Characterization of an NLRP1 from Zebrafish Reveals a Unique Sequential Activation Mechanism Underlying Inflammatory in This information is current as Ancient Vertebrates of September 28, 2021. Jiang-yuan Li, Ke Gao, Tong Shao, Dong-dong Fan, Chong-bin Hu, Cen-cen Sun, Wei-ren Dong, Ai-fu Lin, Li-xin Xiang and Jian-zhong Shao J Immunol published online 27 August 2018 Downloaded from http://www.jimmunol.org/content/early/2018/08/26/jimmun ol.1800498 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2018/08/26/jimmunol.180049 Material 8.DCSupplemental

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 27, 2018, doi:10.4049/jimmunol.1800498 The Journal of Immunology

Characterization of an NLRP1 Inflammasome from Zebrafish Reveals a Unique Sequential Activation Mechanism Underlying Inflammatory Caspases in Ancient Vertebrates

Jiang-yuan Li,* Ke Gao,* Tong Shao,* Dong-dong Fan,* Chong-bin Hu,* Cen-cen Sun,* Wei-ren Dong,* Ai-fu Lin,* Li-xin Xiang,* and Jian-zhong Shao*,†

NLRP1 inflammasome is one of the best-characterized inflammasomes in humans and other mammals. However, the existence of this inflammasome in nonmammalian species remains poorly understood. In this study, we report the molecular and functional identification of an NLRP1 homolog, Danio rerio NLRP1 (DrNLRP1) from a zebrafish (D. rerio) model. This DrNLRP1 possesses similar structural architecture to mammalian NLRP1s. It can trigger the formation of a classical inflammasome for the activation

of zebrafish inflammatory caspases (D. rerio [DrCaspase]–A and DrCaspase-B) and maturation of D. rerio IL-1b in a Downloaded from D. rerio ASC (DrASC)–dependent manner. In this process, DrNLRP1 promotes the aggregation of DrASC into a filament with DrASCCARD core and DrASCPYD cluster. The assembly of DrNLRP1 inflammasome depends on the CARD–CARD homotypic interaction between DrNLRP1 and DrASCCARD core, and PYD–PYD interaction between DrCaspase-A/B and DrASCPYD cluster. The FIIND domain in DrNLRP1 is necessary for inflammasome assembly. To understand the mechanism of how the two DrCaspases are coordinated in DrNLRP1 inflammasome, we propose a two-step sequential activation model. In this model, the

recruitment and activation of DrCaspase-A/B in the inflammasome is shown in an alternate manner, with a preference for http://www.jimmunol.org/ DrCaspase-A followed by a subsequent selection for DrCaspase-B. By using morpholino oligonucleotide–based knockdown assays, the DrNLRP1 inflammasome was verified to play important functional roles in antibacterial innate immunity in vivo. These observations demonstrate that the NLRP1 inflammasome originated as early as in teleost fish. This finding not only gives insights into the evolutionary history of inflammasomes but also provides a favorable animal model for the study of NLRP1 inflammasome- mediated immunology and diseases. The Journal of Immunology, 2018, 201: 000–000.

nflammasomes are cytoplasmic molecular platforms that and AIM2, and ASC-independent types, such as mammalian trigger the activation of inflammatory caspases (such as NLRP1b and NLRC4 (5–8). by guest on September 28, 2021 I caspase-1/4/5/11) and processing of proinflammatory Among the numerous inflammasomes that have been identified cytokines (such as IL-1b and IL-18) (1, 2). Members of the in humans and other mammalian species, the NLRP1 inflam- NOD-like receptor (NLR) family, including NLRP1, NLRP3, masome is the first and most extensively studied because of its NLRP6, NLRP7, and NLRC4, and the -associated critical roles in innate immunity and pathogenesis of various speck-like containing caspase activation and recruit- diseases(9,10).NLRP1,alsoknownasNALP1,DEFCAP,and ment domain (ASC) adaptor protein are core components of the CARD7, was initially identified from human (Homo sapiens) inflammasomes that link microbial and endogenous signals to macrophages (11). The human NLRP1 (HsNLRP1) is structur- effector caspase-1 (3, 4). After being activated in mammalian ally characterized by the presence of an N-terminal cells, ASC is used to form a single, compact speck structure in (PYD), a NAIP, CIIA, HET-E, and TP1 (NACHT) nucleotide- the cytoplasm, which is important for the oligomerization and binding domain, a leucine-rich repeat (LRR) domain, a function autohydrolyzation of caspase-1 in certain inflammasomes. to find domain (FIIND), and a C-terminal caspase recruitment According to the requirement of ASC, inflammasomes are domain (CARD) (12–14). Differing slightly from HsNLRP1, the classifiedintoASC-dependent,suchasmammalianNLRP3 murine NLRP1s lack the N-terminal PYD but retain the other

*College of Life Sciences, Key Laboratory for Cell and Engineering of Zhejiang YuHangTang Road, Hangzhou 310058, China. E-mail addresses: shaojz@zju. Province, Zhejiang University, Hangzhou 310058, People’s Republic of China; and edu.cn (J.-z.S.) and [email protected] (L.-x.X.) †Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for The online version of this article contains supplemental material. Marine Science and Technology, Qingdao 266071, People’s Republic of China Abbreviations used in this article: Ac-WEHD-AFC, acetyl-Trp-Glu-His-Asp-AFC; ORCIDs: 0000-0003-3020-6264 (T.S.); 0000-0001-9987-2842 (W.-r.D.). Ac-YVAD-AFC, acetyl-Tyr-Val-Ala-Asp-amido-4-trifluoromethylcoumarin; ASC, Received for publication April 4, 2018. Accepted for publication July 26, 2018. apoptosis-associated speck-like protein containing caspase activation and recruitment domain; CARD, caspase recruitment domain; Co-IP, coimmunoprecipitation; DPP9, This work was supported by grants from the National Natural Science Foundation of dipeptidyl dipeptidase 9; DrASC, D. rerio ASC; DrCaspase, Danio rerio caspase; China (31630083, 31372554, 31472298, 31572641, 31272691), Stem Cell and Trans- DrIL-1b, zebrafish IL-1b; DrNLRP1, D. rerio NLRP1; FIIND, function to find lational Research, the National Key Research and Development Program of China domain; hpf, hour postfertilization; HsNLRP1, human NLRP1; ID, identifier; (2016YFA0101001), the Open Fund of the Laboratory for Marine Biology and Bio- LRR, leucine-rich repeat; MDP, muramyl dipeptide; ML, maximum likelihood; technology, Qingdao National Laboratory for Marine Science and Technology, MmNLRP1, Mus musculus NLRP1b; MO, morpholino oligonucleotide; NACHT, Qingdao, China (OF2017NO02), and the Zhejiang Major Special Program of Breed- NAIP, CIIA, HET-E, and TP1; NCBI, National Center for Biotechnology Informa- ing (2016C02055-4). tion; NLR, NOD-like receptor; PAMP, pathogen-associated molecular pattern; PDB, The sequences presented in this article have been submitted to GenBank (http://www. ; PYD, pyrin domain; qRT-PCR, quantitative real-time PCR; ROS, ncbi.nlm.nih.gov/genbank/) under accession number MH118554. reactive oxygen species; RSR, relative survival rate.

Address correspondence and reprint requests to Prof. Jian-zhong Shao and Ó Assoc. Prof. Li-xin Xiang, College of Life Sciences, Zhejiang University, 866 Copyright 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800498 2 IDENTIFICATION OF AN NLRP1 INFLAMMASOME FROM ZEBRAFISH domains, including the C-terminal CARD (15). The N-terminal Male and female zebrafish with body lengths of 3–4 cm and weights of PYD of HsNLRP1 was reported to be an autoinhibitory domain 0.5–1.0 g that exhibited healthy appearance and activity were used for the for the regulation of HsNLRP1 inflammasome activation (16, 17). study. Zebrafish embryos were collected at different stages of embryonic development according to previously established protocols (30). The ex- In general, the human and murine NLRP1s directly recruit caspase-1 periments were conducted in accordance with legal regulations and ethical by homotypic CARD–CARD interaction, allowing mammalian approval. NLRP1s to activate the inflammatory caspases and the downstream Bacterial strain IL-1b maturation in an ASC-independent manner (18, 19). Although their occurrence and existence in humans and other An Edwardsiella tarda (TL5m) strain isolated from Trionyx sinensis was mammalian species are numerously investigated, those of kindly provided by Prof. J. Shen (Zhejiang Institute of Freshwater Fish- eries, Huzhou, China). The E. tarda strain was inoculated from 15% (v/v) inflammasomes in ancient vertebrates, such as teleost fish, remain glycerol stock cultures stored at 280˚C, and it was grown in tryptic soy poorly understood. Several previous studies have shown that there are broth (0.85% tryptone, 0.15% Soytone, 0.25% NaCl, 0.125% K2HPO4, and two homologs of proinflammatory caspase, namely Danio rerio 0.125% glucose, pH 7.5). The liquid cultures were cultured for 8 h in a Caspase (DrCaspase)–A (Caspy) and DrCaspase-B (Caspy2) in 30˚C shaker to reach the exponential phase (OD600 = 0.6) (31). CFU assay of the bacteria was performed by plating dilutions of the culture on tryptic zebrafish (20, 21). The pro–IL-1b in fish (as well as in soy agar. amphibian and bird) lack a conserved caspase-1 recognition site, implying the existence of a unique mechanism for IL-1b maturation Molecular cloning in lower vertebrates (22, 23). In fact, both DrCaspase-A and The Genome and Expressed Sequence Tags databases maintained by the DrCaspase-B participate in the cleavage of zebrafish pro–IL-1b National Center for Biotechnology Information (NCBI), the University of

