RIPK3 cleavage is dispensable for necroptosis inhibition but restricts NLRP3 inflammasome activation

Caspase-8 activity is required to inhibit necroptosis during embryogenesis in mice. In vitro studies have suggested that caspase-8 directly cleaves RIPK1, CYLD and the key necroptotic effector kinase RIPK3 to repress necroptosis. However, recent studies have shown that mice expressing uncleavable RIPK1 die during embryogenesis due to excessive apoptosis, while uncleavable CYLD mice are viable. Therefore, these results raise important questions about the role of RIPK3 cleavage. To evaluate the physiological significance of RIPK3 cleavage, we generated Ripk3D333A/D333A mice harbouring a point mutation in the conserved caspase-8 cleavage site. These mice are viable, demonstrating that RIPK3 cleavage is not essential for blocking necroptosis during development. Furthermore, unlike RIPK1 cleavage-resistant cells, Ripk3D333A/D333A cells were not significantly more sensitive to necroptotic stimuli. Instead, we found that the cleavage of RIPK3 by caspase-8 restricts NLRP3 inflammasome activation-dependent pyroptosis and IL-1β secretion when Inhibitors of APoptosis (IAP) are limited. These results demonstrate that caspase-8 does not inhibit necroptosis by directly cleaving RIPK3 and further underscore a role for RIPK3 in regulating the NLRP3 inflammasome.


INTRODUCTION
Necroptosis is activated when the activity of caspase-8 is compromised [1,2].This lytic form of cell death is characterised by cell swelling and the rupture of cellular membranes.Intracellular contents, released upon membrane permeabilisation, are thought to act as damage-associated molecular patterns (DAMPs) which can trigger immune and inflammatory responses [3].Consequently, necroptosis is believed to serve as a backup cell death pathway to eliminate infected cells and alert the immune system when pathogens inhibit caspase-8-mediated apoptosis [4].
The rescue of the midgestational lethality to birth of catalytically inactive caspase-8 mice, Casp8 C362A/C362A , by Mlkl -/-mice supports the idea that caspase-8 must cleave other proteins to prevent necroptosis activation during embryogenesis [23,24].However, recent findings have challenged our knowledge of caspase-8's necroptotic substrates and their cleavage sites.Mice expressing CYLD D215A develop normally, suggesting that cleavage of CYLD at D215 is not essential to repress necroptosis [25].cFLIP, another caspase-8 substrate that regulates necroptosis, did not undergo lethal necroptosis when its cleavage was genetically blocked in mice [25,26].Furthermore, we and others have shown that rather than just inhibiting necroptosis, caspase-8-mediated RIPK1 cleavage is a mechanism to dissociate complex II/RIPoptosome to block apoptosis and autoinflammation [25,[27][28][29][30]. Therefore, RIPK3 might be the primary necroptotic caspase-8 substrate supporting the current model of necroptosis repression.However, RIPK3 cleavage has only been demonstrated in overexpression systems [31].This prompted us to investigate its physiological role using cleavage resistant RIPK3 mutant mice.Our study revealed that, surprisingly, the cleavage of RIPK3 at D333 by caspase-8 does not inhibit necroptosis during development.Interestingly, we found that RIPK3 restricts NLRP3-dependent pyroptosis and IL-1β secretion by limiting caspase-1 activation when IAPs are inhibited.
We next hypothesised that the cleavage of RIPK3 might nevertheless inhibit necroptosis upon inflammatory challenges.To test this hypothesis, we examined the response of Ripk3 D333A/ D333A cells to TNF and TLR-induced cell death.In contrast to the Casp8 -/-and non-cleavable RIPK1 cells (Ripk1 D325A/D325A ) [9,25,27,30,35], Ripk3 D333A/D333A cells did not exhibit sensitivity to TNF alone (Fig. 2a, b).Caspase-8 has been proposed to suppress RIPK3-dependent necroptosis following TLR3/4 engagement [36,37].However, we found that the Ripk3 D333A/D333A bone marrow-derived macrophages (BMDMs) did not undergo cell death after LPS (TLR4 ligand) or poly(I:C) (TLR3 ligand) treatment (Fig. 2a).Despite expectations that non-cleavable RIPK3 would have an enhanced scaffolding function and increase sensitivity to TNF and TLR-induced necroptosis when caspase-8 is inhibited, we did not observe accelerated cell death in Ripk3 D333A/D333A cells compared to wild-type cells, when treated with IDUN plus low doses of TNF/Smac-mimetic, LPS or poly(I:C) (Fig. 2a, b).Consistently, we did not detect significant changes in the phosphorylation status of RIPK1, RIPK3 and MLKL between wild-type and Ripk3 D333A/D333A cells in response to necroptotic stimuli, even at early time points (Fig. 2c, d).

