MLKL Activation Triggers NLRP3-Mediated Processing and Release of IL-1β Independently of Gasdermin-D

This article is featured in In This Issue, p.1763

In addition to the canonical cell suicide pathway of apoptosis, it is now understood that necroptosis and pyroptosis represent alternative forms of programmed cell death (1). Unlike apoptosis, pyroptosis and necroptosis are characterized by cellular swelling and rupture (2, 3). Pyroptosis is triggered by inflammasome- or pathogen-associated molecular pattern–driven activation of the inflammatory caspases, caspase-1 and caspase-11 (caspases-1, 4, and 5 in humans). The NLRP3 inflammasome is the best studied inflammasome and is minimally composed of NLRP3, ASC, and caspase-1. A wide array of stimuli has been shown to activate NLRP3, with disrupted ion homeostasis and potassium efflux recently proposed as a unifying stimulus. Inflammasome assembly leads to activation of caspase-1, which cleaves and activates the recently identified cell death effector gasdermin-D (GSDMD) (4, 5). GSDMD forms pores in the cell membrane, causing disruption of its integrity and triggering release of cytosolic proteins (6, 7). Concurrent with the activation of pyroptotic cell death, inflammasome-mediated activation of caspase-1 also leads to the cleavage and activation of the cytokines IL-1β and IL-18. Pyroptosis thereby promotes inflammation through a combination of caspase-mediated processing of IL-1β and IL-18, as well as GSDMD-mediated release of these cytokines (3).

Similar to pyroptosis, necroptosis is a lytic cell death program. Necroptosis is defined by activation of the receptor interacting protein kinase (RIPK)1 and RIPK3 to form an oligomeric necrosome, which leads to the phosphorylation and activation of the effector pseudokinase mixed lineage kinase domain-like (MLKL) (8). Once activated, MLKL multimerizes and translocates to the cell membrane, where it triggers ion release via pore formation, leading to cell swelling and rupture (9–11). Execution of the necroptotic program has also been associated with the processing of IL-1β through diverse mechanisms (12). These include production of reactive oxygen species (13), altered mitochondrial dynamics (14), and death-independent, RIPK-mediated effects (15) leading to activation of the NLRP3 inflammasome, as well as by direct processing of IL-1β by caspase-8 associated with the necrosome (16–19). Studies have also indicated that MLKL activation could promote NLRP3 inflammasome activation (16); however, this phenomenon has been difficult to characterize because experimental induction of necroptosis requires caspase inhibition, which in turn prevents NLRP3-mediated caspase-1 activation and the production of bioactive IL-1β.

To circumvent this issue we created a system by which MLKL could be directly activated independent of upstream signals. Using this system, we demonstrate that MLKL activation alone is sufficient to trigger NLRP3 inflammasome activation, caspase-1–dependent IL-1β processing, and IL-1β release. We found that MLKL-mediated membrane disruption is sufficient to trigger release of inactive pro–IL-1β upon necroptotic cell death, but that potassium efflux and components of the NLRP3 inflammasome are required to cleave IL-1β during this process. Notably, the release of bioactive IL-1β upon MLKL activation occurs independently of the pyroptotic effector GSDMD. MLKL activation is therefore sufficient for both the processing and release of IL-1β, in a cell-intrinsic manner. Although these findings do not exclude additional connections between RIPK signaling and IL-1β processing, they demonstrate an inherent mechanistic link between execution of the necroptotic program, NLRP3 inflammasome activation, and IL-1β release.

