Active Caspase-1 Induces Plasma Membrane Pores That Precede Pyroptotic Lysis and Are Blocked by Lanthanides

A rapidly induced propidium influx is triggered downstream of inflammasome activation but upstream of pyroptotic cell lysis

Previous studies have assayed the uptake of cationic DNA-intercalating fluorescent dyes after the activation of a canonical inflammasome complex as an indicator of caspase-1–induced PM permeabilization or pyroptosis (19, 28, 29). Normally, these dyes are impermeant to an intact PM. However, upon perturbation of normal PM barrier function, these dyes can access the nucleus, intercalate with DNA, and fluoresce. PM permeabilization can occur through: 1) frank lysis, 2) the gating of resident large-pore channels, or 3) the insertion of large protein pores that can accommodate the molecular dimensions of these dyes. Case et al. (29) previously used a real-time, kinetic assay of Pro²⁺ (molecular mass 415 Da) influx to track the progression of pyroptosis in Legionella-infected macrophages. We adapted a similar protocol to investigate the nature and kinetics of caspase-1–induced PM permeabilization during NLRP3 and Pyrin inflammasome activation in murine BMDMs.

To activate the NLRP3 inflammasome, we stimulated BMDMs with the bacterial ionophore NG, which functions as a K⁺/H⁺ exchanger after insertion in the PM and organellar membranes. Insertion of NG into the PM results in a decrease in cytosolic [K⁺], which is a necessary signal for NLRP3 inflammasome complex assembly (30, 31). We previously reported that after an ∼12-min delay, NG-stimulated murine bone marrow–derived dendritic cells (BMDCs) exhibit robust and rapid caspase-1–dependent Pro²⁺ influx (25). WT BMDMs stimulated with NG displayed similarly rapid Pro²⁺ influx after a 10- to 12-min delay with ∼60% of the BMDMs accumulating dye by 45 min (Fig. 1A). Pro²⁺ influx was absent in Nlrp3^−/−, Asc^−/−, and Casp1^−/− (also deficient in caspase-11) BMDMs (Fig. 1A), demonstrating that Pro²⁺ uptake is dependent on NLRP3 inflammasome components and caspase-1 activation.

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FIGURE 1.

A rapidly induced propidium influx is triggered downstream of inflammasome activation but upstream of pyroptotic cell lysis. (A) WT, Asc^−/−, Casp1^−/−, and Nlrp3^−/− BMDMs were primed with LPS (1 μg/ml) for 4 h before stimulation with NG (10 μM) for 45 min, and the change in PM permeability to Pro²⁺ (1 μg/ml) and subsequent accumulation of fluorescent Pro²⁺ (molecular mass 416 Da) complexed with DNA was quantified every 3 min. A 5-min baseline fluorescent read was performed before stimulation, and Pro²⁺ fluorescence was expressed as a percentage of maximum fluorescence after adding digitonin (50 μg/ml). These data represent the mean ± SE of 6–12 replicates from 4 (WT) or 2 (Asc^−/−, Casp1^−/−, and Nlrp3^−/−) independent experiments. (B) LPS-primed WT, Asc^−/−, Casp1^−/−, and Nlrp3^−/− BMDMs were stimulated with TcdB (0.4 μg/ml) for 60 min, and Pro²⁺ fluorescence was quantified every 4 min as described in (A). These data represent the mean ± SE of three to nine replicates from three (WT), two (Asc^−/− and Casp1^−/−), or one (Nlrp3^−/−) independent experiment. (C) LPS-primed WT BMDMs were stimulated with NG or (D) TcdB in the presence or absence of the cytoprotectant glycine (5 mM). At the indicated times, supernatants were assayed for LDH activity, which was used as an indicator of lytic LDH release. The absorbance values were expressed as a percentage of maximum absorbance after Triton X-100–induced permeabilization of unstimulated LPS-primed cells. These data represent the mean ± SE of four replicates from two independent experiments. (E) LPS-primed WT BMDMs were stimulated with NG for 45 min or (F) TcdB for 60 min in the presence or absence of 5 mM of glycine, and Pro²⁺ fluorescence was quantified every 3 or 4 min, respectively. These data represent the mean ± SE of six replicates from two independent experiments.

To address whether this caspase-1–dependent PM permeability pathway is conserved among canonical inflammasome platforms, we also used a Pyrin inflammasome model. To activate the Pyrin inflammasome, we stimulated BMDMs with TcdB. TcdB is one of the main virulence factors involved in the pathogenesis of a C. difficile bacterial infection (32, 33), a major cause of hospital-acquired, antibiotic-associated infectious diarrhea and pseudomembranous colitis (34, 35). TcdB-induced Pyrin inflammasome activation requires effective internalization of the toxin into the cytosol followed by TcdB-dependent glycosylation and inactivation of Rho-GTPases (36). After an ∼20-min delay, WT BMDMs stimulated with TcdB exhibited rapid and robust Pro²⁺ influx with ∼40% of the cells accumulating dye by 60 min (Fig. 1B). Pro²⁺ influx was almost completely suppressed in Asc^−/− and Casp1^−/− BMDMs throughout the 60-min TcdB stimulation, but remained intact in Nlrp3^−/− BMDMs (Fig. 1B); this demonstrates that TcdB-induced Pro²⁺ uptake is dependent on Pyrin inflammasome components.

We next determined whether caspase-1–induced PM permeabilization indicates a prelytic event, like pore/channel opening, or cell lysis. Early studies by Cookson and colleagues (19, 37, 38) found that the cytoprotectant glycine protected Salmonella-infected macrophages from end-stage lysis but did not suppress uptake of ethidium⁺, a smaller (314 Da) DNA-intercalating dye. We have also reported the use of glycine to suppress caspase-1–induced lysis of macrophages stimulated with maitotoxin (39) or extracellular ATP (40). Notably, millimolar concentrations of glycine have also been used to characterize maitotoxin and palytoxin-induced changes in PM permeability of endothelial cells (41, 42), as well as the pyroptotic responses in macrophages (19). In all of these models of regulated cell death, glycine was shown to prevent or greatly delay end-stage lysis (19, 41, 42).

