Binding to Iron Quercetin Complexes Increases the Antioxidant Capacity of the Major Birch Pollen Allergen Bet v 1 and Reduces Its Allergenicity

Abstract

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.

Nutrition in chronic inflammatory conditions: Bypassing the mucosal block for micronutrients

“…Iron deficiency is therefore linked to C‐reactive protein, CRP, and low‐grade inflammation, 68,282–284 with pro‐inflammatory signatures reported in the monocytic cells in children 66 and infants 67 with iron deficiency, while increasing the labile iron pool is associated with an immature, regulatory macrophage phenotype 146,154 (Figure 4).…”

“…These include seed storage proteins 141 which include the large prolamin superfamily 142–144 comprised of lipid transfer proteins, 2S albumins, cereal prolamins, and the cupins consisting of globulins (legumin‐like proteins) and conglutins (vicilin‐like proteins) as well as pathogenesis‐related proteins 141 . From animal sources animal proteins such as albumins, lipocalins, transferrins, and globulins are similarly serving as carrier for nutrients binding to carbohydrates, 145 minerals, 146–159 lipids, 160–163 phenolics, 146,154–156 and vitamins 149–153 and often play a regulatory role in host defense.…”

Nutritional Provision of Iron Complexes by the Major Allergen Alt a 1 to Human Immune Cells Decreases Its Presentation

“…Indeed, quercetin has per se a light-yellow colour, which results in a spectrum peaking with a single maximum at about 340 nm at physiological pH. In contrast, when in complex with iron (at a pH > 6.5), the colour turns brownish, and due to the interaction of the catechol-group with the d-orbitals of ferric iron, a second maximum, usually about 450 nm, appears [17,21,24]. Binding to FeQ2 could be proven because this second absorption maximum diminished concentration dependently upon the addition of Alt a 1 (empty protein, blue lines) (Figure 2a).…”

“…There seems to exist also a pH and ligand dependency [16], in which pH over 6.5, as well as the presence of a ligand, favors oligomerization to tetramers/hexamers. The proposed ligand for Alt a 1 is thought to have a quercetin core [14], which is a flavonoid with known iron-binding capacity [17][18][19][20][21][22], making Alt a 1 very likely a scavenger of iron complexes. Indeed, quercetin can serve as an iron source for commensal and opportunistic microbial pathogens [23], and its anti-oxidative and ROS-scavenging properties are reported to be greater in complex with iron than without iron [24][25][26].…”

“…We previously suggested that Alt a 1 shares some features with lipocalins [35], such as beta-lactoglobulin [17,20,35,36] and LCN2 [37], as well as the pathogenesis-related protein Bet v 1 [35,36], which are both protein families involved in nutritional immunity and capable of binding to iron when complexed to siderophores or flavonoids.…”