Regulation of NKT cell-mediated immune responses to tumours and liver inflammation by mitochondrial PGAM5-Drp1 signalling

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1 Reeive Mr Aepte Aug Publishe 8 Sep DOI:.8/nomms97 Regultion of NKT ell-meite immune responses to tumours n liver inflmmtion by mitohonril PGAM-Drp signlling Young Jun Kng, Bo-Rm Bng, Kyung Ho Hn, Lixin Hong, Eun-Jin Shim, Jinhui M, Rihr A. Lerner & Motoyuki Otsuk OPEN The reeptor-interting protein kinse (RIPK) plys ruil roles in progrmme nerosis n innte inflmmtory responses. However, little is known bout the involvement of RIPK in NKT ell-meite immune responses. Here, we emonstrte tht RIPK plys n essentil role in NKT ell funtion vi tivtion of the mitohonril phosphtse phosphoglyerte mutse (PGAM). RIPK-meite tivtion of PGAM promotes the expression of ytokines by filitting nuler trnslotion of NFATn ephosphoryltion of ynmin-relte protein (Drp), GTPse is essentil for mitohonril homoeostsis. Ripk / mie show reue NKT ell responses to metstti tumour ells, n both eletion of RIPK n phrmologil inhibition of Drp protets mie from NKT ell-meite inution of ute liver mge. Colletively, the results ientify ruil role for RIPK-PGAM-Drp/NFAT signlling in NKT ell tivtion, n further suggest tht RIPK-PGAM signlling my meite rosstlk between mitohonril funtion n immune signlling. Deprtment of Immunology n Mirobil Siene, The Sripps Reserh Institute, L Joll, Cliforni 97, USA. Deprtment of Cell n Moleulr Biology, The Sripps Reserh Institute, L Joll, Cliforni 97, USA. Deprtment of Gstroenterology, Grute Shool of Meiine, University of Tokyo, Tokyo -, Jpn. Corresponene n requests for mterils shoul be resse to Y.J.K. (emil: ykng@sripps.eu). NATURE COMMUNICATIONS :87 DOI:.8/nomms97 & Mmilln Publishers Limite. All rights reserve.

2 NATURE COMMUNICATIONS DOI:.8/nomms97 Reent stuies hve emonstrte tht number of genes n moleules n regulte the signlling pthwys tht inue nerosis. Reeptor-interting protein kinse (RIPK), is require s n upstrem regultor of neroptosis, the spse-inepenent form of nerosis. The relese of mge-ssoite moleulr pttern moleules (DAMPs) by neroptoti ells in turn tivtes innte immune ells, suh s mrophges n DCs, to proue proinflmmtory ytokines. Thus, RIPK-meite neroptosis hs been suggeste to be n tive inititor of inflmmtion inue by DAMPs relese from infete or mge ying ells, n hs been implite s n unerlying pthogeni mehnism for number of inflmmtory iseses. Although we o not yet hve omplete unerstning of the inuers n finl exeutioners of neroptosis, the pseuokinse mixe-linege kinse omin-like (MLKL) n the mitohonril phosphtse phosphoglyerte mutse (PGAM) hve reently been ientifie s key regultors of RIPK-meite signlling. The intertion of MLKL n RIPK ours through RIPK- epenent phosphoryltion of the C-terminl kinse-like omin of MLKL,. MLKL hs been shown to be inispensble for the inution of neroptosis, s MLKL-efiient mie re protete ginst nerosis-relte erulen-inue ute pnretitis 7. PGAM is reruite to the RIPK/RIPK nerosome, where it is phosphorylte by RIPK. PGAM further ephosphoryltes n tivtes ynmin-relte protein (Drp), GTPse tht regultes mitohonril fission n the proution of mitohonril retive oxygen speies (mtros). mtros lso ply role in ell eth by nerosis 8 n rive proinflmmtory ytokine expression in mrophges n T-ells 9,. However, inhibition of ROS proution by ntioxints oes not ffet ell eth, initing tht PGAM my regulte RIPK-epenent signlling inepenently of ROS genertion. Reent stuies hve provie eviene tht RIPK inepenently inues inflmmtion without promoting ell eth, lthough the mehnism(s) by whih this ours is not ler. Mitohonri re involve in wie rnge of biologil proesses, suh s genertion of energy, biosynthesis of essentil moleules n progrmme ell eth, n hve lso been implite in the tivtion n ontrol of innte n ptive immune responses. The mitohonril protein MAVS (mitohonril ntivirl signlling protein, lso known s IPS-, Crif, or Vis) is essentil for the tivtion of ntivirl innte immunity, n bnorml MAVS tivity is often ssoite with the evelopment n progression of inflmmtion in stetoheptitis, systemi lupus erythemtosus n hroni obstrutive pulmonry isese 8. Aitionlly, mtros generte by TLR signlling in mrophges tivte NF-kB, MAPK, n proinflmmtory ytokine proution n les to enhne bteriil responses 9. In T-ells, tivtion of TCR signlling-epenent mitohonril metbolism rpily upregultes mtros n inreses proution of IL-, expnsion of ntigen-speifi CD8 þ T-ells, n inution of n effetive memory response. However, the regultory role of mitohonri in other immune ell types hs not been fully eluite. Type nturl killer T-ells (NKT ells) re CD-restrite, lipi ntigen-retive, n immunoregultory T-lymphoytes. NKT ells rpily proue pro- n nti-inflmmtory ytokines upon stimultion, llowing them to regulte ellulr immune responses in bro spetrum of iseses,. For exmple, NKT ells promote the immune response to tumours n mirobil infetions, while suppressing immune responses in utoimmune iseses suh s ute heptitis n rthritis. Furthermore, NKT ell tivtion n be hrmful to the host in some inflmmtory iseses suh s theroslerosis. Despite their proxil funtions in ifferent isese onitions, NKT ells lerly ply ruil role in regulting the pthogenesis of vrious iseses. Although RIPK-epenent signlling in the regultion of inflmmtory responses hs been stuie extensively in innte immune ells, little is known bout its ontribution to the tivtion of NKT ells. In this stuy, we emonstrte tht RIPK-meite signlling regultes the tivtion of NKT ells in mouse moels of melnom n ute inflmmtory liver injury. Our results inite tht RIPK tivtes PGAM, whih in turn regultes the ephosphoryltion of NFAT n Drp to inue the expression of proinflmmtory ytokines, suggesting tht RIPK-PGAM-Drp signlling my ply key role in the rosstlk between mitohonril funtion n signlling for NKT ell tivtion. Results RIPK regultes tivtion of NKT ells. Reent stuies hve suggeste tht RIPK n ontribute to physiologil n pthologil immunity inepenently of its involvement in neroptosis. A previous stuy hs shown tht RIPK oes not regulte the tivtion of B ells, T-ells or mrophges. Consistent with this, we hve not etete n effet of Ripk efiieny on ytokine proution, tivtion of NF-kB n MAPK signlling, or prolifertion in TCR-stimulte T-ells, LPS-trete B ells n TLR-stimulte peritonel mrophges uner the onitions of our experiments (Supplementry Fig. ). Thus, our results suggest tht RIPK signlling oes not ply role in T-ells, B ells n mrophges. In this stuy, we ske whether RIPK plys role in the tivtion of NKT ells in vivo n in vitro. The glyolipi -gltosyl ermie (-GlCer) is potent tivtor of NKT ells n inues proution of regultory n inflmmtory ytokines,. Inee, -GlCermeite stimultion of wil-type (WT) liver leukoytes signifintly inrese expression of the proinflmmtory ytokines IFN-g, TNF n IL- t both the mrna n protein levels (Fig.,b). Unlike our observtions with T-ells, B ells n mrophges, liver leukoytes isolte from Ripk / mie showe signifintly reue proution of ytokines ompre with WT ells (Fig.,b), suggesting tht RIPK is ritil for the NKT ell ytokine expression n relese by -GlCer tretment. We next exmine the tivtion of signlling pthwys known to be essentil for RIPK-epenent ytokine proution in NKT ells. Although phosphoryltion n egrtion of IkB n phosphoryltion of ERK were not etete, the mgnitue n kinetis of p8 n JNK phosphoryltion in -GlCer-trete WT n Ripk-efiient liver leukoytes were omprble (Fig. ). These t inite tht RIPK oes not iretly regulte MAPK-epenent signlling for inflmmtory ytokine expression in NKT ells. Of note, we foun tht the frequeny of NKT ells in spleen n liver of Ripk / mie ws omprble to tht of WT mie (Supplementry Fig. ). Consistent with previous report 7, B % of totl lymphoyte popultion in isolte hepti leukoytes ws CD þ lymphoyte, n % ws CD þ CD/7 lign tetrmer þ NKT ells. Although % ws ifferent types of ells suh s mrophge n B ell, we oul exlue their involvement beuse we use NKT ell-speifi lign to inue the ell tivtion. We lso exmine the requirement for RIPK in the NKT ell hybriom line DN.D. Cells were infete with lentiviruses enoing ontrol or Ripk-trgeting, n speifi gene knokown (KD) ws verifie by immunoblotting using nti- RIPK Ab (Supplementry Fig. A). As we h observe with the primry NKT ells, Ripk KD signifintly reue -GlCerstimulte proution of IFN-g, TNF n IL- ompre with NATURE COMMUNICATIONS :87 DOI:.8/nomms97 & Mmilln Publishers Limite. All rights reserve.

