Effect of estrogen on host-pathogen interactions in ex vivo and in vitro models of the inflammatory phase of age-related impaired healing

El Mohtadi, Mohamed (2019) Effect of estrogen on host-pathogen interactions in ex vivo and in vitro models of the inflammatory phase of age-related impaired healing. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Chronic wounds in the elderly often become infected, leading to substantial morbidity and mortality. Impaired healing in the elderly is mediated by age-related changes in steroid hormones, particularly declining levels of estrogen with increasing age. Although the anti-inflammatory activity of estrogen has been defined, very little is known about the effects of estrogen deprivation (ageing processes) on bacterial clearance. The aim of this study was to determine the effect of ageing (estrogen deprivation) on the ability of in vitro human U937-derived macrophages and ex vivo human peripheral blood monocyte (HPBM)-derived macrophages to eliminate bacteria via phagocytosis. Host-pathogen assays were used to measure macrophage-mediated phagocytosis of two major wound pathogens, methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, under in vitro and ex vivo conditions that model estrogen levels in the elderly, young adults and following exogenous estrogen supplementation. Epifluorescence, confocal and scanning electron microscopy were used to visualise host-pathogen interactions and protein mediators of phagocytosis were measured by immunoblotting. Estrogen at concentrations typical of youth or supraphysiological levels significantly (P<0.05) increased the phagocytosis and effective killing of MRSA and P. aeruginosa in a dose-dependent manner compared to estrogen deprivation with significantly enhanced clearance of bacteria by M1 macrophages compared to M2 or M0 macrophages. Epifluorescence, confocal and scanning electron microscopy confirmed estrogen increases co-localisation of fluorescent GFP-S. aureus or mCherry-P. aeruginosa within macrophages and promotes bacterial internalisation. Activation of estrogen receptor (ER)-alpha (ER-α) mirrored the stimulatory effect of estrogen on phagocytosis whilst ER-α antagonism significantly (n=6; P<0.05) blocked the phagocytic effect of estrogen. In contrast, activation of ER-beta (ER-β) had no significant (n=6; P>0.05) effect on phagocytosis, confirming estrogen mediates bacterial clearance via specifically through ER-α. Immunoblotting analysis demonstrated that enhanced phagocytosis by estrogen is associated with altered levels of mediators involved in the actin cytoskeleton of phagocytes including increased levels of FAK, Rac1, Cdc42 and RhoG, but reduced levels of RhoA. Collectively the findings suggest estrogen may promote the resolution of wound bacterial infections during youth but this protection is lost as estrogen levels decline with increasing age, resulting in increased propensity and progression of wound infections in the elderly. Thus, novel wound dressings that provide local estrogen supplementation or selective activation of ER-α and/or specific targeting of downstream mediators of the actin cytoskeleton may provide effective treatment options for infected wounds in the elderly.