Epithelial microbial sensing through the non-canonical inflammasome modulates airway type 2 immunity

Epithelial microbial sensing through the non-canonical inflammasome modulates airway type 2 immunity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Epithelial microbial sensing through the non-canonical inflammasome modulates airway type 2 immunity Erin Gordon, Maya Kotas, Jahanvi Kumar, Louis Sharp, Carter Supple, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8003045/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Asthma is often characterized by type 2 inflammation triggered by environmental aeroallergens. Epidemiological studies link respiratory microbial infections, including intracellular bacteria, to both asthma initiation and exacerbations, yet the underlying molecular mechanisms remain poorly understood. Airway epithelial cells sense both microbes and allergens, and recent studies show that common aeroallergens induce type 2 inflammation through mechanisms including protease activity and pore-forming toxins that converge on epithelial cell damage, IL-33 secretion, and mitogen-activated protein kinase (MAPK) signaling. Host defense against microbes also involves endogenous membrane pore formation through inflammasome activation and gasdermin-mediated pyroptosis, releasing interleukin (IL)-1β and IL-18 to orchestrate bacterial killing. However, whether airway epithelial microbial sensing interacts with allergen-mediated cell death mechanisms to regulate type 2 immunity through analogous membrane pore formation and IL-33 release remains unknown. Using human airway epithelial cells and mouse models, we demonstrate that activation by intracellular lipopolysaccharide (LPS) initiates caspase-4-dependent pyroptotic cell death, IL-33 secretion, and a MAPK transcriptional program. Non-canonical pyroptotic cell death is enhanced by protease allergen through calcium signaling, and mice lacking caspase-4 show reduced IL-33 secretion and diminished innate type 2 responses to airway allergen challenge. Purified LPS from different bacterial species engage the non-canonical inflammasome with differing intensity, suggesting that microbiome composition likely influences this disease mechanism. Asthmatic patients show increased airway expression of caspase-4 and its effector molecule gasdermin D compared to healthy controls. This study identifies non-canonical inflammasome-dependent IL-33 expression and secretion as a novel link between bacterial detection and type 2 inflammation, providing a potential mechanism for how airway bacterial dysbiosis might influence asthma development and offering new insights into the interplay between microbial sensing and allergic responses in airways. Biological sciences/Cell biology/Cell signalling/Extracellular signalling molecules Biological sciences/Immunology/Innate immunity/Pattern recognition receptors Biological sciences/Immunology/Innate immune cells/Innate lymphoid cells Biological sciences/Cell biology/Cell death/Necroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Full Text Additional Declarations (Not answered) Supplementary Files primersequencesTableS1.pdf Table S1 Primer sequences CRISPRguidesTableS2.pdf Table S2 Crispr guide sequences FigS1beas2b.tif Figure S1 FigS2papandmsepithelialexpressionofIL33.tif Figure S2 FigS3fulluncroppedwesterns.pdf Figure S3 Uncropped Western Blots Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8003045","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":539707204,"identity":"98e08ac0-f1ba-4dd1-9073-08dcccf2a9cc","order_by":0,"name":"Erin 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07:17:38","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":27875,"visible":true,"origin":"","legend":"","description":"","filename":"primersequencesTableS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8003045/v1/53803d8d3b011e69ef387846.pdf"},{"id":95895944,"identity":"c03ced78-607f-4a28-96e9-979d1cab8c63","added_by":"auto","created_at":"2025-11-14 