
==== Front
101573691
39703
Cell Rep
Cell Rep
Cell reports
2211-1247

39116210
10.1016/j.celrep.2024.114609
nihpa2019541
Article
FDA-approved disulfiram inhibits the NLRP3 inflammasome by regulating NLRP3 palmitoylation
Xu Jie 1*
Pickard Joseph M. 1
Núñez Gabriel 12*
1 Department of Pathology and Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, MI 48109, USA
2 Lead contact
AUTHOR CONTRIBUTIONS

J.X. and G.N. conceptualized the project. J.X. and J.M.P. conducted the investigation. G.N. acquired funding. J.X. and G.N. wrote the paper with input from J.M.P.

* Correspondence: jiexu@med.umich.edu (J.X.), gabriel.nunez@umich.edu (G.N.)
9 9 2024
27 8 2024
07 8 2024
13 9 2024
43 8 114609114609
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
SUMMARY

The NLRP3 inflammasome is dysregulated in autoinflammatory disorders caused by inherited mutations and contributes to the pathogenesis of several chronic inflammatory diseases. In this study, we discovered that disulfiram, a safe US Food and Drug Administration (FDA)-approved drug, specifically inhibits the NLRP3 inflammasome but not the NLRC4 or AIM2 inflammasomes. Disulfiram suppresses caspase-1 activation, ASC speck formation, and pyroptosis induced by several stimuli that activate NLRP3. Mechanistically, NLRP3 is palmitoylated at cysteine 126, a modification required for its localization to the trans-Golgi network and inflammasome activation, which was inhibited by disulfiram. Administration of disulfiram to animals inhibited the NLRP3, but not NLRC4, inflammasome in vivo. Our study uncovers a mechanism by which disulfiram targets NLRP3 and provides a rationale for using a safe FDA-approved drug for the treatment of NLRP3-associated inflammatory diseases.

Graphical Abstract

In brief

Xu et al. demonstrate that FDA-approved disulfiram inhibits NLRP3 inflammasome activation in mouse and human macrophages and in models of sepsis and peritonitis. They show that zDHHC5-mediated palmitoylation of NLRP3 at cysteine 126, a modification required for appropriate NLRP3 subcellular localization and inflammasome activation, is inhibited by disulfiram.
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pmcINTRODUCTION

Inflammasomes play essential roles in innate immunity. These multimeric protein complexes are activated in the presence of microbial and danger signals to initiate the host immune response. Activation of inflammasomes is induced by host pattern recognition receptors (PRRs) that sense endogenously derived damage-associated molecular patterns, molecular motifs conserved in microbes, and cellular activities induced by pathogens.1 The most extensively characterized inflammasome is activated by NLRP3, a PRR belonging to the nucleotide-binding oligomerization domain and leucine-rich repeat-containing (NLR) protein family. In mouse macrophages, following a priming signal induced by microbial ligands or cytokines such as tumor necrosis factor alpha (TNF-α) or interleukin (IL)-1β, NLRP3 can be activated by several stimuli, including nigericin, ATP, bacterial toxins, and particulate matter.1 In response to these stimuli, NLRP3 recruits the adaptor protein ASC, leading to caspase-1 activation, which enables the processing of IL-1β, IL-18, and gasdermin D (GSDMD). The cleaved N terminus of GSDMD forms pores in the plasma membrane, inducing pyroptosis and promoting the extracellular release of processed IL-1β and IL-18.1 Because NLRP3 activation causes or contributes to the pathogenesis of several inflammatory disorders,2 there is a need for developing safe and specific drugs that target the NLRP3 inflammasome.

The NLRP3 inflammasome is regulated by several post-translational modifications.1 A recently described modification is the palmitoylation of NLRP3, which promotes NLRP3 degradation through chaperone-mediated autophagy.3 Protein palmitoylation occurring on cysteine residues is catalyzed by members of the zinc-finger DHHC-type containing (zDHHC) family of palmitoyl S-acyltransferases.4,5 Several zDHHCs including zDHHC3, -5, -7, and -12 mediate NLRP3 palmitoylation at different cysteine amino acid residues to regulate inflammasome activation.3,6–8 Palmitoylation can regulate not only protein stability but also protein localization and intracellular trafficking.4,5

US Food and Drug Administration (FDA)-approved disulfiram, a drug used to treat chronic alcoholism, showed anti-inflammatory effects in several rodent models.9 Disulfiram inhibits GSDMD pore formation associated with pyroptosis but not NLRP3 inflammasome activation10; however, another report showed that its active metabolite, bis(diethyldithiocarbamate)-copper, can inhibit NLRP3 inflammasome activation.11 Thus, further studies are warranted to determine whether disulfiram inhibits the NLRP3 inflammasome and the inflammasome specificity of the drug, and more importantly, to understand the mechanism by which the drug regulates the NLRP3 inflammasome. In this study, we report that disulfiram inhibits the NLRP3 inflammasome, but not the NLRC4 or AIM2 inflammasome, in vitro and in vivo. Notably, we show that disulfiram acts by inhibiting the palmitoylation of NLRP3 at the cysteine 126 residue (Cys126), which is required for NLRP3 localization at the trans-Golgi network (TGN) and inflammasome activation.

