Allergen protease-activated stress granule assembly and gasdermin D fragmentation control interleukin-33 secretion

Interleukin-33 (IL-33), an epithelial cell-derived cytokine that responds rapidly to environmental insult, has a critical role in initiating airway inflammatory diseases. However, the molecular mechanism underlying IL-33 secretion following allergen exposure is not clear. Here, we found that two cell events were fundamental for IL-33 secretion after exposure to allergens. First, stress granule assembly activated by allergens licensed the nuclear-cytoplasmic transport of IL-33, but not the secretion of IL-33. Second, a neo-form murine amino-terminal p40 fragment gasdermin D (Gsdmd), whose generation was independent of inflammatory caspase-1 and caspase-11, dominated cytosolic secretion of IL-33 by forming pores in the cell membrane. Either the blockade of stress granule assembly or the abolishment of p40 production through amino acid mutation of residues 309–313 (ELRQQ) could efficiently prevent the release of IL-33 in murine epithelial cells. Our findings indicated that targeting stress granule disassembly and Gsdmd fragmentation could reduce IL-33-dependent allergic airway inflammation. Sun and colleagues describe that the secretion of interleukin-33 is dependent on a p40 N-terminal fragment of gasdermin D, whose generation is independent of inflammatory caspase-1 and caspase-11.

Interleukin-33 (IL-33), an epithelial cell-derived cytokine that responds rapidly to environmental insult, has a critical role in initiating airway inflammatory diseases. However, the molecular mechanism underlying IL-33 secretion following allergen exposure is not clear. Here, we found that two cell events were fundamental for IL-33 secretion after exposure to allergens. First, stress granule assembly activated by allergens licensed the nuclear-cytoplasmic transport of IL-33, but not the secretion of IL-33. Second, a neo-form murine amino-terminal p40 fragment gasdermin D (Gsdmd), whose generation was independent of inflammatory caspase-1 and caspase-11, dominated cytosolic secretion of IL-33 by forming pores in the cell membrane. Either the blockade of stress granule assembly or the abolishment of p40 production through amino acid mutation of residues 309-313 (ELRQQ) could efficiently prevent the release of IL-33 in murine epithelial cells. Our findings indicated that targeting stress granule disassembly and Gsdmd fragmentation could reduce IL-33-dependent allergic airway inflammation.
gasdermin family member that controls the release of inflammatory IL-1β produced through the caspase-1/8/11-dependent pathway 19 , was cleaved into a p40 N-terminal form (p40 NT-Gsdmd) through a caspase-independent mechanism. The generation of the p40 NT-Gsdmd fragment promoted the secretion of IL-33 from the cytosol into the extracellular space without the apparent occurrence of cell death. These observations provide two potential targets for interfering with IL-33-dependent inflammatory immune response following allergen protease exposure.

Allergen stimulates IL-33 secretion and Gsdmd fragmentation.
Immunofluorescence staining in unstimulated murine lungs indicated the typical expression of IL-33 in alveolar type II (AT2) lung epithelial cells with surfactant protein C (SPC + ) (Extended Data Fig. 1a). In humans, IL-33 was preferentially expressed in airway epithelium both in inflamed lungs from people with asthma and in noninflamed lung tissues (Extended Data Fig. 1b). Murine AT2 MLE-12 cells expressing IL-33 endogenously and human epithelial A549 cells expressing human influenza hemagglutinin (HA, the amino acid residues 98-106)-tagged human IL-33 with a carboxy-terminal fusion with green fluorescent protein (GFP) (A549-IL-33-GFP) were used to trace IL-33 secretion in vitro (Extended Data Fig. 2a,b). The smallest nuclear localization sequence (NLS) linked with GFP (A549-NLS-GFP) was