Abstract
Wound healing is a complex process, with non-healing wounds representing a substantial clinical and economic burden. Macrophages and the regulatory factors they produce are key to this process, with pro-inflammatory macrophages preventing wound infection and anti-inflammatory macrophages promoting angiogenesis, collagen deposition, and wound contraction, therefore aiding in closure of the wound. Thus, successful transition from pro- to anti-inflammatory states is vital for successful wound healing.
Traditionally, Māori have utilised a mucilage from the black tree fern Cyathea medullaris (mamaku) for wound healing and gastrointestinal issues. This mucilage has been shown to contain a polysaccharide consisting of a mannopyranose and glucuronopyranosyl acid backbone, with short oligosaccharide side chains. As botanical polysaccharides have been previously shown to influence macrophage polarisation, this thesis aimed to investigate the effect of this polysaccharide-containing mucilage on macrophages.
Mamaku fronds were harvested in winter and autumn during Māwharu (rising full moon) of Maramataka (the Māori lunar calendar) and were prepared into a mucilage following traditional methods. Scanning electron microscopy was utilised to observe the structure of the mucilage, revealing a honeycomb cellulose network at lower magnification and networks of strings and blobs, likely to reflect polysaccharide chains, at higher magnification. Characterisation of polysaccharide content revealed no differences between frond sections in total carbohydrate or uronic acid contents. Total carbohydrate content was similar with mamaku age, but significant differences were observed between harvest seasons.
The THP-1-derived macrophage model was then validated, with four polarisation states induced by literature-guided stimuli and assessed for cytokine production, surface marker expression, and gene expression. These included maintenance in growth media for resting M(-) macrophages, interferon-gamma (IFN-g) and lipopolysaccharide (LPS) for pro-inflammatory M(IFNg+LPS) macrophages, and interleukin (IL)-4 and IL-13 or IL-10 for anti-inflammatory/pro-reparative M(IL-4+IL-13) and M(IL-10) macrophages. These demonstrated induction of a pro-inflammatory phenotype by IFN-g and LPS. However, neither IL-4 and IL-13 or IL-10 stimulation consistently induced anti-inflammatory cytokines and growth factors, surface markers, or gene expression, except VEGF.
A mamaku mucilage concentration of up to 0.125% total carbohydrate was tolerated by the THP-1-derived macrophages, as demonstrated by manual cell counting following 48 hours of treatment. This concentration was utilised for all subsequent assays. Cytokine production was assessed in all macrophage states, across multiple treatment regimens of pre-treatment, co-treatment, and post-treatment. Across all treatments pro-inflammatory cytokine production was decreased in M(IFNg+LPS) macrophages, but anti-inflammatory cytokine and growth factor production was not increased. Effects on pro-inflammatory cytokine production by M(IL-4+IL-13) and M(IL-10) macrophages was variable, with no changes to anti-inflammatory cytokine production. The angiogenic chemokines IL-8 and monocyte chemoattractant protein 1 (MCP-1) were increased across all treatments, suggesting a novel mechanism of action from what was hypothesised. The effect of mamaku mucilage on surface marker expression was not consistent with an anti-inflammatory/pro-reparative phenotype, with increased pro-inflammatory markers and variable effects on anti-inflammatory markers in M(-) macrophages and decreases in all markers observed on M(IFNg+LPS) macrophages. Pro-inflammatory marker expression increased on M(IL-4+IL-13) macrophages, with no change to anti-inflammatory markers, and no change any markers on M(IL-10) macrophages. Finally, gene expression was assessed to validate the findings at the protein level. Interestingly, IL-8 mRNA was consistent with protein production, with increased expression observed across the macrophage subsets. However, M(IFNg+LPS) gene expression did not correlate with protein levels, with increased pro- and anti-inflammatory gene expression. It was suggested that this discrepancy could be occurring due to binding of cytokines, similar to sulfated polysaccharides derived from algae. A competitive cytokine binding assay using macrophage conditioned media revealed decreases in CXCL10, IL-1b, TNF-a, IL-6, IL-17A, and VEGF upon mamaku treatment, suggesting a novel mechanism of action. Other polysaccharide controls were also tested, including alginate, fucoidan, heparin, and heparan sulfate. Each demonstrated differing degrees of cytokine binding, with heparan sulfate binding the fewest cytokines.
Overall, this work demonstrates that mamaku does impact macrophage polarisation to elicit its wound healing activity. It does so by inducing production of the pro-angiogenic chemokines IL-8 and MCP-1, which could promote new blood vessel formation to allow delivery of oxygen and nutrients to a wound to aid in healing. Furthermore, mamaku mucilage acts as a cytokine-binding agent, which would allow for regulation of the wound microenvironment. This study provides the first evidence of mamaku acting on macrophages, and further work should be conducted to elucidate the mechanisms behind the effects observed here. Furthermore, this study provides scientific evidence to support the use of mamaku as a wound therapy in the clinic.