Single-cell RNA sequencing combined with functional experiments reveal that CD11d+ NK cell-derived exosomes suppress angiogenesis involved in the pathology of adenomyosis

Background: Adenomyosis is a common gynecological disorder characterized by the presence of endometrial-like tissue in myometrium. The exact cause of adenomyosis remains unclear, but immune dysfunction is believed to play roles in its development. Currently, there are no reports on the cellular-level changes of adenomyosis from the perspectives of intrinsic and extrinsic types.Methods: Eutopic endometrium and ectopic endometrium tissues from patients with internal adenomyosis (AM_in) and external adenomyosis (AM_ex) were collected for single-cell RNA sequencing (scRNA-seq), HE and immunofluorescence staining. Tube formation, wound assay, and EdU assay were used to assess the angiogenic regulatory role of CD11d+ NK cells.Results: The single-cell transcriptome profiles of lesion tissues from AM-in and AM-ex patients showed large differences in cellular composition. The fibroblasts constituted the primary cellular component of the endometrium, comprising 11 distinct subpopulations characterized by varying numerical and functional features in three types endometrium. A newly cell-type of NK cells was identified, CD11d+ NK cells, showing reduced abundance in AM lesion tissues relative to normal and demonstrating a potential relationship with angiogenesis. Clinical samples of endometrial tissue confirmed a reduction of CD11d+ NK cells abundance in the adenomyosis group, accompanied by increased angiogenesis (evidenced by CD31 staining). Compared to HUVECs cultured alone, the angiogenic ability of HUVECs treated with NK cell-conditioned medium was significantly decreased, however, this inhibitory effect was significantly lost when GW4869 was used to deplete exosome from the NK cell-conditioned medium. A total of 175 unique proteins in CD11d+ NK cell-derived exosomes were identified.Conclusion: CD11d+ NK cell-derived exosomes suppressed angiogenesis in adenomyosis. This study offers a cellular-level understanding of the pathological mechanisms underlying the growth of endometrial ectopia from the perspective of NK cells.