also constructed for control analysis 20 . The IL-33-GFP signal in the nucleus was recorded for 30 min after exposure to 5 µg of papain with a live-cell imaging recording system ( Fig. 1a and Supplementary Videos 1 and 2). IL-33-GFP was detected in A549-IL-33-GFP cells at 10 min and 30 min after papain stimulation, with a significant loss of GFP signal at 30 min. At the same time, NLS-GFP did not present a noticeable GFP fluorescence signal change in the nucleus (Fig. 1a,b), and this occurred concurrently with the appearance of two N-terminal (NT) fragments of Gsdmd near 40 kDa (Fig. 1c,d). This observation could be replicated in MLE-12 cells that ectopically expressed NT Flag-tagged mouse Gsdmd (MLE-12-Flag-Gsdmd) (Extended Data Fig. 2c,d). In A549-IL-33-GFP cells, papain exposure led to the generation of a p35 NT human gasdermin D (GSDMD) fragment (Extended Data Fig. 2e), also observed in A549 cells expressing NT Flag-tagged GSDMD (Extended Data Fig. 2e,f). Papain stimulated the release of IL-33 and the appearance of Gsdmd fragments in a time-dependent manner (Fig. 1e,f). Although papain induced the generation of two NT-Gsdmd fragments of around 40 kDa in molecular weight, the appearance of the bottom 40-kDa band did associate with IL-33 secretion and the upper 43-kDa band did not (Fig. 1c,e). The bottom 40-kDa fragment was thus defined as p40 NT-Gsdmd. Because Gsdmd cleavage usually leads to cell death, we investigated whether cell death happened after protease exposure. MLE-12 cells stimulated with papain for 30 min showed no significant PI uptake or annexin V staining (Fig. 1g) and no apparent death morphology at this stage (Supplementary Video 1) compared with tumor necrosis factor-α (TNF-α) and SM-164 (hereafter, TS) stimulated MLE-12 cells. Three hours after the removal of papain, papain-induced p40 NT-Gsdmd disappeared, and IL-33 secretion fell back to the baseline (Fig. 1h), indicating that the papain-induced generation of p40 NT-Gsdmd was reversible. Even with a higher papain stimulation dose (100 µg), LDH was nearly undetectable in the supernatant (Fig. 1i). The induction of phosphorylated mixed lineage kinase domain-like protein (p-Mlkl)-mediated necroptosis by the mixture of TNF-α, SM-164 and Z-VAD-FMK (hereafter, TSZ) or caspase-mediated apoptosis by TS did not contribute to IL-33 release in MLE-12 cells (Fig. 1i). These observations indicated that papain stimulation did not activate classical cell death pathways,  such as apoptosis and necroptosis, but induced the activation of a p40 NT-Gsdmd fragment and IL-33 release.
Papain activates caspase-1/11-independent Gsdmd fragmentation. Gsdmd can be cleaved by caspase-1 and caspase-11 through both the canonical and noncanonical activation of the inflammasome, which induces IL-1β release in multiple cell types, including macrophages 21 . Because macrophages can be an efficient source of IL-33 upon inflammatory stimulation 5,22 , we compared Gsdmd cleavage during caspase-1/11 and papain stimulation in murine bone marrow-derived macrophages (BMMs). After priming with bacterial lipopolysaccharides (LPS), which leads to the production of pro-IL-1β, we added papain, ATP or nigericin (Nig) as a second stimulatory signal. Stimulation with ATP or Nig generated the cleaved p20 caspase-1 and a 35-kDa fragment of NT-Gsdmd (Fig. 2a). The p35 NT-Gsdmd displays a high binding affinity to cell membrane-associated lipids with pore-forming capability 23 , promoted the release of IL-1β and LDH (Fig. 2a,b), and the release of LDH, suggesting that cell membrane rupture and cell death happened in this stage. In contrast, papain stimulation did not promote the activation of p20 caspase-1 (Fig. 2a) and did not permit the secretion of IL-1β into the supernatant (Fig. 2b), but induced the generation of p40 NT-Gsdmd and release of IL-33 in BMMs (Fig. 2a,b). This process did not result in apparent LDH release (Fig. 2a).