PMA (Sigma-Aldrich, St. Louis, MO, P1585-1MG) was dissolved in DMSO to a concentration of 1 mM and used at a concentration of 100 nM. LPS-EB Ultrapure (InvivoGen, San Diego, CA, tlrl-3pelps) was dissolved in endotoxin-free water to a concentration of 1 mg/ml and used at a concentration of 1 μg/ml. Doxycycline hyclate (Sigma-Aldrich, D9891-5G) was dissolved in sterile water to a concentration of 2 mg/ml and used at a concentration of 2 μg/ml. Nigericin (Sigma-Aldrich, N7143-5MG) was dissolved in ethanol to a concentration of 10 mM and used at a concentration of 10 μM. AP1 (Clontech Laboratories, Mountain View, CA, also called B/B homodimerizer, catalog no. 635059) was dissolved in ethanol to a concentration of 100 μM and used at a concentration of 100 nM. KCl was made at a stock concentration of 500 mM and filter sterilized. Hygromycin B (Fisher Scientific, Hampton, NH, BP29521MU) was dissolved in sterile water to a concentration of 50 mg/ml and filter sterilized. Puromycin dihydrochloride was from Life Technologies (Grand Island, NY, A1113803). Recombinant human TNF-α (PeproTech, Rocky Hill, NJ, 300-01B) was reconstituted in PBS to a concentration of 100 μg/ml and used at a concentration of 100 ng/ml. zVAD (SM Biochemicals, Anaheim, CA, SMFMK001) was dissolved in DMSO to a concentration of 50 mM and used at a concentration of 50 μM. BV6 (Smac mimetic/IAP antagonist) was a gift from D. Vucic (Genentech, San Francisco, CA). Recombinant human IL-1β (Life Technologies, PHC-0815) was reconstituted in deionized water to a concentration of 0.1 mg/ml (250,000 U/ml). Anti-human IL-1β–IgG (InvivoGen, mabg-hil1b-3) was reconstituted in 1 ml of sterile water to a concentration of 0.1 mg/ml.

For CRISPR/Cas9 gene targeting we used a lentivirus provided by Dr. D.B. Stetson (20). The guide RNA target sites are listed below. Vesicular stomatitis virus protein G pseudotyped, self-inactivating lentivirus was prepared by polyethylenimine (Polysciences, Warrington, PA, 23966-2) transfection of 293T cells with 1.5 μg of pseudotyped vesicular stomatitis virus protein G, 3 μg of psPAX-2, and 6 μg of CRISPR/Cas9 lentiviral vector. Lentiviral supernatants were collected 48 h after transfection and concentrated by centrifugation at 8500 × g overnight at 4°C. THP-1 cells were transduced with lentivirus and selected with 1 μg/ml puromycin for 5 d. Gene targeting was evaluated by RFLP using restriction sites that overlapped the CRISPR targeting sites, as well as Western blot for endogenous protein. Cas9 targeting sequences used were: CASP1, 5′-AAGCTGTTTATCCGTTCCAT-3′; ASC, 5′-CGACGCCATCCTGGATGCGC-3′; NLRP3, 5′-CTGCAAGCTGGCCAGGTACC-3′; RIPK3, 5′-CTCGTCGGCAAAGGCGGGTT-3′; GSDMD, 5′-CGGCCTTTGAGCGGGTAGTC-3′.

MLKL-2xFV chimeric constructs, here called activatable MLKL (acMLKL), were created by cloning full-length human MLKL upstream of a tetraglycine linker followed by two FKBP^F36V domains. These sequences were cloned into the pSLIK lentiviral vector (21) with hygromycin resistance expression. Full-length human ASC fused to mCherry was cloned into the pRRL lentiviral vector (gift from D. Rawlings) downstream of an MND promoter and upstream of a T2A-puromycin resistance cassette. Lentivirus was made as described above and used to transduce THP-1 CRISPR cell lines and selected with 800 μg/ml of hygromycin for 5 d. MLKL expression was assessed by doxycycline treatment for 24 h followed by Western blot for FKBP and MLKL. THP-1 cells were maintained in RPMI 1640 (Fisher Scientific, SH30027FS) supplemented with 10% FCS (Sigma-Aldrich, 0926-500 Ml), 10 mM HEPES (Fisher Scientific, SH3023701), 0.05 mM 2-ME (EMD Millipore, 444203-250ML), 1 mM sodium pyruvate (Sigma-Aldrich, S2636-100ML), 29.2 g/l glutamine (Fisher Scientific, SH3003402), 10,000 U/ml penicillin, and 10,000 μg/ml streptomycin (Fisher Scientific, SV30010) and grown at 37°C in 5% CO₂.