(pro–DrIL-1b) but with different specificities. The DrCaspase-A first California Santa Cruz, and Ensembl were used to predict NLRP1 homolog Downloaded from cleaves the pro–DrIL-1b at the D104 residue; then, the pro–DrIL-1b in zebrafish as previously described (32). Total RNA was isolated from ∼ zebrafish embryos and tissues by using an RNAiso Plus kit (Takara Bio). was converted into a partially processed form of 20 kDa. There- The cDNAs of DrNLRP1 were amplified by RT-PCR according to the after, the 20 kDa mediate-formed zebrafish IL-1b (DrIL-1b)was homologous sequences predicted before. Full-length encoding sequence of further cleaved by DrCaspase-B at the D122 and transformed into a DrCaspase-A, DrCaspase-B, DrASC and D. rerio IL-1b (DrIL-1b) were fully processed mature form with a molecular mass of ∼18 kDa (24). generated on the basis of the published sequences in the NCBI genome database (accession no. NM_131505.2, NM_152884.2, NM_131495.2, However, whether the activation of DrCaspase-A and DrCaspase-B AY340959.1). The primers used in cloning are listed in Supplemental http://www.jimmunol.org/ depends on an upstream inflammasome is still unclear. According Table I. PCR products were purified and inserted into the pGEM-T to several studies on zebrafish, the effector caspases, such as EASY vector (Promega) as previously described (33). DrCaspase-A, are recruited by D. rerio ASC (DrASC) through the Bioinformatics analysis PYD domain. This finding suggests that DrASC can only be asso- ciated with NLRs by the CARD domain left over from the two do- The Map Viewer in the NCBI was used for retrieving the genome assemblies and locations (34). By comparing DrNLRP1 cDNAs with genome se- mains (25). Among the NLR family members, NLRP1 is the only quences, gene organizations (intron/exon boundaries) were elucidated and member that contains a C-terminal CARD domain (26). Thus, drawn by using GeneMapper 2.5. Multiple alignment of DrNLRP1 was whether an NLRP1 homolog and an NLRP1 inflammasome exist in analyzed by using the Clustal X program (version 2.0). Phylogenetic trees zebrafish is an interesting topic of research. Moreover, the maturation were constructed by using MEGA 5.0 with the neighbor-joining or max- by guest on September 28, 2021 of DrIL-1b relies on the close cooperation between DrCaspase-A and imum likelihood (ML) method (35). The ML tree was also constructed using IQ-TREE web server (http://iqtree.cibiv.univie.ac.at) with default DrCaspase-B, thus posting the question of how these two caspases settings (36). The potential functional motifs were predicted using the were coordinated in such an NLRP1 inflammasome. This coordina- Pfam 31.0 and Conserved Domains Database of NCBI. The domain tion is a unique feature distinct from that of mammalian inflamma- structures of DrNLRP1 were analyzed using SWISS-MODEL, and the somes, in which usually only one caspase (caspase-1) is included. potential DrNLRP1 protein structure was predicted using I-TASSER (37). The tertiary structural figures were reviewed and colored in PyMOL In the current study, we identified an NLRP1 homolog, D. rerio software. The evolutionary correlation was assessed by coevolutional co- NLRP1 (DrNLRP1) and a DrNLRP1-associated inflammasome efficient, which was performed by Pearson correlation analysis in SPSS from zebrafish (27, 28). DrNLRP1 was characterized by a number (version 22) software (38). of conserved structural architectures and functional roles in Plasmid constructions DrASC nucleation, inflammasome formation, proinflammatory caspases activation, and DrIL-1b maturation, which were typi- The open reading frame of DrNLRP1 was inserted into pCMV (Stratagene) cally seen in mammalian NLRP1 counterparts. We also found that to construct eukaryotic expression vectors with Flag- and HA-tags. The encoding sequences of DrNLRP1 with deletion of NACHT, FIIND, and the DrNLRP1 inflammasome plays an important role in the innate CARD domains were constructed into pCMV-Tag2B and were named defense against bacterial infection. Importantly, we proposed a as pCMV-DrNLRP1-DNACHT, pCMV-DrNLRP1-DFIIND, and pCMV- sequential activation model to explain how a pair of proin- DrNLRP1-DCARD. In addition, the DrASC, DrCaspase-A, DrCaspase-B, flammatory caspases (DrCaspase-A and DrCaspase-B) is orga- and DrIL-1b precursor (pro–DrIL-1b) and their mutants were also con- structed into pCMV or pcDNA3.1 (Invitrogen) vectors with Flag/Myc/ nized and activated in DrNLRP1 inflammasome instead of one HA-tags at the N/C termini of the recombinant proteins. The primers (caspase-1) in mammals. In this model, the recruitment and acti- used for construct generation are shown in Supplemental Table I. The vation of the two DrCaspases in DrNLRP1 inflammasome may be quick mutation site-directed mutagenesis kit (Beyotime) was used to in an alternate manner, with a preference for DrCaspase-A, fol- construct a quinary mutant of DrCaspase-A (named as DrCaspaseA-5DA), 106 282 293 294 295 lowed by a subsequent selection of DrCaspase-B. To our knowl- in which five asparagic acids (D ,D ,D ,D , and D ) were substituted by alanines. Plasmids for transfection and microinjection were edge, this study is the first to identify an inflammasome from prepared free of endotoxin by using the Endo-Free Plasmid Mini Kit II teleost fish, providing a cross-species understanding of the evo- (Omega Bio-Tek). lutionary history of inflammasome immunology. Quantitative real-time PCR for expression analysis Materials and Methods The transcripts of DrNLRP1 and its coordinator (DrASC, DrCaspase-A, Experimental fish and embryo DrCaspase-B) in zebrafish tissues and embryos were analyzed via quantitative real-time PCR (qRT-PCR) on a Mastercycler ep realplex in- Wild-type AB zebrafish (D. rerio) were bred and maintained in circulating strument (Eppendorf). In brief, all PCR experiments were performed in a water at 28˚C under standard conditions as previously described (29). The total volume of 10 ml by using a SYBR Premix Ex Taq kit (Takara Bio). fish were held in the laboratory for at least 2 wk before the experiments. The reaction mixtures were incubated for 2 min at 95˚C, followed by The Journal of Immunology 3

