Determining the cellular and molecular mechanisms by which MGN3 mediates innate immunity in a diabetic (hyperglycaemic) model of an infected chronic wound.

Shah, Sana (2025) Determining the cellular and molecular mechanisms by which MGN3 mediates innate immunity in a diabetic (hyperglycaemic) model of an infected chronic wound. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Chronic wounds, particularly the diabetic foot ulcer (DFU), pose significant healthcare challenges, leading to poor patient outcomes because of their susceptibility to reoccurrence and infection. Macrophages play a pivotal role in DFU pathology, with chronic hyperglycaemia (CH) leading to immune dysfunction, chronic inflammation and elevated infection risk. Recent evidence suggests biobran (MGN-3), an immunomodulatory agent derived from rice bran, may be able to mediate the detrimental effects of CH on macrophages. Thus, this study investigated the cellular and molecular mechanisms by which MGN-3 mediates innate immunity in a diabetic (hyperglycaemic) model of an infected DFU. Wildtype and CD33 knockout (CD33-KO) U937-derived M1-like macrophages were used to assess the impact of hyperglycaemia (15, 20, and 30mM glucose-supplementation) and 24-hour treatment with MGN-3 (0.5, 1.0, 2.0 mg/mL) on macrophage function, including the phagocytic clearance of wound pathogens (methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PAO1)), cell viability/apoptosis and the regulation of inflammatory pathways. Fluorescently-tagged bacteria (GFP-S. aureus and mCherry-P. aeruginosa) were used to visualise host- pathogen interactions by epifluorescence, confocal and scanning electron microscopy (SEM). The effect of hyperglycaemia and MGN-3 on cell viability/apoptosis was determined by a colorimetric formazan-based cell viability assay and propidium iodide (PI)/annexin flow cytometry. The potential regulation of CD14/TLR-4 and CD33 pathways by MGN-3 was interrogated through receptor inhibition/KO and associated adaptor molecule (MYD88 and TRIF) inhibition, measuring receptor expression via flow cytometry and immunoassays, and receptor localisation by confocal microscopy. Furthermore, nuclear factor-kappa B (NF-κB) transcription factor activation and the production of inflammatory markers (interferon beta (IFN-β), tumour necrosis factor- alpha (TNF-α), reactive oxygen species (ROS) and nitric oxide (NO) were evaluated by confocal microscopy and immunoassays respectively. Hyperglycaemia induced a significant (P<0.05; n=12) impairment in the phagocytosis of MRSA and PA01 by M1 macrophages compared to euglycaemic conditions, which was partially reversed by 24-hour MGN-3 or lipopolysaccharide (LPS) exposure. Moreover, gentamicin protection assay (GPA) investigations confirmed MGN-3 and LPS increased both the internalisation and killing of MRSA and PA01. Significant differences (P<0.05) in cell population distribution were observed between M1 macrophages under euglycaemic (11mM) and hyperglycaemic (30mM) conditions. M1 macrophages cultured under euglycemia showed 95% viability, while hyperglycaemic conditions reduced this to 76% (P<0.001, n=3). Hyperglycaemic exposure significantly increased early apoptosis (3.4%), late apoptosis (14%), and necrosis (6.6%) (P<0.01, n=3) compared to euglycemia. Additionally, euglycaemic M1 macrophages displayed a significantly higher proportion of cells in the G0/G1 (P=0.031, n=4) and S phases (P=0.02, n=4), indicative of quiescence. Hyperglycaemic conditions, however, significantly elevated the number of M1 macrophages in the G2/M phase (P<0.001, n=4), suggesting that high glucose levels promote cell cycle progression. In the absence of MGN-3/LPS, hyperglycaemia significantly (p<0.05; n=3) dampened IFN- β but had little effect on TNF-α secretion from M1 macrophages. Moreover, significant increases in IFN-β (p<0.05; n=3) and TNF-α (p<0.05; n=3) secretion by M1 macrophages following MGN-3 supplementation was lower than levels observed following LPS stimulation. TLR-4, MYD88 and TRIF