07:17:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":168840,"visible":true,"origin":"","legend":"\u003cp\u003eNon-canonical inflammasome-dependent pyroptosis and IL-33 secretion in airway epithelial cells. (A) Western blot from lysates of primary human airway epithelial cells cultured for 16 hours following electroporation of LPS or dA:dT (or sham). (B) Western blot of GSDMD and GAPDH from lysates of BEAS-2B cells treated with IC LPS and dA:dT for 3 hours or nigericin for 1 hour. (C) Glucose 6-phosphade dehydrogenase (G6PD) activity measured in media from BEAS-2B cells treated with IC LPS, dA:dT, or nigericin for 16 hours. (D) Western blot of IL-33 in concentrated supernatant from IL-33 OE BEAS-2B or GSDMDcrispr BEAS-2B cells. (E) IL-33 activity measured by secreted embryonic alkaline phosphatase from HEK-Blue- IL33 cells treated with media from IL-33 OE BEAS-2B stimulated for 16 hours with IC LPS. (F) Gene expression for mitogen activated protein kinase (MAPK) target genes in BEAS-2B cells treated with IC LPS or dA:dT and nigericin for 4 hours. (G) IL33 gene expression in BEAS-2B cells treated with IC LPS or MAPK inhibitor (MAPKi, trametinib) for 4 hours. (H) Western blot of GSDMD and GAPDH from cellular lysates from CRISPR- BEAS-2B cell lines treated with IC LPS for 3 hours. (I) Propidium iodide (PI) cell uptake over time in CRISPR- BEAS-2B cell lines with indicated knockdowns. (J) IL-33 activity measured by secreted embryonic alkaline phosphatase from HEK-Blue-IL33 cells treated with media from IL33 OE CRISPR- BEAS-2B stimulated for 16 hours with IC LPS. (K) Gene expression for MAPK target genes and IL33 from CRISPR-BEAS-2B stimulated for 4 hours with IC LPS. Error bars represent SEM. p values represent ordinary ANOVA with Sidak correction.\u003c/p\u003e","description":"","filename":"Binder11.png","url":"https://assets-eu.researchsquare.com/files/rs-8003045/v1/e6d3c570be154b09622d4840.png"},{"id":96242007,"identity":"a8202414-4a44-4c56-b5a6-b88110a938cc","added_by":"auto","created_at":"2025-11-19 07:11:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":83281,"visible":true,"origin":"","legend":"\u003cp\u003eCaspase4-dependent epithelial pyroptosis and IL-33 secretion are modulated by papain via intracellular calcium (A) Propidium iodide (PI) uptake at 6 hours in cells treated with IC LPS and papain. (B) GFP ELISA on conditioned media from IL-33 OE BEAS-2B cells treated with IC LPS and papain or heat inactivated (HI) papain for 16 hours. (C) IL-33 activity measured by secreted embryonic alkaline phosphatase from HEK-Blue-IL33 cells treated with media from IL-33OE BEAS-2B cells treated with IC LPS, papain and HI papain for 16 hours. (D) Intracellular calcium measured in BEAS-2B cells following thapsigargin, papain, or papain and BTP2. (E) PI uptake at 6 hours in BEAS-2B cells treated with IC LPS, papain and BTP2. (F) GFP ELISA on conditioned media from IL-33 OE BEAS-2B cells treated with IC LPS, papain and BTP2 for 16 hours. (G) PI uptake in BEAS-2B cells lines treated with IC LPS and calcium chloride. (H) GFP ELISA on conditioned media from IL33OE BEAS-2B cells treated with IC LPS and calcium chloride 16 hours. (I) IL-33 activity measured via HEK-Blue-IL33 cells in media from IL-33OE BEAS-2B cells treated with IC LPS and calcium chloride for 16 hours. (J) Western blot of GSDMD and GAPDH from cellular lysates from BEAS-2B cells treated with IC LPS and calcium chloride for 3 hours. P value represents two-way ANOVA with Tukey correction. (K) Western blot of BAL showing IL-33 protein in wildtype mice treated with 3 days of intratracheal PBS or papain. (L) Eosinophil and ILC2 counts from lungs of wildtype or Casp4-/- mice treated with 3 days of intratracheal PBS or papain. Error bars indicate SEM. P values indicated for two group unpaired, parametric t-test or four group ordinary one-way ANOVA with Tukey correction.