Anti-FKBP12 (Thermo Fisher Scientific, Waltham, MA, PA1-026A) was used to stain for the presence of MLKL-2xFV after activation with doxycycline and AP1. DAPI (Thermo Fisher Scientific, 62248) was used to stain the nucleus. Staining was performed as previously described (22).

Cell death assays were carried out using an IncuCyte bioimaging platform as previously described (23). Briefly, 1 × 10⁵ THP-1 cells per well were seeded in triplicate in 24-well plates and primed with PMA, and LPS or doxycycline was added as indicated. The next day cells were treated with nigericin or AP1 in the presence of a 100 nM concentration of the cell-impermeable DNA-binding fluorescent dye Sytox Green (Life Technologies, S7020). Control cells were treated with 100 nM of the cell-permeable fluorescent dye Sytox Green (Life Technologies, S7559), which allows quantification of the total number of cells present to calculate percentage cell death (23). Results depicted are representative of at least three independent experiments.

The following Abs were used: anti-MLKL clone 3H1 (gift from W. Alexander), anti-RIPK3 (Novus Biologicals, Littleton, CO, NBP2-24588), anti–caspase-1 p10 (C-20) (Santa Cruz Biotechnology, Dallas TX, sc-515), anti-actin clone C4 (EMD Millipore, MAB1501), anti-FKBP12 (Thermo Fisher Scientific, PA1-026A), anti–IL-1β (H-153) (Santa Cruz Biotechnology, sc-7884), anti-GSDMDC1 (64-y) (Santa Cruz Biotechnology, sc-81868), anti-NLRP3 (Adipogen, San Diego, CA, AG-20B-0014), and anti-ASC (TMS1) (MBL International, Woburn, MA, D086-3). Secondary Abs were purchased from Santa Cruz Biotechnology (mouse sc-2005, rat sc-2006, and rabbit sc-2313).

These Abs were used for Western blotting of proteins harvested from whole-cell lysates and quantitated by a BCA protein assay (Thermo Fisher Scientific, PI23277). Supernatants were collected from 5 × 10⁴ THP-1 cells, 5 h after treatment in serum-free RPMI 1640, and concentrated by Amicon Ultra-0.5 filters (membrane of 3 kDa) (EMD Millipore, UFC500324). Proteins were separated using SDS-PAGE precast gels (Invitrogen, Grand Island, NY) by standard protocols. Detection was accomplished using ECL Western blotting substrate (Thermo Fisher Scientific, PI32209) and either standard autoradiography film (Pierce, Rockford, IL) or an electronic luminescence detection platform (ChemiDoc XRS+ system, 170-8265; Bio-Rad Laboratories, Hercules, CA).

For the IL-1β bioassay, HEK-blue IL-1β cells (InvivoGen, hkb-il1r) were cultured according to the manufacturer’s instructions. For each assay, 5 × 10⁴ THP-1 cells were seeded in 96-well plates and primed with PMA and LPS and indicated cells were treated with doxycycline for 24 h. The next day cells were treated with either nigericin or AP1 in fresh complete media. Cell supernatants were harvested 5 h after treatment and diluted 1:10 in complete RPMI 1640. Three microliters of diluted supernatants was used to stimulate secreted embryonic alkaline phosphatase (SEAP) production from 5 × 10⁴ HEK-blue cells seeded in 96-well plates in 200 μl of complete DMEM for 24 h. Five microliters of HEK-blue supernatant was assessed for SEAP activity in 200 μl of QUANTI-blue (InvivoGen, rep-qb1) and incubated for 2 h at 37°C. Absorbance was read at 630 nM with a multimode microplate reader (Synergy HT; BioTek). For each experiment three replicates for each experimental condition were assayed. A standard curve for each experiment was constructed using recombinant human IL-1β and used to calculate and report units per milliliter of IL-1β. Error bars represent SD from the mean of a minimum of three independent wells. Statistical significance was calculated by a Student t test using GraphPad Prism software. Each result depicted is representative of at least three independent experiments.