We adapted these protocols to dissociate lytic from nonlytic propidium uptake by BMDMs in response to NLRP3 and Pyrin inflammasome activation. Stimulation of WT BMDMs with NG or TcdB in the presence of 5 mM of glycine prevented the lytic release of the large macromolecule LDH (140 kDa) (Fig. 1C, 1D), but permitted the influx of Pro²⁺ with a similar kinetic as in the absence of glycine (Fig. 1E, 1F). This temporal dissociation between the uptake of Pro²⁺ and the release of LDH in the presence of glycine indicates that Pro²⁺ influx reflects an event before cell lysis, such as the insertion or opening of a “pyroptotic pore.”

NLRP3 and Pyrin inflammasome activation licenses the opening of a large, nonselective cation- and anion-permeable pyroptotic pore

We next characterized the permeability properties of this putative pyroptotic pore. Previous studies by Cookson and colleagues (19) on pyroptosis in Salmonella-infected macrophages indicated the accumulation of 1.1- to 2.4-nm diameter pores that facilitated the influx of molecules in the 500–1450 Da mass range. A range of DNA-intercalating cationic dyes with different masses, charges, and shapes can be used to probe the dimensions of pyroptotic pores (Supplemental Table I). To define the pores induced by NLRP3 or Pyrin inflammasomes, we assessed permeability to the larger and more highly charged EthD-2 dye (EthD⁴⁺; molecular mass 785 Da). After an ∼10- to 12-min delay, WT BMDMs stimulated with NG exhibited a rapid and robust EthD⁴⁺ influx that was absent in Casp1^−/− BMDMs; this was similar to the WT BMDM Pro²⁺ influx response, albeit with a slower rate of dye uptake (Fig. 2A). WT BMDMs stimulated with TcdB also exhibited rapid and robust EthD⁴⁺ influx after an ∼20-min delay similar to the Pro²⁺ influx profile (Fig. 2B).

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FIGURE 2.

NLRP3 and Pyrin inflammasome activation licenses the opening of a large, nonselective cation- and anion-permeable pyroptotic pore. (A) LPS-primed WT and Casp1^−/− BMDMs were stimulated with NG (10 μM) for 45 min, and Pro²⁺ (molecular mass 416 Da) and EthD⁴⁺ (molecular mass 788 Da, 2 μM) fluorescence were quantified every 3 min as described in Fig. 1. These data represent the mean ± SE of five to six replicates from two independent experiments. (B) LPS-primed WT BMDMs were stimulated with TcdB (0.4 μg/ml) for 60 min, and Pro²⁺ and EthD⁴⁺ fluorescence were quantified every 4 min. These data represent the mean ± SE of six replicates from two independent experiments. (C) LPS-primed WT BMDMs were stimulated with NG for 45 min or (D) TcdB for 60 min in the presence or absence of 5 mM of glycine, and EthD⁴⁺ fluorescence was quantified every 3 or 4 min, respectively. These data represent the mean ± SE of five to six replicates from two independent experiments. (E) LPS-primed WT and Casp1^−/− BMDMs were stimulated with NG or (F) TcdB for 60 min, and the change in cytosolic [Ca²⁺] was determined using the fluo-4-AM (1 μM) Ca²⁺ indicator dye. A 10-min baseline read was taken before stimulation. These data represent the mean ± SE of six replicates from two independent experiments. (G) LPS-primed WT and Casp1^−/− BMDMs were stimulated with NG and TcdB in the presence or absence of 5 mM of glycine for 60 min, and the cytosolic [Ca²⁺] was determined as described in (E) and (F). These data represent the mean ± SE of three to four replicates from two independent experiments. (H) LPS-primed WT and Casp1^−/− BMDMs were stimulated with NG in the presence or absence of 5 mM of glycine. At the indicated times, supernatants were assayed for extracellular [adenine nucleotide] by first rephosphorylating ATP metabolites to ATP and then using a luciferase-based assay to quantify extracellular [ATP]. These data represent the mean ± SE of four replicates from two independent experiments. *p < 0.05.

We verified that the inflammasome-induced permeability to EthD⁴⁺ was also a prelytic event by assessing EthD⁴⁺ permeability in the presence of glycine. After the stimulation of WT BMDMs with NG or TcdB, the EthD⁴⁺ uptake profiles were similar in the presence and absence of glycine (Fig. 2C, 2D); this indicated that the induced pyroptotic pore is permeable to the larger EthD⁴⁺ dye, and thus accommodates large organic molecules in the 800-Da mass range. In contrast, Fink and Cookson (19) observed that EthD⁴⁺ did not permeate the pyroptotic pore induced in Salmonella-infected macrophages. This discrepancy might indicate that pores of varying dimensions can accumulate during pyroptotic induction by different inflammasome subtypes or that pyroptotic pore induction and its dimensions vary dynamically depending on the rate and extent of active caspase-1 accumulation. Indeed, Gaidt et al. (4) recently reported that stimulation of human monocytes with LPS alone induced distinct NLRP3-dependent inflammasomes that did not drive pyroptotic lysis, whereas stimulation of the same monocytes with LPS plus NG resulted in robust NLRP3 inflammasome-dependent pyroptosis.

Given the pyroptotic pore’s permeability to large organic cations, we next characterized its ability to act as a conduit for inorganic cations or organic anions. To assess the permeability of the pore to Ca²⁺, we loaded fluo-4 Ca²⁺ indicator dye into BMDMs before acute induction of NLRP3 or Pyrin inflammasomes. WT BMDMs stimulated with NG exhibited a modest and gradual increase in cytosolic [Ca²⁺] after a 10- to 12-min delay, and this response was absent in Casp1^−/− BMDMs (Fig. 2E). We previously reported similar caspase-1–dependent increases in cytosolic [Ca²⁺] in NG-stimulated BMDCs and demonstrated that this involved influx of extracellular Ca²⁺ rather than mobilization of intracellular Ca²⁺ stores (25). WT BMDMs stimulated with TcdB also demonstrated a gradual increase in cytosolic [Ca²⁺] after an ∼20-min delay that was greatly suppressed in Casp1^−/− BMDMs (Fig. 2F). The onset of Ca²⁺ influx temporally correlated with the onset of Pro²⁺ influx in both inflammasome models; this suggests that the increase in cytosolic [Ca²⁺] is predominantly mediated by the induced pyroptotic pore. That the Ca²⁺ influx profiles of WT BMDMs in response to NG or TcdB were similar in the presence and absence of glycine (Fig. 2G) indicates that Ca²⁺ influx, like Pro²⁺ and EthD⁴⁺ uptake, reflects a prelytic change in PM permeability.

