Atypical Pro Caspase 8 Complex Mediates − a RIPK3

A RIPK3−Caspase 8 Complex Mediates Atypical Pro−IL-1β Processing Kenta Moriwaki, John Bertin, Peter J. Gough and Francis Ka-Ming Chan This information is current as of September 25, 2021. J Immunol 2015; 194:1938-1944; Prepublished online 7 January 2015; doi: 10.4049/jimmunol.1402167 http://www.jimmunol.org/content/194/4/1938 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A RIPK3–Caspase 8 Complex Mediates Atypical Pro–IL-1b Processing

Kenta Moriwaki,* John Bertin,† Peter J. Gough,† and Francis Ka-Ming Chan*

Caspase 8, the initiator caspase for death receptor–induced apoptosis, functions as a negative regulator of receptor interacting protein kinase 3 (RIPK3), an essential factor for TNF-, TLR3-, and TLR4-induced necroptosis. In certain situations, caspase 8 can also participate in pro–IL-1b processing. However, the biochemical complex that mediates caspase 8–mediated processing is not deﬁned. In this study, we show that RIPK3 is crucial for caspase 1– and caspase 8–mediated pro–IL-1b and pro–IL-18 processing in bone marrow–derived dendritic cells (BMDCs) in response to LPS stimulation. Caspase 8–mediated pro–IL-1b processing requires intact RIPK1, RIPK3, TRIF, and FADD. In response to LPS, a complex that contains RIPK1, RIPK3, FADD, and caspase 8 is formed. Surprisingly, RIPK3-speciﬁc kinase inhibitors strongly enhanced caspase 8 activation and pro–IL-1b processing in

LPS-stimulated BMDCs. However, studies in BMDCs expressing the kinase-inactive RIPK3-K51A mutant or RIPK1-K45A mutant Downloaded from showed that the kinase activity of neither RIPK1 nor RIPK3 is required for LPS-induced caspase 8 activation and IL-1b secretion. Hence, RIPK3 is an unexpected positive regulator of caspase 8 activity that promotes IL-1b maturation in BMDCs. The Journal of Immunology, 2015, 194: 1938–1944.

nterleukin-1b is an inﬂammatory cytokine that is closely rodentium,orEscherichia coli also induced caspase 8–dependent

associated with acute and chronic inflammation and has IL-1b secretion (5–7). In addition, stimulation with Fas ligand, che- http://www.jimmunol.org/ I emerged as a therapeutic target for various systemic and motherapeutic agents, or endoplasmic reticulum stress elicited IL-1b local inflammatory diseases (1). In response to pattern recognition secretion in a caspase 8–dependent manner in LPS-primed macro- receptor stimulation, APCs such as dendritic cells (DCs) and phages or DCs (8–11). However, the biochemical mechanism that macrophages produce pro–IL-1b through NF-kB–dependent stimulates caspase 8–mediated pro–IL-1b processing is undefined. transcription. Full maturation and secretion of IL-1b require Receptor interacting protein kinase (RIPK)3 is a serine/threonine caspase 1–mediated proteolytic processing. Caspase 1 is the en- kinase that is crucial for necroptosis in response to ligand binding to zymatic component of a macromolecular structure termed the TNFR-like death receptors, TLR3 and TLR4 (12). RIPK3 interacts inflammasome, which also contains the adaptor protein ASC and with its upstream activator RIPK1 via the receptor-interacting a sensor protein such as NLRP3 or AIM2. protein homotypic interaction motif (RHIM) to form an amyloid- by guest on September 25, 2021 Caspase 8 is the initiator caspase for death receptors in the TNFR like complex termed the necrosome to signal for necroptosis family. It is recruited to the receptor through binding to its upstream downstream of TNF receptor-like death receptors (13). The kinase adaptor Fas-associated death domain protein (FADD). Interest- activities of RIPK3 and RIPK1 are critical for stabilizing the ingly, recent studies show that caspase 8 can also regulate IL-1b necrosome and to promote necroptosis. In contrast, RIPK3 inter- expression by promoting de novo synthesis of pro–IL-1b and acts with another RHIM-containing adaptor Toll/IL-1R domain– processing of pro–IL-1b into its cleaved mature form (2). For containing adapter inducing IFN-b (TRIF) to mediate TLR3- and instance, in DCs, fungi and mycobacteria stimulate Dectin-1– TLR4-induced necroptosis (14, 15). Interestingly, both RIPK1 and mediated and caspase 8–dependent IL-1b secretion (3, 4). Infec- RIPK3 are cleaved and inactivated by caspase 8 (16, 17). Hence, tion of macrophages with Salmonella, Yersinia, Citrobacter necroptosis is optimally induced when the FADD–caspase 8 complex is inactivated (18). Although necroptosis is the most prominent function of RIPK3, *Department of Pathology, Program in Immunology & Microbiology, University of Massachusetts Medical School, Worcester, MA 01655; and †Pattern Recognition several studies showed that RIPK3 could also promote nonnecrotic Receptor Discovery Performance Unit, Immuno-Inflammation Therapeutic Area, signaling under certain conditions. For example, RIPK3 drives IL- GlaxoSmithKline, Collegeville, PA 19422 1b secretion in LPS-primed macrophages or DCs when cellular Received for publication August 22, 2014. Accepted for publication December 7, inhibitor of apoptosis (cIAP) proteins or caspase 8 are depleted 2014. (19–21). In addition, in cells that lack TAK1, RIPK3 promotes This work was supported by National Institutes of Health Grant AI083497 (to TNF-induced apoptosis (22). Mice genetically engineered to ex- F.K.-M.C.). K.M. was supported by postdoctoral fellowships from the Uehara Memo- D161N-KD rial Foundation and the Japan Society for the Promotion of Science. press the kinase-dead RIPK3 mutant D161N (Ripk3 Address correspondence and reprint requests to Dr. Francis Ka-Ming Chan, Depart- mice) died at midgestation because of hyperactivation of caspase ment of Pathology, University of Massachusetts Medical School, 368 Plantation 8 and excessive apoptosis (23). However, because the RIPK3- Street, Worcester, MA 01605. E-mail address: [email protected] dependent effects on IL-1b and apoptosis were detected in The online version of this article contains supplemental material. highly manipulated experimental systems, it remains unclear Abbreviations used in this article: BMDC, bone marrow–derived DC; CHX, cyclo- whether RIPK3 has physiological functions beyond necroptosis. heximide; cIAP, cellular inhibitor of apoptosis; DC, dendritic cell; FADD, Fas- b associated death domain protein; GSK, GlaxoSmithKline; IAP, inhibitor of apoptosis; In this study, we show that RIPK3 promotes IL-1 secretion RHIM, receptor-interacting protein homotypic interaction motif; RIPK, receptor through assembly of an alternative FADD–caspase 8–activating interacting protein kinase; SMAC, second mitochondria–derived activator of caspase; complex. This necroptosis-independent effect of RIPK3 also TRIF, Toll/IL-1R domain–containing adapter inducing IFN-b; WT, wild-type. requires TRIF and RIPK1. In contrast to necroptosis, the kinase Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 activities of RIPK1 and RIPK3 are dispensable for assembly of www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402167 The Journal of Immunology 1939 this complex and IL-1b secretion. In fact, RIPK3 kinase inhibitors NF-kB pathway, and a second signal that causes inflammasome facilitate conformational change that is conducive for assembly of activation and caspase 1–dependent processing of pro–IL-1b. this alternative caspase 8–activating complex. These results reveal However, LPS alone was sufficient to induce a low level of IL-1b RIPK3 as a positive regulator of nonapoptotic caspase 8 activation secretion in DCs (27). Surprisingly, we found that LPS-induced and highlight the complex interplay between RIPK3 and caspase 8 IL-1b secretion was strongly suppressed in Ripk32/2 BMDCs in cell death-dependent and independent signaling. (Fig. 1A, Supplemental Fig. 1A, 1B) (28). LPS alone did not lead to secretion of the related cytokine IL-18 (Supplemental Fig. 1B, Materials and Methods 1C), although pro–IL-18 was constitutively expressed in BMDCs Mice (Supplemental Fig. 1D). Stimulation of the inflammasome with b Ripk32/2 (V. Dixit, Genentech), Trif2/2, Myd882/2 (E. Lien, University of ATP led to greater level of secreted IL-1 and detectable IL-18 Massachusetts Medical School), Casp12/2Casp112/2 (K. Rock, University secretion that were also dependent on intact RIPK3 (Supplemental of Massachusetts Medical School), RIPK1-K45A (Ripk1K45A-KD), and RIPK3- Fig. 1C). Genetic deletion of caspase 1, Nlrp3, Asc (27), or in- K51A kinase-dead (Ripk3K51A-KD) knock-in mice (GlaxoSmithKline hibition of caspase 1 with the inhibitor z-YVAD-fmk only par- fl/fl 2/2 [GSK]) were used (24, 25). Fadd :Cd11c-Cre (dcFadd )micewere tially suppressed LPS-induced IL-1b secretion (Fig. 1B). This provided by A. Winoto (University of California Berkeley) (26). Wild-type (WT) mice from Ripk3+/2 intercross were used. All mice were housed in suggests that RIPK3 is responsible for caspase 1–dependent and specific pathogen-free facility at University of Massachusetts Medical independent pathways of pro–IL-1b processing. School. All animal experiments were approved by the institutional animal Besides caspase 1 activation, LPS also triggered caspase 8 ac- care and use committee. tivation in WT BMDCs (Fig. 1C, Supplemental Fig. 1D). Strik- 2 2 Reagents ingly, caspase 8 activation was also abolished in Ripk3 / Downloaded from BMDCs (Fig. 1C, Supplemental Fig. 1D) (28). RIPK3 did not LPS (Invivogen), z-VAD-fmk, z-YVAD-fmk, z-IETD-fmk, Necrostain-1 b (Nec-1), MG-132 (Enzo Life Sciences), cycloheximide (CHX; Sigma- affect the priming of BMDCs by LPS, because pro–IL-1 in- 2/2 Aldrich), and N-acetyl-L-cysteine (Calbiochem) were used. RIPK3 ki- duction 1 h after LPS treatment was normal in Ripk3 BMDCs nase inhibitors GSK’840, GSK’872, and GSK’843 were provided by GSK (Fig. 1C, Supplemental Fig. 1D). However, the level of intracel- and have been described in previous publications (15, 25). lular pro–IL-1b was higher in Ripk32/2 BMDCs than in WT

Generation and stimulation of bone marrow–derived DCs BMDCs 6 h after LPS treatment (Supplemental Fig. 1E). These http://www.jimmunol.org/ results are most consistent with impaired cleavage of pro–IL-1b in Bone marrow cells were cultured for 1 wk with 10 ng/ml GM-CSF and 2/2 5 ng/ml IL-4 to generate bone marrow–derived DCs (BMDCs). Faddfl/fl and Ripk3 BMDCs. Consistent with a deficiency in cleavage of Faddfl/fl:Cd11c-Cre (dcFadd2/2) bone marrow cells were differentiated pro–IL-1b, the caspase 8–specific inhibitor z-IETD-fmk partially 2 2 2 into BMDCs by GM-CSF alone. Ripk1+/ and Ripk1 / newborn liver suppressed LPS-induced IL-1b secretion, but not pro–IL-1b in- cells were differentiated to DCs with GM-CSF and IL-4. After stimula- duction (Fig. 1D, Supplemental Fig. 1F). Coadministration of tion, culture media and cells were used for ELISA, protein, and cell death analyses (CellTiter-Glo Luminescent Cell Viability Assay; Promega). The caspase 1 and caspase 8 inhibitors further abolished the residual untreated sample is defined as having a value of 0% cell death. IL-1b and IL-1b secretion (Fig. 1D), indicating that both caspases contrib- IL-18 were measured by ELISA sets from BD Biosciences and R&D uted to IL-1b secretion by LPS-treated BMDCs. Systems, respectively. IAP proteins are natural inhibitors of RIPK3-dependent IL-1b by guest on September 25, 2021 Western blotting and immunoprecipitation secretion (19, 20). However, LPS treatment did not alter cIAP1/2 expression in BMDCs (Supplemental Fig. 1G), indicating that Whole-cell extracts were prepared in radioimmunoprecipitation assay b buffer. Protease inhibitor mixture (Roche) and phosphatase inhibitor RIPK3-dependent IL-1 secretion was not due to loss of cIAP mixture (Sigma-Aldrich) were included in lysis buffer. Secreted proteins in expression. Although it is an initiator caspase for apoptosis, cas- culture media were precipitated by trichloroacetic acid. For immunopre- pase 8 activation did not cause cell