RESULTS

Disulfiram inhibits NLRP3 inflammasome activation

To examine whether disulfiram affects the NLRP3 inflammasome, we stimulated lipopolysaccharide (LPS)-primed mouse bone marrow-derived macrophages (BMDMs) with ATP to induce NLRP3 activation in the presence of vehicle control or disulfiram at several concentrations (Figure S1A). While 10 μM disulfiram exerted an inhibitory effect, NLRP3 activation induced by ATP was completely blocked by 50 μM disulfiram, as assessed by the detection of the processed p20 subunit of caspase-1 and the cleaved GSDMD fragment. Further experiments showed that disulfiram, applied 20 min before ATP stimulation, inhibited caspase-1 p20 and GSDMD processing (Figure 1A), as well as IL-1β and IL-18 release (Figures 1B and S1B). Importantly, NLRP3 expression was unaffected (Figure 1A), and the release of TNF-α and IL-6 was reduced to a lesser degree by disulfiram treatment (Figures S1C and S1D). To further investigate whether disulfiram also negatively regulates the process of LPS priming in addition to NLRP3 activation, we co-treated macrophages with LPS and disulfiram for 3 h (Figure S2). We found that disulfiram at 10 μM, a concentration that inhibits the NLRP3 inflammasome (Figure S1A), had a minimal effect on NLRP3 or pro-IL-1β expression, while at 30 μM, it reduced the expression of NLRP3 and pro-IL-1β (Figure S2A) as well as the release of TNF-α and IL-6 (Figures S2B and S2C). The latter results are consistent with previous studies showing that disulfiram can inhibit TLR4-MD-2 signaling.12

We next tested whether the inhibitory effect of disulfiram was limited to ATP-mediated NLRP3 activation. We stimulated LPS-primed BMDMs with an array of agonists to induce NLRP3 activation in the presence of vehicle or disulfiram (Figure 1C). Strikingly, we found that disulfiram abolished caspase-1 p20 and GSDMD processing (Figure 1C) as well as IL-1β production (Figure 1D) induced by all tested NLRP3 agonists including nigericin, imiquimod, gramicidin, L-leucyl-L-leucine methyl ester, and silica. In contrast, TNF-α secretion was modestly affected by disulfiram (Figure S3A). These results suggest that disulfiram inhibits the NLRP3 inflammasome through a mechanism that acts proximally to NLRP3 activation.

We then investigated whether disulfiram also inhibits other inflammasomes. We stimulated LPS-primed BMDMs with poly(dA:dT) or Salmonella to activate the AIM2 or NLRC4 inflammasome, respectively. In the presence of 30 μM disulfiram, the formation of caspase-1 p20 and the GSDMD cleavage product induced by poly(dA:dT) or Salmonella was comparable to that observed with vehicle control, while that induced by nigericin was blocked by disulfiram (Figure 1E). Likewise, nigericin-mediated IL-1β release was abrogated by disulfiram, whereas there was little or no effect of disulfiram on IL-1β production induced by poly(dA:dT) or Salmonella infection (Figure 1F). As controls, TNF-α secretion induced by the same stimuli was marginally affected by disulfiram (Figure S3B). Collectively, these results indicate that disulfiram specifically inhibits the NLRP3 inflammasome, but not the AIM2 or NLRC4 inflammasome, in BMDMs.

Disulfiram inhibits the NLRP3 inflammasome in mouse and human cells

We then tested whether disulfiram also inhibits NLRP3 activation in other cell types. In mouse peritoneal macrophages, disulfiram abolished GSDMD processing (Figure 2A) and IL-1β release (Figure 2B) induced by ATP and nigericin but not poly(dA:dT) that activates the AIM2 inflammasome. Similarly, in human THP-1 monocytic cells, disulfiram reduced GSDMD processing (Figure 2C) and IL-1β release (Figure 2D) induced by nigericin and imiquimod. In contrast, disulfiram enhanced IL-1β production triggered by poly(dA:dT) (Figure 2D), although the mechanism for the enhanced response is unclear. In both cell types, TNF-α secretion was not affected by disulfiram (Figures S4A and S4B) except in peritoneal macrophages in which disulfiram increased TNF-α production after AIM2 activation induced by poly(dA:dT) (Figure S4A). Collectively, these results indicate that disulfiram specifically inhibits NLRP3 activation in both mouse macrophages and human monocytic cells.

Mutations of NLRP3 cause cryopyrin-associated periodic syndromes (CAPSs).13 To investigate the effect of disulfiram on CAPS-associated NLRP3 mutations, we used BMDMs from knockin mice (Nlrp3fl(D301N)/+Esr1-Cre) in which the expression of an aspartate 301-to-asparagine (D301N) substitution, which corresponds to the D303N human NLRP3 mutation that causes CAPSs, is inducible by tamoxifen.14 After treatment of BMDMs with tamoxifen, we found that LPS alone was sufficient to trigger IL-1β release in Nlrp3fl(D301N)/+Esr1-Cre macrophages that is consistent with previous studies,11 which was abolished by disulfiram (Figure 2E). In contrast, TNF-α release was modestly affected by disulfiram (Figure S4C).

Disulfiram possesses a disulfide bond that can be reduced by free thiol groups of proteins. This reaction covalently modifies cysteine residues on target proteins.15 We investigated next whether the inhibition of the NLRP3 inflammasome by disulfiram is through the modification of cysteine residues. To this end, we pre-treated LPS-primed BMDMs with N-acetyl cysteine (NAC), a derivative of cysteine with better solubility, and stimulated NLRP3 with nigericin in the presence of a vehicle control or disulfiram. Strikingly, NAC reversed the inhibitory effect of disulfiram on the NLRP3 inflammasome, as assessed by the processing of p20 and GSDMD (Figure 2F), as well as IL-1β release (Figure 2G). Given the antioxidant activity of NAC, we assessed next whether its role as an antioxidant could play a part in its ability to reverse the inhibitory effect of disulfiram on the NLRP3 inflammasome. To assess this, we tested two other antioxidants, glutathione (GSH) and vitamin C. GSH, a tripeptide, γ-l-glutamyl-l-cysteinyl-glycine, has a free thiol group in its cysteine residue, while vitamin C does not. Like NAC, GSH reversed the inhibitory activity of disulfiram, whereas vitamin C did not, as assessed by p20 formation and GSDMD cleavage (Figure 2F), as well as IL-1β release (Figure 2G). TNF-α secretion remained comparable in the presence or absence of the antioxidants (Figure S4D). Collectively, the results suggest that the ability to reverse the inhibitory effect of disulfiram on NLRP3 lies not on the antioxidant function but on whether the reagent can saturate disulfiram through the presence of a free thiol group on cysteine residues.

