NLRP3 licenses NLRP11 for inflammasome activation in human macrophages

Intracellular sensing of stress and danger signals initiates inflammatory innate immune responses by triggering inflammasome assembly, caspase-1 activation and pyroptotic cell death as well as the release of interleukin 1β (IL-1β), IL-18 and danger signals. NLRP3 broadly senses infectious patterns and sterile danger signals, resulting in the tightly coordinated and regulated assembly of the NLRP3 inflammasome, but the precise mechanisms are incompletely understood. Here, we identified NLRP11 as an essential component of the NLRP3 inflammasome in human macrophages. NLRP11 interacted with NLRP3 and ASC, and deletion of NLRP11 specifically prevented NLRP3 inflammasome activation by preventing inflammasome assembly, NLRP3 and ASC polymerization, caspase-1 activation, pyroptosis and cytokine release but did not affect other inflammasomes. Restored expression of NLRP11, but not NLRP11 lacking the PYRIN domain (PYD), restored inflammasome activation. NLRP11 was also necessary for inflammasome responses driven by NLRP3 mutations that cause cryopyrin-associated periodic syndrome (CAPS). Because NLRP11 is not expressed in mice, our observations emphasize the specific complexity of inflammasome regulation in humans.

G ermline-encoded, cytosolic pattern recognition receptors sense infectious and sterile stress signals and play a key role in mounting an inflammatory response that eradicates infections and facilitates wound healing and homeostasis. A consequence of intracellular pattern recognition is the activation of caspase-1 within the inflammasome 1,2 , and NOD-like receptor (NLR) family pyrin domain containing 3 (NLRP3) is a prominent inflammasome sensor of microbial patterns, self-derived danger signals and environmental cues [3][4][5] . Excessive or mutation-driven NLRP3 responses cause a wide range of inflammatory diseases 6 , including CAPS, which is caused by gain-of-function mutations in NLRP3 (ref. 7 ). NLRP3 consists of an N-terminal PYD, a central NAIP [neuronal apoptosis inhibitor protein], C2TA [class 2 transcription activator, of the MHC], HET-E [heterokaryon incompatibility] and TP1 [telomerase-associated protein 1] (NACHT) domain and C-terminal leucine-rich regions (LRRs). The NACHT has ATPase activity and is bound to the LRRs and/or the PYD to maintain an inactive conformation 8,9 . Once this autoinhibition is released, NLRP3 oligomerizes, and its PYD nucleates polymerization of the adaptor protein ASC, which serves as an amplification mechanism and proceeds in a prion-like, self-perpetuating manner, establishing a temporal-spatial threshold control [10][11][12] . Polymerized ASC filaments eventually assemble into the characteristic single macromolecular aggregate (speck) 13,14 . ASC polymerization in turn nucleates caspase-1 polymerization by caspase recruitment domain (CARD)-CARD interactions, resulting in its induced, proximity-mediated activation 15 . Caspase-1 is ultimately responsible for the induction of pyroptosis through the cleavage of gasdermin D (GSDMD) and subsequent GSDMD pore formation, maturation and release of the proinflammatory cytokines IL-1β and IL-18 and the release of danger signals, including IL-1α, HMGB1 and polymerized ASC particles [16][17][18] . NLRP3 inflammasome activation proceeds in two steps. Priming includes the upregulation of inflammasome components, including NLRP3 and the substrate IL-1β, a metabolic shift from oxidative phosphorylation to glycolysis and the post-translational modifications of NLRP3, ASC and caspase-1 (refs. [3][4][5]. NLRP3 is activated by diverse stimuli 3 , and K + efflux has been proposed as the unifying mechanism for NLRP3 activation 19 . Protein oligomerization is a common mechanism for the activation of innate immune signaling and NLRP3 oligomerization, and particularly the downstream ASC polymerization, are key events in inflammasome activation 10,11 . Among the NLRP3 regulatory proteins, NEK7 promotes inflammasome activation by bridging two NLRP3 molecules, which is insufficient to induce NLRP3 oligomerization [20][21][22][23] . GBP5 enables NLRP3-ASC binding in response to soluble, but not crystalline, agonists 24 , implying that other crucial, yet-unknown cofactors are necessary for NLRP3 oligomerization, inflammasome assembly and activation. To date, the precise mechanism, especially in humans, remains unclear.

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complex only assembled after NLRP3 inflammasome activation, which required NLRP11 PYD . NLRP11 was also necessary for the release of IL-1β induced by the CAPS-associated NLRP3 mutant NLRP3 R260W , which placed NLRP11 at an essential step in human NLRP3 inflammasome assembly and activation. Our study therefore provides important insights into NLRP3 inflammasome regulation in human macrophages.