40 cycles of 15 s at 95˚C, 15 s at 60˚C, and 20 s at 72˚C. The relative (100 ng/ml) (46). Forty-eight hours later, cells were fixed with 4% expression levels were calculated using the 22ΔCt and 22ΔΔCt method with paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100, b-actin for normalization. Each PCR trial was run in triplicate parallel and blocked with 2% BSA at 37˚C for 1 h. The cells were then incubated reactions and repeated three times. The related primers are listed in with primary Abs (rabbit anti-Myc along with mouse anti-Flag) at 4˚C Supplemental Table I. The primer efficiency was checked. overnight. After washing with PBS, the cells were incubated with sec- ondary FITC-conjugated anti-rabbit Abs (Santa Cruz Biotechnology) and Preparation of polyclonal Ab Alexa Fluor 594–conjugated anti-mouse (Life Technologies) in accor- dance with the manufacturer’s instructions. The cells were also incu- Ab against DrIL-1b was produced by an antigenic epitope-based protocol bated with 0.1% DAPI (Invitrogen) for the sake of nucleus staining. as previously described (39). Briefly, the epitope sequences of DrIL-1b Images were captured under a two-photon laser-scanning confocal were predicted through ABCpred, BepiPred, and IEDB online software. microscope (LSM710; Carl Zeiss, Jena, Germany) at 3200 and 3630 The hydrophilic and Ag indices were evaluated by using DNAStar. The magnification (47). DrNLRP1-dependent DrASC nucleation was quan- predicted epitope peptide of DrIL-1b (DRKDTERIINFELC) was chemi- tified by calculating the DrASC speck-forming rate [(number of cells cally synthesized and coupled to keyhole limpet hemocyanin (KLH) at a with specks/number of all the cells) 3 100%]. More than 100 cells with ratio of 10:10 mg (carrier/peptide) by InvivoGen. Subsequently, 6-wk-old DrASC specks from immunofluorescence images were counted in each New Zealand White rabbits with a weight of 1.5–2.0 kg were immunized experimental group. with the synthetic peptides (0.5 mg/kg) in CFA or IFA four times (40). Antiserum was collected after the last immunization when Ab titer reached Electroporation for ZF4 cells transfection above 1:10,000 as determined by microplate-based ELISA. The specificity of the Ab was further validated through Western blot analysis. Zebrafish ZF4 cells were cultured at 28˚C in DMEM-F12 cell culture medium (HyClone) with 110 mg/ml sodium pyruvate (Corning) and 10% Constitution of DrNLRP1 inflammasome in HEK293T cells FBS (Bovogen Biologicals). After being digested by 0.25% trypsin 5 (Thermo Fisher Scientific), the cell numbers were counted by using a HEK293T cells were seeded into six-well plates at 5 3 10 per well in Downloaded from hemocytometer. Approximately 2 3 106 cells were suspended in 200 mlof DMEM culture medium (HyClone) with 10% FBS (Bovogen Biologicals) at DMEM-F12 in a 0.4-cm electroporation cuvette, and 20 mg of plasmid 37˚C in 5% CO . After 24 h, cells were transfected with plasmids expressing 2 DNA was added (48). After electroporation (square wave pulses, 270 V, pro–DrIL-1b (800 ng), DrCaspase-A (200 ng), DrCaspase-B (200 ng), 25 ms, MicroPulser electroporator; Bio-Rad), the cells were suspended DrASC (200 ng), and DrNLRP1 (400 ng) by using polyethylenimine (PEI) into 1 ml of DMEM-F12 medium and transferred onto two wells of a (Invitrogen) (41). Twenty-four to forty-eight hours later, cell lysates were 24-well plate containing coverslips. At 30 h posttransfection, ZF4 cells used for Western blot or caspase fluorogenic assay. were fixed with 2% paraformaldehyde and analyzed by immunofluores- Caspase assay with fluorogenic substrates cence staining as described above. http://www.jimmunol.org/ HEK293T cells (one well in a six-well plate) or zebrafish embryos Intracellular bacterial infection/stimulation model (∼20 embryos) were harvested and lysed with 100 ml of Caspase Cell Lysis For examination of DrCaspases activation in vivo, a bacterial infection/ Buffer (Enzo Life Sciences). A total of 100 mg of protein lysate was added stimulation model was developed by using zebrafish embryos. For this, to the Caspase Assay Buffer (Enzo Life Sciences) containing 100 mM E. tarda, an intracellular virulent pathogen for various aquatic animals, of acetyl-Tyr-Val-Ala-Asp-amido-4-trifluoromethylcoumarin (Ac-YVAD- was cultured and reached the exponential phase (OD = 0.6) with a AFC) (specific to DrCaspase-A in zebrafish) or acetyl-Trp-Glu-His-Asp- 600 concentration of 109 CFU/ml. The bacteria solution was centrifuged at AFC (Ac-WEHD-AFC) (specific to DrCaspase-B in zebrafish) (Alexis, 1700 3 g for 3 min, and E. tarda cells were harvested in the precipitate San Diego, CA) as described (20, 24, 25, 42, 43). After incubation at 37˚C and resuspend by PBS (pH 7.4). Six and forty-eight hours post- for 2 h, the cleavage of caspase-type-specific substrate emitted a fluores- 8 fertilization (hpf), zebrafish embryos were exposed to 1 3 10 CFU/ml by guest on September 28, 2021 cent signal that was measured with excitation at 400 nm and emission at E. tarda in a 10-cm dish (49). After immersion in the bacterial suspen- 505 nm on a Synergy H1 Hybrid Reader (BioTek Instruments). The acti- sion for 40 min to 4 h, the embryos were collected and detected the vation proportion of caspases was calculated by [(experimental group 2 activation of DrCaspases by Ac-YVAD/WEHD-AFC and the maturation control group)/control group] 3 100%. The control group included lysates of DrIL-1b by the rabbit anti–DrIL-1b polyclonal Ab. DNA from from cells that received no transfection. E. tarda was extracted with a bacterial DNA preparation kit (Omega Western blot analysis Bio-Tek). DNA (200 pg/embryo), LPS (Escherichia coli O55:B5, 2 ng/ embryo; Sigma-Aldrich), and muramyl dipeptide (MDP) (2 ng/embryo; HEK293T cells or zebrafish embryos were treated with cell lysis buffer for InvivoGen) were microinjected into 6-hpf embryos (50). After 40 min, Western blot and IP (Beyotime) containing protease inhibitor mixture embryos were collected, and the activation of DrCaspases and the (Roche) (44). The proteins were separated by 12% SDS-PAGE and then maturation of DrIL-1b were detected. transferred onto PVDF transfer membranes (Millipore Sigma). The blots were blocked with 5% nonfat dry milk (BioBasic), following by incu- Visualization of DrNLRP1 inflammasome in vivo bating with mouse anti-Flag/Myc/HA mAbs (Abcam). After adding HRP-conjugated goat anti-rabbit/mouse IgG Ab (Abcam), the objective In vivo examination of DrNLRP1 inflammasome in response to bacterial proteins were visualized with ECL reagents (GE Healthcare) by a digital infection was conducted in zebrafish embryos. For this procedure, gel image analysis system (Tanon). The grayscale quantization of the pCMV-Tag2B-DrNLRP1 and pCMV-Myc-DrASC were comicroinjected protein stripe was performed by ImageJ software. into the one-cell stage embryos at a concentration of 100 pg/embryo. Then, the 48-hpf zebrafish larvae were infected by E. tarda (1 3 108 Coimmunoprecipitation assay CFU/ml) for 4 h, collected, and sliced into 6-mm thick frozen sections by a freezing microtome (CM1950; Leica) (44). For visualization of Coimmunoprecipitation (Co-IP) was performed to detect the interaction among DrNLRP1 inflammasome, immunofluorescent staining was performed on DrNLRP1, DrASC, DrCasapse-A, and DrCaspase-B. For this, HEK293T the sections as described above. cells were plated in 10-cm dishes (Corning) and were cotransfected of 6 mg recombinant eukaryotic plasmids or empty vector as negative control. At 48 h Morpholino oligonucleotide and capped mRNA posttransfection, cells were lysed with precooling cell lysis buffer as described above. Lysates were incubated with mouse or rabbit Abs (1:200 dilution) at The morpholino oligonucleotides (MOs) against DrNLRP1 mRNA 4˚C overnight. The next day, the mixture of cell lysates and Ab was incubated (NLRP1-MO: 59-TGAGGTCAGTGGGTTTGATTGGACA-39)and with 50 ml protein A–agarose beads (Roche) for 4 h. The beads were washed DrCaspase-A mRNA (CaspaseA-MO: 59-CCATGTTTAGCTCAGGGCG- three times with lysis buffer, and the obtained samples were analyzed by CTG-39) and the standard control MO (59-CTCTTACCTCAGTTACAAT- Western blot assay (45). Expression of the transfected plasmids was also TTATA-39) were designed and synthesized by Gene Tools (Philomath, analyzed in the whole cell lysates as an input control. OR). The MOs were dissolved with nuclease-free H2O to 1 mM as stock solutions (51). For MO-resistant mRNA synthesis, DrNLRP1, DrCaspase- Immunofluorescence analysis of DrNLRP1-dependent A, or DrCaspaseA-5DA full-length cDNA sequence was constructed into DrASC nucleation the pcDNA3.1 vector by using the primers as shown in Supplemental Table I. The capped mRNA was synthesized using the mMESSAGE kit A density of 1 3 105 HEK293T cells per well was seeded on coverslips (Ambion), purified with Mini Quick Spin RNA columns (Roche), and then in a 24-well plate. After 24 h, cells were transfected with plasmids solubilized in DEPC water. The one-cell stage embryo was microinjected expressing DrNLRP1 (400 ng/ml), DrASC (100 ng/ml), and DrCaspases with NLRP1-MO (1.5–4.5 ng) or CaspaseA-MO (3.0 ng) for gene 4 IDENTIFICATION OF AN NLRP1 INFLAMMASOME FROM ZEBRAFISH knockdown and MO-resistant mRNA (200 pg) for the sake of rescue. The any residue) and the Mg2+-binding site (Walker B motif; hhhhD/E, phenotype of zebrafish embryos were visualized via an inverted micros- where h is a hydrophobic residue). Moreover, the His552 residue of copy (Zeiss Axiovert 40 CFL; Carl Zeiss). DrNLRP1 and the adjacent motif (FxHxxxQEF), which contribute to Functional evaluation of DrNLRP1 inflammasome in innate ATP or ADP binding and protein oligomerization, were also con- antibacterial immunity served with mammals (Supplemental Fig. 1). The LRR domain in DrNLRP1 contains a cap helix followed by six LRR, with each repeat The functional role of DrNLRP1 inflammasome in innate immunity was bearing a structural unit containing a b-strand and an a-helix with a evaluated through its antibacterial activity in zebrafish embryo model. For this evaluation, the DrNLRP1 or DrCaspase-A knockdown, rescue, and conserved amino acid sequence motif (LxxLxLxxN/CxL). The LRR control embryos were challenged with E. tarda (1 3 108 CFU/ml) at 6 or 48 form a well-defined “horseshoe”-shaped structural scaffold (Fig. 3C), hpf. Mortality in each group was monitored during the 12 h period at one which is believed to be for ligand recognition. In DrNLRP1 FIIND interval. The relative survival rate (RSR) was calculated using the following domain, there are Phe1109 and Ser1110 corresponding to the autolytic formula: RSR (%) = (survival rate of the infected group/survival rate of the 1212 1213 3 cleavage sites (Phe and Ser )inHsNLRP1 for its activation. mock PBS-administered control group) 100%. Infection experiments were 1186 performed in triplicate with 100 embryos per group. However, the residue His in HsNLRP1 that plays an important role during the posttranslational modification event cannot be found in Statistical analysis DrNLRP1 (Supplemental Fig. 1). The CARD domain of DrNLRP1 The data in the study were presented as the mean 6 SD of each group. comprises a helical bundle with six antiparallel a-helices (H1 to H6) Statistical significance between experimental and control groups was (Fig. 3D). Same as the mammalian counterparts, there are two con- assessed by two-tailed Student t test and was considered at *p , 0.05, servative and charged residues in DrNLRP1 CARD domain , , **p 0.01, and ***p 0.001. The sample number for each group of (Supplemental Fig. 1). The negatively charged Asp1290 is on the Downloaded from zebrafish exceeded 20–100 embryos. More than 100 cells with immu- slightly concave surface formed by the H2 and H3 helices, whereas nofluorescence speck were counted for quantifying DrASC nucleation. 1318 All experiments were replicated at least three times. the positively charged Arg is on the convex surface formed by the H1 and H4 helices (Fig. 3D), which may contribute to the highly Results specific homotypic interaction of CARD domains through mutual recognition between concave and convex surfaces. Identification of DrNLRP1 gene http://www.jimmunol.org/ With HsNLRP1 NACHT domain or Mus musculus NLRP1b Phylogenetic analysis of the components of DrNLRP1 (MmNLRP1) full-length sequences as queries, a homologous NLRP1 inflammasome complex gene (DrNLRP1) was retrieved from zebrafish genome and Other potential components of DrNLRP1 inflammasome complex, Expressed Sequence Tags databases by using Genscan and including DrASC, DrCaspase-A, DrCaspase-B, and DrIL-1b,were BLAST programs. Moreover, by searching DrASC CARD se- cloned from zebrafish cDNA libraries in this study. The structure of quence in zebrafish database, DrNLRP1 was also the first one in DrASC, which is composed of conservative PYD and CARD do- the result list (Fig. 1A). DrNLRP1 gene comprised 13 exons and mains, is similar to those in mammals. However, DrCaspase-A and 12 introns and was located on 2 within a 19.2 kb DrCaspase-B contained PYD domains in their N-terminal regions, genomic fragment (Fig. 1B). Genes adjacent to HsNLRP1 locus which was distinct from the mammalian counterpart (caspase-1) by guest on September 28, 2021 (i.e., SDHC, PFN1, HES6, UBA52, and PER2) at chromosome 17 that contains the conventional CARD domain. The structure of were found to be clustered around DrNLRP1 gene, although DrIL-1b is overall similar to mammals, but two inflammatory several gene loci, such as UBA52 and PER2, were in a reverse cleavage sites (104D-X and 122D-X) in DrIL-1b were observed order (Fig. 1C). These observations indicated an overall coinci- compared with only one site in mammalian IL-1b (Fig. 4A). As a dent chromosomal synteny of NLRP1 gene on human and result, shown in Fig. 4B, both DrCaspase-A and DrCaspase-B be- zebrafish genomes. In order to further analyze the phylogenic long to the branch of inflammatory caspases in the phylogenetic relationship of DrNLRP1, the ML tree was constructed with tree, whereas these two caspases cannot be completely clustered bootstrap analysis (1000 replicates) and SH-aLRT branch test. The together with mammalian caspase-1 or caspase-4/5/11. Thus, DrNLRP1 was found to be grouped with mammalian NLRP1 DrCaspase-A and DrCaspase-B belong to the inflammatory caspase counterparts with high bootstrap support (Fig. 2), verifying that family and are the paralogs of caspase-1/4/5/11 instead of the DrNLRP1 is with regard to vertebrate NLRP1. The orthologs. Furthermore, the phylogenetic trees of caspase-1 and cloned DrNLRP1 cDNA consists of 5278 bp comprising a 183 bp IL-1b possessed the same topological structure (Supplemental Fig. 59-untranslated region, a 1027 bp 39-untranslated region, and a 2A, 2B). By counting the evolutionary distance and performing the 4068 bp open reading frame that encodes 1355 aa (Supplemental Pearson correlation analysis, the coevolutional coefficient between Fig. 1, GenBank accession no. MH118554). According to the caspase-1 and IL-1b was calculated as 0.925 (p , 0.01; a value above results, the identification of DrNLRP1 gene is reliable, and exceeding 0.8 denotes a distinct correlation relationship), meaning this DrNLRP1 was subjected to further characterization. that the two genes are relatively correlated in the evolutionary process (Supplemental Fig. 2C). Moreover, the coevolutional co- Structural characterization of DrNLRP1 protein efficient of ASC and NLRP1 in vertebrates is 0.901 (p , 0.01) DrNLRP1 protein was predicted to have 1355 aa with a putative (Supplemental Fig. 2D), and the two conservative and charged molecular mass of 154.1 kDa. It possesses an NACHT (246–463 aa), residues in DrNLRP1 CARD domain are also found in DrASC an LRR (752–913 aa), a FIIND (1003–1233 aa), and a CARD (1269– CARD domain (Fig. 3C), indicating that those two genes are cor- 1350 aa) domain, which is similar to the domain architecture of related through CARD domains in evolutionary history. All results mammalian NLRP1s (Fig. 1A). By SWISS-MODEL program, the indicate that the components of DrNLRP1 inflammasome are individual domain (NACHT, LRR, and CARD) and the combined conserved and coevolutional during vertebrate evolution. domain (NACHT plus LRR domains) of DrNLRP1 apparently exhibit overall conserved tertiary structures along with those of HsNLRP1 Involvement of DrNLRP1 in DrCaspase-A and and MmNLRP1 (Fig. 3A, 3B). The DrNLRP1 NACHT domain DrCaspase-B activation contains hallmark motifs of mammalian NLRP1s, such as the ATP/ To evaluate whether a functional DrNLRP1 inflammasome can exist in GTPase-specific P-loop (Walker A motif; GxxGxGKS/T, where x is cells, the involvement of DrNLRP1 in DrCaspase-A and DrCaspase-B The Journal of Immunology 5

