AMPHIREGULIN MEDIATES IMMUNE-SUPPRESSIVE FUNCTIONS IN AMNIOTIC CELLS/TISSUE VIA THE COX-2/PGE2/EP4 AXIS

Inflammation plays a crucial role in initiating tendon regeneration by coordinating immune cells recruitment. Nevertheless, an immune imbalance often results in tissue fibrosis or chronic inflammation, compromising the recovery of tendon homeostasis after injury. Amniotic epithelial cells (AEC), collected from amniotic membranes (AM) at middle gestation, exhibit a remarkable tendon pro-regenerative influence, particularly in mitigating the early inflammatory response. However, their clinical application is limited due to a lack of knowledge of the underlying molecular mechanisms. This study aims to verify whether the interplay between the COX-2/PGE2/EP4 and the Amphiregulin (AREG)/EGFR axes plays a role in mediating the crosstalk among immune cells and AEC/AM, as observed for other stem and cancer cell models. Results reveal, for the first time, that AREG is constitutively secreted by AEC or AM, exerting a powerful immunosuppressive effect by inhibiting PBMCs-PHA activation (up to 90%) and the NFAT pathway in CD3/CD28-stimulated Jurkat cells (up to 60%). Furthermore, this protein proved to be a key soluble mediator for blunting acute inflammatory responses in vivo, experimentally induced in Tg(lyz:DsRED2) zebrafish larvae. Notably, AEC and AM release of AREG can be enhanced by external inputs such as pro-inflammatory (LPS) and stretching. Of note, these stimuli do not directly affect AREG synthesis. Instead, they firstly interfere with COX-2 expression and, in turn, via PGE2/EP4 signaling stimulate AREG secretion. These findings offer valuable insights for targeted cell-free therapeutic interventions, exploiting the paracrine influence of AEC/AM to enhance host-protective responses through inflammation control, thus promoting tissue regeneration.