For IL-1β ELISA, ELISA was performed using the human IL-1β ELISA Ready-SET Go! kit (eBioscience). For each experiment three replicates for each experimental condition were assayed. Error bars represent SD from the mean of a minimum of three independent wells. Statistical significance was calculated by a Student t test using GraphPad Prism software. Each result depicted is representative of at least three independent experiments.

To study the effects of MLKL activation on inflammasome formation, we created a form of MLKL that could be directly activated using a small-molecule ligand (24, 25). To do this, we fused full-length human MLKL to two FKBP^F36V domains to create an activatable version of MLKL that could be induced to oligomerize and thereby become activated in the presence of a dimerization drug (Fig. 1A). We refer to this activatable form of MLKL as acMLKL, and to the dimerization ligand as AP1. Because constitutive exogenous expression of MLKL can reduce cell viability, we cloned this construct into the pSLIK tetracycline-inducible lentiviral vector (21) and expressed it in cells of the human monocyte line THP-1. We found that in these cells, addition of doxycycline led to induction of MLKL expression (Fig. 1B, Supplemental Fig. 1A), and that subsequent addition of AP1 led to relocalization of MLKL-2xFV to the cell membrane and to cell death (Fig. 1B, 1C), consistent with MLKL-mediated membrane disruption.

To explore the activation of the NLRP3 inflammasome by this construct, we used CRISPR/Cas9 technology to delete the inflammasome effector caspase-1 in cells expressing acMLKL (Supplemental Fig. 1B). We also created a similar cell line lacking the kinase RIPK3, which acts as an endogenous activator of MLKL and has been shown to be capable of inducing IL-1β cleavage through activation of caspase-8 under certain circumstances (16–19) (Supplemental Fig. 1C). As expected, cells lacking caspase-1, but not those lacking RIPK3, were resistant to pyroptosis induced by the canonical NLRP3-activating stimulus of LPS priming followed by nigericin treatment (Fig. 1D). Similarly, cells lacking RIPK3, but not those lacking caspase-1, were resistant to necroptosis induced by treatment with the canonical necroptosis-activating stimulus, comprised of TNF-α, a caspase inhibitor (zVAD), and SMAC mimetic (BV6) (Fig. 1E). Importantly, both RIPK3-deficient and caspase-1–deficient cells underwent loss of membrane integrity and cell death with equivalent kinetics upon direct activation of acMLKL (Fig. 1F, Supplemental Fig. 1D). This indicates that we are able to effectively ablate effectors of pyroptosis or necroptosis in human monocytes, and that our acMLKL construct allows induction of cell death independently of the canonical pyroptotic or necroptotic pathways.

In the course of these experiments, we noted that acMLKL-induced cell death occurred with a reduced magnitude compared with that induced by canonical pyroptotic or necroptotic stimuli. We suspected that this was due to heterogeneous expression of the acMLKL construct in our THP-1 cells, a common challenge with this cell type. Confirming this, we found that acMLKL activation caused ∼25% of our THP-1 cells to die, but that surviving cells were insensitive to restimulation with AP1 (Supplemental Fig. 1D). Nonetheless, we reasoned that the cell death response observed upon acMLKL activation was sufficient to allow us to study the effect of MLKL activation on assembly of the NLRP3 inflammasome.

To test the effect of MLKL activation on IL-1β processing, we primed THP-1 cells expressing acMLKL with LPS and then induced either caspase-1– or MLKL-dependent death via treatment with nigericin or AP1, respectively. Upon blotting for IL-1β in the supernatants of these cells, we found that, as expected, nigericin treatment triggered robust cleavage of IL-1β. However, MLKL activation also triggered release of cleaved IL-1β into the supernatants of cells treated with AP1 (Fig. 2A). Cleaved IL-1β was absent from identically treated THP-1 cells lacking caspase-1, indicating that the IL-1β processing observed upon MLKL activation was caspase-1–dependent. Lysates from these cells demonstrated robust accumulation of pro–IL-1β following LPS treatment, but IL-1β processing was absent in these lysates, indicating that the cleaved IL-1β that we observed in supernatants from these cells was not preformed in intact cells, but rather was produced during cell death (Supplemental Fig. 1E).