3 NATURE COMMUNICATIONS DOI:.8/nomms97 ARTICLE INF-γ (ng ml ) e IFN-γ (ng ml ) IFN-γ (fol inution) α-glcer TNF (pg ml ) +/+ / +/+ / +/+ / RIPK RIPK RIPK 8 9 RIPK +/+ RIPK /.. Ne-s (μm) NS NS Ne-s (μm) + TNF (ng ml ) TNF (fol inution) 9 IL- (pg ml ) (h) NS p-p8α p-jnk RIPK GAPDH Ne-s (μm) NS Ne-s (μm) + b Fol hnges of gene expression Fol mrna inution RIPK +/+ RIPK / IFN-γ TNF 8 RIPK RIPK 9 f % of PI-positive ells 9 IL- (h) RIPK RIPK GAPDH zvad +zvad zvad +zvad RIPK RIPK KD Figure RIPK regultes NKT ell tivtion. (,b) RIPK-epenent expression of ytokines in NKT ells. Leukoytes isolte from the livers of WT (Ripk þ / þ )orripk / mie were trete with or -GlCer ( ng ml ). Culture superntnts were ollete t h n IFN-g n TNF onentrtions were mesure by ELISA (), or ells were hrveste t h for quntifition of Ifng n Tnf mrna levels by qpcr (b). () Ativtion of MAPK signlling in NKT ells. WT or Ripk / liver leukoytes were trete with -GlCer ( ng ml ) for the inite times, n phosphoryltion of p8 n JNK ws nlyse by immunoblotting. GAPDH serve s loing ontrol. (e) Inution of RIPK n RIPK in NKT ells. DN.D ells trete with or -GlCer ( ng ml ) for h n of Ripk n Ripk mrna levels were mesure by qpcr nlysis. DN.D ells were trete with -GlCer ( ng ml ) for inite times, n expression of RIPK n RIPK ws nlyse by immunoblotting. GAPDH serve s loing ontrol. (e) RIPK-epenent NKT ell tivtion oes not require RIPK tivity. Liver leukoytes were inubte with or -GlCer ( ng ml ) n the inite onentrtions of the RIPK inhibitor Ne-s for h. Culture superntnts were ollete t h for quntifition of IFN-g n TNF levels by ELISA, or ells were hrveste t h for etermintion of mrna levels by qpcr. Gene expression ws normlize to levels in ells trete with or meium. Gph mrna levels were use s n internl ontrol. (f) Inution of ell eth. or Ripk KD DN.D ells were preinubte with or zvad ( mm), n trete with -GlCer ( ng ml ) for 8 h. Cells were hrveste n inubte with PI, n vibility ws ssesse by FACS nlysis. Dt re the mens±s.e.m. (n ¼ ). Po., Po. with the Mnn Whitney U-test; NS, not signifint. Results re representtive of three inepenent experiments. The immunoblotting experiments were repete with new smples from iniviul experiments. ontrol -expressing DN.D ells (Supplementry Fig. B,C). Phosphoryltion of p8 n JNK ws omprble between -GlCer-trete ontrol n Ripk KD DN.D ells while egrtion of IkB n phosphoryltion of ERK were not etete, whih is similr to liver leukoytes (Supplementry Fig. B,C). RIPK is known to regulte RIPK tivtion, n both kinses show elevte expression uring ell eth-ssoite inflmmtion 8,9. We foun tht mrna n protein levels of both kinses were signifintly inrese in -GlCer-trete DN.D ells (Fig. ); however, tretment with the RIPK-speifi inhibitor nerosttin-s (Ne-s) i not signifintly reue -GlCer-stimulte expression of IFN-g or TNF mrna n protein (Fig. e). These results inite tht, espite its inrese expression, RIPK oes not ply role in RIPK-epenent tivtion of ytokine proution. Next, we exmine whether RIPK regulte neroptosis uring the tivtion of NKT ells beuse RIPK signlling plys key role in neroptosis in other types of ells. To etermine whether the role of neroptosis, NKT ells were trete with -GlCer plus pn-spse inhibitor zvad-fmk (zvad) n vibility ws nlyse by flow ytometry fter 8 h. -GlCer NATURE COMMUNICATIONS :87 DOI:.8/nomms97 & Mmilln Publishers Limite. All rights reserve.

4 NATURE COMMUNICATIONS DOI:.8/nomms97 tretment i not signifintly inue ell eth in ontrol n Ripk KD NKT ells. The ition of zvad i not ffet the vibility of ontrol n RIPK KD ells, n neroptosis ws not observe (Fig. f). These results suggest tht RIPK regultes the tivtion of NKT inepenently of progrmme ell eth. RIPK promotes NKT ell-meite nti-tumour immunity. NKT ells re ruil prtiipnts in the nti-tumour immune response, ting both iniretly through the proution of IFN-g n iretly through inution of tumour ell lysis. Aministrtion of -GlCer trgets only NKT ells, n mny investigtors hve use syntheti -GlCer, or its vrints to inue strong NKT ell nti-tumour immune response in mie. We use the mouse B melnom moel to exmine the requirement for RIPK in NKT ell responses to tumours,. For this, WT or Ripk / mie were injete first i.v. with B melnom ells n then i.p. with either or -GlCer, n tumour noules in the lungs were exmine. We etete similr number of tumour noules in the lungs of -injete WT n Ripk / mie (Fig. n Supplementry Fig. ). However, ministrtion of -GlCer signifintly reue the number of noules in WT mie but h no effet in Ripk / mie (Fig. ). -GlCer tretment inrese the serum onentrtions of IFN-g n TNF in WT n Ripk / mie, but the levels were higher in WT ompre with Ripk / mie (Fig. b), n intrellulr expression of IFN-g ws signifintly lower in liver leukoytes isolte from -GlCer-trete Ripk / mie thn from similrly trete WT mie (Fig. ). Also, reution of the tivtion mrker CD9 inution ws sttistilly signifint in liver leukoytes of -GlCer-trete Ripk / mie (Fig. ). Colletively, these results strongly suggest tht RIPK plys ruil role in the NKT ell-meite nti-tumour immune response. RIPK efiieny ttenutes NKT ell-meite inflmmtion. Given the mrke effets of RIPK efiieny on NKT ell proution of TNF n IFN-g in the melnom moel, we next exmine RIPK involvement in moel of inflmmtory tissue mge. Previous stuies hve shown tht NKT ells ontribute to the evelopment of ute liver mge, s eviene by the resistne to ute Con A-inue heptitis of J8 / n CD / mie, whih lk NKT ells,. Moreover, ministrtion of -GlCer lone inues liver injury by tivting NKT ells. We exmine the role of RIPK in NKT-meite liver inflmmtion by injetion of -GlCer into WT or Ripk / mie followe by exmintion of serum lnine minotrnsferse (ALT) levels n liver pthology. Notbly, lthough -GlCer mrkely inrese serum ALT levels n liver inflmmtory ell infiltrtes in WT mie, liver injury ws signifintly lower in Ripk / mie (Fig.,), initing tht bltion of Ripk protete ginst ute liver mge. Furthermore, -GlCer-injete Ripk / mie showe substntilly reue levels of IFN-g n TNF protein in the serum n mrna in the liver ompre with WT mie (Fig. b,), onsistent with the effets of Ripk bltion on NKT ell tivtion. The inrese in TNF levels preee tht of IFN-g, s previously note,, n this ws observe whether -GlCer ws injete i.p. or i.v. (Figs b n b). To onfirm these results, we use the ConA-inue ute liver injury moel. As ws observe in -GlCer-injete mie, Ripk efiieny signifintly reue the Con A-stimulte inrese in serum ALT n sprtte minotrnsferse (AST) onentrtions (Fig. ). Con A-inue liver mge ws lso less severe in the Number of tumour noules b RIPK +/+ RIPK / 9 9 +/+ / +/+ / RIPK RIPK (h) (h) IFN-γ (ng ml ) TNF (ng ml ). ±.. ±. 9. ±.. ±. RIPK +/+ RIPK +/+ Cell number. ±.. ±. Cell number.7 ±.7.9 ±.8 RIPK / RIPK / IFN-γ CD9 Figure RIPK expresse in NKT ells is require for the nti-tumour immune response. () Enumertion of tumour noules in the lungs of WT (n ¼ ) or Ripk / mie (n ¼ 7). Animls were injete i.v. with B melnom ells ( ells) n then injete i.p. with or -GlCer ( mg per mouse). Lungs were ollete n tumour noules were ounte ys lter. (b) Proution of inflmmtory ytokines in WT or Ripk / mie (n ¼ ). Animls were injete i.p. with or -GlCer ( mg per mouse), n bloo smples were ollete t the inite times for mesurement of IFN-g n TNF by ELISA. (,) Ativtion of NKT ells in WT or Ripk / mie (n ¼ ). Animls were injete i.p. with or -GlCer ( mg per mouse), n hepti leukoytes were isolte h lter for FACS nlysis of intrellulr IFN-g levels () n CD9 surfe expression () in CD þ mcd/7 lign tetrmer þ NKT ells. Numbers re the perentge of ells within the inite gtes. Dt re the mens±s.e.m. (n ¼ ). Po., Po. with the Mnn Whitney U-test; NS, not signifint. Results re representtive of two or three inepenent experiments. NATURE COMMUNICATIONS :87 DOI:.8/nomms97 & Mmilln Publishers Limite. All rights reserve.