RIPK3 cleavage does not limit the apoptotic scaffolding function of RIPK3
RIPK3 has been proposed to act as a scaffold within complex II/ Ripoptosome to increase apoptosis [27,[38][39][40][41].We therefore examined whether non-cleavable RIPK3 amplifies its scaffolding function, thereby increasing the susceptibility of Ripk3 D333A/D333A cells to apoptosis.Cells were treated with TNF in combination with Smac-mimetic (TS), TAK1 inhibitor (TTAK1i) or Cycloheximide (TC), all known to induce complex II formation.In BMDMs, genetic inhibition of RIPK3 cleavage did not alter their sensitivity to TS, TTAK1i or TC-induced apoptosis when compared to wildtype cells (Fig. 3a).Consistent with this, cleavage and activation of caspase-8 and caspase-3 were comparable between wild-type and Ripk3 D333A/D333A BMDMs upon TS and TC treatments (Fig. 3b).Similarly, Ripk3 D333A/D333A MDFs were not more sensitive to TTAK1i or TC-induced apoptosis and caspase-8 and -3 processing, indicative of activation, mirrored that of the wildtype cells upon TC (Fig. 3c, d).However, a slight increase in cell death and caspase-8 and -3 activation was observed in Ripk3 D333A/D333A MDFs treated with TS compared to wild-type MDFs (Fig. 3c, d).This prompted us to assess if non-cleavable RIPK3 enhances complex II formation in response to TS in fibroblastic cells.
Complex II formation is thought to be transiently formed and swiftly inactivated by various molecular checkpoints to prevent TNF toxicity [25,27,30,[42][43][44][45][46][47][48].One such checkpoint involved the cleavage of RIPK1 by caspase-8, which participates in the dissociation of complex II [27].To determine if non-cleavable RIPK3 could also contribute to the stabilisation of complex II, we immunoprecipitated RIPK1 and used non-cleavable RIPK1 cells (Ripk1 D325A/+ ) as a positive control.As we previously reported in Ripk1 D325A/+ cells [27], complex II was stabilised upon TS treatment as evidenced by the recruitment of caspase-8, FADD and RIPK1 (Fig. 3e lane 11 & f lane 8).In contrast, in both wild-type and Ripk3 D333A/D333A cells, complex II could not be detected upon TS treatment (Fig. 3e, f).Similarly, the addition of IDUN did not increase the recruitment of caspase-8 or FADD in Ripk3 D333A/D333A cells compared to wild-type cells (Fig. 3e, f).Altogether, these findings indicate that the blockade of RIPK3 cleavage does not increase its scaffolding function.
Blocking RIPK1 cleavage does not further sensitise Ripk3 D333A/ D333A mice to cell death Our results demonstrated that cleavage of RIPK3 at D333 does not prevent necroptosis or limit its scaffolding apoptotic function.However, it could be argued that the cleavage of RIPK1 in experiments performed with 2 independent biological replicates.BMDMs (e) and MDFs (f) were treated with either 100 ng ml −1 TNF and 1 μM Smac-mimetic compound A (TS); or with 100 ng ml −1 TNF, 500 nM Smac-mimetic compound A, and 5 μM caspase inhibitor IDN-6556 (TSI).BMDMs were treated with TS for 3 h and with TSI for 1.5 h, while MDFs were treated with TS for 1.5 h and with TSI for 1 h.Immunoprecipitation was performed using anti-RIPK1 antibody.Results are representative of 2 independent experiments performed with 2 independent biological replicates.
It has been reported that ectopic expression or forced dimerisation of RIPK3 can activate NF-κB [56][57][58][59].We hypothesised that noncleavable RIPK3 might accumulate upon TLR treatment and, similar to RIPK3-overexpressing cells, increase NF-κB activation and inflammasome priming.However, treatment with LPS or poly(I:C) did not affect NF-κB activation (Fig. 5d).Furthermore, the levels of NLRP3, caspase-1 and pro-IL-1β were comparable between wildtype and Ripk3 D333A/D333A after LPS treatment (Fig. 5e).This suggested that the increased IL-1β in non-cleavable RIPK3 cells compared with wild type cells was not due to a priming defect.
Our in vitro results demonstrate that genetic blockade of RIPK3 cleavage dominantly exaggerates TLR-induced NLRP3 inflammasome-dependent IL-1β secretion.We therefore challenged Ripk3 D333A/D333A mice intraperitoneally with sublethal doses of LPS or poly (I:C) and measured serum IL-1β levels after 2 h.Consistent with our in vitro findings, Ripk3 D333A/D333A mice had markedly enhanced serum IL-1β levels when compared to wildtype mice (Fig. 6d), confirming that cleavage of RIPK3 is critical to limit TLR-induced NLRP3 inflammasome activity.Collectively, our data demonstrate that cleavage of RIPK3 is not essential to limit necroptosis but rather represses TLR-induced NLPR3-dependent IL-1β production when IAPs are in low abundance or inhibited.