death in LPS-treated WT cipitation, anti-RIPK3 (Prosci) or anti–caspase 8 (Enzo Life Sciences) Abs BMDCs. However, inhibition of de novo synthesis of prosurvival were used. Western blotting was performed with anti-RIPK1 (BD Bio- molecules with CHX led to LPS-induced cell death that was de- sciences), RIPK3 (Prosci), FADD (provided by A. Winoto), caspase 8 (Enzo Life Sciences), MyD88 (eBiosciene), IL-1b, cIAP1/2 (R&D Sys- pendent on RIPK3 (Fig. 1E). These results indicate that, besides tems), GFP (Roche), caspase-1 (Santa Cruz), and IL-18 (BioVision) Abs. caspase 1, RIPK3 also stimulates caspase 8–mediated pro–IL-1b Anti-HSP90 Ab (BD Biosciences) was used as a loading control. processing in LPS-treated BMDCs. Moreover, in contrast to death Lentivirus transfection receptor–induced apoptosis, LPS-induced caspase 8 activation did not result in cell death. Various mutants of mouse RIPK3 were cloned into pTRIPZ vector (Open Biosystems). The 293T cells were transfected by LV-GFP pLKO.1 (addgene RIPK3 mediates assembly of an alternative FADD–caspase 8 25999), LV-Cre pLKO.1 (addgene 25997), or mRIPK3/pTRIPZ in com- complex in a TRIF-dependent manner bination with pMD2.G and psPAX2 to generate lentivirus. On day 4 during BMDC differentiation, floating cells were collected; plated in media MyD88 is required for signaling by all TLR family receptors except containing lentivirus, 10 mg/ml polybrene, GM-CSF, and IL-4; and incu- TLR3, whereas TRIF is a unique signal adaptor for TLR3 and bated for an additional three days prior to stimulation. Lentivirus carrying 2/2 2/2 TLR4. LPS-induced caspase 8 activation was abolished in Trif RIPK3 or its mutant genes was transduced to Ripk3 3T3 cells, and then 2/2 cells were selected for puromycin resistance. To induce RIPK3 expression, BMDCs (Fig. 1F). In contrast, Myd88 BMDCs exhibited cells were treated with 1 mg/ml doxycycline. normal caspase 8 activation. These results are consistent with a Statistical analysis previous report that in macrophages, TRIF is required for caspase 8–dependent IL-1b secretion downstream of TLR3 or TLR4 and The p values were calculated using unpaired t test with Welch’s correction. CHX stimulation (2). Moreover, these results also indicate that The p values ,0.05 were considered statistically significant. TRIF acts upstream of RIPK3 to promote caspase 8 activation. Our recent work shows that RIPK3 promotes caspase 1 acti- Results vation in BMDCs through reactive oxygen species production (28). b LPS induces RIPK3-dependent caspase 8 activation and IL-1 However, unlike caspase 1, the reactive oxygen species scavenger secretion in BMDCs N-acetyl-L-cysteine did not block LPS-induced caspase 8 activa- IL-1b production generally requires two distinct signals, as fol- tion (Fig. 2A). This indicates that RIPK3 controls caspase 1 and lows: a first signal that elicits pro–IL-1b expression through caspase 8 activity through different mechanisms. Binding to the 1940 RIPK3 ENHANCES CASPASE 8 ACTIVATION Downloaded from http://www.jimmunol.org/

FIGURE 1. RIPK3 promotes caspase 1– and caspase 8–dependent IL-1b secretion in BMDCs. (A) RIPK3 is required for LPS-induced IL-1b secretion in BMDCs. IL-1b secretion by WT and Ripk32/2 BMDCs stimulated with LPS for 6 h was determined by ELISA (n = 4). (B) Caspase 1 is partially responsible for RIPK3-dependent IL-1b secretion. WT BMDCs were pretreated with 10 mM z-YVAD-fmk (YVAD) for 1 h and stimulated with LPS for 4 h (n = 4). IL-1b secretion was determined by ELISA. (C) RIPK3 is critical for LPS-induced caspase 8 activation. Cell lysates from WT and Ripk32/2 BMDCs stimulated with LPS for 1 h were subjected to Western blot analysis. (D) Caspase 1 and caspase 8 cooperate to mediate optimal IL-1b secretion. WT BMDCs pretreated with 10 mM YVAD and/or 10 mM z-IETD-fmk (IETD) for 1 h were stimulated with 50 ng/ml LPS for 6 h (n = 4). IL-1b secretion was determined by ELISA. (E) Inhibition of protein synthesis converts the LPS-induced RIPK3 signal to one that causes apoptosis. WT and Ripk32/2 BMDCs pretreated with CHX for 1 h were stimulated with LPS for 14 h. Cell death was determined by measuring intracellular ATP level (n = 3). (F) RIPK3-

