or depletion (Fig. d). These results led us to propose tht exosome seretion prevents the errnt tivtion of DDR pthwys, y exreting hrmful ytoplsmi DNA of nuler origin from the ells. Exosome seretion prevents errnt innte immune response. These oservtions then rised the question of how the ytoplsmi umultion of nuler DNA provokes the DDR in norml humn ells. Cytoplsmi DNA is reportedly deteted y DNA sensors s dnger signl, resulting in the tivtion of the innte immune response, suh s the type I IFN pthwy 9,. Moreover, it hs een reported tht the IFN pthwy n provoke the DDR y elevting the intrellulr levels of ROS in HDFs 9. These findings prompted us to exmine if the inhiition of exosome seretion provokes the DDR, through the tivtion of the innte immune response y the ytoplsmi umultion of nuler DNA in norml humn ells. Indeed, the IFN pthwy ws strikingly tivted when exosome seretion ws inhiited, oinident with the elevtion of the intrellulr levels of ROS in HDFs (Fig. e). Notly, these phenomen were ttenuted when Dnse ws overexpressed in HDFs (Fig. e), somewht onsistent with previous oservtion tht mie lking Dnse gene spontneously produe high levels of type I IFNs nd show emryoni lethlity tht is resued y removing IFN reeptor. Moreover, the tivtion of DDR pthwys nd the onset of poptosis were sustntilly diminished when ROS prodution ws inhiited y N-etyl-L-ysteine (NAC) tretment in HDFs with or depletion (Fig. ). Similr results were lso oserved when STING, ytoplsmi dsdna sensor 9, or its tivtor GAS, ws knoked down, using previously vlidted sirna oligos in HDFs with or depletion (Fig. 7 nd Supplementry Fig. ). Tken together, lthough other mehnisms my lso e involved, the simplest explntion of our dt is tht exosome seretion preserves ellulr homeostsis y loking the errnt tivtion of the innte immune response vi preventing the ytoplsmi umultion of hrmful nuler DNA, t lest to some extent in norml humn ells. It should lso e noted tht, s seen in murine firolsts (Supplementry Fig. 9), the depletion of using hydrodynmis-sed in vivo trnsfetion provoked the redution of exosome seretion nd the errnt tivtion of the DDR in mouse liver (Fig. ), implying tht this mhinery my ontriute more rodly to tissue homeostsis in vivo. Exosomes prevent the virl hijking of ellulr mhinery. Finlly, to further lrify the iologil signifine of our findings, we nlysed whether exosome seretion ould lso expel exogenous DNA, suh s virl DNA, from ells. To this end, HDFs were infeted with reominnt denovirus enoding NATURE COMMUNICATIONS :7 DOI:./nomms7

6 ARTICLE NATURE COMMUNICATIONS DOI:./nomms7 αdsdna (MVE) αdsdna (Exosome) DNA mount (fluoresene units),, nm nm DNA size (p) X. X 9 9 (RPKM)... Genomi DNA... Y (Chromosome numer) Y (Chromosome numer) Density (g ml ). d. (RPKM).. Exosoml DNA.... TSG CD CD Histone H Histone H 7 9 Totl onentrtion (prtiles per ml) Frtion no..e+ E+ E+ GRM7 Reltive mounts of exosoml DNA, qpcr GPC,, 7 9 Frtion no. Figure Exosomes ontin hromosome frgments. () trnsmission eletron mirosopy mirogrph of MVE in pre-senesent TIG- ells following immuno-gold lelling for dsdna. Gold prtiles re depited s lk dots. Right imge shows digitlly zoomed re of exosome. (,) Exosoml DNA isolted from pre-senesent TIG- ells were sujeted to size distriution nlysis using Eletrophoresis Bionlyzer system () or to deep sequening nlysis (). Genomi DNA of TIG- ells re lso sujeted to deep sequening nlysis, s ontrol (). The red ount of eh -k in ws normlized to RPKM nd orreted y the mppility (). (d) Purified exosomes from pre-senesent TIG- ells were sujeted to surose density-grdient seprtion followed y western lotting using ntiodies shown right, NnoSight nlysis () for quntittive mesurement of isolted exosome prtiles nd quntittive PCR nlysis for detetion of genomi DNA frgments (GRM7 nd GPC). NATURE COMMUNICATIONS :7 DOI:./nomms7

