Characterization and molecular functions of the IL-17 – IL-36 signaling axis in keratinocytes and consequences for psoriasis

角质形成细胞中 IL-17 – IL-36 信号轴的特征和分子功能以及银屑病的后果

• 批准号: 408794211
• 负责人: Professor Dr. Bernhard Lüscher
• 金额: --
• 依托单位: Institut für Biochemie und Molekularbiologie
• 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/408794211?language=en
• 关键词: Characterization; molecular; functions; IL; 17

Psoriasis is a chronic inflammatory skin disease affecting about 2% of the population. Typical for the disease is epidermal hyperplasia associated with hyperproliferation and abnormal differentiation of keratinocytes. The immune system is closely associated with psoriasis, e.g. TH17 cells modulate keratinocyte function through IL-17 as well as other cytokines. Moreover, IL-36 cytokines are closely linked to psoriasis. These cytokines activate signaling pathways and control genes that encode proteins associated with keratinocyte differentiation and skin barrier formation, and factors involved in immune cell recruitment and activation. We have studied the effects of IL-17A on primary normal human epidermal keratinocyte (NHEKs) both in monolayer cultures and 3-dimensional (3D) organotypic skin models. One of the genes that is strongly activated is IL36G, which encodes IL-36, an IL-1 family cytokine. IL-36 is thought to be secreted by an unconventional protein secretion pathway and requires N-terminal processing for activation. IL-36 interferes with NHEK differentiation in 3D models and in the mouse ear. This suggests that IL-36 cytokines are effectors of IL-17. In support, IL-17A and IL-36 cooperate in controlling gene transcription and affecting mouse skin development. We propose an IL-17A – IL-36 feedforward loop, aggravating the psoriatic phenotype. Therefore, we want to understand how these two cytokines cooperate both in HaCaT keratinocytes and in NHEKs. For this we will study the signaling pathways and determine how the signals are integrated at target promoters. We will measure signaling molecules, binding of transcription factors to DNA, chromatin status, polymerase II activity, and transcription rates. Finally, post-transcriptional cooperativity will be determined by analyzing RNA stability. Together, these studies will clarify how IL-17A and IL-36 cooperate in controlling target genes. Moreover, we want to understand how the processing and secretion of IL-36 is regulated. We will use fusion proteins of IL-36 with a biotin ligase to modify interacting proteins. Biotinylated interactors will be purified using streptavidin and identified by mass spectrometry. This approach will define even short-term interacting proteins and provide us with leads to define the relevant unconventional protein secretion pathway(s). We will also stimulate cells through pattern recognition receptors to define signals that promote IL-36 secretion. The functional relevance of IL-36 will be further studied in the mouse using ear injection and imiquimod models, in which antagonistic IL-36 receptor fusion proteins as well as newly defined factors involved in IL-36 secretion and activation will be evaluated. Together, we expect from these studies to obtain molecular insight into the activation and processing of IL-36, how it cooperates with IL-17A and what its contribution is to altered keratinocyte function and differentiation in the context of psoriasis.

牛皮癣是一种慢性炎症性皮肤病，影响约2%的人口。该疾病的典型特征是与角质形成细胞的过度增殖和异常分化相关的表皮增生。免疫系统与银屑病密切相关，例如TH 17细胞通过IL-17以及其他细胞因子调节角质形成细胞功能。此外，IL-36细胞因子与银屑病密切相关。这些细胞因子激活信号通路和控制编码与角质形成细胞分化和皮肤屏障形成相关的蛋白质的基因，以及参与免疫细胞募集和激活的因子。我们研究了IL-17 A对单层培养和三维（3D）器官型皮肤模型中的原代正常人表皮角质形成细胞（NHEKs）的影响。被强烈激活的基因之一是IL 36 G，其编码IL-36，一种IL-1家族细胞因子。IL-36被认为是通过非常规的蛋白质分泌途径分泌的，并且需要N-末端加工来活化。IL-36干扰3D模型和小鼠耳中的NHEK分化。这表明IL-36细胞因子是IL-17的效应物。作为支持，IL-17 A和IL-36在控制基因转录和影响小鼠皮肤发育方面合作。我们提出IL-17 A-IL-36前馈回路，加重银屑病表型。因此，我们想了解这两种细胞因子如何在HaCaT角质形成细胞和NHEK中合作。为此，我们将研究信号通路，并确定信号是如何整合在目标启动子。我们将测量信号分子、转录因子与DNA的结合、染色质状态、聚合酶II活性和转录速率。最后，转录后协同性将通过分析RNA稳定性来确定。总之，这些研究将阐明IL-17 A和IL-36如何合作控制靶基因。此外，我们想了解IL-36的加工和分泌是如何调节的。我们将使用IL-36与生物素连接酶的融合蛋白来修饰相互作用的蛋白质。将使用链霉亲和素纯化生物素化的相互作用物，并通过质谱法鉴定。这种方法甚至可以定义短期相互作用的蛋白质，并为我们提供定义相关非常规蛋白质分泌途径的线索。我们还将通过模式识别受体刺激细胞，以确定促进IL-36分泌的信号。将使用耳注射和咪喹莫特模型在小鼠中进一步研究IL-36的功能相关性，其中将评价拮抗性IL-36受体融合蛋白以及新定义的参与IL-36分泌和活化的因子。总之，我们期望从这些研究中获得对IL-36的激活和加工的分子见解，它如何与IL-17 A合作，以及它对银屑病背景下角质形成细胞功能和分化的改变的贡献。