Disulfiram suppresses the formation of ASC specks and pyroptosis triggered by NLRP3 activators

Activation of NLRP3 and other inflammasome sensors induces the formation of a supramolecular structure termed an ASC speck, which is composed of ASC oligomers.16,17 Stimulation of BMDMs with NLRP3 activators including ATP, nigericin, gramicidin, and imiquimod triggered the formation of ASC specks in the macrophage cytosol, which was abolished by incubation with disulfiram (Figures 3A and 3B). However, disulfiram treatment did not affect the formation of ASC specks induced by stimulation with poly(dA:dT) that activates AIM2 or Salmonella that activates the NLRC4 inflammasome (Figures 3A and 3B). Inflammasome activation results in pyroptosis, a lytic form of cell death characterized by cell swelling, bubble-like protrusions, nuclear rounding, and nuclear condensation.18 As expected, stimulation of BMDMs with nigericin induced pyroptosis (Figures 3C and 3D). Disulfiram treatment inhibited nigericin-induced pyroptosis in a dose-dependent manner (Figures 3C and 3D). We further confirmed the inhibitory effect of disulfiram against pyroptosis by measuring lactate dehydrogenase (LDH) release triggered by ATP (Figure 3E). In summary, disulfiram inhibits the formation of ASC specks and pyroptosis induced by stimuli that activate the NLRP3 inflammasome.

zDHHC5-mediated palmitoylation of NLRP3 is important for NLRP3 activation

We utilized enzyme-catalyzed proximity labeling with an engineered biotin ligase, Turbo-ID, to identify NLRP3-interacting proteins in macrophages. In agreement with previous studies,19–21 the analyses revealed that NEK7 interacts with NLRP3 (Figure S5A). In addition, proximity labeling detected zDHHC5, a member of the zDHHC family of palmitoyl S-acyltransferases, as an interactor of NLRP3 (Figure S5A). To confirm this interaction, we immunoprecipitated NLRP3 and immunoblotted the protein immunoprecipitates with an anti-zDHHC5 antibody. As shown in Figure 4A, NLRP3 interacted with endogenous zDHHC5. To test whether NLRP3 can be palmitoylated by zDHHC5, we performed an acyl-biotin exchange assay in macrophages transduced with non-targeting control short hairpin RNA (shRNA) or shRNA targeting zDHHC5. The signal that appeared upon hydroxylamine treatment indicated that NLRP3 undergoes palmitoylation through thioester linkages (Figure 4B). Importantly, the level of palmitoylation decreased in cells in which zDHHC5 expression was knocked down (Figure 4B), suggesting that zDHHC5 is important for NLRP3 palmitoylation. To investigate the role of zDHHC5 in NLRP3 activation, we stimulated LPS-primed macrophages with nigericin to induce NLRP3 activation in the presence or absence of treatment with zDHHC5 shRNA. In macrophages in which zDHHC5 expression was knocked down, nigericin-induced processing of caspase-1 p20 and IL-1β release was inhibited, while Salmonella-induced NLRC4 activation was not affected (Figures 4C and 4D). For both nigericin- and Salmonella-treated macrophages, the levels of TNF-α remained comparable in the presence or absence of zDHHC5 shRNA (Figure S5B). In addition, we found that zDHHC5 was important for potassium efflux independent NLRP3 activation, as zDHHC5 knockdown impaired imiquimod-induced inflammasome activation (Figure S5C). Taken together, these results suggest that zDHHC5 can mediate NLRP3 palmitoylation that is important for NLRP3 activation.

To determine whether the palmitoyl S-acyltransferase activity of zDHHC5 is required for the regulation of the NLRP3 inflammasome, we pre-treated LPS-primed BMDMs with 2-bromopalmitate (2-BP), a pan-zDHHC inhibitor, prior to activating NLRP3 with nigericin. Pre-treatment with 2-BP for 45 and 90 min inhibited nigericin-induced caspase-1 p20 processing and IL-1β release (Figures 4E and 4F). The decrease was specific in that 2-BP pre-treatment had no or a minimal effect on TNF-α production (Figure S5D). These results suggest that zDHHC-mediated palmitoylation of NLRP3 is important for NLRP3 activation.

Palmitoylation at Cys126 promotes NLRP3 activation and is inhibited by disulfiram

Because both palmitoylation and disulfiram modify cysteine residues (Figures 2F and 2G), we asked whether disulfiram could compete with the palmitoylation process for certain protein cysteine residues. To address this question, we performed an acyl-biotin exchange assay to assess the level of NLRP3 palmitoylation in the presence of disulfiram. The results showed that disulfiram inhibits NLRP3 palmitoylation (Figure 4G). The interaction between NLRP3 and NEK7 is essential for NLRP3 activation in mice.20,21 We thus assessed whether disulfiram inhibits the NLRP3-NEK7 interaction by regulating palmitoylation. To this end, we performed pull-down experiments in Nlrp3−/− macrophages reconstituted with an SFP-tagged NLRP3 (NLRP3-SFP). As shown in Figure S6A, the same amount of NEK7 was pulled down by NLRP3-SFP in the presence or absence of disulfiram. These results suggest that disulfiram does not inhibit the NLRP3 inflammasome by impairing the interaction of NLRP3 with NEK7.

NLRP3 localizes at the perinuclear TGN.22 Consistent with previous studies, we found co-localization of NLRP3 and TGN38, a transmembrane protein marker for TGN (Figure S6B). Notably, disulfiram treatment disturbed the TGN localization of NLRP3, resulting in the cytosolic distribution of NLRP3 (Figure 4H).

