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

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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.