Introduction: Amniotic epithelial cells (AECs) possess remarkable immunomodulatory and regenerative properties, largely mediated through paracrine signaling. Recent advances highlight the relevance of both soluble factors and extracellular vesicles (EVs), including microvesicles (MVs), in immune regulation. However, the precise molecular mediators and mechanisms underlying these effects remain partially understood, hindering the clinical translation of AEC-based therapies. Our integrated research aims to dissect the role of  AEC soluble and MV fraction in activating immunemodulatory pathways, identifying Amphiregulin (AREG) as a central effector of MVs fraction mediating AEC immune cell communication.

Methods: Ovine AECs and amniotic membranes (AMs) were exposed to inflammatory stimulus (LPS) before assessing dynamic immunoregulatory responses. The MV fraction was characterized using label-free quantitative lipidomics and proteomics. Functional assays included co-culture of AEC-characterized MVs with PBMCs and Jurkat reporter cells. Mitochondrial content and transfer were evaluated via fluorescence labeling and confocal microscopy, in vitro and in vivo (Zebrafish caudal fin cut model). Gene silencing of AREG a differential expressed protein of MVs fraction was used to assess its functional role in modulate immune cell activation, and exogenous supplementation validated its rescue potential.

Results: AREG was identified as a key molecule released in a PGE2-dependent manner upon COX-2 activation, initiating a positive feedback loop via the EGFR axis that enhances AEC responsiveness to inflammatory cues.  AREG secretome composition suppressed T-cell NFAT activation and PBMC proliferation through a TGF-β-mediated mechanism. Parallelly, MVs fraction is esatblished to be the main responsible of this immunemodulatory activity that inhibit proliferation but even act by delivering active mitochondria inducing in immune cells  mitochondrial hyperpolarization before triggering apoptosis. Proteomic analysis revealed that MVs are selectively enriched of AREG that drammatically increases after  inflammatory stimulation. Silencing AREG disrupted mitochondrial packaging and abolished MV-induced targetting into immuno cell by reducing the immunomodulatory influence, while AREG supplementation enable to restore partial function.

Conclusion: Our findings establish AREG as a master regulator of AEC-derived immunomodulation, mediating mainly MVs communication mechanisms. Through coordinated action along the COX-2/PGE2/EGFR axis, AEC MVs  is enriched of AREG in response to inflamatory stimuli  by increasing their cargo critical for mitochondrial delivery. AREG enables AEC-MVs to dynamically respond to environmental stimuli and execute context-specific immune suppression. These insights lay the groundwork for next-generation cell-free therapies based on amniotic derivatives, offering novel avenues for treating immune-mediated disorders and advancing regenerative medicine.