Masson Pine pollen (Pinus massoniana) activate HD11 chicken macrophages invitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Masson Pine pollen (Pinus massoniana; MP) are used in Traditional Chinese Medicine to treat gut conditions. Early in vivo work supports this claim and suggests interaction of the material with the gastrointestinal immune system. AIM OF THE STUDY: The present study tested if and how MP material activates HD11 chicken macrophages in vitro using material from different production sites and harvest years. MATERIAL & METHODS: We applied twelve batches of MP from different Chinese production sites and harvest years. Materials were subjected to LAL tests (endotoxic activity), GC-MS (fatty acid analysis), and plate techniques (microbiological background, antimicrobial activity). Furthermore, HD11 chicken macrophages were challenged (6 h, 37 °C) with MP or LPS (E. coli O111:B4), respectively, to quantify nitric oxide (NO) production and immune gene expression (RT-qPCR). RESULTS: MP material promoted strong signals in LAL tests and contained significant amounts of 3-hydroxydodecanoic acid and 3-hydroxymyristic acid, irrespective of processing, harvest year, or origin. The pollen material activated HD11 chicken macrophages, which was confirmed by spikes of NO release and k-means cluster analysis of TLR-signaling pathway gene expression data. Response of NO production to Log2-titration of MP and LPS-treated media was in any case linear and significant. The response was reduced by polymyxin-B (PMB) and the inhibition was twice as strong for LPS than MP. No or minor microbiological background was detected on the majority of MP samples. Three samples showed presence of spoilage microorganisms and Gram-negative bacteria, but this did not correlate to LAL data or bacterial DNA counts. No antimicrobial activity of MP was evident. CONCLUSION: Pollen of the Masson Pine activated HD11 chicken macrophages in vitro, which is likely partially due to a background of bacterial LPS associated with the pollen material. However, as most of the effect (appr. 80%) could not be blocked by PMB this is certainly due to other stimuli. We hypothesize that polysaccharides and oligosaccharides of the pollen matrix have the potential to interact with certain immune receptors presented on the plasma membrane of chicken macrophages.

The lipid composition of Legionella dumoffii membrane modulates the interaction with Galleria mellonella apolipophorin III.

Apolipophorin III (apoLp-III), an insect homologue of human apolipoprotein E (apoE), is a widely used model protein in studies on protein-lipid interactions, and anti-Legionella activity of Galleria mellonella apoLp-III has been documented. Interestingly, exogenous choline-cultured Legionella dumoffii cells are considerably more susceptible to apoLp-III than non-supplemented bacteria. In order to explain these differences, we performed, for the first time, a detailed analysis of L. dumoffii lipids and a comparative lipidomic analysis of membranes of bacteria grown without and in the presence of exogenous choline. (31)P NMR analysis of L. dumoffii phospholipids (PLs) revealed a considerable increase in the phosphatidylcholine (PC) content in bacteria cultured on choline medium and a decrease in the phosphatidylethanolamine (PE) content in approximately the same range. The interactions of G. mellonella apoLp-III with lipid bilayer membranes prepared from PLs extracted from non- and choline-supplemented L. dumoffii cells were examined in detail by means of attenuated total reflection- and linear dichroism-Fourier transform infrared spectroscopy. Furthermore, the kinetics of apoLp-III binding to liposomes formed from L. dumoffii PLs was analysed by fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy using fluorescently labelled G. mellonella apoLp-III. Our results indicated enhanced binding of apoLp-III to and deeper penetration into lipid membranes formed from PLs extracted from the choline-supplemented bacteria, i.e. characterized by an increased PC/PE ratio. This could explain, at least in part, the higher susceptibility of choline-cultured L. dumoffii to G. mellonella apoLp-III.