In an effort to measure IL-1β release following MLKL activation in a more sensitive and quantitative manner, we performed ELISA analysis on supernatants from LPS-primed THP-1 cells treated with either nigericin or the MLKL activator AP1 (Fig. 2B). As expected, nigericin treatment led to robust IL-1β release, which was partially blocked by deletion of caspase-1. However, ELISA analysis demonstrated equivalent IL-1β release from cells killed in an MLKL-dependent manner, irrespective of the presence of caspase-1. Notably, these results mirror the relative amounts of cell death observed in these cell lines upon nigericin treatment (partial protection upon ablation of caspase-1, Fig. 1C) and MLKL activation (equivalent death with or without caspase-1, Fig. 1E). Because ELISA analysis does not discriminate between processed and unprocessed IL-1β, we speculated that this assay was measuring release of both forms of IL-1β following cell death. We concluded that traditional ELISA analysis was not providing an accurate measure of the release of processed, bioactive IL-1β.

To measure the presence of processed, bioactive IL-1β produced upon MLKL activation, we used a reporter cell line responsive to human IL-1β. This cell line lacks TNF- and TLR-responsive pathways, but it expresses the human IL-1 receptor along with a construct encoding the SEAP under the control of an NF-κB–responsive promoter. IL-1β activity can thereby be quantified using a colorimetric assay for SEAP activity. We confirmed this using recombinant human IL-1β, which produced robust SEAP activity (Supplemental Fig. 2A), and which was blocked by an IL-1R antagonist Ab (Supplemental Fig. 2B).

Using these reporter cells calibrated with recombinant human IL-1β, we were able to measure the bioactivity of IL-1β release from THP-1 cell lines. We found that MLKL activation in these cells led to the release of active IL-1β to a level similar to that observed upon nigericin treatment (Fig. 2C). In both cases, the production of active IL-1β required caspase-1. Cotreatment of our reporter cells with THP-1 lysates and an IL-1R antagonist Ab blocked SEAP production, confirming that the observed activity resides with IL-1β (Supplemental Fig. 2C). We therefore concluded that MLKL activation triggers caspase-1–dependent IL-1β processing and bioactivity. To gain kinetic insight into the production of bioactive IL-1β upon MLKL activation, we assessed the levels of IL-1β bioactivity present in the supernatants of THP-1 cells following acMLKL activation. We found that peak IL-1β activity was present in the supernatants of these cells between 4 and 6 h after MLKL activation (Fig. 2D), a time point that corresponds to robust induction of cell death by MLKL (Fig. 1C). Notably, the observed bioactivity of IL-1β decreased at later time points, likely due to degradation of this cytokine in cellular supernatants. We therefore selected 5 h after AP1 addition as a representative time point for the assessment of IL-1β production by these cells, and used this time point for subsequent analyses.

We hypothesized that membrane disruption caused by MLKL activated the NLRP3 inflammasome, leading to IL-1β processing by this complex. To test this idea, we created THP-1 cell lines lacking either NLRP3 or the essential adapter ASC using CRISPR/Cas9-mediated gene targeting (Supplemental Fig. 1A, 1B). In these cells, activation of acMLKL triggered cell death with magnitude and kinetics comparable to wild-type (WT) THP-1 cells (Fig. 3A), confirming that MLKL-mediated death does not depend on ASC or NLRP3. Furthermore, these cells were resistant to cell death triggered by LPS plus nigericin, consistent with a model in which nigericin-mediated potassium efflux activates the NLRP3 inflammasome, causing caspase-mediated cell death (Fig. 3B). Notably, this model differs from a recent report indicating that nigericin-induced necrotic membrane rupture and cell death occur upstream of NLRP3 inflammasome activation (26).

