Immunomodulatory Effects of Ligand-binding to Pollen Allergens

Allergic diseases represent one of the major disorders of the human immune system; still the intrinsic features of allergens, which initiate the specific TH2 immune response, are poorly understood. Highly homologous proteins/allergens, i.e. birch pollen Bet v 1 and chestnut pollen Cas s 1, both expressed at high levels in the respective pollen, show striking differences concerning their allergenicity in humans. Several studies have also shown that pollen grains themselves not only function as allergen carriers, but also as sources of bioactive allergy- promoting, thus TH2-polarizing substances. For example, pollen-associated lipid mediators (PALMs), i.e. phytoprostanes, have been identified in allergenic pollen and shown to bias towards a TH2 immune response. Recently, also LPS, which was specified as pollen-inherent compound in preliminary studies, has been demonstrated to influence TH cell development in vivo in a dose dependent manner. Further, direct interaction of lipopolysaccharides (LPS) with the inhalant allergen Der p 2 from house dust mite was associated with the activation of toll-like receptor (TLR) 4, thus demonstrating an auto-adjuvant potential of allergen sources. Of note, the ability of binding hydrophobic molecules is a frequently observed property of allergenic molecules, and amongst others also described for the members of the Bet v 1 allergen family. Thus, evidence has been accumulating supporting the view that "allergenicity" of pollen from certain species may result from particular interactions of proteins displaying "allergenic features" with per se "non?allergenic" Th2?inducing adjuvants. In this project, we will focus on the identification of these interactions by using Fagales pollen allergy as disease model. In a first set of experiments we plan to screen aqueous Fagales pollen exudates by mass spectrometry for possible binding partners of the respective major allergens to identify natural ligands of allergens belonging to the Bet v 1 family. In parallel, we will test already described TH2 polarizing Fagales pollen-derived hydrophobic molecules for direct interactions with recombinant Fagales allergens and allergen derivatives. Thereafter, we will investigate the interplay of these molecules with pollen?derived compounds followed by a detailed physicochemical (structure, stability, oligomerization status, ligand?binding) analysis. Moreover, the immunologic effects (endolysosomal stability, antibody?binding, antigen?uptake and activation of antigen?presenting) resulting from the interaction of allergens or allergen derivatives with hydrophobic pollen components will be investigated in detail. In an in vivo model, we plan to analyze the immune responses at the humoral and cellular level after sensitizing mice with allergen-ligand complexes to study effects on the allergenic activity. The proposed experiments will allow us to investigate protein-inherent allergy promoting mechanisms at the molecular level and to explore the intrinsic or extrinsic nature of allergen-ligand interactions.

Which factors render harmless proteins into dangerous allergens? Are allergenic proteins per se risk factors for our immune system or may extrinsic factors such as ligand molecules modify the immunologic properties of allergens? Allergologists have been concerned with these questions for decades and until today they cannot be readily answered. Allergen sources such as pollen, dust mites, or food release a plethora of proteins and we get in contact with these antigens on a daily basis. Nevertheless, our immune system does not necessarily overreact towards these proteins. In allergic patients, however, the immune system shows an exacerbated reaction by inducing the production of allergen-specific IgE antibodies. When comparing the IgE binding profiles of allergic patients, it is remarkable that the only very few proteins within an allergen source bind the majority of IgE antibodies. Moreover, these patterns are highly conserved among patients. Therefore, in the current project we sought to look for extrinsic factors influencing the allergenicity of protein antigens. Many allergens can bind ligand molecules. Such ligands can either be derived from the allergen source itself or from its environment. Whenever allergens get in contact with such ligand molecules, the proteins can bind the cargo, which will eventually influence the immunologic properties of the allergen. In our project, we focused on two allergen families, namely Bet v 1-related proteins, which represent major allergens in birch pollen and pollen of related tree species, and group 2 dust mite allergens. Members of both families are ligand-binding proteins. Initially, we identified the major dust mite allergen Der p 2 as model antigen and designed surface variants of the protein, which displayed essentially the same structure as the parental wild-type allergen. The mutants were produced as recombinant proteins, characterized extensively, and their ligand-binding properties were compared to wild-type Der p 2. Next we investigated the ligand binding properties of the major birch pollen allergen Bet v 1 in detail. Therefore, six either pollen-derived or microbial ligands were selected. In our experiments we found that Bet v 1 produced in the lab under highest quality standards was immunologically quite inactive, whereas in complex with ligands this pattern changed. However, not only immunogenicity but also immune polarization was modified by ligand molecules. Together with collaboration partners we could solve the structure of Bet v 1 in complex with several ligands, and we were able to assign the structure of the low allergenic Bet v 1-homologue from beech by nuclear magnetic resonance (NMR) spectroscopy. All these studies led to the conclusion that a combination of immune-polarizing compounds with proteins capable of interacting with those ligands might be necessary to significantly influence the immunologic properties of these antigens eventually turning them into allergenic molecules.