To assess whether the pyroptotic pore is also permeable to anions, particularly intracellular metabolites such as ATP, the ECM from WT and Casp1^−/− BMDMs stimulated with NG was assayed for total adenine nucleotide (ATP+ADP+AMP) content. The collected samples were treated with a mixture of myokinase, pyruvate kinase, and phosphoenolpyruvate to convert extracellular AMP and ADP to ATP, and a luciferase-based assay was used to determine [ATP]. We were particularly interested in whether the pyroptotic pore is permeable to ATP because this nucleotide is an important danger-associated molecular pattern that activates innate immune cells for support of adaptive immune responses (43, 44) and also promotes leukocyte chemotaxis (45). In the absence or presence of glycine, NG-stimulated WT BMDMs progressively released comparable amounts of adenine nucleotides after an ∼10-min delay, and this release was absent in Casp1^−/− BMDMs (Fig. 2H). This indicates that adenine nucleotides are released through a caspase-1–induced pore rather than as a secondary consequence of cell lysis. Taken together, these results indicate that caspase-1 activation induces a large nonselective cation- and anion-permeable pore in the macrophage PM that precedes overt cell lysis.

Gsdmd is required for caspase-1 induction of both the prelytic pyroptotic pores and the subsequent pyroptotic lysis

Because Gsdmd was recently identified as a downstream target of caspase-1/11 with the resulting N-terminal cleavage product being necessary to execute pyroptotic cell death (13, 14, 16), we hypothesized that Gsdmd was also required to induce the pyroptotic pore/channel. He et al. (16) used end-point fluorescence imaging to demonstrate NG-stimulated Pro²⁺ staining in control but not Gsdmd-deficient murine macrophages. However, those single-time-point images at 60 min did not distinguish between prelytic versus postlytic dye accumulation. We used Gsdmd-targeting guide RNAs (gRNAs) and iBMDMs to generate CRISPR-Cas9 Gsdmd^−/− iBMDM cell lines (Fig. 3A). Pooled iBMDM clones generated with two separate gRNAs (Gsdmd G1 and Gsdmd G2) lacked immunoreactive Gsdmd protein and were used for subsequent experiments (Fig. 3B). In response to NG stimulation, LPS-primed Gsdmd^{−/− G1} and Gsdmd^{−/− G2} iBMDMs exhibited no Pro²⁺ uptake at early time points, whereas the parental WT iBMDMs and WT iBMDMs transduced with a nontargeting gRNA (NTG) displayed robust Pro²⁺ uptake that was markedly suppressed by the pan-caspase inhibitor zVAD (Fig. 3C). As expected, Gsdmd was also necessary for downstream lysis because Gsdmd^{−/− G1} and Gsdmd^{−/− G2} iBMDMs exhibited complete suppression of LDH release after 30 or 60 min of NG stimulation (Fig. 3D); such blockade of pyroptosis is consistent with previous findings (13, 16). We also verified that glycine retards the lytic release of LDH 30 min post-NG stimulation in WT iBMDMs similarly to its effect on WT primary BMDMs (Supplemental Fig. 1A). These data demonstrate that Gsdmd is required to induce the upstream prelytic pyroptotic pore/channel that mediates Pro²⁺ influx and eventually leads to end-stage pyroptotic cell lysis.

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FIGURE 3.

Gsdmd is required for caspase-1 induction of both the prelytic pyroptotic pores and the subsequent pyroptotic lysis. (A) The gRNAs used to generate CRISPR-Cas9 Gsdmd^−/− iBMDMs. (B) Gsdmd was immunoprecipitated from untreated WT NTG, Gsdmd^−/− Guide 1 (G1: four pooled clones), and Gsdmd^−/− Guide 2 (G2: two pooled clones) iBMDM whole cell lysates were incubated overnight with anti–GSDMDC-1 and then with protein G-Sepharose. Immunoprecipitated samples were probed for Gsdmd and IgG H chain, and soluble cell lysates were probed for actin. These data are representative of results from one experiment. (C) LPS-primed WT iBMDMs in the presence or absence of 50 μM of zVAD and LPS-primed NTG, Gsdmd^−/− Guide 1 (Gsdmd^{−/− G1}), and Gsdmd^−/− Guide 2 (Gsdmd^{−/− G2}) were stimulated with NG (10 μM) for 45 min, and Pro²⁺ fluorescence was quantified every 3 min as described in Fig. 1. These data represent the mean ± SE of four replicates from two independent experiments. (D) LPS-primed WT, Gsdmd^{−/− G1}, and Gsdmd^{−/− G2} iBMDMs were stimulated with NG for 30 and 60 min, and the supernatants were subsequently assayed for LDH activity as described in Fig. 1. These data represent the mean ± SE of four replicates from two independent experiments. ***p < 0.001, *p < 0.05. (E) Gsdmd^{−/− G1} iBMDMs were stimulated with NG (10 μM) for 2 or 3 h, and Pro²⁺ or YoPro²⁺ fluorescence was quantified every 3 min as described in Fig. 1. Where indicated, the assay medium was supplemented with 50 μM of zVAD-fmk, 50 μM of zDEVD-fmk, or 30 μM of trovafloxacin. These data represent the mean ± SE of four replicates from two independent experiments.

