Neutrophils Aggravate Inflammation and NEC-like Lesions in NEC Intestinal Organoids

Neutrophils Aggravate Inflammation and NEC-like Lesions in NEC Intestinal Organoids | 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 Neutrophils Aggravate Inflammation and NEC-like Lesions in NEC Intestinal Organoids Deirdre Vincent, Kim Heuer, Laia Pagerols Raluy, Johanna Hagens, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4511166/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 Necrotizing enterocolitis (NEC) is a leading cause of neonatal death and long-term morbidity, involving complex pathophysiology including prematurity, abnormal bacterial colonization, and ischemia-reperfusion injury, partially mediated by neutrophils. However, development of targeted therapies for NEC is hindered by the limitations of current animal models. Thus, this study aimed to develop a human intestinal organoid model for NEC to investigate its pathophysiology, understand neutrophil involvement, and bridge animal and human research. Organoid cultures were established from human neonatal intestinal samples with NEC (n = 7) and without gut inflammation (controls, n = 7), treated with lipopolysaccharide (LPS), and/or cocultured with neutrophils. Flow cytometry quantified neutrophil survival (PI/Annexin), activation (CD11b/CD66b), and TLR-4 expression, as well as organoid TLR-4 expression and apoptosis markers. NEC status and neutrophil recruitment were analyzed using immunofluorescence. After LPS administration, NEC organoids showed significantly increased TLR-4 expression, intestinal apoptosis markers, and NEC scores compared to controls, with more pronounced differences after neutrophil addition. Neutrophil activation markers were elevated when cocultured with both NEC and control organoids, but TLR-4 expression increased only with NEC organoids. The findings suggest that epithelial cells from NEC patients have a heightened innate TLR-4 expression upon LPS stimulation, potentially contributing to NEC development. LPS stimulation resulted in more pronounced NEC-like lesions in NEC organoids, which was exacerbated by neutrophils. This model demonstrates that neutrophils might contribute to NEC manifestation and maintenance, and that NEC organoids can reflect disease aspects, potentially aiding in the development of targeted therapies. Biological sciences/Immunology/Inflammation/Acute inflammation Biological sciences/Immunology/Inflammation/Sepsis Biological sciences/Cell biology/Mechanisms of disease Biological sciences/Immunology/Translational immunology Full Text Additional Declarations Table 1 is available in the Supplementary Files section. Supplementary Files Vincentetal.Table1.xlsx Table 1. Clinical and demographic data of the patients used for organoid development. 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4511166","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":312913492,"identity":"16bf8042-cac8-4c66-9ce3-c5fcd2184603","order_by":0,"name":"Deirdre 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Clinical and demographic data of the patients used for organoid development.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Vincentetal.Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4511166/v1/35fb26561fef827a34745250.xlsx"}],"financialInterests":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e","formattedTitle":"Neutrophils Aggravate Inflammation and NEC-like Lesions in NEC Intestinal Organoids","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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-4511166/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4511166/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNecrotizing enterocolitis (NEC) is a leading cause of neonatal death and long-term morbidity, involving complex pathophysiology including prematurity, abnormal bacterial colonization, and ischemia-reperfusion injury, partially mediated by neutrophils. However, development of targeted therapies for NEC is hindered by the limitations of current animal models. Thus, this study aimed to develop a human intestinal organoid model for NEC to investigate its pathophysiology, understand neutrophil involvement, and bridge animal and human research. Organoid cultures were established from human neonatal intestinal samples with NEC (n\u0026thinsp;=\u0026thinsp;7) and without gut inflammation (controls, n\u0026thinsp;=\u0026thinsp;7), treated with lipopolysaccharide (LPS), and/or cocultured with neutrophils. Flow cytometry quantified neutrophil survival (PI/Annexin), activation (CD11b/CD66b), and TLR-4 expression, as well as organoid TLR-4 expression and apoptosis markers. NEC status and neutrophil recruitment were analyzed using immunofluorescence. 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This model demonstrates that neutrophils might contribute to NEC manifestation and maintenance, and that NEC organoids can reflect disease aspects, potentially aiding in the development of targeted therapies.\u003c/p\u003e","manuscriptTitle":"Neutrophils Aggravate Inflammation and NEC-like Lesions in NEC Intestinal Organoids","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-26 11:45:33","doi":"10.21203/rs.3.rs-4511166/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":"78c1dcf5-973f-4ad4-ac02-9f000b0999a4","owner":[],"postedDate":"June 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":33078636,"name":"Biological sciences/Immunology/Inflammation/Acute inflammation"},{"id":33078637,"name":"Biological sciences/Immunology/Inflammation/Sepsis"},{"id":33078638,"name":"Biological sciences/Cell biology/Mechanisms of disease"},{"id":33078639,"name":"Biological sciences/Immunology/Translational immunology"}],"tags":[],"updatedAt":"2024-07-02T07:01:15+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-26 11:45:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4511166","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4511166","identity":"rs-4511166","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}