Abstract P3167: Identification Of Macrophage-specific Markers On Extracellular Vesicles In A Transgenic Mouse Model And Humans

Abstract

Extracellular Vesicles (EVs) are key mediators of intercellular communication. Macrophages, a prominent type of immune cells, are known to release EVs with distinct contents and functions. The ExoMap mice model provides a platform to investigate tissue-specific EV subpopulations and the complexity of EV heterogeneity. CD169 and CD68 markers are specific to macrophages and have been associated with immune diseases, including Sarcoidosis. Double Transgenic vav-cre/ExoMap mice and appropriate controls were used to assay for immune cell derived EVs. Expression of cre in the vav-expressing cells, leads to expression of humanized CD81 (hsCD81) and mNeonGreen in the immune cells and in their EVs. Size Exclusion Column and immuno-pulldown (IP) with anti-hsCD81 or anti-CD169/CD68 antibodies were used to isolate macrophage-specific EVs. 3 miRNAs (mir223, mir374c, mir412) specific to macrophages were tested using Droplet Digital PCR (ddPCR). To design our human pilot study, we chose patients with active sarcoidosis (n=10), latent sarcoidosis (n=10), healthy controls (n=10) to compare the level of expression of specific immune markers in their blood. Bone marrow cells prepared from vav-cre/ExoMap mice were found to express mNeon Green and hsCD81. IP combined with vesicle flow cytometry defined the presence of hsCD81/mNeonGreen EVs in the plasma of double transgenic mice. Expression of CD68 and CD169 was robust on the EVs isolated using IP with hsCD81 antibodies from Double Transgenic mice. These results are consistent with the immune specificity of the vav-cre expression. Using human pooled plasma, we detected expression of CD68 on the EVs isolated using IP with CD169 antibody, suggesting the presence of both markers on macrophage specific EVs. Our findings support these markers as conserved between mouse and human samples, indicating their potential as universal markers of macrophage-derived EVs. Ongoing experiments aim to comprehensively characterize individual macrophage-derived EVs using the quantitative single molecule localization microscopy (qSMLM). Together, our findings provide a valuable resource for future studies of macrophage-EV communication and could serve in the development of EV-based diagnostic and therapeutic strategies.