Utilizing High-Throughput Screening of dHL-60 Inflammatory Behaviors to Evaluate Honey-Derived Molecules’ Potential as Immunomodulatory Biomaterial Additives

Evan N Main, James C Huang, Gary L Bowlin Department of Biomedical Engineering, University of Memphis, Memphis, TN, 38152, USACorrespondence: Gary L Bowlin, Email [email protected]: The ability of honey varieties to both prevent infection and prevent a dysregulated immune reaction to an implanted biomaterial provides a strong case for their viability as biomaterial additives. However, the mechanisms, components, and concentration ranges underlying honey’s anti-inflammatory activity remain incompletely understood. The present study sought to screen serial concentrations of the common flavonoid and phenolic components: pinobanksin, pinocembrin, chrysin, and methyl syringate to determine concentrations best to reduce intracellular reactive oxygen species (ROS) activity and neutrophil extracellular trap (NET) release (NETosis) in a differentiated HL60 (dHL60) model.Methods: HL60s were differentiated into neutrophil-like cells (dHL60s) using a validated protocol, and were cultured with concentrations ranging from 1 nM to 1 mM of each flavonoid and from 10 μM to 2 mM of methyl syringate. NETosis and ROS were measured via Sytox Orange staining and DCFH-DA assay.Results: ROS activity was moderately inhibited by chrysin but increased by methyl syringate. Flavonoids failed to reduce NET levels. Methyl syringate significantly reduced NETosis in a dose-dependent manner, but increased ROS.Discussion: The present study provides proof of concept for the honey-derived phenolic compound methyl syringate as a therapeutic candidate to reduce neutrophil-mediated inflammation, specifically NETosis, in response to implanted biomaterials. Methyl syringate is highlighted because NETosis can profoundly affect the progression of downstream inflammatory responses in neutrophils arriving at the site and in other cell types within the microenvironment. Furthermore, it proposes a high-throughput method to screen for potential therapeutic compounds prior to primary neutrophil investigations, thereby addressing the current lack of neutrophil-targeted drug discovery efforts. Keywords: biomaterial additives, HL60, host-biomaterial response, NETosis, neutrophil, ROS