7 NATURE COMMUNICATIONS DOI:./nomms7 ARTICLE +Vetor +Flg-Dnse Flg d DNA dmge foi positive ells (%) +Vetor +Flg-Dnse Reltive mount of exosomes per ell Exosome Reltive levels of ytoplsmi nuler DNA. +Vetor +Vetor +Flg-Dnse +Flg-Dnse CD CD Tsg GRM7(Chr) FGFR(Chr) GPC(Chr) e Reltive levels of IFNβ mrna Reltivelevels of ROS Reltive mounts of poptoti ells +Vetor IFNβ ROS Apoptosis +Flg-Dnse Figure Overexpression of Dnse ttenuted the effets of or knokdown in HDFs. Pre-senesent TIG- ells were infeted with retrovirus enoding flg-tgged Dnse (lnes ) or empty vetor (lnes ). After seletion with puromyin, ells were trnsfeted with indited sirna oligos nd then sujeted to western lotting using ntiodies shown right (), NnoSight nlysis () for quntittive mesurement of isolted exosome prtiles nd western lotting using ntiodies ginst nonil exosome mrkers shown right (exosome) (), isoltion of ytoplsmi frtion followed y quntittive PCR (qpcr) nlysis of hromosoml DNA (), immunofluoresene stining for mrkers of DNA dmge (g-hax [red], pst/q (green) nd,-dimidino--phenylindole (lue)) (d), qpcr nlysis of IFN gene expression (e), nlysis of intrellulr ROS levels (e) or to poptosis nlysis t dy (e). ws used s loding ontrol (). The histogrms indite the perentge of nulei tht ontin more thn foi positive for oth g-hax nd pst/q stining (d). At lest ells were sored per group (d). The representtive dt from three independent experiments re shown. For ll grphs, error rs indite men±s.d. of triplite mesurements. (Po.. Po.; one-wy nlysis of vrine). green fluoresent protein (GFP), with or without the inhiition of exosome seretion (Fig. 9 e). Indeed, the denovirl DNA ws exreted from the infeted ells y exosomes (Fig. 9d). Interestingly, the levels of virlly enoded protein expression were strikingly inresed when exosome seretion ws inhiited, s judged y the GFP expression levels (Fig. 9,e), suggesting tht exosome seretion lso trgets infeted virl DNA for exretion from ells. We thus next tested if this mhinery funtions in preventing virus prodution in infeted ells. Indeed, the inhiition of exosome seretion resulted in drmti inrese in the prodution of infetious denovirus in HEK9 ells (Fig. 9f i), inditing tht exosome seretion lso plys n importnt role in preventing the virl hijking of ellulr mhinery, lthough other mehnisms re lso likely to e involved (see model in Fig. ). These results re somewht similr to those oserved in ltent Epstein Brr virus-infeted ells where sorting nd seretion of pro-inflmmtory virl RNA vi exosomes prevent tivtion of IFN- pthwy. Colletively, these results reveled n dditionl mehnism for the ntivirl tivity of exosomes, further illustrting the iologil signifine of the exosome-medited removl of hrmful DNA from ells. Disussion Exosome seretion hd initilly een proposed s mehnism to mintin ellulr homeostsis, y removing exess or osolete moleules from ells,9. However, emerging evidene hs reveled tht the seretion of exosomes lso plys importnt roles in mediting ell-to-ell ommunition, y tivting NATURE COMMUNICATIONS :7 DOI:./nomms7 7

8 ARTICLE NATURE COMMUNICATIONS DOI:./nomms7 NAC: DNA dmge foi positive ells (%) + Reltive levels of ROS Reltive mounts of poptoti ells NAC: + d * NAC: + NAC: + Figure Redution of ROS levels ttenuted the effets of or knokdown in HDFs. Pre-senesent TIG- ells were trnsfeted with vlidted sirna oligos indited t the top of the pnel for two times t dy intervls in the presene or sene of mm N-etyl ysteine. These ells were then sujeted to western lotting using ntiodies shown right (), nlysis of intrellulr ROS levels (), immunofluoresene stining for mrkers of DNA dmge (g-hax (red), pst/q (green) nd,-dimidino--phenylindole (lue)) () or to poptosis nlysis (d). The histogrms indite the