FIGURE 1. Molecular characterization of DrNLRP1 gene. (A) Schematic of the search process in the molecular cloning of DrNLRP1 gene. Dashed boxes represent the domains whose sequences were used as queries for the zebrafish database search. (B) Exon/intron organization of DrNLRP1 gene. Exons and introns are indicated by black boxes and lines, respectively. (C) Gene synteny and chromosomal location analysis of HsNLRP1 and DrNLRP1 and genes adjacent to NLRP1 loci on human chromosome 17 (top) and zebrafish chromosome 2 (bottom). Arrows indicate gene orientation. Downloaded from activation was initially detected in HEK293T cells that naturally were transfected into the cells in different combinations. The show minimal expression of NLRP1 and other inflammasome activation of DrCaspase-A and DrCaspase-B was first exam- component molecules. For this procedure, the expression con- ined by using DrCaspase-A/B–specific fluorescent substrate (Ac- structs for DrNLRP1, DrASC, DrCaspase-A, and DrCaspase-B YVAD/WEHD-AFC). Slight activation of DrCaspase-A/B was http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. Phylogenetic analysis of the relationship of NLRP1 between fish and other species. The ML tree was constructed by IQ-TREE web server with default settings and the autoselected substitution model. The credibility of each branch was estimated by bootstrap analysis (1000 replicates) and SH-aLRT branch test. The accession numbers for the sequences included in the phylogenetic analysis are listed as follows: H. sapiens NLRP1, AAH51787.1; Pan troglodytes NLRP1, JAA27823.1; Canis lupus familiaris NLRP1, XP 005619937.1; Ailuropoda melanoleuca NLRP1, XP 011215410.2; Hipposideros armiger NLRP1, XP 019520352.1; Physeter catodon NLRP1, XP 007102896.1; Rattus norvegicus NLRP1, NP 001139227.2; M. musculus NLRP1, NP 001155886.1; Phascolarctos cinereus NLRP1, XP 020831609.1; Charadrius vociferus NLRP1, KGL99904.1; Leptosomus discolor NLRP1, KFQ03745.1; Amazona aestiva NLRP1, KQL45381.1; Chelonia mydas NLRP1, EMP24960.1; Xenopus tropicalis NLRP1, XP 004911151.1; Xenopus laevis NLRP1, XP 018095534.1; DrNLRP1, XP 009297081.1; Fundulus heteroclitus NLRP1, JAR78043.1; Maylandia zebra NLRP1, XP 023011105.1; Branchiostoma belcheri NLRP1, XP 019646667.1; Strongylocentrotus purpuratus NLRP1, XP 011666919.1; Hyalella Azteca NLRP1, XP 018018216.1; Orbicella faveolata NLRP1, XP 020600786.1. 6 IDENTIFICATION OF AN NLRP1 INFLAMMASOME FROM ZEBRAFISH Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. Structural characterization of DrNLRP1 protein. (A) Tertiary structures of the individual domains (NACHT, LRR, and CARD) of NLRP1s among human, mouse, and zebrafish predicted by SWISS-MODEL with crystal structures of NACHT (PDB ID: 5irl.1.A), LRR (PDB ID: 4im6.1.A), and CARD (PDB ID: 3kat.1.A) as models. (B) Tertiary structures of the combined domain (NACHT plus LRR domains) of NLRP1s among human, mouse, and zebrafish modeled by SWISS-MODEL with 5irm.1.B as template. (C) Domain architecture and tertiary structure of DrNLRP1 modeled by I-TASSER. The top five threading templates are 5irlA, 5irmA, 5irl, 4kxfB, and 4kxfK. (D) Analysis of the structure of the CARD domain in DrNLRP1. The top five threading templates used by I-TASSER are 2l9 mA, 3katA, 1dgnA, 2l9m, and 1z6tA. The multiple alignments of DrNLRP1, HsNLRP1, MmNLRP1, and DrASC are presented beside the charged residues (Asp1290 and Arg1318) of CARD domain. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. Characterization of the components of DrNLRP1 inflammasome complex. (A) Schematic of the components of NLRP1 inflammasome complex in mammals (Mal.) and zebrafish. (B) Phylogenetic analysis of the relationship of inflammatory caspases from Arthropoda to Vertebrata. The phylogenetic tree was constructed by MEGA (version 5.0) using the ML method. The apoptotic caspases of vertebrates served as outgroups. The reliability of each node was estimated by bootstrapping with 2000 replications. The number before each species name indicates the order of caspase. For instance, 1-Homo_sapiens represents caspase-1 in human. observedinthecellstransfectedwithpCMV–DrCaspase-A/B DrASC preferentially activate DrCaspase-A when DrCaspase-A alone or cotransfected only with pCMV–DrASC. However, and DrCaspase-B were expressed simultaneously. Next, Western when DrCaspase-A/B and DrASC were coexpressed with blot analysis was performed to further characterize the in- DrNLRP1, the protease activity of DrCaspase-A/B was signifi- volvement of DrNLRP1 in DrCaspase-A/B proteolytic activa- cantly upregulated by ∼75% for DrCaspase-A and 50% for tion. Results showed that the 45/47 kDa pro–DrCaspase-A/B DrCaspase-B in a DrNLRP1 dose-dependent manner (Fig. 5A, was activated and self-cleaved into a 35 kDa protein (p35, the 5B). Notably, when DrNLRP1 was coexpressed with DrCaspase- cleavage product of active DrCaspase-A/B) if cells were coex- A/B without DrASC, DrCaspase-A/B was still not activated. pressed with DrNLRP1 and DrASC; the p35 product of As shown in Fig. 5C, the DrNLRP1- and DrASC-mediated ac- DrCaspase-A was relatively stronger than that derived from tivation of DrCaspase-A was higher by 40% than that of DrCaspase-B (Fig. 5D, 5E). By contrast, no cleavage of the DrCaspase-B. This increment indicates that DrNLRP1 and precursor of DrCaspase-A/B was detected in cells without the 8 IDENTIFICATION OF AN NLRP1 INFLAMMASOME FROM ZEBRAFISH Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. DrNLRP1 activates DrCaspase-A and DrCaspase-B in a DrASC-dependent manner in HEK293T cells. (A) DrNLRP1 and DrASC activate DrCaspase-A detected by specific Ac-YVAD-AFC fluorescent substrate. (B) DrNLRP1 and DrASC activate DrCaspase-B detected by specific Ac-WEHD- AFC fluorescent substrate. (C) DrNLRP1 activates DrCaspase-A and DrCaspase-B simultaneously, which was detected by Ac-YVAD-AFC or Ac-WEHD- AFC fluorescent substrates. (D and E) Western blot assay of the autohydrolyzation of DrCaspase-A (D) and DrCaspase-B (E) when coexpressed with DrNLRP1 and DrASC. Caspase activity was detected and expressed as the fold induction over the control as described in Materials and Methods. Each data point shows the mean 6 SD with three replicates. *p , 0.05, **p , 0.01.

