Pollen lipidomics: lipid profiling exposes a notable diversity in 22 allergenic pollen and potential biomarkers of the allergic immune response

Abstract

Background/aim: Pollen grains are the male gametophytes that deliver sperm cells to female gametophytes during sexual reproduction of higher plants. Pollen is a major source of aeroallergens and environmental antigens. The pollen coat harbors a plethora of lipids that are required for pollen hydration, germination, and penetration of the stigma by pollen tubes. In addition to proteins, pollen displays a wide array of lipids that interact with the human immune system. Prior searches for pollen allergens have focused on the identification of intracellular allergenic proteins, but have largely overlooked much of the extracellular pollen matrix, a region where the majority of lipid molecules reside. Lipid antigens have attracted attention for their potent immunoregulatory effects. By being in close proximity to allergenic proteins on the pollen surface when they interact with host cells, lipids could modify the antigenic properties of proteins.

Methodology/principal findings: We performed a comparative pollen lipid profiling of 22 commonly allergenic plant species by the use of gas chromatography-mass spectroscopy, followed by detailed data mining and statistical analysis. Three experiments compared pollen lipid profiles. We built a database library of the pollen lipids by matching acquired pollen-lipid mass spectra and retention times with the NIST/EPA/NIH mass-spectral library. We detected, identified, and relatively quantified more than 106 lipid molecular species including fatty acids, n-alkanes, fatty alcohols, and sterols. Pollen-derived lipids stimulation up-regulate cytokines expression of dendritic and natural killer T cells co-culture.

Conclusions/significance: Here we report on a lipidomic analysis of pollen lipids that can serve as a database for identifying potential lipid antigens and/or novel candidate molecules involved in allergy. The database provides a resource that facilitates studies on the role of lipids in the immunopathogenesis of allergy. Pollen lipids vary greatly among allergenic species and contain many molecules that have stimulatory or regulatory effects on immune responses.

Figure 1. A typical total ion chromatogram (TIC) of trimethylsilyl (TMS)-derivatives of lipid molecular species obtained after separation of lipids extracted from six species of grass, weed, and tree pollen.

The conditions for lipid analysis were as described in Methods.

Figure 2. Heat map visualization comparing cytokine expression profiles of DC/NKT cells stimulated with lipid compounds in vitro.

Several lipid compounds stimulated DC and DC/NKT cells and produced distinct cytokine patterns, including pro-inflammatory cytokines through toll-like receptor (TLR)-mediated DC activation. The co-culture with NKT cells augmented the inflammatory immune response to several lipid compounds. 1×10⁵ autologous immature dendritic cells (WT.B6 DC and MyD88^−/− DC) were left untreated or were stimulated with FAs and n-alkanes (1 µg/ml) or aliphatic alcohol, sterols, or other lipid compounds (5 µg/ml) or αGalCer (100 ng/ml) for 12 h in 96-well U-bottomed plates. Where indicated, 1×10⁵ purified NKT cells were added for an additional 36 h. Monocultures with DCs remained in culture for 48 hours. To exclude the possibility that the secreted cytokines are induced by entotoxin contamination, we measured endotoxin levels in pollen lipids by using limulus amebocyte lysate to confirm the absence of detectable levels of endotoxin. Pro-inflammatory (TNF-α) and pro-allergic (IL-13), regulatory (IL-10) and proliferatory (IL-2) levels in cell-free culture supernatants were then measured by use of ELISA. IL-10 is not shown in the heat map. The heat map represents ∼36 lipid compounds that are clustered into 4 groups based on their lipid classes (FAs, n-alkanes, alkanols, sterols, and controls) shown on the left of the heat map. Cytokines were clustered with the names shown on the top of the heat map. Each raw corresponds to a single lipid compound, and each column represents an independent condition. The heat map color scale corresponding to the relative expression of the cytokine relative to the minimum and maximum of all values is shown on the right. Black and blue indicating the lowest levels, brown and red indicating the highest levels, and green, yellow and orange indicating median levels relative expression of cytokines (average concentration pg/ml). Results are representative of two independent experiments.

Figure 3. Effect of different lipid stimuli on TNF-α response of T cells.

For analysis of cytokines by intracellular staining, conventional T cells harvested after culture in vitro were stimulated with 1 µg/ml lipid molecules for 24 to 36 hours in 6-well plates. After treatment with 10 µg/mL GolgiPlug, a protein transport inhibitor containing brefeldin A (eBiosciences) during the final 6 to 12 hours of stimulation, the cells were stained for surface markers and anti-mouse TNF-α as described in Materials and Methods. Flow cytometry was performed on a FACSCanto II instrument (BD Biosciences) and analyzed using FlowJo software (Tree Star).