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

促炎细胞因子IL-12和IL-23在脊髓损伤后继发性损伤中的作用

• 批准号: 10427136
• 负责人: Karin Swartz
• 金额: --
• 依托单位: CLEMENT J. ZABLOCKI VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10427136
• 关键词: Adaptive Immune System; Affect; Anti-Inflammatory Agents; Antibodies; Apoptosis; Blood; Cells; Characteristics; Code; Contusions; Data; Demyelinations; Deposition; Development; Devices; Edema; Environment; Enzyme-Linked Immunosorbent Assay; Erythrocytes; Event; Family; Flow Cytometry; Goals; Growth Factor; Hemoglobin; Hemorrhage; Histologic; Histopathology; Immune; Immune response; Immunofluorescence Immunologic; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Injury; Innate Immune System; Interleukin-12; Intervention; Knockout Mice; Lead; Lesion; Life; Lipid Peroxidation; Locomotor Recovery; Lymphocyte; Magnetic Resonance Imaging; Measurement; Measures; Mediating; Messenger RNA; Microglia; Military Personnel; Modeling; Mouse Strains; Mus; Oral; Outcome; Pathology; Patients; Phagocytosis; Pharmacology; Phenotype; Play; Production; Proteins; Reactive Oxygen Species; Recovery; Recovery of Function; Role; Secondary to; Sensory; Severities; Signal Transduction; Signal Transduction Pathway; Signaling Molecule; Site; Soldier; Spinal Cord; Spinal Cord Contusions; Spinal Cord Lesions; Spinal cord injury; Testing; Time; Tissues; Trauma; Up-Regulation; Walking; Western Blotting; Wild Type Mouse; autocrine; behavior test; behavioral outcome; cell type; chemokine; combat zone; cytokine; disability; excitotoxicity; experimental study; functional loss; healing; high risk; immune activation; improved; inhibitor; injured; insight; interleukin-23; macrophage; military veteran; neuron loss; neutralizing antibody; neutrophil; novel; paracrine; receptor; repaired; response; small molecule; small molecule inhibitor; tool; transcription factor; transcriptome sequencing; translational approach; treadmill

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.

这项研究的主要目的是调查和调节脊髓后的炎症反应 伤害（SCI），以最大程度地减少组织损伤并促进允许治愈的环境 和维修。 SCI在平民和军事人群中都是一个重大问题，也是任何有助于 这些可怕的伤害的改善可能对那些人的质量和数量产生深远的影响 患者和家庭受到影响。 我们建议评估SCI后促炎性细胞因子IL-12和IL-23的作用，以及效果 在减少组织损伤和促进功能恢复时缺乏或抑制IL-12和IL-23。这 SCI后的主要组织损伤是由创伤本身发生的，次要损害是由随后引起的 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。 次要损害对病理产生了重大贡献，从而对功能的严重程度产生了重大贡献 缺陷。 SCI后的炎症加剧并延长。活化的小胶质细胞和血液来源 巨噬细胞是SCI后受伤组织中的主要免疫细胞类型之一。红细胞（RBC） 由于创伤引起的出血和巨噬细胞，存在于SCI的位置 获得促炎的表型。 IL12B正在编码IL-12和IL-23的共享P40亚基 RBC吞噬作用强烈上调。 IL-12和IL-23都是适应性的主要监管机构， 由多种细胞类型表达的先天免疫系统，并启动和维持免疫 通过诱导促炎性细胞因子的产生和 调节炎症细胞反应。因此，它们可能是SCI后炎症的关键目标。我们的 初步数据表明，SCI后IL-12P40的IL-12P40上调，并且在IL-中更好地恢复了IL- 12p40不足的小鼠。 因此，我们提出了三个目标，以研究SCI后IL-12和IL-23的作用：（1）我们建议 表征小鼠在mRNA和蛋白质中SCI后IL-12和IL-23及其受体的表达 水平并确定表达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，并相关 IL-12和IL-23表达水平的出血。 （2）我们建议评估IL-12和 SCI后IL-23通过比较IL-12P40，IL-23P19和IL-12RB2基因敲除小鼠与野生型对照。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-12P40中和抗体处理。实验会 使用中等挫伤损伤的挫伤损伤模型（IH撞击器设备）完成 IL-12和/或IL-23的缺失或抑制作用将通过功能评估（BMS得分，梯子步行，跑步机 分析，感官测试）和MRI，组织病理学和流式细胞术以检测次级的程度 损害，以及炎症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 次要损伤的调节剂，包括活性氧的调节剂和脂质的调节剂 过氧化。信号转导途径的蛋白质印迹将提供有关机制的洞察力 菌株之间的差异是介导的。 这项研究将为SCI后IL-12和IL-23的作用提供见解。使用MRI评估组织 损伤和小分子或抗体的处理增加了翻译价值。这些实验 可能导致开发一种新的治疗方法，以减少炎症介导的二次组织 SCI后损坏并促进功能恢复。