Toll-Like Receptor-Mediated Photoreceptor Mitochondrial DNA Damage

描述(申请人提供)：自身免疫性疾病的组织损伤以前被归因于T细胞和炎性细胞的渗透以及此后释放的细胞因子。天然免疫中Toll样受体(TLRs)的激活产生与获得性免疫相似的促炎细胞因子，并导致线粒体DNA损伤。直到最近，这种TLRS病理一直被忽视。在多种全身性疾病中，TLRs的激活参与了中枢神经系统脱髓鞘、器官缺血/再灌注损伤和老年性黄斑变性的发病机制。在这一应用中，在远端注射热致死分支杆菌诱导的TLR4，启动了视网膜中神经细胞的氧化应激和线粒体DNA损伤。TLR4的上调也被发现主要与视网膜小胶质细胞有关。基于这些初步的发现，提出了以下的具体目标：1.检测TLR和TNF-1及其受体在具有先天免疫反应的小鼠视网膜中的表达和定位。2.利用不同的TLRs、MyD88和其他接头蛋白敲除动物，确定TLR上调以及与MyD88相互作用对线粒体氧化应激的体内影响。3.利用仅表达小胶质细胞MyD88的骨髓嵌合体，确定骨髓来源的视网膜小胶质细胞TLR上调在视网膜神经细胞线粒体氧化应激中的作用。4.通过上调TLRs在视网膜中定位氧化应激介导的线粒体DNA损伤和半胱氨酸天冬氨酸氨基转移酶，并证明T细胞介导的适应性免疫反应增强了视网膜损伤。设计的研究方法紧扣着这些特定的目标。在先天免疫中，TLRs、转录因子(如NFkB)、细胞因子(如TNF-1)和iNOS上调(特异性目标1)，并可能与视网膜小胶质细胞共定位(特异性目标1)。然而，与其他视网膜免疫细胞的共同定位也将被测试(特定目标1)。TLRs与小胶质细胞的关联将由骨髓嵌合小鼠进一步证实，在嵌合小鼠中，视网膜小胶质细胞将是骨髓来源的(特定目标3)。由TLRs激活介导的线粒体DNA损伤将通过使用各种野生型和敲除来测试，包括适配器蛋白敲除(特定目标2)。此外，将在各种视网膜细胞中寻找DNA损伤和半胱氨酸天冬氨酸酶的位置(特定目标4)。一旦阐明，这些关于TLR激活初始事件的结果可能在自身免疫性疾病和TLR4激活引起的神经细胞特异性DNA损伤方面具有全球意义。公共卫生相关性：眼内炎症，或称葡萄膜炎，是视网膜感光细胞变性致盲的主要原因。这项申请涉及先前被忽视的早期光感受器损伤的方面，仅由先天免疫启动。通过阐明Toll样受体激活导致视网膜损伤的机制，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.