Gsdmd cleavage requires the protease activity of papain. Papain is a proteolytic enzyme with cysteine protease activity. The p40 NT-Gsdmd and IL-33 release from MLE-12 cells were suppressed when papain was incubated with the irreversible cysteine protease inhibitor E-64 (Fig. 3a), or when papain was heat-inactivated at 100 °C for 10 min (Fig. 3a). When MLE-12 cells were incubated with other allergen-derived active enzymatic components, including those from the Aspergillus oryzae, Bacillus licheniformis, HDM extracts and the purified HDM proteases Der p and Der f at different concentrations, all tested allergen proteases (except for Alternaria alternata (Extended Data Fig. 3a)) activated the generation of p40 NT-Gsdmd and induced IL-33 secretion in a dose-dependent manner ( Fig. 3b), suggesting that most allergen proteases share a common protease stress-sensing pathway leading to p40 NT-Gsdmd activation and IL-33 release. We did not detect any Gsdmd fragmentation and IL-33 release after stimulation with nonenzymatic allergens, including ovalbumin, alum, the toll-like receptor (TLR) 2/6 agonist fibroblast-stimulating lipopeptide-1 (FSL-1), the endoplasmic reticulum stress-inducer palmitic acid and the membrane-permeable poly-l-arginine (Fig. 3c). Other stressorsincluding oxidative stress induced by hydrochloric acid or hydrogen peroxide; chemical stress induced by inorganic salts of sodium, calcium or magnesium; peptide agonists of protease-activated receptors, including PAR2/1; and temperature-associated cold shock (4 °C) or heat shock (42-44 °C)-did not induce p40 NT-Gsdmd activation (Extended Data Fig. 3b-d). These results showed that lung epithelial cells possessed unique stress-sensing pathways for exogenous allergen proteases that lead to IL-33 secretion.
Allergen proteases activate SG assembly. To define whether SG assembly participated in allergen protease-induced IL-33 release in the airway epithelium, we analyzed the localization of the Ras-GTPase-activating protein G3BP1, which functions as a molecular switch that triggers the assembly of SGs by forming a multimer 24 in A549-IL-33-GFP cells. Arsenite, an eIF2α phosphorylation-dependent stimulator 25 , induced typical G3BP1 puncta in the A549 cells (Fig. 4a), which indicated effective SG assembly. Following translocation to the cytoplasm, IL-33 colocalized with the G3BP1 puncta (Fig. 4a). Papain stimulation also induced the formation of G3BP1 puncta and the colocalization between G3BP1 puncta and IL-33 ( Fig. 4a,b). Arsenite promoted the nucleocytoplasmic transfer of IL-33, but did not induce IL-33 secretion (Fig. 4a,c), whereas papain induced the direct secretion of IL-33 from the nucleus to the extracellular space (Fig. 4c). Next, we collected cell culture supernatants and whole-cell lysis (WCL) from murine airway epithelial MLE-12 cells stimulated with arsenite or papain, and used ultracentrifugation to isolate SGs 26 . In the arsenite-treated cells, IL-33 was associated with the SG condensates, similar to the SG core components such as TIA1, eIF4A1 and Hsp90 ( Fig. 4d). At the same time, IL-33 was enriched in SG condensates and was detected in the supernatant in the papain-treated cells (Fig. 4d). Although papain stimulation induced TIA degradation in WCL when compared with unstimulated MLE-12 cells, TIA1 and other typical SG proteins (including eIF4A1 and Hsp90) were enriched in the SG fractions of papain-stimulated cells (Fig. 4d). Immunoprecipitation of G3BP1 from cell extracts also indicated an increased interaction between G3BP1 and IL-33 in papain-treated and arsenite-treated MLE-12 cells compared with unstimulated cells (Extended Data Fig. 3e). SG assembly is triggered by the bulk inhibition of translation initiation and can be dependent or independent of p-eIF2α 27 . Ectopically expressed IL-33 migrated from the nucleus to the cytosol in HEK293T cells after stimulation with arsenite, which is eIF2α dependent, or d-sorbitol, which is eIF2α independent (Extended Data Fig. 3f). Mass spectrometry analysis 28 of the IL-33 interaction network with immunoprecipitation in HEK293T cells indicated that arsenite and papain stimulations promoted the enrichment of several common non-membrane-bound, organelle assembly-associated components, including G3BP1, KPNB1 and ATXN2L (Supplementary Table 1, Extended Data Fig. 4). Stimulation of MLE-12 cells with arsenite or d-sorbitol, or other stimuli reported to induce SG assembly, did not induce p40 NT-Gsdmd or the secretion of IL-33, even after extended (2 h) stimulation (Extended Data Fig. 5a-c). These observations indicated that IL-33 was transported into the cytoplasms and associated with the SG in papain-stimulated airway epithelial cells, but the assembly of SG did not contribute to IL-33 secretion.
