Phleum pratense Pollen-Derived Di-galactosyldiacylglycerols Promote Pro-allergic Responses in Mice

Nestor González Roldán¹Lars P. Lunding²Yukari Fujimoto³Sylvia Düpow⁴Dominik Schwudke⁵Michael Wegmann²Katarzyna Anna Duda^4*

• ¹Pollen Laboratory, Department of Biological and Environmental Sciences, University of Gothenburg, Gothenburg, Sweden
• ²Research Group of Lung Immunology, Research Center Borstel, Leibniz Lung Center, Research Center Borstel, Airway Research North (ARCN), German Center for Lung Research (DZL), Borstel, Germany
• ³Laboratory of Biomolecular Chemistry, Department of Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Japan
• ⁴Research Group of Biofunctional Metabolites and Structures, Research Center Borstel, Leibniz Lung Center, Research Center Borstel, Airway Research North (ARCN), German Center for Lung Research (DZL), Borstel, Germany
• ⁵Research Group of Bioanalytical Chemistry, Research Center Borstel, Leibniz Lung Center, Research Center Borstel, German Center for Infection Research (DZIF), Borstel, Germany

Grass pollen triggers nearly 30% of bronchial allergic asthma cases. While most research focuses on pollen allergens, pollen lipids may also influence allergic reactions. Previous studies demonstrated that Timothy grass (TG, Phleum pratense) lipids, such as phytoprostanes, can activate immune cells, promoting pro-allergic responses. However, the role of water-insoluble pollen glycolipids in allergic airway inflammation remains unclear. Thus, this study aimed to isolate and characterize glycolipids from TG pollen and evaluate their bioactivity in allergic airway inflammation. Lipids were extracted from the water-insoluble pollen fraction, separated by silica gel, and fractionated by HPLC. GC-MS, HR ESI-MS, and NMR confirmed the presence of di-galactosyldiacylglycerol (DGDG). The biological activity of fractions containing DGDG (3 and 4) and synthetic DGDG variants was tested in vitro in murine and human cell systems and in vivo in mice. Fraction 4 induced strong proliferation of murine NKT cells and upregulated CD69 expression in human NKT cells. Synthetic DGDG variants (DGDG-1, -2, and -3) with defined acylation profile stimulated robust NKT cell proliferation, with DGDG-2 and -3 increasing IL-13 production, one of the key Th2 cytokines. In vivo, only these variants caused lung inflammation marked by eosinophil infiltration but did not increase airway resistance. This study reveals for the first time the structure-dependent role of DGDG of TG pollen grains in immune cell recognition in the context of allergic inflammation. Our data may pave the way for therapies targeting lipid components in combination with protein allergens.