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Amphiregulin drives mitochondrial vesicle packaging and delivery for immune modulation

ABSTRACT: 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.

INSTRUMENT(S):

ORGANISM(S): Ovis Aries

TISSUE(S): Cell Culture

SUBMITTER: Adrián Cerveró Varona

LAB HEAD: Hélder A. Santos

PROVIDER: PXD074552 | Pride | 2026-07-18

REPOSITORIES: Pride

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			Action	DRS
	17319_1_1_CTR_S1-A1_1.d.zip	Other
	17321_1_2_siCIITA_S1-B1_1.d.zip	Other
	17323_1_3_LPS_S1-C1_1.d.zip	Other
	17325_1_4_siCIITA_LPS_S1-D1_1.d.zip	Other
	17327_2_1_CTR_S1-E1_1.d.zip	Other

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Project description:Microvesicles (MV) are small membrane-bound particles comprised of exosomes and various sized extracellular vesicles. These are released by a number of cell types. Microvesicles have a variety of cellular functions from communication to mediating growth and differentiation. Microvesicles contain proteins and nucleic acids. Previously, we showed that plasma microvesicles contain microRNAs (miRNAs). Based on our previous report, the majority of peripheral blood microvesicles are derived from platelets while mononuclear phagocytes, including macrophages, are the second most abundant population. Here, we characterized macrophage-derived microvesicles and whether they influenced the differentiation of naM-CM-/ve monocytes. We also identified the miRNA content of the macrophage-derived microvesicles. We found that RNA molecules contained in the macrophage-derived microvesicles were transported to target cells, including monocytes, endothelial cells, epithelial cells and fibroblasts. Furthermore, we found that miR-223 was transported to target cells and was functionally active. Based on our observations, we hypothesize that microvesicles bind to and activate target cells. Furthermore, we find that microvesicles induce the differentiation of macrophages. Thus, defining key components of this response may identify novel targets to regulate host defense and inflammation. We used GeneChip microarrays to examine changes in gene expression induced by MV in primary monocyte-derived macrophages (MDM) and in THP1 cells, and compare this to cells treated with GM-CSF and PMA, respectively. All experiments were done in triplicates. Primary monocytes were collected from buffy coats (BC). The freshly isolated monocytes from three donors (Mono1-3) were either treated with GM-CSF or subjected to RNA isolation. Following treatment, MVs were isolated from the GM-CSF-treated macrophage cultures. RNA was isolated from the remaining cells for profiling (GM1-3). The isolated MVs were then used to treat new BC monocytes for 24 h (BC-GMCSF-MV24). A fraction of the new BC monocytes was subjected to RNA extraction for profiling (BC1-3). For THP1 cells, they were treated with either DMSO or PMA to produce MVs. The MVs were collected and the remaining cells lyzed for RNA extraction and profiling (DMSO1-3 and PMA1-3). The collected MVs from the DMSO or PMA-treated THP1 cells were incubated with new THP1 for 24 h and designated DMSO-MV24 or PMA-MV24. We had a total of 24 samples.

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Amphiregulin drives mitochondrial vesicle packaging and delivery for immune modulation

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