Duel to the death: a metabolic switch underlies thymus regeneration

From the Dudakov Lab, Clinical Research Division

As far as your body goes, there is perhaps no better example of ‘punching above your weight’ than the thymus, a small lymphoid organ nestled behind your sternum which is responsible for training your body’s immature T-cells to become effective pathogen-fighting warriors. While the thymus is crucial for effective immunity, it’s also quite vulnerable to a range of insults—infection, shock, or radiation and chemotherapy, to name a few. True to its underdog nature, however, the thymus has a secret weapon against these threats: an exquisite ability to regenerate itself following damage. And while we don’t fully understand the molecular pathways underlying the regenerative capacity of the thymus, we do know that this capacity decreases rapidly as we age, taking our immune systems with it.

The members of the Dudakov Lab in the Clinical Research Division at Fred Hutch are thymus experts, devoted to figuring out how the thymus regenerates following damage and using this knowledge to bolster regeneration and improve immune function during aging or after damaging therapies. A recent study from the lab, led by Dr. Sinéad Kinsella, describes interesting links between the metabolism of thymic cells and their regeneration following damage.

One fact about the thymus that may be confusing to the newcomer is that it trains T-cells using both positive selection (promoting proliferation of T-cells which are effective at recognizing pathogens) and negative selection (culling T-cells which show sub-par effectiveness or recognize the body’s own tissue). Negative selection often takes the form of apoptosis—a type of regulated cell death—which means that considerable apoptotic cell death is a normal aspect of thymic function. Previous research from Dr. Kinsella had shown that this normal apoptosis is sensed as a regulator of thymus regeneration: depletion of apoptotic cells (due, for example, to acute damage) leads to the activation of regenerative signaling events, which in turn promotes thymic regeneration. This lead Dr. Kinsella and team to an intriguing follow-up question: if the thymus normally supports lots of cell death, how do thymus cells (‘thymocytes’) die following damage?

By giving mice a sub-lethal dose of radiation to trigger thymic damage (and regeneration) and subsequently extracting thymocytes for analysis, the team made a surprising initial discovery. Though the simplest assumption would be that thymic damage causes more apoptosis, Kinsella and colleagues found that radiation damage triggers an altogether distinct cell death pathway in thymocytes, called pyroptosis. In contrast to apoptosis, pyroptosis is much more inflammatory—dying cells express membrane pores and expel a variety of inflammatory signals known as damage-associated molecular patterns (DAMPs). To test the hypothesis that pyroptosis promotes thymic regeneration, the team cocultured thymocytes with neighboring thymic cells, induced pyroptosis in the thymocytes, and found that doing so was sufficient to trigger the neighboring thymic cells to express Foxn1, a marker for thymic regeneration!

A schematic illustration of a thymus regenerative pathway depicting a metabolic switch in thmyocytes which causes them to secrete ATP to neighboring thymic cells, leading to their regeneration
A schematic depicting metabolic regulation of thymocyte cell death, and the downstream signaling pathway leading to regenerative signaling in neighboring thymic epithelial cells (TECs) Image courtesy of Dr. Sinéad Kinsella

So, if damage was causing thymocytes to suddenly adopt a new pathway of cell death, what might cause this switch? For this, the team had a prime suspect: the mitochondrion. Classical studies implicate mitochondria in the regulation of apoptosis, and more recent results have linked the ubiquitous organelle to pyroptosis, prompting the team to examine their damaged thymocytes for evidence of mitochondrial changes. Indeed, they found that upon damage, thymocytes unexpectedly ramped up their mitochondrial activity in a manner coincident with the spike in pyroptosis. A few more metabolic assays gave them more clues, specifically that 1.) this ramp-up increased the production of mitochondrial reactive oxygen species, and 2.) these reactive oxygen species precipitated pyroptosis in these cells. Importantly, they were able to recapitulate these results ex vivo by feeding cultured thymocytes metabolic precursors to stimulate mitochondrial activity and subsequently block increased pyroptosis using an antioxidant drug.

Now that they uncovered an interesting metabolic mechanism behind the switch to pyroptosis in damaged thymocytes, Kinsella and colleagues turned back to another tempting question: how does increased pyroptosis in thymocytes trigger thymic regeneration? From previous work, they suspected that a DAMP secreted by dying thymocytes acted as a signaling molecule—but which molecule was it? After testing some candidate molecules, they found their culprit- extracellular ATP. As it turns out, pyroptotic thymocytes secrete ATP into the extracellular environment, which the team showed is recognized by a receptor on thymic epithelial cells called P2Y2, inducing these epithelial cells to upregulate Foxn1—the regeneration marker we introduced earlier. As a cherry on top, Kinsella and colleagues tested the in vivo relevance of their findings by administering a P2Y2 agonist (molecule which binds the P2Y2 receptor similarly to ATP) to mice before inducing thymic damage. Reassuringly, this treatment enhanced Foxn1 expression and thymic regeneration in these mice following radiation damage.

For Dr. Kinsella, this study represents a satisfying bridge between basic and translational biology. “Even though I’m an immunologist by training, metabolism is a bit of a passion of mine—the fact that this mechanism underlies thymic regeneration is interesting to me, and that our findings have potential relevance concerning regeneration in vivo is all the more gratifying.” Regarding the sometimes-disparate fields of metabolism and immunology, Kinsella comments that the underlying biology speaks for itself—closer collaboration between immunologists and metabolism researchers will prove that these findings of hers are only the tip of the iceberg.

The spotlighted research was funded by the National Institutes of Health, the National Cancer Institute, the American Society of Hematology, the DKMS Foundation for Giving Life, the Cuyamaca Foundation, the Bezos Family Foundation, and the American Society for Transplantation and Cellular Therapy.

Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium member Dr. Jarrod Dudakov contributed to this study.

Kinsella, S., Evandy, C. A., Cooper, K., Cardinale, A., Iovino, L., deRoos, P., Hopwo, K. S., Smith, C. W., Grenadier, D., Sullivan, L. B., Velardi, E., Dudakov, J. 2023. Damage-induced pyroptosis drives endogenous thymic regeneration via induction of Foxn1 by purinergic receptor activation. Biorxiv. https://doi.org/10.1101/2023.01.19.524800.