Development of an Ex Vivo Human Whole Blood Platform for Quantifying Bacteria-Induced NETosis

Abstract

This study focused on developing a human whole blood sepsis model using S. aureus. Methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA), obtained from a previous human abscess study, were first characterized through growth curve analysis and Protein A production, using an ELISA assay, across multiple media conditions. We evaluated two outcome measures, red blood cell hemolysis and NETosis. S. aureus produces several hemolysins; however, it was not known if human red blood cells would be sensitive to them. There was no commercially available ELISA assay for these hemolysins, so we used a functional hemolysis assay. Despite optimizing the assay, we were not able to detect significant hemolysis. NETosis measurements were attempted using a commercially available H3.1 nucleosome-based ELISA kit. For unclear reasons this assay showed NETosis predominantly in the negative controls and not in response to the bacteria. We then switched to a kinetic fluorescence-based NETosis assay using SYTOX Green described by Zukas et al. We optimized the protocol by using hirudin as the anticoagulant and a SYTOX Green concentration of 5.0 uM. As expected, MRSA produced a greater degree of NETosis than MSSA. MRSA likely produces more Protein A than MSSA and we demonstrated that adding Protein A to MSSA, but not MRSA, increased the NETosis. Overall, this work suggests that human whole blood, exposed to S. aureus, can be used to model NETosis which is seen in severe sepsis. The NETosis response needs to be confirmed using complementary assays and that additional outcomes consistent with clinical sepsis modeled. If successful, the platform will provide a controlled framework for studying bacterial-induced sepsis response in a physiologically relevant ex vivo human model.