The zebrafish NLRP3 inflammasome has functional roles in ASC-dependent interleukin-1β maturation and gasdermin E–mediated pyroptosis

Abstract

The NLR family pyrin domain containing 3 (NLRP3) inflammasome is one of the best-characterized inflammasomes in humans and other mammals. However, knowledge about the NLRP3 inflammasome in nonmammalian species remains limited. Here, we report the molecular and functional identification of an NLRP3 homolog (DrNLRP3) in a zebrafish (Danio rerio) model. We found that DrNLRP3's overall structural architecture was shared with mammalian NLRP3s. It initiates a classical inflammasome assembly for zebrafish inflammatory caspase (DrCaspase-A/-B) activation and interleukin 1β (DrIL-1β) maturation in an apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)-dependent manner, in which DrNLRP3 organizes DrASC into a filament that recruits DrCaspase-A/-B by homotypic pyrin domain (PYD)–PYD interactions. DrCaspase-A/-B activation in the DrNLRP3 inflammasome occurred in two steps, with DrCaspase-A being activated first and DrCaspase-B second. DrNLRP3 also directly activated full-length DrCaspase-B and elicited cell pyroptosis in a gasdermin E (GSDME)-dependent but ASC-independent manner. These two events were tightly coordinated by DrNLRP3 to ensure efficient IL-1β secretion for the initiation of host innate immunity. By knocking down DrNLRP3 in zebrafish embryos and generating a DrASC-knockout (DrASC−/−) fish clone, we characterized the function of the DrNLRP3 inflammasome in anti-bacterial immunity in vivo. The results of our study disclosed the origin of the NLRP3 inflammasome in teleost fish, providing a cross-species understanding of the evolutionary history of inflammasomes. Our findings also indicate that the NLRP3 inflammasome may coordinate inflammatory cytokine processing and secretion through a GSDME-mediated pyroptotic pathway, uncovering a previously unrecognized regulatory function of NLRP3 in both inflammation and cell pyroptosis. The NLR family pyrin domain containing 3 (NLRP3) inflammasome is one of the best-characterized inflammasomes in humans and other mammals. However, knowledge about the NLRP3 inflammasome in nonmammalian species remains limited. Here, we report the molecular and functional identification of an NLRP3 homolog (DrNLRP3) in a zebrafish (Danio rerio) model. We found that DrNLRP3's overall structural architecture was shared with mammalian NLRP3s. It initiates a classical inflammasome assembly for zebrafish inflammatory caspase (DrCaspase-A/-B) activation and interleukin 1β (DrIL-1β) maturation in an apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)-dependent manner, in which DrNLRP3 organizes DrASC into a filament that recruits DrCaspase-A/-B by homotypic pyrin domain (PYD)–PYD interactions. DrCaspase-A/-B activation in the DrNLRP3 inflammasome occurred in two steps, with DrCaspase-A being activated first and DrCaspase-B second. DrNLRP3 also directly activated full-length DrCaspase-B and elicited cell pyroptosis in a gasdermin E (GSDME)-dependent but ASC-independent manner. These two events were tightly coordinated by DrNLRP3 to ensure efficient IL-1β secretion for the initiation of host innate immunity. By knocking down DrNLRP3 in zebrafish embryos and generating a DrASC-knockout (DrASC−/−) fish clone, we characterized the function of the DrNLRP3 inflammasome in anti-bacterial immunity in vivo. The results of our study disclosed the origin of the NLRP3 inflammasome in teleost fish, providing a cross-species understanding of the evolutionary history of inflammasomes. Our findings also indicate that the NLRP3 inflammasome may coordinate inflammatory cytokine processing and secretion through a GSDME-mediated pyroptotic pathway, uncovering a previously unrecognized regulatory function of NLRP3 in both inflammation and cell pyroptosis. Inflammasomes are cytosolic multiprotein complexes that assemble in response to exogenous microbial invasions and endogenous damage signals (1Franchi L. Muñoz-Planillo R. Núñez G. Sensing and reacting to microbes through the inflammasomes.Nat. Immunol. 