5 NATURE COMMUNICATIONS DOI:.8/nomms97 ARTICLE 8 RIPK +/+ RIPK / ALT (IU L ) RIPK +/+ RIPK / Fol hnges of gene expression IFN-γ TNF b IFN-γ (ng ml ) TNF (ng ml ).... RIPK +/+ RIPK / ND Hourse post injetion RIPK +/+ RIPK / Figure RIPK regultes -GlCer-inue NKT ell-meite inflmmtory responses in vivo. (,b) Groups of WT or Ripk / mie (n ¼ ) were injete i.v. with or -GlCer ( mg per mouse), n bloo smples were ollete h lter for mesurement of ALT levels () or, n h lter for mesurement of IFN-g n TNF levels (b). () qpcr nlysis of Ifng n Tnf mrna levels in the livers of WT or Ripk / mie (n ¼ ) t h fter injetion of or -GlCer ( mg per mouse). Gene expression ws normlize to levels in the livers of -injete mie. () H&E stining of liver speimens from WTor Ripk / mie prepre h fter injetion of or -GlCer. Brs ¼ mm. Dt re the mens±s.e.m. Po., Po. with the Mnn Whitney U-test; ND, not etete. Results re representtive of two to four inepenent experiments. Ripk / mie, s emonstrte by the reution in inflmmtory ell infiltrtes n poptoti ells (Fig. b,). Other inflmmtory mrkers signifintly reue in the Ripk / mie inlue IFN-g n TNF protein levels in the serum n mrna levels in the liver, n surfe CD9 expression n ytokine mrna levels in liver leukoytes (Fig. g). Colletively, these results onfirm the ritil role plye by RIPK in NKT ell tivtion uring ute liver inflmmtion. While Ripk-efiient mie re less sensitive thn WT mie to liver mge use by -GlCer or Con A, they re eqully s sensitive to LPS/GlNinue mge (Supplementry Fig. ) 7. Beuse innte immune ells ply n importnt role in the LPS/GlN injetion moel, n RIPK oes not regulte TLR-meite innte immune responses, this observtion supports the speifi involvement of RIPK in NKT ell-meite immune responses. Furthermore, RIPK tivtion is inepenent of Fs signlling-meite poptosis uring ute liver injury (Supplementry Fig. ) 8. TNF plys n importnt role in the eth of heptoytes uring liver injury, n onsistent with this, mie lking TNFR re resistnt to Con A-inue liver injury n inflmmtion 7. To etermine whether the ttenution of -GlCer- or Con A-inue liver mge observe in Ripk / mie is ue to lk of RIPK expression in heptoytes, we iretly nlyse TNF-inue ell eth of Ripk-efiient primry heptoytes or heptoyte ell lines. However, we foun no ifferenes in the suseptibility of WT n Ripk-efiient heptoytes to TNF-inue eth (Fig. h,i). These t suggest tht RIPK expresse in NKT ells, not in heptoytes, plys ritil role in NKT ell-meite ute liver inflmmtion n injury. Next, we teste the role of RIPK in the inution of neroptosis in heptoytes. Tretment with TNF- þ CHX n zvad i not show ny signifint effet on the vibility of ontrol n Ripk KD heptoytes (Fig. j), initing tht RIPK-meite neroptosis i not ply role in TNF-inue heptoyte ell eth. Previous stuies emonstrte tht RIPK plye ritil role in the inution of progrmme nerosis in mny types of ells 9,. However, we i not observe ny signifint hnges in TNF-inue ell eth in Ripk / primry heptoytes n the Ripk KD heptoyte ell line. This le us to hypothesize tht the bsl expression level of Ripk in heptoytes ws too low to regulte the inution of ell eth, whih ws not ffete by eletion or silening Ripk. Thus, we ompre the expression levels of RIPK in mouse tissues by qpcr nlysis. Enogenous mrna levels of Ripk in hert, intestine, lung n spleen were higher thn those in some tissues suh s brin, liver n musle (Supplementry Fig. A), whih is onsistent with the expression ptterns of Ripk in humn tissues. Therefore, we onlue tht Ripk efiieny in heptoytes oes not ontribute to ttenution of ute liver mge, n TNF-inue ell eth in heptoytes is not regulte by RIPK. RIPK in NKT ells is ritil for ute liver injury. To onfirm the role of RIPK in NKT ells uring ute liver mge, we generte BM himeri mie of the following groups NATURE COMMUNICATIONS :87 DOI:.8/nomms97 & Mmilln Publishers Limite. All rights reserve.

6 NATURE COMMUNICATIONS DOI:.8/nomms97 ConA ConA ALT (IU L ),,,, 8, +/+ / RIPK AST (IU L-),,, +/+ / RIPK bripk+/+ RIPK / RIPK +/+ RIPK / IFN-γ (ng ml ) g i Reltive RIPK mrna levels to gph l IFN-γ (ng ml ) 7 ConA (h) WT WT. ±.9 RIPK +/+ RIPK / RIPK +/+ CD9 TNF-α (ng ml ) C # # # RIPK WT RIPK / RIPK / WT TNF (pg ml ).. ConA, ConA (h) 8. ± Cell vibility (%) WT WT e TNF-α (ng ml ) Fol hnges of gene expression. ±. C # # # RIPK WT RIPK / RIPK / WT RIPK / CD9 RIPK +/+ RIPK / IFN-γ ConA j Cell vibility (%) TNF 8. ± 8.7 TNF + TNF h Cell vibility (%) TNF 8 f ConA Fol inution of gene expression RIPK +/+ IFN-γ RIP / RIPK +/+ RIPK / + TNF RIPK Cont RIPK KD zvad (μm) k ALT (IU L ) TNF RIPK +/+ TNF (ngml ),,,, RIP / WT WT WT RIPK / WT RIPK / m WT WT WT RIPK / RIPK / WT Figure RIPK promotes Con A-inue liver inflmmtion. () Groups of WT or Ripk / mie (n ¼ ) were injete i.v. with Con A, n bloo smples were ollete 8 h lter for mesurement of ALT n AST levels. (b) H&E stining of liver speimens from - or Con A-injete mie. Brs ¼ mm. () TUNEL stining of liver speimens from - or Con A-injete mie. Brs ¼ mm. () IFN-g n TNF levels in bloo smples ollete from WT or Ripk / mie (n ¼ 8) t or h fter injetion of Con A. (e,f) qpcr nlysis of Ifng n Tnf mrna levels in the livers (e) or liver leukoytes (f) ofwtorripk / mie (n ¼ ) t h fter injetion of Con A. Gene expression ws normlize to the levels in livers or liver leukoytes from -trete mie. (g) Ativtion of liver leukoytes from WTor Ripk / mie (n ¼ ) prepre t h fter injetion of or Con A. Expression of CD9 in CD þ CD/7 lign tetrmer þ ells ws nlyse by FACS. Numbers re the perentge of ells within the inite gtes. (h) Heptoytes from WT or Ripk / mie were inubte with yloheximie ( mgml ) n the inite onentrtions of TNF for h, n ell vibility ws mesure using the WST- ssy. (i) Hep heptoytes were infete with lentiviruses enoing ontrol or three Ripk (# ), n KD effiieny ws exmine by qpcr (left pnel) (n ¼ ). Cells were inubte with yloheximie n TNF for h before ell vibility ws mesure (right pnel) (n ¼ ). (j) or Ripk KD Hep ells were inubte with CHX, TNF- n zvad ( mm) s inite. Cell vibility ws evlute fter h. (k,l) RIPK in NKT ells ontributes to the ute liver injury. BM himeri mie were generte WT-WT, WT-Ripk / n Ripk / -WT (onor-reipient). After weeks, mie were injete i.v. with ConA, n bloo ALT levels ( h) (k) or ytokine levels ( h for TNF n h for IFN-g) were mesure (l). (m) H&E stining of liver speimens from Con A-injete mie. Brs ¼ mm. Dt re the mens±s.e.m. Po. with the Mnn Whitney U-test. Results re representtive of t lest three inepenent experiments. NATURE COMMUNICATIONS :87 DOI:.8/nomms97 & Mmilln Publishers Limite. All rights reserve.

