The Role of Bacterial Toxins in Human Skin Disease

 描述(申请人提供)：特应性皮炎(AD)是美国17%的儿童和近3%的成年人中最常见的慢性炎症性皮肤病。它与严重的发病率和职业残疾有关。最近的研究强调了细胞因子环境在AD病理生物学中的重要性，金黄色葡萄球菌的定植/感染导致了这种常见疾病的严重/恶化。AD患者皮肤表皮屏障蛋白表达明显减少，抗菌肽产生水平降低，导致皮肤屏障功能和抗菌宿主防御功能异常。在大多数AD中，这些变化是由抑制表皮角质形成细胞分化引起的。然而，导致AD皮肤角质形成细胞分化不足的分子事件却知之甚少。本次竞争性更新R01拨款申请(5 R01 AR41256-25)的总体目标将是描述与AD皮肤环境相关的细胞因子IL-4/IL-13和IL-22以及葡萄球菌毒素抑制AD皮肤角质形成细胞分化导致表皮屏障异常的初始分子和细胞事件。我们的新的初步数据表明，IL-4/IL-13和IL-22通过不同的信号通路干扰角质形成细胞的早期分化，其机制是通过影响钙信号转导/动员，激活Wnt/β-catenin途径，同时抑制Notch发育途径。我们提供的证据表明，葡萄球菌脂磷壁酸(LTA)选择性地激活了Notch调节的Ankyrin重复蛋白(NRARP)的表达，该蛋白干扰了Notch途径并抑制了早期角质形成细胞分化标志物的表达。最后，我们证明了AD皮肤细胞因子环境和葡萄球菌产品在抑制角质形成细胞分化方面相互协同和互补。该建议的具体目的将是：1)通过检测在IL-4/IL-13和IL-22与IFNG环境中分化的角质形成细胞的基因和蛋白质表达谱，建立AD皮肤免疫反应改变皮肤角质形成细胞分化的分子机制；分析受IL-4/IL-13调控的钙结合蛋白SMOC1和STC2在角质形成细胞钙动员和信号转导中的作用；评估干扰钙结合蛋白的表达和功能如何控制角质形成细胞分化的早期阶段，并影响这些细胞中的Wnt5a/β-catenin信号通路。2)为了研究葡萄球菌毒素对角质形成细胞分化的影响，我们将检测LTA对控制角质形成细胞分化的Notch信号通路的干扰。我们将评估LTA诱导的NRARP在这一过程中的作用。3)探讨AD免疫应答与葡萄球菌LTA在体内角质形成细胞分化过程中的协同作用。我们将评估AD患者皮肤中角质形成细胞的分化计划与皮肤细胞因子环境和金黄色葡萄球菌定植的关系，通过以下方法检测皮肤中不同层角质形成细胞的转录图谱 激光捕获显微解剖和RNAseq，利用AD患者的原代角质形成细胞评估3D器官型皮肤培养中角质形成细胞的分化，并检测调控NRARP的钙结合蛋白SMOC1、STC2和Notch在这些培养物中的差异表达如何影响角质形成细胞的分化。这些研究可能会发现新的选择性治疗方法，可以改变AD皮肤早期的角质形成细胞分化程序，为恢复和开发新的方法来增强AD的表皮屏障功能提供机会。

 DESCRIPTION (provided by applicant): Atopic dermatitis (AD) is the most common chronic inflammatory skin disease in the general population affecting 17% of children and nearly 3% of adults in the U.S. It is associated with significant morbidity and occupational disability. Recent studies have highlighted the importance of cytokine environment in the pathobiology of AD and Staphylococcus aureus colonization/infection contributing to the severity/exacerbation of this common disease. The skin of AD patients has significantly reduced epidermal barrier protein expression and low levels of antimicrobial peptide production, which result in abnormalities in the skin barrier function and antimicrobial host defense. In the majority of AD, these changes are caused by inhibition of epidermal keratinocyte differentiation. However, the molecular events that result in lack of keratinocyte differentiation in AD skin are poorly understood. The overall goal of this competing renewal of R01 grant application (5 R01 AR41256-25) will be to delineate the initial molecular and cellular events by which IL-4/IL-13 and IL-22, cytokines associated with AD skin environment, and staphylococcal toxins inhibit keratinocyte differentiation in AD skin leading to epidermal barrier abnormalities. Our new preliminary data indicates that both IL-4/IL-13 and IL-22 through different signaling pathways interfere with early differentiation events in keratinocytes by affecting Ca2+ signaling/mobilization, activating Wnt/beta-catenin pathway, while inhibiting Notch developmental pathway. We present evidence that staphylococcal lipoteichoic acid, LTA, selectively activates the expression of Notch- Regulated Ankyrin Repeat Protein, NRARP, that interferes with Notch pathway and inhibits the expression of early keratinocyte differentiation markers. Finally, we demonstrate that AD skin cytokine environment and staphylococcal products synergize and complement each other in the inhibition of keratinocyte differentiation. The specific aims of this proposal will be: 1) To establish the molecular mechanisms by which the immune response in AD skin alters differentiation of skin keratinocytes by examining gene and protein expression profiling in keratinocytes differentiated in the IL-4/IL-13 vs IL-22 vs IFNg environment; analyzing the role of the Ca2+- binding proteins, SMOC1 and STC2, regulated by IL-4/IL-13 in Ca2+ mobilization and signaling in keratinocytes; evaluating how interference with Ca2+ binding proteins expression and function may control early stages of keratinocyte differentiation and affect Wnt5a/beta-catenin signaling pathway in these cells. 2) To investigate the effects of staphylococcal toxins on keratinocyte differentiation In this aim, we will examine LTA interference with Notch signaling pathway, which controls keratinocyte differentiation. We will assess the role of LTA-induced NRARP in this process. 3) To investigate the synergistic effects of the AD immune response and staphylococcal LTA, on the keratinocyte differentiation program in vivo. We will assess keratinocyte differentiation program in the skin of AD patients in relationship to skin cytokine environment and colonization with S. aureus, examine transcriptional profile of different layers of keratinocytes in the skin by laser capture microdissection and RNAseq, evaluate keratinocyte differentiation in 3D organotypic skin cultures utilizing primary keratinocytes of AD patients and examine how differential expression of Ca2+ binding proteins SMOC1, STC2 and Notch regulating NRARP in these cultures influences keratinocyte differentiation. These studies will likely identify novel selective therapeutic approaches that can alter the keratinocyte differentiation program in AD skin at its earliest stages, providing the opportunity to restore and develop new approaches to enhance epidermal barrier function in AD.