Fighting fire with fire: Extracellular vesicles from pyroptotic mesenchymal stromal cells can dampen inflammation during sepsis

Main text

Mesenchymal stromal cells (MSCs) have garnered widespread attention for their potential use as a therapeutic to treat a vast range of inflammatory diseases such as graft-versus-host disease, type 1 diabetes, multiple sclerosis, and Crohn’s disease.¹ Current consensus has postulated that the mechanism underpinning MSCs’ therapeutic action is via the secretion of cytokines, chemokines, and subcellular particles such as extracellular vesicles (EVs) from MSCs.² Notably, since the discovery that infused MSCs do not engraft and can undergo apoptosis to exert immunoregulation,^3,4 the importance of MSC cell death and factors that are released during the progression of apoptosis is of particular interest to the field. In a recent study by Huang et al. published in this issue of Molecular Therapy, MSCs were shown to release specialized EVs while undergoing a lytic form of programmed cell death known as pyroptosis.⁵ Importantly, these EVs were shown to protect B cells from undergoing pyroptosis in a mouse model of sepsis and, consequently, limit the severe inflammatory responses and tissue injuries associated with sepsis. The findings from this study highlight the intriguing possibility that MSCs undergoing different forms of programmed cell death can release distinct EV subsets that exhibit unique therapeutic properties.

EVs can mediate intercellular communication through transfer of biomolecules such as phospholipids, microRNAs and signaling proteins that are packaged within or on the vesicles. It is well recognized that EVs generated from healthy MSCs can have many immunomodulatory and regenerative properties.² More recently, EVs derived from dying cells such as those from apoptotic MSCs (denoted as ApoEVs) has also been shown to exhibit similar functional properties.⁶ In this study from Huang and colleagues, the authors described for the first time the ability of MSCs to generate EVs during pyroptosis. Although there are limited reports on the ability of MSCs undergoing pyroptotic cell death in vivo, Huang and colleagues characterized how the commonly used pyroptosis induction method (i.e., LPS priming and nigericin treatment) could trigger MSC pyroptosis under in vitro settings and subsequently generate exosome-like EVs (denoted as pyroEVs). It is worth nothing that while the formation of pyroEVs from immune cells such as macrophages and monocytes undergoing pyroptosis has been described previously,^7,8 this study was able to identify MSC-derived pyroEVs to exhibit distinct features, in particular the distribution of ASC (apoptosis-associated speck-like protein containing CARD) on the vesicle surface. Interestingly, in contrast to the formation of a single large (∼0.5–1 μm) distinct ASC speck in immune cells undergoing pyroptosis, many smaller ASC-speck-like structures were detected in pyroptotic MSCs. Whether such localization of ASC in pyroptotic MSCs could aid the trafficking of ASC to the surface of pyroEVs remains to be defined.

One of the most remarkable observations from this study is that the administration of a single dose of MSC-derived pyroEVs was able to protect mice from tissue injury in a cecal ligation and puncture model of sepsis. Since pyroptosis is often associated with the release of proinflammatory factors, it is intriguing that EVs generated from pyroptotic cells could exhibit such protective properties in the early inflammatory phase of sepsis. Whether this could represent an important mechanism to limit the tissue-damaging effects of excessive pyroptotic cell death remains to be fully defined. Moreover, whether such protective effects are limited to pyroEVs generated from MSCs warrants further investigation. Nevertheless, Huang and colleagues described that the presence of ASC on MSC-derived pyroEVs is critical to prevent B cells from undergoing pyroptosis in the spleen and bone marrow during sepsis. The authors hypothesized that the EV-associated ASC could possibly function by binding to a Toll-like receptor (TLR), thus limiting TLR4 signaling in B cells and subsequent pyroptotic cell death.

Generally, the use of live-cell-derived EVs as cell-free therapeutics have been preferable due to characteristics such as low immunogenicity, targeting capabilities, long retention times, bioengineerability, and scalability. In contrast, ApoEVs have not been extensively explored as therapeutics due to potential disadvantages such as their large diversity in size (ranging from ∼100 nm to >5 μm in diameter) and content heterogeneity, as well as vesicle instability for the larger ApoEVs. Nevertheless, infusion of ApoEVs generated from dying MSCs do exhibit therapeutic benefits in models of tissue injury.⁶ Thus, whether MSC-derived ApoEVs, and possibly pyroEVs as described in this study, could also be developed as cell-free therapeutics is of particular interest to the field. Future investigations in this research area would require determining whether a specific subset of EVs generated from dying MSCs are mediating the therapeutic effects. As shown in this study by Huang and colleagues, variability in size distribution and contents of EVs isolated from pyroptotic MSCs was observed, with the isolated pyroEVs displaying several size peaks between ∼90 and 730 nm and only ∼20% harboring ASC. Such heterogeneity in the EV preparations from dying cells is likely a result of EVs being released during the different stages of cell death progression, including the period prior to cell death (i.e., healthy and stressed cells), after commitment to cell death, and lastly membrane lysis. Moreover, it would be of interest to directly compare EVs generated from healthy MSCs and MSCs undergoing apoptosis or pyroptosis in the described mouse model of sepsis. Notably, it has been shown previously that ApoEVs generated from apoptotic MSCs could limit tissue damage in sepsis through a Fas/Fas-ligand-dependent mechanism.⁹

Taken together, Huang and colleagues have demonstrated an intriguing approach by “fighting fire with fire” using EVs produced during inflammatory cell death to treat a condition characterized by mass inflammation. Although the mechanism underpinning the therapeutic effects of pyroEVs remains to be fully defined, it presents new opportunities to better understand fundamental MSC-EV biology and capabilities, thus expanding our repertoire of potential cell-free therapies in the future.