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• W2078252006 abstract "Caspase-8 deficiency in certain cells prompts chronic inflammation. One mechanism suggested to account for this inflammation is enhanced signaling for necrotic cell death, mediated by the protein kinases RIPK1 and RIPK3 that caspase-8 can cleave. We describe an activity of caspase-8 in dendritic cells that controls the initiation of inflammation in another way. Caspase-8 deficiency in these cells facilitated lipopolysaccharide-induced assembly and function of the NLRP3 inflammasome. This effect depended on the functions of RIPK1 and RIPK3, as well as of MLKL and PGAM5, two signaling proteins recently shown to contribute to RIPK3-mediated induction of necrosis. However, although enhancement of inflammasome assembly in the caspase-8-deficient cells shares proximal signaling events with the induction of necrosis, it occurred independently of cell death. These findings provide new insight into potentially pathological inflammatory processes to which RIPK1- and RIPK3-mediated signaling contributes. Caspase-8 deficiency in certain cells prompts chronic inflammation. One mechanism suggested to account for this inflammation is enhanced signaling for necrotic cell death, mediated by the protein kinases RIPK1 and RIPK3 that caspase-8 can cleave. We describe an activity of caspase-8 in dendritic cells that controls the initiation of inflammation in another way. Caspase-8 deficiency in these cells facilitated lipopolysaccharide-induced assembly and function of the NLRP3 inflammasome. This effect depended on the functions of RIPK1 and RIPK3, as well as of MLKL and PGAM5, two signaling proteins recently shown to contribute to RIPK3-mediated induction of necrosis. However, although enhancement of inflammasome assembly in the caspase-8-deficient cells shares proximal signaling events with the induction of necrosis, it occurred independently of cell death. These findings provide new insight into potentially pathological inflammatory processes to which RIPK1- and RIPK3-mediated signaling contributes. Mice with caspase-8-deficient dendritic cells overproduce IL-1β in response to LPS These deficient dendritic cells show enhanced LPS-induced NLRP3 inflammasome assembly This caspase-8-deficiency effect depends on RIPK1 kinase activity and on RIPK3 RIPK1 and RIPK3 mediate this effect cell autonomously, with no dendritic cell death The caspases are evolutionarily conserved cysteine proteases that cleave specific substrate proteins downstream of aspartate residues. They were initially known for two physiological functions: induction of inflammation by the processing of precursors of proinflammatory cytokines such as IL-1β, an activity mediated by caspase-1 and some other “proinflammatory caspases,” and induction of apoptotic cell death. The latter function is triggered by a number of “initiator caspases” that are activated through interaction of their N-terminal “prodomain” with a distinct upstream receptor complex. However, for such activation to lead to cell death, the initiator caspases must first cleave and thus activate other caspases (“effector caspases”) that lack prodomains. These in turn cleave a wide range of specific cellular proteins, thereby triggering the apoptotic program (reviewed in (Riedl and Shi, 2004Riedl S.J. Shi Y. Molecular mechanisms of caspase regulation during apoptosis.Nat. Rev. Mol. Cell Biol. 2004; 5: 897-907Crossref PubMed Scopus (1567) Google Scholar)). Cells possess mechanisms that can restrict the function of initiator caspases in a way that withholds massive activation of other caspases. Recent findings showed that when the initiator caspases act in this restricted manner, they can initiate additional, nonapoptotic functions (van Raam and Salvesen, 2012van Raam B.J. Salvesen G.S. Proliferative versus apoptotic functions of caspase-8 Hetero or homo: the caspase-8 dimer controls cell fate.Biochim. Biophys. Acta. 2012; 1824: 113-122Crossref PubMed Scopus (76) Google Scholar). Caspase-8 was originally identified as an initiator of apoptotic cell death in response to death-inducing TNF family receptors, to which it is recruited by binding to the adaptor protein FADD (also known as MORT1) (Boldin et al., 1996Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death.Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2109) Google Scholar; Muzio et al., 1996Muzio M. Chinnaiyan A.M. Kischkel F.C. O’Rourke K. Shevchenko A. Ni J. Scaffidi C. Bretz J.D. Zhang M. Gentz R. et al.FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death—inducing signaling complex.Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2739) Google Scholar). It was later found to serve a variety of other functions as well (Ben Moshe et al., 2008Ben Moshe T. Kang T.B. Kovalenko A. Barash H. Abramovitch R. Galun E. Wallach D. Cell-autonomous and non-cell-autonomous functions of caspase-8.Cytokine Growth Factor Rev. 2008; 19: 209-217Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar; Kang et al., 2004Kang T.B. Ben-Moshe T. Varfolomeev E.E. Pewzner-Jung Y. Yogev N. Jurewicz A. Waisman A. Brenner O. Haffner R. Gustafsson E. et al.Caspase-8 serves both apoptotic and nonapoptotic roles.J. Immunol. 2004; 173: 2976-2984Crossref PubMed Scopus (310) Google Scholar; Varfolomeev et al., 1998Varfolomeev E.E. Schuchmann M. Luria V. Chiannilkulchai N. Beckmann J.S. Mett I.L. Rebrikov D. Brodianski V.M. Kemper O.C. Kollet O. et al.Targeted disruption of the mouse Caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally.Immunity. 1998; 9: 267-276Abstract Full Text Full Text PDF PubMed Scopus (1029) Google Scholar). Notably, in several transgenic mouse models, its deficiency results in chronic inflammation (Ben Moshe et al., 2007Ben Moshe T. Barash H. Kang T.B. Kim J.C. Kovalenko A. Gross E. Schuchmann M. Abramovitch R. Galun E. Wallach D. Role of caspase-8 in hepatocyte response to infection and injury in mice.Hepatology. 2007; 45: 1014-1024Crossref PubMed Scopus (68) Google Scholar; Kovalenko et al., 2009Kovalenko A. Kim J.C. Kang T.B. Rajput A. Bogdanov K. Dittrich-Breiholz O. Kracht M. Brenner O. Wallach D. Caspase-8 deficiency in epidermal keratinocytes triggers an inflammatory skin disease.J. Exp. Med. 2009; 206: 2161-2177Crossref PubMed Scopus (156) Google Scholar; Wallach et al., 2010Wallach D. Kang T.B. Rajput A. Kim J.C. Bogdanov K. Yang S.H. Kovalenko A. Anti-inflammatory functions of the “apoptotic” caspases.Ann. N Y Acad. Sci. 2010; 1209: 17-22Crossref PubMed Scopus (7) Google Scholar), suggesting that besides inducing apoptotic cell death caspase-8 also restricts the signaling for some inflammatory mechanisms. In the attempt to identify the signaling molecules whose activity is restricted by caspase-8, it was found that the inflammation and embryonic death resulting from caspase-8 deficiency involve two structurally related protein kinases, RIPK1 and RIPK3 (Duprez et al., 2011Duprez L. Takahashi N. Van Hauwermeiren F. Vandendriessche B. Goossens V. Vanden Berghe T. Declercq W. Libert C. Cauwels A. Vandenabeele P. RIP kinase-dependent necrosis drives lethal systemic inflammatory response syndrome.Immunity. 2011; 35: 908-918Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar; Kaiser et al., 2011Kaiser W.J. Upton J.W. Long A.B. Livingston-Rosanoff D. Daley-Bauer L.P. Hakem R. Caspary T. Mocarski E.S. RIP3 mediates the embryonic lethality of caspase-8-deficient mice.Nature. 2011; 471: 368-372Crossref PubMed Scopus (760) Google Scholar; Oberst et al., 2011Oberst A. Dillon C.P. Weinlich R. McCormick L.L. Fitzgerald P. Pop C. Hakem R. Salvesen G.S. Green D.R. Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis.Nature. 2011; 471: 363-367Crossref PubMed Scopus (909) Google Scholar). These findings were consistent with reports that excessive activation of these two protein kinases results in inflammation and might play important roles in various pathological processes (Cho et al., 2009Cho Y.S. Challa S. Moquin D. Genga R. Ray T.D. Guildford M. Chan F.K. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation.Cell. 2009; 137: 1112-1123Abstract Full Text Full Text PDF PubMed Scopus (1689) Google Scholar; Degterev et al., 2005Degterev A. Huang Z. Boyce M. Li Y. Jagtap P. Mizushima N. Cuny G.D. Mitchison T.J. Moskowitz M.A. Yuan J. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury.Nat. Chem. Biol. 2005; 1: 112-119Crossref PubMed Scopus (2024) Google Scholar; Duprez et al., 2011Duprez L. Takahashi N. Van Hauwermeiren F. Vandendriessche B. Goossens V. Vanden Berghe T. Declercq W. Libert C. Cauwels A. Vandenabeele P. RIP kinase-dependent necrosis drives lethal systemic inflammatory response syndrome.Immunity. 2011; 35: 908-918Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar; He et al., 2009He S. Wang L. Miao L. Wang T. Du F. Zhao L. Wang X. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha.Cell. 2009; 137: 1100-1111Abstract Full Text Full Text PDF PubMed Scopus (1618) Google Scholar; Mocarski et al., 2012Mocarski E.S. Upton J.W. Kaiser W.J. Viral infection and the evolution of caspase 8-regulated apoptotic and necrotic death pathways.Nat. Rev. Immunol. 2012; 12: 79-88Google Scholar; Smith et al., 2007Smith C.C. Davidson S.M. Lim S.Y. Simpkin J.C. Hothersall J.S. Yellon D.M. Necrostatin: a potentially novel cardioprotective agent?.Cardiovascular drugs and therapy / sponsored by the International Society of Cardiovascular Pharmacotherapy. 2007; 21: 227-233Crossref PubMed Scopus (269) Google Scholar; Zhang et al., 2009Zhang D.W. Shao J. Lin J. Zhang N. Lu B.J. Lin S.C. Dong M.Q. Han J. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis.Science. 2009; 325: 332-336Crossref PubMed Scopus (1419) Google Scholar). Ex vivo studies showed that excessive activation of RIPK1 and RIPK3 might result in necrotic cell death (Hitomi et al., 2008Hitomi J. Christofferson D.E. Ng A. Yao J. Degterev A. Xavier R.J. Yuan J. Identification of a molecular signaling network that regulates a cellular necrotic cell death pathway.Cell. 2008; 135: 1311-1323Abstract Full Text Full Text PDF PubMed Scopus (787) Google Scholar; Holler et al., 2000Holler N. Zaru R. Micheau O. Thome M. Attinger A. Valitutti S. Bodmer J.L. Schneider P. Seed B. Tschopp J. Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule.Nat. Immunol. 2000; 1: 489-495Crossref PubMed Scopus (1441) Google Scholar; Oberst et al., 2011Oberst A. Dillon C.P. Weinlich R. McCormick L.L. Fitzgerald P. Pop C. Hakem R. Salvesen G.S. Green D.R. Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis.Nature. 2011; 471: 363-367Crossref PubMed Scopus (909) Google Scholar). A series of molecular events contributing to the induction of such death was recently disclosed: RIPK1 phosphorylates and thus activates RIPK3 (Cho et al., 2009Cho Y.S. Challa S. Moquin D. Genga R. Ray T.D. Guildford M. Chan F.K. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation.Cell. 2009; 137: 1112-1123Abstract Full Text Full Text PDF PubMed Scopus (1689) Google Scholar; He et al., 2009He S. Wang L. Miao L. Wang T. Du F. Zhao L. Wang X. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha.Cell. 2009; 137: 1100-1111Abstract Full Text Full Text PDF PubMed Scopus (1618) Google Scholar; Zhang et al., 2009Zhang D.W. Shao J. Lin J. Zhang N. Lu B.J. Lin S.C. Dong M.Q. Han J. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis.Science. 2009; 325: 332-336Crossref PubMed Scopus (1419) Google Scholar). The latter phosphorylates the protein kinase homolog MLKL and the protein phosphatase PGAM5 (Sun et al., 2012Sun L. Wang H. Wang Z. He S. Chen S. Liao D. Wang L. Yan J. Liu W. Lei X. Wang X. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase.Cell. 2012; 148: 213-227Abstract Full Text Full Text PDF PubMed Scopus (1668) Google Scholar; Wang et al., 2012Wang Z. Jiang H. Chen S. Du F. Wang X. The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways.Cell. 2012; 148: 228-243Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar; Zhao et al., 2012Zhao J. Jitkaew S. Cai Z. Choksi S. Li Q. Luo J. Liu Z.G. Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis.Proc. Natl. Acad. Sci. USA. 2012; 109: 5322-5327Crossref PubMed Scopus (601) Google Scholar). PGAM5 can activate the mitochondrial fission factor Drp1 and the resulting derangement of mitochondrial function probably contributes to the necrotic death of the cells (Wang et al., 2012Wang Z. Jiang H. Chen S. Du F. Wang X. The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways.Cell. 2012; 148: 228-243Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar). It was suggested that the inflammation caused by caspase-8 deficiency in vivo is mediated by proinflammatory cellular components that are released when cells die by necrosis as a result of this deficiency (reviewed in (Wallach et al., 2011Wallach D. Kovalenko A. Kang T.B. ‘Necrosome’-induced inflammation: must cells die for it?.Trends Immunol. 