perentge of nulei tht ontin more thn foi positive for oth g-hax nd pst/q stining (). At lest ells were sored per group (). The representtive dt from three independent experiments re shown. For ll grphs, error rs indite men±s.d. of triplite mesurements. (*Po.. Po.. Po.; one-wy nlysis of vrine). vrious signlling pthwys in ells with whih they fuse nd intert 7. Despite onsiderle progress in understnding how ell-to-ell ommunition is implemented y exosomes 7,,7, fr less is known out how exosome seretion mintins ellulr homeostsis in exosome-sereting ells. In this study, we provide evidene tht the inhiition of exosome seretion, phrmologilly or y RNAi, tivtes the ATM/ATR-dependent DDR in oth senesent nd non-senesent norml humn ells. This response is t lest prtly due to the umultion of nuler DNA frgments in the ytoplsm, sine the redution of ytoplsmi nuler DNA y the overexpression of Dnse or the inhiition of the STING/GAS ytoplsmi DNA sensor y RNAi sustntilly llevited this response. Although other mehnisms my lso e involved, the simplest explntion of our dt is tht exosome seretion preserves ellulr homeostsis y loking the errnt tivtion of the DDR vi preventing the ytoplsmi umultion of hrmful nuler DNA, t lest to some extent in norml ells (see model in Fig. ). This mehnism ppers to eome more importnt in senesent ells, presumly euse nuler DNA tends to umulte in the ytoplsm in senesent ells 7 (see lso Supplementry Fig. g). However, neither nor nor nsmse funtions exlusively in exosome seretion,,9. For exmple, is known to ply key roles in ytokineti sission. Thus, it is possile tht dditionl mehnisms my lso e involved in the tivtion of the DDR pthwy in our experimentl setting. Nevertheless, we oserved extly the sme effets when we loked the funtions of these proteins (Figs nd, Supplementry Figs nd ) nd other proteins (Tsg (ref. 7), R7 (ref. ) or Slp (ref. )) known to e involved in exosome iogenesis or seretion in HDFs (Supplementry Fig. ). Moreover, we did not see sustntil inrese in the frequeny of multinulete ells, sign of ytokineti filure, in HDFs with depletion (Supplementry Fig. ). Furthermore, the purified exosomes ontined genomi DNA frgments (Fig. ) nd hd the potentil to provoke the DDR in reipient norml humn ells, depending on the mounts of dded exosomes (Supplementry Fig. ). Thus, lthough we nnot yet ompletely rule out the possiility tht dditionl mehnism(s) my lso e involved, it is most likely tht exosome seretion mintins ellulr homeostsis y exreting hrmful ytoplsmi DNA, t lest to some extent, in norml ells. It is lso worth noting tht neither poptosis nor nerosis ws oserved in ontrol pre-senesent HDFs (Fig., lne ), preluding the possiility tht the genomi DNA frgments oserved in our exosome frtions originted from poptoti odies. Along similr line, the inhiition of poptosis y Z-VAD, pn spse inhiitor, did not hve ny impt on the pperne of the DDR in pre-senesent HDFs treted with exosome inhiitors (Supplementry Fig. ). Colletively, these results indite tht the DDR provoked y the lokge of exosome seretion is not simply onsequene of the uptke of poptoti DNA frgments through the endoytosis of poptoti odies in HDFs. It hs een shown tht the defiieny of Dnse leds to umultion of dmged self DNA nd indution of pro-inflmmtory ytokine pthwys in murine ells. Moreover, removl of dmged self DNA y Dnse ws shown to require utophgy-medited delivery of the DNA to lysosomes. These notions, in onjuntion with very reent oservtion tht prevention of utophgy-lysosome fusion inreses exosome seretion, imply tht exosome seretion nd utophgy my t in omplementry mnner to remove pro-inflmmtory DNA from ells (see model in Fig. ). The ovious remining questions re the origins of the exosoml DNAs nd how re they generted. Notly, ells in G phse of the ell-yle re more resistnt to the inhiition of exosome seretion, s ompred to those in the proliferting NATURE COMMUNICATIONS :7 DOI:./nomms7