7 NATURE COMMUNICATIONS DOI:.8/nomms97 ARTICLE (onor-reipient): WT-WT, WT-Ripk /,nripk / -WT. Mie were injete i.v. with ConA, n ALT, IFN-g n TNF levels in the ser were mesure. ConA inrese serum ALT levels n infiltrtion of inflmmtory ells in the livers of WT-WT n WT-Ripk / mie. However, liver injury ws signifintly lower in Ripk / -WT mie (Fig. k,m), initing tht RIPK in NKT ells plys ruil role in the inution of ute liver inflmmtion. Furthermore, Ripk / -WT mie showe substntilly reue levels of IFN-g n TNF in the ser ompre with WT-WT n WT-Ripk / mie (Fig. l). Colletively, our result inites tht RIPK regultes the inflmmtory responses of NKT ells in ute liver inflmmtion while RIPK is ispensble in heptoytes. PGAM is the ownstrem regultor of RIPK signlling. MLKL n PGAM re both iretly tivte by RIPK n re key regultors of RIPK-meite signlling,,8. Aitionlly, RIPK phosphoryltion of MLKL is require for RIPK- epenent neroptosis, n PGAM lso interts with the RIPK. We explore the involvement of these ownstrem meitors in NKT ell proinflmmtory funtions. Beuse liver leukoytes n DN.D NKT ells showe the sme response to -GlCer tretment (Fig. n Supplementry Fig. ), we generte DN.D NKT ells expressing Mlkl or Pgm s to knok own the genes (Fig.,, respetively), n exmine -GlCer-stimulte expression of ytokines. IFN-g, TNF n IL- mrna n protein levels were omprble between ontrol n Mlkl KD DN.D ells (Fig. b,) but were signifintly lower in -GlCer-stimulte Pgm KD ells ompre with ontrol ells (Fig. e,f). These results inite tht PGAM, but not MLKL, is n essentil ownstrem meitor of RIPK-epenent NKT ell tivtion. We further teste whether PGAM regulte the inution of neroptosis in -GlCer-trete NKT ells. Tretment with -GlCer n zvad i not show ny signifint ifferene in vibility between ontrol n Pgm KD DN.D ells (Fig. g), initing tht PGAM i not ply role in neroptosis. Colletively, these results implite PGAM s key meitor of RIPK-meite tivtion of NKT ells. RIPK-PGAM signlling regultes the tivtion of NFAT. Previous stuies hve shown tht PGAM inues stresstivte MAPK signlling pthwys by ephosphorylting n tivting the poptosis signl-regulting kinse (ASK), whih in turn tivtes ownstrem p8 n JNK MAPKs. We investigte the involvement of MAPK pthwys in RIPK- PGAM-meite inflmmtory responses by exmining ASK, p8 n JNK phosphoryltion in -GlCer-trete ontrol n Pgm KD DN.D ells (Fig. ). However, tivtion of these enzymes ws omprble in both ell types, initing tht the ASK-MAPK se is not involve in RIPK-PGAM- meite NKT ell tivtion. Expression of proinflmmtory ytokines in NKT ells is inue by the trnsription ftor NFAT, whih is ephosphorylte in the ytoplsm n trnslotes to the nuleus to initite gene trnsription. Exmintion of nuler extrts inite tht -GlCer tretment mrkely inrese trnslotion of NFAT in ontrol DN.D ells but h only moest effet on Pgm KD ells (Fig. b), impliting PGAM in the regultion of NFAT tivity. To onfirm this result, CRISPR/ Cs9-meite gene trgeting metho ws use to elete Pgm in DN.D ells. Consistent with Pgm KD result, nuler trnslotion of NFAT ws signifintly inrese in -GlCertrete ontrol NKT ells, but not in Pgm KO ells (Fig. ). Beuse nuler trnslotion of NFAT is lso ontrolle by the C þ /lmoulin-epenent phosphtse lineurin,we exmine the effet of the lineurin inhibitor FK on PGAM-regulte tivtion of NFAT. Intriguingly, FK not only reue the proution of IFN-g n TNF by ontrol ells, substntiting the importne of NFAT tivtion in ytokine proution in NKT ells, but lso further reue ytokine proution in Pgm KD ells (Fig. ). These t thus suggest tht PGAM n lineurin inepenently regulte the tivtion of NFAT. We lso teste whether PGAM plye role in T-ells. or Pgm KD Jurkt T-ells were inubte with nti- CD/CD8 Abs, n ytokine proution ws exmine. Unlike NKT ells, IL- n IL- levels were omprble between TCRstimulte ontrol n Pgm KD Jurkt ells, while proution of IFN-g ws signifintly reue in Pgm KD Jurkt ells ompre with ontrol ells (Supplementry Fig. 7A,B). Aitionlly, nuler trnslotion of NFAT ws omprble between nti-cd/8 Abs-trete ontrol n Pgm KD ells (Supplementry Fig. 7C). These t suggest tht PGAM funtions ifferentilly in T ells n NKT ells. Drp regultes NKT ell tivity inepenently of NFAT. The PGAM substrte Drp is GTPse tht regultes mitohonril fission n hs been shown to ply role in the expression of ytokines in mrophges n T-ells 9,. Therefore, we next teste whether Drp ontributes to RIPK-PGAM-meite inflmmtory ytokine expression in NKT ells. Drp tivity n trnslotion to the mitohonri is regulte by ephosphoryltion. We observe tht ephosphoryltion of Drp serine 7 ws inrese by -GlCer tretment in ontrol DN.D ells, but not in Ripk KD or Pgm KD ells (Fig. 7), emonstrting tht RIPK-PGAM signlling regultes the phosphoryltion stte of Drp in NKT ells. In ition, Drp tivity ws essentil for -GlCerstimulte ytokine proution. Inhibition of Drp by meite KD or tretment with the smll moleule inhibitor Mivi- signifintly reue ytokine proution in -GlCertrete NKT ells (Fig. 7b,), suggesting tht PGAM-Drp signlling plys ruil role in NKT ell tivtion. Next, we ompre the role of Drp in the tivtion of T-ells n NKT ells. A stuy hs shown tht Drp regultes ytokine proution in PMA þ ionomyin-trete T-ells. Consistently, inhibition of Drp by Mivi- resulte in the reue proution of ytokines n nuler trnslotion of NFAT in nti-cd/8 Abs-trete mouse T-ells (Supplementry Fig. 8). Although Drp regulte the tivtion of both T-ell n NKT ells, the upstrem signlling pthwys suh s RIPK n PGAM tht regulte the Drp tivity re ifferent. Therefore, these results suggest tht RIPK-PGAM-Drp signlling se is ruil in NKT ell-meite immune responses. We further teste whether Drp ffete the inution of ell eth of NKT ells. Tretment with -GlCer i not inue signifint ell eth in ontrol n Drp KD DN.D ells, n inution of neroptosis ws not ffete (Fig. 7), initing tht Drp oes not ply role in the inution of ell eth in NKT ells. Reent stuies hve suggeste tht, in ition to the involvement of Drp in mitohonril fission n fusion, ephosphoryltion of Drp by tivte PGAM inues mtros genertion 8,. Beuse mtros n t s signlling moleules for inution of ytokine gene expression 9,, we ske whether PGAM n/or Drp regultes mtros genertion n subsequent tivtion of NFAT in NKT ells. However, bsl n -GlCer-stimulte levels of mtros were omprble in ontrol n Pgm KD DN.D ells (Fig. 7e). Furthermore, NATURE COMMUNICATIONS :87 DOI:.8/nomms & Mmilln Publishers Limite. All rights reserve.

