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

融合蛋白(FP)包括TLR5配体鞭毛蛋白和病原体来源的抗原，已被证明能有效地诱导中和抗体，并被认为是治疗感染的合适工具。在自己的研究中，以OVA为模型，我们发现rFlaA：OVA FP疫苗预防OVA诱导的小鼠胃肠道过敏。临床相关的主要花粉变应原艾草的Art v 1和桦木的Bet v 1证实了其对吸入性过敏的预防作用。我们展示了不同的FP，但不是非结合组分的混合物，能够在各自的实验小鼠模型中预防已建立的过敏原特异性TH2反应和过敏反应。这些效应伴随着强烈的TH1免疫反应，诱导过敏原特异性IgG2a和抑制-IgE的产生。为了探索FLAA结合物的佐剂作用，我们初步研究了FLAA结合物对MDCs的激活作用，发现：(1)FLAS诱导MDCs成熟和激活，同时伴随着促炎和抗炎细胞因子的分泌；(2)诱导抗炎的MDC产生IL-10，在体外介导TH1(干扰素-和TH2(IL-4，IL-5，IL-13))细胞因子的抑制；(3)rFlaA刺激MDCs：Betv1通过激活mTOR复合体而导致代谢活性增加；(4)mTOR激活通过rFlaA：Betv1刺激MDCs诱导抗炎IL-10的分泌，而不是促炎细胞因子的分泌。因此，FP诱导的促炎和抗炎细胞因子的分泌可能受到不同的信号通路的调节。在拟议的研究项目中，我们打算进一步剖析FP在细胞代谢、TLR信号和炎症小体激活之间的界面上的免疫调节通路。整个工作计划包括两个工作包：在WP1中，我们将阐明(I)哪些细胞类型有助于体内含有rFlaA的FP的免疫调节潜力，以及(Ii)FP介导的免疫细胞激活与小鼠和人类DC中不同代谢途径之间的相互作用。在WP2中，我们将通过两种策略阐明鞭毛蛋白传感通路在观察到的促炎和抗炎免疫反应和APC代谢激活中的作用：(I)沉默/抑制NLRC4炎症体活性和(Ii)通过修改的FlaA：Betv1融合蛋白选择性靶向TLR5或NLRC4。综上所述，我们将探索鞭毛蛋白FP激活先天和获得性免疫反应的机制。剖析FP诱导的促炎和抗炎免疫反应的机制以及参与免疫代谢效应可能会改进干预策略，降低副作用的风险。

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