Pi Is 0021925817499200

ARTICLE cro
The zebrafish NLRP3 inflammasome has functional roles in

ASC-dependent interleukin-1␤ maturation and gasdermin
E–mediated pyroptosis
Received for publication, November 5, 2019, and in revised form, December 2, 2019 Published, Papers in Press, December 18, 2019, DOI 10.1074/jbc.RA119.011751
X Jiang-Yuan Li‡, Yue-Yi Wang‡, Tong Shao‡, Dong-Dong Fan‡, Ai-Fu Lin‡, Li-Xin Xiang‡1, and Jian-Zhong Shao‡§2
From the ‡College of Life Sciences, Key Laboratory for Cell and Gene Engineering of Zhejiang Province, Zhejiang University,
Hangzhou 310058 and the §Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science
and Technology, Qingdao 266071, People’s Republic of China
Edited by Dennis R. Voelker
The NLR family pyrin domain containing 3 (NLRP3) inflam- teleost fish, providing a cross-species understanding of the evo-
masome is one of the best-characterized inflammasomes in lutionary history of inflammasomes. Our findings also indicate
humans and other mammals. However, knowledge about the that the NLRP3 inflammasome may coordinate inflammatory
NLRP3 inflammasome in nonmammalian species remains lim- cytokine processing and secretion through a GSDME-mediated
ited. Here, we report the molecular and functional identification pyroptotic pathway, uncovering a previously unrecognized reg-
of an NLRP3 homolog (DrNLRP3) in a zebrafish (Danio rerio) ulatory function of NLRP3 in both inflammation and cell
model. We found that DrNLRP3’s overall structural architec- pyroptosis.
ture was shared with mammalian NLRP3s. It initiates a classical
inflammasome assembly for zebrafish inflammatory caspase
(DrCaspase-A/-B) activation and interleukin 1␤ (DrIL-1␤) mat- Inflammasomes are cytosolic multiprotein complexes that
uration in an apoptosis-associated speck-like protein contain- assemble in response to exogenous microbial invasions and
ing a caspase-recruitment domain (ASC)-dependent manner, in endogenous damage signals (1, 2). Inflammasomes act as a
which DrNLRP3 organizes DrASC into a filament that recruits central platform to activate inflammatory caspases such as
DrCaspase-A/-B by homotypic pyrin domain (PYD)–PYD inter- Caspase-1, which processes the proinflammatory cytokines
actions. DrCaspase-A/-B activation in the DrNLRP3 inflam- interleukin 1␤ (IL-1␤)3 and IL-18 for their maturation, and
masome occurred in two steps, with DrCaspase-A being acti- hydrolyze gasdermin D to generate an N-terminal fragment to
vated first and DrCaspase-B second. DrNLRP3 also directly induce membrane perforation, cytokine release, and cell pyrop-
activated full-length DrCaspase-B and elicited cell pyroptosis tosis (3, 4). Among numerous inflammasomes identified in
in a gasdermin E (GSDME)-dependent but ASC-indepen- mammals, NLRP3 inflammasome is the most extensively stud-
dent manner. These two events were tightly coordinated by ied because of its crucial functional roles in innate immunity
DrNLRP3 to ensure efficient IL-1␤ secretion for the initiation of and pathogenesis of various diseases (5, 6). NLRP3 is a family
host innate immunity. By knocking down DrNLRP3 in zebrafish member of nucleotide-binding domain and leucine-rich repeat
embryos and generating a DrASC-knockout (DrASCⴚ/ⴚ) fish (LRR)-containing proteins (NLRs) (7). The mammalian NLRP3
clone, we characterized the function of the DrNLRP3 inflam- is structurally characterized by the presence of an N-terminal
masome in anti-bacterial immunity in vivo. The results of our pyrin domain (PYD), a central adenosine triphosphatase
study disclosed the origin of the NLRP3 inflammasome in (ATPase) domain known as NACHT, a domain associated with
NACHT in fish and other vertebrates (FISNA) (8), and a C-ter-
This work was supported by National Natural Science Foundation of China minal LRR domain (9). Once activated by a spectrum of stimuli,
Grants 31630083 and 31572641; National Key Research and Development
Program of China Grants 2018YFD0900503 and 2018YFD0900505; Stem
Cell and Translational Research; National Key Research and Development 3
The abbreviations used are: IL-1␤, interleukin 1␤; ASC, apoptosis-associated
Program of China Grant 2016YFA0101001; the Open Fund of the Labora- speck-like protein containing a caspase-recruitment domain; CaspaseB-
tory for Marine Biology and Biotechnology; Qingdao National Laboratory 4DA, mutant of DrCaspase-B in which four asparagine acids (Asp130,
for Marine Science and Technology, Qingdao, China, Grant OF2017NO02; Asp137, Asp308, and Asp314) were substituted by alanine; DrASC, D. rerio
the Open Funding Project of the State Key Laboratory of Bioreactor Engi- ASC; DrCaspase, D. rerio caspase; DrIL-1␤, D. rerio IL-1␤; DrNLRP3, D. rerio
neering; and the Zhejiang Major Special Program of Breeding Grant NLRP3; DrGSDME, D. rerio GSDME; FCM, flow cytometry; FISNA, domain
2016C02055-4. The authors declare that they have no conflicts of interest associated with NACHT in fish and other vertebrates; FRAP, fluorescence
with the contents of this article. recovery after photobleaching; GSDMD, gasdermin D; GSDME, gasdermin
This article contains Figs. S1–S5 and Table S1. E; gRNA, guide RNA; hpf, hour post-fertilization; HsNLRP3, H. sapiens
The nucleotide sequence(s) reported in this paper has been submitted to the NLRP3; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; LRR,
GenBankTM/EBI Data Bank with accession number(s) MN088121, XM_ leucine-rich repeat; MDP, muramyl dipeptide; MmNLRP3, M. musculus NLRP3;
005170077.3, and NM_001001947.1. MO, morpholino oligonucleotide; NCBI, National Center for Biotechnology
1
To whom correspondence may be addressed: College of Life Sciences, Zhe- Information; NLRP3, NLR family pyrin domain containing 3; NLR, nucleotide-
jiang University, 866 YuHangTang Rd., Hangzhou 310058, China. Tel.: binding domain and leucine-rich repeat-containing proteins ORF, open read-
86-571-88206582; Fax: 86-571-88206582; E-mail: [email protected]. ing frame; PI, propidium iodide; PDB, Protein Data Bank; PYD, pyrin domain;
2
To whom correspondence may be addressed: College of Life Sciences, Zhe- ROI, region of interest; RSR, relative survival rate; LRR, leucine-rich repeat; Ab,
jiang University, 866 YuHangTang Rd., Hangzhou 310058, China. Tel.: antibody; DMEM, Dulbecco’s modified Eagle’s medium; Ac, acetyl; AFC,
86-571-88206582; Fax: 86-571- 88206582; E-mail: [email protected]. amido-4-trifluoromethylcoumarin; gRNA, guide RNA.
This is an Open Access article under the CC BY license.

1120 J. Biol. Chem. (2020) 295(4) 1120 –1141
© 2020 Li et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of an NLRP3 inflammasome from zebrafish
NLRP3 combines with the ASC (apoptosis-associated speck- cross-species understanding of the evolutionary history of NLRP3
like protein containing a caspase-recruitment domain) adaptor inflammasome from teleost fish to mammals.
protein to form a speck-shaped inflammasome, which recruits
Results
and activates Caspase-1 for IL-1␤ maturation (10, 11). Despite
numerous studies on mammals, the occurrence of NLRP3 Molecular characterization of DrNLRP3 and DrGSDME
inflammasome in ancient vertebrates remains poorly under- Through a systematic search in Ensemble by BLASTN, one
stood. The identification of NLRP3 inflammasome in lower NLRP3 (DrNLRP3) and two DrGSDMEa/b candidate genes
vertebrates, particularly in primitive teleost fish, will contribute were retrieved from the zebrafish genome database. The
to cross-species understanding of NLRP3-mediated biology DrNLRP3 gene is located within a 10.0-kb genomic fragment
throughout vertebrate evolution. on zebrafish chromosome 1 with eight exons and seven introns.
Several previous studies identified two proinflammatory The DrGSDMEa/b gene is distributed within a 15.2/13.8-kb
caspases, namely DrCaspase-A (Caspy) and DrCaspase-B genomic fragment on chromosome 19/16 with 9/9 exons and
(Caspy2), from zebrafish (12). These two caspases engage in 8/8 introns. Genes adjacent to DrNLRP3 and DrGSDMEa/b
proIL-1␤ cleavage with different specificities in an ASC-depen- loci share an overall conserved chromosome synteny with those
dent manner (13). The zebrafish proIL-1␤ (proDrIL-1␤) has of humans and other mammalian species (Fig. 1, A and G). The
two unique cleavage sites instead of one conserved Caspase-1 cDNA of DrNLRP3 consists of a 64-bp 5⬘-untranslated region
autoproteolytic site as observed in mammals. proDrIL-1␤ is (5⬘-UTR), a 3243-bp 3⬘-UTR, and a 3345-bp open reading
first cleaved by DrCaspase-A into a partially processed 20-kDa frame (ORF) that encodes 1114 amino acids (GenBankTM
intermediate form at the Asp104 residue, and then cleaved again accession no. MN088121). The cDNA of DrGSDMEa/b con-
into a fully processed 18-kDa mature form by DrCaspase-B at tains a 296/90-bp 5⬘-UTR, a 184/841-bp 3⬘-UTR, and a 1566/
the Asp122 residue (13, 14). The catalytic domain of DrCaspase- 1419-bp ORF that encodes 521/472 amino acids (GenBankTM
A/B shares the highest homology with that of human Caspase- accession no. XM_005170077.3/NM_001001947.1).
1/5, with a sequence similarity of 54/57% (15). In humans, DrNLRP3 and DrGSDMEa/b proteins share similar domain
Caspase-4/5 seems to function similarly to mouse Caspase-11 and tertiary architectures with mammalian counterparts (Fig.
as shown by their abilities to cleave gasdermin D (GSDMD) and 1, B and C). DrNLRP3 contains an N-terminal pyrin domain
thereby induce cell pyroptosis after they are activated by intra- (PYD), a central nucleotide-binding domain (NACHT), a
cellular lipopolysaccharide (LPS) (16). Thus, DrCaspase-A and domain associated with NACHT in fish and other vertebrates
DrCaspase-B are likely the orthologs of Caspase-1 and Caspase- (FISNA), a series of leucine-rich repeats (LRR), and a unique
4/5/11 in mammals (12, 15, 17). Recently, a canonical NLRP1 C-terminal B30.2 domain (Figs. 1B and Fig. S1A). The PYD
inflammasome has also been identified from zebrafish; it can domain comprises five antiparallel ␣-helices and one conserv-
ative truncated helix. The NACHT domain possesses Walker A
initiate DrCaspase-A/-B activation in a sequential recruitment
(ATP/GTPase-specific P-loop) and Walker B (Mg2⫹-binding
manner (18). In addition, DrCaspase-B (caspy2) also senses
site) motifs. Eight LRRs with conserved LXXLXLXXN/CXL
cytosolic LPS through its pyrin domain, which mediates pyrop-
motifs form a well-defined “horseshoe”-shaped scaffold in
tosis in zebrafish fibroblasts via a Caspase-4/5/11-like activity
which the B30.2 domain is located without any complete ␣-he-
as observed in mammalian noncanonical inflammasomes (19).
lix or ␤-sheet architecture (Fig. S1, A and D). DrGSDMEa/b
In mammals, multiple gasdermin proteins, including GSDMD,
consists of an N-terminal domain with the most conservative
GSDMA, GSDMB, GSDMC, and GSDME, were found to
␤-sheets (␤1–␤11) and ␣-helices (␣1–␣4), an aspartic acid–
induce pyroptosis; among them, GSDMD is selectively cleaved
cleaved site in a long-loop structure, and a C-terminal domain
by Caspase-4/5/11 to liberate an N-terminal effector fragment
with seven helices (␣5–␣11) (Fig. 1, E and F). The C terminus
from the C-terminal inhibitory domain (16, 20, 21). The N-ter-
harbors conserved hydrophobic residues, such as Ile296/Leu294,
minal fragment oligomerizes in the cell membrane to form a Ala420/Ala269, and Leu431/Leu280 on ␣5, ␣8, and ␣9 helices, to
10 –16-nm diameter pore through which mature-formed IL-1␤ form nonpolar surfaces that interact with the N-terminal
and IL-18 are secreted (16, 22). With the accumulation of mem- domain for an auto-inhibitory regulation (Fig. S1B). Key pore-
brane pores, cells ultimately undergo membrane rupture and forming residues, such as Lys7/Lys7, Lys39/Lys39, Lys50/Lys50,
pyroptosis (23, 24). In zebrafish, however, only two gasdermin E Thr101/Thr99, Arg136/Arg134, and Ser143/Ser141 also exist in the
homologs (DrGSDMEa/b, also known as DFNA5a/b) were iden- N-terminal domain, which are conserved from fish to mam-
tified to exert a pore-forming effect by their activated N-terminal mals. Phylogenetic analysis shows that DrNLRP3 and
domains; and DrGSDMEa that possesses a Caspase-3 cleavage DrGSDMEa/b are clustered to their homologs with a high boot-
motif induces pyroptosis after chemotherapy drugs are adminis- strap probability (Fig. 1, H and I). In addition, DrNLRP3 and
tered (25–27). Thus, whether DrGSDMEa/b acts as a substrate DrGSDMEa/b are extensively expressed in zebrafish adult tis-
cleaved by DrCaspase-A/B to induce pyroptosis as mammalian sues such as head, kidney, spleen, gill, and intestine and in
GSDME did remains to be elucidated. In this study, we identified embryos (Fig. 1, J and K).
an NLRP3 inflammasome from zebrafish and revealed its unique
characteristics in DrCaspase-A/B activation, DrIL-1␤ maturation, DrNLRP3 initiates DrCaspase-A/-B activation in different
DrGSDMEa/b cleavage, and pyroptotic cell death. Our results manners
showed the molecular and functional characterizations of an early The functional role of DrNLRP3 was first examined by its
NLRP3 inflammasome in an ancient vertebrate, which provides a initiation activity for DrCaspase-A/-B in HEK293T cells that
J. Biol. Chem. (2020) 295(4) 1120 –1141 1121