8 NATURE COMMUNICATIONS DOI:.8/nomms97 MLKL KD MLKL GAPDH b Fol hnges of gene expression 9 IFN-γ TNF IL- NS NS NS MLKL KD MLKL KD MLKL KD IFN-γ (pg ml ) e IFN-γ (pg ml ) Ctrl KD MLKL TNF (pg ml ) Ctrl KD MLKL IL- (pg ml ) PGAM KD # # TNF (pg ml ) 9 Ctrl KD MLKL IL- (pg ml ) PGAM KD C # # PGAM GAPDH f Fol hnges of gene expression 9 (ng ml ) (ng ml ) (ng ml ) IFN-γ TNF IL- 8 C # # C # # C # # PGAM KD PGAM KD PGAM KD 9 g % of PI-positive ells zvad +zvad zvad +zvad PGAM PGAM KD Figure Phosphtse PGAM regultes NKT ell tivtion. ( ) MLKL is ispensble for NKT ell tivtion. DN.D ells were infete with lentiviruses enoing ontrol or Mlkl, n KD effiieny ws ssesse by immunoblotting using nti-mlkl Ab. GAPDH level ws mesure s loing ontrol (). qpcr nlysis of Ifng, Tnf n Il mrna levels in ontrol or Mlkl KD ells trete with meium or -GlCer ( ng ml ) for h (n ¼ ) (b). () IFN-g, TNF n IL- proution by ontrol or Mlkl KD ells trete with meium or the inite onentrtion of -GlCer for h (n ¼ ). ( f) Involvement of PGAM in RIPK-epenent tivtion of NKT ells. DN.D ells were infete with lentiviruses enoing ontrol or Pgm s, n KD effiieny ws ssesse by immunoblotting using nti-pgam Ab. GAPDH level ws mesure s loing ontrol (). or PGAM KD ells trete with meium or -GlCer ( ng ml ), n ulture superntnts were ollete h for nlysis of IFN-g, TNF n IL- proution (e) (n ¼ ) or ells were hrveste t h for qpcr nlysis of Ifng, Tnf n Il mrna (f) (n ¼ ). (g) Inution of ell eth. or Pgm KD DN.D ells were preinubte with zvad ( mm) or not, n trete with -GlCer ( ng ml ) for 8 h. Cells were hrveste n inubte with PI, n vibility ws ssesse by FACS nlysis (n ¼ ). Dt re the mens±s.e.m. Absene of error brs inites tht they fll within the symbols. Po., n Po. with the Mnn Whitney U-test. N. S. ¼ not signifint. Results shown re the representtive of three inepenent experiments. The immunoblotting experiments were repete with new smples from iniviul experiments. nuler trnslotion of NFAT stimulte by -GlCer ourre normlly in Drp KD or Mivi--trete ells (Fig. 7f), even though ytokine proution ws inhibite (Fig. 7b,). From these results, we onlue tht PGAM-Drp signlling regultes ytokine expression inepenently of mtros tivity. We lso ske whether Drp is ephosphorylte by lineurin in NKT ells, s hs been shown in other ell types. Inee, Drp ephosphoryltion in -GlCer-trete ells ws reue by the ition of FK (Fig. 7g). Colletively, these t suggest tht in NKT ells (i) lineurin regultes NFAT n Drp tivities inepenently of RIPK-PGAM-meite signlling n (ii) PGAM regultes NFAT n Drp tivities inepenently of lineurin tivtion n mtros proution. Inhibition of Drp tivity protets the ute liver injury. Our in vitro results hve shown tht Drp plys regultory role in the tivtion of NKT ells. To test this in vivo, we exmine the effet of the Drp inhibitor Mivi- on NKT ell-meite 8 NATURE COMMUNICATIONS :87 DOI:.8/nomms97 & Mmilln Publishers Limite. All rights reserve.

9 NATURE COMMUNICATIONS DOI:.8/nomms97 ARTICLE PGAM PGAM KD PGAM ontrol PGAM KD.. (h) (h) 8 p-ask NE NFAT p-p8α CE NFAT p-jnk GAPDH b PGAM KO PGAM GAPDH NE CE PGAM KO (h) NFAT NFAT IFN-γ (pg ml )..... PGAM KD FK (nm) TNF (pg ml ).... PGAM KD FK (nm) Figure PGAM regultes nuler trnslotion of NFAT in NKT ells. (,b) or Pgm KD DN.D ells were trete with -GlCer ( ng ml ) for the inite times before ells were hrveste. Whole ell lystes were immunoblotte for pask, p8 n pjnk (), n nuler or ytoplsmi extrts were immunoblotte for nuler trnslotion of NFAT (b). GAPDH serve s loing ontrol. () Pgm KO effiieny by CRISPR/Cs9 metho in DN.D ws ssesse immunoblotting using nti-pgam Ab. GAPDH level ws mesure s loing ontrol. or Pgm KO ells were trete with -GlCer ( ng ml ) for the inite times, n nuler or ytoplsmi extrts were immunoblotte for nuler trnslotion of NFAT. () PGAM n lineurin inepenently regulte ytokine proution by NKT ells. IFN-g n TNF proution by ontrol or Pgm KD DN.D ells ws ssesse fter h tretment with -GlCer n the inite onentrtion of FK. Dt re the mens±s.e.m. (n ¼ ). Po. with the Mnn Whitney U-test. Results re representtive of t lest three inepenent experiments. The immunoblotting experiments were repete with new smples from iniviul experiments. ute liver mge inue by -GlCer injetion. Animls ministere Mivi- prior to -GlCer h signifintly lower serum ALT, TNF n IFN-g levels ompre with the vehile ()-injete mie (Fig. 8,b). Aitionlly, mie in whih Drp tivity ws inhibite showe less severe liver pthology, s inite by reue inflmmtory ell infiltrtion (Fig. 8). These results support role for Drp in the tivtion of NKT ells uring ute liver inflmmtion n injury. TCR signlling regultes RIPK-meite NKT ell tivtion. Finlly, we investigte whether TCR-epenent signlling regultes RIPK-PGAM-Drp-meite NKT ell tivtion. For this, -GlCer-stimulte ytokine proution ws nlyse in DN.D ells expressing speifi for phospholipse C g (Plg), protein kinse C y (Pkq) or Vv (Fig. 9). Proution of IFN-g n TNF ws signifintly reue by Plg n Vv KD, but not by Pkq KD (Fig. 9b), initing tht NKT ell ytokine proution ours inepenently of PKCy tivity. We lso exmine ephosphoryltion of Drp in these ells n foun tht Plg or VvKD signifintly reue -GlCerinue Drp ephosphoryltion, wheres Pkq KD h no effet (Fig. 9). It shoul be note tht we were unble to iretly ssy RIPK tivity in NKT ells, beuse publishe methos for in vitro kinse ssys of n enogenous RIPK re not well estblishe but the tivity of overexpresse RIPK in HEK 9T ells hs been teste, whih is not omptible with our stuy bout the tivtion of NKT ell-speifi signlling pthwy. In ition, n nti-phospho-ripk ntiboy is not vilble. Nevertheless, the results of the ytokine proution n Drp ephosphoryltion ssys re onsistent with importnt roles for PLCg n Vv, but not PKCy, in RIPK-PGAM-Drp- meite inflmmtory responses of NKT ells. TCR-meite PLCg n Vv- signlling tivtes TGF-btivte kinse (TAK), whih plys n essentil role in integrting TCR signlling for the regultion of T-ell evelopment n tivtion. TAK ts upstrem of RIPK to regulte its tivity 7, n tivtion of TAK requires the pter protein TAK-bining protein (TAB) 8,9. We therefore teste the involvement of these moleules in regulting RIPK-PGAM- Drp signlling in NKT ells by nlysing the ytokine response of Tk KD n Tb KD ells (Fig. 9). Interestingly, we foun tht wheres IFN-g levels were signifintly reue by Tk or Tb KD, TNF proution ws reue only in Tb KD ells (Fig. 9e,f). Aitionlly, -GlCer-inue ephosphoryltion of Drp ws bolishe by Tb KD but ws only moestly reue by Tk KD (Fig. 9g). Finlly, we exmine NFAT tivtion n foun tht -GlCer-inue nuler trnslotion of NFAT ws signifintly reue by Tb KD, wheres Tk KD ws without effet (Fig. 9h). Tken together, these t inite tht TAB plys n essentil role in RIPK-PGAM-meite tivtion of NKT ells. Colletively, the results presente here revel ritil role for TCR-epenent RIPK-PGAM-Drp signlling in regulting NKT ell-meite immune responses (Fig. 9i). This signlling xis requires PLCg, Vv- n TAB n inues the expression of proinflmmtory ytokines by filitting the nuler trnslotion of NFAT n ephosphoryltion of Drp. Given tht PGAM is lolize t the outer membrne of the mitohonri, our results suggest tht this phosphtse my ply pivotl role in oorinting rosstlk between mitohonril funtion n NATURE COMMUNICATIONS :87 DOI:.8/nomms & Mmilln Publishers Limite. All rights reserve.