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ELISA (Extended Data Fig. 1b). IL-1β release was strongly impaired in both NLRP11 KD cells compared to two non-targeting shRNA control (Ctrl KD ) cells (Extended Data Fig. 1b). IL-6 is secreted independently of inflammasome activation and was not affected (Extended Data Fig. 1b). Reduced IL-1β release was not a result of impaired transcription of IL1B, as indicated by quantitative real-time polymerase chain reaction (PCR) (Extended Data Fig. 1c). Moreover, release of IL-1β, but not IL-6, was reduced in primary human macrophages with short interfering RNA (siRNA) silenced NLRP11 (NLRP11 siRNA ) compared to control siRNA (Ctrl siRNA ) ( Fig.  1b and Extended Data Fig. 1d), indicating NLRP11 contributed to the efficient activation of the NLRP3 inflammasome. Because NLRP11 knock down and silencing did not completely abolish NLRP11 expression, we used CRISPR-Cas9 to knock out NLRP11 in THP-1 cells. Sequencing of two independent clones (NLRP11 KO#1 NLRP11 KO#2 ) indicated a deletion of 4 bp and 172 bp adjacent to the start ATG (Extended Data Fig. 2a), resulting in a frame shift and the introduction of a premature stop codon after amino acid 13 or 17, respectively (Extended Data Fig. 2b). To prevent the expression of various splice forms predicted for NLRP11 (Extended Data Fig.  2c), including the ones using an alternative start site downstream of the PYD, we additionally used CRISPR-Cas9 to target the NACHT/ NAD in NLRP11 KO#1 and NLRP11 KO#2 cells. A 229-bp deletion caused a frame shift (Extended Data Fig. 2d) and premature stop (Extended Data Fig. 2e) in both cell lines leading to complete loss of NLRP11 expression (Fig. 1c), without impacting the expression of NLRP3, ASC or caspase-1 (Extended Data Fig. 2f). THP-1 cells with an shRNA-mediated knockdown of ASC (ASC KD ) and THP-1 cells with CRISPR-Cas9-generated deletion of NLRP3 (NLRP3 KO ), CASP1 (CASP1 KO ), CASP4 (CASP4 KO ) and Cas9 Ctrl cells were used as controls (Extended Data Fig. 2f,g) 13,25 . Activation of primed NLRP11 KO cells with soluble (nigericin, ATP) and crystalline (silica) NLRP3 activators, as well as K + efflux, resulted in loss of IL-1β (Fig.  1d,e) and IL-18 ( Fig. 1f) release compared to Cas9 Ctrl cells. IL-1β and IL-18 release was also lost upon noncanonical activation of the NLRP3 inflammasome following transfection of lipopolysaccharide

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NAtuRE ImmuNoLogY response to nigericin compared to Myc control (Ctrl Myc ) cells, without affecting the release of IL-6 ( Fig. 1k). Collectively, these data strongly indicated that NLRP11 was required for NLRP3-mediated IL-1β and IL-18 release in human macrophages.

NLRP11 is required for NLRP3-mediated caspase-1 activation.
Next, we directly assessed caspase-1 activity in Ctrl KD cells, which showed robust activation of caspase-1 in response to nigericin (Fig.  2a) and poly(dA:dT) (Fig. 2b), whereas NLRP11 KD cells showed markedly reduced caspase-1 activation in response to nigericin ( Fig. 2a and Extended Data Fig. 4), but not to poly(dA:dT) (Fig.  2b). Nigericin-mediated caspase-1 activation was also reduced in NLRP11 siRNA transfected human macrophages compared to Ctrl siRNA transfected cells (Fig. 2c), whereas nigericin-mediated caspase-1 activation was enhanced in NLRP11 Myc cells compared to Ctrl Myc cells (Fig. 2d). Accordingly, the release of the cleaved, p20 form of active caspase-1 was diminished, and the subsequent proteolytic cleavage of the caspase-1 substrate GSDMD was abolished in primed NLRP11 KO cells after nigericin treatment (Fig. 2e), but not after poly(dA:dT) transfection (Fig. 2f). Pyroptosis was also defective in NLRP11 KO cells comparable to NLRP3 KO cells (Fig. 2g). Detection of ASC and NLRP3 was strongly reduced in the culture SNs of primed and nigericin-activated NLRP11 KD cells compared to Ctrl KD cells but was maintained in NLRP11 KD THP-1 cell lysates ( Fig. 2h), suggesting ASC and NLRP3 were retained inside the cells and not released by pyroptosis. Overall, NLRP11 regulated NLRP3-dependent caspase-1 activation, GSDMD cleavage, pyroptosis and the release of inflammasome particles.

