Enhancing therapeutic potential of olfactory ensheathing cells in spinal cord injury by phagocytosis

Under normal physiological condition the body needs to fight bacterial infection, clear old dead or dying cells as well as regenerate new ones. Phagocytosis is the immunological process required for cells to digest and destroy particulates thereby maintaining a systemic homeostasis. Phagocytosis involves four steps of recognition, engulfment, formation of phagosome, degradation and elimination. In the nervous system, the phagocytes are the glial cells which support the system framework, primarily microglia. Spinal cord injury is an irreparable nervous system injury and restoration of function is very difficult. One strategy which could aid repair and regeneration is to enhance the phagocytic process thereby removing cell debris and bacteria from the injury site. Glial cells are involved in phagocytosis and clearing the spinal cord injury area. Understanding this process may improve strategies to develop therapy, particularly those that involve cell transplantation such as peripheral nerve glia. Within the nervous system, the main phagocytic glia, which has been studied, in the central nervous system (CNS) are astrocytes and microglia, and in the peripheral nervous system (PNS), the main glia are Schwann cells for most nerves, with olfactory ensheathing cells (OECs) in the olfactory nerve. In this study, my first objective was to compare the CNS and PNS cells like astrocytes, microglia, OECs and trigeminal Schwann cells (TgSCs) for their antimicrobial response to bacterial infection (S. aureus). Results showed live bacteria could be isolated from all glia after 24 h in culture, and microglia, OECs and TgSCs exhibited better protection against intracellular S. aureus survival than astrocytes. All glial types responded to the bacteria by cytokine secretion, but overall, OECs secreted the lowest level of cytokines. Debris clearance is crucial for neural regeneration and OECs have the ability to phagocytose both bacteria and cell debris. OECs and SCs are being considered as cell transplantation therapies in spinal cord injury for the purpose of repairing the damaged nervous system. Thus, understanding the genetic expression in between OECs and SCs during the process of phagocytosis of bacteria (S. aureus) and axonal debris is essential. Results showed OECs responded to S. aureus with protein modification and phosphorus metabolic processes involved with immune response, and leukocyte mediated immunity signalling mechanisms for bacterial engulfment. SCs responded to cell debris with pathways associated with actin filament-based processes, cytoskeleton organization, and FC gamma receptor dependent phagocytosis. These findings demonstrate the differences between OECs and SCs during phagocytic responses to bacteria and cell debris. Overall, OECs expressed a low gene expression profile compared to SCs. Discovering novel compounds that can stimulate OECs to phagocytose bacteria and cell debris more efficiently may improve the therapeutic potential of the cells. Potentially, if OECs could be pre-stimulated with a drug to enhance OEC phagocytosis, that could be useful in spinal cord injury regeneration. Therefore to identify compounds that would enhance the phagocytosis of glial cells, drug discovery assays were performed. Following a preliminary compound screening, a synthetic compound and a natural compound was identified. For a third compound, a FDA approved drug was selected to test their effects on glial cells. As the process and analyses, required to identify compounds, that stimulate glial cell phagocytic activity has not been previously established, development and optimization of a protocol for phagocytosis detection was performed. Phagocytosis measurement in glial cells based on live imaging and fixed widefield imaging was performed to make a replicable drug discovery testing method which could be used on cell lines and primary cells both. Using this method results like cytotoxicity, morphological changes and phagocytosis of various targets can be reliably performed. Testing the compounds with the protocol, demonstrated that significant stimulation of phagocytosis activity could be detected. All three compounds tested showed positive phagocytosis increase on cell lines. The synthetic compound showed positive increase in phagocytosis on primary OECs and TgSCs but not in astrocytes and microglia for S. aureus pHrodo BioParticles. Testing further, using the synthetic compound, in the presence of bacterial stimulus, showed no significant upregulation in cytokines but some positive upregulation in phagocytotic genes, in the compound stimulated wells against controls. The natural compound showed increase in phagocytosis only in microglia but not in other glial cells (OECs, TgSCs and astrocytes). The FDA compound Liraglutide showed significant increase in phagocytosis in all glial cells (astrocytes, microglia, OECs and TgSCs). Testing the synthetic compound on the phagocytosis of Beta (β) amyloid peptide, in OECs, astrocytes and microglia, showed an increase in peptide uptake in compound stimulated OECs as compared to non-stimulated ones. Astrocyte and microglia did not show any difference. In conclusion, this thesis gives us an understanding of the difference between CNS and PNS phagocytosis. While the different glial cells share many properties, OECs exhibit several characteristics which make them favourable candidates for transplantation therapies. Compound testing revealed that phagocytic activity can be stimulated and opens up avenues for identification of compounds that could be used to enhance therapeutic activity of glial cells in SCI and possibly in Alzheimer’s diseases research.