10 NATURE COMMUNICATIONS DOI:.8/nomms97 RIPK KD (h) p-drp (ser7) Drp PGAM KD (h) p-drp (ser7) Drp b Drp Drp GAPDH IFN-γ (pg ml ) 8 Ctrl Drp Ctrl Drp Ctrl Drp TNF (pg ml ) IL- (pg ml ) IFN-γ (pg ml ) Meium Mivi- (μm) + TNF (pg ml ) Meium Mivi- (μm) + IL- (pg ml ) Meium Mivi- (μm) + % of PI-positive ells. zvad +zvad zvad +zvad Drp Drp KD e Cell number PGAM PGAM KD Fluoresene (MitoSOX) Unstine Stine, unstimulte Stine, -trete -trete ells PGAM KD f Drp KD (h) NFAT HDAC None Mivi- (μm) NFAT HDAC g Meium FK.. α-gslcer (h) p-drp (Ser7) Drp Figure 7 Regultion of RIPK-PGAM signling in NKTells. () RIPK-PGAM signlling regultes the ephosphoryltion of Drp. or Ripk KD DN.D ells were trete with -GlCer, n ell lystes were immunoblotte with ntiboies to totl Drp n Drp (Ser7). (b,) Drp is involve in ytokine proution by NKT ells. (b) DN.D ells were infete with lentiviruses enoing ontrol or Drp, n KD effiieny ws exmine by immunoblotting (left pnel). IFN-g, TNF n IL- proution by ontrol or Drp KD ells ws ssesse h fter tretment with or -GlCer (right pnels, n ¼ ). () As esribe for B exept ells inubte with Mivi- for min before ition of or -GlCer (n ¼ ). () Inution of ell eth. or Drp KD DN.D ells were preinubte with zvad ( mm) or not, n trete with or -GlCer ( ng ml ) for 8 h. Cells were hrveste n inubte with PI, n vibility ws ssesse by FACS nlysis (n ¼ ). (e) Proution of mtros in -GlCer-trete NKTells. or Pgm KD DN.D ells were preinubte with MitoSOX for min, n trete with -GlCer for min. Cells were wshe, n nlyse by FACS (n ¼ ). (left pnel) ells±-glcer; (mile pnel) Pgm KD ells±-glcer; n (right pnel) overly of -GlCer-trete ontrol n Pgm KD ells. (f) (left pnel) or Drp KD ells were inubte with -GlCer for the inite times, n nuler extrts were immunoblotte with ntiboies to NFAT. HDAC serve s loing ontrol for nuler extrts. (right pnel) Cells were trete s in () n nuler extrts were immunoblotte with ntiboies to NFAT or HDAC. (g) Dephosphoryltion of Drp by lineurin. DN.D ells were preinubte with FK ( mm) for h, trete with -GlCer for inite times, n ell lystes were then immunoblotte with ntiboies to pdrp (Ser7) to etete Drp ephosphoryltion. Dt re the mens±s.e.m. In () bsene of error brs inites tht they fll within the symbols. Po., Po. with the Mnn Whitney U-test. Results re representtive of two or three inepenent experiments. The immunoblotting experiments were repete with new smples from iniviul experiments. ytokine proution by NKT ells in iseses suh s ner n ute inflmmtory isorers. Disussion Mitohonri re essentil for iverse biologil proesses, inluing energy genertion, ell eth n biosynthesis of essentil moleules. Reent stuies hve shown tht mitohonri re lso involve in regulting innte immune ell- n T-ellmeite immune responses. However, the regultory roles of mitohonri in other immune ells, suh s NKT ells, remin unler. In this stuy, we hve shown tht the RIPK-epenent mitohonril phosphtse PGAM regultes NKT ell tivtion by ephosphoryltion of NFAT n Drp, whih strongly suggests previously unreognize role for mitohonri in the regultion of NKT ell-meite immunity. RIPK-RIPK signlling plys ruil role in the inution of neroptosis. In mrophges, tivtion of TLR n TLR signlling in the presene of the pn-spse inhibitor z-vad-fmk rives RIPK-RIPK omplex-epenent NATURE COMMUNICATIONS :87 DOI:.8/nomms97 & Mmilln Publishers Limite. All rights reserve.

11 NATURE COMMUNICATIONS DOI:.8/nomms97 ARTICLE ALT (IU L ), 9 Mivi- b IFN-γ (ng ml ) (h) Mivi- Mivi- progrmme nerosis. Although RIPK tivity is ispensble for TCR-epenent T-ell tivtion n TLR response in mrophges, the speifi roles for RIPK signlling in innte immunity hve reently been ientifie. In mrophges, the RIPK-RIPK signlling tivtes formtion of the NLRP inflmmsome in response to RNA virus infetion. Aitionlly, RIPK in DCs plys ritil role in injuryinue inflmmtion n tissue repir. However, we showe here tht RIPK is not require for NKT ell tivtion; therefore, RIPK signlling for tivtion of NKT ell-meite immune responses ours inepenently of RIPK. NKT ells re involve in the regultion of rnge of physiologil n pthologil immune responses, inluing nti-tumour, nti-mirobil n utoimmune responses,. In the B melnom metstsis n ute liver injury moels exmine here, Ripk efiieny severely ompromise the proution of inflmmtory ytokines by tivte NKT ells, suggesting tht RIPK-meite signlling likely plys n importnt pthophysiologil role in regulting NKT ell responses to both enogenous ntigens n exogenous pthogens. Our results re onsistent with previous observtions showing tht eletion of RIPK reues ell eth n tissue mge in inflmmtory isorers suh s pnretitis,,. Abltion of RIPK protets mie ginst TNF-inue heptitis s well s liver injury n inflmmtion resulting from ethnol-inue tivtion of neroptosis,. In ition, it hs been shown tht Ripk mrna is present in methionine-holine-efiient iet, humn non-loholi stetoheptitis in liver, n heptorinogenesis,, impliting tht there my be little role in helthy liver, but RIPK funtion n be inue in isese liver. TNF (ng ml ) (h) Figure 8 Inhibition of Drp tivity meliortes ute liver inflmmtion. (,b) Two groups of WT mie (n ¼ eh) were injete i.p. with or Mivi- ( mg per kg boy weight) n then i.v. with -GlCer ( mg per mouse). Bloo smples were ollete t h for mesurement of ALT levels () or t n h for mesurement of IFN-g n TNF levels (b). () H&E stining of liver speimens ollete h fter tretment s in (,b). Brs ¼ mm. Dt re the mens±s.e.m. Po., Po. with the Mnn Whitney U-test. The results of this n other stuies inite tht RIPK expression levels re ell-type epenent; Ripk mrna levels re high in lymphoytes, monoytes, n NK ells, but re very low in tissues suh s brin, musle n liver. Although RIPK expression is upregulte in mny ell types unergoing RIPK- meite ell eth n inflmmtion,,8,9, we hve foun tht RIPK levels in heptoytes remin unhnge uring ute liver injury while the protein levels inrese in liver leukoytes. Sine heptoytes ontin low bsl levels of RIPK, speifi eletion or silening of Ripk in these ells i not ffet inution of ell eth. Aitionlly, pretretment of Ne-s signifintly reue ytokine proution by -GlCer-trete NKT ells. Sine Ne-s is potent RIPK inhibitor, whih hs weker inolemine,-ioxygense inhibition