Characterization and molecular functions of the IL-17 – IL-36 signaling axis in keratinocytes and consequences for 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表皮增生伴角化细胞增生和异常分化。免疫系统与银屑病密切相关，如TH17细胞通过IL-17及其他细胞因子调节角化细胞功能。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并控制编码与角质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素。我们在单层培养和三维（3D）器官型皮肤模型中研究了IL-17A对原代正常人表皮角质形成细胞（NHEKs）的影响。其中一个被强烈激活的基因是IL36G，它编码IL-36，一种IL-1家族细胞因子。IL-36被认为是通过一种非常规的蛋白质分泌途径分泌的，需要n端加工才能激活。IL-36在3D模型和小鼠耳中干扰NHEK分化。这表明IL-36细胞因子是IL-17的效应器。因此，IL-17A和IL-36共同调控基因转录，影响小鼠皮肤发育。我们提出一个IL-17A - 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是如何在HaCaT角质形成细胞和nhek中合作的。为此，我们将研究信号通路并确定信号如何在目标启动子上整合。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活性和转录率。最后，通过分析RNA稳定性来确定转录后的协同性。总之，这些研究将阐明IL-17A和IL-36如何协同控制靶基因。此外，我们想了解IL-36的加工和分泌是如何被调节的。我们将使用IL-36的融合蛋白与生物素连接酶来修饰相互作用的蛋白。生物素化的相互作用物将用链霉亲和素纯化，并用质谱法鉴定。这种方法甚至可以定义短期相互作用的蛋白质，并为我们定义相关的非常规蛋白质分泌途径提供线索。我们还将通过模式识别受体刺激细胞来定义促进IL-36分泌的信号。IL-36的功能相关性将通过耳注射和咪喹莫特模型在小鼠中进一步研究，其中将评估拮抗IL-36受体融合蛋白以及参与IL-36分泌和激活的新定义因子。总之，我们期望从这些研究中获得IL-36的激活和加工的分子洞察力，它如何与IL-17A合作，以及它在牛皮癣背景下改变角质细胞功能和分化的贡献。

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.