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

• Applicants: Dr. Stephan Scheurer; Privatdozent Dr. Stefan Schülke
• Subject Area: Clinical Immunology and Allergology
• Term: from 2018 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 408055700

Fusion proteins (FP) containing the TLR5 ligand flagellin and pathogen-derived antigens have been shown to efficiently induce neutralizing antibodies and were suggested as suitable tools to combat infections. In own studies, using Ova as a model, we showed that vaccination with a rFlaA:Ova FP prevented Ova-induced gastrointestinal allergy in mice. The preventive effect on inhalant allergies was confirmed with the clinically relevant major pollen allergens Art v 1 from mugwort and Bet v 1 from birch. We showed the different FPs, but not the mixture of the non-conjugated components, to be capable to prevent both established allergen specific TH2 responses and allergic sensitization in respective experimental mouse models. These effects were accompanied by strong TH1 immune responses, the induction of allergen-specific-IgG2a and suppression of -IgE production. However, the underlying subcellular mechanism by which the FPs exert the strong immune activating effects remains to be elucidated.To explore the adjuvant effect of FlaA-conjugates we initially investigated the activation of APCs and found: (1) FPs to induce maturation and activation of mDCs, accompanied by the secretion of both pro- and anti-inflammatory cytokines, (2) the induction of anti-inflammatory IL-10-producing mDC to mediate the suppression of TH1 (IFN- and TH2 (IL-4, IL-5, IL-13) cytokines from allergen specific CD4+ T cells in vitro; (3) the stimulation of mDCs with rFlaA:Betv1 to result in an increased metabolic activity, mediated by an activation of the mTOR complex, and (4) the mTOR activation to induce anti-inflammatory IL-10 secretion by rFlaA:Betv1-stimulated mDCs, but not pro-inflammatory cytokine secretion. Therefore, FP-induced pro- and anti-inflammatory cytokine secretion are likely regulated by different signaling pathways.In the proposed research project, we intend to further dissect the immune modulating pathways of FPs at the interface between cell metabolism, TLR signaling, and inflammasome activation. The overall work program comprises two work packages: In WP1 we will elucidate (I) which cell types contribute to the immune modulating potential of rFlaA-containing FPs in vivo and (II) the interplay between FP-mediated immune cell activation and different metabolic pathways in both mouse and human DCs. In WP2 we will elucidate the contribution of both flagellin sensing pathways to the observed pro-and anti-inflammatory immune response and activation of APC metabolism by two strategies: (I) silencing/suppression of NLRC4 inflammasome activity and (II) selective targeting of TLR5 or NLRC4 by modified FlaA:Betv1 fusion proteins. Taken together, we will explore the mechanisms by which flagellin FPs activate innate and adaptive immune responses. Dissecting the mechanisms contributing to FP-induced pro- and anti-inflammatory immune responses and the engagement of immune metabolic effects likely will improve intervention strategies and reduce the risk of side effects

• DFG Programme: Research Grants