Unlike arsenite, which promoted eIF2α phosphorylation when compared with unstimulated cells, stimulation with papain did not alter the amount of eIF2α or p-eIF2α, but increased the degradation of eIF4A1, which inhibits SG assembly 29 , when compared with arsenite-treated or unstimulated MLE-12 cells (Fig. 4e). Treatment with integrated stress response inhibitor (ISRIB), which inhibits SG assembly downstream of eIF2α 30 , did not affect p40 NT-Gsdmd activation and IL-33 release (Extended Data Fig. 5d). Actinomycin D, a potent inhibitor of both cell-intrinsic and cell-extrinsic EIF4A inhibitors that helps to relieve the function of EIF4A 31-34 , impaired IL-33 secretion and blunted p40 NT-Gsdmd fragmentation in MLE-12 cells (Fig. 4f), indicating that papain stimulation triggered a p-eIF2α-independent SG assembly pathway. All tested allergen proteases activated SG assembly into G3BP1 puncta in MLE-12 cells (Fig. 4g). These data indicated that protease-activated SG assembly was necessary for controlling the nucleocytoplasmic transport of IL-33.
p40 NT-Gsdmd contributes to IL-33 secretion. Next, we investigated whether p40 NT-Gsdmd facilitated the extracellular secretion of cytosolic IL-33. We used the online pro-protein conversion prediction tool ProP 1.0 to predict potential cleavage sites in Gsdmd (ref. 35 ). Aside from the amino acid (aa) sites within the aa 1-276, which is generated by caspase-1/8/11 cleavage, we obtained six putative cleavage sites in arginine (R) and lysine (K), among which R311, K394 and K409 were chosen for further analysis (Extended Data Fig. 6). We constructed three murine Gsdmd fragments (Gsdmd 1-311 , Gsdmd 1-394 and Gsdmd 1-409 ) with a Flag-tagged N terminus, the caspase-1/11-processed Gsdmd 1-276 as a positive control and the full-length form of Gsdmd (Gsdmd FL ) as a nonfunctional control (Fig. 5a). After 12 h of expression in HEK293T cells, Gsdmd 1-311 induced less LDH release than pyroptotic Gsdmd 1-276 (Fig. 5b). To further define their function in controlling IL-33 release, we cotransfected these fragments and C-terminal HA-tagged mature IL-33 (aa 109-266, without the N-terminal nuclear localization signal peptide) into HEK293T cells. Compared with Gsdmd FL , the expression of pyroptotic Gsdmd 1-276 and Gsdmd 1-311 induced IL-33 release with high efficiency, whereas the other expressed fragments did not (Fig. 5c,d). Next, we introduced point mutations and deletions close to R311 into full-length mouse Gsdmd and lentivirally expressed them in MLE-12 cells. We found that amino acid mutation or deletion of residues 309-313 (ELRQQ) blocked the generation of p40 NT-Gsdmd and prevented IL-33 release in papain-stimulated MLE-12 cells (Fig. 5e), which suggested that this sequence was necessary for the generation of p40 NT-Gsdmd.