inhibition led to a significant (p<0.01; n=3) reduction in TNF-α secretion whereas only TLR-4 and TRIF inhibition led to a significant (p<0.01; n=3) reduction in IFN-β production. Similarly, confocal analysis showed MGN-3 significantly (p<0.05; n=3) stimulated the translocation of NF-κB p65 in M1 macrophages but in a somewhat moderate manner compared to the more pronounced NF-κB translocation induced by LPS under hyperglycaemic conditions. Hyperglycaemia resulted in a significant (p<0.05; n=3) increase in ROS production by M1 macrophages. Unlike LPS that exaggerated ROS levels even further (p<0.01, n=3) MGN-3 significantly (p<0.05; n= 3) dampened glucose-induced ROS production, suggesting MGN-3 mitigates oxidative stress under elevated glycaemic conditions. TLR-4, MYD88 and TRIF inhibition led to a significant (p<0.05; n=3) reduction in ROS production under hyperglycaemic conditions following treatment of M1 macrophages with MGN-3. Moreover, MGN-3 supplementation significantly (p<0.01; n=7) restored glucose-induced suppression of NO production, with TRIF and MYD88 inhibition, but not TRL4 inhibition, significantly (p<0.01; n=7) blocking NO production (p<0.01; n=7). The significantly increased (p<0.05; n=12) bacterial clearance observed in CD33-KO M1 macrophages suggests that CD33 may act as a negative regulator of phagocytosis in M1 macrophages. Stimulation of CD33-KO M1 macrophages with MGN-3 significantly (p<0.05; n=12) boosted their phagocytic capacity further. CD33-KO M1 macrophages secreted significantly (p<0.05; n= 3) elevated TNF-, ROS and NO production together with increased NF-κB activity, indicating the absence of CD33 leads to heightened inflammatory responses. MGN-3 effectively fine-tuned immune responses in CD33-KO M1 macrophages, producing moderate enhancements in TNF-, ROS and NO production, and NF-κB activity. Moreover, confocal microscopy revealed close proximity and potential interactions between CD33 and CD14 in M1 macrophages upon stimulation with MGN-3. Flow cytometry analysis showed MGN-3 downregulated CD33 expression whereas CD33 expression was increased by inhibition of LPS-induced TLR-4 and downstream MYD88/TRIF-mediated signalling. Similarly, CD14 expression was increased by CD33 KO, indicating a reciprocal relationship may exist between these two receptors in terms of immune activation. Flow cytometry results demonstrated that neither LPS nor MGN-3 influence mCD14 levels under euglycemia conditions. However, hyperglycaemia induced significant (p<0.05; n= 3) increases in mCD14 when M1 macrophages were stimulated with LPS but not MGN-3. Intriguingly, concurrent treatment of M1 macrophages with MGN-3 and LPS significantly (P<0.01) reversed the hyperglycaemia-induced effects on LPS-mediated mCD14 expression, suggesting MGN-3 was competing with LPS for the same receptor(s). Furthermore, LPS and MGN-3 significantly (P<0.01; n=3) increased sCD14 levels under glycaemic conditions >20mM glucose whereas MGN-3 significantly (P<0.05; n=3) decreased sCD14 levels in M1 macrophages at lower glucose concentrations, indicating a potential role for MGN-3 in modulating sCD14 production in response to the glucose environment. In summary, MGN-3 mitigated hyperglycaemia-induced immune impairment in M1 macrophages, resulting in enhanced bacterial clearance and increased inflammatory signalling under hyperglycaemic conditions. However, the inflammatory response induced by MGN-3 appeared controlled, unlike the overly pronounced stimulation of inflammation induced by LPS. This suggests MGN-3 may restrain excessive inflammation whilst enhancing the suppressed phagocytic and bacterial killing activities induced by CH. Moreover, these investigations have identified CD14-TRL4, MYD88/TRIF and CD33 as key interconnected signalling mechanisms through which the immunomodulatory actions of MGN-3 are mediated in M1 macrophages. Collectively, the findings of this study highlight MGN-3 as a promising therapeutic agent for the management of DFU infections that warrants further investigation.