2011; 32: 505-509Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar)). Here we describe an additional functional consequence of caspase-8 deficiency that is likely also to contribute to inflammation. Dendritic cells (DCs) deficient in caspase-8 are shown to be hyperresponsive to induction of assembly of the NLRP3 inflammasome, a signaling complex that, through activation of caspase-1, mediates processing of the precursors for proinflammatory mediators such as IL-1β (Schroder and Tschopp, 2010Schroder K. Tschopp J. The inflammasomes.Cell. 2010; 140: 821-832Abstract Full Text Full Text PDF PubMed Scopus (4115) Google Scholar). Similarly to the signaling for necrotic cell death, the enhanced activation of the inflammasome as a result of caspase-8 deficiency depends on the functions of RIPK1, RIPK3, MLKL, and PGAM5. This activation, however, occurs independently of cell death. To investigate the functional roles of caspase-8 in DCs, we generated mice with specific deficiency of caspase-8 in these cells (Casp8fl/−:Itgax-Cre mice; “C8− mice”). The mice were born with normal Mendelian segregation and appeared to develop normally. However, their responses to various pathological challenges revealed that they display greatly enhanced vulnerability to the lethal effect of injected bacterial endotoxin (lipopolysaccharide [LPS]; Figures 1A and 1B ). Unlike in the widely used galactosamine-sensitization model (Galanos et al., 1979Galanos C. Freudenberg M.A. Reutter W. Galactosamine-induced sensitization to the lethal effects of endotoxin.Proc. Natl. Acad. Sci. USA. 1979; 76: 5939-5943Crossref PubMed Scopus (843) Google Scholar), in which mice can be protected from the lethal effect of a low dose of injected LPS by blocking of TNF-induced liver damage but not by blocking IL-1, death of the C8− mice after LPS injection was not associated with liver damage (Figure 1C; see also Figure S1 available online) and was not affected by injection of TNF antibody but could be prevented by the injection of IL-1 receptor antagonist (IL-1RA; Figure 1A). Examination of the sera of the LPS-injected mice showed that the increase in IL-1β in these mice was much higher than in wild-type (WT) mice (Figure 1D). In contrast, induction of TNF by LPS was minimally affected by a caspase-8 deficiency at the time of peak TNF elevation, though at later times it was higher than in WT mice (Figure 1E). Injection of IL-1RA decreased the serum concentrations of both IL-1β and TNF (Figures 1F and 1G). We assessed the involvement of RIPK1 and RIPK3 in the enhanced response of the C8− mice to LPS and found that injection of necrostatin-1 (Nec1), an inhibitor of RIPK1 kinase function (Degterev et al., 2008Degterev A. Hitomi J. Germscheid M. Ch’en I.L. Korkina O. Teng X. Abbott D. Cuny G.D. Yuan C. Wagner G. et al.Identification of RIP1 kinase as a specific cellular target of necrostatins.Nat. Chem. Biol. 2008; 4: 313-321Crossref PubMed Scopus (1468) Google Scholar), delayed the lethal effect of LPS (Figure 1B) and suppressed both the excessive induction of IL-1β and the later excessive increase in TNF (Figures 1H, 1I). Moreover, on RIPK3-deficient background, caspase-8 deficiency in the DCs did not result in enhanced vulnerability to LPS (Figure 1B) or in an enhanced LPS-induced increase in serum IL-1β or TNF (Figures 1J and 1K). Anatomical and histological analyses revealed complex cellular changes in the lymphoid systems of these mice. In both the RIPK3-expressing and the RIPK3-deficient C8− mice, spleens and lymph nodes were greatly enlarged. However, the cellular basis for these size increases differed. In the mice expressing RIPK3, the increased spleen size (Figure 2A; Figure S2A) was not associated with a gross change in splenic microarchitecture (Figure 2B). It was also not