Effect And Elementary Study Of Mechanism Of Kynurenine On Humoral Immune Response

There is a seriously effect of autoimmune diseases on human health. The data showed that the incidence of autoimmune diseases is gradually trend to increase. However, there are many side effects of the currently-used therapeutic strategies, including immunosuppressive agents, cytokines, and TNF-a antagonists, such as causing tumors, infection, as well as delayed dysfunction, and especially reduced targeting effectiveness. Thus, a novel strategy is required to improve therapeutic effects. Vaccination against autoimmune diseases is a feasible strategy because vaccines induce immune response memory and the antigen specificity. Recovering tolerance or down-regulating the immune response to non-harmful antigens is a better ideal way to treat autoimmune diseases through administrating a mixture of special antigen with adjuvant that decreases the strength of the immune response. Recent medical advances have included determining the self-antigens for various autoimmune diseases. However, no suitable adjuvant is available to direct the immune response toward tolerance or suppression. Thus, it is difficult to find novel tolerogenic/suppressive adjuvants and to understand their biological mechanisms of action for a successful new therapeutic strategy.Kynurenine (Kyn), an endogenous substance, is safer than exogenous substances when considered as a vaccine adjuvant. In this thesis, firstly, we evaluated whether Kyn could serve as a novel tolerogenic or suppressive adjuvant to decrease the humoral immune responses against hepatitis A virus (HAV) in the ICR mouse model in vivo. The data showed that Kyn significantly decreased HAV immunogenicity when co-administered with HAV and that the effect appeared throughout the test period. B cells play a key role in the humoral immune response. In addition to the experimental animal model, B cell terminal differentiation also can be studied successfully in vitro, because B cells are capable of both immunoglobulin class switch recombination and differentiation toward antibody-secreting plasma cells in response to a TD stimulus or a T1-related signal such as lipopolysaccharide (LPS). B cell responses in vitro thus provide a controlled system to investigate the humoral immune response on both a cellular and molecular level. Subsequently, the function of Kyn as a novel suppressive adjuvant in vitro was evaluated by stimulating B cells with the mixture of Kyn and LPS. The results showed that Kyn (100μM/1000μM) impaired IgM generation compared to that induced by LPS alone. These findings suggest that Kyn is able to attenuate the antigenic immunogenicity, and that can serve as a novel and effective suppressive adjuvant for vaccines.MicroRNAs play important and broad roles in a wide variety of biological processes, including embryogenesis, carcinogenesis, proliferation and apoptosis, differentiation and signal transduction. The interaction between microRNA and its target gene is a regulation of gene expression except for ’Central Dogma of Molecular Biology’. It has been estimated that30-60%of the genes in a given genome are regulated in this manner. Furthermore, several microRNAs were identified involving in the progress of B cell terminal differentiation. We provide the hypothesis that some microRNA involves in the progress of Kyn-mediated suppressive humoral immune responses induced by LPS. In this study, we analyzed the contribution of microRNAs to the process of humoral immune responses suppressed by Kyn, and found that, in the presence of Kyn, the level of microRNA30b (miR30b) was significantly decreased compared to that in the corresponding control group. We also demonstrated that miR30b played a critical role in the process of Kyn-mediated suppression of IgM responses induced by LPS.In the RNA-induced silencing complex, microRNA down-regulates the expression of the target gene either by direct degradation or destabilization and eventually suppresses translation of the target through binding to the target sequence in the3’UTR via the2-7nucleotide seed region. Our results demonstrated that human Bach2and human Foxo3were novel targets of miR30b through Dual-Luciferase Reporter Assay System. Next, the results were further strengthened by reducing the protein levels of human Bach2and human Foxo3by miR30b in normal human B cells through Western blot assay. At same time, we also demonstrated that there are effects of miR30b on the protein levels of human Bach2and human Foxo3in the process of Kyn-mediated suppression of IgM responses induced by LPS.Bach2, a transcriptional repressor, is well known for the regulation of B cell terminal differentiation. Foxo3is one of the members of the FOXO transcription factor family, exists in a variety of cells such as T cell, B cell and dendritic cell, and plays a role in many physiological processes, including cell survival, proliferation, apoptosis, metabolism, emergency response, aging, maintaining homeostasis and immune response. Since there is no commercial human Foxo3 protein, we successfully constructed recombination plasmid of pcDNA3.1(+)-human Foxo3, and evaluated that the effect of Foxo3on the process of IgM responses induced by LPS in normal human B cells treated with Kyn or miR30b on the basis of it. The data of ELISA showed that human Foxo3plays an important role in the process of miR30b-mediated humarol immune responses.In conclusion, our data demonstrate that Kyn can suppress the humoral immune responses induced by special antigen and attenuate antigenic immunogenicity. In B cells in vitro, miR30b plays an important role in Kyn-mediated suppression of IgM responses induced by LPS. Bach2and Foxo3were novel targets of miR30b. In addition, we also successfully constructed the recombination plasmid of pcDNA3.1(+)-human Foxo3, and demonstrate that Foxo3plays an important role in the process of miR30b-mediated humarol immune responses. These findings suggest that Kyn can serve as a novel and effective suppressive adjuvant for vaccines.