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研究和调节脊髓损伤后的炎症反应。 损伤(SCI)，以将组织损伤降至最低，并促进允许愈合的环境 并进行修复。脊髓损伤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这些人的生活质量和数量产生深远的影响 受影响的患者和家属。 我们建议评估促炎细胞因子IL-12和IL-23在脊髓损伤后的作用和影响 IL-12和IL-23的缺失或抑制在减少组织损伤和促进功能恢复方面的作用。这个 脊髓损伤后的原发组织损伤是由创伤本身造成的，继发性损伤是由继发性损伤引起的 事件，包括但不限于出血、炎症、浮肿、脂质过氧化和兴奋性毒性。 继发性损伤对病理和功能损害的严重性有显著影响。 赤字。脊髓损伤后的炎症反应加剧并延长。活化的小胶质细胞与血源性 巨噬细胞是脊髓损伤后损伤组织中的主要免疫细胞类型。红细胞(RBC) 存在于创伤出血所致的脊髓损伤部位，以及吞噬红细胞的巨噬细胞 获得一种促炎表型。IL12b编码IL-12和IL-23的共享p40亚基，是 被红细胞吞噬作用强烈上调。IL-12和IL-23都是适应性和 先天免疫系统，由各种类型的细胞表达，并启动和维持免疫 以自分泌或旁分泌的方式通过诱导促炎细胞因子和 调节炎症细胞反应。因此，它们可能是脊髓损伤后炎症反应的关键靶点。我们的 初步数据显示，脊髓损伤后脊髓组织中IL-12p40表达上调，IL-12p40恢复较好 12p40缺陷小鼠。 因此，我们提出了三个目标来研究IL-12和IL-23在脊髓损伤后的作用：(1)我们建议 小鼠脊髓损伤后IL-12、IL-23及其受体在mRNA和蛋白水平的表达 并鉴定表达IL-12、IL-23及其受体的细胞类型。我们将使用早期核磁共振 量化出血的测量，通过测量组织中的血红蛋白来确认这一点，并相互关联 出血与IL-12、IL-23表达水平相关。(2)我们建议评估IL-12和 比较IL-12p40、IL-23p19和IL-12RB2基因敲除小鼠与野生型对照小鼠脊髓损伤后IL-23的变化。使用这些 基因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)使用后SCI 用IL-12/IL-23小分子抑制剂和IL-12p40中和抗体治疗。实验将会 采用挫伤模型(IH冲击器装置)制作中度挫伤模型并观察其影响 将对IL-12和/或IL-23缺失或抑制进行功能评估(BMS评分、阶梯行走、跑步机 分析、感官测试)，并通过MRI、组织病理学和流式细胞术检测继发性 损伤，炎性细胞成分和表型的变化。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 二次损伤的调节剂，包括活性氧产生的调节剂和脂质调节剂 过氧化。信号转导通路的蛋白质印迹将提供对 菌株之间的差异是通过中介实现的。 本研究将为探讨IL-12和IL-23在脊髓损伤后的作用提供依据。磁共振成像技术在组织评估中的应用 损伤和小分子或抗体的治疗增加了翻译价值。这些实验 可能导致开发一种新的治疗方法来减少炎症介导的继发性组织 损伤并促进脊髓损伤后的功能恢复。

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.