dependent caspase 8 activation requires an intact TRIF. Cell lysates from BMDCs of the indicated genotypes stimulated with LPS for 1 h were subjected to by guest on September 25, 2021 Western blot analysis. Results shown are mean 6 SEM. *p , 0.05. adaptor FADD is a requisite step for autocleavage and activation pared with the cells transduced with GFP vector (Fig. 2D [com- of caspase 8 in response to death receptor stimulation (29). In- pare lanes 6 and 8], Supplemental Fig. 2B). In contrast, WT and terestingly, we detected constitutive interaction between caspase 8 Ripk1K45A-KD BMDCs transduced with control GFP lentivirus and FADD in BMDCs (Fig. 2B, Supplemental Fig. 2A). The in- exhibited equal caspase 8 activation and IL-1b secretion (Fig. 2D teraction between FADD and caspase 8 was not changed upon [compare lanes 2 and 6], Supplemental Fig. 2B). Consistent with LPS treatment (Fig. 2B), suggesting that LPS causes a qualitative, results from Ripk1K45A-KD BMDCs, the RIPK1 kinase inhibitor but not quantitative change in FADD–caspase 8 interaction. This Nec-1 also did not affect TLR4-induced caspase 8 activation is in contrast to TNF- and CHX-stimulated 3T3 fibroblasts, which (Supplemental Fig. 2C). These results indicate that, whereas an showed inducible interaction between FADD and caspase 8 intact RIPK1 is essential for TLR4-induced caspase 8 activation, (Supplemental Fig. 2A). its kinase activity is dispensable. Moreover, our results suggest Because RIPK3 was required for LPS-induced caspase 8 acti- that LPS triggers formation of a RIPK1–RIPK3–FADD–caspase 8 vation (Fig. 1C), we examined RIPK3 recruitment to the caspase 8 complex. This complex is reminiscent of the apoptosis complex complex. Indeed, LPS significantly enhanced the interaction of driven by the kinase-inactive RIPK3 mutant D161N (23). How- RIPK3 with caspase 8 and FADD (Fig. 2C). RIPK1 is a RHIM- ever, recruitment of RIPK1 to the RIPK3–FADD–caspase 8 and death domain–containing adaptor that facilitates death re- complex in LPS-treated BMDCs was weak (see below), in part ceptor–induced apoptosis and necroptosis. We reasoned that because of a reduction in overall RIPK1 expression (Fig. 2D). The RIPK1 might interact with RIPK3 via the RHIM and FADD or reduction in cellular RIPK1 was not due to caspase-mediated caspase 8 via the death domain to mediate caspase 8 activation. To cleavage or proteasome-dependent degradation (Supplemental ascertain this possibility, we took advantage of Ripk1K45A-KD Fig. 2D), but might be caused by translocation to detergent- mice, which express a knock-in version of kinase-inactive RIPK1 insoluble compartment (30). (24). Because exon 3 of the Ripk1K45A-KD allele is flanked by loxP RIPK3 kinase inhibitor enhanced LPS-induced caspase 8 sites, we can specifically delete Ripk1 by Cre-mediated recombi- K45A-KD activation and IL-1b secretion nation in BMDC cultures. We transduced WT or Ripk1 BMDCs with lentivirus expressing Cre or GFP as control and The results shown above indicate that RIPK3 licenses activation observed significant reduction of RIPK1 expression in Ripk1K45A-KD, of the FADD–caspase 8 complex. In necroptosis, RIPK3 kinase but not WT BMDCs transduced with Cre-expressing lentivirus activity is essential for assembly of a signaling complex termed (Fig. 2D, compare lanes 3 and 7). In these cells, LPS-induced the necrosome (31). To address whether RIPK3 kinase activity is caspase 8 activation and IL-1b secretion were suppressed com- similarly required for LPS-induced caspase 8 activation, we used The Journal of Immunology 1941

