Fasciola hepatica Soluble Antigen (FhAg)-Induced NETs Under Hypoxic Conditions Exert Cytotoxic Effects on Hepatic Cells In Vitro

<i>Fasciola hepatica</i> is a parasitic trematode that causes fasciolosis in sheep, provoking a decrease in their reproductive capacity, weight gain, meat and milk production, and wool quality. In the pathogenesis of <i>F. hepatica</i>, the penetration and migration of parasitic stages through the liver provoke intense inflammatory immune responses and tissue damage. The aim of this study was to investigate the cytotoxic effects of <i>Fascila hepatica</i>-induced ovine NETs in exposed hepatocytes in vitro, and to analyze whether <i>F. hepatica</i> antigens (<i>Fh</i>Ag) trigger the release of ovine NETs under hypoxic conditions as well as the roles of matrix metalloproteinase-9 (MMP-9) and CD11b in this cellular process in vitro. Here, isolated ovine PMNs were co-cultured with <i>Fh</i>Ag under hypoxia (5% O₂) and NETs were visualized via immunofluorescence analyses, confirming their classical characteristics. The quantification of NETs in response to <i>Fh</i>Ag in hypoxic conditions significantly enhanced the formation of anchored and cell-free NETs (<i>p</i> < 0.01), and NADPH oxidase (NOX) inhibitor diphenylene iodonium (DPI) significantly reduced their production (<i>p</i> < 0.05). Furthermore, the cytotoxic effect of NETs on hepatic cells was determined by using a live/dead-staining with Sytox Orange, thereby demonstrating that <i>Fh</i>Ag-induced NETs are cytotoxic for hepatic cells (<i>p</i> = 0.001). We additionally analyzed PMN supernatants to determine the enzymatic activity of MMP-9, observing that <i>Fh</i>Ag exposure enhances MMP-9 release in ovine PMNs (<i>p</i> < 0.05) but not in bovine PMNs. Interestingly, by using flow cytometric analysis, we determined that the exposure of PMNs to <i>Fh</i>Ag does not increase the CD11b surface expression of ovine PMNs. This could be an effect of the activation of other surface receptors or transcription factors involved in <i>F. hepatica</i>-induced NETosis. Consequently, we hypothesize that <i>F. hepatica</i>-induced NETs play a role in the pathogenesis of fasciolosis, contributing to liver tissue damage if released in an uncontrolled manner.