We next used these cells, as well as THP-1 cells lacking RIPK3 (described in Fig. 1), to assess the contribution of the NLRP3 inflammasome and the RIPK3 necrosome to IL-1β activation following MLKL-induced cell death. As expected, both cleaved and pro–IL-1β were found in the supernatants of THP-1 cells following treatment with LPS plus nigericin, and the release of both species was abolished upon ablation of NLRP3 or ASC (Fig. 3C). These findings are consistent with the idea that IL-1β is released upon cell death, because cells lacking NLRP3 or ASC fail to undergo pyroptosis upon stimulation with LPS plus ATP (Fig. 3B). In contrast, upon activation of MLKL in these cells, we observed that pro–IL-1β was released irrespective of the presence of ASC, NLRP3, or RIPK3 (Fig. 3D). However, cleaved IL-1β was absent from the supernatants of cells lacking ASC or NLRP3 following MLKL activation. MLKL activation thus allows pro–IL-1β release via membrane disruption independent of the NLRP3 inflammasome. However, when the NLRP3 inflammasome is intact, MLKL activation also induces cleavage and activation of IL-1β during MLKL-mediated cell death.

We next confirmed the activation of IL-1β by the NLRP3 inflammasome using our reporter cell system. We found that, as expected, LPS plus nigericin treatment triggered secretion of bioactive IL-1β, and that this activity required both NLRP3 and ASC (Fig. 3E). MLKL activation led to a similar release of bioactive IL-1β, and this effect was also NLRP3- and ASC-dependent. Neither nigericin-induced nor MLKL-induced secretion of bioactive IL-1β was altered in the absence of RIPK3 (Fig. 3F). This indicates that formation of putative RIPK3–caspase-8 complexes, which can mediate IL-1β processing in some conditions (16–18), does not contribute to IL-1β activation downstream of MLKL. Taken together, these data indicate that MLKL activation promotes formation of the NLRP3 inflammasome and caspase-1–mediated cleavage of IL-1β during MLKL-dependent cell death.

To directly assess activation of the inflammasome in cells undergoing MLKL-dependent cell death, we used fluorescence microscopy to visualize formation of the ASC speck, a protein aggregate that accompanies inflammasome formation and pyroptosis. To do this, we expressed a fusion protein comprised of ASC and mCherry in acMLKL-expressing THP-1 cells and then assessed ASC speck formation using the IncuCyte Zoom fluorescent imaging platform. As expected, upon induction of NLRP3 activation with LPS plus nigericin in the presence of the cell-impermeable nuclear dye Sytox Green, we observed ASC foci associated with the Sytox-positive nuclei of pyroptotic cells (Fig. 4A). Notably, when these cells were instead LPS primed, then killed by activation of acMLKL, we also observed ASC foci associated with dying cells (Fig. 4B).

We next quantified the frequency with which acMLKL-dependent cell death caused observable ASC foci. As previously observed, acMLKL activation caused less cell death than did LPS plus nigericin. However, upon quantification we found that MLKL-dependent cell death led to observable ASC focus formation with a similar frequency to treatment with LPS plus nigericin (Fig. 4C). Taken together, these data indicate that MLKL-dependent cell death triggers ASC oligomerization and inflammasome formation with an efficiency comparable to that of canonical activators of the NLRP3 inflammasome.

Potassium efflux has been proposed as a unifying mechanism of NLRP3 inflammasome activation (27), and nigericin activates this pathway by acting as a potassium ionophore. Consistent with this finding, we found that eliminating the potassium gradient across the cell membrane via addition of 40 mM KCl to our culture media greatly reduced the pyroptosis-inducing effect of nigericin (Fig. 5A). However, the death of THP-1 cells following MLKL activation was not affected by increased levels of KCl in culture media, indicating that MLKL-dependent cell death does not require an intact potassium gradient across the plasma membrane (Fig. 5B).

We hypothesized that MLKL activation and subsequent cell membrane disruption could trigger potassium efflux, which would be necessary for NLRP3 activation. Consistent with this idea, we found that increased extracellular potassium levels greatly reduced the appearance of cleaved IL-1β in the supernatants of cells treated with nigericin or in cells killed via MLKL activation (Fig. 5C). Also consistent with this hypothesis, increased extracellular potassium eliminated the bioactivity of IL-1β released upon nigericin treatment or MLKL activation (Fig. 5D). From these findings, we conclude that MLKL-mediated membrane disruption triggers potassium efflux that, although not required for MLKL-induced cell death, is necessary to trigger NLRP3 inflammasome assembly and IL-1β cleavage during MLKL-induced cell death.