NLRP11 is a component of the NLRP3 inflammasome.
To determine whether NLRP11 was a part of the NLRP3 inflammasome, we stained untreated or primed and nigericin-activated cells for NLRP11 and found redistribution of diffuse NLRP11 into characteristic 'speck'-like aggregates, which colocalized with ASC and NLRP3 upon NLRP3 activation (Fig. 3a), suggesting all three proteins formed a complex. When transiently transfected in HEK293 cells, NLRP11, ASC and NLRP3 colocalized to the characteristic ASC aggregates within concentric layers (Fig. 3b). A cross-section view indicated NLRP11 localized between the ASC core and peripheral NLRP3 (Fig. 3b), similar to the spatial organization reported for NLRP3 and NLRC4 (ref. 28 ). To directly test whether NLRP11 interacted with the NLRP3 inflammasome, we transduced NLRP11-Flag into NLRP11 KO cells (NLRP11 Flag cells), to allow the specific detection and selection of different levels of NLRP11 expression (Extended Data Fig. 5a). In these cells, NLRP3 copurified NLRP11 Flag and ASC following priming and nigericin activation, whereas NLRP3 did not bind to NLRP11 Flag in primed cells (Fig. 3c). To test whether NLRP11 interacted with NLRP3, ASC or both, we co-expressed NLRP11 and ASC in HEK293 cells. NLRP11 colocalized with the ASC aggregates, which are induced spontaneously after expression in HEK293 cells, in the absence of NLRP3 (Fig. 3d), suggesting that ASC could be bridging NLRP11 to the NLRP3 inflammasome. To test whether NLRP11 recruited ASC independently of NLRP3, we immunoprecipitated ASC from primed and nigericin-activated NLRP3 KO cells expressing NLRP11 Flag (Extended Data Fig. 5b) and observed that ASC interacted with NLRP11 Flag even in the absence of NLRP3 (Fig. 3e). These data suggest that NLRP11 interacted with ASC independently of NLRP3 in response to nigericin.

ASC recruitment and polymerization requires the NLRP11 PYD .
Both ASC and NLRP11 contain a PYD, which is known to mediate homotypic interactions. Indeed, the PYD of ASC (ASC PYD ) was sufficient to copurify the NLRP11 PYD , at levels comparable to the established ASC PYD -NLRP3 PYD interaction (Fig. 4a). However, the NLRP11 PYD did not copurify the NLRP3 PYD (Fig. 4b).
To understand the role of the NLRP11 PYD , we transduced NLRP11 KO cells with NLRP11 lacking the PYD (NLRP11 ΔPYD-Flag ), selected for comparable expression to full-length NLRP11 Flag (Extended Data Fig. 5a) and immunoprecipitated ASC. Although ASC coimmunoprecipitated NLRP11 Flag , it did not copurify NLRP11 ΔPYD-Flag in primed and nigericin-activated cells (Fig. 4c), suggesting that the NLRP11 PYD was required for the NLRP11-ASC interaction. Primed and nigericin-activated NLRP11 ΔPYD-Flag cells also failed to secrete IL-1β and IL-18 compared to NLRP11 Flag cells, even at increased expression of NLRP11 ΔPYD-Flag (Fig. 4d), indicating that the NLRP11 PYD was required for NLRP3 inflammasome-mediated cytokine release. Primed and nigericin-activated NLRP11 ΔPYD-Flag cells also showed increased secretion of TNF compared to NLRP11 Flag cells, comparable to ASC KD cells (Fig. 4d) and reminiscent of the effect observed in NLRP11 KO cells (Fig. 1h), suggesting that the NLRP11 PYD was also important for this non-NLRP3 inflammasome-mediated effect on NF-κB 26,27 . ASC polymerization is believed to be nucleated by activated NLRP3 10,11 . However, because NLRP11 interacted with ASC and was necessary for NLRP3 inflammasome activation, we investigated whether NLRP11 contributed to the recruitment and polymerization of ASC. In HEK293 ASC-EGFP cells transfected with NLRP3 at levels that promoted only limited ASC polymerization, coexpression of NLRP11 greatly enhanced ASC polymerization (Fig. 4e). Comparable results were obtained by immunoblot assays following nonreversible crosslinking of cell lysates from above cells. Expression of NLRP3 alone promoted the formation of dimeric and oligomeric ASC, whereas coexpression of NLRP3 and NLRP11 synergistically induced strong ASC polymerization (Fig. 4f). Expression of NLRP11 alone did not efficiently nucleate ASC polymerization (Fig. 4f). Primed and nigericin-activated NLRP11 KO cells (Fig. 4g) or NLRP11 KD cells (Extended Data Fig. 5c) were completely defective in ASC polymerization, similar to NLRP3 KO cells, without affecting the expression of total ASC in the cells. Only NLRP11 Flag cells, but not NLRP11 ΔPYD-Flag cells, promoted nigericin-induced ASC polymerization (Fig. 4h). These results indicated that NLRP3 can nucleate ASC polymerization when overexpressed but that both NLRP3 and NLRP11 are required to induce ASC polymerization in THP-1 cells in a manner dependent on the NLRP11 PYD .

