Abstract
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.
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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.
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Acknowledgements
We thank F. Shao (National Institute of Biological Sciences, China) for helpful suggestions on the study and for providing Gsdmd-deficient mice and cell lines. This work was supported by the Ministry of Science and Technology of China (2018YFA0507402), the National Natural Science Foundation of China (81761128009 and 32000667), Shanghai Science and Technology Innovation Action (21ZR1470600), the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2022264), the Affiliated Hospital of Guangdong Medical University ‘Clinical Medicine+’ CnTech Co-operation Project (CLP2021B001 and CLP2021B017), the Discipline Construction Project of Guangdong Medical University (4SG21231G), the Project of Zhanjiang City (2018A01025, 2020A01016 and 2021A05052) and the Natural Science Foundation of Guangdong Province, China (2021A1515011062 and 2022A1515011731).
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W.C., S.C., C.Y., Y.Z., R.Z. and M.C. designed and performed the experiments. S. Zhong, W.F., S. Zhu, D.Z., X. Lu, J. Zhang, Y.H., L.Z., X. Li, D.L., Y.F., H.l., Z.L., L.M., J.H., L.G. and J. Zhu provided protocols and suggestions. W.C., S.C. and C.Y. performed the data analysis and prepared figures. M.C., B.W. and D.W. provided clinical samples. W.C., B.S. and Y.Z. interpreted the results and wrote the manuscript. B.S., B.W., D.W. and Y.Z. supervised the project.
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Nature Immunology thanks Hirohito Kita and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Ioana Visan, in collaboration with the Nature Immunology team.
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Extended data
Extended Data 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.
Extended Data Fig. 2 Construction of IL-33 and Gsdmd constitutively expressive airway epithelial cells.
a, Immunoblot analysis of whole-cell lysis (WCL) of constructed C-terminal HA-tagged IL-33 expressing A549 with Lenti-virus overexpression. b, Microscopy of the immunofluorescence stained IL-33 (green) and DAPI (blue) in A549-IL-33 cells without stimulations. Scale bars (50 μm upper, 10 μm below). c, Immunoblot analysis of constructed N-terminal Flag-tagged Gsdmd expressing MLE-12. d, Immunoblot analysis of MLE-12-flag-Gsdmd cells treated with papain (0 µg, 5 µg, 10 µg and 50 µg per well) for 30 min. e, Immunoblot analysis of WCL and culture supernatants of constructed IL-33 expressing A549 after treatment with papain (0 µg, 1 µg, 5 µg and 10 µg) for 30 min. f, Immunoblot analysis of WCL of N-terminal Flag-tagged GSDMD expressing A549 cells exposed to papain (0 µg, 5 µg, 10 µg and 50 µg) for 30 min. The red arrow marked fragment represents the activated form of p35 NT-GSDMD. Asterisk marked nonspecific fragment were not discussed in our work. Data are representative of two independent experiments with similar results.
Extended Data Fig. 3 Stimulators that do not contribute to Gsdmd fragmentation and IL-33 release.
a, Immunoblot analysis of whole-cell lysis (WCL) and culture supernatants of MLE-12 cells treated with A. alternata (0.1, 1, 5, 10, 50 µg/well) for 30 min. b, Immunoblot analysis of WCL of MLE-12 cells treated with Ctrl (without stimulation); Pap (10 µg/well papain) for 30 min; 500 mM HCl, 500 mM H2O2, 5 mM NaCl, 5 mM CaCl2, 5 mM MgCl2, and 5 mM ZnCl2 for 1 h. c, Immunoblot analysis of WCL from MLE-12 cells treated with Pap (10 µg/well papain for 30 min), and the temperatures of 4, 42 and 44 °C for 1–6 h. d, Immunoblot analysis of WCL of MLE-12 cells treated with Ctrl (without stimulation); Pap (10 µg/well papain for 30 min); agonist for PAR-1 (TFLLR-NH2 trifluoroacetate salt, 0.1-50 µg/mL for 1 h), and agonist for PAR-2 (2-furoyl-LIGRLO-amide, 0.1–50 µg/mL for 1 h). e, Immunoblot analysis of G3BP1-immunoprecipitation samples from MLE-12 cells treated with Ctrl (without stimulations), Pap (10 µg/mL papain for 30 min), and AS (0.5 mM arsenite for 1 h). f, Immunofluorescent analyses of IL-33 from HEK293T cells with C-terminal HA-tagged IL-33 expression following the treatment of arsenite (0.5 mM) and d-sorbitol (0.5 mM) for 1 h. Data are representative of two independent experiments with similar results.