2012; 13 (22430785): 325-33210.1038/ni.2231Crossref PubMed Scopus (781) Google Scholar, 2Winsor N. Krustev C. Bruce J. Philpott D.J. Girardin S.E. Canonical and noncanonical inflammasomes in intestinal epithelial cells.Cell Microbiol. 2019; 21 (31265745): e1307910.1111/cmi.13079Crossref PubMed Scopus (29) Google Scholar). 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The identification of NLRP3 inflammasome in lower vertebrates, particularly in primitive teleost fish, will contribute to cross-species understanding of NLRP3-mediated biology throughout vertebrate evolution. interleukin 1β apoptosis-associated speck-like protein containing a caspase-recruitment domain mutant of DrCaspase-B in which four asparagine acids (Asp130, Asp137, Asp308, and Asp314) were substituted by alanine D. rerio ASC D. rerio caspase D. rerio IL-1β D. rerio NLRP3 D. rerio GSDME flow cytometry domain associated with NACHT in fish and other vertebrates fluorescence recovery after photobleaching gasdermin D gasdermin E guide RNA hour post-fertilization H. sapiens NLRP3 lactate dehydrogenase lipopolysaccharide leucine-rich repeat muramyl dipeptide M. musculus NLRP3 morpholino oligonucleotide National Center for Biotechnology Information NLR family pyrin domain containing 3 nucleotide-binding domain and leucine-rich repeat-containing proteins open reading frame propidium iodide Protein Data Bank pyrin domain region of interest relative survival rate leucine-rich repeat antibody Dulbecco's modified Eagle's medium acetyl amido-4-trifluoromethylcoumarin guide RNA. Several previous studies identified two proinflammatory caspases, namely DrCaspase-A (Caspy) and DrCaspase-B (Caspy2), from zebrafish (12Masumoto J. Zhou W. Chen F.F. Su F. Kuwada J.Y. Hidaka E. Katsuyama T. Sagara J. Taniguchi S. Ngo-Hazelett P. Postlethwait J.H. Núñez G. Inohara N. Caspy, a zebrafish caspase, activated by ASC oligomerization is required for pharyngeal arch development.J. Biol. Chem. 2003; 278 (12464617): 4268-427610.1074/jbc.M203944200Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). These two caspases engage in proIL-1β cleavage with different specificities in an ASC-dependent manner (13Boudinot P. Reis M.I. do Vale A. Pereira P.J. Azevedo J.E. Dos Santos N.M. Caspase-1 and IL-1β processing in a teleost fish.PLoS ONE. 2012; 7 (23226286): e5045010.1371/journal.pone.0050450Crossref PubMed Scopus (62) Google Scholar). 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Commun. 2018; 9 (30076291): 305210.1038/s41467-018-04984-1Crossref PubMed Scopus (31) Google Scholar). In mammals, multiple gasdermin proteins, including GSDMD, GSDMA, GSDMB, GSDMC, and GSDME, were found to induce pyroptosis; among them, GSDMD is selectively cleaved by Caspase-4/5/11 to liberate an N-terminal effector fragment from the C-terminal inhibitory domain (16Shi J. Zhao Y. Wang K. Shi X. Wang Y. Huang H. Zhuang Y. Cai T. Wang F. Shao F. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death.Nature. 2015; 526 (26375003): 660-66510.1038/nature15514Crossref PubMed Scopus (2873) Google Scholar, 20Ding J. Wang K. Liu W. She Y. Sun Q. Shi J. Sun H. Wang D.C. Shao F. Pore-forming activity and structural autoinhibition of the gasdermin family.Nature. 2016; 535 (27281216): 111-11610.1038/nature18590Crossref PubMed Scopus (1271) Google Scholar, 21Kayagaki N. Stowe I.B. Lee B.L. O'Rourke K. Anderson K. Warming S. Cuellar T. Haley B. Roose-Girma M. Phung Q.T. 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With the accumulation of membrane pores, cells ultimately undergo membrane rupture and pyroptosis (23de Vasconcelos N.M. Van Opdenbosch N. Van Gorp H. Parthoens E. Lamkanfi M. Single-cell analysis of pyroptosis dynamics reveals conserved GSDMD-mediated subcellular events that precede plasma membrane rupture.Cell Death Differ. 2019; 26 (29666477): 146-16110.1038/s41418-018-0106-7Crossref PubMed Scopus (169) Google Scholar, 24Evavold C.L. Ruan J. Tan Y. Xia S. Wu H. Kagan J.C. The pore-forming protein gasdermin D regulates interleukin-1 secretion from living macrophages.Immunity. 