Suppression of autophagy and HCK signaling promotes PTGS2high FCGR3- NK cell differentiation triggered by ectopic endometrial stromal cells.

Impaired NK cell cytotoxic activity contributes to the local dysfunctional immune environment in endometriosis (EMS), which is an estrogen-dependent gynecological disease that affects the function of ectopic endometrial tissue clearance. The reason for the impaired cytotoxic activity of NK cells in an ectopic lesion microenvironment (ELM) is largely unknown. In this study, we show that the macroautophagy/autophagy level of endometrial stromal cells (ESCs) from EMS decreased under negative regulation of estrogen. The ratio of peritoneal FCGR3- NK to FCGR3+ NK cells increases as EMS progresses. Moreover, the autophagy suppression results in the downregulation of HCK (hematopoietic cellular kinase) by inactivating STAT3 (signal transducer and activator of transcription 3), as well as the increased secretion of the downstream molecules CXCL8/IL8 and IL23A by ESCs, and this increase induced the upregulation of FCGR3- NK cells and decline of cytotoxic activity in ELM. This process is mediated through the depression of microRNA MIR1185-1-3p, which is associated with the activation of the target gene PTGS2 in NK cells. FCGR3- NK with a phenotype of PTGS2/COX2high IFNGlow PRF1low GZMBlow induced by hck knockout (hck-/-) or 3-methyladenine (3-MA, an autophagy inhibitor)-stimulated ESCs accelerates ESC's growth both in vitro and in vivo. These results suggest that the estrogen-autophagy-STAT3-HCK axis participates in the differentiation of PTGS2high IFNGlow PRF1low GZMBlow FCGR3- NK cells in ELM and contributes to the development of EMS. This result provides a scientific basis for potential therapeutic strategies to treat diseases related to impaired NK cell cytotoxic activity. ABBREVIATIONS: anti-FCGR3: anti-FCGR3 with neutralizing antibody; Ctrl-ESC: untreated ESCs; CXCL8: C-X-C motif chemokine ligand 8; ectoESC: ESCs from ectopic lesion; ELM: ectopic lesion microenvironment; EMS: endometriosis; ESCs: endometrial stromal cells; eutoESC:eutopic ESCs; HCK: hematopoietic cellular kinase; HCK(OE): overexpression of HCK; IFNG: interferon gamma; IL23A (OE): overexpression of IL23A; KLRK1: Killer cell lectin like receptor K1; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; 3 -MA: 3-methyladenine; 3-MA-ESC: 3-MA-treated ESCs; MIR1185-1-3p+: overexpression of HsMIR1185-1-3p; NK: natural killer; normESCs: normal ESCs; Rap-ESC:rapamycin-treated ESCs; PCNA: proliferating cell nuclear antigen; PF: peritoneal fluid; SFKs: SRC family of cytoplasmic tyrosine kinases; si-HCK: silencing of HCK; siIL23A: silencing of IL23A; USCs: uterus stromal cells.