(32). This necrotic cell death was suppressed by GSK’872 and GSK’843, but not by the human RIPK3-specific inhibitor GSK’840 (25). Surprisingly, GSK’872 and GSK’843 significantly increased LPS-induced IL-1b secretion in a dose-dependent manner (Fig. 3B). The enhanced IL-1b secretion requires TLR4 signaling, because the inhibitors did not induce IL-1b secretion without LPS (Fig. 3C). The effect of the inhibitors was RIPK3 specific, because neither GSK’872 nor GSK’843 increased IL-1b secretion in Ripk32/2 BMDCs (Fig. 3B). The RIPK3 kinase inhibitor-induced increase in IL-1b secretion was not driven by caspase 1, because enhanced LPS-induced IL-1b secretion was also detected in RIPK3 inhibitor-treated Casp12/2Casp112/2 BMDCs (Fig. 3D, filled bars). FADD is an essential adaptor of caspase 8 activation. DC- specific Fadd-deficient (dcFadd2/2) mice developed systemic inflammation associated with elevated proinflammatory cytokines, including IL-1b (26). BMDCs generated from dcFadd2/2 mice FIGURE 2. RIPK3 promotes formation of an alternative caspase 8– showed significantly increased IL-1b secretion upon treatment activating complex. (A) RIPK3 mediates caspase 8 activation independent with LPS (Fig. 4A) (26). The enhanced IL-1b secretion was due to Downloaded from of ROS production. WT BMDCs pretreated with NAC for 1 h were increased pro–IL-1b induction and caspase 1, but not caspase 8, stimulated with LPS for 1 h. Cell lysates were subjected to Western blot activation, because caspase 8 activation was abolished in analysis. (B and C) LPS induces assembly of a complex among RIPK3, 2/2 2/2 dcFadd BMDCs (Fig. 4B) (21). Moreover, the RIPK3 inhibitor FADD, and caspase 8. Cell lysates from WT and Ripk3 BMDCs GSK’872 did not enhance IL-1b secretion or caspase 8 activation stimulated with LPS for 1 h were subjected to immunoprecipitation with 2 2 in dcFadd / BMDCs (Fig. 4A, 4B). Because GSK’872 only (B) anti-caspase 8 or (C) anti-RIPK3 Abs, followed by Western blot

b http://www.jimmunol.org/ analysis. IP, immunoprecipitate; WCE, whole-cell extract. (D) An intact enhanced caspase 8 activation, but not pro–IL-1 expression and RIPK1, but not its kinase activity, is required for LPS-induced caspase 8 caspase 1 activation (Fig. 4C), these results indicate that the activation. WT and Ripk1K45A-KD BMDCs were transduced with lentivirus RIPK3 inhibitor enhanced pro–IL-1b processing through caspase expressing GFP or Cre and subsequently stimulated with LPS for 1 h. Cell 8, but not caspase 1. In addition to FADD, RIPK1, and RIPK3, lysates were subjected to Western blot analysis. LPS-induced caspase 8 activation also requires an intact TRIF, but not MyD88 (Supplemental Fig. 2E). the recently described RIPK3-specific kinase inhibitors GSK’872 Consistent with the specific stimulation of caspase 8–mediated and GSK’843 (15, 25). Treatment with the second mitochondria– pro–IL-1b processing, GSK’872 augmented LPS-induced inter- derived activator of caspase (SMAC)–mimetic LBW242 and the action of RIPK3 with FADD and caspase 8 (Fig. 4D). Specifically, pan-caspase inhibitor z-VAD-fmk induced autocrine TNF pro- GSK’872 significantly enhanced LPS-induced RIPK1 recruitment by guest on September 25, 2021 duction and RIPK3-dependent necroptosis in BMDCs (Fig. 3A) to RIPK3 (Fig. 4D). Moreover, deletion of RIPK1 by Cre-

FIGURE 3. RIPK3 kinase inhibitor enhances LPS- induced IL-1b secretion. (A) RIPK3 kinase inhibitors protect BMDCs against necroptosis. WT and Ripk32/2 BMDCs pretreated with indicated concentration (mM) of RIPK3 kinase inhibitors for 1 h were stimulated with 10 mM z-VAD-fmk (zVAD) and 0.1 mM LBW242 for 14 h to induce necroptosis (n = 3). (B and C) RIPK3 kinase inhibitors increase LPS-induced IL-1b secretion. WT or Ripk32/2 BMDCs pretreated with the indicated RIPK3 inhibitors (mM) for 1 h were stimulated with 50 ng/ml LPS for 6 h (n = 3). IL-1b secretion was determined by ELISA. (D) The effect of RIPK3 inhibitor on IL-1b secretion is not mediated by caspase 1 or caspase 11. WT and Casp12/2Casp112/2 BMDCs pretreated with GSK’872 for 1 h were stimulated with LPS for 6 h (n = 4). IL-1b secretion was determined by ELISA. Results shown are mean 6 SEM. *p , 0.05. 1942 RIPK3 ENHANCES CASPASE 8 ACTIVATION