\u003c/p\u003e","description":"","filename":"Binder12.png","url":"https://assets-eu.researchsquare.com/files/rs-8003045/v1/fba97ea0113ed54147966102.png"},{"id":96242715,"identity":"69006dbf-ad7b-47c4-a28a-343d3d117382","added_by":"auto","created_at":"2025-11-19 07:14:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":71407,"visible":true,"origin":"","legend":"\u003cp\u003eLPS from different gram-negative bacteria differ in ability to activate non- canonical inflammasome and IL-33 secretion in airway epithelia. (A) Western blot of GSDMD and GAPDH from cellular lysates from BEAS-2B cells treated for 3 hrs with purified LPS from three different bacterial species: Escherichia coli 55:B5 (E.coli), Pseudomonas aeruginosa 10.22 (Ps.A) and Klebsiella pneumonia (Kleb). (B) Western blot of IL-33 in concentrated supernatant from BEAS-2B cells treated for 16 hrs with IC LPS from different bacterial species. (C) Propidium iodide (PI) cell uptake over time in BEAS-2B cells lines treated with IC LPS from different bacterial species. (D) Propidium iodide (PI) cell uptake at 6 hours in CRISPR-BEAS-2B cells lines treated with IC LPS from different bacterial species. (E) MAPK and (F) IL33 gene expression from BEAS-2B cells treated for 4 hours with IC LPS from different bacterial species.\u003c/p\u003e","description":"","filename":"Binder13.png","url":"https://assets-eu.researchsquare.com/files/rs-8003045/v1/05025a0a6093ad95a2666ffa.png"},{"id":96242614,"identity":"9f8490b1-d489-4456-8b8c-7acf76db2bfc","added_by":"auto","created_at":"2025-11-19 07:13:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":29658,"visible":true,"origin":"","legend":"\u003cp\u003eNon-canonical inflammasome components are upregulated in asthma. (A) Gene expression of CASP4, GSDMD, NLRP3, and CASP1 in airway epithelial brushings in healthy controls and mild asthmatics. (B) Gene expression of CASP4, GSDMD, NLRP3, and CASP1 in paired airway epithelial brushings from mild asthmatics at baseline and after 8 weeks of treatment with twice daily inhaled steroid. P values for A. represent unpaired, parametric t-test. P values for B. represent paired, parametric t-test. ICS, inhaled corticosteroid.\u003c/p\u003e","description":"","filename":"Binder14.png","url":"https://assets-eu.researchsquare.com/files/rs-8003045/v1/1b8bb6fa0b1b62e046b59c2c.png"},{"id":99316824,"identity":"e4749042-7722-4768-9f71-19f766585401","added_by":"auto","created_at":"2025-12-31 16:29:17","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":797594,"visible":true,"origin":"","legend":"Article File","description":"","filename":"il33Casp4manuscript2025.10.31CDDwordcount.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8003045/v1_covered_ec86f0ac-f741-4633-aa0f-1ee7200a0520.pdf"},{"id":95895946,"identity":"ea508588-c65d-4c45-88a7-cb918583cd7d","added_by":"auto","created_at":"2025-11-14 07:17:37","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":27875,"visible":true,"origin":"","legend":"Table S1 Primer sequences","description":"","filename":"primersequencesTableS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8003045/v1/70b6cd267d3a0909882b232f.pdf"},{"id":95895941,"identity":"aecf35fd-69c9-445c-97df-38ad5e8627f5","added_by":"auto","created_at":"2025-11-14 07:17:37","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":39608,"visible":true,"origin":"","legend":"Table S2 Crispr guide sequences","description":"","filename":"CRISPRguidesTableS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8003045/v1/0489809f02489bcce141118b.pdf"},{"id":95895955,"identity":"8404cdd6-7e4c-48ee-b1fd-1e04cadd1c3d","added_by":"auto","created_at":"2025-11-14 07:17:38","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":7361400,"visible":true,"origin":"","legend":"Figure S1","description":"","filename":"FigS1beas2b.tif","url":"https://assets-eu.researchsquare.com/files/rs-8003045/v1/65a1cf1f3e5608537b7edc6d.tif"},{"id":96242656,"identity":"d4bc38f0-fbab-4699-8da1-cdbc7034c8fc","added_by":"auto","created_at":"2025-11-19 07:13:55","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":5683660,"visible":true,"origin":"","legend":"Figure S2","description":"","filename":"FigS2papandmsepithelialexpressionofIL33.tif","url":"https://assets-eu.researchsquare.com/files/rs-8003045/v1/1642d4b856ba4d8570a21e6d.tif"},{"id":95895958,"identity":"40c72766-1825-4620-8e3e-78edca9115ca","added_by":"auto","created_at":"2025-11-14 07:17:38","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":49567409,"visible":true,"origin":"","legend":"Figure S3 Uncropped Western Blots","description":"","filename":"FigS3fulluncroppedwesterns.