Bet v 1 is the major allergen in birch pollen to which up to 95% of patients sensitized to birch respond. As a member of the pathogenesis-related PR 10 family, its natural function is implicated in plant defense, with a member of the PR10 family being reported to be upregulated under iron deficiency. As such, we assessed the function of Bet v 1 to sequester iron and its immunomodulatory properties on human immune cells. Binding of Bet v 1 to iron quercetin complexes FeQ2 was determined in docking calculations and by spectroscopy. Serum IgE-binding to Bet v 1 with (holoBet v1) and without ligands (apoBet v 1) were assessed by ELISA, blocking experiments and Western Blot. Crosslinking-capacity of apo/holoBet v 1 were assessed on human mast cells and Arylhydrocarbon receptor (AhR) activation with the human reporter cellline AZ-AHR. Human PBMCs were stimulated and assessed for labile iron and phenotypic changes by flow cytometry. Bet v 1 bound to FeQ2 strongly with calculated Kd values of 1 nm surpassing affinities to quercetin alone nearly by a factor of 1000. Binding to FeQ2 masked IgE epitopes and decreased IgE binding up to 80% and impaired degranulation of sensitized human mast cells. Bet v 1 facilitated the shuttling of quercetin, which activated the anti-inflammatory AhR pathway and increased the labile iron pool of human monocytic cells. The increase of labile iron was associated with an anti-inflammatory phenotype in CD14+monocytes and downregulation of HLADR. To summarize, we reveal for the first time that FeQ2 binding reduces the allergenicity of Bet v 1 due to ligand masking, but also actively contributes anti-inflammatory stimuli to human monocytes, thereby fostering tolerance. Nourishing immune cells with complex iron may thus represent a promising antigen-independent immunotherapeutic approach to improve efficacy in allergen immunotherapy.

Center for Plant Biotechnology and Genomics Biotechnology Department ETSIAAB CBGP Universidad Politécnica de Madrid 28040 Madrid Spain

Center of Pathophysiology Infectiology and Immunology Institute of Pathophysiology and Allergy Research Medical University of Vienna 1090 Vienna Austria

Comparative Medicine The Interuniversity Messerli Research Institute of the University of Veterinary Medicine Vienna Medical University Vienna and University of Vienna 1210 Vienna Austria

Department of Biosciences University of Salzburg 5020 Salzburg Austria

Department of Cell Biology and Genetics Faculty of Science Palacky University 78371 Olomouc Czech Republic

Division of Pharmacology Utrecht Institute for Pharmaceutical Sciences Faculty of Science Utrecht University 3584 CG Utrecht The Netherlands

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