Figure 1. Molecular and structural identification of DrNLRP3 and DrGSDME. A, gene synteny and chromosomal location analysis of genes adjacent to
NLRP3 loci on human chromosome 1 (top) and zebrafish chromosome 1 (bottom). Arrows indicate gene orientation. B, schematic of the domain architecture of
HsNLRP3 and DrNLRP3. C, DrNLRP3 and HsNLRP3 tertiary structures predicted by SWISS-MODEL with crystal structures of NACHT and LRR (PDB code 4kxf.3.A)
as models. D, DrNLRP3 domain architecture and tertiary structure modeled by I-TASSER. The top five threading templates are 5irmA, 6npva, 4kxfK, 6b5bA, and
6eqoA. E, schematic of the domain architecture of HsGSDME and DrGSDMEa/b. The predicted cleaved sites in DrGSDMEa and DrGSDMEb are 253SEVD256 and
244
FEID247, respectively. F, tertiary structures of full-length HsGSDME and DrGSDMEa/b protein predicted by SWISS-MODEL with crystal GSDMA3 structures
(PDB code 5b5r.1.A) as model. G, gene synteny and chromosomal location analysis of genes adjacent to HsGSDME and DrGSDMEa/b loci on human chromo-
some 1 (top) and zebrafish chromosome 19/16 (bottom). Arrows indicate gene orientation. H and I, phylogenetic analysis of the relationship of NLRP3 and
GSDME between fish and other species. The phylogenetic tree was constructed by MEGA (version 5.0) by using the maximum likelihood method. Each node
reliability was estimated by bootstrapping with 2000 replications. J, quantitative RT-PCR analysis of the expression patterns of DrNLRP3 and DrGSDMEa/b
genes in adult zebrafish tissues. K, expression patterns of DrNLRP3 and DrGSDMEa/b genes in zebrafish embryos at different developmental stages. The relative
expression levels of the genes was calculated by the 2⫺⌬Ct method with ␤-actin for normalization. Each data point shows the mean ⫾ S.D. with three replicates
representative of three independent experiments.
naturally have minimal expression of inflammasome compo- DrCaspase-B activation. The DrNLRP3-⌬LRR mutants
nents. As expected, DrNLRP3 and DrASC coexpression signif- restrained DrCaspase-A but not DrCaspase-B activation.
icantly augmented (p ⬍ 0.01) the DrCaspase-A/-B activity in a Conversely, DrNLRP3-⌬B30.2 did not influence either
DrNLRP3 dose-dependent manner (Fig. 2, A and B). However, DrCaspase-A or DrCaspase-B (Fig. 2C). Western blot analysis
the DrNLRP3 and DrCaspase-A/-B coexpression in the absence showed the self-cleavage of 45/47-kDa pro-DrCaspase-A/-B
of DrASC dramatically promoted (p ⬍ 0.001) DrCaspase-B but into a 35-kDa hydrolytic product (p35) if pro-DrCaspase-A/-B
not DrCaspase-A activation, with a maximum up-regulation of was coexpressed with DrNLRP3 and DrASC (Fig. 2, D and E).
up to 180% (Fig. 2B). The DrNLRP3-⌬PYD and DrNLRP3- By contrast, no p35 was detected without the coexpression of
⌬NACHT (lacking both NACHT and FISNA) mutant proteins either DrNLRP3 or DrASC. The DrNLRP3 mutants that lacked
significantly impaired (p ⬍ 0.01 or p ⬍ 0.001) DrCaspase-A and PYD, NACHT-FISNA, and LRR domains failed to induce pro-
1122 J. Biol. Chem. (2020) 295(4) 1120 –1141

Figure 2. DrNLRP3 involvement in the activation of DrCaspase-A and DrCaspase-B. A, DrNLRP3 and DrASC activate DrCaspase-A detected by the specific
Ac-YVAD-AFC fluorescent substrate. Each data point shows the mean ⫾ S.D. with three replicates. *, p ⬍ 0.05. B, DrNLRP3 and DrASC activate DrCaspase-B
detected by specific Ac-WEHD-AFC fluorescent substrate. Each data point shows the mean ⫾ S.D. with three replicates (*, p ⬍ 0.05; **, p ⬍ 0.01; ***, p ⬍ 0.001).
C, DrNLRP3 and its domain-lacking mutants simultaneously activate DrCaspase-A and DrCaspase-B detected by Ac-YVAD-AFC or Ac-WEHD-AFC fluorescent
substrates. Data are representative of three independent experiments as mean ⫾ S.D. (*, p ⬍ 0.05; ***, p ⬍ 0.001). D and E, Western blotting assay of
DrCaspase-A (D) and DrCaspase-B (E) auto-hydrolyzation when coexpressed with DrNLRP3 and DrASC. F, Western blotting assay of the DrCaspase-A and
DrCaspase-B auto-hydrolyzation when coexpressed with DrNLRP3 mutants and DrASC. G, IP assay shows the DrNLRP3 interaction with DrASC through the PYD
domain. HEK293T cells were transfected with pCMV-Tag2B–DrNLRP3/DrNLRP3-⌬PYD and pCMV-HA-DrASC for 48 h. Cell lysates were immunoprecipitated
with rabbit anti-Flag Ab and analyzed by Western blotting by using mouse anti-Flag or anti-HA against DrNLRP3 or DrASC, respectively (top panel). Expression
of the transfected plasmids was analyzed with anti-Flag or anti-HA Ab in the whole-cell lysates (bottom panels). H, coIP assay shows the protein–protein
interactions among DrCaspase-A (CasA-5DA), DrASC (DrASC-⌬PYD/⌬CARD), and DrNLRP3 (WT). I, coIP assay reveals the protein–protein interactions among
DrCaspase-B (CasB-4DA), DrASC (DrASC-⌬PYD/⌬CARD), and DrNLRP3 (WT). The results are representative of three independent experiments. Caspase activity
was detected and expressed as the fold induction over the control as described under “Experimental procedures.”
J. Biol. Chem. (2020) 295(4) 1120 –1141 1123

