Inhibition of red blood cell-derived small extracellular vesicles release by calpeptin attenuates neutrophil inflammation

Transfusion of aged red blood cells (RBCs) is associated with adverse outcomes in critically ill patients. Extracellular vesicles (EVs), as potential risk factors for transfusion-related immunomodulation (TRIM), play a critical role in the development of sepsis. However, little is known about the effects and mechanisms of red blood cell-derived small extracellular vesicles (RBC-sEVs) that aggravate inflammation and cause poor prognosis in septic patients after RBC transfusion. This study characterized RBC-sEVs from human and mouse leukoreduced packed RBC units and their role in promoting neutrophilic inflammation in vitro and in vivo. We found that RBC-sEVs induce host inflammatory responses in a dose-dependent manner. These vesicles significantly upregulate the expression of TLR4/NF-κB, resulting in increased inflammatory cytokines, and cause dysfunction of umbilical vein endothelial cell (HUVEC). Furthermore, RBC-sEVs significantly aggravated CLP-induced septic mice inflammation by promoting the production of inflammatory cytokines, exacerbating lung injury, and reducing survival rate of CLP mice, all these effects were alleviated by treatment with the EV inhibitor calpeptin. Our findings indicate that RBC-sEVs induce host inflammatory responses, which may contribute to adverse outcomes in septic patients receiving RBC transfusions, whereas calpeptin may alleviate these pro-inflammatory effects.