Toll-Like Receptor-Mediated Photoreceptor Mitochondrial DNA Damage

描述（由申请人提供）：自身免疫性疾病中的组织损伤先前归因于T细胞和炎性细胞浸润以及此后释放的细胞因子。先天免疫中Toll样受体（TLR）的激活产生与适应性免疫相似的促炎细胞因子，并造成线粒体DNA损伤。这种TLR病理学直到最近才被忽视。在各种系统性疾病中，TLR活化与CNS脱髓鞘、器官缺血/再灌注损伤和年龄相关性黄斑变性的发病机制有关。在该应用中，由注射在远端部位的热灭活分枝杆菌诱导的TLR 4引发视网膜中的神经元细胞氧化应激和线粒体DNA损伤。TLR 4的上调也被发现主要与视网膜小胶质细胞有关。基于这些初步研究结果，提出了以下具体目标：1。确定TLR和TNF-1及其受体在具有先天免疫应答的小鼠视网膜中的表达和定位。2.使用各种TLR、MyD 88和其他衔接蛋白敲除动物，确定TLR上调和与MyD 88相互作用对线粒体氧化应激的体内影响。3.使用表达限于小胶质细胞的MyD 88的骨髓嵌合体，确定骨髓来源的视网膜小胶质细胞TLR上调在视网膜神经元细胞线粒体氧化应激中的作用。4.局部氧化应激介导的线粒体DNA损伤和半胱天冬酶在视网膜与TLR的上调，并证明视网膜损伤与T细胞介导的适应性免疫反应的增强。所设计的研究方法与这些具体目标密切相关。在先天性免疫中，TLR、转录因子（如NF κ B）、细胞因子（如TNF-1）和iNOS上调（特异性目的1），并且可能共定位于视网膜小胶质细胞（特异性目的1）。然而，还将测试与其他视网膜免疫细胞的共定位（具体目标1）。TLR与小胶质细胞的关联将通过骨髓嵌合小鼠进一步证实，其中视网膜小胶质细胞将是骨髓来源的（具体目标3）。将通过使用各种野生型和敲除（包括衔接蛋白敲除）来测试TLR激活介导的线粒体DNA损伤（具体目标2）。此外，将在各种视网膜细胞中寻找DNA损伤和半胱天冬酶的位点（具体目标4）。一旦澄清，TLR激活的初始事件的这些结果可能在自身免疫性疾病和TLR 4激活的神经元细胞中的特异性DNA损伤中具有全球意义。公共卫生相关性：眼内炎症或葡萄膜炎是视网膜感光细胞变性导致失明的主要原因。这个应用程序处理以前被忽视的方面，早期感光细胞损伤单独先天免疫启动。通过阐明Toll样受体激活引起视网膜损伤的机制，我们可以推进治疗干预的设计，以达到预期的效果。

DESCRIPTION (provided by applicant): Tissue damage in autoimmune disease has previously been attributed to T-cell and inflammatory cell infiltration and to the cytokines released thereafter. Activation of Toll-like receptors (TLRs) in innate immunity generates similar proinflammatory cytokines as those of adaptive immunity and inflict mitochondrial DNA damage. Such TLRs pathology has been overlooked until recently. Among various systemic diseases, TLRs activation has been implicated in the pathogenesis of CNS demyelination, ischemia/reperfusion injury of the organs, and age-related macular degeneration. In this application, TLR4 induced by heat-killed mycobacteria injected at distant sites initiates neuronal cell oxidative stress and mitochondrial DNA damage in the retina. The TLR4 upregulation was also found to associate primarily with the retinal microglia. Based on these preliminary findings, the following specific aims are proposed: 1. Determine the expression and localization of TLR and TNF-1 and its receptors in the mouse retina with an innate immune response. 2. Determine the in-vivo effects of TLR upregulation and interaction with MyD88 on mitochondrial oxidative stress, using various TLRs, MyD88, and other adaptor proteins knockout animals. 3. Determine the role of bone marrow derived retinal microglia TLR upregulation in the retinal neuronal cell mitochondrial oxidative stress, using bone marrow chimeras that express MyD88 limited to the microglia. 4. Localize oxidative stress-mediated mitochondrial DNA damage and caspases in the retina with upregulation of TLRs and demonstrate augmentation of retinal damage with T-cell-mediated adaptive immune response. The research methods designed are closely adhered to these specific aims. In innate immunity, TLRs, transcription factors (such as NFkB), cytokines (such as TNF-1), and iNOS are upregulated (specific aim 1) and are likely to co-localize to retinal microglia (specific aim 1). However, co-localization with other retinal immune cells will be also tested (specific aim 1). The association of TLRs to microglia will be further substantiated by bone marrow chimeric mice in which the retinal microglia will be bone marrow derived (Specific aim 3). The mitochondrial DNA damage mediated by TLRs activation will be tested by using various wild types and knockouts, including adaptor protein knockouts (specific aim 2). Further, the site of DNA damage and caspases will be sought in various retinal cells (specific aim 4). Once clarified, these results on initial events of TLR activation might have a global implications in autoimmune diseases and specific DNA damage in neuronal cells from TLR4 activation. PUBLIC HEALTH RELEVANCE: Intraocular inflammation, or uveitis, is a leading cause of blindness from retinal photoreceptor cell degeneration. This application deals with the previously overlooked aspect of early photoreceptor damage initiated by the innate immunity alone. By clarifying the mechanism of retinal damage rendered by Toll-like receptor activation, we can advance the design of therapeutic intervention to achieve the desired outcome.