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Immune modulating mechanisms of flagellin A (FlaA)-fusion proteins at the interface between cell metabolism, TLR signaling, and inflammasome activation

Subject Area Clinical Immunology and Allergology
Term from 2018 to 2023
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 408055700
 
Final Report Year 2023

Final Report Abstract

Adjuvants conjugated to antigens, including fusion proteins including the TLR5- and NLRC4-ligand flagellin, have been repeatedly described to have strong immune modulating properties, making them also interesting candidates for novel approaches in allergen-specific immunotherapy. However, the molecular mechanisms underlying the adjuvant activity of these proteins were less investigated. Own initial results suggested a recombinant flagellin:antigen fusion protein incorporating the major birch pollen allergen Bet v 1 (rFlaA:Betv1) to induce the secretion of both pro- and anti-inflammatory cytokines from dendritic cells (DCs) which was not achieved by the equimolar mixture of both single proteins. Therefore, the present projects aimed (I) to understand the crosstalk between molecules classically associated with immune cell activation (TLR5, NLRC4) and immune cell metabolism, (II) to identify and characterize cell types activated by the fusion protein, and (III) to generate either flagellin:allergen fusion proteins with preferential TLR5- or NLRC4-binding activity or inflammasomedeficient cell lines to dissect the contribution of these pathways to the overall immune responses induced by the fusion protein. The results from human and mouse studies showed flagellin-containing fusion proteins to modulate immune responses by activating many types of immune cells, including DCs, macrophages, B cells, and epithelial cells. The interaction between cell metabolism and immune function was analyzed in detail using mouse DCs and macrophages. Here, the effector function of rFlaA:Betv1-activated mDCs was found to mainly rely on glycolysis with fatty acid synthesis contributing to rFlaA:Betv1-mediated cytokine secretion, the production of anti-microbial molecules, and the modulation of T cell responses. These results further underline the importance to study the contribution of immune cell metabolism to the overall immune responses induced by certain stimuli. Furthermore, for the first time the induction of regulatory B cells by a potential allergen therapeutic and a dual NLRP3- and NLRC4-inflammasome activation by the fusion protein in human macrophages were shown. While some results obtained in the projects have emerged as common features in all cell types activated by the fusion protein, e.g. the MAPK- and NFκB-dependent activation of mDCs, macrophages, epithelial cells, and B cells resulting in both pro- and anti-inflammatory cytokine secretion, other effects of the fusion protein were found to be more cell type-specific. For example, the observed immune cell activation was partially TLR5-dependent in mouse and human macrophages while being unexpectedly TLR5-independent in mouse mDCs, epithelial cells, and B cells. Finally, both projects have greatly improved our understanding of both the cell types and molecular mechanisms contributing to the immune modulating properties of flagellin:antigen fusion proteins. The new findings, considering the immune metabolic and adjuvant mediated effects will provide valuable insights for future development of adjuvants, allergen therapeutics, and vaccines.

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