Development of an In-Vitro Model to Study the Effect of Shear and Biomaterial on Neutrophil Extracellular Trap Release by Blood Neutrophils and HL-60 Cells
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Cardiovascular biomaterials are essential for the support of damaged or diseased cardiovascular tissue and structures. Their applications include stents, heart valves, vascular grafts, and pacemakers. The success of these biomaterials is crucial for improving patient outcomes and enhancing quality of life. Shear stress, the frictional force exerted by flowing blood on the surface of cardiovascular biomaterials, can directly influence the performance and stability of these materials. Studying the impact of shear stress can aid in the development of improved biomaterials. Neutrophils (PMNs) are a type of white blood cell that play a crucial role in the body’s immune system and defend against infection. In addition to their conventional role in phagocytosing and killing pathogens, neutrophils employ another defense mechanism known as neutrophil extracellular traps (NETs). These web-like structures are released by neutrophils to immobilize and eliminate microbes and are composed of DNA as well as antimicrobial proteins. With the introduction of a biomaterial into the body, neutrophils respond by releasing inflammatory mediators and forming NETs. Previous studies have revealed that NETs form on the surface of a variety of biomaterials, however, the effect of shear on NETosis in the presence of a biomaterial has not been examined in blood neutrophils or in the neutrophil-like HL-60 cell line model. In this study, an in-vitro model was developed to assess the effect of shear on NETosis in the presence of silicone, a common biomaterial. Neutrophils isolated from healthy donor blood and differentiated HL-60s were exposed to either static or dynamic conditions. Following exposure, NETs and neutrophil activation were measured using a FACSCalibur flow cytometer and the silicone surface was characterized by immunofluorescence. Preliminary HL-60 results indicate that two populations of cells are present during analysis: healthy and damaged. With the inclusion of both populations during analysis, results show that shear, in the presence of a plasma-coated biomaterial, increases the number of cells undergoing NETosis and results in a small, elevated, NET marker release as shown by expression levels of the antimicrobial protein myeloperoxidase (MPO) and citrullinated histone (citH3). In blood-isolated neutrophils, shear also increases the number of cells undergoing NETosis, however there is a minimal change in NET marker expression. For both cell types, immunofluorescence staining reveals that NETs largely aggregated on the silicone surface, presenting the need to further explore and quantify NETosis on the biomaterial. When the complement system is inactivated under shear conditions by ethylenediaminetetraacetic acid (EDTA), the NET signal is reduced in both HL-60 cells and blood neutrophils. Using the developed model, results support the idea that shear modulates NETosis in the presence of a biomaterial and may play a role in the distribution of NETs between the material surface and circulation. Given the difference in NETosis response between HL-60 cells and PMNs, the HL-60 cell line may not be an accurate and appropriate model to investigate NET and material interactions under shear. To better characterize the mechanisms involved in NETosis on the biomaterial and under shear, the silicone surface and the aggregates found therein should be further analyzed.
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Andreea Palage (2023). Development of an In-Vitro Model to Study the Effect of Shear and Biomaterial on Neutrophil Extracellular Trap Release by Blood Neutrophils and HL-60 Cells. UWSpace. http://hdl.handle.net/10012/19602