Trim32 regulation of PKC-zeta in atopic dermatitis

项目摘要 特应性皮炎（AD）是一种先天免疫受损的炎性皮肤病， 易受细菌和病毒感染。尽管已经鉴定出了这些基因中的遗传多态性， 先天免疫信号通路，如TLR 1/2/6/9和NOD 1/2，先天免疫缺陷在 AD的发病机制仍然知之甚少。在这项研究中，我们提供了新的证据来支持 先天免疫缺陷在Th 2活化和AD发病机制中的作用 模特作为对咪喹莫特局部应用的响应，咪喹莫特是一种用于已建立的免疫缺陷综合征的Toll样受体激动剂。 在银屑病模型中，我们观察到Trim 32缺陷小鼠反而发展出AD样表型，包括 嗜酸性粒细胞和肥大细胞的真皮浸润，Th 2细胞因子产生增加，血清IgE升高， 与Trim 32-感受态野生型同窝仔中预期的Th 17应答相比。基于此，我们 假设Trim 32（一种具有天然抗病毒活性的E3泛素连接酶）调节宿主体内的稳态 此外，Trim 32途径蛋白质或功能性的缺陷可导致对感染的应答， Th 2特应性表型。在寻找Trim 32底物蛋白可能负责特应性特征，在这些 在小鼠和人类AD中，我们为Trim 32调节蛋白激酶PKC β提供了新的证据， 信号级联的Stat 6激活剂，最终导致T细胞中IL-4/IL-5的表达和B的IgE产生 小鼠过敏模型中的细胞。因此，我们建议解决Trim 32/ PKC β轴在调节中的作用， 在小鼠模型中负责AD样疾病发展的分子信号事件， 与人AD患者样品中发现的验证平行。鉴于我们的发现，我们建议 以下目的：1）确定Trim 32在调节PKC β介导的IL-4/Stat 6信号通路中的作用， 小鼠模型; 2）研究体内稳态负调控轴的分子和结构基础， Trim 32和PKC β轴;和3）评估Trim 32和PKC β轴与AD严重程度和AD 人类对病毒感染的易感性。我们的研究将结合多重免疫组织化学图像 用于分析人类皮肤活检的细胞计数法和先进的成像技术， 通过超分辨率显微镜、相关光学显微镜和电子显微镜以及分子生物学技术， 结构分析本研究将为更好地理解细胞凋亡的分子机制提供新的见解。 Th 2极化是AD病理学的基础，有望改善对患者疾病严重程度的预测， 治疗反应，并提出新的干预策略，以恢复健康的免疫和皮肤 AD患者的体内平衡。

PROJECT SUMMARY Atopic dermatitis (AD) is an inflammatory skin disease with compromised innate immunity as indicated by susceptibility to bacterial and viral infection. Despite the identification of genetic polymorphisms in genes in the innate immune signaling pathways, such as TLR1/2/6/9 and NOD1/2, the role of defective innate immunity in AD pathogenesis remains poorly understood. In this study, we provide new evidence to support the contribution of defective innate immunity in Th2 activation and AD pathogenesis with Trim32 knockout mice as a model. In response to topical application of imiquimod, a toll-like receptor agonist used in an established psoriasis model, we observed that Trim32-deficient mice instead developed AD-like phenotypes, including dermal infiltration of eosinophils and mast cells, increased Th2 cytokine production, and elevated serum IgE, compared to the expected Th17 response in Trim32-competent wild type littermates. Based upon this, we hypothesize that Trim32 (an E3 ubiquitin ligase with innate anti-viral activity) regulates homeostasis in the host response to infection and, further, that defects in Trim32 pathway proteins or functionality can predispose to Th2 atopic phenotypes. In seeking Trim32 substrate proteins potentially responsible for atopic features in these mice and in human AD, we provide novel evidence for Trim32 regulation of protein kinase PKCζ, a known Stat6 activator of a signaling cascade culminating in IL-4/IL-5 expression in T cells and IgE production by B cells in allergy models in mice. We therefore propose to address the role of Trim32/ PKCζ axis in the regulation of molecular signaling events that are responsible for the development of AD-like disease in mouse models, in parallel to validation of findings in human AD patient samples. In view of our discoveries, we propose the following aims: 1) Define the role of Trim32 in regulating a PKCζ mediated IL-4/Stat6 signaling pathway in mouse models; 2) Investigate the molecular and structural basis of a homeostatic negative regulatory axis of Trim32 and PKCζ; and 3) Evaluate the Trim32 and PKCζ axis in association with AD severity and AD susceptibility to viral infection in humans. Our studies will incorporate Multiplex Immunohistochemistry Image Cytometry for analysis of human skin biopsies and advanced imaging technologies to identify localization of interacting proteins by super resolution microscopy, correlative light and electron microscopy, and molecular structure analysis. This study will provide new insight for better understanding of the molecular mechanisms of Th2 polarization underlying AD pathology and promises to improve prediction of patient severity of disease and treatment response and to suggest novel intervention strategies to restore healthy immune and skin homeostasis in AD patients.