associated with an overall increase in cell number (Figure 2C), although an increase in number was seen in some specific cells, including stromal cells (Figure 2D), erythrocytes (Figure 2E), and several kinds of myeloid cells (Figures 2F and 2G, also see Figures S2B–S2D). The increase in spleen size was apparently due to a marked increase in the spleen’s collagen content (Figure S2E). In contrast, in Ripk3 null mice the increase in spleen size as a result of caspase-8 deficiency in the DCs (Figure 2H; Figure S2A) was associated with altered microarchitecture and a marked increase in cell number of the lymphoid organs when the mice grew older (Figures 2I and 2J), resulting from accumulation of CD4−CD8−CD3+ B220+ T lymphocytes (Figures 2K and 2L; Figures S2F–S2H). These lymphocytes were found, like the DCs, to be deficient in caspase-8 (Figures S2I and S2J). Accumulation of such caspase-8-deficient cells in C8− mice, whose Casp8 deletion was dictated by their mating to a mouse strain expressing Cre under control of the Itgax (CD11c) promoter, is consistent with the reported expression of the latter transgene (though only in a proportion of the cells) in several leukocyte types in addition to DCs, including T lymphocytes (Caton et al., 2007Caton M.L. Smith-Raska M.R. Reizis B. Notch-RBP-J signaling controls the homeostasis of CD8- dendritic cells in the spleen.J. Exp. Med. 2007; 204: 1653-1664Crossref PubMed Scopus (615) Google Scholar). It is also consistent with studies showing that CD4−CD8−CD3+ B220+ T lymphocytes accumulate in mice with T cell-specific or ubiquitous deficiency of caspase-8 on a Ripk3 null background (Kaiser et al., 2011Kaiser W.J. Upton J.W. Long A.B. Livingston-Rosanoff D. Daley-Bauer L.P. Hakem R. Caspary T. Mocarski E.S. RIP3 mediates the embryonic lethality of caspase-8-deficient mice.Nature. 2011; 471: 368-372Crossref PubMed Scopus (760) Google Scholar; Oberst et al., 2011Oberst A. Dillon C.P. Weinlich R. McCormick L.L. Fitzgerald P. Pop C. Hakem R. Salvesen G.S. Green D.R. Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis.Nature. 2011; 471: 363-367Crossref PubMed Scopus (909) Google Scholar). Results of bromodeoxyuridine labeling tests in vivo suggested that caspase-8 deficiency in DCs led to an enhanced turnover of these cells in the spleen (Figure S2K). However, whereas their numbers in the lymph nodes were found to increase, their amounts in the spleen remained unchanged (Figure S2D). The enhanced induction of IL-1β by LPS observed in the C8− mice might reflect a direct impact of caspase-8 deficiency on IL-1β generation by these cells. Alternatively, it could be an indirect consequence of the complex cellular changes observed in these mice. To determine which of these two possibilities is correct, we examined the effect of caspase-8 deletion in DCs ex vivo on the generation of IL-1β in these cells. Casp8 was effectively deleted in bone-marrow progenitor cells of C8− mice when the cells were induced (by culturing with GM-CSF) to differentiate to DCs (Figure S3). The regulation of IL-1β generation in the caspase-8-deficient cells appeared to be different from that in the WT. Normally, induction of IL-1β secretion by cultured DCs requires their exposure to dual stimulation, first by a priming agent thought to trigger the synthesis of several proteins that participate in this process, and then by an activating agent. LPS usually exerts only the priming effect, and further stimulation must be provided by an activating agent such as ATP. However, DCs that were rendered deficient in caspase-8 also produced IL-1β in response to LPS treatment alone (although less effectively than when treated with a combination of LPS and ATP) (Figures 3A and 3B ). The same was found for the generation of IL-18 (Figure 3C). In contrast, the generation of TNF and IL-6, two cytokines whose secretion can be induced even in WT DCs merely by treatment with LPS, was not affected by caspase-8 deficiency (Figures 3D and 3E). DCs derived from WT bone-marrow cells in which caspase-8 had been knocked down by small-interfering RNA (siRNA) treatment also produced increased amounts of IL-1β in response to stimulation with