Exploring the immunoregulatory properties of New Zealand honeys

Abstract

Honey is a sweet and readily assimilable carbohydrate compound produced by bees using nectar from flowers. It possesses several properties, including anti-bacterial, anti-fungal, immunostimulatory, anti-inflammatory, antioxidant and gastroprotective activities. Medical honey is useful in the healing of wounds and burns, in particular New Zealand manuka honey, which is widely used for this purpose in hospitals in Western countries. There is a resurgence in the use of medical honey to heal wounds due to the growing clinical problem of antibiotic-resistant bacteria. Manuka honey dressings have been approved for use as medical devices by the Food and Drug Administration (FDA), in the treatment of wounds and burns. Nevertheless, our poor understanding of the bioactives in honey, their properties, and their distribution within different floral varieties of honey, is a major obstacle to optimizing the application of honey for medical purposes. This study investigated the immunomodulatory properties of honey. A better understanding of how honey affects the immune response will lead to advances in its application to treat wounds and infections. The aim of this study was to investigate the mechanisms by which honey exerts its immunomodulatory activities, and to identify the bioactives responsible. Honey arabinogalactans proteins (AGPs) were partially characterized for their ability to stimulate the release of tumour necrosis factor (TNF)-α from macrophages during my MSc study, and remained a major focus of this study. A central question addressed in various sections of the thesis was “what contribution does lipopolysaccharide (LPS) make to the immunostimulatory activity of honey?” LPS and honey AGPs were shown to share various properties including binding to polymyxin B (PmB), heat stability, and inhibition by lactoferrin which confounds analyses. In contrast, irradiation reduced the immunostimulatory activity of LPS, but had no effect on the immunostimulatory activity of honey. The latter could arise if honey contains components that protect against the effects of radiation. New Zealand kanuka, manuka and clover honeys and LPS induced the release of TNF-α, interleukin (IL)-1β, and IL-6 from monocytes, whereas LPS induced the release of a far greater number of different cytokines and growth factors, suggesting LPS is less selective in its mechanism of activation. The active moiety of kanuka honey AGPs could be released by phospholipase C from kanuka honey AGPs that had been bound to polymixin B (PmB) conjated agarose, unlike that of LPS where the active component is the lipid A moiety that cannot be cleaved by phospholipase C. This study quantified the levels of AGPs in different floral varieties of New Zealand honeys, revealing that kanuka honey has the highest AGP content, followed by manuka, and then clover honey. Royal Jelly had no detectable AGP content. The three New Zealand honeys stimulated the release of very similar levels of TNF-α from blood monocytes. In contrast, Royal Jelly failed to stimulate TNF-α release. Honey AGPs were shown to be structurally different from gum arabic AGPs, as evidenced by their different migration patterns in crossed gel electrophoresis. Apisimin, a short 5.8 kDa polypeptide secreted from the head glands of honey bees, was identified as a novel immunostimulatory component of honey. Quantification using especially prepared anti-apisimin antisera revealed that kanuka honey has the highest content of apisimin, followed by manuka, and then clover honey. Apisimin synergized with honey AGPs to stimulate the release of TNF-α from fresh blood leukocytes. Thus, while honey AGPs and apisimin individually have comparatively low immunostimulatory activity, when combined together their activity provides a considerable contribution to the overall immunostimulatory activity of honey. In contrast, apisimin did not synergize with gum arabic AGPs. New Zealand commercial honeys and the “international” honeys were compared for their ability to stimulate monocytes. All the honeys, except for a Malaysian “amber honey” stimulated the release of TNF-α from monocytes. However, they differed greatly in their immunostimulatory activity presumably due to their content of different amounts of bioactives. Amber honey appeared to lack proteins altogether, including the major apalbumins, suggesting it had been adulterated or concocted. This study compared the gel clot and chromogenic Limulus amoebocyte lysate (LAL) assays for their ability to measure endotoxin levels in Medihoneys. The data revealed that endotoxin levels obtained using the chromogenic method were overall much lower (up to 45-fold less) than those obtained using the gel clot assay. The reason for such large differences in measurement warrants further investigation as it is an issue that is highly relevant for the quality control of medical honeys. New Zealand honeys were shown to stimulate TNF-α release from monocytes in a Toll-like receptor (TLR)1/2-dependent fashion, and also potentially in a TLR-4-dependent fashion if the marked effects of the TLR4 antagonists CLI-095 and LPS-RS are due to antagonism rather than cytotoxicity. Finally, the honey components methylglyoxal (MGO) and chrysin were shown to be potent immune suppressors capable of antagonizing the immunostimulatory activities of honey AGPs and apisimin. Thus, the overall immunoregulatory property of a particular honey depends upon a balance between its components that are immunostimulatory and those that are immunosuppressive. A pilot study was designed to determine ability of commercial honey products to release cytokines and growth factors from the oral cavity, and to deplete the caries-forming bacteria Streptococcus mutans and Lactobacilli. Ethics approval was obtained to test the response of 40 participants to the honey products. A practise run involving just two volunteers was undertaken prior to performing the trial in order to become familiar with the treatment and analysis procedures. Whilst the assays may need optimising, the data indicated that commercial honey and olive leaf products marketed by Comvita have no marked effect on the mucocal immunity of the oral cavity (refer to Appendix I). For this reason the intended pilot study was not undertaken. The data gained from this thesis are important for identifying and understanding the bioactive components in honey and their mechanism of action. The results could have use in improving wound healing and other applications.