9 NATURE COMMUNICATIONS DOI:./nomms7 ARTICLE sirna(): sirna(): Reltive levels of IFNβ mrna Reltive levels of ROS sirna(): STING IFNβ ROS STING Reltive numer of ells sirna(): sirna(): STING Dys sirna(): STING Figure 7 Depletion of STING ttenuted the effets of or knokdown in HDFs. Pre-senesent TIG- ells were trnsfeted with two different sets of vlidted sirna oligos indited t the top of the pnel for three times t dy intervls. These ells were then sujeted to western lotting using ntiodies shown right (), ell prolifertion nlysis () nd quntittive PCR nlysis of IFN gene expression or to nlysis of intrellulr ROS levels (). ws used s loding ontrol (). The representtive dt from three independent experiments re shown. For ll grphs, error rs indite men±s.d. of triplite mesurements. (Po.. Po.; one-wy nlysis of vrine). phse in pre-senesent HDFs (Supplementry Fig. ). Thus, it is very likely tht exosoml DNA frgments re generted y the onservtive homologous reomintion tht ours preferentilly in the lte S, G nd M phses of the ell-yle in pre-senesent ells. These oservtions lso suggest tht the ell-yle sttus my determine whether the inhiition of exosome seretion in pre-senesent ells drives the ells into poptosis or senesene-like ell-yle rrest. However, euse senesent ells re non-prolifertive, exosoml DNA frgments re likely to e generted y different mehnism(s) in these ells. This ide is somewht onsistent with reent oservtions tht dmged nuler DNA tends to move into the ytoplsm,,7. It is lso worth mentioning tht t lest ertin proportion of the exosoml DNA ws ound to histones (Supplementry Fig. ), nd histones re reportedly undnt in exosomes,7 (see lso Fig. d). Thus, it is tempting to speulte tht nuler DNA my e loded into exosomes through histones. In summry, while the preise mehnisms underlying the nuler DNA loding into exosomes require further lrifition, our results support model in whih exosome seretion mintins ellulr homeostsis y removing hrmful ytoplsmi DNA from ells, t lest to some extent, in ertin types of norml ells (see model in Fig. ). These results revel n unexpeted role of exosome seretion, nd provide new insights into the mintenne of ellulr homeostsis in norml ells, opening up new possiilities for its ontrol. Methods Cell ulture. TIG- ells,, HEK-9T ells, HeL ells nd UOS ells were otined from Jpnese Cner Reserh Resoures Bnk (JCRB) nd mouse emryoni firolsts (MEFs) were estlished from dy. mouse emryos. These ells were ultured in Duleo s Modified Egle s medium supplemented with % foetl ovine serum. HRPE ells (Lonz In.) nd HEK ells (Cell Applitions In.) were ultured ording to the mnufturer s instrutions. Erly pssge TIG- ells (o popultion doulings) were used s growing ells, nd lte pssge TIG- ells (7 popultion doulings) tht esed prolifertion were used s replitive senesent ells. For retrovirl infetion, TIG- ells were rendered sensitive to infetion y eotropi retroviruses. Cells were then infeted with reominnt retroviruses enoding Rs V (in pbe puro ), DNse (in pmrx puro 9 ), nd sirna-resistnt nd (in pmrx puro). MEFs were infeted with reominnt retroviruses enoding shrna ginst (in pretrosuper puro ). After puromyin seletion, pools of drug-resistnt ells were nlysed 7 dys fter infetion. In some experiments, ells were treted with n N-SMse inhiitor GW9 (SIGMA), Spiroepoxide (Snt Cruz), doxoruiin (Wko), Z-VAD-FMK (Promeg) or N-etyl-L-ysteine (SIGMA). We hve onfirmed the sene of myoplsm ontmintion in our tissue ulture ells. Cell prolifertion ssy. Cells were plted on mm dishes with mm grids (Thermo Fisher Sientifi). The numer of ells in eh grid ws ounted every dy, nd the reltive numer of ells ws lulted sed on n djusted ell numer t dy set t.. Apoptosis ssy. Apoptoti ells were wshed with inding uffer nd stined with n fluoresein isothioynte-annexin V solution using n poptoti/helthy ells detetion kit (PromoKine). After n inution t room temperture for min, fluoresene ws mesured with Wll ARVO Multilel ounter (PerkinElmer Co., Ltd.). NATURE COMMUNICATIONS :7 DOI:./nomms7 9