Previous work showed that four consecutive lysine residues (residues 127–130, the KKKK motif) within a polybasic region between the Pyrin and NACHT domains are required for the perinuclear TGN localization of NLRP3.22 Because we found that NLRP3 palmitoylation regulates its subcellular localization, we hypothesized that palmitoylation may occur on a cysteine residue adjacent to the KKKK motif. We found that Cys126, which is conserved across species, resides next to the lysine-rich motif (Figure 4I). To determine whether Cys126 of NLRP3 is important for inflammasome activation, we replaced NLRP3 Cys126 with a serine amino acid residue. We found that the NLRP3 mutant (C126S) lost its TGN localization and became cytosolically distributed (Figure 4J), suggesting that the KKKK motif is not sufficient to mediate TGN localization of NLRP3. Importantly, the C126S mutation impaired NLRP3 activation, as indicated by reduced levels of caspase-1 p20 and IL-1β release triggered by stimulation with ATP or nigericin (Figures 4K and 4L). In contrast, TNF-α release was comparable in macrophages expressing the wild type and C126S NLRP3 mutant (Figure S6C). In addition, we found that Cys126 palmitoylation is important for potassium efflux independent of NLRP3 activation, as there was impaired imiquimod-induced inflammasome activation in macrophages harboring the C126S mutation (Figure S6D). We then compared the levels of palmitoylation between wild type and the NLRP3 C126S mutant in macrophages. As shown in Figures 4M and 4N, macrophages harboring the C126S NLRP3 mutant displayed lower palmitoylation levels than cells expressing the wild-type protein. Because there is residual palmitoylation signal in the Cys126 NLRP3 mutant, the results suggest that NLRP3 is also palmitoylated at additional cysteine residues, consistent with recently published studies.3,11,22,23 Unlike that observed in wild-type NLRP3, palmitoylation of the C126S mutant was not further decreased in macrophages treated with disulfiram (Figures 4M and 4N), suggesting that disulfiram inhibits palmitoylation primarily at Cys126. Recently, it was reported that zDHHC7 also palmitoylates NLRP3 at Cys126.22 Consistent with this observation, we found that the levels of NLRP3 palmitoylation decreased with zDHHC7 knockdown (Figures S6E and S6F). Notably, disulfiram could further lower the level of NLRP3 palmitoylation in cells with zDHHC7 knockdown (Figures S6E and S6F), suggesting that palmitoylation mediated by other zDHHC enzyme(s) is also inhibited by disulfiram. We then overexpressed zDHHC5 in cells expressing wild-type NLRP3 or the C126S NLRP3 mutant. While overexpression of zDHHC5 resulted in increased palmitoylation in wild-type NLRP3, the same increase was not observed in cells harboring the C126S mutation (Figures S6G and S6H). Since Cys837/Cys838 (Cys834/Cys835 in the mouse) was shown to be palmitoylated by zDHHC5,11 we created a construct encoding NLRP3 harboring triple replacements of Cys126, Cys834, and Cys835 for serine amino acid residues. When the triple NLRP3 mutant was co-expressed with zDHHC5, the level of palmitoylation was further decreased in the triple mutant compared to the single C126S NLRP3 mutant (Figures S6G and S6H). Taken together, the results indicate that NLRP3 palmitoylation at Cys126 is important for TGN localization and NLRP3 activation. Furthermore, disulfiram inhibits the NLRP3 inflammasome by interfering with NLRP3 palmitoylation at Cys126.

Disulfiram inhibits NLRP3 inflammasome activation in vivo

We next assessed the effect of disulfiram on NLRP3 inflammasome activation in vivo. Mice were stimulated intraperitoneally with LPS, a model that induces IL-1β production via the NLRP3 inflammasome in vivo.24,25 Administration of LPS induced IL-1β production in serum and the peritoneal cavity, which was markedly reduced in mice treated with disulfiram (Figures 5A and 5C). In contrast, the production of TNF-α in serum and the peritoneal cavity was comparable between the vehicle- and disulfiram-treated mice (Figures 5B and 5D). Furthermore, IL-1β production induced by intraperitoneal injection of monosodium urate (MSU) crystals, which is largely mediated by NLRP3,26 was reduced in mice treated with disulfiram (Figure S7A). In contrast, IL-6 production was comparable in mice treated with vehicle or disulfiram (Figure S7B). In addition, disulfiram abolished neutrophil recruitment in the peritoneal cavity induced by MSU injection (Figure S7C). To test whether disulfiram specifically targets the NLRP3 inflammasome, we infected mice with Salmonella, a model that induces IL-1β production via the NLRC4 inflammasome.27 Salmonella infection triggered IL-1β and TNF-α production in serum and the peritoneal cavity, which was not affected by treatment of the animals with disulfiram (Figures 5E–5H). These data suggest that disulfiram specifically inhibits NLRP3 inflammasome activation in vivo.

DISCUSSION

In this study, we find that zDHHC5 promotes NLRP3 palmitoylation and activation, with palmitoylation at Cys126 being critical for the TGN localization and activation of the NLRP3 inflammasome. Chen et al. reported that the KKKK motif between the Pyrin and NACHT domains mediates NLRP3 localization at the TGN.22 Our results suggest that in addition to the KKKK motif, Cys126 palmitoylation is also required for proper localization of NLRP3, as the C126S NLRP3 mutant that exhibits impaired palmitoylation did not localize to the TGN and displayed a cytosolic distribution. Previous work showed that zDHHC12-mediated palmitoylation inhibits NLRP3 activation through chaperone-mediated autophagy.3 While our manuscript was under submission, another study was published showing that zDHHC5 enables NLRP3 palmitoylation and activation, consistent with our results.6 However, instead of Cys126, these authors reported that Cys837 and Cys838 of NLRP3 were palmitoylated.6 In agreement with our results, two additional studies reported that NLRP3 palmitoylation occurs at Cys126.7,8 One study suggested that zDHHC3/5/7 mediates NLRP3 palmitoylation,8 while the other study showed that zDHHC7 is responsible for NLRP3 palmitoylation at Cys126.7 The inconsistency regarding which zDHHC mediates NLRP3 palmitoylation may be due to redundancy among the 23 zDHHC members or the use of different cells.3,6–8