DrCaspase-A/-B hydrolyzation (Fig. 2F). Coimmunoprecipita- underlying the PYD–PYD and CARD–CARD homotypic inter-
tion (co-IP) assay revealed the protein–protein interaction action are conserved from fish to mammals.
among DrNLRP3, DrASC, and DrCaspase-A/-B (Fig. 2, G–I).
The DrNLRP3-⌬PYD and DrASC-⌬PYD mutants lacked such DrNLRP3-DrASC inflammasome recruits DrCaspase-A/-B in a
an ability, whereas DrASC–⌬CARD maintained the activity sequential manner
(Fig. 2, H and I). Overall, DrNLRP3 could activate pro- Immunofluorescence results revealed perfect DrCaspase-
DrCaspase-A/-B by self-hydrolyzation in a DrASC-dependent A/-B colocalization with the DrASC speck, suggesting
manner and initiate pro-DrCaspase-B activation in a DrASC- DrNLRP3-DrASC inflammasome as a platform for DrCaspase-
independent manner without undergoing self-hydrolyzation. A/-B recruitment (Fig. 4, A and B). Nevertheless, DrCaspase-A
The pro-DrCaspase-A/-B self-hydrolyzation depends on the and DrCaspase-B are almost independently localized in an
association between DrNLRP3 and DrASC via the PYD–PYD inflammasome, and only one inflammasome can be assembled
homotypic interaction in which NACHT and LRR domains in one cell (Fig. 4C). The percentage of DrCaspase-A–
were included. associating cells (⬃20%) was higher than that of DrCaspase-B–
associating cells (⬃7%). By introducing two chimera bPYD–
DrNLRP3 triggers DrASC nucleation and inflammasome CasA and aPYD–CasB caspases in which the PYD of
formation DrCaspase-A and DrCaspase-B was replaced by each other,
these two caspases were still separately colocalized with the
A DrASC nucleation (i.e. speck formation) assay was per-
DrASC speck, whereas the percentage of aPYD–CasB-associat-
formed in HEK293T cells to observe whether DrNLRP3 could
ing cells (⬃28%) exceeded that of bPYD–CasA-associating cells
organize an inflammasome. When DrNLRP3 or DrASC was
(⬃6%). This finding indicated that the privilege of DrCaspase-A
expressed alone in cells, the fluorescence was a weak signal that
was deprived by DrCaspase-B when their PYD domains were
diffused throughout the cell (Fig. 3A and Fig. S2, A and B). With
exchanged (Fig. 4D). Given that DrASC PYD shared a higher
the DrNLRP3 and DrASC coexpression, DrNLRP3 associates
similarity (82.92%) to DrCaspase-A PYD compared with that of
DrASC into a speck structure with a size of 1.84 ⫾ 0.55 ␮m in
DrCaspase-B (55.46%) (Fig. S1C), the priority of DrCaspase-A
diameter (Fig. 3, B and C). In some cases, it was observed that into DrNLRP3-DrASC inflammasome might be determined by
DrNLRP3 formed an outer ring around DrASC (Fig. S2C). This the high degree of similarity, which provided strong hydropho-
speck organization was also observed in zebrafish ZF4 cells bic (from Leu16, Leu21, Ile49, Val57, and Ile75) and surface charge
(Fig. S2D). The DrNLRP3-⌬PYD, DrNLRP3-⌬NACHT, and (from Arg22, Lys23, Glu43, and Asp50) effects on the homotypic
DrNLRP3-⌬LRR mutants blocked the speck organization, PYD–PYD interaction. The fluorescence recovery after photo-
whereas the DrNLRP3-⌬B30.2 did not have an influence (Fig. bleaching (FRAP) assay revealed that DrCaspase-A/-B dis-
3D and Fig. S2E). The expression of truncated DrASC-⌬PYD or played fluorescence recovery in foci within the inflammasome
DrASC-⌬CARD induced filament (ASCCARD or ASCPYD) for- in ⬃150 s after photobleaching (Fig. 4, E, F, and H). This out-
mation instead of speck organization (Fig. 3E). DrASCPYD come supported the dynamic recruitment of DrCaspase-A/-B
rather than the DrASCCARD filament could be colocalized with into the inflammasome. By contrast, DrNLRP3 and DrASC did
DrNLRP3 (Fig. 3, F and G). Functionally, only DrNLRP3 and not show fluorescence recovery (Fig. 4, G and H). This obser-
DrASC coexpressed in cells could activate DrCaspase-A (Fig. vation indicated that DrNLRP3-DrASC inflammasome main-
3H). This activity was competitively inhibited by introducing tained a stable structure that remained unmoved once it is orga-
DrASC-⌬PYD but not DrASC-⌬CARD in a ratio-dependent nized. Collectively, DrCaspase-A and DrCaspase-B were
manner (Fig. 3I). Thus, the DrNLRP3-DrASC inflammasome dynamically and sequentially recruited into the DrNLRP3-
formation might be started by a linker DrASC, which was asso- DrASC inflammasome, with preference for DrCaspase-A, fol-
ciated with the DrNLRP3 disk via the PYD–PYD interaction lowed by a replacement of DrCaspase-B after DrCaspase-A was
and recruited another DrASC via the CARD–CARD interac- released from the inflammasome.
tion to form a DrASC filament. The DrASC filament was com-
posed of a DrASCCARD core and DrASCPYD cluster. The latter DrNLRP3 contributes to proDrIL-1␤ maturation in a DrASC-
finally recruited DrCaspase-A/-B via the PYD–PYD association dependent manner
(Fig. S3, A and B). From the tertiary structures of ASCPYD and The above results showed that DrNLRP3 acted as an initiator
ASCCARD cores, six PYDs or four CARDs interacted with each that organized a DrASC-dependent DrNLRP3 inflammasome,
other by PYD–PYD or CARD–CARD homotypic interaction to leading to the self-cleavage and activation of DrCaspase-A/-B.
form a circular helix in one layer of the filament core (Fig. S3C). This phenomenon might further contribute to proDrIL-1␤
This phenomenon explained that the DrASCPYD filament was maturation. For clarification, DrNLRP3, DrASC, DrCaspase-
thicker than the DrASCCARD filament under a confocal micro- A/-B, and proDrIL-1␤ were coexpressed in HEK293T cells in
scope (Fig. 3, E–G). With the alignment of DrASC, MmASC, different combinations, and proDrIL-1␤ maturation was deter-
and HsASC, some surface electrostatic amino acids, including mined through Western blot analysis. As expected with the
Glu13, Lys21, Arg38, Arg41, Asp48, Asp51, and Asp54 in ASCPYD DrNLRP3, DrASC, and DrCaspase-A/-B coexpressions, pro-
and Arg125, Glu130, Asp134, Tyr146, Arg150, Arg160, and Asp191 in DrIL-1␤ (31 kDa) was cleaved into an 18-kDa mature form,
ASCCARD that perform important roles in mammalian PYD or accompanied by the pro-DrCaspase-A/-B (45/47 kDa) activa-
CARD fibrillation, are also highly conserved in zebrafish (Fig. tion through self-cleavage into a p35 product (Fig. 5A). How-
S3, C and D). This condition suggested that the mechanisms ever, proDrIL-1␤ was partially processed into a 20-kDa product
1124 J. Biol. Chem. (2020) 295(4) 1120 –1141

Figure 3. Aggregation of DrASC-dependent DrNLRP3 inflammasome. A, transient transfection of pCMV-Myc-DrASC or pCMV-Tag2B-DrNLRP3 in HEK293T
cells, and diffuse fluorescent signals were detected in the cells. B, DrNLRP3 and DrASC coexpression in HEK293T cells. Flag-DrNLRP3 signal (red) and Myc-DrASC
signal (green) accumulate in the same speck. C, confocal microscopy image of DrNLRP3-DrASC speck in ZF4 cells transfected with pCMV-Myc-DrASC and
pCMV-Tag2B-DrNLRP3 by electroporation. D, statistics of DrASC speck-forming rates induced by DrNLRP3 and its mutants. More than 100 cells with DrASC
speck were counted in each experimental group to quantify DrNLRP3-dependent DrASC nucleation. Fig. S2E shows the original immunofluorescence images.
Data are representative of three independent experiments as mean ⫾ S.D. (**, p ⬍ 0.01). E, immunofluorescence examination of DrASCPYD/CARD filament when
transiently transfected by Myc-tagged DrASC-PYD (DrASC-⌳CARD) or DrASC-CARD (DrASC-⌳PYD) in HEK293T cells. F and G, with the DrNLRP3 and DrASC
coexpression, DrNLRP3 was colocalized with the DrASCPYD filament (F) but not with the DrASC CARD filament (G). H, DrNLRP3 coexpressed with DrASC-⌳CARD
or DrASC-⌳PYD cannot activate DrCaspase-A when being detected by the specific Ac-YVAD-AFC. Each data point shows the mean ⫾ S.D. with three replicates
(*, p ⬍ 0.05). I, DrASC-⌳PYD but not DrASC-⌳CARD interacts with the linker ASC and inhibits the DrCaspase-A activation by DrNLRP3 and DrASC. Images were
captured under a laser-scanning confocal microscopy (Zeiss LSM-710; original magnification, ⫻630; scale bars represent 5 or 10 ␮m). The results are represen-
tative of three independent experiments as mean ⫾ S.D. (*, p ⬍ 0.05).
J. Biol. Chem. (2020) 295(4) 1120 –1141 1125

Figure 4. Recruitment of DrCaspase-A/-B into DrNLRP3-DrASC inflammasome in a sequential manner. A and B, DrNLRP3-HA, DrASC-Myc, and DrCaspase-
A-Flag (A) or DrCaspase-B-Flag (B) coexpression in HEK293T cells elicited the DrCaspase-A/-B colocalization with the DrASC speck. C, DrNLRP3-HA, DrASC-HA,
DrCaspase-A-Myc, and DrCaspase-B-Flag coexpression in HEK293T cells elicited the formation of DrCaspase-B (white arrowheads) or DrCaspase-A (white arrows)
specks in the cells. D, coexpression of DrNLRP3-HA, DrASC-HA, aPYD–CasB-Flag, and bPYD–CasA-Myc in HEK293T cells elicited the formation of aPYDCasB
(white arrowheads) or bPYDCasA (white arrows) specks in the cells. E–G, FRAP of DrNLRP3 inflammasome. Bleaching was performed after HEK293T cells stably
expressing DrNLRP3–GFP, DrASC–GFP, DrCaspase-A–RFP, or DrCaspase-B–RFP. Time-lapse micrographs of DrNLRP3–DrASC–DrCaspase-A (E), DrNLRP3–
DrASC–DrCaspase-B (F), and DrNLRP3–DrASC (G) punctum formation after bleaching. Arrows indicate punctum. Scale bar, 5 or 10 ␮m. These images are
representative of at least 10 photobleached cells mentioned previously. H, fluorescence intensities of DrNLRP3–DrASC and DrCaspase-A/-B specks over the
time course of 5 min after bleaching. Each data point shows the mean ⫾ S.D. with at least three replicates.
1126 J. Biol. Chem. (2020) 295(4) 1120 –1141