To identify the specific cleavage site of the p35 NT-GSDMD fragment in human cells, we cloned the GSDMD fragments GSDMD 1-290 , GSDMD 1-320 and GSDMD 1-327 and the pyroptosis-mediating fragment GSDMD 1-275 into tetracycline-responsive expression vectors expressing GFP (Fig. 5f) and expressed them in HEK293T cells. The 35-kDa GSDMD 1-290 induced LDH to release at lower levels than the pyroptotic GSDMD 1-275 fragment (Fig. 5g). Following tetracycline-induced expression, GSDMD 1-290 presented high efficiency in promoting human mature IL-33 release into the supernatant, similar to GSDMD 1-275 , and other constructs did not possess this function (Fig. 5h). To confirm the specific GSDMD cleavage site, we constructed Flag-tagged GSDMD expression vectors with several mutations close to aa 290 and transfected them in GSDMD-deficient HeLa cells. Mutation of aa 288-292 (GLRAE) into AAAAA blocked the generation of the p35 NT-GSDMD following papain exposure (Fig. 5i), suggesting that the cleavage site for p35 NT-GSDMD was located in this region. Deleting both R290 and L289 also effectively prevented the generation of p35 NT-GSDMD (Fig. 5i). Together, these results indicated that human GSDMD 1-290 promoted the secretion of cytosolic IL-33 into the supernatant.

Gsdmd contributes to type 2 inflammatory immune responses.
To determine the expression of GSDMD in asthmatic human lung tissue, we collected bronchial samples from 17 people with asthma and 6 people without asthma (Extended Data Fig. 7). Asthmatic lung tissue presented increased inflammatory cell infiltration, as well as hyperplasia of the mucous glands, compared with the nonasthmatic controls with hematoxylin and eosin (H&E) staining (Fig. 6a). GSDMD was mainly expressed in the pulmonary airway epithelium of both noninflamed and inflamed asthmatic lung tissues (Fig. 6a) and presented typical expression in areas with mucous gland hyperplasia (Fig. 6a). Expression of GSDMD in the lung tissues also showed significant positive correlation with the amount of IL-33 secreted into the bronchoalveolar lavage (BAL) and the amount of IgE in the serum from people with asthma (Fig. 6b,c), suggesting that higher GSDMD expression in the lung tissue in people with asthma might contribute to increased IL-33 secretion and potentially promote the allergic immune response. In mice, Gsdmd expression was weak in the unstimulated wide-type murine airway epithelium, including peribronchial and AT2 cells (Fig. 6d). Intranasal HDM stimulation increased bronchial immune cell infiltration and significantly upregulated the expression of Gsdmd in the lung tissue in mice (Fig.  6d-f). These observations indicated the association between Gsdmd expression and type 2 inflammation in humans and mice.  inflammation, among which epithelial cell-derived IL-33, IL-25 and thymic stromal lymphopoietin (TSLP) were shown to be important in initiating type 2 inflammation 36 . To investigate the contribution of IL-33 in vivo during allergen protease exposure, we evaluated the secretion of inflammatory cytokines in the BAL fluid (BALF) after papain exposure in mice. Using a multiple-cytokine detection system, we analyzed the amount of interferon-γ (IFN-γ), IL-10, IL-13, IL-17A, IL-25, IL-1β, IL-33, IL-4, IL-5 and IL-9 in the BALF 3 h after papain inhalation. IL-33 had a markedly high production (Fig. 7a), whereas IL-25 and TSLP displayed no significant change after papain exposure (Fig. 7b). Kinetic analysis indicated that the secretion of IL-33 into the BALF peaked at 3 h after papain exposure (Fig. 7b), indicating that IL-33 was released early during lung inflammation. Analysis of BALF from wild-type and Gsdmd-deficient (Gsdmd −/− ) mice 3 h after papain exposure indicated that Gsdmd deficiency inhibited the early release of IL-33 (Fig. 7c,d) and resulted in elevated IL-33 retained in the lung tissue homogenates, with no effect on the expression of IL-33 messenger RNA after papain stimulation (Fig. 7e). To determine whether Gsdmd interfered with IL-33-ST2 signaling, we intraperitoneally challenged Gsdmd-deficient mice with recombinant murine mature IL-33 (rIL-33, 25 µg) for 4 continuous days. rIL-33 induced an inflammatory immune response in both wild-type and Gsdmd −/− mice, which showed no significant difference in inflammatory cell infiltration (Extended Data Fig. 8a-e) and IL-5 + or IL-13 + ILC2s (Extended Data Fig. 8f-h), indicating that Gsdmd did not interfere with ST2 signaling. These results indicated that Gsdmd controlled cellular events before IL-33 secretion.

Gsdmd modulated HDM-induced chronic airway inflammation.
