Identification and functional analysis of TNFR2-induced signaling complexes

TNFR2诱导的信号复合物的鉴定和功能分析

• 批准号: 310944718
• 负责人: Professor Dr. Harald Günther Wajant
• 金额: --
• 依托单位: Abteilung für Molekulare Innere Medizin
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/310944718?language=en
• 关键词: Identification; functional; analysis; TNFR2; induced

In the expired project, we made among others the following findings and progresses: i) We demonstrated in caspase-inhibited macrophages that TNFR2 enables necroptosis induction by sensitizing the cells for endogenous TNF/TNFR1-triggred necroptosis by depletion of complexes of TRAF2 with cIAP1 or cIAP2. ii) We identified furthermore a novel cooperative pathway in which TNFR2 and TNFR1 cooperate to stimulate RIPK1 kinase activity-dependent gene induction and which bifurcates from necroptosis signaling upstream of RIPK3 and MLKL. Notably, TRAF1 and A20, two components of the TNFR2 and TNFR1 signaling complexes, are targets of this pathway. iii) We found that sharpin antagonizes necroptotic TNFR2-TNFR1 cooperation. iv) We developed various novel TNFR2 agonists and demonstrated by their help that stimulation of TNFR2 alone is sufficient to expand Tregs in vitro and in vivo and to promote their suppressive activity. Notably, while the profound effects of selective TNFR2 stimulation in macrophages were mainly dependent on the ability of TNFR2 to modulate the quality of TNFR1 signaling, the effects of TNFR2 stimulation on Tregs were found to be so far independent from the endogenous TNF/TNFR1 axis. Based on these achievements the current project proposal pursues now the following objectives: i) Follow up analysis of the newly identified TNFR2- and caspase-restricted TNFR1-induced RIPK1 kinase activity-dependent gene inductive signaling pathway. We will clarify how controls TNFR2 this pathway at the molecular level. We will further identify target genes of this pathway and evaluate its relevance for the inflammatory response against TNF and/or TNF-inducing PAMPs in macrophages expressing viral or bacterial caspase-8 inhibitors. ii) Identification and characterization of TNFR2 activities and functions in macrophages which are independent from the endogenous TNF-TNFR1 axis. We will therefore analyze TNF- and TNFR1-deficient macrophages with respect to TNFR2-induced signaling events and their relevance for survival and activity of macrophages. iii) Evaluation of the TNFR1-TNFR2 crosstalk in Tregs and CD8+ T-cells. We will address this issue by comprehensive TNFR1/2 costimulation experiments in the presence and absence of caspase inhibition. iv) The mid- and long-term activities of TNF are the integrated result of the effects of various feed-back mechanisms triggered by the two TNF receptors. We will evaluate such crosstalk mechanisms which act on the level of the TNF receptor signaling complexes. For this purpose, we will analyze the effect of TNFR1 and TNFR2 priming on signaling complex formation by subsequently stimulated TNF receptors. Especially, we will investigate whether TRAF1, A20 or yet unknown factors are of relevance in such scenarios.

在已到期的项目中，我们取得了以下发现和进展：i）我们在 caspase 抑制的巨噬细胞中证明，TNFR2 通过消除 TRAF2 与 cIAP1 或 cIAP2 的复合物，使细胞对内源性 TNF/TNFR1 触发的坏死性凋亡敏感，从而能够诱导坏死性凋亡。 ii)我们进一步鉴定了一种新的协同途径，其中TNFR2和TNFR1协同刺激RIPK1激酶活性依赖性基因诱导，并且该途径从RIPK3和MLKL上游的坏死性凋亡信号转导中分叉。值得注意的是，TRAF1 和 A20（TNFR2 和 TNFR1 信号复合物的两个成分）是该通路的靶标。 iii) 我们发现 Sharpin 拮抗坏死性 TNFR2-TNFR1 的合作。 iv) 我们开发了各种新型 TNFR2 激动剂，并通过它们的帮助证明，单独刺激 TNFR2 足以在体外和体内扩增 Tregs 并促进其抑制活性。值得注意的是，虽然巨噬细胞中选择性 TNFR2 刺激的深远影响主要取决于 TNFR2 调节 TNFR1 信号传导质量的能力，但迄今为止发现 TNFR2 刺激对 Tregs 的影响与内源性 TNF/TNFR1 轴无关。基于这些成就，当前的项目提案追求以下目标： i) 对新发现的 TNFR2 和 caspase 限制的 TNFR1 诱导的 RIPK1 激酶活性依赖性基因诱导信号通路进行后续分析。我们将阐明如何在分子水平上控制 TNFR2 这条通路。我们将进一步鉴定该途径的靶基因，并评估其与表达病毒或细菌 caspase-8 抑制剂的巨噬细胞中针对 TNF 和/或 TNF 诱导 PAMP 的炎症反应的相关性。 ii) 巨噬细胞中独立于内源性 TNF-TNFR1 轴的 TNFR2 活性和功能的鉴定和表征。因此，我们将分析 TNFR2 诱导的信号事件及其与巨噬细胞存活和活性的相关性，从而分析 TNF 和 TNFR1 缺陷型巨噬细胞。 iii) Tregs 和 CD8+ T 细胞中 TNFR1-TNFR2 串扰的评估。我们将通过在存在和不存在 caspase 抑制的情况下进行全面的 TNFR1/2 共刺激实验来解决这个问题。 iv) TNF的中长期活性是两种TNF受体触发的多种反馈机制综合作用的结果。我们将评估作用于 TNF 受体信号复合物水平的串扰机制。为此，我们将分析 TNFR1 和 TNFR2 引发对随后刺激的 TNF 受体形成的信号复合物的影响。特别是，我们将调查 TRAF1、A20 或未知因素在这种情况下是否相关。