LPS alone (Figure 3F). Besides LPS, several other pathogen-derived compounds known to prime DCs for IL-1β generation also induced, independently of an activating agent, IL-1β generation in the caspase-8-deficient DCs but not in the normal cells (Figure 3G). Nec1 effectively blocked IL-1β induction in the C8− cells by LPS as well as by other pathogen-derived compounds (Figure 3G) but did not affect its induction by combined treatment with LPS and ATP (Figure 3H) or the generation of TNF by either the WT or the mutant cells (Figure 3I). DCs deficient in both caspase-8 and RIPK3 did not produce IL-1β in response to LPS (Figure 3J). Thus, the enhanced generation of IL-1β that occurs as a result of caspase-8 deficiency in the DCs is not secondary to the complex cellular changes imposed by this deficiency in vivo but reflects a direct impact of caspase-8 on the functions of RIPK1 and RIPK3 within these cells. The need for dual stimulation to generate IL-1β in DCs is due to participation of two distinct groups of mechanisms in this process. Priming of the cells initiates the synthesis of cytoplasmic IL-1β precursor molecules (pro-IL-1β) and some of the molecules required for their maturation. Activation of the cells triggers the assembly of “inflammasomes”—macromolecular complexes in which pro-caspase-1 is activated by self-processing, allowing caspase-1 to process the pro-IL-1β molecules. Activation of the inflammasome is coupled to IL-1β secretion from the cells by an unknown mechanism (Schroder and Tschopp, 2010Schroder K. Tschopp J. The inflammasomes.Cell. 2010; 140: 821-832Abstract Full Text Full Text PDF PubMed Scopus (4115) Google Scholar). We quantified the pro-IL-1β transcripts and protein and found that both were low in caspase-8-deficient DCs, just as they were in WT cells, and that the transcription and translation of pro-IL-1β were induced by LPS to the same extent in both (Figures 4A and 4B ). In both C8− and WT cells, LPS also induced a similar mild increase in NLRP3, a component of the inflammasome that participates in IL-1β induction by LPS (Figure 4B). These findings suggest that the greater production of IL-1β by the caspase-8-deficient cells does not reflect enhanced synthesis of the proteins mediating this process. Production of the proteolytically processed, active form of caspase-1 occurred in the WT DCs only after their combined treatment with LPS and ATP. In contrast, in caspase-8-deficient cells accumulation of the active form of the enzyme was induced by treatment with LPS alone (Figure 4C). Consistent with this observation, LPS alone was sufficient to induce the caspase-8-deficient but not the WT cells to secrete the proteolytically processed IL-1β generated by the active form of caspase-1 (Figure 4D). These findings implied that in cells deficient in caspase-8, LPS induces not only the synthesis of pro-IL-1β but also activation of the enzyme that cleaves it—a process that in the WT cells is mediated by the NLRP3 inflammasome. As with the generation of IL-1β in WT cells, its generation in the caspase-8-deficient DCs in response to LPS alone was decreased when NLRP3 or ASC (an adaptor protein that associates with NLRP3 and caspase-1 within the inflammasome) was knocked down (Figures 4E–4G), suggesting that here too IL-1β processing is mediated by the inflammasome. Further evidence for the induced assembly of these proteins came from our finding that treatment of caspase-8-deficient DCs with LPS, as well as treatment of caspase-8-expressing cells with LPS+ATP (though not with LPS alone), resulted in assembly of ASC and caspase-1 into a form that could not be extracted with a solution containing 1% of the detergent NP40. Nec1 treatment blocked such assembly when it was induced by LPS in the caspase-8-deficient cells, but did not affect that induced by LPS+ATP (Figure 4H). LPS also induced assembly of some NLRP3 into a detergent-insoluble form. However, the portion of NLRP3 converted to an insoluble form was much lower than that of ASC (not shown). Part of the caspase-1 that accumulated in the detergent-insoluble fraction was the mature, proteolytically processed form, suggesting that the protein