To further investigate the function of Gsdmd in allergic airway inflammation, we administered HDM extracts intranasally for 16 noncontinuous days in Gsdmd −/− and wild-type mice to induce chronic asthmatic airway inflammation. We found less inflammatory infiltration into the bronchus, less mucus production in the airways (Fig. 8a) and lower numbers of infiltrated eosinophils and ILC2s in the lungs (Fig. 8b and Extended Data Fig. 9a,b) in the Gsdmd −/− mice than in the wild-type mice. The secretion of inflammatory cytokines, including IL-5 and IL-13, in BALF was also significantly reduced in the Gsdmd −/− mice compared with the wild-type mice (Fig. 8c). In the papain-induced acute airway inflammation model (5 µg of papain for 5 continuous days), Gsdmd −/− mice had blunted airway inflammation infiltration (Fig. 8d-f). The infiltration of alveolar eosinophil and ILC2s was decreased (Fig. 8g), and the numbers of activated IL-5-producing or IL-13-producing ILC2s were significantly lower (Fig. 8h) in the Gsdmd −/− mice than in the wild-type mice. The secretion of the inflammatory cytokines IL-5 and IL-13 into the BALF was also impaired (Fig. 8i). Together, these data indicated that Gsdmd deficiency in mice alleviated both HDM-induced chronic and papain-induced acute airway inflammatory responses by limiting the secretion of IL-33, suggesting that Gsdmd played a fundamental role in the pathogenesis of type 2 immunity in vivo.

Discussion
Here, we show that two cellular events regulated IL-33 secretion sequentially in the context of allergen protease exposure. First, the activation of SG assembly in airway epithelial cells licensed the nucleocytoplasmic transport of IL-33 but did not control its secretion. Second, the cleavage of Gsdmd at aa 309-313 (ELRQQ) generated p40 NT-Gsdmd, which promoted the cytosolic IL-33 to cross the membrane into the extracellular space. Our investigation also noted that GSDMD had a relatively high expression in the human airway epithelium and was associated with increased IL-33 secretion into BALF in people with asthma, suggesting that GSDMD may be involved in the pathogenesis of asthma.  IL-10  IL-13  IL-17A  IL-25  IL-1β  IL-33  IL-4  IL-5  IL-9 Time (h) p.i. We show that various allergen-derived proteases, including plant-derived papain, fungal allergen Aspergillus oryzae, HDMs, HDM-derived Der p and Der f, and subtilisin protease from Bacillus species, activated SG assembly in airway epithelium, which was fundamental for the translocation of IL-33 from the nucleus into the cytosol. SGs undergo fusion, fission and flow in mammalian cell cytosol cycles, and their assembly is highly dynamic 37 . The activation of various cell death signaling pathways in lung airway epithelial cells, including caspase-mediated apoptosis (stimulated with TS) and p-Mlkl-dependent necroptosis (stimulated with TSZ), was insufficient to induce IL-33 release in airway epithelial cells, suggesting that IL-33 secretion was independent of these pathways. In our model, we observed the cessation of IL-33 secretion and the loss of Gsdmd fragmentation within 3 h after removing allergen protease, which indicated the dynamic regulation of IL-33 secretion through SG assembly in living cells.
Gsdmd-mediated pore formation has been reported in several cell types, including immune cells such as macrophages and neutrophils, and stromal cells such as nasal epithelial cells 38,39 . The cleavage of Gsdmd at D276 by caspase-1, caspase-8 or caspase-11 has been reported during various viral and bacterial infections. However, caspase-1 and caspase-11 double deficiency and the pan-caspase inhibitor Z-VAD-FMK did not inhibit the release of IL-33 and the generation of p40 NT-Gsdmd, indicating that this process was independent of the caspase family. Mutations in the aa 309-313 (ELRQQ) in Gsdmd efficiently prevented the cleavage of Gsdmd into the functional p40 fragment and blocked IL-33 release in the airway epithelial cells. Similarly, the human residues L289-R290 were needed to generate the functional NT-GSDMD fragment. Thus, the cleavage of leucine and arginine might provide clues for enzymes associated with this process. The enzymes involved in the allergen protease stress-sensing pathway that contributed to the generation of p40 NT-Gsdmd have yet to be determined.