Figure 5. DrNLRP3 contribution to the proDrIL-1␤ maturation in a DrASC-dependent manner. HEK293T cells were transfected with a pcDNA3.1–DrIL-1␤
construct alone or with pCMV–DrNLRP3, pCMV–DrNLRP1, pCMV–DrASC, pCMV–DrCaspase-A, and pCMV–DrCaspase-B. At 24 h post-transfection, immunoblot
analysis was performed on the cell lysates with mouse anti-Flag or anti-Myc monoclonal Ab. A, ProDrIL-1␤ cleavage triggered by activated DrCaspase-A and
DrCaspase-B. B, ProDrIL-1␤ cleavage triggered by DrNLRP3–DrASC-activated DrCaspase-A alone. C, ProDrIL-1␤ cleavage triggered by DrNLRP3–DrASC-acti-
vated DrCaspase-B alone. Blots were re-probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. The results are representative
of three independent experiments, as described under “Experimental procedures.” Bar charts under A–C showed the relative density of the cleavage product
of DrIL-1␤ in the blots. Each data point shows the mean ⫾ S.D. with three replicates.
(the first cleavage product at the Asp104 residue of proDrIL-1␤) activated DrCaspase-B alone failed in the proDrIL-1␤ cleavage
by the activated DrCaspase-A alone (Fig. 5B). This product (Fig. 5C). In the absence of DrNLRP3 and/or DrASC, no any
coexisted with several other mid-forms (25–30 kDa) known as mature/partial forms of proDrIL-1␤ (18/20 kDa) and
the cleavage products of some other proteases existing in cells, DrCaspase-A/-B (35 kDa) were detected. These findings indi-
including neutrophil elastase, proteinase 3, cathepsins G/D, cated that DrNLRP3-DrASC inflammasome contributed to
granzyme A, and matrix metalloproteinases. By contrast, the pro-DrCaspase-A/-B activation through self-hydrolyzation,
J. Biol. Chem. (2020) 295(4) 1120 –1141 1127

which leads to proDrIL-1␤ maturation. Interestingly, the DrCaspase-A. Furthermore, 11 basic amino acids
DrIL-1␤ maturation could be enhanced when DrNLRP1 was (Lys17, 28, 39, 44, 73 and Arg21, 37, 46, 70, 77, 80) and 11 acidic
introduced into the DrNLRP3 inflammasome (Fig. 5A). In this amino acids (Asp6, 45, 48, 51, 60 and Glu9, 13, 15, 39, 41, 82) are con-
case, a chimera inflammasome with DrNLRP3 and DrNLRP1 served in PYDs of DrNLRP3 and DrCaspase-B (Fig. S1D). The
was detected, which perfectly encircled the ASC speck (Fig. S4, ratio of negatively-charged residues to positively charged resi-
A–D). Functionally, DrNLRP3 was found to preferentially acti- dues in PYDs is 1:2 for DrNLRP3 and DrCaspase-A but 1:1 for
vate DrCaspase-B, and DrNLRP1 preferred DrCaspase-A (Fig. DrNLRP3 and DrCaspase-B. This result means that isoelectric
S4, E and F). When DrNLRP3 associates with DrNLRP1, the repulsion hardly exists between the PYDs of DrNLRP3
DrCaspase-A and DrCaspase-B activation was enhanced syn- and DrCaspase-B compared with that of DrNLRP3 and
chronously (Fig. S4, G and H). This functional compensation DrCaspase-A. This phenomenon explains a strong interaction
might contribute to the enhancement of DrIL-1␤ maturation. between DrNLRP3 and DrCaspase-B. Structurally, the PYD of
DrNLRP3 is much more similar to that of DrCaspase-B rather
DrNLRP3 organizes DrCaspase-B to activate DrGSDMEa/b in a than DrCaspase-A (Fig. S1E). This condition is beneficial to the
DrASC-independent manner association of DrNLRP3 with DrCaspase-B through the PYD–
Although the DrNLRP3-DrASC inflammasome contributed PYD interaction. Indeed, Co-IP assay clearly shows the interac-
to DrIL-1␤ maturation in cells, a minimal amount of mature tion between DrNLRP3 and DrCaspase-B but not between
DrIL-1␤ was detected in the supernatant out of the cells. This DrNLRP3-⌬PYD and DrCaspase-B-⌬PYD mutants (Fig. 6I).
phenomenon might be attributed to the absence of GSDMD/ With the lack of PYD and NACHT domains, DrNLRP3 cannot
GSDME mediated membrane perforation, which was essential interact and activate DrCaspase-B, which decreases the
for cell pyroptosis and IL-1␤ secretion. Thus, DrGSDMEa/b DrGSDMEb-mediated cytotoxicity, whereas the lack of LRR
was introduced in the study. As expected, DrGSDMEa/b coex- and B30.2 domains does not affect cytotoxicity (Fig. 6, J and K).
pression with DrNLRP3, DrASC, and DrCaspase-A/-B signifi- The DrNLRP3-⌬LRR mutant inhibits DrASC nucleation and
cantly induced pyroptosis, as determined by cytotoxicity pro-DrCaspase-A/-B hydrolyzation but maintains the activa-
accompanied with lactate dehydrogenase (LDH) release and tion DrCaspase-B in full length (Figs. 2, C and F, and 3D), fur-
pyroptotic morphology (Fig. 6, A and B). DrASC and ther demonstrating that DrNLRP3 organizes full-length
DrCaspase-A removal from the components did not impair DrCaspase-B to activate DrGSDMEa/b in a DrASC-inde-
pyroptosis, whereas DrCaspase-B withdrawal significantly pendent manner.
inhibited pyroptosis. Strikingly, the cells that expressed
DrNLRP3, DrCaspase-B, and DrGSDMEa/b induced signifi- DrNLRP3 coordinates DrIL-1␤ maturation and cell pyroptosis
cant pyroptosis, in which DrGSDMEb had a higher activity Given that DrIL-1␤ maturation occurs in a DrNLRP3-
(⬎50%) than DrGSDMEa (Fig. 6A). In this case, DrGSDMEa/b DrASC– dependent manner, whereas DrGSDMEa/b triggers
was cleaved into an N-terminal product by full-length DrIL-1␤ release and cytotoxicity/pyroptosis through a DrASC-
DrCaspase-B without undergoing self-cleavage in the absence independent way, the tight coupling of these two events was
of DrASC. This phenomenon was evidenced by the observation further explored. DrCaspaseA-5DA and DrCaspaseB-4DA
that a quaternary mutant DrCaspase-B (Caspase-B– 4DA) also mutants that lack autocleavage sites were introduced to exclude
cleaved DrGSDMEa/b into an N-terminal product (Fig. 6C). the DrNLRP3-DrASC– dependent reaction that depends on
This Caspase-B– 4DA was generated by using alanine to substi- DrCaspase-A/-B autocleavage but retains DrASC-independent
tute the four potential autocleavage sites (Asp130, 137, 308, 314) to reaction that is independent of DrCaspaseB cleavage. When
prevent DrCaspase-B from self-cleavage. The full-length cells were coexpressed with a combination of DrNLRP3,
DrCaspase-B activity was also examined using DrCaspase-B DrASC, DrCaspase-A, DrCaspase-B, proDrIL-1␤, and
fluorescent substrate and DrGSDMEa/b-mediated pyroptosis DrGSDMEb, a complete proDrIL-1␤ cleavage product (18 kDa)
(Fig. 6, D and E). In addition, the DrCaspase-B P20 and P10 occurred, accompanied by the DrIL-1␤ secretion and LDH
combination showed weak DrGSDMEb cleavage and pyropto- release (Fig. 7). When DrCaspase-B was replaced by CaspaseB-
sis (Fig. 6, F and G). These findings indicated that the associa- 4DA in the combination, the proDrIL-1␤ was only cleaved into
tion of DrNLRP3 with full-length DrCaspase-B was sufficient the 20-kDa–mediated form that was merely processed by the
for DrGSDMEa/b activation to elicit pyroptosis. DrNLRP3 activated DrCaspase-A in a DrNLRP3-DrASC– dependent
PYD shared an 89.33% similarity with DrCaspase-B PYD, and manner (Fig. 7A). This process declined the DrIL-1␤ secretion
this value was higher than those of DrASC (55.46%) and but did not influence the LDH release, suggesting that
DrCaspase-A (49.76%) (Fig. S1C). Consequently, DrNLRP3 was DrGSDMEb-mediated perforation occurred in a DrCaspaseB
supposed to preferentially associate with DrCaspase-B without noncleavage manner that is DrASC-independent (Fig. 7, B and
intervention of DrASC and DrCaspase-A through a more C). This finding means that the DrASC-dependent DrIL-1␤
homotypic PYD–PYD interaction. As a support, the chimera maturation and DrASC-independent DrIL-1␤ secretion and
DrCaspase-B (aPYD–CasB), whose PYD was replaced with that cell pyroptosis were coordinated by DrNLRP3. The results from
of DrCaspase-A, weakly interacted with DrNLRP3 and trig- other combinations provide further support for this conclu-
gered DrGSDMEb-mediated pyroptosis (Fig. 6H). sion. For example, when DrCaspase-A was replaced by Caspa-
In multiple alignment analysis, three basic amino acids seA-5DA, DrIL-1␤ could not be cleaved and released, although
(Lys37, 44 and Arg70) and six acidic amino acids (Asp6, 45, 48, 51, 60 cytotoxicity still appeared. A similar result was also observed
and Glu41) are conserved in PYDs of DrNLRP3 and when DrCaspase-A and DrCaspase-B were replaced by Caspa-
1128 J. Biol. Chem. (2020) 295(4) 1120 –1141

J. Biol. Chem. (2020) 295(4) 1120 –1141 1129

Figure 7. Coordination of DrNLRP3 inflammasome between mature-formed DrIL-1␤ secretion and cell pyroptosis. A, HEK293T cells were transfected
with a pcDNA3.1-DrIL-1␤ construct with pCMV-DrNLRP3, pCMV-DrASC, pCMV-DrCaspase-A (pCMV-CasA-5DA), pCMV-DrCaspase-B (pCMV-CasB-4DA), and
pCMV-DrGSDMEb. Immunoblot analysis was performed at 24 h post-transfection on the cell lysates to detect the proDrIL-1␤ cleavage triggered by DrNLRP3
inflammasome. B, supernatants from the indicated cells were analyzed for cell death, as measured by the LDH release. C, levels of soluble IL-1␤ in culture
supernatants were analyzed by ELISA by using DrIL-1␤ polyclonal antibody. Each data point shows the mean ⫾ S.D. with three replicates. **, p ⬍ 0.01;
***, p ⬍ 0.001.
seA-5DA and CaspaseB-4DA together or with the DrASC DrGSDMEb expression did not influence the DrIL-1␤ matura-
removed from the combination. Without the DrNLRP3 expres- tion but markedly decreased the cytotoxicity and DrIL-1␤
sion, no detectable cytotoxicity, DrIL-1␤ maturation, and release. A schematic diagram for the two types of DrNLRP3
secretion were observed. Meanwhile, the absence of inflammasome is presented in Fig. 8.
Figure 6. DrNLRP3 activates DrCaspase-B to cleave DrGSDMEa/b in a DrASC-independent manner. A, HEK293T cells were transfected with different
combinations of plasmids of DrNLRP3, DrASC, DrCaspase-A/-B, and DrGSDMEa/b. Supernatants from the indicated cells were analyzed for cell death, as
measured by LDH release. B, images were taken after 4.5 ␮M PI were added to the indicated cells. The dyed cells indicate the loss of plasma membrane integrity
and exhibit pyroptotic-like features. Scale bar, 10/100 ␮m. C, DrGSDMEa/b cleavage by DrNLRP3 and DrCaspase-B (or the mutant CasB-4DA) was analyzed by
immunoblotting. D, DrNLRP3 activating DrCaspase-B or CasB-4DA was detected by specific Ac-WEHD-AFC fluorescent substrate. E, supernatants from the
indicated cells were analyzed for cell death, as measured by the LDH release. F, supernatants from the indicated cells coexpressed with different combinations
of DrNLRP3 and DrCaspase-B (including CaspB-P35, CaspB-P20, and CaspB-P10) were analyzed for cell death, as measured by LDH release. G, DrGSDMEb
cleavage by DrNLRP3 and DrCaspase-B in different combinations of CaspB-P35, CaspB-P20, and CaspB-P10 was analyzed by immunoblotting. H, supernatants
from the HEK293T cells transfected with PCMV-GSDMEb/Caspase-B/aPYD–CasB/NLRP3 were analyzed for cell death, as measured by LDH release. I, protein–
protein interactions between DrNLRP3 and DrCaspase-B but not their PYD-lacking mutants. J, DrNLRP3 and mutants activate DrCaspase-B detected by specific
Ac-WEHD-AFC fluorescent substrate. K, supernatants from the indicated cells were analyzed for cell death, as measured by the LDH release. All the above results
are representative of at least three independent experiments, and error bars denote the S.D. of triplicate wells. *, p ⬍ 0.05; **, p ⬍ 0.01; ***, p ⬍ 0.001.
1130 J. Biol. Chem. (2020) 295(4) 1120 –1141