expression of either DrASC or DrNLRP1. These results verify (Fig. 6A). However, the activated DrCaspase-B alone was ineffective for the functional role of DrNLRP1, which activates both DrCaspase-A the pro–DrIL-1b cleavage (Fig. 6B). When pro–DrIL-1b was coex- and DrCaspase-B in an ASC-dependent manner, with a preference pressed with DrCaspase-A, DrCaspase-B, DrASC, and DrNLRP1, in for activating DrCaspase-A to some extent. which all the DrNLRP1 inflammasome components were orchestrated in the complex, the pro–DrIL-1b canbecleavedintoan18kDamature DrNLRP1 inflammasome contributes to form (Fig. 6C). This outcome indicates that a functional DrNLRP1 pro–DrIL-1b maturation inflammasome occurs in cells, finally contributing to pro–DrIL-1b Based on the above results, DrNLRP1 is an initiator for organiz- maturation. ing a DrNLRP1–DrASC–DrCaspase-A/B–mediated inflammasome (referred to as DrNLRP1 inflammasome), which may further contribute DrNLRP1 triggers the formation of a classical inflammasome to the maturation of DrIL-1b. For clarification, the eukaryotic expres- complex in vitro sion plasmids of DrNLRP1, DrASC, DrCaspase-A, DrCaspase-B, and Given that ASC nucleation (also called ASC speck) is a convenient pro–DrIL-1b were cotransfected into HEK293T cells in different upstream readout for inflammasome activation and can be easily combinations. As shown in Fig. 6, the pro–DrIL-1b protein (31 kDa) observed under light microscopy, the DrNLRP1-triggered assembly expressed in cells coexisted with several midforms (∼25–30 kDa), of inflammasome complex was detected by the formation of DrASC which were the cleavage products of several other proteases existing in speck through immunofluorescence examination. When DrASC or cells, including neutrophil elastase, proteinase 3, cathepsins G and D, DrNLRP1 was expressed alone, the fluorescence was a weak signal granzyme A, and matrix metalloproteinases. The pro–DrIL-1b can be that diffused throughout the cell (Fig. 7A). With the coexpression of partially processed into a 20 kDa product (the first cleavage product at DrASC and DrNLRP1, the fluorescent signal of DrASC became one D104 residue of pro–DrIL-1b)bytheactivatedDrCaspase-A alone bright spot in a paranuclear area (Fig. 7B). In the amplified images, The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. DrNLRP1 inflammasome contributes to partial maturation of pro–DrIL-1b. HEK293T cells were transfected with a pcDNA3.1–DrIL-1b construct alone or with pCMV–DrNLRP1, pCMV–DrASC, pCMV–DrCaspase-A, and pCMV–DrCaspase-B. At 24 h posttransfection, immunoblot analysis was performed on the cell lysates with mouse anti-Flag or anti-Myc mAb. (A) Cleavage of pro–DrIL-1b triggered by DrNLRP1–DrASC acti- vated DrCaspase-A alone. (B) Cleavage of pro–DrIL-1b triggered by DrNLRP1–DrASC activated DrCaspase-B alone. (Figure legend continues) 10 IDENTIFICATION OF AN NLRP1 INFLAMMASOME FROM ZEBRAFISH the DrASC speck reached a size of ∼1–2 mmindiameter,and evolutionary issue, which results in a slight difference of ASC DrNLRP1 formed an outer ring around DrASC. These results in- filaments between fish and mammals. dicate that DrNLRP1 promotes DrASC-aggregate speck formation. As further support, Co-IP assay was performed to examine the DrNLRP1–DrASC complex recruits DrCaspase-A protein–protein interaction involved in DrASC nucleation. Interac- and DrCaspase-B tion clearly existed between DrNLRP1 and DrASC but not Given that ASC nucleation provides a platform for the recruitment of among the CARD-lacking mutants (DrNLRP1–DCARD and caspase-1, the correlation between DrASC and DrCaspase-A/B was DrASC–DCARD) (Fig. 8A). This observation suggests that further examined. The Co-IP assay showed that the interaction DrNLRP1 interacts with DrASC through CARD domains, trig- clearly existed between DrASC and DrCaspase-A (Fig. 10A). gering DrASC nucleation to form a classical ASC-dependent However, the interaction existing between DrASC and DrCaspase-B inflammasome. Furthermore, the DFIIND and DCARD mutants was slightly detectable (Fig. 10B) and occurred only with the of DrNLRP1 blocked the DrNLRP1-dependent DrASC nucle- coexpression of DrNLRP1 (Fig. 10C). These results suggest that ation, whereas the DNACHT mutant did not influence the for- DrASC exhibits preference for DrCaspase-A association. According mation of DrASC speck (Fig. 8B, Supplemental Fig. 3). Similarly, to immunofluorescence analysis, both DrCaspase-A and DrCaspase- the DNACHT mutant can activate DrCaspase-A/B, whereas the B were perfectly colocalized with DrASC depending on the coex- DFIIND and DCARD mutants cannot (Fig. 8C, 8D). These results pression of DrNLRP1 (Fig. 11). Nevertheless, when DrCaspase-A suggest that instead of the NACHT domain, the FIIND and CARD and DrCaspase-B were coexpressed with DrNLRP1 and DrASC in domains of DrNLRP1 contribute to the nucleation of DrASC and the cells, these two DrCaspases were almost independently localized in activation of DrCaspase-A/B. Finally, zebrafish ZF4 cell line was the inflammasome, and only a few DrCaspase-A– and DrCaspase- Downloaded from used to verify the DrNLRP1-dependent DrASC nucleation. Typical B–colocalized inflammasomes can be detected (Fig. 12A). DrNLRP1–DrASC specks clearly existed in ZF4 cells, which were Moreover, the percentage of the DrCaspase-A–associating cells ∼ coexpressed with DrNLRP1 and DrASC (Supplemental Fig. 3C). ( 23%) was higher than those of the DrCapsase-B–associating ∼ The ZF4 cells that owned the DrNLRP1–DrASC speck presented cells ( 9%). These observations suggest that DrCaspase-A and pyroptotic morphology with crushed nuclei and irregular membranes. DrCaspase-B may be sequentially recruited into the DrNLRP1–

DrASC complex, with a preferential recruitment of DrCaspase-A http://www.jimmunol.org/ CARD DrASC -mediated DrASC nucleation first, followed by the replacement of DrCaspase-B after DrCas- The ASC nucleation in mammals is started by an ASC filament pase-A was self-cleaved and released from the inflammasome. As with ASCPYD core and ASCCARD cluster, the ASCPYD core a support for this concept, the detection of DrCaspase-A– and combines with NLRPs, and the ASCCARD cluster recruits caspase- DrCaspase-B–colocalized inflammasomes may represent an in- 1. For understanding the mechanism of DrASC nucleation, two termediate form during the alternate transition of these two cas- mutant DrASC proteins that contained only the PYD domain pases in the inflammasome (Fig. 12A, 12B). However, the (named as DrASC–PYD) and the CARD domain (named as percentage of this DrCaspase-A/B–colocalized inflammasome was DrASC–CARD) were constructed. Interestingly, overexpression extremely low, suggesting rapid sequential replacement, which was of DrASC–PYD or DrASC–CARD in cells induces the filament only detectable in a narrow window period. by guest on September 28, 2021 (ASCPYD or ASCCARD) formation instead of the speck construc- tion (Fig. 9A, 9B). The results represented the assembly of in- Sequential activation of DrCaspase-A and DrCaspase-B by complete DrASC that can be induced by the interaction between PYD–PYD interaction PYD–PYD or CARD–CARD domains. The ASCCARD filament, For further support, the possible mechanism underlying the se- not the ASCPYD filament, was colocalized with DrNLRP1 upon quential activation of DrCaspase-A and DrCaspase-B in DrNLRP1 the introduction of DrNLRP1 (Fig. 9C, 9D), suggesting that inflammasome was explored. In multiple alignment analysis, the DrNLRP1 is associated with DrASC by their CARD–CARD in- sequence identity of the PYD domains between DrASC and teraction. However, the formation of DrASCCARD filament (with DrCaspase-A was 82.92%, and the identity of the domains between or without introducing DrNLRP1) cannot activate DrCaspase-A DrASC and DrCaspase-B was 55.46%. Thus, we supposed that the or DrCaspase-B (Fig. 9E). Thus, the typical DrASC nucleation in preferential association of DrCaspase-A with the DrNLRP1 DrNLRP1 inflammasome comprised the DrASCCARD core and inflammasome may be determined by the increased sequence ho- DrASCPYD cluster, the latter of which contributes to the recruit- mology between the PYD of DrASC and DrCaspase-A. To clarify ment and activation of DrCaspases by their PYD–PYD interac- this hypothesis, two chimera molecules (bPYD-CasA and aPYD- tion. Next, the tertiary structures of ASCPYD core (Protein Data CasB) were constructed, in which the PYD domains of DrCaspase-A Bank [PDB] identifier [ID]: 2N1F) and ASCCARD core (PDB ID: and DrCaspase-B were replaced by each other. After bPYD-CasA, 5FNA) were analyzed between zebrafish and mammalian coun- aPYD-CasB, DrASC, and DrNLRP1 were coexpressed in terparts (Fig. 9F). The Asn14 and Arg38 residues in ASCPYD and HEK293T cells, both bPYD-CasA and aPYD-CasB were colocalized the Asp26, Arg54, and Val33 residues in ASCCARD are predicted to with the DrASC speck individually (Fig. 12B, Supplemental Fig. be highly conserved from fish to mammals, suggesting that these 3D); these two caspases were capable of self-cleaving into a amino acids may perform important functional roles in the ASC 35 kDa cleavage product (Fig. 6D). However, the percentage of the fibrillation. However, the mammalian ASC’s Tyr59 residue, which aPYD-CasB-DrASC speck-containing cells (∼18%) exceeded that of plays an essential role in the PYD-homotypic interaction, was the bPYD-CasA-DrASC speck-forming cells (∼6%), and the self- replaced with Phe59 in DrASC. This replacement implies an cleavage activity of aPYD-CaspB was more apparent than that of

(C) Cleavage of pro–DrIL-1b triggered by activated DrCaspase-A and DrCaspase-B. (D) Cleavage of pro–DrIL-1b triggered by activated aPYDCasB and bPYDCasA, which had the exchanged PYD domains. Blots were reprobed for GAPDH as a loading control. Bar charts in (C) and (D) showed the relative density of the cleavage product of DrCaspases and DrIL-1b in the blots. The results are representative of three independent experiments, as described in Materials and Methods. The Journal of Immunology 11

FIGURE 7. Colocalization of DrNLRP1 and DrASC. (A) Transient transfection of pCMV-Myc-DrASC or pCMV- Tag2B-DrNLRP1 in HEK293T cells and diffuse florescent signals were detected in the cells. (B) Transient transfec- tion of pCMV-Myc-DrASC and pCMV-Tag2B-DrNLRP1 in HEK293T cells. Flag-DrNLRP1 signal (red) and Myc-DrASC signal (green) accumulate in the same speck (white arrow- heads). Scale bar, 10 mm. Images were captured under a laser-

scanning confocal microscopy (LSM-710; original magnification Downloaded from 3630; Carl Zeiss). The results are representative of three independent experiments. http://www.jimmunol.org/