Disulfiram has been approved by the FDA for the treatment of chronic alcoholism.15 It functions by irreversibly inhibiting aldehyde dehydrogenase,28 resulting in acetaldehyde accumulation after alcohol consumption, which leads to discomfort and amplified hangover.15 In addition, disulfiram is effective in the treatment of cocaine addiction by inhibiting dopamine beta-hydroxylase.15,29 Recently, it was reported that disulfiram inhibits GSDMD pore formation by covalently modifying Cys191 of GSDMD.10 In the liposome leakage assay of that report, the authors applied disulfiram to purified GSDMD, which resulted in cleaved GSDMD being unable to be incorporated into liposome membranes.10 In addition to the purified GSDMD in vitro system, the authors pre-treated mice with disulfiram and challenged the mice intraperitoneally with LPS, a model that induces IL-1β production via the NLRP3 inflammasome.10 However, these in vivo studies could not distinguish whether NLRP3 or GSDMD is targeted by disulfiram. In our study, we included control experiments using Salmonella infection, which induces IL-1β production via the NLRC4 inflammasome.27 Since GSDMD pore formation is a common downstream event shared among different inflammasomes, it is expected that disulfiram will inhibit all inflammasomes if GSDMD is the main target of disulfiram. However, our results showed that IL-1β induced by LPS and MSU crystals, two stimuli that trigger NLRP3 activation in vivo, but not Salmonella infection, was inhibited by disulfiram, suggesting that the drug targets the NLRP3 inflammasome instead of GSDMD to regulate IL-1β production. Consistent with these results, we found that disulfiram specifically inhibits NLRP3 but not other inflammasomes in macrophages. In addition, our results show that GSDMD cleavage is potently inhibited by disulfiram, further supporting the notion that disulfiram acts on NLRP3 upstream of GSDMD pore formation. Overall, our study provides a rationale for considering the treatment of inflammatory diseases characterized by aberrant NLRP3 activation with FDA-approved disulfiram or improved disulfiram derivatives.

Limitations of the study

We provide evidence that NLRP3 palmitoylation at Cys126 is required for its TGN localization and inflammasome activation, which was inhibited by disulfiram. However, we could not detect the NLRP3 modification on Cys126 mediated by disulfiram through mass spectrometry. This may be due to the limited quantity of NLRP3 protein, the polycationic nature or the length of the digested peptide of interest or other factors. We found that disulfiram inhibits the NLRP3 inflammasome, but not the NLRC4 inflammasome, in vivo. However, disulfiram could also affect other signaling pathways unrelated to NLRP3 activation in vivo. Thus, additional studies are needed to further determine the signaling events affected by disulfiram and, more importantly, whether our findings can be extended to the human system.

STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

RESOURCE AVAILABILITY

Lead contact

Further information and requests for reagents should be directed to and will be fulfilled by the Lead Contact, Gabriel Núñez (gabriel.nunez@umich.edu).

Materials availability

All unique reagents generated in this study are available from the lead contact with a completed Material Transfer Agreement.

Data and code availability

MS data generated in this study have been deposited to the ProteomeXchange Consortium via the PRIDE31 partner repository with the dataset identifier PXD054126 and are publicly available as of the date of publication.

This paper does not report original code.

Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Mice

C57BL/6 mice were purchased from Jackson Laboratories and maintained in our specific pathogen-free facility. Both male and female mice were used for this study. Age- and sex-matched 7- to 9-week-old mice after co-housing were used for in vivo studies, and 6- to 12-week-old mice were used for in vitro studies. Animal studies were approved by the University of Michigan Committee for the Care and Use of Laboratory Animals.

LPS treatment and Salmonella infection

For LPS-induced NLRP3 inflammation in vivo, mice were pre-treated with disulfiram (50 mg/kg) or vehicle sesame oil by intraperitoneal injection 24 and 4 h before intraperitoneal LPS challenge (20 mg/kg, from Escherichia coli 0111:B4; Sigma). IL-1β and TNF-α in serum and peritoneal lavage were measured by ELISA 12 h after LPS challenge.

For Salmonella infection, Mice were pre-treated with disulfiram (50 mg/kg) or vehicle by intraperitoneal injection 24 and 4 h before intraperitoneal infection of Salmonella enterica serovar Typhimurium strain 14028 (CFU 6×107). IL-1β and TNF-α in serum and peritoneal lavage were measured by ELISA 6 h post Salmonella infection.

MSU-induced peritonitis

Mice were pre-treated with disulfiram (50 mg/kg) or vehicle sesame oil by intraperitoneal injection 24 and 4 h before intraperitoneal MSU challenge (2 mg per mouse). IL-1β, IL-6 and the number of neutrophil in the peritoneal cavity were measured after MSU challenge.

Cell and bacterial culture

BMDMs were prepared by differentiating bone marrow cells from the tibia and femur bones of C57BL/6 for 7 days in 10% FBS IMDM (GIBCO) supplemented with 30% L929-cell supernatant, non-essential amino acids (NEAA), sodium pyruvate and antibiotics (penicillin/streptomycin). L929-cell and immortalized BMDM (iBMDM) were cultured in 10% FBS IMDM (GIBCO) supplemented with NEAA, sodium pyruvate and antibiotics (penicillin/streptomycin). Salmonella Typhimurium Strain 14028 was cultured in Luria-Bertani media at 37°C.

METHOD DETAILS

Inflammasome activation and cytokine measurement

Macrophages were plated in 12-well plates (0.5 × 106 cells per well, scaled down for 24-well plates). Culture medium was replaced with 0.5 mL serum-free IMDM per well after overnight culture. Cells were then primed with 200 ng/mL ultrapure LPS for 4 h, followed by stimulation with PBS (mock), ATP (5 mM, 40 min), nigericin (5 μM, 40 min), imiquimod (20 μg/mL, 1 h), gramicidin (0.5 μM, 1 h), LLOMe (2 μM, 1 h), Nano-Silica (200 μg/mL, 1 h), Samonella (MOI 30, 1 h), or poly (dA:dT) (1 μg/mL, 4 h). After stimulation, cell lysates were processed for immunoblotting analysis; culture supernatants were collected for cytokine measurement with ELISA kits (R&D system).

Identification of NLRP3-interacting proteins

Nlrp3−/− iBMDMs were reconstituted with empty lentiviral vector (pHIV-EGFP) or a vector expressing a TurboID-fused NLRP3. Proximity labeling with TurboID was performed following a published protocol.23 Biotinylated proteins enriched on streptavidin beads were processed through on-bead digestion and analyzed by liquid chromatography mass spectrometry according to the previously described methods.32

Immunoprecipitation

Cells were lysed in HEPES lysis buffer (20 mM HEPES, pH 7.4, 100 mM sodium chloride, 1% Triton X-100, EDTA-free Roche protease inhibitor cocktail). Cell lysates were clarified by centrifugation (15,000g) at 4°C for 10 min. Pre-cleared cell lysates were incubated with anti-FLAG antibody or control IgG at 4°C for 3 h. The proteins bound by antibody were pulled down by protein G beads and subjected to immunoblotting analysis.