Figure 8. Schematic for the functional roles of DrNLRP3 inflammasome in DrASC-dependent and DrASC-independent manners.
In vivo examination of DrNLRP3 inflammasome LPS and H2O2 but not by muramyl dipeptide (MDP), bacte-
DrNLRP3 inflammasome was evaluated in vivo in the rial DNA, and ATP, although the latter exhibited stimulatory
zebrafish embryo model. The DrNLRP3 inflammasome occur- effects on DrCaspase-A/-B activation at varying degrees in
rence is optimized at 6 hpf embryos or 72 hpf larvae with the embryos without DrNLRP3 knockdown (Fig. 9, K and L).
Edwardsiella tarda immersion infection (108 CFU/ml) for 1– 4 This finding suggests the contribution of LPS and cellular
h as determined by significantly increased DrCaspase-A/-B oxidation to DrNLRP3 inflammasome activation. Next, cell
activity at those times (Fig. 9, A and B). DrNLRP3 overexpres- pyroptosis and DrIL-1␤ maturation, two downstream events
sion alone in embryos with infection only augmented endog- of the DrNLRP3 inflammasome, were examined. The pyrop-
enous DrCaspase-B activation. However, DrNLRP3 coex- tosis occurrence was optimized in 72 hpf embryos with
pression with DrASC promoted both DrCaspase-A and E. tarda infection (108 CFU/ml) for 4 h. Therein, the expres-
DrCaspase-B activation, accompanied by an increased sion of DrNLRP3, DrCaspase-B, and DrGSDMEb genes but
DrIL-1␤ maturation (Fig. 9C). These observations support not apoptosis-associated genes (such as Fas/FasL and Bcl2)
the existence of DrASC-dependent and DrASC-independent was significantly (p ⬍ 0.001) augmented, and considerable
types for DrCaspase-A/-B activation and DrIL-1␤ matura- cells presented pyroptotic morphology (Figs. 9M and 10A).
tion. Structurally, DrNLRP3- and DrASC-colocalized specks FCM analysis showed that the percentage of the PI-positive
were clearly observed in the embryos (Fig. 9D). DrNLRP3 cells increased from 8.80 to 34.8% in the embryos upon infec-
knockdown significantly inhibited (p ⬍ 0.05) DrCaspase- tion. This value decreased to 19.7% in DrNLRP3 morphants.
A/-B activation upon E. tarda infection. This inhibition was This result implied the involvement of DrNLRP3 in pyrop-
rescued by MO-resistant mRNAs in a dose-dependent man- tosis (Fig. 10B). However, DrIL-1␤ maturation was sup-
ner (Fig. 9, E, F, H, and I and Fig. S5A). Correspondingly, pressed in DrCaspase-A and DrCaspase-B morphants (Fig.
DrNLRP3 knockdown increased the mortality of embryos 10C), whereas cell pyroptosis declined in DrCaspase-B mor-
with E. tarda infection, which was restored by MO-resistant phants (13.2%) but not in DrCaspase-A morphants (30.6%)
mRNAs (Fig. 9, G and J). In addition, DrCaspase-A/-B acti- (Fig. 10D). This result indicated that pyroptosis depends on
vation was abrogated in DrNLRP3 morphants stimulated by DrCaspase-B rather than DrCaspase-A. Furthermore, a
J. Biol. Chem. (2020) 295(4) 1120 –1141 1131

DrASC mutant was generated by CRISPR/Cas9, resulting in E and F), whereas pyroptosis was not inhibited by the DrASC
transcripts with a nonsense codon within the first exon (Fig. knockout (Fig. 10G). This phenomenon supported the
S5, B–D). DrCaspase-A/-B activation and DrIL-1␤ matura- notion that DrIL-1␤ maturation is DrASC-dependent, and
tion were diminished in the DrASC knockout larvae (Fig. 10, cell pyroptosis is DrASC-independent.
1132 J. Biol. Chem. (2020) 295(4) 1120 –1141

Figure 10. In vivo examination of DrNLRP3 inflammasome in DrASC-independent manner. A, images were taken after PI was added to the 72-hpf
zebrafish larval cells infected by E. tarda (108 CFU/ml) for 4 h. The dyed cells exhibit pyroptotic-like features. Scale bar, 10 ␮m. The sample number for each
group was 20 –100 zebrafish larvae, and each image is representative of three independent experiments. B, flow cytometry with PI detected the cell pyroptosis
in 72-hpf zebrafish larvae (n ⫽ 100) with E. tarda immersion infection (108 CFU/ml) for 4 h. C, DrCaspase-A (CasA-MO) or DrCaspase-B (CasB-MO) knockdown
decreased the DrCaspase-A/-B activation and the DrIL-1␤ maturation in zebrafish embryos (n ⫽ 100) after E. tarda infection. Data are representative of three
independent experiments as mean ⫾ S.D. (**, p ⬍ 0.01). D, flow cytometry with PI detected the cell pyroptosis in CasA-MO or CasB-MO zebrafish larvae (n ⫽
100) with E. tarda immersion infection (108 CFU/ml) for 4 h. E and F, DrASC knockout (ASC-KO) decreased the DrCaspase-A/-B activation (E) and the DrIL-1␤
maturation (F) in zebrafish embryos (n ⫽ 100) after E. tarda infection. Data are representative of three independent experiments as mean ⫾ S.D. (*, p ⬍ 0.05).
G, flow cytometry with PI detected the cell pyroptosis in ASC-KO zebrafish larvae (n ⫽ 100) with E. tarda immersion infection (108 CFU/ml) for 4 h.
Discussion tive chromosomal synteny, gene organization, protein domain

In this study, we have identified an NLRP3 homolog architecture, and tertiary structure to mammalian NLRP3s,
(DrNLRP3) from zebrafish, which shares an overall conserva- except for a B30.2 domain that is unique in fish (28). This
Figure 9. In vivo determination of DrNLRP3 inflammasome. A and B, fluorogenic substrate detection of the DrCaspase-A/-B activation in 6-hpf embryos (A)
or 72 hpf larvae (B) after E. tarda infection at 108 CFU/ml for 0 – 6 h. The sample number for each group was 20 –100 zebrafish embryos or larvae, and each data
point shows the mean ⫾ S.D. with three replicates. C, DrNLRP3 and DrASC coexpression in vivo increased the DrCaspase-A/-B activation level and promoted the
DrIL-1␤ maturation under E. tarda infection in 72-hpf larvae (n ⫽ 100). Data are representative of three independent experiments as mean ⫾ S.D. (*, p ⬍ 0.05;
**, p ⬍ 0.01; ***, p ⬍ 0.001). D, Flag-tagged DrNLRP3 and Myc-tagged DrASC coexpression in vivo triggers the DrNLRP3-DrASC speck nucleation in 72-hpf
zebrafish larvae (n ⫽ 100). E and H, DrNLRP3 knockdown by DrNLRP3-MO decreased the DrCaspase-A/-B activation in 6-hpf embryos (n ⫽ 50) (E) or 72 hpf larvae
(n ⫽ 50) (H) after E. tarda infection for 40 min or 4 h. Each data point shows the mean ⫾ S.D. with three replicates (*, p ⬍ 0.05; **, p ⬍ 0.01). F and I, MO-resistant
DrNLRP3 mRNA rescued the DrCaspase-A/-B activation in 6-hpf embryos (n ⫽ 50) (F) or 72-hpf larvae (n ⫽ 50) (I) after E. tarda infection for 40 min or 4 h. Each
data point shows the mean ⫾ S.D. with three replicates (*, p ⬍ 0.05; **, p ⬍ 0.01). G and J, RSRs of 6-hpf embryos (G) or 72-hpf larvae (J) after E. tarda infection
at 106 CFU/ml for 12 h. Zebrafish embryos were microinjected with standard MO (Control), DrNLRP3-MO (NLRP3-MO), or both with the corresponding mRNA
(NLRP3-(MO⫹mRNA)). Mortality in each group was monitored during the 1-h period at one interval. The results are performed in triplicate with 100 embryos per
group. K and L, evaluation of bacterial LPS, MDP, and DNA and cellular metabolites H2O2 and ATP for the DrNLRP3 inflammasome activation in 6-hpf embryos
(n ⫽ 50) via in vivo knockdown assay. Each data point shows the mean ⫾ S.D. with three replicates (***, p ⬍ 0.001). M, fold change of mRNA levels of the genes
involved in pyroptosis and apoptosis in zebrafish 72-hpf larvae (n ⫽ 20) after E. tarda infection for 4 h. The fold change of the relative expression levels was
calculated by the 2⫺⌬⌬Ct method with ␤-actin for normalization. Data are representative of three independent experiments as mean ⫾ S.D. (*, p ⬍ 0.05; **, p ⬍
0.01; ***, p ⬍ 0.001).
J. Biol. Chem. (2020) 295(4) 1120 –1141 1133