2018; 48 (29195811): 35-44.e610.1016/j.immuni.2017.11.013Abstract Full Text Full Text PDF PubMed Scopus (563) Google Scholar). In zebrafish, however, only two gasdermin E homologs (DrGSDMEa/b, also known as DFNA5a/b) were identified to exert a pore-forming effect by their activated N-terminal domains; and DrGSDMEa that possesses a Caspase-3 cleavage motif induces pyroptosis after chemotherapy drugs are administered (25Busch-Nentwich E. Söllner C. Roehl H. Nicolson T. The deafness gene dfna5 is crucial for ugdh expression and HA production in the developing ear in zebrafish.Development. 2004; 131 (14736743): 943-95110.1242/dev.00961Crossref PubMed Scopus (57) Google Scholar, 26Rogers C. Fernandes-Alnemri T. Mayes L. Alnemri D. Cingolani G. Alnemri E.S. Cleavage of DFNA5 by caspase-3 during apoptosis mediates progression to secondary necrotic/pyroptotic cell death.Nat. Commun. 2017; 8 (28045099): 1412810.1038/ncomms14128Crossref PubMed Scopus (690) Google Scholar, 27Wang Y. Gao W. Shi X. Ding J. Liu W. He H. Wang K. Shao F. Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin.Nature. 2017; 547 (28459430): 99-10310.1038/nature22393Crossref PubMed Scopus (1189) Google Scholar). Thus, whether DrGSDMEa/b acts as a substrate cleaved by DrCaspase-A/B to induce pyroptosis as mammalian GSDME did remains to be elucidated. In this study, we identified an NLRP3 inflammasome from zebrafish and revealed its unique characteristics in DrCaspase-A/B activation, DrIL-1β maturation, DrGSDMEa/b cleavage, and pyroptotic cell death. Our results showed the molecular and functional characterizations of an early NLRP3 inflammasome in an ancient vertebrate, which provides a cross-species understanding of the evolutionary history of NLRP3 inflammasome from teleost fish to mammals. Through a systematic search in Ensemble by BLASTN, one NLRP3 (DrNLRP3) and two DrGSDMEa/b candidate genes were retrieved from the zebrafish genome database. The DrNLRP3 gene is located within a 10.0-kb genomic fragment on zebrafish chromosome 1 with eight exons and seven introns. The DrGSDMEa/b gene is distributed within a 15.2/13.8-kb genomic fragment on chromosome 19/16 with 9/9 exons and 8/8 introns. Genes adjacent to DrNLRP3 and DrGSDMEa/b loci share an overall conserved chromosome synteny with those of humans and other mammalian species (Fig. 1, A and G). The cDNA of DrNLRP3 consists of a 64-bp 5′-untranslated region (5′-UTR), a 3243-bp 3′-UTR, and a 3345-bp open reading frame (ORF) that encodes 1114 amino acids (GenBankTM accession no. MN088121). The cDNA of DrGSDMEa/b contains a 296/90-bp 5′-UTR, a 184/841-bp 3′-UTR, and a 1566/1419-bp ORF that encodes 521/472 amino acids (GenBankTM accession no. XM_005170077.3/NM_001001947.1). DrNLRP3 and DrGSDMEa/b proteins share similar domain and tertiary architectures with mammalian counterparts (Fig. 1, B and C). DrNLRP3 contains an N-terminal pyrin domain (PYD), a central nucleotide-binding domain (NACHT), a domain associated with NACHT in fish and other vertebrates (FISNA), a series of leucine-rich repeats (LRR), and a unique C-terminal B30.2 domain (Figs. 1B and Fig. S1A). The PYD domain comprises five antiparallel α-helices and one conservative truncated helix. The NACHT domain possesses Walker A (ATP/GTPase-specific P-loop) and Walker B (Mg2+-binding site) motifs. Eight LRRs with conserved LXXLXLXXN/CXL motifs form a well-defined “horseshoe”-shaped scaffold in which the B30.2 domain is located without any complete α-helix or β-sheet architecture (Fig. S1, A and D). DrGSDMEa/b consists of an N-terminal domain with the most conservative β-sheets (β1–β11) and α-helices (α1–α4), an aspartic acid–cleaved site in a long-loop structure, and a C-terminal domain with seven helices (α5–α11) (Fig. 1, E and F). The C terminus harbors conserved hydrophobic residues, such as Ile296/Leu294, Ala420/Ala269, and Leu431/Leu280 on α5, α8, and α9 helices, to form nonpolar surfaces that interact with the N-terminal domain for an auto-inhibitory regulation (Fig. S1B). Key pore-forming residues, such as Lys7/Lys7, Lys39/Lys39, Lys50/Lys50, Thr101/Thr99, Arg136/Arg134, and Ser143/Ser141 