FIGURE 4. RIPK3 kinase activity is dispensable for LPS-induced caspase 8 activation. (A and B) FADD is required for RIPK3-dependent caspase 8 activation. BMDCs generated from Faddfl/fl and Faddfl/fl:Cd11c-Cre (dcFadd2/2) bone marrow cells were pretreated with GSK’872 for 1 h and then stimulated with LPS for (A) 6hor(B) 1 h. (A) IL-1b secretion was determined by ELISA (n = 2). (B) Cell lysates were subjected to Western blot analysis. (C and D) RIPK3 kinase inhibitor specifically enhances caspase 8, but not caspase 1 activation. WT BMDCs pretreated with GSK’872 for 1 h were stimulated with LPS for 1 h. Cell lysates were subjected to (C) Western blot analysis or (D) immunoprecipitation with anti-RIPK3 Ab, followed by Western blot analysis. IP, immunoprecipitate; WCE, whole-cell extract. (E) RIPK3 kinase inhibitor enhances caspase 8 activation through RIPK1. WT and Ripk1K45A-KD BMDCs were transduced with lentivirus Downloaded from expressing GFP or Cre and subsequently treated with GSK’872 for 1 h, followed by LPS for 1 h. Cell lysates were subjected to Western blot analysis. (F and G) RIPK3 kinase activity is essential for necroptosis, but not IL-1b secretion. (F) WT and Ripk3K51A-KD BMDCs were treated with 10 mM z-VAD-fmk (zVAD) and 0.1 m

M LBW242 for 14 h to induce necroptosis (n = 3). http://www.jimmunol.org/ Cell death was determined by measuring intracellular ATP level. The upper panel shows protein expression level of RIPK3. (G) WT and Ripk3K51A-KD BMDCs pretreated with GSK’872 for 1 h were stimulated with LPS for 6 h (n = 3). IL-1b secretion was determined by ELISA. Results shown are mean 6 SEM. by guest on September 25, 2021 mediated recombination in Ripk1K45A-KD BMDCs repressed the autocrine TNF production and necroptosis (32). Consistent with GSK’872-mediated enhancement of caspase 8 activation (Fig. 4E). the essential role of RIPK3 kinase activity in necroptosis, The residual caspase 8 activation by GSK’872 was most likely due Ripk3K51A-KD BMDCs were protected from SMAC-mimetic and to incomplete deletion of RIPK1 (Fig. 4E, lane 10, second panel). zVAD-induced necroptosis (Fig. 4F). In contrast to the RIPK3 Indeed, GSK’872-mediated enhancement of caspase 8 activation inhibitors, LPS-induced IL-1b secretion was not increased in was completely abrogated in Ripk12/2 DCs generated from Ripk3K51A-KD BMDCs (Fig. 4G). In fact, Ripk3K51A-KD BMDCs Ripk12/2 newborn liver cells (Supplemental Fig. 2F). The RIPK1 secreted reduced level of IL-1b (Fig. 4F). However, the reduced kinase inhibitor Nec-1 failed to reverse the enhanced caspase 8 IL-1b secretion was most likely caused by reduced RIPK3 activation by the RIPK3 inhibitor (Supplemental Fig. 2G). Hence, expression in Ripk3K51A-KD BMDCs (Fig. 4F). Importantly, the enhanced RIPK3-dependent caspase 8 activation and pro–IL-1b GSK’872 still enhanced IL-1b secretion by LPS-treated processing by RIPK3 kinase inhibitors require an intact RIPK1, but Ripk3K51A-KD BMDCs (Fig. 4G). These results strongly suggest not its kinase activity. that RIPK3 kinase activity is not required for LPS-induced caspase 8 activation. Rather, the RIPK3 kinase inhibitors promote IL-1b RIPK3 kinase activity is not required for LPS-induced caspase secretion by inducing a conformational change that is amenable for 8 activation assembly of the alternative caspase 8 complex. The results with the RIPK3 inhibitors are surprising given the Although the low doses of the RIPK3 kinase inhibitor we used in positive role of RIPK3 in pro–IL-1b processing. One possible BMDCs did not cause any toxicity (Supplemental Fig. 3A), higher explanation for these results is that RIPK3 phosphorylates and concentrations of GSK’872 enhanced caspase 8 activation and inhibits an unknown substrate that is required for switching on apoptosis in RIPK3-positive cells (25). Strikingly, the apoptosis caspase 8 activity. In support of this model, mice