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

Abstract 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. Methods 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). Results 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 S1P 2 and S1P 3 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. Conclusions 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.