DISCUSSION
The proximity of caspase-8, RIPK1 and RIPK3 within complex II/ RIPoptosome, as well as the rescue of Casp8-deficient and catalytically inactive mice by loss of Ripk3 or Mlkl supports the notion that caspase-8 cleaves RIPK3 to prevent necroptosis.However, unlike the non-cleavable RIPK1 mice, our findings reveal that the genetic blockade of RIPK3 cleavage at D333 does not lead to uncontrolled lethal necroptosis.These results suggest the existence of other molecular mechanisms that may substitute or cooperate with RIPK3 cleavage in suppressing necroptosis.One proposed mechanism could be the regulation of RIPK3's necroptotic function through ubiquitylation [61][62][63][64][65][66][67].Therefore, it is plausible that an E3 ligase or a deubiquitinase cleaved by caspase-8 inhibits RIPK3-dependent necroptosis.While CYLD could be a potential candidate a recent study showed that its cleavage is not required to inhibit necroptosis during embryogenesis [25].If there is redundancy, the generation of Ripk3 D333A/D333A Cyld D215A/D215A double mutant mice will allow this hypothesis to be tested.
Akin to necroptosis, genetically blocking RIPK3 cleavage had little to no impact on its apoptotic scaffolding function.This is somewhat surprising given that the increased activation of caspase-8 observed in uncleavable RIPK1 cells was markedly reduced on a Ripk3 -/-background [25,27].These findings raise the question of why the RIPK3 cleavage site is conserved during evolution, even in organisms that do not have the capability to undergo necroptosis.Our finding that RIPK3 cleavage plays a role in limiting IL-1β secretion offers a potential explanation.These results reinforce the previously described involvement of RIPK3 in driving NLPR3 inflammasome activation [49][50][51][52][53][54][55].Significantly, our study provides new insights by revealing that RIPK3 cleavage is a negative feedback mechanism to restrain excessive NLPR3 inflammasome activation in situations when IAPs are scarce.These findings hold relevance for individuals with XIAP deficiency, wherein degradation of cIAPs through the TLR-TNFR2 pathway could trigger RIPK3-dependent inflammasome activation [55,68].Furthermore, our results shed light on the role of caspase-8 and RIPK3 in development and why the loss of Ripk3 extends the lifespan of catalytically inactive caspase-8 (Casp8 C362A/C362A ) mice when compared to the loss of Mlkl [23,24].We can conclude that caspase-8 exerts repression over necroptosis during embryogenesis through multiple mechanisms and restrain pyroptosis after birth via cleavage of RIPK3.
In non-cleavable RIPK3 BMDMs, we did not observe an increase in caspase-8 processing in response to LPS/Smac-mimetic, suggesting that non-cleavable RIPK3 did not enhance RIPoptosome formation.This aligns with our results when cells were treated with TS or TSI treatment.However, it's worth noting RIPK3 can drive mitochondrial respiration and ROS production [15], and ROS have previously been linked to RIPK3-driven NLRP3 responses [51].This may explain the increased TLR-RIPK3-NLRP3 signalling observed in RIPK3 D333A mutant macrophages, although this idea requires further investigation.

Cell death
MDFs were plated at 1 × 10 4 cells per well (96-well flat-bottom plate, Greiner) in complete DMEM on the same day as treatment.BMDMs were plated at 1 × 10 5 cells per well (96-well) in complete DMEM supplemented with 20% L929 conditioned media, either the day before or on the same day as the treatment.Media were replaced and cells were labelled with SPY505-DNA (Spirochrome SC-101, 1:1000 dilution) for 1 h at 37 °C, 10% CO 2 before imaging and appropriate treatment compounds were added 5 min prior to the initiation of imaging.Percentage cell death was assayed every 30 min or 1 h by time-lapse imaging using the IncuCyte live cell analysis imaging (Sartorius) for a total of 16-18 h under 5% CO 2 and 37 °C conditions.Dead cells were identified by propidium iodide (PI, 0.25 μg ml −1 ) staining and the percentage of cell death was calculated as the ratio of PI-positive cells to total cells stained with SPY505-DNA (Spirochrome SC-101).

