The development of a direct co-culture based  diabetic foot ulcer model  with application in drug screening

Diabetic foot ulcers (DFU) are wounds that are hard to heal. Secondary to hyperglycaemia, both chronic inflammation and defective phagocytosis have been identified as contributing factors to the non-healing status of DFU. In this work, both inflammation and defective phagocytosis were modelled in vitro for a DFU model using pHRODO bioparticles under optimized pathophysiological conditions using a direct co-culture approach. The pHRODO bioparticles are chemically killed microorganisms (e.g. E. coli, S. aureus) conjugated to the pH-sensitive pHRODO dye, that solely fluoresces within the acidic lysosomes where phagocytosis occurs. The application of pHRODO bioparticles as both proinflammatory stimuli and phagocytic cargos is believed to be novel for in vitro wound modelling. Previous studies have limited the use of pHRODO bioparticles to quantify phagocytic activity without considering their proinflammatory potential. The direct co-culture based DFU model was developed by identifying which ratio of diabetic fibroblasts to THP-1 derived macrophages, exposed to pHRODO bioparticles, for an optimal stimulation time, under a certain oxygen level, and a certain nutrient level (based on foetal bovine serum concentration), exhibited both significant inflammation and reduced phagocytic ability. Inflammation was confirmed via TNF-α and MCP-1 quantification using ELISA. Phagocytic activity, derived from emitted fluorescence of ingested pHRODO bioparticles, was quantified using an automated, whole-well, fluorescent imaging system. Work in this thesis has shown both significant inflammation and reduced phagocytic activity for a 1:4 ratio of diabetic fibroblasts to THP-1 derived macrophages upon four-hour incubation with 200 μg/ml pHRODO green S. aureus bioparticles under hypoxia, with a 2% foetal bovine serum concentration ideally – when compared to the in vitro healthy wound counterpart. Then, the developed direct co-culture based DFU model was tested against four drugs that have previously been shown to improve phagocytosis in vitro for other inflammatory-related diseases; namely: Dexamethasone, saracatinib, brepocitinib and Bay11-7085. Dexamethasone is a common and potent anti-inflammatory glucocorticoid for the treatment of variety of immune-related diseases. Saracatinib was recently proposed as a positive reference to identify novel drugs that improve microglial phagocytic activity for Alzheimer’s disease treatment. The dual Jak1/Tyk2 inhibitor, brepocitinib, reversed the inhibitory effect of lipopolysaccharide on myelin phagocytosis by murine Raw264.7 macrophages. Bay 11-7085, a NF-κB inhibitor, blocks TNFα-induced IκB-α phosphorylation and results in suppressed proinflammatory cytokine release and improved phagocytosis in vitro. In the present doctoral work, mild blockade of inflammation significantly increased phagocytosis using either Dexamethasone or Bay11-7085 in the in vitro DFU model (using both 10% FBS and normoxia, which also demonstrated impaired phagocytosis yet mimicking first-line treatment of growth factor-and oxygen-based therapies). On the other hand, saracatinib significantly repressed phagocytosis and brepocitinib had no stimulatory effect on phagocytosis. Overall, this work presents a direct co-culture based DFU model based on optimized exposure to pHRODO bioparticles, nutrient and oxygen levels, that can be further validated for conducting preliminary high throughput screening of phagocytosis-modulating drugs for DFU treatment.

Funder

Science Foundation Ireland

Publisher

University of Galway

Rights

Attribution-NonCommercial-NoDerivatives 4.0 International

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Collections

University of Galway Theses (PhD Theses)