Doxorubicin (Dox) is an effective antineoplastic agent used to treat cancers, but its use is limited as Dox induces adverse cardiotoxic effects. Dox-induced cardiotoxicity (DIC) can lead to heart failure and death. There is no study that investigates whether embryonic stem cell-derived exosomes (ES-Exos) in DIC can attenuate inflammation-induced pyroptosis, pro-inflammatory M1 macrophages, inflammatory cell signaling, and adverse cardiac remodeling. For this purpose, we transplanted ES-Exos and compared with ES-cells (ESCs) to examine pyroptosis, inflammation, cell signaling, adverse cardiac remodeling, and their influence on DIC induced cardiac dysfunction. Therefore, we used C57BL/6J mice ages 10 ± 2 weeks and divided them into four groups (n = 6–8/group): Control, Dox, Dox + ESCs, and Dox + ES-Exos. Our data shows that the Dox treatment significantly increased expression of inflammasome markers (TLR4 and NLRP3), pyroptotic markers (caspase-1, IL1-β, and IL-18), cell signaling proteins (MyD88, p-P38, and p-JNK), pro-inflammatory M1 macrophages, and TNF-α cytokine. This increased pyroptosis, inflammation, and cell signaling proteins were inhibited with ES-Exos or ESCs. Moreover, ES-Exos or ESCs increased M2 macrophages and anti-inflammatory cytokine, IL-10. Additionally, ES-Exos or ESCs treatment inhibited significantly cytoplasmic vacuolization, myofibril loss, hypertrophy, and improved heart function. In conclusion, for the first time we demonstrated that Dox-induced pyroptosis and cardiac remodeling are ameliorated by ES-Exos or ESCs.

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 fibroblast-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. NEW & NOTEWORTHY Doxorubicin (Dox)-induced cardiotoxicity is mediated through increased oxidative stress, apoptosis, and necrosis. We report for the first time as per the best of our knowledge that Dox initiates Toll-like receptor 4 and pyrin domain containing-3 inflammasome formation and induces caspase-1-mediated inflammatory pyroptotic cell death in H9c2 cells. Moreover, we establish that inflammation and pyroptosis is inhibited by embryonic stem cell-derived exosomes that could be used as a future therapeutic option to treat Dox-induced cardiotoxicity.

Doxorubicin (Dox) is an effective anticancer drug. Unfortunately, it causes cardiac and muscle toxicity due to increased oxidative stress and inflammation; however, it remains unknown whether Dox induces "pyroptosis" - an inflammation-mediated cell death. We investigated whether Dox induces pyroptosis in mouse soleus muscle (Sol 8) cells in vitro and to show the protective effect of embryonic stem cell exosomes (ES-exos) on pyroptosis. Dox and inflammation-induced in vitro model was generated. Pyroptosis was confirmed using immunohistochemistry (with putative markers caspase-1, IL-1β, and pro-inflammatory cytokine IL-18) and Western blotting of caspase-1 and IL-1β. The results show significant increase in the expression of caspase-1, IL-1β, and IL-18 following treatment with Dox, which was inhibited by ES-exos but not mouse embryonic fibroblast exosomes. Moreover, GW4869 compound inhibited functional activity of ES-exos, suggesting these vesicles are key players in the inhibition of pyroptosis. These results suggest that Dox induces inflammatory pyroptosis in Sol 8 cells, which is attenuated by ES-exos in vitro.

Neuraminidase (NA) of human influenza H3N2 virus has evolved rapidly and been accumulating mutations for more than half-century. However, biophysical constraints that govern the evolutionary trajectories of NA remain largely elusive. Here, we show that among 70 natural mutations that are present in the NA of a recent human H3N2 strain, >10% are deleterious for an ancestral strain. By mapping the permissive mutations using combinatorial mutagenesis and next-generation sequencing, an extensive epistatic network is revealed. Biophysical and structural analyses further demonstrate that certain epistatic interactions can be explained by non-additive stability effect, which in turn modulates membrane trafficking and enzymatic activity of NA. Additionally, our results suggest that other biophysical mechanisms also contribute to epistasis in NA evolution. Overall, these findings not only provide mechanistic insights into the evolution of human influenza NA and elucidate its sequence-structure-function relationship, but also have important implications for the development of next-generation influenza vaccines.

Doxorubicin (Dox) is a potent antineoplastic drug used to treat various cancers. Unfortunately, Dox administration is limited due to induction of adverse cardiotoxicity (DIC), which is mediated through oxidative stress, ER stress, and inflammation. However, it is unknown whether Dox induces an inflammasomne‐mediated cell death, called “pyroptosis” in heart. Pyroptosis occurs via activation of TLR‐4 receptor and generation of NLRP3 inflammasome in response to damage‐associated pattern molecules (DAPMs). This would follow‐up with stimulation of caspase‐1 cascade and secretion of pro‐inflammatory cytokines from the dead cells. This study is undertaken to determine whether Dox generates pyroptosis in cardiac cell culture model. Furthermore, to elucidate the protective effects of embryonic stem cell‐derived exosomes (ES‐Exos) in inhibiting pyroptotic cell death. For this purpose, we designed a cell culture model using H9c2 cadiomyoblasts. To generate DIC, H9c2 were exposed to Dox (2 μM for 24 hrs) to induce pyroptosis and accordingly treated with exosomes (10 μg for additional 24 hrs). Cells were assigned into 4 groups: Control, Dox, Dox+ES‐Exos, and Dox+MEF‐Exos (as negative control). According to western blot analysis, immunohistochemical staining and ELISA data, Dox administration caused significant increase in expression of inflammasome markers, TLR‐4 and NLRP3, (p<0.05), pyroptotic markers, caspase‐1, IL1‐β, Caspase‐11, and gasdermin‐D, (p<0.05), as well as pro‐inflammatory cytokines, TNF‐α and IL‐6, (p<0.05) in H9c2 cells. Conversely, increased pyroptosis and inflammation post‐Dox treatment were attenuated significantly upon ES‐Exos treatment in H9c2 cells (p<0.05). However, MEF‐Exos treatment did not make significant changes vs Dox group (non‐significant vs Dox). Overall, our data shows Dox induces pyroptosis and inflammation within cardiac cells, which can be inhibited following treatment with ES‐exosomes. The significance of this study is a new mechanistic insight on the pathophysiological role of NLRP3 inflammasome‐mediated pyroptosis in Dox‐induced cardiotoxicity.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neuraminidase (NA) of human influenza H3N2 virus has evolved rapidly and been accumulating mutations for more than half-century. However, biophysical constraints that govern the evolutionary trajectories of NA remain largely elusive. Here, we show that among 70 natural mutations that are present in the NA of a recent human H3N2 strain, >10% are deleterious for an ancestral strain. By mapping the permissive mutations using combinatorial mutagenesis and next-generation sequencing, an extensive epistatic network is revealed. Biophysical and structural analyses further demonstrate that certain epistatic interactions can be explained by non-additive stability effect, which in turn modulates membrane trafficking and enzymatic activity of NA. Additionally, our results suggest that other biophysical mechanisms also contribute to epistasis in NA evolution. Overall, these findings not only provide mechanistic insights into the evolution of human influenza NA and elucidate its sequence-structure-function relationship, but also have important implications for the development of next-generation influenza vaccines.