Kavanagh, AoifeAoifeKavanagh2025-10-302025-10-302025 the A2025http://hdl.handle.net/10197/29601Introduction: Efferocytosis is the clearance of apoptotic cells phagocytic engulfment, with inadequate clearance halting inflammation resolution and being linked to autoimmune and chronic inflammatory conditions (Cai et al.,2017; Doran et al.,2020; Gerlach et al.,2020; Jorge et al.,2022; Li et al.,2024). Specialised pro-resolving lipid mediators, LXA4, have been shown to enhance efferocytosis. Synthetic mimetics of LXA4, AT-01-KG and AT-02-CT, have been developed and shown to emulate several anti-inflammatory and pro-resolving actions of LXA4, however their effect on efferocytosis has yet to be rigorously tested (de Gaetano et al.,2019; de Gaetano et al.,2021; Galvão et al.,2021; Rago et al.,2024). In this study, the ability of AT-01-KG and AT-02-CT to mimic the efferocytosis enhancing effects LXA4 was evaluated using a novel imaging assay, IncuCyte Live-cell system, to measure efferocytosis through the percentage of macrophages engulfing apoptotic cells, the rate of apoptotic cell uptake, and the number of unengulfed apoptotic cells remaining. Methods: A cell-line based model of efferocytosis was created using macrophages, derived from Phorbol 12-myristate 13-acetate-treated THP-1 cells, and neutrophil-like cells, derived from dimethyl sulfoxide treated HL-60 cells, rendered apoptotic via staurosporine. Macrophages were pretreated with AT-01-KG or AT-02-CT [concentrations 1pM-1μM] for 30 minutes prior to co-incubation with apoptotic cells. Live-cell imaging was utilised to track efferocytosis over a 10-hour co-incubation period. Results: Images were successfully captured using the live-cell imaging assays and subsequently analysed to track apoptotic cell engulfment. Using the aforementioned criteria for evaluation of efferocytosis, neither pretreatment with AT-01-KG nor AT-02-CT, resulted in a statistically significant enhancement of efferocytosis, nor did the endogenous control LXA4, which had been shown to be effective in other studies of efferocytosis enhancement using primary based macrophages. Discussion: The cell-line based model used in this study did not show significant enhancement of efferocytosis in any of the pretreatment conditions, including LXA4, which had been shown in multiple previous studies to have induced significant enhancement of efferocytosis when used to pretreat macrophage cells derived from primary cell sources (Godson et al.,2000; Mitchell et al., 2002; O'Sullivan et al., 2007; Reville et al., 2006). The utilisation of a cell-line based model in this study may have been unsuitable for examining enhancement of efferocytosis due to the baseline rate of efferocytosis being far greater than baseline rates shown in previous studies that utilised primary-cells. These primary cell-based models may have been better suited to highlight any enhancing effect that pretreatment may have had on efferocytosis. The likelihood of the cell-line based model being effectively saturated prior to pretreatments, was further supported by the lack of efferocytosis enhancement in response to the positive control BMS-986235, a FPR2 selective agonist. While future experiments would benefit from the live-cell-imaging model used, enhancement of efferocytosis may be better observed in cells with lower baseline rates of efferocytosis, due either to a lower number of apoptotic cells present, or through the use of primary monocyte derived macrophages that have been utilised by similar studies in the past.enInflammation resolutionEfferocytosisChronic inflammationMaster Thesishttps://creativecommons.org/licenses/by-nc-nd/3.0/ie/