Sphingosine 1-phosphate-driven interplay between neutrophils and endometriotic cells fuels fibrosis in endometriosis: insights into the role of neutrophil extracellular traps

Background Endometriosis is a chronic disease affecting millions of women worldwide, characterized by the presence of ectopic endometrial-like tissue. While the formation of neutrophil extracellular traps (NETs) has been linked to endometriosis progression, the underlying implicated molecular mechanisms remain poorly understood.

The presence of NETs was evaluated using immunofluorescence and the extent of fibrosis was assessed using Masson’s trichrome staining in patient endometriotic lesions (n = 10) in comparison with the endometrium of healthy women (n = 10). Human epithelial and stromal endometriotic cells were used to evaluate the fibrotic effect of NETs. The involvement of the signaling of sphingosine 1-phosphate (S1P) in the fibrotic effect of NETs was assessed by genetic and pharmacologic approaches. Conditioned media from endometriotic cells where S1P metabolism or export were modulated, were employed to evaluate their effect on neutrophil NETosis. The plasma levels of the NET marker (MPO) and S1P were measured in patients (n = 30) and controls (n = 20).

Immunofluorescence and immunohistochemistry analysis of patient-derived lesions revealed a significant increase in NET markers and collagen deposition compared to controls. NET treatment induced epithelial-mesenchymal transition and fibrosis in endometriotic epithelial as well as stromal cells. Mechanistically, in endometriotic cells NETs modulated the S1P signaling axis by increasing the expression of sphingosine kinase-1 (SK1), one of the two isoforms responsible for S1P biosynthesis, the S1P transporter Spns2, and S1P2 and S1P3 receptors. Notably, the pro-fibrotic effect of NETs was abrogated when SK1 or Spns2 were specifically silenced. To add further layers of complexity, conditioned media from endometriotic cells where S1P metabolism or export were modulated, potently influenced neutrophil NETosis demonstrating that endometriotic cells stimulated neutrophils to release NETs via S1P, creating a pathological feedback loop. The relevance of the S1P/NET axis in endometriosis was supported by the positive correlation between S1P and MPO plasma levels in patients but not in healthy controls.

Our findings identify a novel S1P-driven bidirectional crosstalk between neutrophils and endometriotic cells that fuels the progression of fibrosis. These results highlight the S1P signaling pathway as a promising non-hormonal therapeutic target for the treatment of endometriosis since interfering with S1P metabolism and export could be extremely beneficial not only for counteracting fibrogenesis but also NETosis. Similar content being viewed by others Abbreviations - BSA: - Bovine serum albumin - CIB1: - Calcium integrin binding protein 1 - citH3: - citrullinated histone H3 - CM: - Conditioned medium - DMEM: - Dulbecco’s modified eagle medium - E-CAD: - E-caderin - EEC: - Endometriotic epithelial cells - EMT: - Epithelial–mesenchymal transition - ESC: - Immortalized human stromal endometriotic cells - F12: - Nutrient mixture f-12 ham - FBS: - Fetal bovine serum - FN: - Fibronectin - GAPDH: - Glyceraldehyde-3-phosphate dehydrogenase - IF: - Immunofluorescence analysis - IHC: - Immunohistochemistry - IS: - Internal standard (IS) - LC-MS/MS: - Liquid chromatography tandem-mass spectrometry - MPO: - Myeloperoxidase - MTT: - 3-[4,5-dimethylthiazol–2-yl]-2,5-diphenyltetrazolium bromide) - NE: - Neutrophilic elastase - NETs: - Neutrophil extracellular traps - PBS: - Phosphate buffer solution - PMA: - Phorbol-12-myristate-13-acetate - RA: - Retinoic acid - S1P: - Sphingosine 1-phosphate - S1PR: - Sphingosine 1-phosphate receptor - SK: - Sphingosine kinase - SPL: - S1P lyase - SPP: - S1P phosphatase - Spns2: - Spinster homolog 2 - TGLN: - Transgelin - αSMA: - αsmooth muscle actin

We took advantage of the newly established Molecular Medicine Facility of the Department of Experimental and Clinical Biomedical Sciences “Mario Serio” at the University of Florence. The Facility was supported by a grant from the Italian Ministry of Education University and Research (MIUR) after the Department was awarded as one of the 180 Departments of Excellence in Italy. This paper is dedicated to the memory of our co-author, Prof. Asgerally T. Fazleabas, who passed away after the resubmission of the revised version of this manuscript. We are deeply grateful for his invaluable dedication to this project. Funding The work was supported by Fondi di Ateneo (ex 60%) to C.B., C.D. and F.P., by PRIN 2022 PNRR D.D. 1409 14/9/2022 National Recovery and Resilience Plan, Mission 4—Component 2, Investment 1.1 funded by the European Union—NextGeneration EU—CUP_B53D23024590001 to C.B. and C.G., by Fondo di Beneficenza Intesa Sanpaolo to C.B. Author information Authors and Affiliations Corresponding author Ethics declarations Ethics approval and consent to participate The study was conducted under the approval of Institutional Review Board (number 13742), and in accordance with the Declaration of Helsinki. Informed consent was obtained from all participants. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Additional information Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Information Below is the link to the electronic supplementary material. Rights and permissions Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. About this article Cite this article Prisinzano, M., Seidita, I., Bertilacchi, M.S. et al. Sphingosine 1-phosphate-driven interplay between neutrophils and endometriotic cells fuels fibrosis in endometriosis: insights into the role of neutrophil extracellular traps. J Transl Med (2026). https://doi.org/10.1186/s12967-026-08971-9 Received: Accepted: Published: DOI: https://doi.org/10.1186/s12967-026-08971-9