During prolonged NLRP3 inflammasome activation in the absence of Gsdmd, macrophages eventually divert to apoptosis (16). Apoptotic caspase-7 is a substrate of caspase-1 (46) and, in the absence of Gsdmd, macrophages accumulated cleaved caspase-3/7 and active caspase-8 within several hours after assembly of caspase-1 inflammasomes. He et al. (16) also reported that Gsdmd-null macrophages accumulated nuclear Pro²⁺ after 3 h of NG stimulation. The dye accumulation in the Gsdmd-deficient cells may reflect lytic uptake caused by secondary necrosis and/or prelytic influx through other large-pore ion channels, such as pannexin-1 (Panx1). Núñez and colleagues (47) recently described an alternative pyroptotic pathway initiated by caspase-11–dependent cleavage of Panx1 channels in murine macrophages. We used NG-stimulated Gsdmd-deficient iBMDMs to define alternative modes by which accumulation of active caspase-1 can alter PM permeability. After an ∼60-min delay, the cells began to accumulate Pro²⁺, and this response was completely suppressed by the pan-caspase inhibitor zVAD or caspase-3–selective inhibitor DEVD, but not the Panx1 blocker trovafloxacin (Fig. 3E). Activated Panx1 channels have low permeability to Pro²⁺ but high permeability to YoPro²⁺, which has the same charge but smaller mass (375 Da) compared with Pro²⁺ (Supplemental Table I) (27, 48–50). In response to NG, Gsdmd-null iBMDMs displayed a delayed and zVAD-sensitive increase in YoPro²⁺ accumulation (Fig. 3E). Notably, trovafloxacin produced a 2-fold decrease in the rate of YoPro²⁺ uptake. This suggests that caspase-3/7–gated Panx1 channels contribute to the altered PM permeability of inflammasome-activated macrophages under conditions of suppressed pyroptosis.

Lanthanides coordinately suppress both the caspase-1–dependent PM permeability change and pyroptotic lysis induced by NLRP3 and Pyrin inflammasome activation

To further characterize the molecular and biophysical properties of the caspase-1/Gsdmd–induced pore and its mechanistic coupling to pyroptotic cell death, we investigated whether the pore could be targeted pharmacologically. Given its nonselective permeability to large organic and inorganic cations and anions, we tested whether activity of the pyroptotic pore might be blocked by lanthanides (Gd³⁺ and La³⁺), which are broadly acting channel inhibitors. Lanthanides are known to inhibit nonselective cation channels (51) and a broad range of large-pore channels (52). WT BMDMs stimulated with NG in the presence of 1 mM of Gd³⁺ or La³⁺ were characterized by modest decreases in the rate of Pro²⁺ influx relative to that observed in lanthanide-free medium (Fig. 4A, 4B). The rate and magnitude of suppression was much greater as the lanthanide concentration increased to 1.2 mM (Gd³⁺: ∼60% mean suppression, La³⁺: 100%) and 1.5 mM (Gd³⁺: ∼90% mean suppression, La³⁺: 100%) (Fig. 4A, 4B). Thus, the inhibitory effects of lanthanides on pyroptosis-induced PM permeabilization were defined by very steep concentration–response relationships with La³⁺ as a modestly more potent suppressor compared with Gd³⁺. We verified that the lanthanides similarly suppressed the pyroptotic PM permeability change induced by pyrin inflammasomes. In these experiments, the BMDMs were treated with increasing concentrations of Gd³⁺ or La³⁺ added 20 min post-TcdB, which is after the toxin has been internalized but before initiation of Pro²⁺ influx. A total of 1 mM Gd³⁺ or La³⁺ induced a marked suppression in the rate and magnitude of Pro²⁺ influx (Gd³⁺: ∼30% mean suppression, La³⁺: ∼60% mean suppression) (Fig. 4C, 4D). The efficacy of blockade was increased as the concentrations were increased to 1.2 mM (Gd³⁺: ∼60% mean suppression, La³⁺: ∼70% mean suppression) and 1.5 mM (Gd³⁺: ∼90% mean suppression, La³⁺: ∼90% mean suppression) (Fig. 4C, 4D). These results demonstrate that the lanthanides suppress pyroptotic Pro²⁺ influx downstream of two distinct inflammasome signaling pathways.

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FIGURE 4.

Lanthanides coordinately suppress both the caspase-1–dependent PM permeability change and pyroptotic lysis induced by NLRP3 and Pyrin inflammasome activation. (A) LPS-primed WT BMDMs were stimulated with NG (10 μM) in the presence or absence of Gd³⁺ (1, 1.2, and 1.5 mM) or (B) La³⁺ (1, 1.2, 1.5 mM) for 45 min, and Pro²⁺ fluorescence was quantified every 3 min as described in Fig. 1. These data represent the mean ± SE of four replicates from two independent experiments. (C) LPS-primed WT BMDMs were stimulated with TcdB (0.4 μg/ml) in the presence or absence of Gd³⁺ (1, 1.2, and 1.5 mM) or (D) La³⁺ (1, 1.2, 1.5 mM) for 60 min, and Pro²⁺ fluorescence was quantified every 4 min. Gd³⁺ and La³⁺ were added 20 min after TcdB (after the toxin has been internalized but before pyroptotic Pro²⁺ influx). These data represent the mean ± SE of two to eight replicates from six independent experiments. (E) LPS-primed WT BMDMs were stimulated with NG for 30 min in the presence or absence of 1.2 mM of Gd³⁺ or La³⁺, and the supernatants were subsequently assayed for LDH activity as described in Fig. 1. These data represent the mean ± SE of four replicates from two independent experiments. (F) LPS-primed WT BMDMs were stimulated with TcdB for 45 min in the presence or absence of 1 mM of Gd³⁺ or La³⁺, and the supernatants were subsequently assayed for LDH activity. Gd³⁺ and La³⁺ were added 20 min after TcdB. These data represent the mean ± SE of four replicates from two independent experiments. (G) LPS-primed WT BMDMs were stimulated as in (E). Pro²⁺ fluorescence was quantified 30 min post-NG. These data represent the mean ± SE of four replicates from two independent experiments. (H) LPS-primed WT BMDMs were stimulated as in (F). Pro²⁺ fluorescence was quantified 45 min post-TcdB. These data represent the mean ± SE of eight replicates from six independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

We next determined whether the lanthanides also suppress the downstream execution of pyroptotic cell death by assaying LDH release as an indicator of lysis. WT BMDMs stimulated with NG for 30 min (a time point corresponding to active pyroptotic Pro²⁺ influx) in the presence of 1.2 of mM Gd³⁺ or La³⁺ released markedly less LDH compared with cells stimulated with NG in lanthanide-free medium (Fig. 4E). In the presence of 1.2 of mM Gd³⁺ or La³⁺, the percent suppression of LDH release (Gd³⁺: 67% mean suppression, La³⁺: 86% mean suppression) was comparable with the percent suppression of NG-induced Pro²⁺ influx (Gd³⁺: 70% mean suppression, La³⁺: 99% mean suppression) over 30-min test periods (Fig. 4E, 4G). Pyroptotic cell death induced by TcdB-stimulated pyrin inflammasome activation was similarly attenuated by the lanthanides (Fig. 4F, 4H). In the presence of 1 mM of Gd³⁺ or La³⁺, the percent suppression of LDH release (Gd³⁺: 48% mean suppression, La³⁺: 58% mean suppression) was comparable with the percent suppression of Pro²⁺ influx (Gd³⁺: 45% mean suppression, La³⁺: 71% mean suppression) (Fig. 4F, 4H). Taken together, these data demonstrate that, similar to the phenotype of Gsdmd-deficient macrophages, inflammasome-activated WT BMDMs are characterized by a profound suppression in both caspase-1–mediated PM permeabilization and downstream pyroptotic cell lysis in the presence of lanthanides.

