UROP Research Mentor Project Submission Portal: Submission #756

Overall Research Project Description Spinal cord injury (SCI) is a medical condition resulting in morbidity and permanent disability that can be caused by a major trauma directly to the spinal cord or vertebrae, degenerative disease or spinal infection. SCI arises from the disruption of nerve axons running through the spinal cord which leads to the loss of sensory and motor function. Following the initial trauma, a cascade of secondary events will transpire, including excessive proinflammatory response that includes the activation of CNS resident microglia to pro-inflammatory M1 phenotype, that release inflammatory cytokines, chemokines, and reactive oxygen species (ROS), contributing to further neuronal death and tissue damage. M1 phenotype is dominated by NF-kB that increases phosphodiesterase-4 (PDE4) expression, which rapidly hydrolyses cAMP. cAMP is the main regulator maintaining innate immune cell homeostasis and preventing the M1 phenotype, especially in the presence of anti-inflammatory interleukin 4 (IL4). Elevated cAMP and IL4 levels activate the anti-inflammatory M2 phenotype, that secrete factors associated with tissue repair, wound healing, and neuroprotection. Stimulating intracellular cAMP and IL4 activity remarkably promotes neuronal survival, regeneration, and enhances intrinsic axonal growth.
We propose to investigate the possibility of using engineered extracellular vesicles for therapeutic conversion that could be utilized in SCI. In vitro and in vivo experimental SCI models have shown that combining cAMP elevation with anti-inflammatory cytokines, such as IL4, phenotypically converts pro-inflammatory M1 microglia to M2, which enhances peripheral nerve recovery and regeneration, suppresses inflammation, and stimulates tissue repair and wound healing.
In the experimental set-up, mouse IL4 will be expressed as recombinant murine IL4-R9 in E. coli BL21(DE3) cells and purified by column chromatography. Purified IL4 will be analyzed by gel electrophoresis (SDS-PAGE and native PAGE) and circular dichroism (CD) spectroscopy. Purified IL4-R9 will be loaded into the MSCEVs and verified by Cys5-labelling, and immunostaining with Alexa Fluor 488-labeled anti-IL4 antibody, using TIRF and confocal microscopy.
Mouse microglia cells will be cultured in appropriate conditions and microglial classic activation will be achieved by LPS or IFN-γ induction, after which the expression of pro-inflammatory markers (iNOS, COX-2, IL-1β, TNF-α) is determined in cell lysates by Western blot or ELISA analysis.