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8003045/v1/973f029cbab78ce0c1e3678d.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"Epithelial microbial sensing through the non-canonical inflammasome modulates airway type 2 immunity","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8003045/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8003045/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Asthma is often characterized by type 2 inflammation triggered by environmental aeroallergens. Epidemiological studies link respiratory microbial infections, including intracellular bacteria, to both asthma initiation and exacerbations, yet the underlying molecular mechanisms remain poorly understood. Airway epithelial cells sense both microbes and allergens, and recent studies show that common aeroallergens induce type 2 inflammation through mechanisms including protease activity and pore-forming toxins that converge on epithelial cell damage, IL-33 secretion, and mitogen-activated protein kinase (MAPK) signaling. Host defense against microbes also involves endogenous membrane pore formation through inflammasome activation and gasdermin-mediated pyroptosis, releasing interleukin (IL)-1β and IL-18 to orchestrate bacterial killing. However, whether airway epithelial microbial sensing interacts with allergen-mediated cell death mechanisms to regulate type 2 immunity through analogous membrane pore formation and IL-33 release remains unknown. Using human airway epithelial cells and mouse models, we demonstrate that activation by intracellular lipopolysaccharide (LPS) initiates caspase-4-dependent pyroptotic cell death, IL-33 secretion, and a MAPK transcriptional program. Non-canonical pyroptotic cell death is enhanced by protease allergen through calcium signaling, and mice lacking caspase-4 show reduced IL-33 secretion and diminished innate type 2 responses to airway allergen challenge. Purified LPS from different bacterial species engage the non-canonical inflammasome with differing intensity, suggesting that microbiome composition likely influences this disease mechanism. Asthmatic patients show increased airway expression of caspase-4 and its effector molecule gasdermin D compared to healthy controls. This study identifies non-canonical inflammasome-dependent IL-33 expression and secretion as a novel link between bacterial detection and type 2 inflammation, providing a potential mechanism for how airway bacterial dysbiosis might influence asthma development and offering new insights into the interplay between microbial sensing and allergic responses in airways.","manuscriptTitle":"Epithelial microbial sensing through the non-canonical inflammasome modulates airway type 2 immunity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-14 07:17:33","doi":"10.21203/rs.3.rs-8003045/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"88cf7d07-4a3a-4bed-94fd-3c644591914f","owner":[],"postedDate":"November 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":57399877,"name":"Biological sciences/Cell biology/Cell signalling/Extracellular signalling molecules"},{"id":57399878,"name":"Biological sciences/Immunology/Innate immunity/Pattern recognition receptors"},{"id":57399879,"name":"Biological sciences/Immunology/Innate immune cells/Innate lymphoid cells"},{"id":57399880,"name":"Biological sciences/Cell biology/Cell death/Necroptosis"}],"tags":[],"updatedAt":"2025-12-29T10:55:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-14 07:17:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8003045","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8003045","identity":"rs-8003045","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}