Lanthanides have previously been used to probe inflammatory signaling responses in other inflammasome models. Yang et al. (47) found that Gd³⁺ did not suppress LDH release in their model of caspase-11/Panx1–mediated pyroptosis. However, that study tested only 100 μM of Gd³⁺, a concentration that was also submaximal for blocking Pro²⁺ influx and LDH release in our model of caspase-1/Gsdmd–mediated pyroptosis (Supplemental Figs. 1, 3). Compan et al. (53) used 2 mM of Gd³⁺ or 2 mM of La³⁺ to block IL-1β release and YoPro²⁺ uptake in response to hypotonicity-stimulated NLRP3 inflammasome activation. In that model, the lanthanides were used to block activity of the TRPM7 and TRPV2 ion channels, which functioned as upstream regulators of the volume-sensitive caspase-1 activation response. This contrasts with our use of the lanthanides to block downstream responses to caspase-1 activation by canonical inflammasome stimuli. Lee et al. (54) used 1 mM of extracellular Gd³⁺ as an agonist for the G protein–coupled calcium-sensing receptor that was linked to NLRP3 inflammasome activation. In contrast, we observed no stimulatory effects of the lanthanides per se on pyroptotic signaling or inflammasome activation when added alone in the absence of NG (data not shown). A caveat in interpretation of the Lee et al. finding is that the Gd³⁺ was added to phosphate-containing medium, which can result in formation of insoluble gadolinium phosphate particles, phagocytosis of the particles, and subsequent lysosome destabilization, a known NLRP3 activation stimulus (31).

Interestingly, there was an escape from lanthanide suppression of lytic LDH release as the duration of NLRP3 or pyrin inflammasome stimulation was prolonged (Supplemental Fig. 2A–C). Also, there was a more rapid escape from pyroptotic suppression in the presence of 1 versus 1.2 mM concentrations of Gd³⁺ or La³⁺ during NG stimulation (Supplemental Fig. 2A, 2B). This suggests either a time-dependent loss in efficacy of lanthanide suppression and/or that signaling events downstream of caspase-1/Gsdmd target the integrity of intracellular organelle compartments, as well as the PM to facilitate the execution of pyroptotic cell death.

In contrast with the escape from lanthanide suppression of LDH release with longer duration (60 min) NG stimulation in WT BMDMs (Supplemental Fig. 2A, 2B), Gsdmd^{−/− C1} and Gsdmd^{−/− C2} iBMDMs maintained complete suppression of LDH release at 60 min post-NG stimulation (Fig. 3D). Sustained suppression of lytic death in the absence of Gsdmd further suggests that, in addition to PM pyroptotic pore induction, Gsdmd may target other intracellular signaling responses that contribute to the pyroptotic cell death process.

We verified that lanthanides attenuate caspase-1–dependent pyroptotic signaling in iBMDMs similarly to their actions in primary BMDMs. As in primary WT BMDMs, concentrations of Gd³⁺ or La³⁺ >1 mM markedly attenuated Pro²⁺ influx in WT iBMDMs in response to NG (Supplemental Fig. 1B, 1C). Likewise, 1.2 mM of Gd³⁺ or La³⁺ suppressed downstream lytic LDH release at 30 min post-NG stimulation in WT iBMDMs (Supplemental Fig. 1D). However, at 60 min after NG stimulation, WT iBMDMs exhibited an escape from lanthanide suppression of LDH release similarly to primary WT BMDMs (Supplemental Fig. 1D).

Lanthanides do not block NLRP3 inflammasome activation or IL-1β release, whereas Gsdmd deficiency also does not block NLRP3 inflammasome activation but does block IL-1β release

To test whether the lanthanides might suppress downstream pyroptotic signaling by inhibiting upstream inflammasome assembly or activity, we assayed various indices of inflammasome activation in response to NG (Fig. 5) or TcdB (Supplemental Fig. 2D) stimulation in the presence of Gd³⁺ or La³⁺. After 30 min of NG stimulation in the presence of 1, 1.2, and 1.5 mM of Gd³⁺ or La³⁺ (concentrations that suppress pyroptotic Pro²⁺ influx in a dose-dependent manner), WT BMDMs displayed intact ASC oligomerization (Fig. 5A), suggesting the assembly of an ASC-containing inflammasome complex. Control experiments (data not shown) indicated that treatment of the BMDMs with lanthanides alone produced no stimulatory or inhibitory effects on ASC oligomerization, caspase-1 activation, or IL-1β release.

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FIGURE 5.

Lanthanides do not block NLRP3 inflammasome activation or IL-1β release, whereas Gsdmd deficiency also does not block NLRP3 inflammasome activation but does block IL-1β release. (A) LPS-primed WT BMDMs were stimulated with NG (10 μM) for 30 min in the presence or absence of Gd³⁺ or La³⁺ (0.3, 1, 1.2, and 1.5 mM). The ECM and soluble cell lysates were processed and analyzed on Western blot for the presence of caspase-1 and IL-1β. The detergent insoluble fraction was DSS cross-linked and analyzed on Western blot for the presence of oligomerized ASC. Western blot analysis of the soluble lysate for actin was also performed. These data are representative of results from two experiments. (B) LPS-primed WT BMDMs were treated as in (A) in the presence of 5 mM of glycine. The ECM, soluble lysate, and detergent insoluble fraction were analyzed on Western blot as described in (A). These data are representative of results from two separate experiments. (C) LPS-primed WT, Gsdmd^{−/− G1} (G1), and Gsdmd^{−/− G2} (G2) iBMDMs were stimulated with NG for 30 min. The ECM and soluble lysates were processed and analyzed on Western blot for the presence of caspase-1 and IL-1β. Western blot analysis of the soluble lysate for Gsdmd and actin was also performed. The detergent insoluble fraction of the cell lysates was incubated with the chemical cross-linker DSS and analyzed on Western blot for the presence of ASC monomers, dimers, and oligomers. These data are representative of results from two experiments. Asterisk represents nonspecific band.