expressing the induced by high doses of the RIPK3 kinase inhibitors requires kinase-inactive RIPK3 mutant D161N (Ripk3D161N-KD) were re- a similar RIPK1–RIPK3–FADD–caspase 8 complex (25). There- cently reported to suffer from embryonic lethality due to massive fore, we used this system to further examine the requirement of caspase 8–mediated apoptosis in the yolk sac (23). However, the RHIM, which is essential for necroptosis (13), for RIPK3- mice expressing another kinase-inactive RIPK3 mutant K51A dependent caspase 8 activation. We expressed WT or mutant (Ripk3K51A-KD) were viable (25), suggesting that inhibition of RIPK3 in Ripk32/2 3T3 cells under the control of doxycycline. RIPK3 kinase activity per se does not necessarily induce caspase 8 Expression of WT RIPK3 and RIPK3-D161N, but not RIPK3- activation. To further test the role of RIPK3 kinase activity in K51A, led to caspase 8–dependent apoptosis (25) (Supplemental LPS-induced caspase 8 activation, we generated BMDCs from Fig. 3B). The cell death induced by RIPK3-D161N is apoptotic, Ripk3K51A-KD mice. Treatment of DCs or macrophages with because it was reversed by the pan-caspase inhibitor z-VAD-fmk SMAC mimetics in the presence of caspase inhibition causes (Supplemental Fig. 3C). However, z-VAD-fmk did not rescue cell The Journal of Immunology 1943 death in WT RIPK3-expressing cells due to a switch from apo- formation of the ripoptosome and the necrosome requires deple- ptosis to necroptosis (Supplemental Fig. 3C). Caspase 8–depen- tion of the cellular IAPs. Although depletion of cellular IAPs is dent apoptosis was enhanced by GSK’872 in cells expressing WT not a prerequisite for the caspase 8 complex that cleaves pro–IL- RIPK3, RIPK3-K51A, and RIPK3-D161N (Supplemental Fig. 1b, other adaptors may yet determine the signaling outcome of 3B), indicating that the RIPK3 kinase inhibitor enhanced cas- this alternative caspase 8–activating complex. It is also noteworthy pase 8 activation independent of the inhibition of RIPK3 kinase that the kinase activities of RIPK1 and RIPK3, which are essential activity. In contrast, tetra-alanine substitution in the RHIM abol- for necrosome and ripoptosome assembly, are dispensable for ished caspase 8 activation and apoptosis in WT RIPK3- and the LPS-induced caspase 8 activation. Hence, posttranslational RIPK3-D161N–expressing cells (Supplemental Fig. 3B) (25). In modifications such as ubiquitination, protein phosphorylation, or addition, the effect of GSK’872 was no longer observed in the binding by pathway-specific adaptors may determine the signaling RHIM mutant (Supplemental Fig. 3B). These results indicate that outcome of the RIPK1–RIPK3–FADD–caspase 8 complex. RHIM-mediated interaction is crucial for necroptosis as well as RIPK3-dependent caspase 8 activation and pro–IL-1b processing. Acknowledgments We thank D. Porter (Novartis) for LBW242 and V. Dixit, E. Mocarski, Discussion W. Kaiser, K. Rock, M. Kelliher, E. Lien, and A. Winoto for providing dif- RIPK3 is touted as a major driver of inflammation due to ferent knockout mice used in this study. necroptosis-associated release of intracellular immunogenic con- tents. However, recent evidence shows that RIPK3 can promote Disclosures inflammation independent of necroptosis (33). Specifically, RIPK3 J.B. and P.J.G. are employees of GlaxoSmithKline. Downloaded from positively promotes IL-1b secretion in macrophages or DCs when caspase 8 or IAP proteins are depleted (19–21). In contrast, sev- References b eral recent studies show that RIPK3 is dispensable for IL-1 se- 1. Dinarello, C. A. 2011. Interleukin-1 in the pathogenesis and treatment of in- cretion in macrophages when caspase 8 and IAP functions are flammatory diseases. Blood 117: 3720–3732. preserved (5–9). In this study, we show that RIPK3 functions as an 2. Maelfait, J., E. Vercammen, S. Janssens, P. Schotte, M. Haegman, S. Magez, and R. Beyaert. 2008. 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