Immunoprecipitation
Ten million (1 × 10 7 ) cells were seeded in 15 cm tissue culture dishes and treated accordingly.After the indicated treatments, cells were lysed in DISC lysis buffer (150 mM sodium chloride, 2 mM EDTA, 1% Triton X-100, 10% glycerol, 20 mM Tris, pH 7.5, Roche complete protease inhibitor cocktail, Roche phosSTOP phosphatase inhibitor).Proteins were immunoprecipitated with 20 μl of protein A Sepharose plus 8 μg of RIPK1 antibody (Cell Signaling; 3493) with overnight rotation at 4 °C.Beads were washed four times in DISC and samples eluted by boiling in 60 μl 1× SDS loading dye.

ELISA
Mouse serum and cellular supernatants from BMDMs were analysed by ELISA for IL-1β content (R&D Systems) according to the manufacturer's instructions.

In vivo TLR challenge
Eight-to-twelve-week-old mouse littermates received intraperitoneal injection of either 2 mg kg −1 LPS or 50 μg poly(I:C).Calculations to determine group sizes were not performed, mice were not randomised but were grouped according to genotype.Animal technicians and researchers who performed intraperitoneal injection and ELISA were blinded to the mice genotype and treatment conditions.

Fig. 2
Fig. 2 Genetic blockade of caspase-8-mediated RIPK3 cleavage does not increase necroptosis in vitro.Cell death of BMDMs (a) and MDFs (b) monitored by time-lapse imaging of propidium iodide (PI) staining over 18 h.NT, non-treated; T, 100 ng ml −1 TNF; S, 100 nM Smac-mimetic compound A; I, 5 μM caspase inhibitor IDN-6556; L, 25 ng ml −1 LPS; P, 5 μg ml −1 poly(I:C).Data are represented as mean + SEM of N = 3 independent biological replicates per genotype.Graphs are representative of 3 independent experiments performed with 3 independent biological replicates each time.Western blot of MDFs (c) treated as in (b) for 2 h, and of BMDMs lysates (d) treated as in (a) for the indicated time.Results are representative of 2 independent experiments performed with 2 independent biological replicates.

Fig. 3
Fig. 3 Genetic blockade of caspase-8-mediated RIPK3 cleavage increases apoptosis in vitro.Cell death of BMDMs (a) and MDFs (c) monitored by time-lapse imaging of propidium iodide (PI) staining over 18 h.NT, non-treated; T, 100 ng ml −1 TNF; S, 100 nM Smac-mimetic compound A; TAK1i, 100 nM TAK1 inhibitor; C, 10 μg ml −1 cycloheximide.Data are represented as mean + SEM of N = 3 independent biological replicates per genotype.Graphs are representative of 3 independent experiments performed with 3 independent biological replicates each time.Western blot of BMDMs (b) treated as in (a), and of MDFs (d) treated as in (c).Results are representative of 2 independent experiments performed with 2 independent biological replicates.BMDMs (e) and MDFs (f) were treated with either 100 ng ml −1 TNF and 1 μM Smac-mimetic compound A (TS); or with 100 ng ml −1 TNF, 500 nM Smac-mimetic compound A, and 5 μM caspase inhibitor IDN-6556 (TSI).BMDMs were treated with TS for 3 h and with TSI for 1.5 h, while MDFs were treated with TS for 1.5 h and with TSI for 1 h.Immunoprecipitation was performed using anti-RIPK1 antibody.Results are representative of 2 independent experiments performed with 2 independent biological replicates.

Fig. 4
Fig. 4 Blocking RIPK1 cleavage does not further sensitise Ripk3 D333A/D333A mice to cell death.a Representative pictures of ~300 days old mice with indicated genotypes.Cell death of BMDMs (b) and MDFs (c) monitored by time-lapse imaging of propidium iodide (PI) staining over 16 h (b) or 18 h (c) upon receiving treatments as described in Figs.2a and 3a.Data are represented as mean + SEM of N = 2-3 independent biological replicates per genotype.Graphs are representative of 3 independent experiments performed with 2-3 independent biological replicates each time.