title: Immunological characterization of Man2 as a novel adjuvant and rFlaA:Betv1 as a therapeutic candidate for allergy treatment
Creators: name: Lin, Yen-Ju
Publisher: name: UNSPECIFIED
Year: 2022

description: Type I hypersensitivity disorders are caused by exaggerated Th2-biased, IgE-mediated inflammatory reactions against harmless environmental allergens. The treatment options for type I allergies are limited, and “allergen vaccination,” or allergen-specific immunotherapy (AIT), is currently the only method to re-establish immunological tolerance towards the respective allergens. AIT is performed by repeated administration of allergen extracts shifting allergen-specific Th2 responses towards Th1 responses. However, AIT consists of long treatment schedules, has the risk of inducing anaphylactic side effects, and partially has a low efficacy due to weakly immunogenic allergen molecules. Therefore, novel adjuvants need to be developed for either enhancing or modifying the overall immune responses in AIT. This thesis aimed to evaluate the immune-modulating capacity of (1) β-(1→4)-mannobiose (Man2) as a novel adjuvant on myeloid dendritic cells (mDCs), and the new therapeutic candidate (rFlaA:Betv1, consisting of Listeria monocytogenes flagellin A fused to the major birch pollen allergen Bet v 1) on either (2) epithelial cells or (3) macrophages for type I allergy treatment. 1) The present results have shown that Man2 could induce a mainly “Toll”-like receptor 4 (TLR4)- and partially complement C3a receptor (C3aR)-dependent production of inflammatory cytokines (IL-6, TNF-α, IL-1β, IFN-β, and IL-10) and the upregulation of co-stimulatory molecules (CD40, CD80, and CD86) on mDCs. Besides, Man2 activated mitogen-activated protein kinase (MAPK)-/ nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB)-pathways, and triggered the Warburg Effect in mDCs. Finally, Man2-stimulated mDCs enhanced antigen-specific, T cell-derived IL-2 production, demonstrating that Man2 has potential as a vaccine adjuvant. 2) In our group´s previous studies, rFlaA:Betv1 was shown to induce mDCs activation, but its effects on other cell types remained unknown. The data presented in this thesis demonstrated, that rFlaA:Betv1 could induce a MAPK- and NFκB-dependent, but TLR5-independent secretion of CCL2, CCL20, and IL-6 from lung epithelial cells. Additionally, rFlaA:Betv1 induced a p38-MAPK and cyclooxygenase 2 (COX2)-dependent prostaglandin E2 (PGE2) production from epithelial cells, which could modulate mDC responses by decreasing their IL-12- and TNF-α-production. Therefore, epithelial cells also contribute to the immune-modulating capacity of rFlaA:Betv1, by modulating the responses of antigen-presenting cells (APCs). 3) Besides DCs, macrophages are another important type of APC. This thesis demonstrated, that macrophages could also be activated by rFlaA:Betv1 stimulation. Here, rFlaA:Betv1 induced a Myeloid differentiation primary-response protein 88 (MyD88)-, Jun N-terminal kinases (JNK)-MAPK-, and partially TLR5-dependent inflammatory cytokine secretion as well as a Warburg Effect from bone marrow-derived macrophages (BMDMs). Furthermore, using RNA-Seq and Western Blot analyses, the upregulation of Janus kinase (JAK)-signal transducer and activator of transcription (STAT)-transcription factor hypoxia-inducible factor 1 α (HIF-1α) signaling was identified, which confirmed results from Seahorse Extracellular Flux technology showing, that rFlaA:Betv1 triggered a pronounced metabolic shift towards glycolysis in BMDMs. Finally, rFlaA:Betv1-stimulated BMDMs could suppress the secretion of Th2 cytokines from Bet v 1-specific, Th2-biassed CD4+ T cells while increasing both Th1- and anti-inflammatory cytokine secretion. In summary, the publications presented in this thesis demonstrated that Man2 could be an attractive novel adjuvant, and rFlaA:Betv1 can be an effective therapy for type I allergy treatment. The results in this thesis may contribute to the future development of safe and effective vaccines and therapeutics.