NLRP11 is necessary for the oligomerization of human NLRP3.
Next, we investigated whether NLRP11 directly contributed to NLRP3 oligomerization. Cotransfection of NLRP11 with ASC and

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NAtuRE ImmuNoLogY NLRP3 EGFP in HEK293 cells enhanced NLRP3 EGFP oligomerization (Fig. 5a). Furthermore, NLRP11 cotransfection with dispersed NLRP3 EGFP in HEK293 cells promoted NLRP3 oligomerization in a dose-dependent manner, even in the absence of ASC, excluding any feedback from polymerized ASC (Fig. 5b), indicating that NLRP11 was necessary and sufficient to promote NLRP3 oligomerization. Because expression of NLRP11 ΔPYD in NLRP11 KO cells resulted in defective release of IL-1β and IL-18 (Fig. 4d), suggesting that the NLRP11 PYD may also be required for NLRP3 oligomerization, we tested the ability and requirement of individual NLRP11 domains to promote NLRP3 oligomerization. Comparable expression of NLRP11, but not NLRP11 ΔPYD , resulted in NLRP3 oligomerization in HEK293 cells ( Fig. 5c and Extended Data Fig. 6a). The PYD, NACHT or LRR alone did not support NLRP3 oligomerization (Fig.  5c). NLRP11 lacking the NACHT domain (NLRP11 ΔNACHT ) or the LRR (NLRP11 ΔLRR ) was also defective in inducing NLRP3 oligomerization (Fig. 5c), suggesting that intact NLRP11 was required. To further interrogate this mechanism, we stably restored the expression of NLRP11, NLRP11 ΔPYD , NLRP11 ΔNACHT , NLRP11 ΔLRR , NLRP11 PYD , NLRP11 NACHT and NLRP11 LRR in NLRP11 KO cells and sorted cells for comparable expression (Extended Data Fig. 6b). Primed and nigericin-activated Cas9 Ctrl and NLRP11 KO cells expressing NLRP11, but not NLRP11 KO cells or NLRP11 KO cells expressing any of the other truncated NLRP11 proteins, induced NLRP3 oligomerization, as determined by microscopy and quantification of NLRP3 oligomers using NLRP3 KO cells as a specificity control (Fig. 5d,e). Biochemical analysis using blue native gel electrophoresis also demonstrated the nigericin-induced NLRP3 oligomerization in primed Cas9 Ctrl cells, which was further enhanced in NLRP11 Flag cells ( Fig.  5f) but reduced in NLRP11 KO cells (Fig. 5g) or NLRP11 ΔPYD-Flag cells (Fig. 5h). NLRP3 KO cells were used as a specificity control. This analysis also revealed the oligomerization of NLRP11 itself in primed and nigericin-activated NLRP11 Flag cells (Fig. 5f). Primed and nigericin-activated NLRP11 Flag cells, but not NLRP11 KO cells and NLRP11 KO cells expressing any truncated NLRP11, secreted IL-1β comparable to Cas9 Ctrl cells (Fig. 5i). Collectively, these results demonstrated that intact NLRP11 was necessary for the oligomerization of NLRP3. NLRP11 promotesNLRP3 inflammasome assembly. Next, we investigated whether NLRP11 interacted with NLRP3 using a proximity ligation assay (PLA). A specific PLA signal was detected in primed and nigericin-activated NLRP11 Flag cells, but not in primed cells (Fig. 6a). NLRP3 immunoprecipitation further corroborated the nigericin-dependent interaction of NLRP3 with endogenous NLRP11 in THP-1 cells (Fig. 6b). The reduced expression of NLRP3 and NLRP11 in TCL after prolonged activation (45 min) was likely the result of partially released NLRP3 inflammasome components (Fig. 6b). Accordingly, Flag immunoprecipitation from the SNs of primed and nigericin-activated, but not from primed, NLRP11 Flag cells copurified NLRP3 (Fig. 6c), indicating that NLRP11 and NLRP3 were released as a complex by pyroptosis. To determine whether the NLRP3-NLRP11 interaction occurred independently of ASC, we expressed NLRP11 Flag in ASC KD cells (Extended Data Fig. 5b) and immunoprecipitated NLRP3. NLRP3 coimmunoprecipitated NLRP11 in primed and nigericin-activated NLRP11 Flag cells, and this interaction was also observed in ASC KD cells expressing NLRP11 Flag (Fig. 6d), suggesting that NLRP11 interacted with NLRP3 independently of ASC. NLRP3 oligomerization is mediated by the NACHT domain 8 . In HEK293 cells, the NLRP11 NACHT bound directly to the NLRP3 NACHT , as demonstrated by coimmunoprecipitation of transiently transfected NLRP11 NACHT and NLRP3 NACHT domains (Fig. 6e), but the NLRP11 NACHT also bound to the NLRP12 NACHT and NOD1 NACHT domains (Fig. 6f). Binding was still observed under very stringent conditions in RIPA buffer (Extended Data Fig. 7a) and in buffers with up to 800 mM NaCl (Extended Data Fig. 7b), indicating that the NLRP11 NACHT bound with high affinity to other NACHT domains. To address whether isolated NACHT domains were more easily accessible for interactions in the absence of intramolecular interactions with the LRR and/or the PYD in the intact protein 9,29 , we tested the interaction of the NLRP11 NACHT with full-length NLRP3 and NLRC4 by transient transfection of HEK293 cells. The NLRP11 NACHT coimmunoprecipitated NLRP3 and NLRC4 (Fig. 6g), even though the NLRC4 inflammasome was not affected by NLRP11 (Fig. 1j), confirming previous reports of spontaneous interactions between NLR NACHT domains in HEK293 cells 30 . The NLRP11 NACHT did not interact with pyrin, which lacks a NACHT domain (Fig. 6g). These results suggested that although the NLRP11 NACHT can interact with the NLRP3 NACHT , additional events may facilitate and determine the specificity of these interactions in macrophages. NEK7 promotes NLRP3 oligomerization and ASC polymerization but cannot mediate NLRP3 activation on its own [20][21][22] . NEK7 binds to the LRR and NACHT domains of NLRP3 to bridge two adjacent NLRP3 molecules 23 . NEK7 interacted with NLRP3 in LPS-primed and LPS-primed and nigericin-activated Cas9 Ctrl and NLRP11 KO cells (Extended Data Fig. 7c), indicating that NLRP11 does not mediate the NEK7-NLRP3 interaction. We did not observe substantial NLRP3 localization to the mitochondria in untreated, primed and primed and nigericin-activated Cas9 Ctrl and NLRP11 KO cells (Extended Data Fig. 8a), but, as previously

