Development of a flow cytometry method to investigate reactive oxygen species induced by poly (I:C) in coelomocytes of <i>Eisenia hortensis</i>

Abstract This study investigated cellular effects of polyinosinic–polycytidylic acid [poly (I:C)] in coelomocytes of the earthworm Eisenia hortensis. Poly (I:C), a double-stranded RNA mimetic and Toll-like receptor 3 agonist, is involved in stimulating antiviral interferon and pro-inflammatory responses of innate immunity. Coelomocytes, the white blood cell equivalents in E. hortensis, were extracted, cultured with poly (I:C), and treated with dihydroethidium (DHE) to determine if pro-inflammatory responses were induced in vitro by measuring reactive oxygen species (ROS) production using flow cytometry. DHE is a fluorescent marker used for the detection of hydrogen peroxide, a molecular indicator of intracellular pro-inflammatory events. Hyaline amoebocytes were gated to exclude autofluorescent eleocytes. Fluorescence was measured in a flow cytometer, and data were analyzed using WinList software and Microsoft Excel. Significant results obtained in two independent runs for the majority of earthworms tested suggest that poly (I:C) has the capacity to increase pro-inflammatory immune responses in coelomocytes of E. hortensis, as shown by higher levels of ROS production. Future studies could investigate optimization of poly (I:C) concentration and determination of the effects of poly (I:C) on phagocytosis and signaling pathways involved in the innate immune response in this annelid model.