Embryonic stem cell-derived exosomes inhibit doxorubicin-induced TLR4-NLRP3-mediated cell death-pyroptosis

Abstract

Doxorubicin (Dox)-induced cardiac side effects are regulated through increased oxidative stress and apoptosis. However, it remains unknown whether Dox induces the specific inflammatory-mediated form of cell death called pyroptosis. The current study is undertaken to determine whether Dox induces pyroptosis in an in vitro model and to test the potential of exosomes derived from embryonic stem cells (ES-Exos) in inhibiting pyroptosis. H9c2 cells were exposed to Dox to generate pyroptosis and then subsequently treated with exosomes to investigate the protective effects of ES-Exos. Mouse embryonic fi-broblast-exosomes (MEF-Exos) were used as a cell line control. We confirmed pyroptosis by analyzing the presence of Toll-like receptor 4 (TLR4)-pyrin domain containing-3 (NLRP3) inflammasome that initiates pyroptosis, which was further confirmed with pyroptotic markers caspase-1, IL-1β, caspase-11, and gasdermin-D. The presence of inflammation was confirmed for proinflammatory cytokines, TNF-α, and IL-6. Our data show that Dox exposure significantly (P ± 0.05) increases expression of TLR4, NLRP3, pyroptotic markers (caspase-1, IL-1β, caspase-11, and gasdermin-D), and proinflammatory cytokines (TNF-α and IL-6) in H9c2 cells. The increased expression of inflammasome, pyroptosis, and inflammation was significantly (P ± 0.05) inhibited by ES-Exos. Interestingly, our cell line control, MEF-Exos, did not show any protective effects. Furthermore, our cytokine array data suggest increased anti-inflammatory (IL-4, IL-9, and IL-13) and decreased proinflammatory cytokines (Fas ligand, IL-12, and TNF-α) in ES-Exos, suggesting that anti-inflammatory cytokines might be mediating the protective effects of ES-Exos. In conclusion, our data show that Dox induces pyroptotic cell death in the H9c2 cell culture model and is attenuated via treatment with ES-Exos.