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NAtuRE ImmuNoLogY described 31 , NLRP3-activating stimuli caused the disassembly of the trans-Golgi network (TGN), and NLRP3 localization to the dispersed TGN in Cas9 Ctrl cells and also in NLRP11 KO cells (Extended Data Fig. 8b), indicating that NLRP11 did not affect the intracellular localization of NLRP3. Because the LRR has a key role in assembling mouse NLRP3 oligomers 32 , we tested whether the NLRP11 LRR was involved in the NLRP3-NLRP11 interaction. Transient transfection of HEK293 cells with NLRP11 LRR and NLRP3, NLRC4 (which has a LRR) or pyrin (which lacks a LRR) demonstrated that the NLRP11 LRR coprecipitated NLRP3, but not NLRC4 or pyrin (Fig. 6h). NLRP3 coimmunoprecipitated NLRP11 in primed and nigericin-activated NLRP11 Flag cells, but not in NLRP11 ΔPYD-Flag cells (Fig. 6i). To test whether NLRP11 directly controlled the assembly of the NLRP3 inflammasome, we performed a PLA between NLRP3 Articles NAtuRE ImmuNoLogY and caspase-1. A positive PLA signal was detected in primed and nigericin-activated Cas9 Ctrl cells, but not in NLRP11 KO or ASC KD cells (Fig. 6j). However, expression of NLRP11 cannot compensate for the loss of NLRP3, as NLRP11 overexpression could not induce IL-1β release in primed and nigericin-activated NLRP3 KO cells (Fig.  6k). Collectively, these results showed that NLRP11 interacts with NLRP3 independently of ASC through its NACHT and LRRs, but an intact NLRP11, including the NLRP11 PYD , was nevertheless required for promoting nigericin-induced interactions between NLRP11 and NLRP3.
NLRP11 is necessary for IL-1β release in CAPS. qPCR analysis indicated that NLRP11 mRNA was induced in primed THP-1 cells, similar to the inducible expression of NLRP3 (Fig. 7a). In primed NLRP11 KO cells restored with low or high amounts of NLRP11 protein to mimic the inducible expression of NLRP11-primed cells (Fig.  7b), we observed an NLRP11 concentration-dependent increase in IL-1β secretion in response to nigericin treatment (Fig. 7c), indicating that the activation of the NLRP3 inflammasome was influenced by the amount of NLRP11. In patients with CAPS, myeloid-lineage restricted mutations in NLRP3 and somatic mosaicism 6 allow NLRP3 activation in the absence of an activation signal, and priming alone is sufficient to trigger NLRP3 inflammasome-mediated IL-1β release 33 . The majority of CAPS mutations are localized within the NACHT domain and prevent the autoinhibited conformation of NLRP3 (ref. 34 ). Accordingly, stable expression of the CAPS mutation NLRP3 R260W-EGFP in HEK293 cells resulted in spontaneous oligomerization of NLRP3 (Fig. 7d), whereas wild-type NLRP3 EGFP was distributed diffusely throughout the cells (Fig. 7d). Coexpression of NLRP11 in HEK293 cells further increased the aggregation of NLRP3 R260W-EGFP (Fig. 7d)

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NAtuRE ImmuNoLogY (Fig. 7e), but not in NLRP11 KO cells (Fig. 7e), whereas stable reexpression of NLRP11 Flag in NLRP11 KO cells restored NLRP3 R260W oligomerization in a dose-dependent manner (Fig. 7e). Priming further increased the number of NLRP3 R260W oligomers in Cas9 Ctrl cells, but not in NLRP11 KO cells (Fig. 7e). IL-1β was already released in primed NLRP11 Flag cells, but not in NLRP11 KO cells expressing NLRP3 R260W (Fig. 7f). These results indicated that NLRP11 was also necessary for facilitating oligomerization and promoting IL-1β release from mutant NLRP3 that causes CAPS. Therefore, NLRP11 is an essential component of the NLRP3 inflammasome (Extended Data Fig. 9).