GSDMD was recently described as a key effector of pyroptosis. Cleavage of GSDMD by the inflammatory caspases leads to the formation of GSDMD pores in the plasma membrane (4, 5, 7). These pores represent an essential step in the execution of pyroptosis and secretion of bioactive IL-1β following both canonical and noncanonical inflammasome activation. Because MLKL can also disrupt the plasma membrane, we hypothesized that MLKL-dependent cell death might trigger both IL-1β processing and release independent of GSDMD. To test this idea, we deleted GSDMD from THP-1 monocytes expressing acMLKL (Supplemental Fig. 2D, 2E). Consistent with earlier reports, these cells displayed a defect in the execution of pyroptosis following treatment with nigericin (Fig. 6A). However, upon activation of MLKL, GSDMD-deficient cells died with kinetics similar to those of WT cells (Fig. 6B).

To test the effect of MLKL activation on IL-1β secretion, we carried out bioassay analysis of the supernatants of both normal and GSDMD-deleted THP-1 cells following either treatment with LPS plus nigericin on MLKL activation. We found that deletion of GSDMD led to a clear defect in secretion of bioactive IL-1β following LPS plus nigericin treatment, consistent with a role for GSDMD in the canonical inflammasome–IL-1β pathway (Fig. 6C). However, following MLKL activation, no such defect was observed: GSDMD-deficient cells secreted equivalent levels of bioactive IL-1β to normal THP-1 cells (Fig. 6C). This indicates that MLKL-mediated membrane disruption can both activate the NLRP3 inflammasome and allow the release of processed IL-1β produced by this complex, independent of the effector GSDMD.

Collectively, our findings indicate that activation of the necroptotic effector MLKL is sufficient to trigger assembly of the NLRP3 inflammasome, caspase-1–mediated IL-1β processing, and release of bioactive IL-1β. Bacterial pore-forming toxins are well-described activators of the NLRP3 inflammasome (28), and NLRP3 has been proposed as a sensor of necrotic cell death (26). Our findings are therefore consistent with the idea that MLKL acts as an endogenous toxin-like molecule and that MLKL-mediated membrane disruption and resultant necroptotic cell death trigger the same inflammasome-mediated pathway as bacterial toxin molecules. Notably, this effect is independent of GSDMD, which is required for efficient release of IL-1β during caspase-dependent pyroptosis.

Notably, although we find that MLKL activation is sufficient to cause assembly of the NLRP3 inflammasome and caspase-1–dependent processing of IL-1β, we did not observe that MLKL activation could initiate pyroptosis. As shown in Figs. 1F, 3A, and 6B, the kinetics and magnitude of cell death responses observed upon MLKL activation were similar irrespective of the presence of caspase-1, ASC, NLRP3, or GSDMD. Additional experiments using reduced concentrations of AP1 yielded similar results (data not shown). Thus, in our experimental system, we were not able to observe a phenomenon wherein sublethal MLKL activation led to NLRP3-dependent execution of pyroptosis. This could be because MLKL can only induce sufficient potassium efflux to activate the NLRP3 inflammasome concurrently with complete membrane disruption; that is, NLRP3 inflammasome activation could occur after the point of no return of MLKL-dependent cell death. Additional studies, and a more complete understanding of the nature of MLKL-dependent membrane disruption, may clarify this point.

Several other connections between the necroptotic pathway and inflammasome activation have been reported, including RIPK-mediated induction of reactive oxygen species (13), alterations in mitochondrial dynamics (14), and direct processing of IL-1β by caspase-8 associated with the RIPK1/RIPK3 necrosome (16–19). Our findings do not challenge the existence of these pathways; rather, they indicate that, irrespective of upstream signaling events, activation of MLKL leads to processing and activation of IL-1β. These findings may explain the recently reported defects in NLRP3 inflammasome activation in macrophages lacking MLKL (29). Our findings indicate an inextricable link between the execution of necroptosis and the release of IL-1β, and they implicate bioactive IL-1β and IL-18 as likely effectors of inflammatory responses to the necroptotic death of myeloid cells.

The authors have no financial conflicts of interest.