10 ARTICLE NATURE COMMUNICATIONS DOI:./nomms7 d shrna: Non invsive Invsive.. 7 (photons per seond) μm Reltive mounts of exosome per μg tissue μm BP DAPI shrna: shrna:. DNA dmge foi positive ells (%) Mouse liver shrna: Mouse liver Mouse liver Figure Inhiition of exosome seretion in mouse liver. ICR mie were sujeted to hydrodynmi til vein injetion with plsmid enoding firefly luiferse or smll hirpin RNA (shrna) ginst or ontrol (n ¼ per group). After h, the mie trnsfeted with firefly luiferse were sujeted to in vivo ioluminesent imging for onfirmtion of the trnsfetion effiieny (), nd then other mie were euthnized nd livers were sujeted to western lotting using ntiodies shown right (), NnoSight nlysis () for quntittive mesurement of isolted exosome prtiles () or to immunofluoresene nlysis of liver setion (d). ws used s loding ontrol (). Setion of livers were sujeted to immunofluoresene stining for mrkers of DNA dmge (BP (red) nd,-dimidino--phenylindole (lue)) (d). The histogrms indite the perentge of nulei tht ontin more thn foi positive for BP stining. At lest ells were sored per group. The representtive dt from three independent experiments re shown. For ll grphs, error rs indite men±s.d. of triplite mesurements. (Po.. Po.; one-wy nlysis of vrine). Exosome isoltion from ells. For exosome isoltion, ells were inuted in Duleo s Modified Egle s medium with % foetl ovine serum, depleted of mirovesiles, for h. The ell superntnts were olleted nd entrifuged t g for min to eliminte ells, nd then t,g for min to remove the ellulr deris. The superntnt ws then entrifuged t,g for min, followed y filtrtion through.-mm pore filter (Sigm) to remove the ontminting poptoti odies, shedded vesiles nd ell deris. The olleted superntnt ws then ultrentrifuged t,g for 7 min nd the preipitte ws rinsed with PBS twie, s previously desried,. For surose density-grdient seprtion, purified exosome frtions were further sujeted to surose density-grdient entrifugtion. In rief,. ml of purified exosome frtions were suspended in. ml of. M surose in mm HEPES/NOH t ph 7. nd loded in SW Ti tue (Bekmn Coulter), nd ml of ontinuous surose grdient, from. M to. M surose in mm HEPES/NOH t ph 7., ws lyered on top. Tues were entrifuged t,g for h t C. Ten frtions with equl volumes (. ml) were olleted from the top of the grdient. The density for eh frtion fter ultrentrifugtion ws determined using refrtometer (RX-, Atgo Co.Ltd, Tokyo, Jpn). All frtions were resuspended in ml PBS nd were gin entrifuged t, g for 7 min t C. The wshed pellet ws resuspended in. ml PBS. The olleted frtions were stored t C untilfurthernlysis. The size distriution nd onentrtion of the exosomes were determined y, using NnoSight LM system (NnoSight Ltd.). Exosome isoltion from mouse tissue. Fresh mouse liver setions ( mg) were wshed with ml of PBS nd then inuted with. ml of RPMI- medium (Nli Tesque) inluding ntiiotis (Sigm), t 7 C for h with gittion in CO inutor. The medium ws olleted nd entrifuged t,g for min nd gin,g for min, followed y filtrtion through.