Interaction between birch pollen and Toll-Like Receptors

Principal Investigator Prof. Dr. Fatima FERREIRA Co-Investigators PD Dr. Gabriele GADERMAIERDr. Lorenz AGLAS Wider research context: Allergens have been defined as proteins or other environmental substances that have the capacity to induce the production of IgE antibodies in atopic individuals, thus implying an intrinsic capacity to induce Th2 polarization. Challenging this view, we have showed that purified Bet v 1, the major birch pollen allergen, lacks sensitizing capacity and that its depletion does not affect the capacity of birch pollen extracts to induce IL4-producing Th cells in vivo. These findings, together with our previous work on the birch pollen microbiome, prompted us to suggest that pollen intrinsic adjuvants and extrinsic microbe-derived compounds could critically contribute to pollen allergenicity and sensitization to the Bet v 1 allergen. Hypotheses and Objectives: We now propose that birch pollen (BP) sensitization is initiated upon interactions between the innate immune network and pollen-derived adjuvants co-delivered with Bet v 1 and other allergenic proteins. In addition, we postulate that BP-mediated activation of DCs in vitro and induction of IL- 4-producing Th cells in vivo are partially mediated by Toll-like receptor (TLR) signaling. Our preliminary testing in human embryonic kidney (HEK) reporter cell lines stably transfected with TLR2, 4 or 5, showed their activation by BP extracts. Thus, the overall purpose of the project is to provide a mechanistic understanding of how BP-derived adjuvants and associated bacteria induce DC activation though TLR signaling to create an inflammatory milieu that favors the development of Th2-biased responses to pollen antigens. Approach and methods: To define the TLR signatures in BP, we will use HEK-TLR reporter cells, an extensive panel of TLR agonists and antagonists, as well as cellular assays with human and murine DCs. In addition, transgenic mouse models will help to fully understand and validate the in vitro data obtained from reporter cells and DCs. Innovation and impact: The projects results will significantly help in understanding the role of combined TLR stimulation to the induction of Th2 polarized responses to BP and stimulate further investigations on the initiation of sensitization by other allergenic pollen sources. Ultimately, understanding initiation of sensitization could open new perspectives for prophylactic and therapeutic approaches in allergic diseases. Primary researchers involved: Fatima Ferreira has a solid track record in molecular allergology with important contributions to the characterization of tree and weed pollen allergens, as well as to the development of allergen vaccines. Gabriele Gadermaier is trained in biotechnology and immunology and co-supervised the generation of the preliminary data for this project. Lorenz Aglas is experienced working with DCs and in vivo sensitization/Th2 polarization animal models. Furthermore, the team is very experienced in project management, dissemination of research results to the research community and to the general public.

Allergens have been defined as proteins or other environmental substances that induce IgE antibody production in atopic individuals, implying an intrinsic capacity to trigger Th2 polarization. Challenging this view, we showed that purified Bet v 1, the major birch pollen allergen, lacks sensitizing capacity and that its depletion does not affect the ability of birch pollen extracts to induce IL-4-producing Th cells in vivo. These findings, together with our previous work on the birch pollen microbiome, led us to propose that pollen-intrinsic adjuvants and extrinsic microbe-derived compounds critically contribute to pollen allergenicity and sensitization to Bet v 1. In this project we propose that birch pollen sensitization is initiated through interactions between the innate immune system and pollen-derived adjuvants co-delivered with Bet v 1 and other allergenic proteins. Additionally, we postulate that birch pollen-mediated activation of dendritic cells in vitro and induction of IL-4-producing Th cells in vivo are partially mediated by Toll-like receptor (TLR) signaling. We established an interleukin (IL)-4/interferon- (IFN-) dual-reporter mouse model to investigate the intrinsic ability of several purified major allergens to induce Th1 or Th2 polarization compared to their corresponding whole pollen extracts in vivo. We found that major allergens from birch, grass, mugwort, and ragweed pollen do not exhibit an intrinsic ability to induce Th1 or Th2 polarization in vivo, whereas the whole extracts triggered strong Th2 polarization. While investigating the ability of various pollen extracts to activate extracellular TLRs (TLR1/2, TLR2/6, and TLR4), it became evident that TLR activation-and thus TLR ligand concentration-varied significantly between species and between pollen batches. Our findings demonstrated a critical role for TLR4 in dendritic cell activation by birch pollen in vitro. In vivo, TLR4-deficient mice exhibited reduced allergic inflammation, including diminished eosinophilia and Th2 cytokine secretion. Endotoxin levels in birch pollen extracts influence immune responses but are not the sole determinant of allergenicity. Other pollen-derived components also contribute to allergic sensitization. For instance, intrinsic factors such as Bet v 1 concentration and extrinsic factors such as the pollen microbiome, including bacterial components like LPS, clearly affect pollen allergenicity. Interestingly, we found that self-collected pollen possesses distinct immunogenic properties compared to commercial samples, suggesting a possible role for surface lipids and microbiome composition. In conclusion, our findings contribute to understanding the complex process of allergic sensitization. By investigating sensitization beyond major allergens and their role in immune system stimulation, we uncovered key aspects of allergic sensitization linked to the whole pollen matrix, including pollen-specific microbiome-derived compounds.