bPYD-CaspA. These observations indicated that the priority of DrCaspaseA-5DA majorly predominated the DrASC nucleation, and by guest on September 28, 2021 DrCaspase-A in DrNLRP1 inflammasome was substituted by DrCaspase-B was mostly diffused in the cells (Fig. 12F). Thus, when DrCaspase-B when their PYD domains were exchanged. As a result, DrCaspase-A cannot be autocleaved and released from DrNLRP1 the pro–DrIL-1b in bPYD-CasA, aPYD-CasB, DrASC, and inflammasome, DrCaspase-B lost the chance to combine with the mo- DrNLRP1coexpressedcellscanonlybeprocessedintoafaint18 lecular platform. This result supports for the sequential activation model kDa mature form (Fig. 6D). It indicated that the processing of pro– of DrCaspase-A and DrCaspase-B in DrNLRP1 inflammasome. The DrIL-1b was significantly impaired with the disorder of the se- schematic of the model is presented in Fig. 13. Evidently, DrNLRP1 quential activation of DrCaspase-A and DrCaspase-B in DrNLPR1 proteins combine with DrASC through CARD–CARD interaction and inflammasome. This outcome suggests that the sequence identity of form a DrASC fiber with a DrASCCARD core and a DrASCPYD cluster PYD domains among DrASC and DrCaspase-A/B contributes to the (Fig. 13A). The DrNLRP1–DrASC substructures interact with each preferential association and activation of the two caspases. other to assemble a speck structure in the cytoplasm with a diameter of Structurally, a total of 65 and 38 aa in the PYD domains of 1–2 mm(Figs.7B,13B).TheDrNLRP1–DrASC multiprotein complex DrCaspase-A and DrCaspase-B, respectively, were consistent with recruits DrCaspase-A and DrCaspase-B by PYD–PYD interaction in DrASC (Fig. 12C). These conserved amino acids formed potential a sequential manner. DrCaspase-A is the first to be activated in the interactive interfaces of PYD domains in DrASC, DrCaspase-A, and complex, which cleaves pro–DrIL-1b at D104 into the midformed DrCaspase-B (Fig. 12D). Among these amino acids, a considerable DrIL-1b. The second activated DrCaspase-B cleaves midformed amount of the hydrophobic residues, such as Leu16,Leu21,Ile49,Val57 DrIL-1b at D122 and finally results in DrIL-1b maturation (Fig. 13C). ,andIle75, and surface-charged residues, such as Arg22,Lys23,Glu43, and Asp50,wereobservedinDrASC and DrCaspase-A. It suggests Expression analysis of the components of DrNLRP1 that the hydrophobic and charge effects provided by these residues inflammasome complex may play important roles in homotypic PYD interactions. Notably, To evaluate the existence and functional roles of DrNLRP1 the positively charged Lys23 and hydrophobic Ile49 residues, which inflammasome in vivo, the expression of DrNLRP1, DrASC, were conserved between the DrASC and DrCaspase-A PYD do- DrCaspase-A, and DrCaspase-B was initially analyzed in zebrafish mains, were absent in the DrCaspase-B PYD domain, implying that tissues and embryos by qRT-PCR. All of the genes were ubiqui- the two residues may largely contribute to the differential association tously expressed and distributed similarly in the examined immune- of the two DrCaspases with DrNLRP1 inflammasome. Moreover, relevant tissues, such as spleen, head kidney, gill, and skin five potential autocleavage sites (D106,D282,D293,D294,andD295)of (Fig. 14A). These genes were also constitutively expressed in the DrCaspase-A were predicted, and the asparagic acids in these sites early developmental embryos (2–96 hpf) (Fig. 14B–E). Infection of were substituted by alanine to construct a quinary mutant (named as E. tarda (108 CFU/ml) for 40 min promoted the expression of these DrCaspaseA-5DA) for functional evaluation (Fig. 12E). Although genes in zebrafish embryos as determined at 6 hpf (Fig. 14F). This being coexpressed with DrCaspase-B, DrASC and DrNLRP1, the event was followed by the activation of DrCaspase-A/B and the 12 IDENTIFICATION OF AN NLRP1 INFLAMMASOME FROM ZEBRAFISH Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 8. Functional evaluation of the domains in DrNLRP1. (A) Co-IP assay shows that DrNLRP1 interacts with DrASC through CARD domain. The black lines indicate where parts of the images were joined from a single experiment. (B) The statistics of DrASC speck-forming rates induced by DrNLRP1 and its mutants. More than 100 cells with DrASC speck were counted in each experimental group to quantify DrNLRP1-dependent DrASC nucleation. The original immunofluorescence images are shown in Supplemental Fig. 3B. (C) Function of DrNLRP1 mutants in the activation of DrCaspase-A detected by the specific Ac-YVAD-AFC fluorescent substrate. (D) Function of DrNLRP1 mutants in the activation of DrCaspase-B detected by the specific Ac-WEHD- AFC fluorescent substrate. Values represent the mean 6 SD calculated in triplicate cultures. *p , 0.05, **p , 0.01. maturation of DrIL-1b when detected by caspase-specific fluores- one-cell stage embryos at a concentration of 100 pg/embryo. The cent substrates and anti–DrIL-1b polyclonal Ab (Fig. 14G, 14H). DrNLRP1 inflammasome was clearly observed after 48 hpf when This observation indicates that zebrafish embryo with intracellular stimulated by E. tarda for 4 h, as determined by the typical DrASC bacterial infection is a favorable in vivo model system for the speck structures colocalized with DrNLRP1 (Fig. 15A). More- functional study of DrNLRP1 inflammasome. over, DrCaspase-A and DrCaspase-B activities were significantly upregulated with the overexpression of DrNLRP1 and DrASC, In vivo determination of DrNLRP1 inflammasome accompanied by the markedly increased maturation of DrIL-1b Next, the structural and functional characterizations of DrNLRP1 (Fig. 15B). These findings suggest that DrNLRP1 triggers the for- inflammasome were further examined in zebrafish embryo model to mation of classical inflammasome complexes in vivo, inducing the provide in vivo evidence. For this purpose, direct visualization of the activation of endogenous DrCaspase-A and DrCaspase-B and inflammasome formation and functional evaluation of DrNLRP1 leading to the maturation of DrIL-1b. inflammasome-mediated DrCaspase-A/B activation and DrIL-1b For further clarity, antisense MO-based knockdown assays maturation were initially conducted. The eukaryotic expression were performed in the 6-hpf embryos, in which DrCaspase-A and vectors of DrNLRP1 and DrASC were comicroinjected into the DrCaspase-B were significantly induced (p , 0.01) by E. tarda The Journal of Immunology 13 Downloaded from http://www.jimmunol.org/

FIGURE 9. DrNLRP1 triggers the DrASCCARD-mediated DrASC nucleation. (A) Immunofluorescence examination of DrASCPYD filament when transiently transfected by Myc-tagged DrASC–PYD (DrASC-DCARD) in HEK293T cells. (B) Immunofluorescence examination of DrASCCARD filament when transiently transfected by Myc-tagged DrASC–CARD (DrASC-DPYD) in HEK293T cells. (C and D) Flag-DrNLRP1 was colocalized with the DrASCCARD filament (D) but not with DrASCPYD filament (C). Images were captured under a laser scanning confocal microscope (LSM-710; original 3 E CARD PYD magnification 630; Carl Zeiss). ( ) DrNLRP1 coexpressed with DrASC or DrASC cannot activate DrCaspase-A/B when being detected by the by guest on September 28, 2021 specific Ac-YVAD/WEHD-AFC. (F) Tertiary structure of ASCPYD (PDB ID: 2N1F) and ASCCARD (PDB ID: 5FNA) filaments. The conserved and im- portant residues in the fibrillation of ASCPYD and ASCCARD are colored and listed by the side. The data are the average caspase activity 6 SD of three independent experiments (*p , 0.05, **p , 0.01). immersion infection (108 CFU/ml) for 40 min (Fig. 14G). The by MO-resistant DrCaspase-A mRNA but not by MO-resistant MO against DrNLRP1 was microinjected into the one-cell stage DrCaspase-5DA mRNA (Fig. 16A). Meanwhile, the activation of embryos with various concentrations (1.5–4.5 ng). The activation DrCaspase-B was not decreased in DrCaspase-A morphants or in of DrCaspase-A and DrCaspase-B significantly decreased (from morphants rescued by MO-resistant DrCaspase-A mRNA but was 55 to 15% and from 50 to 25%) in a MO dose-dependent manner significantly reduced (from 50 to 30%; p , 0.05) in the morphants (Fig. 15C, 15D). The impaired DrCaspase-A and DrCaspase-B rescued by DrCaspase-5DA mRNA, encoding a mutant DrCaspase-A activation was notably rescued (p , 0.01) by the MO-resistant that lost the autocleavage sites and occupied the DrNLRP1 DrNLRP1 mRNAs (Fig. 15E). In addition, the deficient pheno- inflammasome (Fig. 16A). This observation is consistent with that type of 48-hpf zebrafish larvae after knockdown was rescued from HEK293T cells, again demonstrating that the recruitment of by the DrNLRP1 mRNAs (Fig. 15F). As a result, DrNLRP1 DrCaspase-B into DrNLRP1 inflammasome does not require the knockdown disturbed the antibacterial immunity, leading to a help of DrCaspase-A but is dependent on the cleavage and significant decrease in survival compared with the control group. release of DrCaspase-A from the inflammasome. Correspond- However, the high mortality of 6- and 48-hpf zebrafish embryos ingly, the percentage of survival of zebrafish embryos with in DrNLRP1 morphants can be restored by the administration of E. tarda infection was dramatically decreased in DrCaspase-A MO-resistant mRNA (Fig. 15G, 15H). These results indicate the morphants, which was restored by the administration of MO- important function of DrNLRP1 inflammasome in the innate resistant DrCaspase-A mRNA but not by CaspaseA-5DA mRNA antibacterial immunity of zebrafish. (Fig. 16B). Similarly, the sequential activation model of DrCaspase-A and Finally, the embryo model was used to explore how DrNLRP1 is DrCaspase-B in DrNLRP1 inflammasome was evaluated by MO- activated by bacterial infection. We chose three typical pathogen- based knockdown and MO-resistant mRNA rescue assays in the associated molecular patterns (PAMPs) from Gram-negative bac- 6-hpf embryos under E. tarda infection as described above. The MO teria (LPS, MDP, and genomic DNA) that function as activators for against DrCaspase-A (3.0 ng) and MO-resistant mRNAs (200 pg) certain mammalian inflammasomes to test whether they can activate was microinjected into the one-cell stage embryos in different DrNLRP1 inflammasome. In vivo stimulation and MO-based combinations. As expected, the activation of DrCaspase-A signifi- knockdown assays were used. Results showed that the activation cantly declined (from 60 to 10%; p , 0.01) in DrCaspase-A of DrNLRP1 inflammasome was overall induced by bacterial LPS, morphants, and the impaired DrCaspase-A activation was rescued MDP and DNA, as determined by the increased activities of 14 IDENTIFICATION OF AN NLRP1 INFLAMMASOME FROM ZEBRAFISH Downloaded from

FIGURE 10. Protein interactions between DrASC and DrCaspases in HEK293T cells. (A and B) DrASC interacts with DrCaspase-A (A) but not DrCaspase-B (B). (C) Protein–protein interactions among DrCaspase-B, DrASC, and DrNLRP1. HEK293T cells were transfected with pCMV-Myc-

DrASC, pCMV-Tag2B–DrCaspase-B, and pCMV-HA-NLRP1 for 48 h. Cell lysates were immunoprecipitated with rabbit anti-Flag Ab and analyzed by http://www.jimmunol.org/ Western blot using mouse anti-Flag, anti-Myc, or anti-HA Ab against DrCaspase-B, DrASC, or DrNLRP1, respectively (top panel). Expression of the transfected plasmids was analyzed with anti-Flag, anti-Myc, or anti-HA Ab in the whole cell lysates (bottom panels).

