Amphiregulin drives mitochondrial vesicle packaging and delivery for immune modulation

Amniotic Epithelial Cells (AECs) exhibit paracrine immunoregulatory functions, however the effectors within their extracellular vesicles (EVs) are poorly defined. Using label-free proteomics and genetic approaches, we profiled the secretome of ovine AECs under basal and lipopolysaccharide (LPS)-mimicking inflammatory stimuli. Microvesicles (MVs) emerged as the main LPS-responsive EVs, selectively enriching 169 proteins involved in immunometabolism and stress signaling. Among these, the Epidermal Growth Factor ligand Amphiregulin (AREG) was enriched in MVs after LPS. AREG silencing (siAREG) abolished mitochondria packaging into MVs impairing their delivery to immune cells. siAREG-MVs exhibited reduced uptake by Peripheral Blood Mononuclear Cells (PBMCs) and T-cells, diminishing their capacity to suppress proliferation and CD3-dependent NFAT activation. Supplementation with exogenous AREG partially restored mitochondrial transfer and immunosuppression, supporting a dual immunoregulatory role for AREG in AEC biology. These findings elucidate the immunomodulatory function of AEC-derived MVs and highlight AREG- mediated vesicular signaling as a promising immunotherapeutic target for cell-free strategies.