Abstract
Lipids are a component of the skin barrier, which is critical for immunological homoeostasis. Atopic dermatitis is a chronic inflammatory skin condition in which damage to the epidermal lipid barrier leads to infiltration of innate and adaptive immune cells with phasic release of T helper (Th) 2 and Th1 cytokines. This inflammatory process causes further epidermal damage and allows exposure to bacterial and fungal pathogens, which in turn exacerbates the inflammation. While topical emulsions containing lipids are used to prevent atopic dermatitis, their therapeutic effectiveness and mechanisms of action remain unclear. Immunosuppressants like corticosteroids give immediate relief, but long-term usage causes undesirable side effects. Macrocystis pyrifera, a brown seaweed found in abundance in New Zealand waters, includes a variety of chemicals, including lipids, that have been demonstrated to affect both barrier dysfunction and inflammatory processes. The hypothesis for this thesis was that a lipid extract from M. pyrifera would provide omega-3 fatty acids to suppress inflammation and promote epidermal barrier function, resulting in a more successful therapy for atopic dermatitis. To begin, a review of the literature was undertaken to determine which lipid classes and fatty acids are most typically demonstrated, as well as the methods used to extract and characterise lipids from brown seaweed. Polar lipids esterified to omega-3 fatty acids such as eicosapentaenoic and stearidonic acids, as well as omega-6 arachidonic acid, were found in brown seaweed and commonly extracted using the Bligh and Dyer & Folch methods. Additionally, the study examined whether lipid extracts or lipids from brown seaweed had the potential to treat atopic dermatitis-related inflammation. In RAW264.7 macrophage and BALB/c mouse models, crude lipid extracts and enriched glycolipids and fatty acids from brown seaweed were shown to exhibit anti-inflammatory potential. However, there was little investigation into the lipid content, mechanism of action, and therapeutic utility of M. pyrifera extracts. This facilitated an investigation of the anti-inflammatory activities of a methanol:chloroform extract prepared from M. pyrifera (Linnaeus) C. Agardh using the Folch method. In human THP-1 monocytes stimulated with the Toll-like receptor-2 agonist lipoteichoic acid (LTA), the extract decreased mRNA and protein levels of interleukin (IL)-1β, IL-8, and monocyte chemoattractant protein (MCP)-1 to varied degrees at non-toxic concentrations. The greatest anti-inflammatory effects were elicited when the extract was applied between 6 h prior to, and 6 h after, the stimuli. Reduced levels of nuclear factor kappalight-chain-enhancer of activated B cells (NF-κB) signalling proteins were observed in extract treated cells, with a significant decrease in the myeloid differentiation factor 88 (MYD88) protein abundance relative to stimulated THP-1 cells. Chromatographic fractionation of the extract yielded forty fractions, of which fraction F25 exhibited the greatest inhibition of MCP-1 production in activated THP-1 cells. Fatty acids abundant within the extract and F25 were identified and tested, individually and in combination, for their anti-inflammatory effects. Lipoteichoic acid-stimulated MCP-1 production was inhibited by myristic acid, palmitoleic acid, and a-linolenic acid, but not by the fatty acid combination. These findings indicate that the fatty acid-rich extract and fraction from M. pyrifera provide anti-inflammatory and cytoprotective effects on human monocytes. Next, the anti -inflammatory properties of the M. pyrifera lipid extract were examined in human keratinocytes stimulated with tumor necrosis factor (TNF)-α. At non-toxic concentrations, the extract reduced mRNA and protein levels of IL-8 and MCP-1 to varying degrees in activated HaCaT cells. The extract elicited the strongest anti-inflammatory effects when applied 2 h prior to the stimulus. Membranes isolated from extract-treated cells showed an increased abundance of omega-3 fatty acids relative to stimulated cells. The presence of docosahexaenoic acid in HaCaT cell membranes and increased mRNA expression of ALOX12 indicate that fatty acid metabolism and production of anti-inflammatory lipid mediators may be induced by extract treatment. In TNF-stimulated cells, the extract also caused physical changes to the HaCaT cell membrane by increasing delocalization of the cell-surface TNF receptor 1 and stimulating production of its signalling protein TNF receptor associated factor 2. These findings suggests that the lipid extract from M. pyrifera also modulates TNF-α-mediated inflammatory signalling in human keratinocytes. The effects of the M. pyrifera lipid extract #2 produced using the Bligh and Dyer method on pro-inflammatory chemokine production and tight junction function in keratinocytes were subsequently investigated in the presence of Th1 and Th2 cytokines. In human HaCaT keratinocyte monolayers stimulated with TNF-α, the extract inhibited the release of MCP-1. The extract also inhibited the release of interferon gamma-induced protein (IP)-10 following stimulation of HaCaT cells with IL-4 and IL-13. Increased claudin-1 protein levels and tight junction formation were observed in IL-4 and IL-13-stimulated HaCaT cells after treatment with the extract. With TNF-α, IL-4, and IL-13 stimulation, three-dimensional cultures of HaCaT cells differentiated at the air-liquid interface showed increased trans-epidermal leakage of lucifer yellow, production of MCP-1 and IP-10, and disruption of claudin-1 tight junctions. Each of these parameters was reduced by the extract treatment. These findings suggest that the M. pyrifera extract has anti-inflammatory and barrier-protective effects on keratinocytes. Finally, the neutral and polar lipids from M. pyrifera were extracted, characterised, and their anti-inflammatory effects evaluated. Fractionation was performed on the M. pyrifera extract, producing one neutral lipid and six polar lipid fractions (F1 - F6). Polar lipids represented 85% of the total lipids, and two glycolipids and seven phospholipids were identified by thin layer chromatography and 31P nuclear magnetic resonance, respectively. A further thirteen phospholipids were observed but were not identified. Gas chromatography-mass spectrometry revealed differences in the fatty acid composition and omega-6:omega-3 ratio between lipid fractions. In human THP-1 monocytes stimulated with LTA, polar lipid fractions F4, F5 and F6 significantly decreased MCP-1 with minimal toxicity. F4 and F5 were rich in phosphatidylglycerol, while F6 contained phosphatidylcholine. In human HaCaT keratinocytes activated by TNF-α, polar lipids F1 and F5 were the most potent inhibitors of MCP-1 production, again with minimal toxicity. Monogalactosyldiacylglycerol was detected in F1. The neutral lipid fraction was tolerated by both cell types but was less potent than other fractions at suppressing MCP-1 production. Fraction 2, which was abundant in digalactosyldiacylglycerol and an unidentified phospholipid, was highly cytotoxic to both THP-1 and HaCaT cells. These results show that polar lipids from M. pyrifera have anti-inflammatory effects on human monocytes and/or keratinocytes and can be separated from polar lipids that are toxic to cells. The potential of M. pyrifera as a source of bioactive lipids for the treatment of atopic dermatitis was proven in this study. The M. pyrifera lipid extract was found to be active on critical skin cells under a variety of stimulatory conditions, limiting epidermal inflammation and barrier dysfunction, implying that it may alleviate atopic dermatitis symptoms. These results contribute to understanding the molecular actions of lipids and their targets. The study addressed critical phases in the development of a novel medication for atopic dermatitis. However, further research is needed to find the ideal M. pyrifera lipid composition, formulation, and dosage regimen before assessing them in animal models or human clinical trials of atopic dermatitis.