| Multiple sclerosis (MS) is a chronic autoimmune and/or inflammatory disease of central nervous system (CNS) in which the body’s own immune system attacks and damages the myelin sheaths around the axons of the brain and spinal cord, leading to demyelization and inflammatory scarring as well as a broad spectrum of signs and symptoms of CNS. Its complex pathogenesis is still not fully understood. Non-selective immune inhibition therapies such as steroid hormone and interferon could incur intolerable adverse effects. So far, there is no known cure for MS yet because the current MS medications have adverse effects that can be poorly tolerated. Therefore, compounds with higher selectivity for MS therapy have been emerged. Experimental autoimmune encephalomyelitis (EAE) is also the prototype for T-cell-mediated autoimmune disease in general, suggesting that EAE has similar mechanisms to MS in humans. It is an animal model of mimicking an inflammatory demyelization process of CNS and widely studied as a most reliable model. The major functions of T cells include T cell activation, differentiation to its subtypes as well as migration and infiltration toward local diseased foci from peripheral lymph organs.My research efforts have been mainly focused on finding small molecules which have displayed therapeutic advantages in EAE model through a selective T cell function modulation. The detailed mechanisms on T cell function modulation and their selectivity have been explicitly explored. Through my research work, not only the regimens with advantages of fewer and less severe adverse effects, but also multiple new targets opportunities among MS disease pathogeneses possibly involved have been revealed for providing better therapies and for providing more accurate MS diagnosis and prognosis.In the first chapter of my research work, the major work started on the screening process to find out a small molecule compound which has a selective inhibitory effect on IFNy productions of T cells without significantly affecting their proliferations. By this way, the decreased IFNy production would only benefit MS without affecting the proportion of modulatory T cells in the entire T cell population and would not disturb the balance of entire body immune system functions at the same time. Fcll is an orally available small molecule compound that we have found through a screening process with such advantages. Fell showed significant therapeutic effects on EAE model in mice. Meanwhile Fell displayed matched effects found both in the EAE model and the in vitro assay without affecting the modulatory T cell population proportion in the entire T cell population. The further research has found that Fell significantly inhibited the IFNy signal transduction pathway selectively without affecting the protein synthesis and degradation and SHP-2 activity in the IFNy signal transduction pathway. The endocytosis of IFNy molecule on the CD4+T cell surface was promoted by Fcll. In vitro kinase assay results showed that Fcll inhibited JAK2 significantly in a dose dependent manner, indicating that the increased endocytosis by Fcll is through JAK2 inhibition to further block the IFNy/STAT1 signal transduction process.The second part of my research proposed a therapeutic method targeting migration of effector T cell into uninflamed CNS. My research work has shown that NSC 87877 at a very low dose,2.5 mg/kg ip completely abolished the incidence of EAE in a mouse EAE model. The therapeutic effects of NSC 87877 on EAE are so dramatic and therefore,worth efforts to further explore its mechanisms for its novelty and therapeutic advantages which have not been seen in the literature up to now. My research has found that NSC 87877 did not affect T cell functions at all both in vitro and in vivo and that NSC 87877 treatment neither affected T cell activation nor differentiation, indicating that the therapeutic effects of NSC 87877 on EAE were not mediated by T cell inhibition. Our results has shown that the mice with selective SHP-2 knock-out had similar vulnerability to EAE compared to the wild type and that SHP-2 knock-out did not affect the differentiation of T-cell in vitro indicating that the therapeutic effects NSC 87877 on EAE were not through SHP-2 inhibition in T cells. By investigating every steps of EAE development stages that we could understand and by examining all related immune cell in EAE, we have found that NSC 87877’s therapeutic effects on EAE were by affecting the effector T cell differentiation process during the migration of effector T cells toward CNS. The inflammatory infiltration of pioneer effector T cells toward uninflamed CNS is an important step for the development of EAE in mice as well as for MS in humans. However, the research on this aspect is currently rare and targeting this path for EAE is a brand new direction for EAE medicine research and development. Nevertheless, our research has found that the effector T cells from NSC 87877 treated EAE model mice had a similar immune response to MOG re-stimulation in the effector T cells from untreated EAE model mice, but not EAE pathogenic. We have found that NSC 87877 did not have effects on chemokines expression stimulated by cytokines in CNS, indicating that NSC 87877’s therapeutic effects are via inhibiting infiltration of effector T cells into uninflamed CNS. By examining levels of gene expressions of effector T cells of EAE treated with NSC 87877, we have found that there were increased expressions of CCR.7, which promotes the homing of effector T cells; that there was increased CXCR7 expression on CD8+T cell surfaces. The increased CXCR7 expression of CD8+T cell could inhibit the chemotactic effects of CXCR4 by competing with its endogenous ligand SDF-1α. Taken the evidence together, we believe that the raised expression of CCR7 on the CD8+ T cells by NSC 87877 could be due to increased PGE2 secretions from CD8+ T cells, suggesting the possibility of PGE2’s therapeutic roles to EAE. Meanwhile, NSC 87877 inhibited the effects of SDF-la/CXCR4 on chemotaxis of effector T cells through up-regulating expressions of CXCR7 in the CD8+ T cells, in a competitive manner and consequently reduced the incident rate of EAE in mice.In summary, my research work all above has proposed and validated two strategic therapeutic pathways on EAE based on selective inhibiton of T cell functions.First, by inhibiting effector T cell IFNγ production without affecting its proliferation to justify disturbed T cell functions of EAE such as what Fcll has displayed in my first part of research work. The advantage of selective effector T cell function inhibition over CsA traditional therapy on EAE is to avoid intolerable adverse effects due to imbalanced immune functions of T cell sub-populations caused by CsA therapy on MS clinically since Fcll’s selectivity was through the inhibition of JAK2 to promote the endocytosis of IFNyR. In another aspect, we have also found that SHP-2 inhibitor NSC 87877 completely abolished the incidence of EAE in mice through selectively inhibiting infiltration of effector T cells into uninflamed CNS. The expression of chemokine receptors were involved in its mechanism. These two strategic regimen researches have provided mechanisms and theories for more effective MS therapies with possible less intolerable side effects from current available MS therapies. |
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