Role of the pro-inflammatory cytokines IL-12 and IL-23 in secondary damage after spinal cord injury

本研究的主要目的是探讨和调节脊髓损伤后的炎症反应， 损伤（SCI），以最大限度地减少组织损伤并促进允许愈合的环境 和修复。SCI在平民和军人中都是一个严重的问题，任何有助于 这些可怕的伤害的改善可以对那些生活质量和数量产生深远的影响， 受影响的患者和家庭。 我们建议评估SCI后促炎细胞因子IL-12和IL-23的作用， 缺乏或抑制IL-12和IL-23在减少组织损伤和促进功能恢复方面的作用。的 SCI后的原发性组织损伤是由创伤本身引起的，继发性损伤是由随后的创伤引起的。 事件，包括但不限于出血、炎症、水肿、脂质过氧化和兴奋性毒性。 继发性损伤对病理学有显著影响，从而对功能性损伤的严重性有显著影响。 赤字SCI后的炎症加重并延长。激活的小胶质细胞和血液来源的 巨噬细胞是SCI后损伤组织中的主要免疫细胞类型。红细胞（RBC） 由于创伤引起的出血和吞噬红细胞的巨噬细胞， 获得促炎表型。IL-12 b编码IL-12和IL-23共有的p40亚基， 通过RBC吞噬作用强烈上调。IL-12和IL-23两者都是适应性和非适应性免疫系统的主调节因子。 先天免疫系统，由多种细胞类型表达，启动和维持免疫 通过诱导促炎细胞因子的产生以自分泌或旁分泌方式应答， 调节炎症细胞反应。因此，它们可能是SCI后炎症的关键目标。我们 初步数据显示SCI后脊髓组织中IL-12 p40的上调和IL-12 p40的更好恢复。 12 p40缺陷小鼠。 因此，我们提出三个目的来研究IL-12和IL-23在SCI后的作用：（1）我们建议， 从mRNA和蛋白质水平分析SCI后小鼠IL-12和IL-23及其受体的表达， 水平，并鉴定表达IL-12、IL-23及其受体的细胞类型。我们将使用早期MRI 测量以量化出血，通过测量组织中的血红蛋白来确认这一点，并将 IL-12和IL-23表达水平。(2)我们建议评估IL-12的功能作用， 通过比较IL-12 p40、IL-23 p19和IL-12 RB 2敲除小鼠与野生型对照，观察SCI后IL-23的表达。使用这些 敲除小鼠将使我们能够区分IL-12和IL-23效应之间的差异。(3)我们将使用后SCI 用IL-12/IL-23小分子抑制剂和IL-12 p40中和抗体治疗。实验将 使用中度挫伤模型（IH撞击器装置）进行， 将对IL-12和/或IL-23的缺乏或抑制进行功能性评估（BMS评分、阶梯步行、跑步机 通过MRI、组织病理学和流式细胞术检测继发性炎症的程度。 损伤以及炎性细胞的组成和表型的变化。RNAseq分析将帮助我们 更好地了解细胞因子和趋化因子，信号分子，生长因子和 继发性损伤的调节剂，包括活性氧产生和脂质代谢的调节剂， 过氧化作用蛋白质印迹的信号转导途径将提供深入了解的机制， 菌株之间的差异是介导的。 这项研究将为SCI后IL-12和IL-23的作用提供深入见解。使用MRI评估组织 用小分子或抗体治疗增加了翻译价值。这些实验 可能导致一种新的治疗方法的发展，以减少炎症介导的继发性组织 损伤后促进功能恢复。

The main objective of this study is to investigate and modulate the inflammatory response after spinal cord injury (SCI), in order to minimize tissue damage and to promote an environment that is permissive for healing and repair. SCI is a significant problem in both civilian and military populations, and any strategies that facilitate improvement in these terrible injuries can have far-reaching effects on quality and quantity of life for those patients and families affected. We propose to assess the role of the pro-inflammatory cytokines IL-12 and IL-23 after SCI and the effects of absence or inhibition of IL-12 and IL-23 in reducing tissue damage and promoting functional recovery. The primary tissue damage after SCI occurs from the trauma itself, and secondary damage is caused by subsequent events, including but not limited to hemorrhage, inflammation, edema, lipid peroxidation and excitotoxicity. Secondary damage contributes significantly to the pathology and thereby to the severity of the functional deficits. Inflammation after SCI is exacerbated and prolonged. Activated microglia and blood-derived macrophages are among the main immune cell types in the injured tissue after SCI. Red blood cells (RBCs) are present at the site of SCI due to trauma-induced hemorrhage and macrophages that phagocytose RBCs acquire a pro-inflammatory phenotype. Il12b, which is coding for the shared p40 subunit of IL-12 and IL-23, is strongly upregulated by RBC phagocytosis. Both IL-12 and IL-23 are master regulators of the adaptive and innate immune system which are expressed by a variety of cell types, and initiate and maintain immune responses in an autocrine or paracrine manner by inducing the production of pro-inflammatory cytokines and regulating inflammatory cell responses. They may therefore be a critical target in post-SCI inflammation. Our preliminary data show an upregulation of IL-12p40 in spinal cord tissue after SCI and better recovery in IL- 12p40 deficient mice. We therefore propose three aims to study the role of IL-12 and IL-23 after SCI: (1) We propose to characterize the expression of IL-12 and IL-23 and their receptors after SCI in mice at the mRNA and protein level and to identify the cell types that express IL-12, IL-23 and their receptors. We will use early MRI measurement to quantify hemorrhage, confirm this by measuring hemoglobin in the tissue, and correlate hemorrhage with IL-12 and IL-23 expression levels. (2) We propose to assess the functional role of IL-12 and IL-23 after SCI by comparing IL-12p40, IL-23p19 and IL-12RB2 knockout mice to wild type controls. Using these knockout mice will allow us to distinguish differences between IL-12 and IL-23 effects. (3) We will use post-SCI treatment with an IL-12/IL-23 small molecule inhibitor and an IL-12p40 neutralizing antibody. Experiments will be done using the contusion injury model (IH Impactor device) with a moderate contusion injury and the effects of absence or inhibition of IL-12 and/ or IL-23 will be assessed functionally (BMS scoring, ladder walk, treadmill analysis, sensory testing) and by MRI, histopathology and flow cytometry to detect the extent of secondary damage, and changes in the composition and phenotype of inflammatory cells. RNAseq analysis will help us gain better understanding on expression of cytokines and chemokines, signaling molecules, growth factors and modulators of the secondary damage, including regulators of reactive oxygen species production and lipid peroxidation. Western blots of signal transduction pathways will provide insight into mechanisms by which differences between the strains are mediated. This study will provide insights into the role of IL-12 and IL-23 after SCI. The use of MRI to assess tissue damage and the treatment with small molecules or antibodies adds translational value. These experiments could lead to the development of a novel treatment approach to reduce inflammation mediated secondary tissue damage and promote functional recovery after SCI.