Transgenic Exosomes for Thymus Regeneration.

Abstract

During senescence, Wnt4 expression is down-regulated (unlike their Frizzled receptors), while PPARgamma expression increases in the thymus. Together, these changes allow for thymic degeneration to occur, observed as adipose involution. However, when restored, Wnt4 can efficiently counteract PPARgamma and prevent thymic senescence from developing. The Wnt-pathway activator miR27b has also been reported to inhibit PPARgamma. Our goal was to evaluate the Wnt4 and miR27b levels of Wnt4-transgenic thymic epithelial cell (TEC)-derived exosomes, show their regenerative potential against age-related thymic degeneration, and visualize their binding and distribution both in vitro and in vivo. First, transgenic exosomes were harvested from Wnt4 over-expressing TECs and analyzed by transmission electron microscopy. This unveiled exosomes ranging from 50 to 100 nm in size. Exosomal Wnt4 protein content was assayed by ELISA, while miR27b levels were measured by TaqMan qPCR, both showing elevated levels in transgenic exosomes relative to controls. Of note, kit-purified TEI (total exosome isolate) outperformed UC (ultracentrifugation)-purified exosomes in these parameters. In addition, a significant portion of exosomal Wnt4 proved to be displayed on exosomal surfaces. For functional studies, steroid (Dexamethasone or DX)-induced TECs were used as cellular aging models in which DX-triggered cellular aging was efficiently prevented by transgenic exosomes. Finally, DiI lipid-stained exosomes were applied on the mouse thymus sections and also iv-injected into mice, for in vitro binding and in vivo tracking, respectively. We have observed distinct staining patterns using DiI lipid-stained transgenic exosomes on sections of young and aging murine thymus samples. Moreover, in vivo injected DiI lipid-stained transgenic exosomes showed detectable homing to the thymus. Of note, Wnt4-transgenic exosome homing outperformed control (Wnt5a-transgenic) exosome homing. In summary, our findings indicate that exosomal Wnt4 and miR27b can efficiently counteract thymic adipose involution. Although extrapolation of mouse results to the human setting needs caution, our results appoint transgenic TEC exosomes as promising tools of immune rejuvenation and contribute to the characterization of the immune-modulatory effects of extracellular vesicles in the context of regenerative medicine.