ompre with the prototype inhibitor Ne- (refs,), our result suggests tht RIPK oes not ply role in RIPK-meite immune response in NKT ells. RIPK tivtes MLKL, kinse ritil for inution of neroptosis in mny ell types. However, MLKL plys little or no role in the inflmmtory responses of innte immune ells, s shown by the lk of effet of Mlkl efiieny on TNF- or LPS-inue tivtion of NF-kB, p8, ERK, JNK n inflmmtory ytokine proution in mrophges,7. Our results exten these observtions by showing tht RIPK- epenent tivtion of PGAM, but not MLKL, plys n essentil role in the regultion of NKT ell tivtion. Although PGAM n tivte ASK by ephosphoryltion of inhibitory sites,, we foun tht ASK-MAPK signlling is not involve in PGAM-meite NKT ell tivtion. However, we i fin tht PGAM regultes the trnsription ftor NFAT, whih is ruil for ytokine proution by NKT ells,,. Interestingly, FK tretment further reue ytokine proution by PGAM KD NKT ells, whih suggests tht PGAM n lineurin inepenently regulte NFAT tivtion. Beuse PGAM is phosphtse n regultes the nuler trnslotion of NFAT like lineurin, it is suggeste tht PGAM regultes the phosphoryltion of NFAT. However, more work is neessry to better unerstn the mehnism by whih PGAM regultes NFAT ephosphoryltion. Despite previous stuy hs inite the role of PGAM in ell eth 8, others hve suggeste tht RIPK-PGAM signlling oes not ply ruil role in the regultion of progrmme nerosis,7,8. Similrly, PGAM-meite NKT ell tivtion oes not inlue the inution of ell eth, supporting the notion tht PGAM oes not regulte the inution of neroptosis. The PGAM substrte Drp regultes mitohonril fission in mny ell types n is lso involve in regulting ytokine proution by mrophges n T-ells 9,. Here, we showe tht Drp inhibition or silening reue NKT ell tivtion, supporting role for Drpin PGAM-meite ytokine proution. The mehnism by whih this might our is unler. Drp inhibition or silening reues mtros genertion, rising the possibility tht Drp tivity is importnt for ROS genertion n tivtion of ownstrem signlling pthwys suh s NF-kB n MAPK. However, Drp KD only moestly suppresses mtros genertion n NF-kB n MAPK tivtion in LPS-trete mrophges 9,. Moreover, we foun tht RIPK-PGAM signlling is not neessry for ROS genertion in -GlCer-trete NKT ells, n Drp tivity oes not iretly regulte NFAT tivtion. Therefore, it seems likely tht RIPK-PGAM-epenent Drp tivity my regulte NKT ell ytokine gene expression inepenently of mtros genertion. Beuse lineurin lso regultes the phosphoryltion sttus of Drp, this phosphtse oul ontribute to NKT ytokine proution by ting s ul regultor of NFAT n Drp. However, the mehnism of Drp NATURE COMMUNICATIONS :87 DOI:.8/nomms97 & Mmilln Publishers Limite. 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12 NATURE COMMUNICATIONS DOI:.8/nomms97 b Reltive mrna level Reltive mrna level 9 Ctrl PLCγ PLCθ Vv TAK TAB IFN-γ (pg ml ) IFN-γ (pg ml ) p-drp (Ser7) Drp TAK KD (h) Ctrl PLCγ PLCθ Vv TNF (pg ml ) TNF (pg ml ) Ctrl PLCγ PLCθ Vv. e 8 f.8... Ctrl TAK Ctrl TAB Ctrl TAK Ctrl TAK s g h TAB KD NFAT HDAC (h) NFAT HDAC i Clineurin NFAT Drp PGAM TCR RIPK IFN-γ (pg ml ) Mitohonrion s PLCγ PLCθ Vv Ctrl TAB NKT ell RIPK ASK p8α JNK Cytokine gene expression TNF (pg ml ) tretment p-drp (Ser7) Drp Ctrl TAB Figure 9 TCR-epenent tivtion of RIPK-PGAM signlling in NKT ells. ( ) Involvement of TCR signling moleules in PGAM-meite NKT ell tivtion. () DN. ells were infete with lentiviruses enoing ontrol or Plg-, Pkq- or Vv-speifi s, n KD effiieny ws ssesse by qpcr nlysis. (b) IFN-g n TNF proution by ontrol n KD ells ws ssesse h fter tretment with -GlCer (n ¼ ). () n KD ells were trete for h with -GlCer ( ng ml ), n Drp ephosphoryltion ws nlyse by immunoblotting with nti-drp (Ser7) Ab. ( g) TAB regultes the RIPK-PGAM-meite NKT ell tivtion. DN. ells were infete with lentiviruses enoing ontrol or Tk- or Tb-speifi s, n KD effiieny ws ssesse by qpcr. Gph levels from eh ell were mesure s n internl ontrol (). (e,f) IFN-g n TNF proution by ontrol n Tk KD ells (e) ortb KD ells (f) ws mesure fter h of -GlCer tretment (n ¼ ). (g) Drp ephosphoryltion ws ssesse in ontrol, Tk KD or Tb KD ells trete for h with -GlCer. (h) Nuler trnslotion of NFAT ws nlyse in ontrol, Tk KD or Tb KD ells t or h fter tretment with -GlCer. (i) A moel of RIPK-PGAM-Drp-meite NKT ell tivtion. Dt re the mens±s.e.m. Po., Po. with the Mnn Whitney U-test. Results re representtive of t lest three inepenent experiments. The immunoblotting experiments were repete with new smples from iniviul experiments. ontribution to NKT ell-meite immune responses is not limite to its role in gene expression, sine inhibition of Drp- meite mitohonril ivision meliortes the pthology of NKT ell-inue ute liver inflmmtion. Therefore, more work is neee to better unerstn the Drp-epenent ellulr tivtion mehnism tht regultes the gene expression. Interestingly, reent stuy revele speifi role for RIPK- RIPK-epenent phosphoryltion of Drp serine in RNA virus-inue tivtion of the NLRP inflmmsome, n nother stuy emonstrte tht ephosphoryltion of Drp by PGAM is ritil for mitohonril fission n energy genertion 9. Our results inite tht ephosphoryltion of serine 7, but not serine, is essentil for ytokine proution by tivte NKT ells. These isrepnt observtions oul be ue to ifferenes in stimuli, ells or pthologil onitions between the stuies. TAK hs previously been shown to regulte RIPK-RIPK signlling 7, n n ssoition between TAK n TAB is ritil for the inution of ytokine gene expression in T-ells n mrophges 8,9. Nevertheless, we foun tht TAB is require for tivtion of TCR-inue RIPK-PGAM signlling for ytokine expression by NKT ells, wheres TAK plys only limite role. In onlusion, we hve emonstrte key role for RIPK- PGAM signlling in the regultion of NKT ell tivtion in isese sttes suh s ner n inflmmtory isorers. Our fining tht the mitohonril phosphtse PGAM regultes NKT ell-meite immune responses suggests tht it my serve s link between mitohonril funtion n TCR signlling in these ells. Our results thus support novel role for the RIPK- PGAM-Drp signlling xis in rosstlk between mitohonril funtion n host immunity. NATURE COMMUNICATIONS :87 DOI:.8/nomms97 & Mmilln Publishers Limite. All rights reserve.