-mm pore filter (Sigm). The superntnt ws then sujeted to ultrentrifugtion t,g for 7 min, nd the preipitte ws rinsed with PBS twie. The size distriution nd onentrtion of the exosomes were determined using NnoSight LM system (NnoSight Ltd.). Quntittive mesurement of isolted exosoml DNA. To redue externl DNA ontmintion, prior to DNA extrtion, exosomes were treted with DNse I (Rohe In.) nd Exonulese III (Tkr In.), ording to the mnufturers instrutions. After het intivtion, the exosoml DNA ws purified y Proteinse K (Wko) tretment. The mount of dsdna ws determined using n Agilent High Sensitivity DNA kit (Agilent Tehnologies) or QuntiFluor dsdna System (Promeg). Cytoplsmi nuler DNA nlysis. Cytoplsmi frtions were otined using n NE-PER Nuler nd Cytoplsmi Extrtion Kit (Thermo Fisher Sientifi). Cytoplsmi DNA ws purified y Proteinse K (Wko) tretment. The mount of nuler DNA ws determined y quntittive rel-time PCR, using three different sets of primers designed for different hromosomes (GRM7, FGFR nd GPC). Deep sequening of exosoml DNA. The deep sequening nlysis ws performed s previously desried. Briefly, ng portions of exosoml DNA nd genomi DNA were shered using Bioruptor USD- th sonitor (Cosmo-Bio; 9 sonitions of s with -s intervls t W). Lirries were prepred ording to the mnufturer s instrutions (Illumin). Fifteen nnogrm of shered DNA ws end-repired using mix of Klenow DNA polymerse, T DNA polymerse nd T polynuleotide kinse (NEB), tiled with n A se using Klenow Frgment exo minus (NEB), nd ligted with the Illumin single-end dptor, using DNA ligtion Kit (TKR). Adptor-ligted frgments of B p were purified using the E-gel SizeSelet system (Life Tehnologies), nd were sujeted to yles of PCR mplifition using KOD FX polymerse (TOYOBO). To remove the remining PCR primers, the mplified produts were further purified using AMPure XP Kits (Bekmn Coulter). Lirries were quntified using BioAnlyzer with High Sensitivity DNA Kit (Agilent) nd sequened using n Illumin Genome Anlyzer IIx to generte 7 -p single-end reds. The sequene reds were ligned to the humn referene genome, UCSC hg inluding single unit of humn riosoml DNA repets, using BWA with defult prmeters rin. Reds with low mpping qulity (o) were disrded, to llow reds mpped to unique genomi positions to e onsidered with onfidene. Possile PCR duplites were removed using smtools. We divided eh hromosome into non-overlpping -k ins nd lulted the numer of mpped reds per kilo se per million reds (RPKM) for eh in. To orret mppility is, ll possile -mer sequenes of the referene genome were mpped k in the sme mnner s the sequened reds nd the RPKM vlue ws divided y the frtion of mpple ses for eh in. NATURE COMMUNICATIONS :7 DOI:./nomms7

11 NATURE COMMUNICATIONS DOI:./nomms7 ARTICLE d Reltive levels of denovirl DNA in exosome sirna trnsfetion. +Ad-GFP Ad-GFP infetion TIG- ells Dy e Smple olletion Mok Mok +Ad-GFP +Ad-GFP GFP μm μm μm μm Reltive mount of exosomes per ell Exosome. +Ad-GFP CD CD Tsg f sirna trnsfetion 9 ells Ad-GFP infetion Dy (g, h) Adenovirus purifition Ad-GFP infetion g h Reltive mount of exosomes per ell Exosome. CD CD Tsg 9 ells Dy i Adenovirus titrtion (i) mm mm mm Virus titer (x ifu per ml) Figure 9 Exosome seretion prevents virl hijking of ellulr mhinery. () Timeline of the experimentl proedure. ( e) Pre-senesent TIG- ells trnsfeted with indited sirna oligos followed y infetion with reominnt denovirus enoding GFP (Ad-GFP) were sujeted to western lotting using ntiodies shown right (), NnoSight nlysis () nd western lotting ginst nonil exosome mrkers for quntittive mesurement of isolted exosome prtiles (), quntittive mesurement of isolted denovirl DNA from exosome using quntittive PCR (d), or to mirosopi nlysis of GFP expression (e). The representtive dt from three independent experiments re shown. (f) Timeline of the experimentl proedure. (g i) 9 ells were trnsfeted with indited sirna oligos followed y infetion with Ad-GFP. These ells were then sujeted to western lotting using ntiodies shown right (g), NnoSight nlysis () nd western lotting ginst nonil exosome mrkers for quntittive mesurement of isolted exosome prtiles (h) or to titrtion of generted Ad-GFP (i). The histogrms