DrNLRP3 can trigger the assembly of a classical inflammasome CasB-4DA) into the experiment. DrGSDMEb possesses a better
speck structure (DrNLRP3-DrASC inflammasome) in a ability to elicit cell pyroptosis compared with DrGSDMEa. The
DrASC-dependent manner, in which DrCaspase-A/-B was LRR domain of DrNLRP3 necessary for DrASC nucleation and
activated by self-cleavage into the p35/p10 subunits and further DrCaspase-A/-B autoproteolysis does not influence the full-
cleaves proDrIL-1␤ at the Asp104/Asp122 residues to generate length DrCaspase-B activation and DrGSDMEb-mediated
an 18-kDa mature-formed DrIL-1␤. The DrNLRP3-DrASC pyroptosis. This phenomenon signifies the existence of a
inflammasome assembly and DrCaspase-A/-B autoproteolytic unique mechanism underlying full-length DrCaspase-B activa-
activation require the PYD, NACHT, and LRR domains of tion. This DrCaspase-B activation might require a conforma-
DrNLRP3 as observed in mammalian NLRP3s, in which the tional transition from an inactive to an active form by some
PYD is believed to recruit ASC, the NACHT bridges oligomer- unknown allosteric triggers. Similar results were also observed
ization, and LRR recognizes stimulus signals (29 –32). Mean- in several other inflammasomes, such as NLRP1, NLRC4, and
while, the B30.2 domain is not functional in inflammasome Nlrp1b, in which Caspase-1/5 was activated without self-cleav-
assembly and DrCaspase-A/-B activation. The DrASC nucle- age (36, 37). For example, by reconstituting Caspase-1 in cells
ation is the key process of DrNLRP3-DrASC inflammasome with a noncleavable mutant, the noncleavable Caspase-1 can be
formation, in which DrNLRP3 first associates with a linker fully activated in response to Nlrp1b activators (38). Consistent
DrASC, which further recruits other DrASCs for their elonga- with this study, the ASC knockout macrophages severely
tion to form a DrASC filament. The PYD cluster of DrASC impaired their ability to process and secrete mature IL-1␤ but
filament finally recruits the PYDs of DrCaspase-A/-B. The promoted rapid cell death as WT macrophages upon bacterial
DrNLRP3-DrASC inflammasome recruits DrCaspase-A/-B infection. In this case, ASC-driven Caspase-1 autoproteolysis is
in a two-step sequential manner, with a preference for differentially required for cytokine maturation and pyroptosis
DrCaspase-A and a subsequent choice for DrCaspase-B. This (36). These observations indicate the universal existence of the
process ensures proDrIL-1␤ maturation that requires cleavage uncleaved caspase activation. Further study is therefore needed
at the Asp104/Asp122 sites by activated DrCaspase-A/-B in turn. to clarify the mechanism behind this type of activation.
This sequential activation is determined by the homotypic How mature-formed IL-1␤ is released from cells is a basic
degree between the PYDs of DrASC and DrCaspases, in which question because of the lack of a signal peptide at the N termi-
the number of conservative hydrophobic and charged amino nus of this molecule. This concern has remained unclear for a
acid residues is included. Notably, the sequential activation of long time until the discovery of the GSDMD/E-mediated pore-
DrCaspase-A/-B is also observed in an NLRP1 inflammasome forming membrane initiated by Caspase-4/5/11- and Caspase-
that we recently identified from zebrafish, suggesting its univer- 3– dependent noncanonical inflammasomes in mammals (16,
sal significance in fish (18). Interestingly, a synergistic effect was 20). This finding suggests a potential correlation between a
detected between DrNLRP3 and DrNLRP1 by forming a chi- noncanonical inflammasome for GSDMD/E activation and a
mera inflammasome, which promoted DrCaspase-A/-B auto- canonical inflammasome for IL-1␤ maturation despite the
proteolytic activation and DrIL-1␤ maturation. The functional poorly understood manual coupling of these two kinds of
collaboration of different inflammasomes was also observed inflammasomes. Our study showed that NLRP3 inflammasome
among AIM2, NLRC4, and NLRP3 in various cells (33–35). alone can perform IL-1␤ maturation and GSDME activation
However, the details of these collaborations remain to be without the help of other inflammasomes. This dual functional
addressed. performance of DrNLRP3 enables a coordinator between DrIL-
Meanwhile, DrNLRP3 possesses the ability to directly recruit 1␤–induced inflammation and DrGSDME-mediated pyropto-
and activate DrCaspase-B, but not DrCaspase-A, without sis. To date, numerous gasdermin family proteins, such as
undergoing autoproteolysis and the help of DrASC. This full- GSDMD, GSDMA, GSDMB, GSDMC, and GSDME, are acti-
length DrCaspase-B activated by DrNLRP3 alone contributes vated by Caspase-4/5/11 and Caspase-3 noncanonical inflam-
to the DrGSDMEa/b cleavage. This situation induces DrIL-1␤ masomes to induce pyroptosis in mammals (4). However, only
secretion and cell pyroptosis. However, the full-length two GSDMEa/b isoforms and a DFNB59 family member exist
DrCaspase-B fails in proDrIL-1␤ cleavage, and the latter in zebrafish and other teleost fish. DrGSDMEa/b becomes the
requires DrCaspase-A/-B autoproteolysis in the presence of major candidate that mediates pyroptosis in fish because
DrASC. Thus, it is evident that DrNLRP3 participates in pro- DFNB59 lacks an Asp cleavage site and cannot be cleaved into
DrIL-1␤ maturation and DrIL-1␤ secretion followed by cell an effective N-terminal domain (4, 20). This finding means that
pyroptosis in two different ways. Specifically, proDrIL-1␤ mat- GSDME is an ancient gasdermin member involved in pyropto-
uration requires DrNLRP3-DrASC inflammasome formation sis during vertebrate evolution. Thus, teleost fish can become
and depends on DrCaspase-A/-B autoproteolytic activation in a an attractive model population to understand the evolutionary
DrASC-dependent manner. However, DrIL-1␤ secretion and correlation between gasdermin proteins and inflammatory
cell pyroptosis require the occurrence of a DrNLRP3– caspases regulated by various noncanonical and/or canonical
DrCaspase-B complex, in which DrCaspase-B was activated in a inflammasomes.
full-length form to cleave DrGSDMEa/b in a DrASC-indepen- The DrNLRP3 inflammasome is finally examined in vivo by
dent manner. The mature-formed DrIL-1␤ can finally be using the zebrafish embryo model. Typical DrNLRP3-DrASC
released from the cell only when these two types are tightly inflammasome was detected in embryos, which triggers
coordinated. These conclusions were supported by introducing DrCaspase-A/-B activation, DrIL-1␤ maturation, and cell
two noncleavable DrCaspase-A/-B mutants (CasA-5DA and pyroptosis under E. tarda infection. These activities can be
1134 J. Biol. Chem. (2020) 295(4) 1120 –1141

Figure 11. Functional substitution of mouse NLRP3 (MmNLRP3) to DrNLRP3 in DrASC nucleation and cell pyroptosis. A, DrASC and DrNLRP3 coexpres-
sion in HEK293T cells elicits DrASC speck aggregation. B, DrASC and MmNLRP3 coexpression instead of DrNLRP3 also elicits DrASC speck aggregation. C,
speck-forming rates of DrNLRP3 or MmNLRP3 with DrASC. Data are representative of three independent experiments as mean ⫾ S.D. (**, p ⬍ 0.01). D, DrNLRP3
or MmNLRP3 directly activates DrCaspase-B and triggers DrGSDMEb-dependent cell pyroptosis. Images were captured under a laser-scanning confocal
microscopy (Zeiss LSM-710; original magnification, ⫻630, scale bars represent 50 or 10 ␮m). The DrCaspase-B activity was detected and expressed as the fold
induction over the control as described under “Experimental procedures.” Each data point shows the mean ⫾ S.D. with three replicates. *, p ⬍ 0.05; **, p ⬍ 0.01;
***, p ⬍ 0.001.
diminished by antisense MO-based DrNLRP3 knockdown and independent but DrCaspaseB-dependent pyroptotic cell death
rescued by MO-resistant mRNAs. Meanwhile, the high mortal- (Fig. 11). This result implies the conservation between fish and
ity of DrNLRP3 morphants with E. tarda infection is restored mammalian NLRP3 inflammasomes throughout vertebrate
by administering MO-resistant mRNAs. These findings verify evolution.
the functional roles of DrNLRP3 inflammasome in antibacterial In conclusion, our study demonstrates the origin of NLRP3
immunity in vivo. Furthermore, by generating a DrASC inflammasome in teleost fish and reveals its role in ASC-depen-
CRISPR mutant (DrASC⫺/⫺) with a 2-bp deletion in zebrafish dent IL-1␤ maturation and GSDME-mediated pyroptosis. This
by using Cas9/gRNA technology, it was found that the finding enriches our current knowledge on NLRP3 inflam-
DrASC⫺/⫺ mutant only abrogates the DrIL-1␤ maturation but masome biology. Given that the NLRP3 inflammasome is also
maintains the ability of cell pyroptosis. This result confirms the closely-associated with various diseases, such as type II diabetes
existence of a DrASC-independent pyroptosis pathway in vivo. and inflammatory bowel disease (41, 42), teleost fish will
Mechanistically, LPS and H2O2 administration into the become a new research model for these kinds of diseases.
embryos significantly activates the DrNLRP3 inflammasome.
This outcome suggests that pathogen-associated molecular Experimental procedures
patterns and cellular metabolic homeostasis (such as redox
state) can elicit the DrNLRP3 inflammasome activation as Experimental fish and embryo
observed in mammalian NLRP3 inflammasomes (39, 40). Inter- WT AB zebrafish (Danio rerio) with body lengths of 3– 4 cm
estingly, the mouse NLRP3 (MmNLRP3) can replace DrNLRP3 and weights of 0.5–1.0 g were maintained in circulating water at
for DrASC-dependent inflammasome aggregation and DrASC- 28 °C under standard conditions. The fish that exhibited
J. Biol. Chem. (2020) 295(4) 1120 –1141 1135

