Effect of Hormone-Driven Ageing on Inflammatory Cell Clearance of Bacteria under Hyperglycemic Conditions

Belboul, Amina (2024) Effect of Hormone-Driven Ageing on Inflammatory Cell Clearance of Bacteria under Hyperglycemic Conditions. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

The diabetic foot ulcer (DFU) represents a substantial clinical burden, frequently complicated by infection and often necessitating amputation, particularly among elderly diabetic patients. The chronic hyperglycaemia associated with diabetes disrupts the wound healing cascade, impairing immune cell phagocytic function despite exacerbating inflammatory responses. The ageing process, driven principally through estrogen deprivation, also leads to impaired wound healing and elevated inflammation. Thus, similar underlying molecular mechanisms may mediate the detrimental effects of hyperglycaemia and hormone-driven ageing, and both factors may synergistically contribute to DFU pathology. To elucidate the effects of hyperglycaemia and hormone-driven ageing on immune cell clearance of bacteria, an in vitro host-pathogen model was established whereby bacterial biofilms were exposed to U937-derived macrophages cultured in increasing concentrations of glucose (11mM, 15mM, 20mM and 30mM) and treated in the presence or absence of estrogen (1x10-7 M). Experiments conducted using the model included cell viability and growth assays, biofilm assays, phagocytosis assays, gene knockout and receptor agonist/antagonist assays, flow cytometry and/or immunoassays, and microscopy assessments of host-pathogen interactions and receptor/transcription factor localisation. High glucose levels were found to promote bacterial proliferation and biofilm formation, compromise macrophage viability and phagocytic function, and elevate macrophage-derived inflammatory cytokine production. Notably, hyperglycaemia impaired macrophage differentiation but skewed macrophage polarization toward a proinflammatory M1 phenotype. Importantly, estrogen supplementation reversed the detrimental effects of hyperglycaemia on M1 macrophage-mediated phagocytosis and bacterial biofilm clearance in a dose-dependent manner. This phagocytic rescue was associated with suppression of tumour necrosis factor-alpha (TNF-α) secretion and nuclear transcription factor-kappa B (NF-κB) activation. Furthermore, estrogen supplementation modulated the expression of key phagocytic mediators in M1 macrophages cultured under hyperglycaemic conditions, with enhanced bacterial clearance associated with reduced CD14 but increased TREM2/ApoE levels. Confocal and scanning electron microscopy confirmed estrogen stimulates the formation of actin cytoskeleton extensions, promotes bacterial internalization, and reduces NF-κB nuclear translocation in M1 macrophages. These findings suggest that reducing CD14 expression, NF-κB nuclear translocation and/or increasing TREM2/ApoE levels might be possible therapeutic approaches to reverse the detrimental effects of hyperglycaemia and estrogen deprivation. Moreover, the beneficial effects of estrogen were mediated through activation of estrogen receptor (ER)-alpha (ER-α) and not ER-beta (ER-), highlighting the potential use of selective ER modulators (SERMs) in wound dressings to enhance macrophage function and dampen inflammation in DFU patients. Further investigations employed a CD33 knockout (CD33KO) cell line (C4) within the model to elucidate the role of CD33, an immunoinhibitory receptor on monocytes/macrophages that is known to negatively regulate both phagocytic activity and inflammation. Interestingly, in line with observations in diabetic patients, hyperglycaemia reduced CD33 levels in CD33+ M1 macrophages but estrogen supplementation had no effect on CD33 levels. However, estrogen supplementation stimulated phagocytosis and reduced TNF-α in CD33KO-derived M1 macrophages cultured under hyperglycaemic conditions. Confocal microscopy, flow cytometry analyses and immunoassays showed estrogen and CD33KO have opposing effects, whereby estrogen increases and CD33KO inhibits TREM2 and ApoE levels in M1 macrophages. Moreover, estrogen reversed the detrimental effects of CD33KO on TREM2/ApoE expression and promoted the co-localization of membrane-bound CD33 (mCD33) and membrane-bound TREM2 (mTREM2) receptors on CD33+ M1 macrophages. These findings provide additional evidence that promoting the TREM2/ApoE axis with estrogen supplementation, ER- activation and/or increasing local CD33 expression in wound macrophages may be a therapeutic strategy to restore macrophage function and reduce inflammation in diabetic/DFU patients. In summary, this thesis gives an insight into the combined detrimental impacts of hyperglycaemia and estrogen deprivation on CD14/NF-κB and CD33-TREM2/ApoE axes. Results consistently indicate estrogen acts through ERα to mitigate the detrimental effects of hyperglycaemia on macrophage function by promoting TREM2/ApoE expression and blocking CD14/ NF-κB signalling to enhance the phagocytic capacity of M1 macrophages and dampen inflammatory cytokine production. Taken together, the investigations point to potential therapeutic interventions that can harness or emulate the beneficial effects of local estrogen supplementation to resolve diabetic foot infections and promote healing in DFU patients.