Innate Immunity in the Pathogenesis of PVL

PVL 发病机制中的先天免疫

• 批准号: 7006510
• 负责人: TIMOTHY VARTANIAN
• 金额: $ 35.09万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7006510
• 关键词: brain injury; cerebrovascular disorders; disease /disorder etiology; disease /disorder prevention /control; gene mutation; gray matter; heat shock proteins; human fetus tissue; immune response; immunocytochemistry; inflammation; microglia; mixed tissue /cell culture; neuroimmunomodulation; neurons; neuropathology; oligodendroglia; postmortem; protein localization; protein protein interaction; receptor expression; recombinant proteins; toll like receptor; white matter

PVL is the major pathologic substrate for neurological morbidity observed in premature infants. Hypoxia-ischemia and maternal-fetal infection are considered two major risk factors for development of PVL. In this project our overall hypothesis is that both hypoxia-ischemia and infection result in heightened innate immunity that in turn leads to injury of developing oligodendrocytes and neurons/axons. We propose that there are two major mechanisms of innate immune activation in PVL: (1) innate immune activation due to infection occurs through pathogen associated molecular patterns (PAMPs) and (2) innate immune activation due to hypoxia-ischemia occurs due to release of heat shock protein 60 (HSP60). In previously published reports and in preliminary data, we have shown that specific PAMPs activate the resident innate immune cells of the CNS (microglia) through toll-like receptors (TLRs). Activation of innate immune cells by PAMPs results in injury to developing oligodendrocytes and neurons/axons in CNS cultures. We have shown that HSP60 is a putative endogenous TLR4 agonist. HSP60 induces TNFalpha and nitric oxide production by microglia from wild-type but not TLR4 or MyD88 mutant mice. HSP60 is released from cells undergoing experimental necrosis or apoptosis. HSP60, like LPS, causes severe injury to axons/neurons in CNS cultures. Since axonal development may be inhibited in PVL, we have also examined mechanisms by which cell injury in PVL could lead to inhibition of axonal growth. We have found that neurons express TLR3 and that double stranded RNA, a TLRS ligand, inhibits neurite extension and causes growth cone collapse. This is the first example of a TLR functioning in neurons. Our preliminary data support the hypothesis that a convergence of innate immune activating mechanisms acting through toll-like receptors on microglia and neurons leads to the oligodendrocyte and axonal defects characteristic of PVL. We will test this hypothesis through the following specific aims: Aim 1. To characterize the developmental expression of TLRs in normal human white matter and in PVL. Hypothesis: TLR expression will be up-regulated in fetal versus white matter and in PVL lesions. Aim 2. To determine whether endogenous activators of microglia cause pre-OL injury in vitro. Hypothesis: (i) HSP60 activates microglia in a TLR4-MyD88 dependent fashion leading to secondary (bystander) injury of pre-OLs. Aim 3. To characterize the interaction of HSP60 with TLR4. Hypothesis: A restricted portion of the HSP-60 primary sequence interacts with TLR4; this interaction is required for activation of TLR4. Aim 4. To determine whether blocking microglial activation will protect against neonatal brain injury in models of PVL. Hypothesis: Specific inhibition of the TLR/MvD88 pathway will improve outcomes in mouse models of PVL.

PVL 是早产儿神经系统发病的主要病理基础。缺氧缺血和母婴感染被认为是PVL发生的两个主要危险因素。在这个项目中，我们的总体假设是缺氧缺血和感染都会导致先天免疫增强，进而导致发育中的少突胶质细胞和神经元/轴突损伤。我们提出PVL中先天免疫激活有两种主要机制：（1）通过病原体相关分子模式（PAMP）发生感染引起的先天免疫激活；（2）由于热休克蛋白60（HSP60）的释放而发生缺氧缺血引起的先天免疫激活。在之前发表的报告和初步数据中，我们已经表明特定的 PAMP 通过 Toll 样受体 (TLR) 激活中枢神经系统（小胶质细胞）的固有先天免疫细胞。 PAMP 激活先天免疫细胞会导致中枢神经系统培养物中发育中的少突胶质细胞和神经元/轴突损伤。我们已经证明 HSP60 是一种推定的内源性 TLR4 激动剂。 HSP60 诱导野生型小胶质细胞产生 TNFα 和一氧化氮，但不诱导 TLR4 或 MyD88 突变小鼠。 HSP60 从经历实验性坏死或凋亡的细胞中释放。 HSP60 与 LPS 一样，会对 CNS 培养物中的轴突/神经元造成严重损伤。由于PVL中轴突发育可能受到抑制，我们还研究了PVL中细胞损伤可能导致轴突生长抑制的机制。我们发现神经元表达 TLR3，而双链 RNA（TLRS 配体）会抑制神经突延伸并导致生长锥塌陷。这是 TLR 在神经元中发挥作用的第一个例子。我们的初步数据支持以下假设：通过小胶质细胞和神经元上的 Toll 样受体发挥作用的先天免疫激活机制的融合导致少突胶质细胞和轴突 PVL 的缺陷特征。我们将通过以下具体目标来检验这一假设： 目标 1. 表征正常人白质和 PVL 中 TLR 的发育表达。假设：与白质相比，胎儿中以及 PVL 病变中 TLR 表达会上调。目标 2. 确定小胶质细胞内源性激活剂是否会在体外引起前 OL 损伤。假设：(i) HSP60 以 TLR4-MyD88 依赖性方式激活小胶质细胞，导致前 OL 的继发性（旁观者）损伤。目标 3. 表征 HSP60 与 TLR4 的相互作用。假设：HSP-60 一级序列的受限部分与 TLR4 相互作用；这种相互作用是 TLR4 激活所必需的。目标 4. 确定是否阻断小胶质细胞 激活将防止 PVL 模型中的新生儿脑损伤。假设：TLR/MvD88 通路的特异性抑制将改善 PVL 小鼠模型的结果。