The Role of the p75ntr in immune cell differentiation and trafficking in traumatic brain injury (TBI)

项目摘要 p75神经营养因子受体（p75 ntr，NGFR 1/TNFRSF 16）在损伤和修复中的作用是复杂的; 与细胞存活和细胞死亡相关，这取决于其与多种辅助受体的相互作用1。 P75 ntr对神经发育很重要，但仅在成人大脑中稀疏表达。但是在CNS之后， 损伤后，p75 ntr在神经元和神经胶质细胞中重新表达。几年前，我们发现NGF的前体形式（前体形式） p75 ntr缺失可保护少突胶质细胞（OL）的凋亡，而p75 ntr缺失可保护OL的凋亡。 脊髓损伤（SCI）后的OL 2.现在有许多研究表明，删除或拮抗 CNS创伤后p75 ntr可以改善SCI和创伤性脑损伤（TBI）后的结局3，4，尽管 5.保护并不总是存在的。我们最近发现一种新的p75 ntr拮抗剂， 保护神经元和OL细胞死亡，并改善神经功能的结果时，给予7天后， 大鼠6和小鼠7中的TBI。治疗减少了血液中促炎单核细胞的数量， 减少小鼠皮质挫伤（CCI）-TBI后CCR 2+单核细胞侵入病变部位 7. P75 ntr在许多外周组织中表达，包括免疫细胞8-11。我们发现p75 ntr 拮抗剂降低LPS对小鼠白细胞的促炎作用7。此外，我们还发现， p75 ntr拮抗剂抑制LPS诱导的炎症单核细胞的产生，表明p75 ntr 参与TBI后的骨髓细胞分化和外周炎症。我们想测试一下那本小说 假说.有两个目标：目标1。我们将评估p75 ntr在外周炎症中的作用。 通过在体外用LPS刺激来自野生型（wt）与p75 ntr缺失小鼠的单核细胞的反应。我们将 测试TLR 4激动剂脂多糖（LPS）对野生型体外髓样细胞分化的影响 (wt)C57 B1/6髓样细胞与来自具有p75 ntr缺失的同窝小鼠的髓样细胞。我们预测p75 ntr的KO 将减少NFkB信号传导。当我们在p75 ntr科斯中检测p75 ntr缺失的体外效应时，我们将 产生具有外周髓样细胞中p75 ntr的选择性条件性敲低的小鼠用于体内研究 为目标2提出。AIM 2.我们将交叉可用的p75 ntr floxed，和骨髓细胞特异性CRE/ERT 小鼠以产生用于测试外周免疫细胞p75 ntr在TBI中的作用的新小鼠系。我们将使用一个 条件遗传学方法检测p75 ntr在髓系细胞中的作用 小鼠12，其具有在骨髓细胞中表达cre的可用转基因，以产生骨髓细胞特异性 p75 ntr缺失（Csfr 1cre/ERT aka CD 115 creER）。这些小鼠将在CCI TBI之前给予他莫昔芬或溶媒 降低p75 ntr在外周髓系细胞中的表达，并且我们将比较对分化的影响。 单/MAC谱系细胞在循环和它们的运输到受伤的大脑。 总之，这些研究集中在TBI后p75 ntr信号传导的外周效应上，并测试新的假设。 外周骨髓细胞中的p75 ntr信号传导是TBI后损伤/修复级联的重要部分。

PROJECT SUMMARY The role of the p75 neurotrophin receptor (p75ntr, NGFR1/TNFRSF16) in injury and repair is complex; p75ntr is associated with both cell survival and cell death, depending upon its interactions with multiple co-receptors 1. P75ntr is important for neural development, but is only sparsely expressed in the adult brain. But after CNS injury, p75ntr is re-expressed in both neurons and glia 1. We showed years ago that the pro-form of NGF (pro- NGF), which binds to p75ntr, can induce oligodendrocyte (OL) apopotosis, and that p75ntr deletion protects OLs after spinal cord injury (SCI) 2. There are now many studies demonstrating that deletion or antagonism of p75ntr after CNS trauma can improve outcomes after SCI and traumatic brain injury (TBI)3,4, although protection is not always found 5. We recently found that a novel p75ntr antagonist provided highly significant protection from neuronal and OL cell death, and improved neurological outcomes when given for 7 days after TBI in both rats 6 and mice 7. Treatment reduced the number of pro-inflammatory monocytes in the blood, and reduced the invasion of CCR2+ monocytes into the lesion site after cortical contusion injury (CCI)-TBI in mice 7. P75ntr is expressed in many peripheral tissues including immune cells 8-11. We found that the p75ntr antagonist reduced the pro-inflammatory effects of LPS on mouse leukocytes in vitro 7. In addition, we found that the p75ntr antagonist inhibited LPS-induced production of inflammatory monocytes, suggesting that p75ntr is involved in myeloid cell differentiation and peripheral inflammation after TBI. We want to test that novel hypothesis. There are two aims: AIM 1. We will assess the role of p75ntr in peripheral inflammatory responses by stimulating monocytes from wild-type (wt) vs p75ntr null mice with LPS in vitro. We will test the effects of the TLR4 agonist lipopolysaccharide (LPS) on myeloid cell differentiation in vitro in wild type (wt) C57Bl/6 myeloid cells vs myeloid cells from littermates with p75ntr deletion. We predict that KO of p75ntr will reduce NFkB signaling. As we are testing the in vitro effects of p75ntr deletion in the p75ntr KOs, we will generate mice with selective conditional knock-down of p75ntr in peripheral myeloid cells for the in vivo studies proposed for aim 2. AIM 2. We will cross available p75ntr floxed, and myeloid cell-specific CRE/ERT mice to yield a new mouse line for testing the role of peripheral immune cell p75ntr in TBI. We will use a conditional genetic approach to examine the role of p75ntr in myeloid cells by crossing the p75ntr floxed mouse12 with available transgenics with cre expression in myeloid cells to produce myeloid cell-specific deletion of p75ntr (Csfr1cre/ERT aka CD115creER). These mice will be given tamoxifen or vehicle prior to CCI TBIs to reduce p75ntr expression in peripheral myeloid cells, and we will compare the effects on the differentiation of mono/MAC lineage cells in the circulation and their trafficking to the injured brain. Together, these studies focus on peripheral effects of p75ntr signaling after TBI & test the novel hypothesis that p75ntr signaling in peripheral myeloid cells is an important part of the post-TBI injury/repair cascade.