Discussion
Here, we identified NLRP11 as an essential component of the NLRP3 inflammasome in human macrophages, which was required for caspase-1 activation, release of IL-1β and IL-18 and pyroptosis. NLRP11 bound to ASC through homotypic PYD-PYD interactions, which was required for nigericin-induced ASC polymerization. NLRP11 also independently interacted with NLRP3, which involved the NLRP11 LRR and the NLRP11 NACHT domains and facilitated NLRP3 oligomerization. The NLRP11 NACHT did not specifically interact with NLRP3 in HEK293 cells. This unspecific affinity of NACHT domains when expressed in HEK293 cells has been reported earlier 30 . In THP-1 cells, the interaction between NLRP11 and NLRP3 occurred after NLRP3 activation. Therefore, we speculate that in macrophages additional unknown signals may be required to confer specificity to NLRP11 NACHT domain interactions. Additional specificity is provided by the NLRP11 LRR , which interacted with NLRP3, but not with NLRC4. Taken together, it is very likely that the NLRP11 LRR and the NLRP11 NACHT domains both contributed to the specific interaction between NLRP3 and NLRP11, reminiscent of the interaction between NLRP3 and NEK7 (ref. 23 ). Other NACHT domain-mediated NLR hetero-oligomerizations have been described, including NLRC4-NLRP3 (refs. 28,35 ), NAIP-NLRC4 (refs. 36,37 ) and Nod2-NLRP1 (ref. 38 ). NLRC5 also interacts with the NLRP3 NACHT to regulate NLRP3 by an unknown mechanism 39 but has more recently been linked to major histocompatibility complex class I transactivation 40,41 . Even though NLRP11 and NLRP3 did not interact through their PYDs, the NLRP11 PYD was still crucial for complex formation, because deletion of the PYD prevented NLRP11 recruitment to the NLRP3 inflammasome, NLRP3 oligomerization and NLRP3 inflammasome responses, which required an intact NLRP11 protein. NLRP3 can nucleate ASC polymerization in vitro, and we observed this ability in HEK293 cells, but only if NLRP3 was overexpressed. Increasing the expression of NLRP3 during inflammasome priming contributes to, but is not sufficient for, inflammasome activation [3][4][5] . Low-level expression of NLRP3 did not nucleate ASC polymerization in HEK293 cells, and even priming-induced elevation of NLRP3 expression was insufficient in THP-1 cells in the absence of NLRP11. NLRP11 was required for NLRP3 inflammasome responses in a dose-dependent manner, but NLRP11 expression was not able to compensate for the loss of NLRP3, indicating that NLRP11 alone could not assemble an inflammasome under these conditions. This mode of activation is unique, because NLRP11 interacted with NLRP3 as well as ASC, and all three were required for NLRP3 inflammasome assembly in THP-1 cells. Other described mechanisms for inflammasome activation require bridging of the NLRP3-ASC interaction by GBP5 24 , or bridging NLRP3 molecules through NACHT-LRR interaction by NEK7 (ref. 23 ). NLRP11 uniquely combines these mechanisms. NLRP11 was required for the response to all tested soluble and crystalline NLRP3 triggers, supporting its essential role within the NLRP3 inflammasome.
NLRP11 is encoded in humans and absent from mice 42 , but whether this mechanism is unique to human macrophages will require additional studies. Nevertheless, several other examples exist for increased complexity of inflammasome regulation in humans, including the family of PYD-and CARD-only proteins 43 . In addition to its function in NLRP3 inflammasome activation, NLRP11 could potentially function as an inflammasome sensor. Arguably, this would require the ability of NLRP11 to nucleate ASC polymerization, and based on our ASC EGFP polymerization assays in HEK293 cells, some NLRP11-mediated ASC polymerization was possible, especially in cells with sufficiently high NLRP11 expression. However, expression of NLRP11 in THP-1 cells failed to polymerize ASC in the absence of activated NLRP3, suggesting that physiological amounts of NLRP3 require the cooperation between NLRP3 and NLRP11, even though macrophages are the cells with the highest expression of NLRP3 (ref. 44 ).
Little is known about NLRP11, and there are conflicting reports on its role in type I interferon (IFN) or NF-κB signaling 26,27,45 . NLRP11 causes degradation of TRAF6 to inhibit TLR-mediated NF-κB activation 27 , and we observed slightly elevated TNF release in NLRP11 KO cells. However, NF-κB-dependent IL-6 release and IL1B transcription were not impacted. NLRP11 also binds to DDX3X and inhibits IFN-β and reduces caspase-1 activity in HEK293T cells 46 . siRNA-mediated silencing of NLRP11 in THP-1 cells slightly elevates Sendai virus-induced IFN-β production and does not affect IL-1β release 45 , but Sendai virus already completely prevents NLRP3 inflammasome assembly 47 . Several other NLRs, including NLRP2, NLRP3, NLRP6, NLRP7, NLRP12 and NLRC5, have been linked to inflammasomes, as well as transcriptional responses through regulating NF-κB, mitogen-activated protein kinase and IFN signaling 48 . Overall, our identification of NLRP11 as an essential adaptor or scaffold for NLRP3 inflammasome assembly and activation provides important insights into the still incompletely understood NLRP3 inflammasome response in humans. NLRP3 is uniquely positioned as a central sensor for infections and cellular stress and has been implicated in a wide range of inflammatory diseases ranging from crystal arthropathies to hereditary autoinflammatory disorders 49 . NLRP11 may provide an important checkpoint control for NLRP3 inflammasome assembly. Intriguingly, NLRP11 is also necessary for NLRP3 inflammasome responses initiated by CAPS-linked NLRP3 mutations, which may have important clinical implications.