healthy appearance and activity were used for the study. Plasmid constructions
Zebrafish embryos were collected at optimized hours post-fer- The full-length encoding sequences of DrNLRP3 and
tilization (6 –96 hpf). The experiments were conducted in DrGSDMEa/b and the partial encoding sequences of DrNLRP3
accordance with legal regulations and ethical approval. with the deletion of PYD, NACHT, LRR, and B30.2 domains
were inserted into pCMV (Stratagene) or pcDNA3.1 (Invitro-
Molecular cloning gen) vectors with Flag/Myc/HA-tags at the N/C termini. The
The Genome and Expressed Sequence Tags (EST) databases mutant constructs were named pCMV-DrNLRP3-⌬PYD,
maintained by the National Center for Biotechnology Informa- pCMV-DrNLRP3-⌬NACHT, pCMV-DrNLRP3-⌬LRR, and
tion (NCBI), the University of California Santa Cruz (UCSC), pCMV-DrNLRP3-⌬B30.2. A quinary mutant of DrCaspase-B
and Ensembl were used to predict and confirm NLRP3 and (named as CaspaseB-4DA) in which four aspartic acids (Asp130,
GSDME homologs in zebrafish, as described previously (43). Asp137, Asp308, and Asp314) were substituted by alanines was
Briefly, with the PYD and NACHT domain sequence of human constructed using a QuikSite-directed mutagenesis kit (Beyo-
NLRP3 as a query, a candidate DrNLRP3 gene named si:dkey- time). DrASC, DrCaspase-A, DrCaspase-B, proDrIL-1␤, and
156m2.3 was retrieved from the zebrafish genome and EST DrNLRP1 encoding constructs and their mutant constructs,
databases by using Genscan and Ensemble (TBLASTN) pro- including DrASC-⌬PYD, DrASC-⌬CARD, aPYD–CasB,
grams. With the full-length sequences of human GSDME or bPYD–CasA, and CaspaseA-5DA, were generated in our previ-
gasdermin D (GSDMD) as queries, three candidate gasdermin ous study. The plasmids for transfection and microinjection
members (i.e. DrGSDMEa, DrGSDMEb, and DrDFNB59) were were prepared free of endotoxin by using endo-free plasmid
predicted from the zebrafish genome database. Given that mini kit II (Omega Bio-tek). The primers used in cloning and
mammalian GSDME can trigger cell pyroptosis, but DFNB59 construct generation are listed in Table S1.
cannot, due to its inability to cleave the N-terminal structure
by lacking an aspartic acid cleavage site, DrGSDMEa and Quantitative real-time PCR
DrGSDMEb were chosen for further identification. Total RNA The transcripts of DrNLRP3, DrGSDMEa/b, DrFas/DrFasL,
was isolated from zebrafish embryos and tissues by using an and DrBcl2 genes in zebrafish tissues and embryos were ana-
RNAiso Plus kit (Takara Bio). The cDNAs of DrNLRP3 and lyzed via quantitative real-time PCR on a Mastercycler EpReal-
DrGSDMEa/b were amplified by RT-PCR according to the ho- plex instrument (Eppendorf). In brief, all PCR experiments
mologous sequences predicted previously. The PCR products were performed in a total volume of 10 ␮l by using a SYBR
were purified and inserted into the pGEM-T easy vector (Pro- Premix Ex Taq kit (Takara Bio). The reaction mixtures were
mega) and sequenced on a 3730xl DNA analyzer (Applied Bio- incubated for 2 min at 95 °C, followed by 40 cycles of 15 s at
systems). The primers used in cloning are shown in Table S1. 95 °C, 15 s at 60 °C, and 20 s at 72 °C. The relative expression
levels were calculated using the 2⫺⌬Ct and 2⫺⌬⌬Ct method with
Bioinformatics analysis ␤-actin for normalization. Each PCR trial was run in triplicate
Genome assemblies and locations of NLRP3 and GSDME parallel reactions and repeated three times. The primers are
genes in human and zebrafish genomes were retrieved from the listed in Table S1, and the efficiency of these primers was
University of California at Santa Cruz (UCSC) genome bioin- checked.
formatics website and Genome Data Viewer in the National
Center for Biotechnology Information (NCBI). Gene organiza- Constitution of DrNLRP3 inflammasome in HEK293T cells
tions (intron/exon boundaries) were elucidated by comparing HEK293T cells were seeded into 6-well plates at 5 ⫻ 105 per
DrNLRP3 and DrGSDMEa/b cDNAs with genome sequences well in DMEM culture medium (HyClone) with 10% fetal
from UCSC, and figures were drawn using GeneMapper 2.5 bovine serum (Bovogen) at 37 °C in 5% CO2. After 24 h, cells
(44). Full-length cDNAs of DrNLRP3 and DrGSDMEa/b were were transfected with plasmids expressing DrNLRP3 (400 ng),
assembled using the CAP3 Sequence Assembly Program (45). DrASC (200 ng), DrCaspase-A (200 ng), DrCaspase-B (200 ng),
Multiple alignments of DrNLRP3 and DrGSDMEa/b with their and proDrIL-1␤ (800 ng) by using polyethyleneimine (Invitro-
mammalian counterparts were analyzed by using the Clustal X gen) (52). After another 24 – 48 h, cell lysates were prepared for
program (version 2.0) (46). Phylogenetic trees were constructed Western blotting or caspase fluorogenic assay.
by using MEGA 5.0 with the maximum likelihood method. The
node values represent the percentage bootstrap confidence Caspase assay with fluorogenic substrates
derived from 1000 replicates (47). The percentage of amino acid HEK293T cells (one well in a 6-well plate) or zebrafish
sequence identity was calculated using the MEGALIGN pro- embryos (⬃20 embryos) with transfection or microinjection
gram from DNASTAR. The potential functional motifs in were harvested and lysed with 100 ␮l of caspase cell lysis buffer
DrNLRP3 and DrGSDMEa/b proteins were predicted by using (Enzo Life Sciences). Lysate protein (100 ␮g) was added to the
the Pfam 31.0 and Conserved Domains of NCBI online software caspase assay buffer (Enzo Life Sciences) containing 100 ␮M
with protein sequence (48). The domain structures of acetyl–Tyr–Val–Ala–Asp–amido-4-trifluoromethylcoumarin
DrNLRP3 and DrGSDMEa/b were analyzed using SWISS- (Ac-YVAD-AFC, specific to DrCaspase-A) or Ac–Trp–Glu–
MODEL, and the whole-protein structure of DrNLRP3 was His–Asp–AFC (Ac-WEHD-AFC, specific to DrCaspase-B)
analyzed by using I-TASSER (49, 50). The tertiary structural (Alexis, San Diego, CA) as described previously (12, 53). After
figures were reviewed and colored in PyMOL software (51). incubation at 37 °C for 2 h, the cleavage of caspase-type–
1136 J. Biol. Chem. (2020) 295(4) 1120 –1141

specific substrate emitted a fluorescent signal that was mea- catalog no. A-11005, Life Technologies, Inc.) according to the
sured with excitation at 400 nm and emission at 505 nm on a manufacturer’s instructions. Finally, the cells were incubated
Synergy H1 Hybrid Reader (BioTek Instruments). The activa- with 0.1% 4⬘,6-diamidino-2-phenylindole (Invitrogen) for
tion level of DrCaspase-A/-B was calculated as follows: ((exper- staining the nucleus. The images were captured under a two-
imental group ⫺ control group)/control group) ⫻ 100%. photon laser-scanning confocal microscope (Zeiss LSM710,
Germany) at ⫻200 and ⫻630 magnifications. Three-dimen-
Western blot analysis sional super-resolution images were captured using a three-
HEK293T cells or zebrafish embryos with designated treat- dimensional structured illumination microscope with the
ments were treated with cell lysis buffer for Western blotting N-SIM System (3D-SIM, Nikon) (55, 56). DrASC nucleation
and immunoprecipitation (Beyotime) containing protease was quantified by calculating the speck-forming rate. More
inhibitor mixture (Roche Applied Science). The proteins were than 100 cells with immunofluorescence speck images were
separated through 12% SDS-PAGE and then transferred onto counted in each experimental group.
polyvinylidene difluoride transfer membranes (Millipore). The
blots were blocked with 5% nonfat dry milk (BBI Life Sciences) Electroporation for ZF4 cell transfection
and incubated with mouse anti-Flag (1:1000, catalog no. Zebrafish ZF4 cells were cultured at 28 °C in a DMEM/F-12
ab125243, Abcam, clone FG4R), anti-Myc (1:1000, catalog no. mixture medium (HyClone) with 110 ␮g/ml sodium pyruvate
ab56, Abcam, clone 9E11), and anti-HA monoclonal Abs (Corning) and 10% fetal bovine serum (Bovogen). The cell
(1:1000, catalog no. ab1424, Abcam, clone 4C12) or rabbit anti- numbers were counted using a hemocytometer after being
Flag (1:5000, catalog no. D110005, BBI Life Sciences) and anti- digested by 0.25% trypsin (Thermo Fisher Scientific). Approx-
Myc (1:5000, catalog no. D110006, BBI Life Sciences) poly- imately 2 ⫻ 106 cells were suspended in 200 ␮l of DMEM/F-12
clonal Abs. The objective proteins were visualized with medium in a 0.4-cm electroporation cuvette and mixed with 20
enhanced chemiluminescence reagents (GE Healthcare) by ␮g of plasmid DNA. After electroporation (square wave pulses,
using a digital gel image analysis system (Tanon) after horse- 270 V, 25 ms, Bio-Rad MicroPulser electroporator), the cells
radish peroxidase– conjugated goat anti-rabbit (1:8000, catalog were suspended into 1 ml of DMEM/F-12 medium and trans-
no. ab6721, Abcam) or mouse IgG Ab (1:8000, catalog no. ferred onto two wells of a 24-well plate containing coverslips.
ab205719, Abcam) was added. The protein strip was subjected ZF4 cells at 30 h post-transfection were fixed with 2% parafor-
to grayscale quantization by using ImageJ software. maldehyde for immunofluorescence imaging of DrASC nucle-
ation and DrNLRP3 inflammasome assembly as described
Co-immunoprecipitation assay above.
Co-IP was performed to detect the interaction among
DrNLRP3, DrASC, and DrCaspase-A/-B. In this process, Fluorescence recovery after photobleaching (FRAP) assay
HEK293T cells were plated in 10-cm dishes (Corning) and FRAP assay was used to evaluate the dynamic recruitment of
cotransfected with 6 ␮g of recombinant plasmids or an empty DrCaspase-A/-B into the DrNLRP3 inflammasome. Enhanced
vector as a negative control. After 48 h, the cells were lysed with GFP–DrASC/DrNLRP3 and RFP–DrCaspase-A/-B fusion pro-
pre-cooling cell lysis buffer (Enzo Life Sciences). The lysates teins were coexpressed in HEK293T cells, and FRAP was per-
were incubated with mouse or rabbit Abs (1:200 dilution) at formed on an LSM710 Airyscan microscope with a 488-nm or
4 °C overnight. The next day, the mixture of the cell lysates and 543-nm laser. The DrCaspase-A/-B in the inflammasome speck
the antibody were incubated with 50 ␮l of protein A-agarose structure was fully or partially photobleached with 100% laser
beads (Roche Applied Science) for 4 h. The beads were washed power. Time-lapse images were acquired over 45 cycles after
three times with lysis buffer, and the obtained samples were bleaching at 3-s intervals (57–59). The fluorescence intensities
analyzed with Western blotting assay. The expression of the of regions of interest (ROIs) were corrected by unbleached con-
transfected plasmids was also analyzed in the whole-cell lysates trol regions and then normalized to prebleached intensities of
as an input control. the ROIs. Data from image analysis were graphed using
GraphPad Prism 7.
Immunofluorescence imaging of DrNLRP3-dependent DrASC
nucleation Morphological examination for pyroptotic cell death
HEK293T cells (1 ⫻ 10 ) were seeded on coverslips in a
5
To examine the morphological changes during pyroptotic
24-well plate per well for 24 h. The cells were transfected with cell death, the images of transfected or stimulated cells were
plasmids expressing DrNLRP3 (400 ng/ml), DrASC (100 captured. 4.5 ␮M PI (Invitrogen) was added to the medium as an
ng/ml), and DrCaspases (100 ng/ml) (54). After 48 h, the cells indicator of cell membrane integrity. The dyed cells exhibit the
were fixed with 4% paraformaldehyde for 10 min, permeabi- loss of plasma membrane integrity and exhibit pyroptotic-like
lized with 0.1% Triton X-100, and blocked with 2% BSA at 37 °C features when captured under a two-photon laser-scanning
for 1 h. The cells were then incubated with primary antibodies confocal microscope (Zeiss LSM710, Germany) at room tem-
(rabbit anti-Myc along with mouse anti-Flag) at 4 °C overnight. perature (19).
After washing with phosphate-buffered saline (PBS), the cells
were incubated with secondary FITC-conjugated anti-rabbit Cytotoxicity assay
antibodies (1:100, catalog no. sc-2359, Santa Cruz Biotechnol- The cytotoxicity triggered by DrNLRP3 inflammasome was
ogy) and AlexaFluor 594 – conjugated anti-mouse (1:1000, monitored by the release of LDH by using a CytoTox 96威 non-
J. Biol. Chem. (2020) 295(4) 1120 –1141 1137

