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。缺氧缺血和母胎感染被认为是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，缺氧缺血和感染导致先天免疫增强，这反过来又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先天性免疫激活有两种主要机制：（1）感染引起的先天性免疫激活通过病原体相关分子模式（PAMPs）发生;（2）缺氧缺血引起的先天性免疫激活由于热休克蛋白60（HSP 60）的释放而发生。在先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CNS（小胶质细胞）的固有免疫细胞。PAMP激活先天免疫细胞导致CNS培养物中发育中的少突胶质细胞和神经元/轴突损伤。我们已经表明，HSP 60是一个假定的内源性TLR 4激动剂。HSP 60诱导野生型小胶质细胞产生TNF α和一氧化氮， TLR 4或MyD 88突变小鼠。HSP 60是从实验性坏死或凋亡的细胞中释放出来的。HSP 60与LPS一样，在CNS培养物中对轴突/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。我们已经发现神经元表达TLR 3，并且双链RNA（TLRS配体）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这样的假设，即通过小胶质细胞和神经元上的toll样受体作用的先天免疫激活机制的会聚导致少突胶质细胞和轴突的凋亡。 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。表征正常人白色物质和PVL中TLR的发育表达。假设：TLR表达在胎儿与白色物质和PVL病变中上调。目标2.确定小胶质细胞的内源性激活剂是否在体外引起前OL损伤。假设：（i）HSP 60以TLR 4-MyD 88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.探讨HSP 60与TLR 4的相互作用。假设：HSP-60一级序列的限制性部分与TLR 4相互作用;这种相互作用是激活TLR 4所必需的。目标4。为了确定阻断小胶质细胞 激活将保护PVL模型中的新生儿脑损伤。假设：TLR/MvD 88通路的特异性抑制将改善PVL小鼠模型的结局。

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.