Acyl-biotin exchange assay

Acy-biotin exchange assays were performed as previously described.33 Cells were lysed in lysate buffer (50 mM Tris-HCl pH 8, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail). Samples were treated with 20 mM N-ethylmaleimide at room temperature with rotation. After centrifugation, the supernatants were precipitated with a methanol-chloroform-water mixture and then resuspended and dissolved in lysate buffer containing 2% SDS. Samples were equally divided into two-halves and treated with 0.7 M hydroxylamine or a vehicle control, respectively. 1 mM biotin-HPDP was added to all samples and incubated with rotation at room temperature. The samples were then precipitated and dissolved in lysate buffer containing 2% SDS and biotin-HPDP (0.1 mM). After incubation with rotation at room temperature, the samples were precipitated and resuspended in lysate buffer containing 2% SDS. Once the samples were dissolved, lysate buffer was added to dilute SDS to 0.4%. Following centrifugation, a portion of the supernatants was kept as input. 40 μL of streptavidin agarose was added to the remaining supernatants and incubated for 1 h at 4°C with rotation. The agarose beads were washed three times with lysate buffer before being processed for SDS-PAGE and western blot analysis.

Immunofluorescence and pyroptosis analysis

Macrophages were plated on coverslips overnight, which had been coated with poly-L-lysine. Cells were treated with vehicle or disulfiram. After washing with PBS, cells were fixed with 4% paraformaldehyde, followed by permeabilization with 0.1% Triton X-100 and blocked with PBS buffer containing BSA. Cells were incubated with anti-FLAG and anti-TGN38, or anti-ASC and then incubated with Alexa-Fluor-conjugated secondary antibody. In some cases, nuclei were stained by DAPI. Images were captured by a Nikon A1 inverted confocal microscope system. For imaging of pyroptotic cells, macrophages were plated on 24-well plate overnight. Cells were primed with 200 ng/mL LPS for 4 h, then stimulated with nigericin in the presence or absence of disulfiram. Images were taken with Olympus DIC microscopy.

QUANTIFICATION AND STATISTICAL ANALYSIS

No statistical methods were used to predetermine sample size or to include or exclude samples. Data are expressed as mean ± SD. Statistical analysis was performed using unpaired two-tailed Student’s t test, Mann-Whitney test, or one-way ANOVA by GraphPad Prism. A p-value less than 0.05 was considered statistically significant.

Supplementary Material

1

ACKNOWLEDGMENTS

We thank Dr. M. Seaman for TGN38 antibody and Dr. H.M. Hoffman and J. Onyuru for Nlrp3fl(D301N)/+ Esr1-Cre and Nlrp3fl(D301N)/+ mouse leg bones. We thank Drs. Y. Lu and D. Neculai for assistance with experiments, Dr. J. Whitfield for ELISA assays, and Dr. V. Basrur for mass spectrometry. We acknowledge the Immune Monitoring Shared Resource, Proteomics Resource Facility, and Microscopy Core at the University of Michigan. This work was funded in part by a National Institutes of Health grant (R37AI063331 to G.N.) and was supported by funds to the University of Michigan core facilities. The illustration for the graphical abstract was created with assistance from BioRender.com.

Figure 1. Disulfiram inhibits caspase-1 activation induced by the activators of the NLRP3 inflammasome in mouse BMDMs

(A and B) LPS-primed BMDMs were treated with DMSO or disulfiram (DSF; the indicated doses, 20 min), followed by stimulation of ATP (5 mM, 40 min). Cell lysates were then analyzed by immunoblotting (A), and IL-1β release was analyzed by ELISA (B). Results are representative of three independent experiments. The bars represent mean ± SD. Results were analyzed by one-way ANOVA. ****p < 0.0001.

(C and D) LPS-primed BMDMs were treated with DMSO or DSF (30 μM, 20 min), followed by stimulation of PBS, nigericin (5 μM, 40 min), imiquimod (20 μg/mL, 1 h), gramicidin (0.5 μM, 1 h), L-leucyl-L-leucine methyl ester (LLOMe; 2 μM, 1 h), or nano-silica (200 μg/mL, 1 h). Cell lysates were then analyzed by immunoblotting (C), and IL-1β release was analyzed by ELISA (D). Results are representative of three independent experiments. The bars represent mean ± SD. Results were analyzed by unpaired two-tailed Student’s t test. ****p < 0.0001.

(E and F) LPS-primed BMDMs were treated with DMSO or DSF (30 μM, 20 min), followed by stimulation of PBS, Samonella (MOI 30, 1 h), poly(dA:dT) (1 μg/mL, 4 h), or nigericin (5 μM, 40 min). Cell lysates were then analyzed by immunoblotting (E), and IL-1β release was analyzed by ELISA (F). Results are representative of three independent experiments. The bars represent mean ± SD. Results were analyzed by unpaired two-tailed Student’s t test. ****p < 0.0001; ns, not significant.

See also Figures S1–S3.

Figure 2. Disulfiram inhibits NLRP3 activation in mouse peritoneal macrophages, human THP-1 cells, and Nlrp3D301N/+ BMDMs

(A and B) LPS-primed peritoneal macrophages were treated with DMSO or DSF (30 μM, 20 min), followed by stimulation with PBS, ATP (5 mM, 40 min), nigericin (5 μM, 40 min), or poly(dA:dT) (1 μg/mL, 4 h). Cell lysates were then analyzed by immunoblotting (A), and IL-1β release was analyzed by ELISA (B). ACC, acetyl-CoA carboxylase. Results are representative of three independent experiments. The bars represent mean ± SD. Results were analyzed by unpaired two-tailed Student’s t test. ****p < 0.0001. ns, not significant.

