TREM2 regulation of macrophages in spinal cord injury and CNS endogenous repair

描述（由申请人提供）：脊髓损伤（SCI）引发神经炎症反应，可加重组织损伤（如神经元死亡、轴突损伤、脱髓鞘）并促进修复（如轴突再生、髓鞘再生、血管重建）。最近，在脊髓损伤部位发现了功能不同的巨噬细胞亚群，即“M1”（促炎）和“M2”（抗炎）细胞，这可能是功能分化的基础。M1巨噬细胞具有神经毒性，并在损伤后数周内主导损伤脊髓。相反，即使存在抑制分子（如CSPG和髓磷脂），M2巨噬细胞也能促进轴突生长，而不伴有神经毒性。不幸的是，M2巨噬细胞仅在损伤部位停留几天，最终被M1巨噬细胞淹没。因此，高M1:M2比值可能解释了为什么受损中枢神经系统的修复相对于周围组织缓慢而低效。我们预测，通过调节对损伤作出反应的巨噬细胞的表型和功能，脊髓修复的效率和程度将得到改善。我们将使用基因功能缺失（基因敲除）和功能获得（慢病毒）技术来验证这一假设，以操纵在常驻小胶质细胞和髓样前体细胞上表达的触发受体-2 （TREM2）的表达，即产生单核细胞源性巨噬细胞（MDMs）的细胞，因为巨噬细胞中过度表达TREM2会诱导M2表型。在Aim 1中，我们将研究TREM2过表达或选择性敲除对小胶质细胞和MDMs的影响如何影响挫伤脊髓损伤后的炎症、运动恢复和解剖学指标的修复。在Aim 2的脊髓背半断损伤模型中，通过慢病毒构建体在MDMs上过表达TREM2，我们将确定TREM2操作是否影响巨噬细胞对髓鞘/轴突吞噬、轴突再生和轴突收缩或损伤轴突“死背”的作用。使用局灶性脊髓内脱髓鞘（溶卵磷脂）模型结合功能方案的获得，我们将在Aim 3中确定操纵巨噬细胞TREM2是否影响脊髓内OPC分化和髓鞘再生。目前的提案概述了原理验证实验，这些实验将促进我们对不同分子信号通路（即TREM2）如何影响脊髓损伤后CNS巨噬细胞功能的自然过程的理解。重要的是，如果这些研究的数据表明，操纵TREM2可以带来解剖学或功能上的好处，而对中枢神经系统的结构或功能几乎没有或没有不良影响，那么开发类似的人体临床试验方案应该是可行的。事实上，骨髓细胞静脉注射（在拟议的研究中使用的主要技术）已经在脊髓损伤患者中进行了试验，没有出现不良反应。此外，酶替代疗法，利用慢病毒转导自体外周血单个核细胞，被证明是安全有效的人类受试者。

DESCRIPTION (provided by applicant): Spinal cord injury (SCI) triggers a neuroinflammatory reaction that can aggravate tissue injury (e.g., neuronal death, axonal injury, demyelination) and promote repair (e.g., axon regeneration, remyelination, revascularization). Recently, functionally distinct subsets of macrophages, i.e., "M1" (pro-inflammatory) and "M2" (anti-inflammatory) cells have been identified at sites of SCI which may underlie the functional dichotomy. M1 macrophages are neurotoxic and dominate the injured spinal cord for several weeks post-injury. In contrast, M2 macrophages promote axon growth, even in the presence of inhibitory molecules (e.g. CSPG and myelin), without concomitant neurotoxicity. Unfortunately, M2 macrophages populate the injury site for only a few days, eventually becoming overwhelmed by M1 macrophages. Thus, the high M1:M2 ratio might explain why repair of the injured CNS is slow and inefficient relative to tissues in the periphery. We predict that the efficiency and magnitude of spinal cord repair will be improved by modulating the phenotype and function of macrophages that respond to the injury. We will test this hypothesis using genetic loss-of-function (knockout) and gain-of-function (lentiviral) techniques to manipulate expression of the triggering receptor expressed on myeloid cells-2 (TREM2) on resident microglia and myeloid precursor cells, i.e., cells that give rise to monocyte-derived macrophages (MDMs), since overexpressing TREM2 in macrophages induces an M2 phenotype. In Aim 1 we will examine how TREM2 overexpression or selective knockout on microglia vs. MDMs effects inflammation, motor recovery and anatomical indices of repair after contusive spinal cord injury. By using lentiviral constructs to overexpress TREM2 on MDMs in a model of dorsal spinal hemisection injury in Aim 2, we will determine if TREM2 manipulation influences macrophage effects on myelin/axon phagocytosis, axon regeneration and axonal retraction or "die-back" of injured axons. Using a model of focal intraspinal demyelination (lysolecithin) in conjunction with the gain of function protocols, we will determine if manipulating macrophage TREM2 affects OPC differentiation and remyelination within the spinal cord in Aim 3. The current proposal outlines proof-of-principle experiments that will advance our understanding of how a distinct molecular signaling pathway, i.e., TREM2, influences the natural course of CNS macrophage function after SCI. Importantly, if data from these studies indicate that manipulating TREM2 confers anatomical or functional benefits with minimal or no adverse effects on CNS structure or function, then it should be feasible to develop similar protocols for human clinical trials. Indeed, intravenous delivery of bone marrow cells (the primary technique to be used in the proposed studies) has already been tried in SCI patients and without adverse effects. Moreover, enzyme replacement therapies, using lentiviral transduction of autologous peripheral blood mononuclear cells, were shown to be safe and effective in human subjects. PUBLIC HEALTH RELEVANCE: The injured spinal cord does not heal properly resulting in chronic paralysis and expensive health care costs for patients with spinal cord injuries. The goal of this proposal is to alter the response to injury to promote increased regeneration, reduced tissue loss, and ultimately increased functional recovery by genetically engineering inflammatory cells that normally respond to injury. Similar therapeutic approaches are being used to cure a variety of neurological diseases; therefore data obtained from our studies could provide the groundwork for a reparative therapy to treat spinal cord injury.