Increasing concentrations of Gd³⁺ or La³⁺ resulted in an enhanced retention of processed caspase-1 in the cell lysate during 30-min incubations with NG (Fig. 5A). The production of processed caspase-1 in the presence of Gd³⁺ and La³⁺ further demonstrated that the lanthanides do not limit NLRP3 inflammasome complex assembly and downstream caspase-1 activation. Notably, at the 1, 1.2, and 1.5 mM concentrations of Gd³⁺ or La³⁺, procaspase-1 was predominantly retained in the cell lysate, whereas in the absence of Gd³⁺ and La³⁺ (or at the 300 μM concentration that does not suppress pyroptosis), some procaspase-1 was released into the ECM (Fig. 5A).

We also examined the effect of lanthanide treatment on the extent of mature IL-1β production as an additional index of inflammasome activation. Western blot analysis revealed that WT BMDMs, stimulated with NG for 30 min in the presence of concentrations (>1 mM) of Gd³⁺ and La³⁺ that inhibit pyroptotic dye uptake, generated and released mature IL-1β in amounts comparable with that observed in the absence of lanthanides (Fig. 5A). This marked production of mature IL-1β in the presence of Gd³⁺ and La³⁺ further demonstrates that the lanthanides do not limit NLRP3 inflammasome activation. Importantly, the additional presence of glycine to attenuate cell lysis did not modulate the effects of the lanthanides on NG-stimulated NLRP3 inflammasome signaling and IL-1β export (Fig. 5B).

The inhibitory effects of lanthanides on pyroptotic signaling responses downstream of inflammasome activation mostly mimicked the effects of Gsdmd knockout with the following notable exception: release of caspase-1 processed mature IL-1β to the extracellular compartment. We confirmed previous findings (16) that Gsdmd deficiency does not limit upstream inflammasome activation in response to NG. WT, Gsdmd^{−/− G1}, and Gsdmd^{−/− G2} iBMDMs were stimulated with NG for 30 min, and the formation of mature caspase-1 and IL-1β and the oligomerization of ASC were assayed by Western blot. The cell lysates of unstimulated WT iBMDMs, but not the Gsdmd^−/− cells, contained full-length 53 kDa Gsdmd that was extensively processed to the 31-kDa N-terminal fragment after NG stimulation; this is consistent with previous reports that active caspase-1 cleaves Gsdmd to produce a 31-kDa N-terminal propyroptotic product (13, 14, 16). Both WT and Gsdmd^−/− iBMDMs exhibited intact ASC oligomerization and robust accumulation of processed caspase-1 and IL-1β (Fig. 5C), indicating that Gsdmd mediates downstream pyroptosis but not upstream NLRP3 inflammasome activation.

Notably, mature IL-1β and caspase-1 were completely retained in the cell lysates of the Gsdmd^−/− iBMDMs but were predominantly released into the ECM fraction of WT iBMDMs (Fig. 5C). Nonlytic IL-1β and caspase-1 release represented a major mode of export because comparable amounts of mature IL-1β and caspase-1 were present in the ECM fractions of WT iBMDMs in the presence or absence of glycine (Fig. 5C). Therefore, the absence of Gsdmd does not simply prevent release of caspase-1 and IL-1β as a consequence of blocked pyroptotic lysis, but also suppresses the nonlytic export of these cytosolic proteins that has been previously described in multiple models of inflammasome function (2, 3).

Whereas NG-stimulated Gsdmd^−/− macrophages were characterized by intracellular retention of both mature IL-1β and the p20 subunit of active capase-1 (Fig. 5C), WT BMDMs treated with NG in the presence of lanthanides retained the active p20 caspase-1 in the cytosol but released mature IL-1β to the ECM. To clarify how the absence of Gsdmd, but not the presence of lanthanides, completely suppressed the release of IL-1β, we further investigated the effect of lanthanide treatment on caspase-1 and IL-1β export. Interestingly, concentrations of Gd³⁺ and La³⁺ that inhibit caspase-1–induced PM permeabilization (1, 1.2, 1.5 mM) also greatly suppressed caspase-1 release but permitted robust IL-1β release in response to 30 min of NG stimulation (Fig. 5A). Also, similar amounts of 20 kDa caspase-1 and mature IL-1β were present in the ECM fraction in the presence and absence of the cytoprotectant glycine, which prevents cell lysis (Fig. 5A, 5B). These data suggest that Gsdmd knockout per se and lanthanide blockade of the Gsdmd-dependent PM permeability change regulate nonlytic caspase-1 and IL-1β export in distinct ways. Lanthanide inhibition of Gsdmd-induced PM permeabilization markedly limits nonlytic caspase-1 release; however, it permits robust IL-1β release, which further underscores an apparent role for Gsdmd in the nonlytic vesicular trafficking and export of IL-1β.

Similarly, after 45 min of TcdB stimulation in the presence of 1 mM of Gd³⁺ or La³⁺ (concentrations that suppress pyroptotic Pro²⁺ influx), WT BMDMs also displayed intact ASC oligomerization and caspase-1 activation (Supplemental Fig. 3). Although the presence of 1 mM of Gd³⁺ or La³⁺ did not prevent the formation of processed caspase-1, the p20 fragment of caspase-1 and procaspase-1 were retained in the cell lysate. Taken together, these data demonstrate that Gd³⁺ and La³⁺ do not suppress NLRP3 and Pyrin inflammasome activation or caspase-1 activity, but rather inhibit the downstream pyroptotic signaling processes.

