The Study Of TLRs Expression In Tumor And The Relationship Between TLRs And Tumor Tolerance

TLRs Play critical roles in the innate recognition of Pathogen-assoeiated molecule Pattems (PAMPs) and activation of acquired immunity. Innate immune cells recognize PAMPs conserved in microbes by TLRs to initiate host immune response against infectious pathogens. The family of TLRs senses conserved strueture found in abroad range of Pathogens, causing innate immune responses that include the Production of inflammatory cytokines, chemokines and interferons. Up to now, more than 10 human TLRs have been discovered, and are conserved between human and mouse. TLRs are thought to be restricted to immune cells, such as dendritic cells (DC), macrophages, T and B lymphocytes etc, which initiate innate and adaptive immune responses through producing cytokines, increasing expression of cell adhesion molecules, and secreting proinflammatory mediators. While optimal production of the proinflammatory cytokines is necessary for eliminating invading pathogens, uncontrolled TLR activation might result in the immunopathological injury to host. One of the most severe conditions is LPS-induced endotoxin shock, in which lethal amount of proinflammatory cytokines are produced and damage soft issues indueed upon LPS challenge. Another issue is the pathogensis of autoimmune diseases. So, the control of TLR signaling is very important for the immune system.In the past years, the studies of TLRs focused on TLRs in the tumor immunity. Including the adjuvant which can enhance the host immunity and recognize the"dangerous signal"that created by tumor cells. Furthermore, in the process of research in the immune system, people found that the TLRs can be expressed by tumor cells, and play important role in the tumor pathonesis. For example, the human breast tumor cell line Cama-1 and melanoma cell line Me260 can both express the TLR3 which can promote the apoptosis of tumor. The TLR4 expressed by human colon tumor cell line MC26 can promote the tolerance of tumor cells. In the mouse breast tumor cell line D2F2, the TLR5 can promote the progression of tumor in the early stage but repress the tumor develop in the late stage. So, the TLR expressed by tumor cells may play important role in the tumor pathonesis. And this may be a new pathway to cure the cancer patient. Our study shows the expression of TLRs in the mouse Lewis lung cancer cell and ascetic 180 cell, to test the expression of TLR in tumor cells and the mechanisms of the promotion of tolerance of tumor cells by the TLRs expressed by tumor cells. In our study: 1. we established the Lewis lung cancer models and ascitic S180 models, separate pure tumor cell with the method of density gradient centrifugation. analyzed the expression of TLR1-9 mRNA by semi-quantitative RT-PCR, and compare the tumor either cultured in vitro or grew in mice. 2. We stimulate the cultured ascitic S180 cells by LPS, and to study the mechanisms of the promotion of tolerance of tumor cells by the TLR4 expressed by tumor cells.Part 1: The expression of TLRs in the cells of Lewis lung cancer and ascitic S180 in vitro and in vivo.The Lewis lung cancer models were established by injected subcutaneously to the right axilla of C57BL/6 mice with subculturing Lewis lung cancer cells. The ascitic S180 models were established by injected subcutaneously to the left abdominal of ICR mice with subculturing ascitic S180 cells. And then separate pure tumor cell with the method of density gradient centrifugation. The expression of TLR1-9 mRNA were analyzed by semi-quantitative RT-PCR. The result shows that:In the group of Lewis lung cancer, TLR1, 5, 6 expressed more in vitro than in vivo; TLR3, 4, 9 show no difference; TLR2, 7 expressed more in vivo; TLR8 neither expressed in vitro nor in vivo. In the group of ascitic S180, TLR1, 2, 5, 6, 9 expressed more in vitro than in vivo; TLR7 expressed more in vivo;TLR3, 8 neither expressed in vitro nor in vivo. In the different stage of cancer, TLR4, 7, 9 expressed more in early stage than late stage. TLR1, 2, 3, 5, 6 expressed more in late stage. TLR8 shows no expression.Conclusion: TLRs are expressed on the Lewis lung cancer cell and ascetic 180 cell, and there are some differences between the two cell lines. Further more, it is also different in vitro and in vivo. So it is in different stage of tumor. Part 2: Mechanisms of the promotion of tumor tolerance by the TLR4 expressed on tumor cells.To study the mechanisms of the promotion of tumor tolerance by the TLR4 expressed on S180 cells, we stimulate the S180 cells 12h with LPS in different concentration ( 0ug/ml, 0.1ug/ml, 1ug/ml, 10ug/ml ). We test the expression of TLR4 on the tumor cells by FACS, the expression of Foxp3, TGF-βand IL-10 by semi-quantitative RT-PCR, and TGF-βprotein by ELISA. And then we Stimulate the S180 cells with 10ug/ml LPS for different time ( 12h, 24h, 36h, 48h ). We test the expression of TLR4 on the tumor cells by FACS, the expression of Foxp3, TGF-βand IL-10 by semi-quantitative RT-PCR, and TGF-βprotein by ELISA. The result shows that:In the Stimulation with LPS in different concentration, TLR4 protein shows no change, the Foxp3 mRNA increase in the concentration of 10ug/ml, the TGF-βand IL-10 mRNA increase with the concentration of LPS. But the TGF-βprotein remains. In the Stimulation with LPS in different time, TLR4 protein shows no change too, the Foxp3 mRNA increase all the time, but there are no difference at the four time point, the TGF-βand IL-10 mRNA increase with the time goes. The TGF-βprotein increase with time too.Conclusion: The activation of TLR4 can promote the expression of Foxp3, TGF-βand IL-10, and then enhance the tolerance of tumor cells.