Characterizing the dynamics of cell death and surface marker changes in tolerized human monocytes treated with epigenetic modifiers

Abstract

Sepsis is a life-threatening condition which can be characterised by dysregulated host immune responses to infection, this disease leads to significant global morbidity and mortality with over 48.9 million cases and 11 million deaths annually. The work done in this dissertation aims to investigate the effects of two epigenetic modifiers, known as squalene and butyrate on human monocytes that are treated with LPS to induce the immune tolerance phenotype. Using PBMCs obtained from healthy donors, monocytes were isolated via MACS and treated with LPS (simulating infection) and varying concentrations of squalene and butyrate to determine the ideal working concentration. Once the working concentrations were established, the cells were treated with LPS, squalene and butyrate and left for 24 and 4-hour periods. FACS analysis was then carried out using 7AAD to assess for cell death and CD14, CD206 and HLA-DR to assess cell surface marker changes. ELISAs were carried out in order to assess the levels of TNF-α. The results showed that both squalene and butyrate exhibited cytoprotective properties, which aligns with the current literature. Cell viability was also retained with both squalene and butyrate showcasing significantly lower levels of cell death when compared to the LPS control. Additionally, Cell surface marker changes were also noted for both treatments, indicating that the cells retained their function as well.