Lanthanides reversibly block the caspase-1–dependent pyroptotic pores and suppress pyroptosis

Given that Gd³⁺ and La³⁺ inhibit caspase-1–dependent PM permeabilization without limiting inflammasome activation, we next investigated the mechanism by which lanthanides target this PM permeability change. Because there is a narrow time window between pyroptotic pore opening and subsequent lysis, we first verified that lanthanides suppress prelytic pyroptotic Pro²⁺ influx before end-stage lysis by stimulating WT BMDMs with NG in the presence of glycine ± 1.5 mM of Gd³⁺ or 1.2 mM of La³⁺. A total of 1.5 mM of Gd³⁺ and 1.2 mM of La³⁺ almost completely suppressed pyroptotic Pro²⁺ influx in the presence of glycine, similar to the responses in the absence of glycine (Fig. 6A, 6B). Interestingly, the lanthanides were more potent as suppressors of pyroptosis-induced Pro²⁺ influx in the presence of glycine than in its absence (Supplemental Fig. 3A–D). For example, 800 μM of Gd³⁺ and La³⁺ partially suppressed the rate and magnitude of NG-induced Pro²⁺ influx in the presence of glycine, but not in the absence of glycine (Supplemental Fig. 3E, 3F). Also, 1 mM of Gd³⁺ and La³⁺ was more efficacious in reducing the rate and magnitude of NG-induced Pro²⁺ influx in the presence of glycine versus in its absence (Supplemental Fig. 3G, 3H). Together these data indicate that lanthanides target the prelytic PM permeability change induced by upstream caspase-1/Gsdmd signaling that may include: 1) gating of large-pore channels already resident in the PM, or 2) insertion of pore-forming proteins into the PM.

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FIGURE 6.

Lanthanides reversibly block the caspase-1–dependent pyroptotic pores and suppress pyroptosis. (A) LPS-primed WT BMDMs were stimulated with NG (10 μM) in the presence or absence of 5 mM of glycine and ± 1.5 mM of Gd³⁺ or (B) 1.2 mM of La³⁺ for 45 min, and Pro²⁺ fluorescence was quantified every 3 min as described in Fig. 1. These data represent the mean ± SE of four to six replicates from two to three independent experiments. (C) LPS-primed WT BMDMs were stimulated with NG in the presence or absence of 1.5 mM of Gd³⁺ or (D) 1.2 mM of La³⁺ for 45 min. At 15 min post-NG stimulation, wells containing WT BMDMs, Pro²⁺, NG, and Gd³⁺/La³⁺ were either washed with PBS (+W) and replaced with fresh NaCl BSS plus 1 μg/ml Pro²⁺ or not washed (−W). Pro²⁺ fluorescence was quantified every 3 min. These data represent the mean ± SE of four to six replicates from two to three independent experiments. (E) LPS-primed WT BMDMs were stimulated with NG in the presence or absence of 1.5 mM of Gd³⁺ or (F) 1.2 mM of La³⁺ for 45 min. Pro²⁺ and lanthanides (Gd³⁺ or La³⁺) were added at different times during NLRP3 inflammasome activation, which included: 1) Pro²⁺ and lanthanides added at the same time as NG (T = 0; blue curve); 2) Pro²⁺ added at the same time as NG, and lanthanides added 20 min post-NG (T = 20; green curve); or 3) lanthanides added 20 min post-NG, and Pro²⁺ added 5 min postlanthanide addition (T = 20*; magenta curve). Pro²⁺ fluorescence was quantified every 3 min. These data represent the mean ± SE of four to six replicates from three (E) or two (F) separate experiments. (G) LPS-primed WT BMDMs were stimulated with TcdB in the presence or absence of 1.5 mM of Gd³⁺ or (H) La³⁺ for 60 min. Pro²⁺ and lanthanides were added at different times during Pyrin inflammasome activation, which included: 1) Pro²⁺ added at the same time as TcdB, and lanthanides added 20 min post-TcdB (T = 20; blue curve); 2) Pro²⁺ added at the same time as TcdB, and lanthanides added 30 min post-TcdB (T = 30; green curve); or 3) lanthanides added 30 min post-TcdB, and Pro²⁺ added 5 min postlanthanide addition (T = 30*; magenta curve). Pro²⁺ fluorescence was quantified every 4 min. These data represent the mean ± SE of two to eight replicates from two independent experiments.

Previous studies have demonstrated that lanthanides can inhibit channels by either acting as competitive pore blockers (55–57) or binding to anionic phospholipids to induce lateral compression of channels (58, 59). Lanthanides have similar cationic radii as Ca²⁺, enabling them to compete for binding within the selectivity filter of Ca²⁺ channels (55–57). Sukharev and colleagues (58) have reported that Gd³⁺ reversibly inhibits the large mechanosensitive channel of E. coli by binding to anionic phospholipids. Gd³⁺ binding then alters phospholipid packing and greatly increases the lateral pressure within the PM, which forces the large mechanosensitive channel to adopt a closed conformation (58).

We designed experiments to determine whether the lanthanides reversibly inhibit the pyroptotic pore/channel. After stimulating WT BMDMs for 15 min with NG in the presence of Pro²⁺ and 1.5 mM of Gd³⁺ or 1.2 mM of La³⁺, the lanthanide-containing ECM were removed and replaced with fresh Pro²⁺-containing media. The removal of Gd³⁺ and La³⁺ rapidly restored the NG-induced Pro²⁺ influx (Fig. 6C, 6D), suggesting that lanthanides reversibly block the Gsdmd-dependent pyroptotic pore/channel.

