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

Abstract

The NLR family pyrin domain containing 3 (NLRP3) inflammasome is one of the best-characterized inflammasomes in humans and other mammals. However, knowledge about the NLRP3 inflammasome in nonmammalian species remains limited. Here, we report the molecular and functional identification of an NLRP3 homolog (DrNLRP3) in a zebrafish (Danio rerio) model. We found that DrNLRP3's overall structural architecture was shared with mammalian NLRP3s. It initiates a classical inflammasome assembly for zebrafish inflammatory caspase (DrCaspase-A/-B) activation and interleukin 1β (DrIL-1β) maturation in an apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)-dependent manner, in which DrNLRP3 organizes DrASC into a filament that recruits DrCaspase-A/-B by homotypic pyrin domain (PYD)-PYD interactions. DrCaspase-A/-B activation in the DrNLRP3 inflammasome occurred in two steps, with DrCaspase-A being activated first and DrCaspase-B second. DrNLRP3 also directly activated full-length DrCaspase-B and elicited cell pyroptosis in a gasdermin E (GSDME)-dependent but ASC-independent manner. These two events were tightly coordinated by DrNLRP3 to ensure efficient IL-1β secretion for the initiation of host innate immunity. By knocking down DrNLRP3 in zebrafish embryos and generating a DrASC-knockout (DrASC^-/-) fish clone, we characterized the function of the DrNLRP3 inflammasome in anti-bacterial immunity in vivo The results of our study disclosed the origin of the NLRP3 inflammasome in teleost fish, providing a cross-species understanding of the evolutionary history of inflammasomes. Our findings also indicate that the NLRP3 inflammasome may coordinate inflammatory cytokine processing and secretion through a GSDME-mediated pyroptotic pathway, uncovering a previously unrecognized regulatory function of NLRP3 in both inflammation and cell pyroptosis.

Keywords: NLRP3; apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC); caspase; gasdermin E (GSDME); inflammasome; innate immunity; interleukin 1β (IL-1β); pattern recognition receptor; pyroptosis; zebrafish.

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 ²⁴⁴FEID²⁴⁷, 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 chromosome 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^−ΔCt method with β-actin for normalization. Each data point shows the mean ± S.D. with three replicates representative of three independent experiments.

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-ΔPYD 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-ΔPYD/ΔCARD), and DrNLRP3 (WT). I, coIP assay reveals the protein–protein interactions among DrCaspase-B (CasB-4DA), DrASC (DrASC-ΔPYD/ΔCARD), 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.”

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 DrASC^PYD/CARD filament when transiently transfected by Myc-tagged DrASC-PYD (DrASC-ΛCARD) or DrASC-CARD (DrASC-ΛPYD) in HEK293T cells. F and G, with the DrNLRP3 and DrASC coexpression, DrNLRP3 was colocalized with the DrASC^PYD filament (F) but not with the DrASC ^CARD filament (G). H, DrNLRP3 coexpressed with DrASC-ΛCARD or DrASC-ΛPYD 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-ΛPYD but not DrASC-ΛCARD 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 μm). The results are representative of three independent experiments as mean ± S.D. (*, p < 0.05).

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 μm. 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.

Figure 5.

DrNLRP3 contribution to the proDrIL-1β maturation in a DrASC-dependent manner. HEK293T cells were transfected with a pcDNA3.1–DrIL-1β 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-1β cleavage triggered by activated DrCaspase-A and DrCaspase-B. B, ProDrIL-1β cleavage triggered by DrNLRP3–DrASC-activated DrCaspase-A alone. C, ProDrIL-1β cleavage triggered by DrNLRP3–DrASC-activated 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-1β in the blots. Each data point shows the mean ± S.D. with three replicates.

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 μm 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 μm. 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.

Figure 7.

Coordination of DrNLRP3 inflammasome between mature-formed DrIL-1β secretion and cell pyroptosis. A, HEK293T cells were transfected with a pcDNA3.1-DrIL-1β 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-1β 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-1β in culture supernatants were analyzed by ELISA by using DrIL-1β polyclonal antibody. Each data point shows the mean ± S.D. with three replicates. **, p < 0.01; ***, p < 0.001.

Figure 8.

Schematic for the functional roles of DrNLRP3 inflammasome in DrASC-dependent and DrASC-independent manners.

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-1β 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^−ΔΔCt method with β-actin for normalization. Data are representative of three independent experiments as mean ± S.D. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

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 μm. 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-1β 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-1β 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.

Figure 11.

Functional substitution of mouse NLRP3 (MmNLRP3) to DrNLRP3 in DrASC nucleation and cell pyroptosis. A, DrASC and DrNLRP3 coexpression 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 μm). 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.