interactions occurring within this fraction correspond to the functional inflammasome (Figure 4H). Consistently with this notion, we found that the inflammasome components within this fraction could be crosslinked to each other with the aid of a reversible short chemical linker (Figure 4I). They could also be crosslinked when we applied the crosslinking agent to intact DCs in which IL-1β generation was induced (Figure S4A). We previously reported that caspase-8 deficiency facilitates induction of interferon and other IRF3-dependent genes by the RIG-I signaling complex (Rajput et al., 2011Rajput A. Kovalenko A. Bogdanov K. Yang S.H. Kang T.B. Kim J.C. Du J. Wallach D. RIG-I RNA helicase activation of IRF3 transcription factor is negatively regulated by caspase-8-mediated cleavage of the RIP1 protein.Immunity. 2011; 34: 340-351Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Induction of such genes by LPS in the DCs was not enhanced by caspase-8 deficiency (Figure S4B). Moreover, neither application of interferon nor blocking of interferon function had any effect on the LPS-induced production of IL-1β by these cells (Figures S4C and S4D), ruling out an autocrine interferon effect on the enhanced production of IL-1β by the caspase-8-deficient DCs. One way that caspase-8 deficiency in the DCs could obviate the need for activating agents such as ATP to induce IL-1β generation is by inducing generation of such activating agents by the DCs themselves. This possibility seemed plausible in view of prior evidence that caspase-8 deficiency facilitates necrotic cell death (Holler et al., 2000Holler N. Zaru R. Micheau O. Thome M. Attinger A. Valitutti S. Bodmer J.L. Schneider P. Seed B. Tschopp J. Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule.Nat. Immunol. 2000; 1: 489-495Crossref PubMed Scopus (1441) Google Scholar; Oberst et al., 2011Oberst A. Dillon C.P. Weinlich R. McCormick L.L. Fitzgerald P. Pop C. Hakem R. Salvesen G.S. Green D.R. Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis.Nature. 2011; 471: 363-367Crossref PubMed Scopus (909) Google Scholar) and extensive evidence that this form of cell death can result in release of cellular components capable of triggering inflammation (Rock and Kono, 2008Rock K.L. Kono H. The inflammatory response to cell death.Annu. Rev. Pathol. 2008; 3: 99-126Crossref PubMed Scopus (639) Google Scholar). To explore the interrelationship between the induction of necrotic cell death and the induction of IL-1β generation in the caspase-8-deficient DCs, we determined the kinetics of IL-1β generation in the DCs and their viability after application of LPS, as well as in response to several other agents previously shown to facilitate RIPK3-mediated necrosis, namely TNF, the pan-caspase inhibitor z-VAD, and BV6, an inhibitor of cIAP1 and cIAP2. Assessment of DC viability by a number of different techniques and determination of their mitochondrial membrane potential revealed no sign of enhanced death in the caspase-8-deficient DCs 3 hr after LPS application, by which time the extent of IL-1β generation was already maximal (Figures 5A and 5B ; Figures S5A–S5D). Initial signs of minor induction of cell death could be discerned in these cells only about 24 hr after application of LPS (Figure 5B). Treatment of the caspase-8-deficient cells with TNF and BV6 together (but not separately) also resulted in induction of IL-1β, with no sign of cell death as late as 24 hr after application (Figures 5C and 5D). However, both LPS treatment and treatment with TNF+BV6, when supplemented with the pan-caspase inhibitor z-VAD, did result in cell death, which was associated with release of unprocessed IL-1β (Figures 5A–5D; Figure S5E). The generation of IL-1β in response to each of these inducers was proportional to the extent of assembly of the" @default.
• W2078252006 created "2016-06-24" @default.
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• W2078252006 date "2013-01-01" @default.
• W2078252006 modified "2023-10-15" @default.
• W2078252006 title "Caspase-8 Blocks Kinase RIPK3-Mediated Activation of the NLRP3 Inflammasome" @default.
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