An investigation of the cellular uptake and mechanisms of action of MGN-3 on inflammatory processes

Tan, Sally (2018) An investigation of the cellular uptake and mechanisms of action of MGN-3 on inflammatory processes. Masters by Research thesis (MSc), Manchester Metropolitan University.

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Abstract

The alarming rate at which pathogens are becoming resistant to conventional therapies is a reason for the urgent need to develop alternative strategies to combat bacterial infections. MGN-3 (biobran) was discovered and named after the three scientists: Maeda, Ghoneum and Ninomiya. It is an enzymatically modified rice bran arabinoxylan, which can enhance the host immune system by mediating macrophage function. The aim of this study was to investigate the effect of MGN-3 on the clearance of methicillinresistant Staphylococcus aureus (MRSA) by human U937 macrophages. The uptake of MGN-3 by U937 macrophages was measured by a phloroglucinol colorimetric assay and visualised by confocal microscopy. An in vitro host-pathogen assay (n=24) was performed following exposure of macrophages to MGN-3 (0.5 to 2.0 mg/ml) and/or lipopolysaccharide (LPS) at 1.0 μg/ml or 5.0 μg/ml for 24 hours. The assay was repeated in the presence of receptor inhibitors, to identify potential molecular mechanisms of action. Bacterial internalisation by U937 macrophages was visualised by scanning electron microscopy (SEM). The study demonstrated approximately 29% of total available MGN-3 was taken up by U937 macrophages. There was a significant (P<0.05) dose-dependent increase in clearance of MRSA by U937 macrophages in the presence of MGN-3 compared to the negative control. SEM confirmed internalisation of MRSA by U937 macrophages following MGN-3 treatment. However, MGN-3 inhibited LPS-induced phagocytosis, suggesting both polysaccharides may compete as ligands for the same receptor(s) but that LPS is more potent at inducing phagocytosis. TLR-4 and dectin-1 receptor inhibition reversed the effects of MGN-3 or LPS treatment, suggesting both MGN-3 and LPS mediate phagocytosis at least in part through these receptors. In conclusion, this study established that MGN-3 significantly promotes phagocytosis of MRSA by U937 macrophages in a dose-dependent manner via TLR-4 and dectin-1. Moreover, MGN-3 mediated LPS-induced phagocytosis, suggesting competition for the same receptor(s) and highlighting the potential of MGN-3 to modulate excessive LPSinduced pro-inflammatory responses. Collectively, these findings may ultimately lead to novel potential therapeutic strategies that can be used alongside antibiotic treatments to combat MRSA infection, thereby reducing the over reliance on antibiotic usage.