It was previously reported that arsenite-induced activation of SG assembly did not contribute to Gsdmd fragmentation in BMMs, and stimulation with arsenite significantly inhibited NLRP3 inflammasome activation and cell death in BMMs 16 . These observations supported that SG assembly is independent of Gsdmd fragmentation, and this is consistent with our observation in airway epithelial cells.
Allergens from Alternaria were shown to trigger the RIPK1caspase-8 ripoptosome, which engaged caspase-3 and caspase-7 and contributed to intracellular maturation and the release of IL-33 in epithelial cells 40 . Although we did not observe caspase-3 activation and cell death during early exposure (30 min) to papain, sustained allergen protease stimulation can contribute to the release of DAMPs, including ATP, from damaged cells, further promoting cell death in bystander cells 1,41,42 . We observed IL-33 secretion without Gsdmd fragmentation in MLE-12 cells exposed to Alternaria for 30 min, indicating that Alternaria uniquely activated a Gsdmd-independent IL-33 secretion pathway. Therefore, additional mechanisms might regulate IL-33 secretion under various stimulations.
Our data highlight a unique role of Gsdmd in controlling cytokine release by switching the two different cleaved forms. The pyroptotic p35 NT-Gsdmd fragment reacts to classic caspase-1/8/11 cleavage that contributes to IL-1β release under typical type 1 inflammatory contexts. The p40 NT-Gsdmd fragment responds to environmental allergen protease stimulation permitting IL-33 secretion without apparent cell death, thus contributing to type 2 disease pathogenesis. Each fragment reacts to different inflammatory cues and amplifies different downstream immune cell activation events.

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Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/ s41590-022-01255-6.
Quantitative real-time PCR. Total RNA was extracted from tissues and cells using TRIzol (Invitrogen). The purified RNA was quantified and reverse-transcribed using the ReverTraAce qPCR RT Kit (TOYOBO). The expression levels of mRNA transcripts were calculated relative to the expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the 2 −ΔΔCT method.

Human samples. Clinical information is summarized in Extended Data
Tissue collection. All tissues were collected after subjects provided informed consent, with the approval of tissue-specific protocols by the Medical Ethical Committee of the Academic Medical Centre, Affiliated Hospital, Institute of Respiratory Diseases, Guangdong Medical College, Zhanjiang, China. Inflamed lung tissue samples were obtained from people with asthma. Uninflamed control lung tissue samples were obtained from adult patients undergoing lung tumor surgery or lung transplantation surgery; tissue samples were obtained at an appropriate distance from the tumor. BALF samples were obtained from patients undergoing bronchoscopy for diagnostic purposes.
Statistical analysis. Statistical significance was analyzed as described in the figure legends, and statistical analyses were performed using GraphPad Prism 6.
Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD033460. Source data are provided with this paper. Fig. 1 | IL-33 is mainly expressed in airway epithelium cells both in humans and mice. a, Microscopy of the immunofluorescent (IF) stained SPC (green) expressing AT2 cells and IL-33(red) expressing cells in lung tissue from WT Balb/c mice without any stimulations. b, Representative microscopy of the immunohistochemically (IHC) stained IL-33 (brown) in airway epithelium respective in inflamed asthma patients and non − inflamed lung tissues. Scale bars 50 μm. Data are representative of two independent experiments. Fig. 4 | Stress granule assembly is involved in IL-33 transportation under allergen protease stimulation. a, Venn diagrams of mass spectrometry analyzed IL-33 interactome from HEK293T cells expressed with C-terminal HA-tagged human IL-33 following the treatment of Ctrl (without stimulations), Pap (10 µg/mL papain for 30 min), and AS (0.5 mM arsenite exposure for 1 h). b, Venn diagrams of proteins with up-regulated interaction with IL-33 under papain and AS stimulation as in a. c, Common proteins associated with non-membrane-bounded organelle assembly were observed with up-regulated interaction with IL-33 as in a. d, Metascape analyzed protein clusters enriched with IL-33 under papain (10 µg/mL for 30 min) exposure context.