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Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/ s41590-022-01220-3.
qPCR. Total RNA was isolated from cells using the E.Z.N.A. total RNA isolation Kit (Omega Bio-tek), incubated with DNAse I and reverse transcribed (Verso cDNA Synthesis Kit, Thermo Scientific). Multiplexed gene expression analysis was performed on an ABI 7300 Real-Time PCR Machine (Applied Biosystems) and Quantstudio 3 (Thermo Scientific) and displayed as relative expression compared to ACTB, using FAM-labeled exon-spanning primers for IL1B (Hs01555410_m1), NLRP11 (Hs00935472_m1), and NLRP3 (Hs00918082_m1) in combination with VIC-labeled primers for ACTB (Hs99999903_m1) (Invitrogen).
ELISA. Cells were seeded into six-well plates (10 6 cells per well) and treated as indicated, and cleared culture SNs were analyzed for IL-1β (Invitrogen), IL-18 (R&D Systems), IL-6 (BD Biosciences) or TNF (Invitrogen) secretion by ELISA according to the manufacturer's instructions.
LDH cytotoxicity assay. LDH activity was determined using the LDH Cytotoxicity Detection Kit (Takara Bio) in freshly collected culture SNs. Cytotoxicity was defined as a percentage of released LDH compared to total LDH activity upon cell lysis with 1% Triton X-100. ASC polymerization assay (crosslinking of TCLs). Cells were rinsed with ice-cold PBS and lysed in 20 mM HEPES, pH 7.4, 100 mM NaCl, 1% NP-40, 1 mM sodium orthovanadate, supplemented with protease inhibitors, followed by shearing with a 27-gauge needle. Insoluble pellets were resuspended in PBS supplemented with 2 mM disuccinimidyl suberate (Pierce) and incubated under rotation at room temperature for 30 min. Samples were centrifuged at 2,348×g for 10 min at 4 °C, and crosslinked pellets and cleared cell lysates were resuspended in Laemmli sample buffer and analyzed by immunoblot for ASC.