indite the virus titre (i). For ll grphs, error rs indite men±s.d. of triplite mesurements. (Po.. Po.; one-wy nlysis of vrine). Eletron mirosopy. Exosomes isolted from TIG- ells were sored to formvr ron-oted nikel grids nd immune-lelled with n nti-cd ntiody (BD, 9), followed y nm of gold-lelled seondry ntiody (British BioCell Interntionl Ltd.). The smples were fixed in % glutrldehyde in. M phosphte uffer, nd % phospho tungsti id solution (ph 7.) ws used for negtive stining. For the oservtion of ellulr dsdna, TIG- ells were plted on the gold disks nd frozen in liquid propne t 7 C. The smples were freeze sustituted with.% glutrldehyde in etone nd % distilled wter t C for dys. After dehydrtion, the smples were emedded into resin (LR White, London Resin Co. Ltd.) nd ultr-thin setioned t nm using n ultrmirotome (Ultrut UCT, Lei). The smples were immunolelled with n nti-dsdna ntiody (Snt Cruz, s-79) in norml got serum nd % BSA, NATURE COMMUNICATIONS :7 DOI:./nomms7

12 ARTICLE NATURE COMMUNICATIONS DOI:./nomms7 DNA virus MVE Exosome follows:, -GCAGCAGAACAAAATCTCGACAACGACGAGGGATTGAA AATCG- (forwrd) nd -CGATTTTCAATCCCTCGTCGTTGTCGAGAT TTTGTTCTGCTGC- (reverse); nd, -CTTTGAAACTAGTGCAG CGAACGGTACGAATATAAGCCAAGC- (forwrd) nd -GCTTGGCT TATATTCGTACCGTTCGCTGCACTAGTTTCAAAG- (reverse). All DNAs were sequened on Geneti Anlyzer (Applied Biosystems) using BigDye Termintor v. Cyle Sequening Kit (Applied Biosystems). Quntittive rel-time PCR. Totl RNA ws extrted from ultured ells using mirvn kit (Thermo Fisher Sientifi), nd then sujeted to reverse trnsription using PrimeSript RT regent kit (TKR). Quntittive rel-time RT-PCR ws performed on StepOnePlus PCR system (Applied Biosystems) using SYBR Premix Ex Tq (TKR). The PCR primer sequenes were listed in Supplementry Tle. The mens±s.d. of three independent experiments re shown. lysosome DNse Cytosol Nuleus DNA DNA dmge STING ROS IFNβ Figure A model of exosome-medited ellulr homeostsis. The exosome seretion elimintes hrmful ytoplsmi DNA from ells. The inhiition of exosome seretion uses the ytoplsmi umultion of nuler DNA, therey using the tivtion of STING, the ytoplsmi DNA sensing mhinery. This event provokes the innte immune response, suh s type I IFN pthwy, leding to the elevtion of the intrellulr levels of ROS. In turn, this tivtes the DDR in norml humn ells. This mhinery my lso ply keys role in preventing virl hijking of host ells y exreting virl DNA from ells. followed y nm gold-lelled seondry ntiody. The grids were pled in % glutrldehyde in. M phosphte uffer nd dried. They were stined with % urnyl ette for min nd Led stin solution (SIGMA). The smples were oserved with trnsmission eletron mirosope (JEM-Plus, JEOL Ltd.) t kv. Digitl imges were otined with CCD mer (VELETA, Olympus Soft imging solutions GmH). Fluoresene mirosopi nlysis. Immunofluoresene nlysis ws performed using ntiodies ginst g-hax (:,, Millipore, -), phosphor-(ser/thr) ATM/ATR sustrte (:, Cell Signling Tehnology, ) nd BP (:,, Snt Cruz, s-7; :,, m, ). DNA ws stined with mg ml,-dimidino--phenylindole (Dojindo). Fluoresene imges were oserved nd photogrphed using n immunofluoresene mirosope (Crl Zeiss),. RNAi. RNAi ws performed y the trnsfetion of sirna oligos using the Lipofetmine RNAiMAX trnsfetion regent (Thermo Fisher Sientifi), ording to the mnufturer s instrutions. The sequenes of the sirna oligos were s follows., : -GAACCUGGAUAAUGAUGAA-. (ref. ): -GCUGCCAAUGGGACAAACA-.R7 (ref. ): -CCCAAAUU CAUCACUACAGUA-. Tsg (ref. ): -CCUCCAGUCUUCUCUCGUC-. ATM : -UGAAGAUGGUGCUCAUAAA-. ATR : -CCUCCGUGAUG UUGCUUGA-. GAS : -GGAAGGAAAUGGUUUCCAA-. MISSION sirna trgeting for Slp/Sytl ( -CUGUUAAUCCACUAUAUGA- )ws purhsed y Sigm Genosys. ON-TARGETplus sirnas (Dhrmon) nd non-trgeting ontrol sirna were used to trget Sting 7 nd. Knokdown