also exist in the N-terminal domain, which are conserved from fish to mammals. Phylogenetic analysis shows that DrNLRP3 and DrGSDMEa/b are clustered to their homologs with a high bootstrap probability (Fig. 1, H and I). In addition, DrNLRP3 and DrGSDMEa/b are extensively expressed in zebrafish adult tissues such as head, kidney, spleen, gill, and intestine and in embryos (Fig. 1, J and K). The functional role of DrNLRP3 was first examined by its initiation activity for DrCaspase-A/-B in HEK293T cells that naturally have minimal expression of inflammasome components. As expected, DrNLRP3 and DrASC coexpression significantly augmented (p < 0.01) the DrCaspase-A/-B activity in a DrNLRP3 dose-dependent manner (Fig. 2, A and B). However, the DrNLRP3 and DrCaspase-A/-B coexpression in the absence of DrASC dramatically promoted (p < 0.001) DrCaspase-B but not DrCaspase-A activation, with a maximum up-regulation of up to 180% (Fig. 2B). The DrNLRP3-ΔPYD and DrNLRP3-ΔNACHT (lacking both NACHT and FISNA) mutant proteins significantly impaired (p < 0.01 or p < 0.001) DrCaspase-A and DrCaspase-B activation. The DrNLRP3-ΔLRR mutants restrained DrCaspase-A but not DrCaspase-B activation. Conversely, DrNLRP3-ΔB30.2 did not influence either DrCaspase-A or DrCaspase-B (Fig. 2C). Western blot analysis showed the self-cleavage of 45/47-kDa pro-DrCaspase-A/-B into a 35-kDa hydrolytic product (p35) if pro-DrCaspase-A/-B was coexpressed with DrNLRP3 and DrASC (Fig. 2, D and E). By contrast, no p35 was detected without the coexpression of either DrNLRP3 or DrASC. The DrNLRP3 mutants that lacked PYD, NACHT-FISNA, and LRR domains failed to induce pro-DrCaspase-A/-B hydrolyzation (Fig. 2F). Coimmunoprecipitation (co-IP) assay revealed the protein–protein interaction among DrNLRP3, DrASC, and DrCaspase-A/-B (Fig. 2, G–I). The DrNLRP3-ΔPYD and DrASC-ΔPYD mutants lacked such an ability, whereas DrASC–ΔCARD maintained the activity (Fig. 2, H and I). Overall, DrNLRP3 could activate pro-DrCaspase-A/-B by self-hydrolyzation in a DrASC-dependent manner and initiate pro-DrCaspase-B activation in a DrASC-independent manner without undergoing self-hydrolyzation. The pro-DrCaspase-A/-B self-hydrolyzation depends on the association between DrNLRP3 and DrASC via the PYD–PYD homotypic interaction in which NACHT and LRR domains were included. A DrASC nucleation (i.e. speck formation) assay was performed in HEK293T cells to observe whether DrNLRP3 could organize an inflammasome. When DrNLRP3 or DrASC was expressed alone in cells, the fluorescence was a weak signal that diffused throughout the cell (Fig. 3A and Fig. S2, A and B). With the DrNLRP3 and DrASC coexpression, DrNLRP3 associates DrASC into a speck structure with a size of 1.84 ± 0.55 μm in diameter (Fig. 3, B and C). In some cases, it was observed that DrNLRP3 formed an outer ring around DrASC (Fig. S2C). This speck organization was also observed in zebrafish ZF4 cells (Fig. S2D). The DrNLRP3-ΔPYD, DrNLRP3-ΔNACHT, and DrNLRP3-ΔLRR mutants blocked the speck organization, whereas the DrNLRP3-ΔB30.2 did not have an influence (Fig. 3D and Fig. S2E). The expression of truncated DrASC-ΔPYD or DrASC-ΔCARD induced filament (ASCCARD or ASCPYD) formation instead of speck organization (Fig. 3E). DrASCPYD rather than the DrASCCARD filament could be colocalized with DrNLRP3 (Fig. 3, F and G). Functionally, only DrNLRP3 and DrASC coexpressed in cells could activate DrCaspase-A (Fig. 3H). This activity was competitively inhibited by introducing DrASC-ΔPYD but not DrASC-ΔCARD in a ratio-dependent manner (Fig. 3I). Thus, the DrNLRP3-DrASC inflammasome formation might be started by a linker DrASC, which was associated with the DrNLRP3 disk via the PYD–PYD interaction and recruited another DrASC via the CARD–CARD interaction to form a DrASC filament. The DrASC filament was composed of a DrASCCARD core and DrASCPYD cluster. The latter finally recruited DrCaspase-A/-B via the PYD–PYD association (Fig. S3, A and B). From the tertiary structures of ASCPYD and ASCCARD cores, six PYDs or four CARDs interacted with each other by PYD–PYD or