Next, Gd³⁺/La³⁺ and Pro²⁺ were added at different times during the course of NLRP3 or Pyrin inflammasome activation to investigate whether lanthanides inhibit the presumed assembly/activation of the pyroptotic pore or the flux of permeant ions through an already assembled/activated pore. When 1.5 mM of Gd³⁺ or 1.2 mM of La³⁺ was added to WT BMDMs during NG-induced pyroptotic Pro²⁺ influx (T = 20; 20 min post-NG), further dye uptake was prevented (Fig. 6E, 6F). Similarly, adding 1.5 mM of Gd³⁺ or La³⁺ to WT BMDMs during TcdB-induced pyroptotic Pro²⁺ influx (T = 30; 30 min post-TcdB) prevented further dye uptake (Fig. 6G, 6H). Also, if 1.5 mM of Gd³⁺ or 1.2 mM of La³⁺ was added after NG-induced pyroptotic pore opening (20 min post-NG) and Pro²⁺ was added 5 min later (T = 20*; Pro²⁺ added 5 min postlanthanide addition), dye uptake was almost completely suppressed (Fig. 6E, 6F). Similarly, adding 1.5 mM of Gd³⁺ or La³⁺ after TcdB-induced pyroptotic pore opening (30 min post-TcdB) and then adding Pro²⁺ 5 min later (T = 30*; Pro²⁺ added 5 min postlanthanide addition) also almost completely suppressed dye uptake (Fig. 6G, 6H). Residual dye uptake could reflect BMDMs that have already progressed to lytic cell death. Because Gd³⁺ and La³⁺ prevented further pyroptotic dye uptake and almost completely prevented any pyroptotic dye uptake if Pro²⁺ was added after Gd³⁺ and La³⁺, direct pore blockade and/or lateral compression of a pore/channel are plausible mechanisms for how lanthanides reversibly inhibit activity of the pyroptotic pore/channel. Using the lanthanides as a tool to characterize the nature of the caspase-1–dependent PM permeability change suggests that Gd³⁺ and La³⁺ may reversibly inhibit an already active Gsdmd-containing pore or pyroptotic pore/channel(s) regulated by Gsdmd.

Panx1, P2X7R, and certain TRP channel family members are not required for caspase-1–dependent pyroptotic pore induction

Given the observed permeability characteristics and lanthanide sensitivity of the pyroptotic pore/channels, we investigated the potential role of known large-pore channels in mediating caspase-1–dependent pyroptosis. For example, Panx1 is an important ATP release channel that adopts a large-pore conformation upon activation and is also permeable to large DNA-intercalating fluorescent dyes (52, 60). Interestingly, Panx1 channels can be gated by apoptotic executioner caspases that excise an autoinhibitory domain from the Panx1 cytosolic C terminus (60). Núñez and colleagues (47) recently reported that Panx1 is also cleaved by caspase-11 to mediate caspase-11–dependent pyroptosis in macrophages that accumulate cytosolic LPS. However, we found that the Panx1 inhibitor trovafloxacin did not suppress NG-induced pyroptotic Pro²⁺ influx in WT BMDMs (Fig. 7A), suggesting that Panx1 is not required for caspase-1/Gsdmd–dependent prelytic pore activation in the context of canonical inflammasome-driven pyroptosis.

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FIGURE 7.

Panx1, P2X7R, and certain TRP channel family members are not required for caspase-1–dependent pyroptotic pore induction. (A) LPS-primed WT BMDMs were stimulated with NG (10 μM) for 45 min in the presence or absence of the Panx1 inhibitor trovafloxacin (30 μM). These data represent the mean ± SE of four replicates from two independent experiments. (B) LPS-primed WT BMDMs were stimulated with NG for 45 min in the presence of the P2X7R antagonists A10606120 (10 μM) or A438079 (10 μM). These data represent the mean ± SE of four replicates from two independent experiments. (C) LPS-primed WT or P2X7R^−/− BMDMs were stimulated with NG or (D) TcdB (0.4 μg/ml) for 45 min. These data represent the mean ± SE of three replicates from one experiment. (A–D) Pro²⁺ fluorescence was quantified every 3 (NG) or 4 (TcdB) min as described in Fig. 1. (E) LPS-primed WT or P2X7R^−/− BMDMs were stimulated with NG. At the indicated times, supernatants were assayed for LDH activity as described in Fig. 1. These data represent the mean ± SE of two replicates from one experiment. (F) LPS-primed WT BMDMs were stimulated with NG for 45 min or (G) TcdB for 60 min in the presence of the TRPV channel inhibitor ruthenium red (RR: 1 μM) or the TRPM7 channel inhibitor NS8593 (30 μM). These data represent the mean ± SE of two to four replicates from two (F) or one (G) independent experiment. (F and G) Pro²⁺ fluorescence was quantified every 3 (NG) or 4 (TcdB) min as described in Fig. 1.

P2X7R is an ATP-gated nonselective cation channel that can adopt a large-pore conformation that is both permeable to large DNA-intercalating fluorescent dyes and inhibited by Gd³⁺ (52). ATP gating of the P2X7R also mediates NLRP3 inflammasome activation by facilitating K⁺ efflux (30, 31, 61). Autocrine activation of P2X7R by ATP released through Panx1 channels has also been implicated in mediating caspase-11–dependent pyroptosis (47). However, we found that the P2X7R antagonists A10606120 and A439079 did not suppress NG-induced Pro²⁺ influx in WT BMDMs (Fig. 7B). Similarly, P2rx7^−/− BMDMs stimulated with NG or TcdB exhibited comparable rates and magnitudes of Pro²⁺ influx with those observed in WT BMDMs (Fig. 7C, 7D). Consistent with this, the absence of P2X7R did not suppress downstream LDH release in response to NG (Fig. 7E). These data suggest that the P2X7R is also not required for the caspase-1– and Gsdmd-dependent prelytic pore induction or ensuing pyroptotic lysis downstream of canonical inflammasomes.

We also evaluated the roles of certain members of the TRP channel family, which are nonselective ion channels in mediating caspase-1/Gsdmd–induced pyroptosis given their expression in hematopoietic cells, sensitivity to lanthanides, activation by multiple cell stressors, and roles in innate immunity (51). Specifically, TRPV2 and TRPM7 have been implicated in NLRP3 inflammasome activation in response to hypotonic stress (53). TRPM7 can also be proteolytically gated by apoptotic caspases and is involved in Fas-induced apoptosis (62). TRPM2 is gated in response to oxidative stress (63), and mitochondrial reactive oxygen species (ROS) production has been implicated in NLRP3 inflammasome activation (64). In particular, mitochondrial ROS–dependent TRPM2 activation was shown to mediate liposome/particulate-induced NLRP3 inflammasome activation (65). We observed that neither the broad TRP channel inhibitor ruthenium red nor the selective TRPM7 inhibitor NS8593 (66, 67) suppressed NG- or TcdB-induced pyroptotic Pro²⁺ uptake (Fig. 7F, 7G). In another recent study (68), we reported that Trpm2^−/− BMDCs stimulated with NG did not exhibit reduced Pro²⁺ influx compared with that in WT cells. Taken together, these experiments indicate that TRPV, TRPM7, or TRPM2 channels are not required components of the caspase-1/Gsdmd–dependent pyroptotic pores.