In a new study, researchers at the University of Pécs, Hungary used cell secretions known as exosomes to regenerate the thymus, one of the most important organs in the body.
The thymus shrinks as we age
The thymus is arguably one of the most critical organs in the body, and it is where new T cells develop before being trained in the lymph nodes in order to become the soldiers of the adaptive immune system. However, as we get older, the thymus starts to shrink, its ability to create new T cells declines, and the immune cell-producing tissue increasingly turns into fat and wastes away; this process is known as thymic involution.
This loss of thymic output results in a decline of the adaptive immune system and is part of the overall decline of the immune system known as immunosenescence. The result of thymic involution is that the immune system can no longer mount an effective defense and is inappropriately activated, leading to dysfunction and persistent inflammation. This inflammation contributes to inflammaging, a chronic smoldering background of low-grade inflammation, which other age-related sources contribute to as well.
The decline of the thymus has been linked to cancer risk, which rises dramatically as we age as part of the immunosenescence model of cancer. Immunosenescence is also strongly correlated with multiple age-related diseases, which is probably no surprise, given that the aged immune system is no longer able to respond effectively or even appropriately to invading pathogens.
Over the years, researchers have engaged in multiple approaches to regenerate the thymus, causing it to regrow and resume efficient T cell production; some of these approaches are currently ongoing. This new study takes a somewhat novel approach, as it encourages the thymus to regrow using exosomes .
What are exosomes?
Exosomes are part of a larger group of cell secretions known as extracellular vesicles (EVs), which are basically membrane-wrapped packages that contain proteins and other molecules, are produced and released by cells, and deliver messages to other cells. When nearby cells intercept these packages, they change their behavior based on the information contained in the EVs.
The vesicles being secreted by healthy cells are referred to by multiple names, including ectosomes, microparticles, and shedding microvesicles. For the purposes of discussion, we use extracellular vesicles as a generic term to describe all secreted vesicles.
EVs can broadly be described as exosomes, microvesicles (MVs), or apoptotic bodies, depending on their cellular origin:
|Origin||Endocytic pathway||Plasma membrane||Plasma membrane|
|Function||Intercellular communication||Intercellular communication||Facilitation of phagocytosis|
|Size||40-120 nm||50-1,000 nm||500-2,000 nm|
|Contents||Proteins and nucleic acids (mRNA, miRNA, and other non-coding RNAs)||Proteins and nucleic acids (mRNA, miRNA, and other non-coding RNAs)||Nuclear fractions, cell organelles|
Extracellular vesicles have attracted significant interest in the scientific community in recent years due to their role in intercellular signaling. It has been known for a long time that cells release vesicles into the extracellular environment during apoptosis. However, the fact that healthy cells also release vesicles into the extracellular environment has only been realized more recently.
These researchers have used EVs, specifically exosomes, the smallest of the types of EVs, to seek out the thymus and encourage thymic tissue regeneration to occur. To achieve this, they focused on activating the transcription factor FOXN1, the master regulator of thymus organogenesis and identity, which ensures that new cells become T-cell producing tissue rather than fat.
FOXN1 is directly targeted by the glycolipoprotein Wnt4 in order to activate it, which makes Wnt4 critical for thymus growth during development and for maintaining this organ during adulthood. However, as we age, our thymic epithelial cells produce less Wnt4, meaning that they begin to lose thymic identity and instead turn into fat cells; this ultimately causes the thymus to begin the process of involution and T cell production to fall.
These researchers opted to target Wnt4 using transgenic thymic epithelial cells modified to overexpress Wnt4. Then, they harvested the exosomes given off by these transgenic cells, which contained elevated levels of Wnt4 and other key components packaged with it. The reason for using exosomes is that, as previous studies have shown, pure Wnt4 loses its ability to activate FoxN1, as it normally travels as part of an exosome in the body.
This study shows that the exosomes collected from the transgenic thymic epithelial cells were able to counter gene expression changes in other thymic epithelial cells exposed to them, thus preserving thymic cell identity and preventing those cells from changing into mesenchymal or adipose cells, which leads to thymic involution and loss of T cell production.
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.
Intriguingly, the exosomes from thymic epithelial cells had, until now, not been shown to be linked to thymus regeneration, and these results show that their contributions to the growth, maintenance and regeneration of the organ are significant. This opens the door to regrowing the thymus via the use of exosomes.
These initial results in mice are promising, and while we should be cautious about their translation to humans, there are grounds for some degree of optimism about the approach. This study’s in vitro and in vivo results suggest that exosomes offer an avenue for regenerating the mouse thymus and that they hold promise for translation to people in the future. There is, of course, more work to be done before we reach this point, but the march towards thymic regeneration continues apace.
 Banfai, K., Garai, K., Ernszt, D., Pongracz, J. E., & Kvell, K. (2019). Transgenic Exosomes for Thymus Regeneration. Frontiers in immunology, 10.