ASC polymerization and NLRP3 oligomerization by immunofluorescence.
HEK293 cells were stably transfected with ASC-EGFP or transiently transfected for NLRP3-EGFP and low-expressing clones were selected by limited dilution to prevent spontaneous aggregation and were grown on poly-lysine-coated coverslips. THP-1 cells were differentiated using PMA (20 nM, 16 h) on coverslips, washed in PBS and rested for 48 h before treatment and stained with mouse monoclonal anti-NLRP3 and secondary anti-mouse Alexa Fluor 488-conjugated antibodies. Cells were processed as described above and ASC and NLRP3 oligomerization was quantified using Fiji and normalized to cell numbers 60 .
PLA. PLA (Duolink PLA, Millipore-Sigma) was performed according to the manufacturer's instructions. Briefly, THP-1 cells were differentiated using PMA (20 nM, 16 h) on coverslips, washed in PBS and rested for 48 h. Following treatment, cells were washed with PBS, fixed with 3.7% paraformaldehyde for 10 min at room temperature, permeabilized with 0.2% Triton X-100 for 10 min at room temperature and washed with PBS. All incubations were performed in a humidified chamber at 37 °C. Cells were blocked with Duolink Blocking Solution for 1 h, followed by incubation with primary antibodies in Duolink Antibody Diluent for 2 h, incubated with PLUS and MINUS PLA probes, washed (Buffer A) at room temperature, incubated with Ligase for 30 min, washed (Buffer A), incubated with polymerase for 100 min, washed (Buffer B) and mounted on slides using a Duolink In Situ Mounting Medium with DAPI. Cells were analyzed by confocal microscopy (Zeiss, LSM 780).
Statistics and reproducibility. All representative results were independently repeated at least three times with similar results, and n indicates the number of biological replicates. Graphs were prepared in Prism 9 (GraphPad) and data are presented as mean values ± s.d. A standard two-tailed unpaired t-test was used for pairwise statistical analysis of all data. Values of P < 0.05 were considered significant (and marked by an asterisk), and P values are listed in the figure legends.
Reporting Summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
All data sets are provided as source data, and additional information is available from the corresponding authors upon reasonable request. Source data of intact immunoblots are included for Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7 Fig. 7. Source data of graphs are included for Fig. 1, Fig. 2, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Extended Data Fig. 1 and Extended Data Fig. 3. Source data are provided with this paper. cells; quantified initial ASC and NLRP3 aggregates in HEK293 cells; contributed to the FLICA assay; performed some of the microscopy of NLRP11 in HEK293 and THP-1 cells; performed the preliminary PYD coimmunoprecipitation experiments and ASC crosslinking in NLRP11 KD and transiently transfected HEK293 cells; contributed to some of the native gels; contributed to the PLA; performed cytokine analysis in NLRP11 KD cells, NLRP11 siRNA transfected cells, NLRP11-expressing NLRP3 KO /NLRP11 Flag cells, NLRP11 KO /NLRP3 R260W cells and some of the NLRP11(lo) and NLRP11(hi) restored cells and in NLRP11 Myc cells; and performed the LDH assays and endogenous interaction of NLRP3 and NLRP11. These contributions are reflected as the only contributor to Figs. 1a,b,d,h,k, 2g,h, 3a,b,d, 4a,e-g, 5a,b, 6b,e,k and 7a-d,f, Extended Data Fig. 1a,b, Extended Data Fig. 3 and Extended Data Fig. 5c and shared contributor to Fig. 1j (panel  1), Fig. 2a-d, Fig. 5f,g and Fig. 6a,j. S.D. performed most of the cytokine analysis in NLRP11 KO , CASP4 KO , CASP1 KO and NLRP3 KO cells; performed the caspase-1 and GSDMD cleavage analysis, inflammasome complex purification assays in NLRP11 KO , NLRP11 Flag , NLRP11 ΔPYD-Flag , NLRP3 KO , ASC KD , NLRP3 KO /NLRP11 Flag , ASC KD /NLRP11 Flag THP-1 cells, the interaction analysis of NLRP3 and NEK7 and ASC crosslinking in NLRP11 Flag and NLRP11 ΔPYD-Flag cells; and contributed or performed the blue native gels, the cytokine analysis in NLRP11 ΔPYD-Flag cells. These contributions are reflected as the only contributor to Figs. 1e, 1f,g, 2e, 3c,e, 4c,d,h, 5h and 6d,i, Extended Data Fig. 2g and Extended Data Fig. 7c and shared contributor to (Figs. 1j (panels 2 and 3) and 5f,g). S.T. generated the THP-1 cell lines restored with varying NLRP11 Flag and NLRP11 ΔPYD-Flag expression all the NLRP11 single domain and single domain deletion restored cells, the NLRP3 KO / NLRP11 Flag and ASC KD /NLRP11 Flag cells, regenerated the NLRP3 R260W cells, sorted cells for comparable expression, performed the expression verification of all THP-1 cell lines except NLRP11 Myc cells and CASP4 KO cells, performed the NLRP3 EGFP oligomerization analysis with all NLRP11 domains in HEK293 cells and THP-1 cells, performed the NLRP3 R260W oligomerization in THP-1 cells, performed the cytokine analysis in NLRP11 domain-expressing cells, performed the NLRP11 complex analysis in the culture SN and performed the microscopy for mitochondria and TGN. These contributions are reflected as the only contributor to Figs. 1c, 5c-e,i, 6c, 7e, Extended Data Fig. 2f, Extended Data Fig. 5a,b, Extended Data Fig. 6a,b and Extended Data Fig. 8a,b. J.C. performed most of the binding studies in HEK293 cells with PYD and NACHT domains, determined specificity and performed all the LRR binding assays. These contributions are reflected as the only contributor to Figs. 4b, 6f,g,h and Extended Data Fig. 7a,b. H.N. performed some of the cloning and supported the generation of lentiviruses and stable cells. E.J. contributed to the caspase-1 activation assays. These contributions are reflected as the only contributor to Fig. 2f. H.K. performed the qPCR analysis. These contributions are reflected as the only contributor to Fig. 1i and Extended Data Fig. 1c. L.H.C. established and characterized the CASP1 KO and CASP4 KO cells, supported the generation of NLRP11 KO cells and contributed to the PLAs. These contributions are reflected as shared contributor to Fig. 6a,j. R.A.R. contributed to the FLICA experiments. These contributions are reflected as shared contributor to Fig. 2a- Corresponding author(s): Christian Stehlik Last updated by author(s): Apr 7, 2022 Reporting Summary Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Research policies, see our Editorial Policies and the Editorial Policy Checklist.

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