Extended Data 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.
Extended Data Fig. 5 Stress granule assembly-associated stimulators do not contribute to Gsdmd fragmentation and IL-33 release.
a–c, Immunoblot analysis of whole-cell lysis (WCL) and culture supernatants of MLE-12 cells treated with following stimulants for 2 h, a arsenite: 0.05, 0.5,1, 5, 50 mM; d-sorbitol: 0.01, 0.05, 0.1, 0.5 mM; b paclitaxel: 0.1, 1, 5, 50 M; vincristine: 0.1, 1, 5, 50 μM; puromycin: 1, 20 µg/mL; c thapsigargin: 0.1, 1, 10 µg/mL. d, Immunoblot analysis of whole-cell lysis (WCL) and culture supernatants of MLE-12 cells with a pre-treatment of ISRIB (10 µM) or 30 min following with the stimulation of 20 µg/mL papain. Data are representative of two independent experiments with similar results.
Extended Data Fig. 6 Predicted propeptide cleavage sites of mGsdmd with ProP-1.0.
Amino acid sequence of murine Gsdmd were analyzed with pro-protein converting prediction online tools ProP-1.0 for predicting of possible cleave sites (https://services.healthtech.dtu.dk/service.php?ProP-1.0). Predicted cleave site with different cleaving score were presented in the table. Sites in black dashed box were those possible for predicted p40 fragment, red colored sites were chosen for functional analyzing.
Extended Data Fig. 7 Human sample’s information.
WBC: white blood cells; EO%: percentages of eosinophils; IgE: detected IgE concentration in the serums.
Extended Data Fig. 8 Gsdmd deficiency does not interfere with IL-33/ST2 downstream events in mice.
a, Flow cytometry analyzed cell numbers of lung total from wide-type and Gsdmd−/− mice treated with recombinant murine IL-33 (or PBS for the control group) for continuous 4 days. b, Flow cytometry analyzed SiglecF+CD11c– eosinophils from BAL fluids as in a. c, Flow cytometry analyzed SiglecF-CD11c–CD11b+ neutrophils from BAL fluids as in a. d, Flow cytometry analyzed SiglecF+CD11c+ macrophages from BAL fluids as in a. e, Flow cytometry analysis of Lin–CD90.2+ST2+ ILC2 from mice lungs as in a. Flow cytometry analyzed cell numbers of IL-5+ ILC2 (f), IL-13+ ILC2 (g) and IL-5+ IL-13+ ILC2 (h) in mice lungs as in a. Results are depicted as means ± SEM, n = 5 using the two-tailed, Mann–Whitney test, and Dunnett’s multiple comparisons test. Data are representative of two independent experiments with similar results.
Extended Data Fig. 9 Flow-gating strategies of mice eosinophils and ILC2 cells in mice.
a, Representative flow cytometry gated scheme of Siglec F+CD11c+ eosinophils in BAL fluid of WT C57BL/6 mice and Gsdmd−/− mice. b, Gating strategy of Lin−CD90.2+ST2+ ILC2 in the lungs.
Supplementary information
Supplementary Video 1
A 30-min time-lapse record of GPF signal in A549-IL-33-GFP cell with exposure to 5 µg of papain in a 3.5-cm-diameter dish.
Supplementary Video 2
A 30-min time-lapse record of GPF signal in A549-NLS-GFP cell with exposure to 5 µg of papain in a 3.5-cm-diameter dish.
Supplementary Table 1
Mass spectrometry analysis of IL-33 interaction networks under various stimulations.
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Chen, W., Chen, S., Yan, C. et al. Allergen protease-activated stress granule assembly and gasdermin D fragmentation control interleukin-33 secretion. Nat Immunol 23, 1021–1030 (2022). https://doi.org/10.1038/s41590-022-01255-6
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DOI: https://doi.org/10.1038/s41590-022-01255-6
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