CARD–CARD homotypic interaction to form a circular helix in one layer of the filament core (Fig. S3C). This phenomenon explained that the DrASCPYD filament was thicker than the DrASCCARD filament under a confocal microscope (Fig. 3, E–G). With the alignment of DrASC, MmASC, and HsASC, some surface electrostatic amino acids, including Glu13, Lys21, Arg38, Arg41, Asp48, Asp51, and Asp54 in ASCPYD and Arg125, Glu130, Asp134, Tyr146, Arg150, Arg160, and Asp191 in ASCCARD that perform important roles in mammalian PYD or CARD fibrillation, are also highly conserved in zebrafish (Fig. S3, C and D). This condition suggested that the mechanisms underlying the PYD–PYD and CARD–CARD homotypic interaction are conserved from fish to mammals. Immunofluorescence results revealed perfect DrCaspase-A/-B colocalization with the DrASC speck, suggesting DrNLRP3-DrASC inflammasome as a platform for DrCaspase-A/-B recruitment (Fig. 4, A and B). Nevertheless, DrCaspase-A and DrCaspase-B are almost independently localized in an inflammasome, and only one inflammasome can be assembled in one cell (Fig. 4C). The percentage of DrCaspase-A–associating cells (∼20%) was higher than that of DrCaspase-B–associating cells (∼7%). By introducing two chimera bPYD–CasA and aPYD–CasB caspases in which the PYD of DrCaspase-A and DrCaspase-B was replaced by each other, these two caspases were still separately colocalized with the DrASC speck, whereas the percentage of aPYD–CasB-associating cells (∼28%) exceeded that of bPYD–CasA-associating cells (∼6%). This finding indicated that the privilege of DrCaspase-A was deprived by DrCaspase-B when their PYD domains were exchanged (Fig. 4D). Given that DrASC PYD shared a higher similarity (82.92%) to DrCaspase-A PYD compared with that of DrCaspase-B (55.46%) (Fig. S1C), the priority of DrCaspase-A into DrNLRP3-DrASC inflammasome might be determined by the high degree of similarity, which provided strong hydrophobic (from Leu16, Leu21, Ile49, Val57, and Ile75) and surface charge (from Arg22, Lys23, Glu43, and Asp50) effects on the homotypic PYD–PYD interaction. The fluorescence recovery after photobleaching (FRAP) assay revealed that DrCaspase-A/-B displayed fluorescence recovery in foci within the inflammasome in ∼150 s after photobleaching (Fig. 4, E, F, and H). This outcome supported the dynamic recruitment of DrCaspase-A/-B into the inflammasome. By contrast, DrNLRP3 and DrASC did not show fluorescence recovery (Fig. 4, G and H). This observation indicated that DrNLRP3-DrASC inflammasome maintained a stable structure that remained unmoved once it is organized. Collectively, DrCaspase-A and DrCaspase-B were dynamically and sequentially recruited into the DrNLRP3-DrASC inflammasome, with preference for DrCaspase-A, followed by a replacement of DrCaspase-B after DrCaspase-A was released from the inflammasome. The above results showed that DrNLRP3 acted as an initiator that organized a DrASC-dependent DrNLRP3 inflammasome, leading to the self-cleavage and activation of DrCaspase-A/-B. This phenomenon might further contribute to proDrIL-1β maturation. For clarification, DrNLRP3, DrASC, DrCaspase-A/-B, and proDrIL-1β were coexpressed in HEK293T cells in different combinations, and proDrIL-1β maturation was determined through Western blot analysis. As expected with the DrNLRP3, DrASC, and DrCaspase-A/-B coexpressions, proDrIL-1β (31 kDa) was cleaved into an 18-kDa mature form, accompanied by the pro-DrCaspase-A/-B (45/47 kDa) activation through self-cleavage into a p35 product (Fig. 5A). However, proDrIL-1β was partially processed into a 20-kDa product (the first cleavage product at the Asp104 residue of proDrIL-1β) by the activated DrCaspase-A alone (Fig. 5B). This product coexisted with several other mid-forms (25–30 kDa) known as the cleavage products of some other proteases existing in cells, including neutrophil elastase, proteinase 3, cathepsins G/D, granzyme A, and matrix metalloproteinases. By contrast, the activated DrCaspase-B alone failed in the proDrIL-1β cleavage (Fig. 5C). In the absence of DrNLRP3 and/or DrASC, no any mature/pa