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

鞭毛蛋白 A (FlaA) 融合蛋白在细胞代谢、TLR 信号传导和炎症小体激活之间界面的免疫调节机制

• 批准号: 408055700
• 负责人: Dr. Stephan Scheurer
• 金额: --
• 依托单位: Abteilung Allergologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/408055700?language=en
• 关键词: Immune; modulating; mechanisms; flagellin; FlaA

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

已证明含有TLR5配体鞭毛蛋白和病原体衍生抗原的融合蛋白（FP）有效地诱导中和抗体，并建议作为对抗感染的合适工具。在自己的研究中，使用OVA作为模型，我们表明使用RFLAA的疫苗接种：OVA FP阻止了小鼠oVA诱导的胃肠道过敏。通过临床相关的大花粉过敏原ART v 1和BET v 1的临床相关大花粉过敏原ART v 1证实了对吸入过敏的预防作用。我们显示了不同的FPS，但没有显示非偶联成分的混合物，能够防止已建立的过敏原特异性TH2响应和在各自的实验小鼠模型中的过敏敏化。这些作用伴随着强烈的Th1免疫反应，过敏原特异性-IGG2A的诱导和-IgE产生的抑制。 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抗炎IL-10产生的MDC介导Th1的抑制（IFN-和TH2（IL-4，IL-5，IL-5，IL-13，IL-13）的细胞因子在体外特异性CD4+ T细胞中的细胞因子；（3）用RFLAA：BetV1刺激MDC的刺激，导致RFLAA：BETV的刺激，并导致了增强的激进性，并且MESTIS逐渐变化，并且（4）激活了（4），并且（4）激活了（4），并且（4）激活了（4），并且（4）激活了（4），并且（4）激活了（4），并且（4）动作（4）激活（4）激活了（4）激进性，并且（4）动作（4）激发了（4）激进分子（4）诱导RFLAA的抗炎IL-10分泌：Betv1刺激的MDC，但没有促炎性细胞因子的分泌，FP诱导的促疾病和抗炎细胞因子的分泌可能会通过不同的信号传导来调节。 TLR信号传导和炎性体激活。总体工作计划包括两个工作包：在WP1中，我们将阐明（i）哪种细胞类型有助于体内含有RFLAA的FPS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DC中不同的代谢途径之间的相互作用。在WP2中，我们将通过两种策略来阐明两种鞭毛蛋白传感途径对观察到的抗炎免疫反应和APC代谢激活的贡献：（i）NLRC4炎症体活性的沉默/抑制和（ii）通过Modifiend Flaa：BETV1 FOTIN的选择性靶向TLR5或NLRC4：BETV1 FOTINS：BETV1 FOTINS：BETIFFIES：BETV1 FOTIN。综上所述，我们将探讨鞭毛蛋白FPS激活先天和适应性免疫反应的机制。解剖导致FP引起的促疾病和抗炎免疫反应的机制以及免疫代谢作用的参与可能会改善干预策略并降低副作用的风险