Engineering Fetal Cell Exosomes to contain HMGB1: Its trafficking and role as an inflammatory activator in uterine cells

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ABSTRACT Feto-maternal paracrine signaling, an inflammatory process, is one of the key initiators of parturition. Recently we reported that senescent fetal tissues at term, specifically fetal membrane cells, generate damage- associated molecular pattern markers (DAMPs) with proinflammatory properties, locally and at distant sites. DAMPs, along with other inflammatory cytokines and chemokines, enhance the inflammatory load to transition quiescent uterine tissues to an active state for parturition. DAMPs can reach distant sites through exosomes (30–150-nm extracellular vesicles) released by senescent fetal cells and cause inflammatory changes by increasing cytokine and chemokine productions in the recipient cells. One of the well-studied DAMPs in term and preterm parturition is high-mobility group box (HMGB)1 protein. HMGB1 is a non-histone nuclear protein, released to the cytoplasm because of nuclear injury due to cellular senescence or other insults. Senescence of fetal amnion epithelial cells (AEC) leads to packaging of HMGB1 in exosomes. HMGB1 concentration is higher in senescent-cell-derived exosomes, which led us to hypothesize that AEC exosomes containing HMGB1 can either systemically or by traversing through tissues reach maternal uterine cells (decidua and myometrium), cause functional changes by enhancing their inflammatory load (NF-kB activation and increased production of cytokines), and contribute to the parturition process. Current studies have not been able to elucidate the functional capabilities of exosome-encapsulated HMGB1 because senescent AEC exosomes traffic contents other than HMGB1. To get a precise functional property of HMGB1 in exosomes, we plan to engineer exosomes to contain a novel protein via the Exosomes for Protein Loading Via Optically Reversible Protein- Protein Interactions (EXPLORs) method. This method is developed to overcome the limitations of conventional exosome-based protein delivery. Using these HMGB1-encapsulated exosomes, we will test HMGB1's functional role in vitro in decidual and myometrial cell cultures and in animal models of pregnancy. Our aims will be Specific Aim #1: to engineer AEC-derived exosomes to specifically contain HMGB1 by integrating a reversible protein-protein interaction module controlled by blue light with the endogenous process of exosome biogenesis. Specific Aim #2: to test the functional role of HMGB1-rich AEC-derived exosomes in in vitro and pregnant animal models. We expect to generate exosomes containing specific cargo and test its functional role in promoting inflammation and parturition. Successful generation of exosomes loaded with specific cargo will introduce a new technology to load exosomes with functionally viable proteins. Future studies will test the usefulness of exosomes with therapeutic proteins that can be used for interventional purposes in adverse pregnancies.