effiieny ws onfirmed y quntittive rel-time PCR. For knokdown of or in MEFs nd mouse liver, the trget sequenes of the shrna used were s follows: mouse, -GAACCUGGAUAAUGAUGAA- (ref. ); ontrol, -CAUUGCUAUAGAGGCAGAU- (ref. ). Plsmids. The epitope tgged DNAs of DNse, nd were loned into the pmrx puro retrovirus vetor,,9. To generte sirna-resistnt nd mutnts, seven- or six-point muttions, whih do not hnge the enoded mino ids, were introdued into the -nuleotide trget regions of the nd DNAs, using Quik Chnge Site-direted Mutgenesis kit (Strtgene). For the onstrution of sirna-resistnt mutnts, the primer sequenes were s Western lotting. For western lotting nlysis, ells or mouse liver tissues were lysed in lysis uffer ( mm Hepes, ph 7., mm NCl, mm EDTA,. mm EGTA, % glyerol,.% Tween, mm -glyerophosphte) with % Protese inhiitor oktil (Nli Tesque),. The protein onentrtion ws determined using DC Protein Assy (Bio-Rd), nd proteins were seprted y SDS polyrylmide gel eletrophoresis nd trnsferred onto polyvinylidene difluoride memrne (EMD Millipore). After loking with % milk, memrnes were proed with primry ntiodies (:,) s follows: nti-h-rs (Snt Cruz, s-9), p (IBL, ), G9 (Cell Signling Tehnology, ), GLP (MBL, D-), DNMT (BD Trnsdution Lortories, ), Lmin B (Snt Cruz, s-7), phospho-p (Ser; Cell Signling Tehnology, 9), ATM (SIGMA, A), ATR (MABI, -), (Proteinteh, ), (Proteinteh, 7), R7 (Proteinteh, ), Slp (Proteinteh, ), STING (Cell Signling Tehnology, 7), GAS (Cell Signling Tehnology, ), Cspse (Cell Signling Tehnology, 9), CD (BD, 9), CD (Cosmo-Bio, SHI-EXO-M), Tsg (Proteinteh, 97), Histone H (m, 79), Histone H (m, ), DNse (ProSi, 9), GFP (Clonteh, ) nd -tuulin (SIGMA, T9). The memrnes were then inuted with seondry ntiodies (GE Helthre; :,) nd visulised with the Chemi-Lumi One regent (Nli Tesque). Full immunolots re provided in Supplementry Fig.. ROS nlysis. To ssess the levels of intrellulr ROS genertion, ells were inuted with mm DCF-DA (Cliohem) t 7 C for min. The pek exittion wvelength for oxidised DCF ws nm nd tht for emission ws nm, mesured y using Wll ARVO Multilel ounter (PerkinElmer Co., Ltd.). Adenovirus infetion. TIG- ells were trnsfeted with sirna oligo nd the next dy ells were infeted with reominnt denovirus (Ad) enoding GFP,t multipliity of infetion of. For virl titrtion nlysis, 9 ells were trnsfeted with sirna oligo nd dys lter ells were infeted with Ad enoding GFP, nd the virus titre ws determined using n Adeno-X Rpid Titer Kit (Clonteh). Animl experiments. Hydrodynmis-sed trnsfetion ws performed using -dy-old mle ICR mie 9. In rief, mg of plsmid enoding firefly luiferse or mg of shrna plsmid ginst or ontrol plsmid, in. ml sline, ws injeted into the til vein of mie over short durtion of 7 s, to filitte the uptke of plsmid DNA in the liver 9. Forty-eight hours lter, mie trnsfeted with luiferse were sujeted to in vivo ioluminesent imging,7 for onfirmtion of the trnsfetion effiieny, nd mie trnsfeted with shrna were euthnized nd the liver setions were sujeted to exosome olletion, western lotting or immunofluoresene nlysis. The smple size used in this study ws determined sed on the expense of dt olletion, nd the requirement for suffiient sttistil signifine. Rndomistion nd linding were not used in this study. Mie with ody weights etween. nd. g t the ge of dys were used for experiments. All niml re ws performed ording to the protools pproved y the Committee for the Use nd Cre of Experimentl Animls of the Jpnese Foundtion for Cner Reserh. Sttistil nlysis. Sttistil signifine ws determined using Student s t-test nd one-wy nlysis of vrine. P vlues o. were onsidered signifint. Dt vilility. Sequening dt of exosoml DNA hs een deposited in the DDBJ sequene red rhive under ession numer DRA. 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