Determinants of birch pollen allergenicity

In course of the allergic sensitization process, genetically susceptible individuals become allergic to a certain allergenic source (e.g. pollen allergens), which results in the expression of allergic symptoms upon re-exposure to the source. In case of birch pollen allergy, allergic patients suffer from seasonal hay fever and even from allergic asthma, influencing their quality of life enormously. The induction of symptoms is caused by proteins found in the pollen, called allergens. One protein in birch pollen, named Bet v 1, is of particular interest since over 95% of all birch pollen allergic patients respond to it. Although the mechanism by which allergens trigger symptoms is well understood (effector phase), less is known regarding the initiation of the allergic response (sensitization phase). It appears that the sensitization is triggered by yet-unidentified pollen compounds rather than by the allergens themselves, which seem to play only a secondary role. Therefore, the primary goal of this project is to dissect the pollen source on a molecular level, to characterize essential pollen compounds and to evaluate their contribution in allergic sensitization. Human and murine epithelial and dendritic cells, two major cell types involved in the sensitization process, will be stimulated with the pollen compounds and compared to the effects caused by a complete, untreated birch pollen extract. In mouse models, the effects of the birch pollen compounds will further be studied regarding their capacity to promote allergic sensitization. By combining human and murine experimental models, we expect to identify birch pollen compounds and immunological pathways that help in understanding the mechanism of allergic sensitization. The generated findings as well as the newly gained mechanistic knowledge will pave the way for the development of prophylactic and therapeutic approaches, which might prevent allergies to birch pollen in the future.

Pollen allergy is the most prevalent chronic disease worldwide, affecting an estimated 4 billion people with symptoms ranging from seasonal rhinitis and conjunctivitis to asthma. Despite its global burden, the precise immunological mechanisms that initiate allergic sensitization, especially the very early events that ultimately drive the pathological immune response, remain poorly understood. This project aimed to close this fundamental knowledge gap using carefully designed mouse models of intranasal pollen exposure. We developed adjuvant-free intranasal sensitization protocols using aqueous extracts of birch (Betula pendula) and ragweed (Ambrosia artemisiifolia) pollen (BPE and RPE), two of the most clinically relevant aeroallergens in Europe. By avoiding external adjuvants, our models closely mimic natural human pollen exposure. Using IL-4 reporter (4Get) and IL-4 knockout mouse strains, we systematically tracked the sequence of immunological events in the lungs, lymph nodes, and blood across multiple time points and experimental conditions. Our results demonstrate that a Th2-polarized immune response, the hallmark of allergic disease, is detectable as early as day 4 after initial pollen exposure, in both the mediastinal lymph nodes and bronchoalveolar lavage. This early Th2 response was followed by eosinophil recruitment to the airways from day 6 onward, a process found to be IL-4-dependent for both pollen types. The two extracts, however, differed markedly in their capacity to elicit a humoral response: RPE induced allergen-specific IgG1 and IgE antibodies after as few as 8 intranasal instillations, while BPE required 14 instillations for IgG1 detection and a 6-week protocol for measurable IgE, pointing to fundamental differences in the immunostimulatory composition of the two pollens. To investigate which pollen-derived compounds drive sensitization, we generated protein-depleted and cysteine protease-inhibited RPE fractions. Strikingly, neither the removal of proteins by proteinase K digestion nor the inhibition of cysteine proteases significantly affected Th2 polarization, airway eosinophilia, or antibody production. This unexpected finding indicates that proteins and proteases are not the primary initiators of Th2 sensitization for ragweed pollen. In parallel, size-exclusion chromatography of BPE revealed that high-molecular-weight fractions consistently induced the strongest Th2 activation in both a dendritic cell-T cell co-culture system and in vivo, implying that non-proteinaceous or large macromolecular compounds are key immunostimulatory components of birch pollen. Collectively, our data suggest that allergic sensitization is initiated in the lung tissue before systemic Th2 commitment occurs, and that innate immune pathways, including alarmins and ILC2-mediated eosinophil recruitment, may play a critical role upstream of the adaptive response. These findings open important avenues for future work focused on the very earliest innate events in pollen-driven allergy, with the long-term goal of identifying novel targets for prevention and immunotherapy.