Amphiregulin shapes mitochondrial delivery and immune modulation by microvescicles.

Amniotic epithelial cells (AECs) exert potent paracrine immunoregulatory functions, yet whether inflammatory cues enhance the biological activity of their extracellular vesicles (EVs) remains unclear. To address this question, we characterized the secretome of ovine AECs under basal and lipopolysaccharide (LPS)-stimulated conditions using label-free proteomics and genetic approaches. Inflammatory stimulation profoundly remodeled the microvesicles (MVs) cargo, enriching 101 proteins associated with metabolic and regulatory pathways. Among the top 5% were two immune-related proteins, including amphiregulin (AREG), an epidermal growth factor receptor ligand involved in immune regulation and tissue remodeling. AREG silencing reduced AREG-positive MVs and was associated with altered mitochondrial cargo content and transfer to immune cells. Consequently, siAREG-MVs showed reduced uptake by PBMCs and T cells, resulting in diminished suppression of proliferation and CD3-dependent NFAT activation. Exogenous AREG partially restored these functions. Collectively, these findings identify inflammation-driven AREG signaling as a key mechanism enhancing the immunomodulatory potential of AEC-derived MVs for cell-free immunotherapy.