DrCaspase-A/B and maturation of DrIL-1b in embryos adminis- DrNLRP1 inflammasome. This finding is supported by a recent tered with PAMPs compared with that of control embryos that did study, proving that the active caspase-1 is a p33/p10 dimer (56). not receive any stimulation (Fig. 16C). By knockdown assay, the Subsequently, the activated DrCaspase-A and DrCaspase-B work activation of DrCaspase-A/B and maturation of DrIL-1b were together in the maturation of DrIL-1b. The above results indicate significantly abrogated (from 50 to 10% for DrCaspase-A, and from that DrNLRP1 inflammasome acts as a platform for the recruit- by guest on September 28, 2021 40 to 10% for DrCaspase-B; p , 0.05) in DrNLRP1 morphants ment and activation of proinflammatory caspases and pro–IL-1b, stimulated by MDP but not by LPS and bacterial DNA (Fig. 16C). which is a hallmark functional performance typically seen in These results suggest that MDP largely contributes to the activation mammalian inflammasome. of DrNLRP1 inflammasome in zebrafish. In addition, other Despite these similarities, DrNLRP1 inflammasome acquires inflammasomes may exist in zebrafish, which can be activated in distinct features from its mammalian counterparts. For example, response to bacterial LPS and DNA stimulation. the inflammatory DrCaspase-A and DrCaspase-B are both PYD- containing caspases instead of CARD-containing caspases in mammals (57), whereas DrNLRP1 owns a CARD that cannot Discussion directly bind with such caspases. Thus, the recruitment of NLRP1 inflammasome is one of the best-characterized inflam- proinflammatory caspases into DrNLRP1 inflammasome needs matory mediators in innate immunity and pathogenesis of various the help of the adaptor DrASC. In other words, DrNLRP1 diseases in mammals (52–54). However, the existence of NLRP1 inflammasome triggers the activation of DrCaspase-A and inflammasome in ancient vertebrates is poorly understood. In this DrCaspase-B in an ASC-dependent way, whereas mamma- study, an NLRP1 homolog (DrNLRP1) was characterized from lian NLRP1 inflammasomes can activate caspase-1 in an ASC- zebrafish. Structurally, this DrNLRP1 shares an overall coincident independent way (58). Accordingly, DrNLRP1 triggers the gene organization and chromosomal synteny, as well as protein aggregation of DrASC into a filament with DrASCCARD core and domain architecture and tertiary structure, with human and mouse DrASCPYD cluster, which is opposite to that of mammalian ASC NLRP1s. With the coexpression of DrNLRP1 or its mutants and filament (5, 59). To explain how DrCaspase-A and DrCaspase-B DrASC in cells, DrNLRP1 is able to trigger the formation of a are coordinated in DrNLRP1 inflammasome, we assumed classical inflammasome complex, as determined by the assembly that the two DrCaspases are recruited and activated in the of the DrASC speck structure under fluorescence microscopy. The inflammasome in a sequential manner, with a preference for Co-IP assay reveals that the assembly of DrNLRP1 inflammasome DrCaspase-A and a subsequent choice for DrCaspase-B. This depends on the CARD–CARD homotypic interaction between sequential activation is determined by the PYD–PYD homotypic DrNLRP1 and DrASC proteins. In addition, both the FIIND and interaction between the DrASC and DrCaspases. Several experi- CARD domains of DrNLRP1 are necessary for DrASC nucle- mental lines apparently support this hypothesis. For instance, ation, which is similar to that of mammalian NLRP1 inflamma- DrCaspase-A and DrCaspase-B always independently exist in some (16, 55). Functionally, DrNLRP1 can trigger the activation DrNLRP1 inflammasome. However, when the autocleavage site of of both DrCaspase-A and DrCaspase-B while being detected by DrCaspase-A is depleted, the DrCaspase-B associated inflamma- specific fluorescent substrates and Western blot assay. The acti- some is rarely visible. The activation of DrCaspase-B is extremely vated DrCaspase-A/B is autohydrolyzed into a p35 product by the impaired in the HEK293T cells and E. tarda–infected embryos, in The Journal of Immunology 15 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 11. Visualization of DrNLRP1–DrASC–DrCaspases inflammasome complex. (A) Diffuse florescent signal (top panel) emerged with the transient transfection of pCMV-Myc-DrASC and pCMV-Tag2B–DrCaspase-A in HEK293T cells. With the coexpression of DrNLRP1, DrASC recruited DrCaspase-A and formed specks in the cells (bottom panels with white arrowheads). (B) Diffuse florescent signal (top panel) emerged with the transient transfection of pCMV-Myc-DrASC and pCMV-Tag2B–DrCaspase-B in HEK293T cells. With the coexpression of DrNLRP1, DrASC also recruited DrCaspase-B and formed specks in the cells (bottom panels with white arrows). Scale bar, 10 mm (LSM-710; original magnification 3630; Carl Zeiss). The results are representative of three independent experiments. which DrCaspase-A is largely replaced by a mutant DrCaspase-A DrCaspase-A, and DrCaspase-B. These results suggest the in- (DrCaspaseA-5DA) without any potential autocleavage sites. volvement of hydrophobic and charge effects in homotypic These findings indicate that the retention of DrCaspase-A in PYD–PYD interactions (60, 61). Further study (such as site- DrNLRP1 inflammasome inhibits the recruitment of DrCaspase-B directed mutagenesis) is needed to clarify this notion. To into the inflammasome. In other words, DrCaspase-B is recruited date, the mechanisms underlying homotypic interactions, such into the inflammasome, followed by the cleavage and release of as PYD–PYD and CARD–CARD interactions and even others, DrCaspase-A from the inflammasome, suggesting that these two are still poorly understood; thus, the DrNLRP1 inflammasome DrCaspases are sequentially activated. In addition, the occurrence is anticipated to be a favorable model system for such kind of the DrCaspase-A–associated inflammasome in cells is more of study. apparent than the DrCaspase-B–associated one, suggesting that The maturation of DrIL-1b depends on the cleavage of pro– DrCaspase-A preferentially associates with the inflammasome. DrIL-1b at D104 and D122 by activated DrCaspase-A and However, this preferential association of DrCaspase-A can be DrCaspase-B in a strict order (24, 62). Thus, we assume that the replaced by DrCaspase-B when the PYD domains of these two sequential activation of DrCaspase-A and DrCaspase-B in caspases are exchanged with each other. Given the higher sim- DrNLPR1 inflammasome enables the orderly and efficiently ilarity of the PYD domain of DrASC with DrCaspase-A com- processing of pro–DrIL-1b. Actually, when the sequential pared with DrCaspase-B, the homotypic degree between the activation of DrCaspase-A and DrCaspase-B was reversed by PYD domains governs the preference. By multiple alignment coexpressing DrNLRP1 and DrASC with bPYD-CasA and aPYD- and homology modeling analyses, a number of hydrophobic and CasB instead of the wild-type DrCaspase-A/B, only extremely charged amino acid residues are conspicuously conserved on faint 18 kDa mature DrIL-1b can be detected. This observation the extensive surfaces of PYD domains among DrASC, means that the processing of pro–DrIL-1b in this experiment is 16 IDENTIFICATION OF AN NLRP1 INFLAMMASOME FROM ZEBRAFISH Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 12. Sequential activation of DrCaspase-A and DrCaspase-B in DrNLRP1 inflammasome. (A) Coexpression of DrNLRP1, DrASC, DrCaspase-A, and DrCaspase-B in HEK293T cells elicited the formation of DrCaspase-A (white arrowheads) or DrCaspase-B (white arrows) specks in the cells. (B) Coexpression of DrNLRP1, DrASC, bPYDCasA, and aPYDCasB in HEK293T cells elicited the formation of bPYDCasA (white ar- rowheads) or aPYDCasB (white arrows) specks in the cells. (C) Amino acid alignments of the PYD domains in DrASC, DrCaspase-A, and DrCaspase- B. (D) Tertiary structures of the PYD domains predicted by SWISS-MODEL. The same residues among DrASC, DrCaspase-A, and DrCaspase-B were red in color. The same residues between DrASC and DrCaspase-A or DrASC and DrCaspase-B were colored by orange or yellow, respectively. (E) Schematic of the five potential cleavage sites (colored by red) of DrCaspase-A. (F) Coexpression of DrNLRP1, DrASC, CaspaseA-5DA, and DrCaspase-B in HEK293T cells triggered the formation of CaspaseA-5DA (white arrowheads) specks but blocked the nucleation of DrCaspase-B in the cells. The rates of cells developing the ASC speck are marked on the upper-left corner of each panel, as conducted by calculating more than 100 cells from immunofluorescence images in each experimental group. Data are represented as the mean 6 SD for three coverslips. Scale bar, 10 mm (LSM-710; original magnification 3630; Carl Zeiss). disorganized and significantly impaired. The prioritized activation This observation indicates that intracellular inflammatory and of DrCaspase-A provides a guarantee for the full maturation of apoptotic signaling pathways may be occurred more indepen- DrIL-1b. Given that the DrCaspase-A– and DrCaspase-B–like dently in ancient vertebrates (at least in cyprinid fish), whose bi- caspases (also referred to caspase-1A and caspase-1B) can be ological significance is worth further study. widely predicted in numerous other teleost fish, the cooperation of Finally, the structural and functional characterizations of the two inflammatory caspases may be universal in fish species. DrNLRP1 inflammasome were determined in zebrafish both Thus, our work may make a favorable guide for such a study in in vitro and in vivo with the help of zebrafish ZF4 cells and teleost species. Notably, two classes of caspase were known in embryos. As expected, typical DrNLRP1–DrASC inflamma- zebrafish and other Cyprinidae fish. Inflammatory caspases, such somes were clearly detected when cells and embryos were as DrCaspase-A and DrCaspase-B, own a PYD domain in their N overexpressed with DrNLRP1 and DrASC. Compared with terminus, whereas the other apoptotic caspases, such as caspase- HEK293T cells containing DrNLRP1 inflammasome exhibiting 3/8/9, own a CARD domain in their N-terminal region (63, 64). normal morphology, ZF4 cells containing DrNLRP1 inflam- The Journal of Immunology 17 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 13. Schematic diagram of DrNLRP1 inflammasome and the proposed sequential activation model for DrCaspase-A/B in DrNLRP1 inflam- masome. (A) DrASC-dependent aggregation of DrNLRP1 inflammasome from side view. (B) DrNLRP1–DrASC–DrCaspase-A/B multiprotein complex and formation of the cytoplasmic speck from top view. (C) Sequential activation of DrCaspase-A and DrCaspase-B in DrNLRP1 inflammasome for the maturation of DrIL-1b in a DrCaspase-A– and DrCaspase-B–directed cleavage order. masome usually present pyroptotic morphology with nuclear by various cellular metabolites, such as ATP, short-chain fatty condensation and plasma membrane rupture (65). This event acids, uric acid, cholesterol crystals, and reactive oxygen spe- may depend on the expression of endogenous pyroptosis-related cies (ROS) (67, 68), the latter of which frequently increasing molecules in ZF4 cells that are activated by DrNLRP1 inflam- during viral and intracellular bacterial invasions. Therefore, we masome. In the embryo model, the DrNLRP1–DrASC nucle- test the potential stimulatory role of ROS in the activation of ation triggers the endogenous DrCaspase-A and DrCaspase-B DrNLRP1 inflammasome. As expected, the activation of activation and DrIL-1b maturation under stimulation of E. tarda DrNLRP1-dependent DrCaspase-A in HEK293T cells is sub- infection. These activities can be diminished by antisense stantially stimulated when the cells are treated with H2O2 MO-based knockdown of DrNLRP1andrescuedbyMO- (0.1 mM) but is inhibited when the cells are treated with resistant mRNAs. In accordance with these observations, the N-acetyl-L-cysteine (5 mM), a widely used ROS scavenger high mortality of DrNLRP1 morphants in E. tarda immersion- (Fig. 16D). These observations suggest that the alteration of infection model is restored by the administration of the MO- cellular metabolic homeostasis (such as redox state) during resistant mRNAs. These results verify the functional roles of bacterial infection may stimulate the activation of DrNLRP1 DrNLRP1 inflammasome in antibacterial innate immunity inflammasome. However, further study is needed to clarify this in vivo. To investigate the mechanism of DrNLRP1 inflamma- notion. some activation in response to bacterial infection in vivo, we In addition to sensing foreign infectious or endogenous danger- administer three typical PAMPs (LPS, MDP, and bacterial DNA) related triggers, NLRP1 inflammasome must avoid spontaneous into zebrafish embryos. The MDP largely contributes to the and aberrant activation to ensure the stabilization of cellular ho- activation of DrNLRP1 inflammasome. This result is consistent meostasis. Therefore, the negative feedback regulatory mechanism with the observation that MDP is a potent stimulator for mam- underlying NLRP1 inflammasome activation is a particular re- malian NLRP1 inflammasome activation (66). Mammalian search focus. A novel endogenous inhibitor of NLRP1 inflam- inflammasomes (such as NLRP1 and NLRP3) can be activated masome, named as dipeptidyl dipeptidase 9 (DPP9), is recently 18 IDENTIFICATION OF AN NLRP1 INFLAMMASOME FROM ZEBRAFISH Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 14. Analyses for the expression of DrNLRP1 inflammasome–related molecules and the activation of DrNLRP1 inflammasome in zebrafish in response to bacterial infection. (A) qRT-PCR analysis of the expression patterns of DrNLRP1 inflammasome-related genes in adult zebrafish tissues. (B–E) Expression patterns of DrNLRP1 (B), DrASC (C), DrCaspase-A (D), and DrCaspase-B (E) in zebrafish embryos at different developmental stages. (F and H) E. tarda infection induced DrNLRP1 inflammasome activation in 6-hpf zebrafish embryo, as examined by the fold change of the mRNA levels (F), the activation of DrCaspase-A/B (G), and the maturation of DrIL-1b (H). Immunoblot analysis was performed on the lysate of embryos for DrIL-1b by using a rabbit polyclonal Ab and was reprobed for GAPDH as a loading control. The relative expression levels (A–E) were calculated using the 22DCt method with b-actin for normalization,16 and the fold change (F) was calculated by the method of 22ΔΔCt. Data points represent the mean 6 SD from three independent experiments (**p , 0.01, ***p , 0.001). identified from diverse primary cell types of humans and mice (Ref. and mouse (78.49%) counterparts and possesses converted N-terminal 69 and F.L. Zhong, K. Robinson, C. Lim, C. R. Harapas, C. Yu, b-barrel and C-terminal S9 hydrolase domains (data not shown), W. Xie, R. M. Sobota, V. B. Au, R. Hopkins, J. E. Connolly, suggesting its potential functional involvement in the negative regu- S. Masters, and B. Reversade, manuscript posted on bioRxiv). This lation of DrNLRP1 inflammasome activation. As a support, the FIIND DPP9 shares a similar domain structure with other DPP-IV family domain in DrNLRP1 is critical for the assembly and activation of members consisting of an N-terminal b-barrel (DPP-IV N) and a DrNLRP1 inflammasome. In summary, the DPP9-mediated C-terminal S9 hydrolase domain, the latter of which binds to the NLRP1 regulatory pathway is likely to exist in zebrafish autocleaving FIIND module and maintains NLRP1 in its mo- and is highly conserved from fish to mammals throughout the nomeric and inactive state. Interestingly, a DPP9 homolog vertebrate evolution. Further clarification on this issue would (D. rerio DPP9) is clearly predicted from zebrafish. This D. rerio aid in understanding the molecular and functional evolu- DPP9 shares high sequence identity with human (79.79%) tionary history of NLRP1 inflammasome. The Journal of Immunology 19 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 15. In vivo structural and functional determinations of DrNLRP1 inflammasome by overexpression or gene knockdown assays. (A) Coexpression of Flag-tagged DrNLRP1 and Myc-tagged DrASC in vivo triggers the nucleation of DrNLRP1–DrASC speck in zebrafish embryos. (B) Coexpression of DrNLRP1 and DrASC in vivo increased the activation level of DrCaspase-A/B and promoted the maturation of DrIL-1b under E. tarda infection. (C and D) Knockdown of DrNLRP1byNLRP1-MOdecreasedtheactivationofDrCaspase-A (C)andDrCaspase-B (D) in 6-hpf embryos after E. tarda (E. tard) infection at 106 CFU/ml for 40 min. (E) MO-resistant DrNLRP1 mRNA rescued the activation of DrCaspase-A/B in 6-hpf embryos after E. tarda infection. (F) The phenotype of 48-hpf zebrafish larvae after gene knockdown and rescue. (G and H) The RSRs of 6-hpf embryos (G) and 48-hpf larvae (H)afterE. tarda infection at 106 CFU/ml for 12 h. Zebrafish embryos were microinjected with standard-MO (Ctrl-MO), NLRP1-MO, or both with the corresponding mRNA [NLRP1 2 (MO + mRNA)]. Data are representative of three independent experiments as mean 6 SD (*p , 0.05, **p , 0.01).