radioactive cytotoxicity assay kit (Promega). After transfection MOs were dissolved with nuclease-free H2O to 1 mM as stock
by the plasmids encoding DrNLRP3, DrASC, DrCaspase-A, solutions (63). For MO-resistant mRNA synthesis, DrNLRP3,
DrCaspase-B/CaspaseB-4DA, and DrGSDMEa/b for 48 h, the DrCaspase-A, and DrCaspaseB cDNA sequences were con-
supernatant (50 ␮l) of the HEK293T cells was collected from structed into the pcDNA3.1 vector by using the primers as
each sample and mixed with the CytoTox 96威 reagent (50 ␮l) shown in Table S1. The capped mRNAs were synthesized using
for enzymatic assay. After a 30-min reaction in an opaque box, the mMESSAGE kit (Ambion), purified with Mini Quick Spin
the stop solution (50 ␮l) was added to each sample. Absorbance RNA columns (Roche Applied Science), and solubilized in
(OD490) was also detected by a Synergy H1 Hybrid Multi-Mode diethyl pyrocarbonate water. The one-cell–stage embryo was
Microplate Reader (BioTek). The cytotoxicity (%) of the effec- microinjected with DrNLRP3-MO (1.5– 4.5 ng) or CaspaseA/
tor cells was calculated following the protocol previously B-MO (4.0 ng) for gene knockdown and MO-resistant mRNAs
described (60, 61). (200 pg) for the sake of rescue.
Intracellular bacterial challenge assay Evaluation of DrNLRP3 inflammasome in antibacterial

immunity
A bacterial challenge assay was performed in zebrafish
embryos by immersion infection with E. tarda TL5m strain, an The functional role of DrNLRP3 inflammasome in innate
intracellular virulent pathogen for various aquatic animals, as immunity was evaluated through its antibacterial activity in the
described previously, to examine in vivo the DrCaspase-A/-B zebrafish embryo model. For this evaluation, the DrNLRP3
activation, DrIL-1␤ maturation, and pyroptosis. For this pro- knockdown, rescue, and control embryos were challenged with
cess, the embryos (6 –72 hpf) were exposed to 1 ⫻ 108 CFU/ml E. tarda (1 ⫻ 108 CFU/ml) at 6 or 72 hpf. Mortality in each
E. tarda in a 10-cm dish for 40 min to 4 h. Then, the embryos group was monitored during the 12-h period at one interval.
were collected to detect the activation of DrCaspases by Ac- The relative survival rate (RSR) was calculated using the follow-
YVAD/WEHD-AFC and the maturation of DrIL-1␤ by the rab- ing formula: RSR (%) ⫽ (survival rate of the infected group/
bit anti-DrIL-1␤ polyclonal Ab prepared in our previous study survival rate of the mock PBS-administered control group) ⫻
(18, 62). DNA from E. tarda was extracted with a bacterial DNA 100%. Infection experiments were performed in triplicate with
preparation kit (Omega Bio-tek). DNA (200 pg per embryo), 100 embryos per group.
LPS (Escherichia coli O55:B5, Sigma, 2 ng per embryo), MDP Generation of DrASC CRISPR knockout line
(InvivoGen, 2 ng per embryo), and ATP (BBI Life Sciences, 20
The gRNA for CRISPR-Cas9 – based gene knockout was
ng per embryo) were microinjected into 6 hpf embryos. Mean-
designed by a website program (CHOPCHOP: http://
while, 10 mM H2O2 was used for immersed stimulus. After 40
chopchop.cbu.uib.no). The target sequence of the first
min, the embryos were collected, and the DrCaspase activation
DrASC exon is GTGTTCACATCAAAAGACGCGG. The
and the DrIL-1␤ maturation were detected.
gRNA was prepared by in vitro transcription by using the
Visualization of DrNLRP3 inflammasome in vivo MEGAscriptTM T7 high-yield transcription kit (Invitrogen)
and purified using the MEGAclearTM kit (Invitrogen). The
DrNLRP3 inflammasome was examined by its occurrence in
mixture of 500 ng/␮l TrueCutTM Cas9 protein v2 (Invitro-
zebrafish embryos under bacterial infection. For this process,
gen) and 300 ng/␮l DrASC-KO gRNA was microinjected
pCMV-Tag2B-DrNLRP3 and pCMV-Myc-DrASC were co-mi-
into one-cell–staged embryos. TheDrASC⫺/⫺ zebrafish
croinjected into the one-cell–stage embryos at a concentration
homozygote was acquired after two generations (64 –66).
of 100 pg per embryo. At 72 hpf, the embryos were challenged
with E. tarda (1 ⫻ 108 CFU/ml) for 4 h, collected, and sliced Flow cytometry analysis for cell pyroptosis
into 6-␮m–thick frozen sections by a freezing microtome The percentage of pyroptotic cells in zebrafish embryos was
(CM1950, Leica). For DrNLRP3 inflammasome visualization, evaluated by a flow cytometer (FACSCalibur or FACSJazz, BD
immunofluorescent staining was performed on the sections as Biosciences). For this process, the 72 hpf WT embryos,
described above. Moreover, DrNLRP3 cooperation with DrNLRP3, the DrCaspase-A/-B knockdown embryos, and the
DrNLRP1 in the inflammasome signaling was characterized in DrASC knockout embryos were challenged with E. tarda (1 ⫻
embryos by co-microinjecting with pEGFP-N1-DrNLRP3 and 108 CFU/ml) by immersion infection for 4 h. The embryonic
pDsRED-C1-DrNLRP1 expression vectors. The DrNLRP3 cells were gently separated from the infected embryos (n ⫽ 100)
(green) and DrNLRP1 (red) colocalization in 72 hpf embryos digested with type IV collagenase (1.0 unit/ml, Sigma, at room
was visualized via an inverted microscope (Zeiss Axiovert 40 temperature for 30 min) by filtration through a 40-␮m strainer
CFL; Carl Zeiss, Jena, Germany). (Falcon, BD Biosciences) and centrifugation at 100 ⫻ g at 4 °C
for 5 min, and then washed three times with PBS by centrifuga-
MOs and capped mRNAs
tion, and labeled by PI according to the manufacturer’s protocol
The MOs against the mRNAs of DrNLRP3 (NLRP3-MO, 5⬘- (Thermo Fisher Scientific). In FCM analysis, at least 10,000
CATCAACCTGTTCATGGCCTCCATTTTC-3⬘), DrCaspase-A cells were acquired from the gate for examination. FlowJo 7.6
(CaspaseA-MO, 5⬘-CCATGTTTAGCTCAGGGCGCTG- software (BD Biosciences) was used for data processing (67, 68).
3⬘), and DrCaspase-B (CaspaseB-MO, 5⬘-AGCTGGGTAATA-
TCCTCCATTTTCT-3⬘) and the standard control MO (Ctrl- Statistical analysis
MO, 5⬘-CTCTTACCTCAGTTACAATTTATA-3⬘) were The data in the study were presented as the mean ⫾ S.D. of
designed and synthesized by Gene Tools (Philomath, OR). The each group. Statistical significance between experimental and
1138 J. Biol. Chem. (2020) 295(4) 1120 –1141

control groups was assessed by two-tailed Student’s t test and 12. Masumoto, J., Zhou, W., Chen, F. F., Su, F., Kuwada, J. Y., Hidaka, E.,
was considered at *, p ⬍ 0.05; **, p ⬍ 0.01 or ***, p ⬍ 0.001. The Katsuyama, T., Sagara, J., Taniguchi, S., Ngo-Hazelett, P., Postlethwait,
J. H., Núñez, G., and Inohara, N. (2003) Caspy, a zebrafish caspase, acti-
sample number for each group of zebrafish exceeded 20 –100
vated by ASC oligomerization is required for pharyngeal arch develop-
embryos or larvae. More than 100 cells with immunofluores- ment. J. Biol. Chem. 278, 4268 – 4276 CrossRef Medline
cence speck were counted for quantifying DrASC nucleation. 13. Boudinot, P., Reis, M. I., do Vale, A., Pereira, P. J., Azevedo, J. E., and Dos
All experiments were replicated at least three times. Santos, N. M. (2012) Caspase-1 and IL-1␤ processing in a teleost fish. PLoS
ONE 7, e50450 CrossRef Medline
14. Koussounadis, A. I., Ritchie, D. W., Kemp, G. J., and Secombes, C. J. ( 2004)
Author contributions—J.-Y. L., Y.-Y. W., D.-D. F., and L.-X. X. data
Analysis of fish IL-1␤ and derived peptide sequences indicates conserved
curation; J.-Y. L. and T. S. software; J.-Y. L., T. S., L.-X. X., and J.-Z. S.
structures with species-specific IL-1 receptor binding: implications for
formal analysis; J.-Y. L., T. S., A.-F. L., L.-X. X., and J.-Z. S. validation; pharmacological design. Curr. Pharm. Des. 10, 3857–3871 CrossRef
J.-Y. L. and Y.-Y. W. visualization; J.-Y. L. and L.-X. X. methodology; Medline
J.-Y. L. and Y.-Y. W. writing-original draft; D.-D. F., L.-X. X., and 15. Li, Y., Li, Y., Cao, X., Jin, X., and Jin, T. (2017) Pattern recognition recep-
J.-Z. S. resources; A.-F. L., L.-X. X., and J.-Z. S. writing-review and tors in zebrafish provide functional and evolutionary insight into innate
editing; L.-X. X. and J.-Z. S. supervision; L.-X. X. and J.-Z. S. funding immune signaling pathways. Cell. Mol. Immunol. 14, 80 – 89 CrossRef
acquisition; L.-X. X. and J.-Z. S. project administration; J.-Z. S. con- Medline
ceptualization; J.-Z. S. investigation. 16. Shi, J., Zhao, Y., Wang, K., Shi, X., Wang, Y., Huang, H., Zhuang, Y., Cai, T.,
Wang, F., and Shao, F. (2015) Cleavage of GSDMD by inflammatory
caspases determines pyroptotic cell death. Nature 526, 660 – 665
Acknowledgments—We thank Prof. Di Wang for sharing the eukary-
CrossRef Medline
otic expression plasmid of mouse NLRP3 and Prof. Jinyu Shen for 17. Sakamaki, K., and Satou, Y. (2009) Caspases: evolutionary aspects of their
providing the E. tarda TL5m strain. We also thank Dr. Wei-ren Dong, functions in vertebrates. J. Fish Biol. 74, 727–753 CrossRef Medline
Cen-cen Sun, and She-long Zhang for their technical support for 18. Li, J. Y., Gao, K., Shao, T., Fan, D. D., Hu, C. B., Sun, C. C., Dong, W. R., Lin,
molecular cloning and two-photon laser confocal-scanning micro- A. F., Xiang, L. X., and Shao, J. Z. (2018) Characterization of an NLRP1
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mechanism underlying inflammatory caspases in ancient vertebrates.
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The zebrafish NLRP3 inflammasome has functional roles in

This is an Open Access article under the CC BY license.

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Discussion tive chromosomal synteny, gene organization, protein domain

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J. Biol. Chem. (2020) 295(4) 1120 –1141 1137

Intracellular bacterial challenge assay Evaluation of DrNLRP3 inflammasome in antibacterial

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