13 NATURE COMMUNICATIONS DOI:.8/nomms97 ARTICLE Methos Mie. C7Bl/ bkgroun WT mie were obtine from the mouse breeing fility t The Sripps Reserh Institute, n Ripk / (C7Bl/ bkgroun) mie were obtine from Dr Dixit (Genenteh, In.). Sex- n ge-mthe mie, usully t 8 weeks of ge, were use for eh experiment. Protools for the use of nimls were pprove by the Institutionl Animl Cre n Use Committee. Regents. The regents n soures were s follows: Con A, LPS from Esherihi oli O:B, n N-gltosmine (GlN) (Sigm-Alrih); TNF (Peproteh); -gltosyl ermie (-GlCer, KRN7) n Mivi- (Enzo Life Sienes); Ne-s (7-Cl-O-Ne-, BioVision); FK (LC Lbs); yloheximie (Clbiohem); phospho-ask(s-99, : ilution), NFAT (s-9, :, ilution), RIPK (s-788, :, ilution), RIPK (s-7, :, ilution), n HDAC (s-78, : ilution) ntiboies (Snt Cruz Biotehnology); nti-gapdh ntiboy (MAB7, :, ilution, Chemion); phospho-p8 (#9), phospho-jnk (#9), phospho-erk (#9) n phospho-drp (Ser7, #87) ntiboies (:, ilution, Cell Signling Tehnology); nti-mlkl ntiboy (APb, :, ilution, Abgent); nti-fs (lone Jo, #, mg per mouse, BD Phrmingen); CD-FITC (#,. mg per ells), CD-PerCP/Cy. (#,. mg per ells), n CD9-PE ntiboies (# 9,. mg per ells) (ebiosiene); n MitoSOX RED (Moleulr Probes). APC-mCD/7 lign tetrmers were generously provie by the Ntionl Institute of Allergy n Infetious Disese MHC Tetrmer Core Fility (Atlnt, GA, USA). Cell ulture. HEK 9T, Hep (mouse heptoyte ell line), Jurkt (humn T-ell line) n B mouse melnom ells were ulture in DMEM supplemente with % FBS. Mouse NKT hybriom ells DN.D were obtine from Dr Benel (University of Chigo) n were mintine in RPMI with % FBS. Cell vibility ws mesure by the WST- ell prolifertion ssy (Rohe) oring to the mnufturer s instrutions. n the ell pellet ws wshe with three times. Purifie heptoytes were resuspene in Willim E meium (Qulity Biologil, In.) supplemente with % FBS n ntibiotis, seee on ollgen-ote ulture pltes ( mgml, Stem Cell Tehnologies) for h t RT, n then wshe with twie before use in experiments. Preprtion of liver leukoytes. Bloo ws remove from the liver by perfusion with ml of HBSS injete through the portl vein. Liver homogentes were inubte with U ml ollgense (Sigm) for min t 7 C, n the igest ws psse through -mm ell striner. Cells were entrifuge t 7 g for min t RT n the pellets were resuspene in % isotoni Peroll ontining U ml of heprin. The suspension ws entrifuge t 7 g for min t RT n RBCs were remove from the non-prenhyml ell pellet with RBC lysis buffer. Flow ytometry. Liver leukoytes were stine with CD-FITC, CD-PerCP/ Cy., CD9-PE n APC-CD lign tetrmers, n subsequently fixe in % prformlehye for nlysis. To ssess intrellulr mtros levels, ells were inubte with MitoSOX ( mm) n -GlCer (. mgml ) for min n wshe before nlysis. Dt were quire with FACSClibur (BD Biosienes) n nlyse with FlowJo (TreeStr, In.). Plsmis n s. Lentivirl expression vetors for mouse RIPK s were obtine from Jihui Hn. The onstruts were esigne bse on the single oligonuleotie RNA interferene tehnology, n lentivirl vetors expressing these s were generte oring to the mnufturer s instrutions (Biosetti). Oligonuleoties sequenes use to generte the s re liste in Supplementry Tble. Pgm sgrna CRISPR/Cs9 All-in-One Lentivetor (Mouse trget ) ws purhse from Applie Biologil Mterils (Ct # K). Melnom metstsis moel. Mie were rnomly injete i.v. with B melnom ells in ( ells per mouse), n or -GlCer ( mg per mouse) ws injete i.p. h lter. After ys, mie were kille n lungs were remove n fixe in Bouin s solution. The number of tumour noules ws ounte uner issetion mirosope in bline mnner. Aute liver injury moels. Mie were rnomly injete i.v. vi the lterl til vein with Con A ( mg per kg boy weight) or -GlCer ( mg per kg). Anti-Fs ntiboies ilute in ( mg per g boy weight) were intrperitonelly injete. LPS ( mg per kg) plus GlN (, mg per kg) ws intrperitonelly ministere into mie. At the inite times, nimls were kille, bloo ws ollete n the livers were surgilly remove in bline mnner. Serum smples were prepre n nlyse for trnsminse n ytokine levels. Genertion of bone mrrow himers. Chimeri mie were generte by totl-boy gmm irrition followe by trnsfer of bone mrrow ells. Briefly, WT n Ripk / reipient mie were rnomly groupe n irrite with ose of 9 Gy n then i.v. injete with ells per mouse from WT n Ripk / mie. Mie were ministere with ntibiotis libitum for weeks. After weeks, mie were injete i.v. with ConA. ALT n AST ssys. Serum ALT n AST levels were quntifie with enzymti ssys (Pointe Sientifi) oring to the mnufturer s instrutions. Histology. Livers were remove, fixe in % prformlehye, n embee in prffin. Tissue setions were stine with H&E or subjete to terminl eoxynuleotiyl trnsferse UTP nik en lbelling (TUNEL) stining oring to the kit mnufturer s instrutions (In Situ Cell Deth Detetion Kit; Rohe). Reverse trnsription n quntittive PCR. Totl RNA from liver or ells ws prepre with TRIzol regent (Invitrogen), n DNA templtes were generte using Supersript III reverse trnsriptse (Invitrogen) with n oligo-t primer, oring to the mnufturer s protool. Rel-time PCR nlysis ws performe using SYBR Green PCR Mster Mix (Applie Biosystems). Expression of the genes of interest ws normlize to the levels of Gph or Atin mrna, n reltive expression levels were lulte oring to the DDC T metho. Primer sequenes use for quntittive PCR re liste in Supplementry Tble. Heptoyte preprtion. Livers were perfuse n igeste by pssing wrm perfusion buffer (HBSS supplemente with mm HEPES n. mm EGTA) n igestion buffer (HBSS supplemente with mm HEPES n. mgml liberse (Rohe)s) sequentilly vi the portl vein. Liver tissue ws further gently igeste n ells were psse sequentilly through -, 7- n -mm ell striners. Heptoytes were isolte by entrifugtion t g for min t RT, Lentiviruses. Reombinnt lentiviruses were pkge in HEK 9T ells by otrnsfetion of -enoing plsmis n helper plsmis suh s prsv-rev, pmdlg n pvsv-g. Western blotting nlysis. Primry liver leukoytes n DN.D ells were wshe n inubte t C for min in lysis buffer ( mm Tris-Cl, ph 7., mm NCl, mm EDTA, % NP-,.% soium eoxyholte, n.% SDS) ontining protese inhibitor oktil (Rohe), mm DTT, n mm PMSF. Liver tissue smples were wshe with ie-ol, resuspene in lysis buffer t C, n homogenize. Cell n liver lystes were inubte for min t Cn entrifuge t, r.p.m. for min t C. For preprtion of nuler extrts, ells were inubte in ml of buffer ( mm HEPES, ph 7.9, mm KCl,. mm EDTA,. mm EGTA, mm DTT n. mm PMSF) t C for min. NP- ws then e to finl onentrtion of.%, n the tube ws vigorously vortexe for s. The homogente ws entrifuge t, g for min t C, the nuler pellet ws resuspene in ml of buffer ( mm HEPES, ph 7.9, mm NCl, mm EDTA, mm DTT n mm PMSF), n the tube ws vigorously vortexe t C for min. The extrt ws entrifuge t,g for min t C. Protein onentrtions in the superntnts were etermine by the Brfor metho. Lystes were resolve by SDS polyrylmie gel, n proteins were trnsferre to membrnes, immunoblotte with the pproprite primry (ilution :,) n seonry ntiboies (ilution :,,), n visulize by hemiluminesene. Imges hve been roppe for presenttion. Full size imges re presente in Supplementry Fig. 9. Mesurement of ytokine onentrtions. IFN-g, TNF n IL- levels in ser or ulture superntnts were quntifie by ELISA (ebiosiene). Sttistil nlysis. Dt were nlyse with the Mnn Whitney U-test. Po. ws onsiere signifint. The sttistil signifine of the results ws lso onfirme by nlysis of vrine test, n the t psse the normlity test. Sttistil nlysis ws performe using Prism softwre (GrphP, Sn Diego). Referenes. Foell, D., Wittkowski, H. & Roth, J. Mehnisms of isese: DAMP view of inflmmtory rthritis. Nt. Clin. Prt. Rheumtol., 8 9 (7).. Cho, Y. S. et l. Phosphoryltion-riven ssembly of the RIP-RIP omplex regultes progrmme nerosis n virus-inue inflmmtion. Cell 7, (9).. Lin, J. et l. A role of RIP-meite mrophge nerosis in theroslerosis evelopment. Cell Rep., ().. Royhowhury, S., MMullen, M. R., Pisno, S. G., Liu, X. & Ngy, L. E. Absene of reeptor interting protein kinse prevents ethnol-inue liver injury. Heptology 7, (). NATURE COMMUNICATIONS :87 DOI:.8/nomms97 & Mmilln Publishers Limite. All rights reserve.