(C and D) LPS-primed THP-1 cells were treated with DMSO or DSF (30 μM, 20 min), followed by stimulation with PBS, nigericin (5 μM, 40 min), imiquimod (20 μg/mL, 1 h), or poly(dA:dT) (1 μg/mL, 4 h). Cell lysates were then analyzed by immunoblotting (A), and IL-1β release was analyzed by ELISA (B). ACC, acetyl-CoA carboxylase. Results are representative of three independent experiments. The bars represent mean ± SD. Results were analyzed by unpaired two-tailed Student’s t test. ***p < 0.001 and ****p < 0.0001.

(E) Tamoxifen-treated BMDMs from Nlrp3fl(D301N)/+ Esr1-Cre or Nlrp3fl(D301N)/+ mice were incubated with both LPS and DMSO/DSF. IL-1β release was analyzed by ELISA. Results are representative of three independent experiments. The bars represent mean ± SD. Results were analyzed by unpaired two-tailed Student’s t test. ****p < 0.0001.

(F and G) LPS-primed BMDMs were treated with PBS, NAC (15 mM, 70 min), GSH (15 mM, 70 min), or vitamin C (15 mM, 70 min) in the presence or absence of DSF (30 μM, 20 min), followed by stimulation of nigericin (5 μM, 40 min). Cell lysates were then analyzed by immunoblotting (G), and IL-1β release was analyzed by ELISA (H). Results are representative of three independent experiments. The bars represent mean ± SD. Results were analyzed by unpaired two-tailed Student’s t test. ****p < 0.0001; ns, not significant.

See also Figure S4.

Figure 3. Disulfiram inhibits ASC speck formation and pyroptosis induced by the activators of NLRP3 inflammasome

(A and B) Representative immunofluorescence images and quantification of endogenous ASC specks (arrowheads) in LPS-primed BMDMs treated with agonists as indicated in the presence of vehicle DMSO or DSF. Cells were stained with anti-ASC (red) and DAPI (blue). Scale bar, 15 μm. Data were analyzed from three combined experiments. The bars represent mean± SD. Results were analyzed by unpaired two-tailed Student’s t test. ***p < 0.001 and ****p < 0.0001. ns, not significant.

(C) Representative differential interference contrast images of LPS-primed BMDMs treated with nigericin in the presence or absence of DSF (2 μM, 10 or 50 μM). Arrowheads indicate cells with features of pyroptosis. Scale bar, 15 μm.

(D) Quantification of BMDMs that underwent pyroptosis in (C). Data were analyzed from three combined experiments. The bars represent mean ± SD. Results were analyzed by one-way ANOVA. ****p < 0.0001. ns, not significant; DSF, disulfiram.

(E) LDH levels in cell culture supernatants of LPS-primed macrophages treated with ATP (5 mM) were analyzed. The bars represent mean ± SD. Results were analyzed by unpaired two-tailed Student’s t test. **p < 0.01.

Figure 4. Palmitoylation at Cys126 promotes NLRP3 activation and is inhibited by disulfiram

(A) Nlrp3−/− immortalized BMDMs (iBMDMs) were reconstituted with FLAG-tagged NLRP3. After LPS priming, NLRP3 was immunoprecipitated, and samples were then analyzed by immunoblotting.

(B) zDHHC5 or control shRNA transduced iBMDMs were primed with LPS, followed by acyl-biotin exchange assay. NLRP3 palmitoylation and protein levels were determined by immunoblotting.

(C and D) zDHHC5 or control shRNA transfected iBMDMs were primed with LPS and treated with nigericin or Salmonella. Cell lysates were analyzed by immunoblotting (C), and IL-1β release was analyzed by ELISA (D). ACC, acetyl-CoA carboxylase.

(E and F) LPS-primed BMDMs were pre-treated with vehicle ethanol, or 2-BP (25 μM, 45 or 90 min) before stimulation with nigericin (5 μM, 40 min). Cell lysates were analyzed by immunoblotting (E), and IL-1β release was analyzed by ELISA (F).

(G) LPS-primed iBMDMs were treated with vehicle DMSO or DSF, followed by acyl-biotin exchange assay. NLRP3 palmitoylation and protein levels were determined by immunoblotting.

(H) Representative immunofluorescence images and quantification of the percentage of cells with NLRP3 perinuclear puncta. Nlrp3−/− iBMDMs reconstituted with NLRP3-FLAG were treated with vehicle (DMSO, left) or 30 μM DSF (right) and then simultaneously stained with both anti-FLAG (green, top) and anti-TGN38 (red, bottom). The arrowheads indicate the perinuclear puncta where NLRP3 and TGN38 co-localized. Scale bar, 10 μm. Data were analyzed from three combined experiments. The bars represent mean ± SD. Results were analyzed by unpaired two-tailed Student’s t test. *p < 0.05.

(I) Domain organization of NLRP3. Amino acid sequences in the polybasic region between the Pyrin and NACHT domains are shown. C126 is highlighted in red.

(J) Representative immunofluorescence images and quantification of the percentage of cells with NLRP3 perinuclear puncta. Nlrp3−/− iBMDMs reconstituted with wild-type (WT) NLRP3 (left), or C126S mutant (C126S; right) with FLAG tag were simultaneously stained with both anti-FLAG (green, top) and anti-TGN38 (red, bottom). Arrowheads indicate the perinuclear puncta where NLRP3 and TGN38 co-localized. Scale bar, 10 μm. Data were analyzed from three combined experiments. The bars represent mean ± SD. Results were analyzed by unpaired two-tailed Student’s t test. **p < 0.01.

(K and L) LPS-primed Nlrp3−/− iBMDMs reconstituted with NLRP3 WT or C126S mutant (C126S) were stimulated with PBS, ATP, or nigericin. Cell lysates were then analyzed by immunoblotting (K), and IL-1β release was analyzed by ELISA (L). Results are representative of three independent experiments. The bars represent mean ± SD. Results were analyzed by unpaired two-tailed Student’s t test. **p < 0.01 and ***p < 0.001.