Collectively, our present study demonstrates that the NLRP1 conserved structural hallmarks and functional roles in inflam- inflammasome originates as early as in teleost fish. As an ancient matory responses, as well as some distinct features from its protein apparatus, NLRP1 inflammasome in zebrafish possesses mammalian counterparts. Given the crucial roles of NLRP1 20 IDENTIFICATION OF AN NLRP1 INFLAMMASOME FROM ZEBRAFISH Downloaded from http://www.jimmunol.org/

FIGURE 16. Sequential activation model of DrCaspase-A/B in DrNLRP1 inflammasome and the mechanism underlying DrNLRP1 inflammasome activation in response to bacterial infection in zebrafish embryos and HEK293T cells. (A) Sequential activation model examined by in vivo knockdown and rescue assays. Zebrafish embryos were microinjected with standard-MO (Ctr-MO), CaspaseA-MO (CasA-MO), CaspaseA-MO with the corresponding mRNA (CasA-mRNA), or CaspaseA-MO with CaspaseA-5DA mRNA (CasA5DA-mRNA). The activation of DrCaspase-A/B was determined in 6-hpf by guest on September 28, 2021 embryos after E. tarda infection at 108 CFU/ml for 40 min. (B) Sequential activation of DrCaspase-A/B evaluated by in vivo antibacterial activity against E. tarda infection through knockdown and rescue assays. Zebrafish embryos received various MOs and MO-resistant mRNAs. RSRs were determined by using 6-hpf embryos after E. tarda infection at 108 CFU/ml for 12 h. (C) Evaluation of bacterial LPS, MDP, and DNA for the activation of DrNLRP1 inflammasome by in vivo knockdown assay. Zebrafish embryos were microinjected with Ctr-MO or NLRP1-MO (P1-MO) and LPS, MDP, or bacterial DNA in different combinations. The activation of DrNLRP1 inflammasome was determined by the increased activity of DrCaspase-A/B using specific Ac- YVAD/WEHD-AFC fluorescent substrate (top) and IL-1b maturation using Western blot (bottom). (D) Evaluation of ROS for the activation of DrNLRP1 inflammasome in HEK293T cells coexpressed with DrASC and DrNLRP1 under treatment of ROS or N-acetyl-L-cysteine. DrNLRP1 inflammasome activation was determined through increased activity of DrCaspase-A by using specific Ac-YVAD-AFC fluorescent substrate. All data are representative of three independent experiments as mean 6 SD (*p , 0.05, **p , 0.01).

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2 3 SUPPLEMENTAL FIGURE 1. Multiple alignments of DrNLRP1 with other orthologs. Amino 4 acid residues shaded in black are completely conserved in all aligned species, whereas residues shaded 5 in gray present similar side chains. The four conserved domains NACHT, LRR, FIIND, and CARD are 6 indicated above the alignment. The consensus sequences of Walk A/B motif and L motif (leucine-rich 7 repeats motif) are underlined. Triangles indicate the important residues in the NACHT, FIIND, and 8 CARD domains.

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10 11 SUPPLEMENTAL FIGURE 2. Co-evolution analysis of caspase-1 and IL-1β or NLRP1 and ASC 12 from low vertebrates to mammals. (A and B) The phylogenetic trees of caspase-1 (A) and IL-1β (B) 13 were constructed by MEGA (version 5) using neighbor-joining method with the bootstrap of 1000 14 replications. (C) Evolutionary correlation (co-evolutional coefficient) between caspase-1 and IL-1β 15 in vertebrates. (D) Evolutionary correlation (co-evolutional coefficient) between NLRP1 and ASC in 16 vertebrates. 17

18 19 SUPPLEMENTAL FIGURE 3. Mutants of DrNLRP1 and their functions in the formation of 20 DrASC specks. (A) Schematic diagram of DrNLRP1 and its mutants that lack the NACHT, FIIND, 21 or CARD domain. (B) Confocal microscopy image of DrNLRP1 (WT or mutants)-dependent DrASC 22 nucleation. (C) Confocal microscopy image of DrNLRP1-DrASC speck in ZF4 cells. (D) Confocal 23 microscopy image of DrCaspase-A (or bPYDCasA or CaspaseA-5DA) and DrCaspase-B (or 24 aPYDCasB) co-localized with DrASC nucleation in HEK293T cells. Images were captured under a 25 laser scanning confocal microscope (Zeiss LSM-710; original magnification, 200× or 630×).