Innate Immunity in the Pathogenesis of PVL

PVL是早产儿神经系统疾病的主要病理基础。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/轴突的损伤。我们认为PVL的先天免疫激活主要有两种机制：(1)感染引起的先天免疫激活通过病原体相关分子模式(PAMPs)发生；(2)由于热休克蛋白60(HSP60)的释放而引起的缺氧缺血先天免疫激活。在以前发表的报道和初步数据中，我们已经证明特定的PAMP通过Toll样受体(Toll-like Receptor，TLRs)激活中枢神经系统(小胶质细胞)的固有免疫细胞。PAMPs激活先天免疫细胞，导致CNS培养中发育中的少突胶质细胞和神经元/轴突的损伤。我们已经证明HSP60可能是一种内源性TLR4激动剂。热休克蛋白60诱导野生型小胶质细胞产生肿瘤坏死因子α和一氧化氮 TLR4或MyD88突变小鼠。热休克蛋白60是从实验性坏死或凋亡的细胞中释放出来的。HSP60和脂多糖一样，对中枢神经系统培养中的轴突/神经元造成严重损伤。由于PVL的轴突发育可能受到抑制，我们还研究了PVL中细胞损伤导致轴突生长抑制的机制。我们发现神经元表达TLR3，TLRS配体双链RNA抑制轴突延伸，导致生长锥体塌陷。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这样一种假设，即通过作用于小胶质细胞和神经元上的Toll样受体的先天免疫激活机制的汇聚导致少突胶质细胞和轴突的形成。 PVL特有的缺陷。我们将通过以下具体目标来验证这一假说：目的1.研究TLRs在正常人脑白质和PVL中的发育表达。假设：TLR在胎儿脑白质和PVL皮损中的表达将上调。目的2.探讨内源性小胶质细胞激活剂是否会在体外引起小胶质细胞损伤。假设：(1)HSP60以TLR4-MyD88依赖的方式激活小胶质细胞，导致前驱OLS继发性(旁观者)损伤。目的3.研究HSP60与TLR4的相互作用。假设：HSP-60初级序列的一个受限部分与TLR4相互作用；这种相互作用是激活TLR4所必需的。目的4.确定阻断小胶质细胞 在PVL模型中，激活将对新生儿脑损伤具有保护作用。假设：特异性抑制TLR/MvD88通路将改善PVL小鼠模型的预后。

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.