ORIGINAL RESEARCH article

Front. Immunol.

Sec. Mucosal Immunity

Volume 16 - 2025 | doi: 10.3389/fimmu.2025.1582526

Neutrophils Aggravate Inflammatory Lesions in Intestinal Organoids from Necrotizing Enterocolitis

Provisionally accepted
  • 1Department of Pediatric Surgery, University Medical Center Hamburg Eppendorf, Hamburg, Germany
  • 2Department of Pediatric Surgery, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany
  • 3Leibniz Institute of Virology (LIV), Hamburg, Hamburg, Germany
  • 4III. Medical Clinic and Polyclinic, University Hospital Hamburg-Eppendorf, Hamburg, Hamburg, Germany
  • 5Department of Medicine 3, Friedrich Alexander University Erlangen-Nuremberg and Universitätsklinikum Erlangen, Erlangen, Germany
  • 6Deutsches Zentrum Immuntherapie, Medizinische Fakultät, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Bavaria, Germany

The final, formatted version of the article will be published soon.

Introduction: 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, the limitations of current animal models hinder the development of targeted therapies for NEC. 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. Methods: Organoid cultures were established from human neonatal intestinal samples with NEC (n=7) and without gut inflammation (controls, n=7), treated with lipopolysaccharides (LPS), and/or cocultured with neutrophils. Flow cytometry quantified neutrophil survival (propidium iodide/Annexin-V), 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. Results: 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. Discussion: 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 were 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.

Keywords: NEC1, neutrophils2, intestinal organoids3, neonate4, TLR-45, innate immune system5, co-culture6

Received: 24 Feb 2025; Accepted: 09 Jun 2025.

Copyright: © 2025 Heuer, Boettcher, Pagerols Raluy, Hagens, Kolman, Bunders, Wesche, Knopf, Herrmann, Reinshagen and Vincent. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Deirdre Kathleen Vincent, Department of Pediatric Surgery, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany

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