Interakcija Th-17 imunskog odgovora i glutamatske ekscitotoksičnosti u patogenezi multiple skleroze : klinička i eksperimentalna studija
Interaction Between Th-17 Immune Response and Glutamate Excitotoxicity in the Pathogenesis of Multiple Sclerosis – Clinical and Experimental Study
AuthorKostić, Miloš S.
Committee membersStojanović, Ivana
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Introduction: Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS) characterized by episodic neurological defects, disseminated in time and neuroanatomical localization. The central role in the development of inflammatory mediated lesions in MS is now attributed to autoreactive, myelin-specific CD4+ T lymphocytes, which are thought to regulate all immune mechanisms that lead to the myelin sheath damage. It was originally thought that these cells belong to the subpopulation of Th-1 cells, however, a number of nowadays studies indicate the importance Th-17 cells in the pathogenesis of the disease. In addition to demyelination, which is still considered to be the main pathological feature of the disease, in the last few years special attention in MS research has been given to neurodegenerative processes, considering that it has been shown that the clinical symptoms of the disease are directly related to the degree of axonal damage and loss. Recent...ly, glutamate excitotoxicity is considered as a possible mechanism of neurodegeneration in MS. This is a phenomenon that occurs when the excessive amount of excitatory neurotransmitter glutamate, overactivates its receptors on the membranes of neurons and oligodendrocytes, which leads to the intracellular accumulation of Ca2+ ions and the consequent cell death. Aims of the Thesis: To examine the association of Th-17 mediated inflammation and glutamate excitotoxicity in the development of MS. To determine the potential molecular mechanisms by which inflammation stimulates glutamate excitotoxicity and the consequent neurodegeneration with special emphasis on the role of astrocytes. Materials and Methods: The study had two integral parts: a clinical part conducted on the human material and experimental part conducted on the primary cultures of rat astrocytes. The clinical study enrolled 79 patients, divided into two groups: a control group and a group of MS patients. The presence of a Th-17 cells in the CNS of the studied subjects was monitored by IL-17A levels in the cerebrospinal fluid (CSF) determined by ELISA (Enzyme Immunosorbent Assay) method; while glutamate level was used to estimate excitotoxic damage by HPLC (High Performance Liquid Chromatography) method. The experimental Тh-17 имунски одговор и глутаматска ексцитотоксичност у патогенези МС др Милош Костић 10 study was carried out on the primary cultures of rat astrocytes. In order to investigate the effects of IL-17A on astrocyte ability to uptake and metabolize glutamate, cell cultures were stimulated with increasing concentrations of recombinant rat IL-17A protein, and then used for gene expression analysis of glutamate transporter (GLAST and GLT-1) and the enzyme glutamine synthetase, by PCR (Polymerase Chain Reaction). Investigation of IL-17A effect on astrocyte ability to release glutamate, was conducted in basal conditions and under conditions of elevated and reduced extracellular Ca2+. Astrocyte cultures were stimulated with the rising concentrations of recombinant rat IL-17A protein, and then glutamate secretion was followed up by the level of glutamate in the cell supernatants by HPLC. Results: Patients suffering from MS had a significantly higher level of IL-17A in the CSF compared to the control subjects (P <0.005), which was directly related to the level of glutamate (rs = 0.368, P <0.05). The level of IL-17A was also directly related to the number of neutrophils in the CSF (rs = 0.426, P <0.05) and blood-brain barrier disruption (rs = 0.440, P <0.05), whereas a negative correlation between CSF IL-17A level and disease duration was observed (rs = -0.466, P <0.01). In the experimental study it has been shown that IL-17A in lower concentrations (10, 25, 50 ng/mL) reduces glutamate transporter and glutamine synthetase gene expression in astrocytes, however, this effect was lost when IL-17A was used in higher dose (100 ng/mL). IL-17A did not significantly modify glutamate secretion in basal conditions, but following Ca2+ stimulation, and after Ca2+ removal from culture medium, IL-17A stimulated glutamate release from astrocytes in dose-dependent manner. Conclusions: IL-17A plays an important role in the complex cytokine network that mediates MS pathogenesis. For the first time direct relationship between Th-17 immune response and glutamate excitotoxicity is reported, which could be a missing link between inflammatory and neurodegenerative processes in MS. Three mechanisms by which IL-17A stimulates extracellular glutamate increase to excitotoxic levels are postulated. Firstly, IL-17A reduces astrocyte ability to uptake and metabolize glutamate and in addition IL-17A also stimulates Ca2+ dependent glutamate release from astrocytes.
Faculty:University of Niš, Faculty of Medicine
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