(M and N) LPS-primed Nlrp3−/− iBMDMs reconstituted with NLRP3 WT or C126S mutant (C126S) were treated with vehicle DMSO or DSF, followed by acyl-biotin exchange assay. NLRP3 palmitoylation and protein levels were determined by immunoblotting. Results are representative of three independent experiments. The bars represent mean ± SD. Results were analyzed by one-way ANOVA. *p < 0.05. ns, not significant.

See also Figures S5 and S6.

Figure 5. Disulfiram inhibits NLRP3 inflammasome activation in vivo

(A–D) Mice were pre-treated with DSF (50 mg/kg) or vehicle sesame oil by intraperitoneal injection 24 and 4 h before intraperitoneal LPS challenge (20 mg/kg). IL-1β and TNF-α in serum and peritoneal lavage were measured by ELISA 12 h post-LPS challenge. Results were analyzed by unpaired two-tailed Student’s t test.

(E–H) Mice were pre-treated with DSF (50 mg/kg) or vehicle sesame oil by intraperitoneal injection 24 and 4 h before intraperitoneal Salmonella infection (6 × 107 colony-forming units (CFUs). IL-1β and TNF-α in serum and peritoneal lavage were measured by ELISA 6 h after Salmonella infection. Results were analyzed by the Mann-Whitney test. Results were from two experiments. The bars represent mean ± SD. ns, not significant; *p < 0.05 and ***p < 0.001.

See also Figures S7.

KEY RESOURCES TABLE REAGENT or RESOURCE	SOURCE	IDENTIFIER	
	
Antibodies	
	
Mouse monoclonal anti-Flag	GenScript	Cat# A00187; RRID:AB_1720813	
Mouse monoclonal anti-NLRP3(Cryo-2)	Adipogen	Cat# AG-20B-0014; RRID:AB_2490202	
Rabbit monoclonal anti-NEK7	Abcam	Cat# ab133514; RRID:AB_2877625	
Rabbit monoclonal anti-GSDMD	Abcam	Cat# ab209845; RRID:AB_2783550	
Mouse monoclonal IgG2b isotype control	Cell signaling	Cat# 53484; RRID:AB_2799435	
Rat polyclonal anti-ASC	Dr. Gabriel Núñez	N/A	
Rabbit polyclonal anti-Caspase-1	Dr. Gabriel Núñez	N/A	
THE™ beta Actin Antibody [HRP]	GenScript	Cat# A00730; RRID:AB_914100	
	
Bacterial and virus strains	
	
Salmonella Typhimurium Strain 14028	Dr. Denise Monack	N/A	
	
Chemicals, peptides, and recombinant proteins	
	
High-capacity streptavidin agarose resin	ThermoFisher	Cat# 20359	
Protein G beads	GenScript	Cat# L00209	
LPS (Escherichia coli 055:B5, Ultrapure)	InvivoGen	Cat# tlrl-pb5lps	
LPS (Escherichia coli O111:B4, for in vivo expt)	Sigma	Cat# L2630	
Poly (dA:dT)	InvivoGen	Cat# tlrl-patc	
ATP	Sigma	Cat# A2383	
Gramicidin	Sigma	Cat# G5002	
L-leucyl-L-leucine methyl ester (LLOMe)	Sigma	Cat# L7393	
Nigericin	EMD Millipore	Cat# 481990	
Imiquimod	InvivoGen	Cat# tlrl-imqs-1	
Nano-SiO2	InvivoGen	Cat# tlrl-sio-2	
N-ethylmaleimide	Sigma	Cat# E3876	
hydroxylamine	Sigma	Cat# 438227	
HPDP-Biotin	Cayman Chemical	Cat# 16459	
Lipofectamine LTX	ThermoFisher	Cat# 15338100	
Lipofectamine 2000	ThermoFisher	Cat# 11668019	
	
Critical commercial assays	
	
Mouse TNFα ELISA Kit	R&D Systems	Cat# DY410-05	
Mouse IL-1 β ELISA Kit	R&D System	Cat# DY401-05	
QuikChange II XL Site-Directed Mutagenesis Kit	Agilent	Cat# 20051	
Cell line Nucleofector kit V	Lonza	Cat# 90279050	
RNeasy Mini kit	Qiagen	Cat# 74104	
	
Deposited data	
	
MS data	This paper	PXD054126	
	
Experimental models: Cell lines	
	
Immortalized bone marrow-derived macrophages	Dr. Gabriel Núñez	N/A	
Nlrp3−/− iBMDM	Dr. Gabriel Núñez	N/A	
	
Experimental models: Organisms/strains	
	
Mouse: C57BL/6J	Jackson Laboratories	Cat# 000664	
	
Recombinant DNA	
	
pHIV-EGFP	Welm et al.30	pHIV-EGFP, Addgene plasmid # 21373	
pHIV-NLRP3-SFP	Dr. Gabriel Núñez	N/A	
pHIV-NLRP3-FLAG	This paper	N/A	
pHIV-NLRP3 C126S-FLAG	This paper	N/A	
pLKO.I-shRNA control	Dr. Gabriel Núñez	N/A	
pLKO.1-zDHHC5 shRNA	This paper	N/A	
pMD2.G	Trono Lab Packaging and Envelope Plasmids (unpublished)	pMD2.G, Addgene plasmid # 12259	
psPAX2	Trono Lab Packaging and Envelope Plasmids (unpublished)	psPAX2, Addgene plasmid # 12260	
	
Software and algorithms	
	
ImageJ	National Institutes of Health	https://imagej.nih.gov	
GraphPad Prism 7.0	GraphPad Software, Inc	https://www.graphpad.com	
	
Other	
	
Nitrocellulose membrane	Fisher Scientific	Cat# WP2HY00010	

Highlights

Disulfiram inhibits NLRP3 inflammasome activation

NLRP3 is palmitoylated at Cys126

NLRP3 palmitoylation at Cys126 is required for TGN localization and inflammasome activation

Disulfiram inhibits NLRP3 palmitoylation at Cys126

DECLARATION OF INTERESTS

The authors declare no competing interests.

SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2024.114609.
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