Roles Of TIM3 In Regulating The Inflammation And Antigen Presentation Of Macrophage

Backgrounds: Tim3, a member of the Tim (T cell immunoglobulin and mucin domain) family , is the first identified to be specifically expressed on CD4+ Th1 but not on Th2 cells. The Tim3 gene encodes a 281-amino acid protein that is expressed on terminally differentiated Th1 cells.This discovery has catalyzed investigation into how Tim3 might negatively regulate Th1 cells and consequently influence the development of organ-specific autoimmunity. A recent study has shown that Tim3 is also constitutively expressed on innate immune cells. By activating innate immune cells, such as dendritic cells (DCs), monocytes, and macrophages, the Tim3 signaling pathway promote a pro-inflammatory response.Objective:To investigate the roles of Tim3 in regulating the activity and phenotype of macrophage. Methods: (1) Recombinant Tim-3 SiRNA vector and control vector were transfected into RAW264.7 cell lines by lipofectamine 2000. After being selected by G418, a RAW264.7 cell lines stably expressing Tim-3 SiRNA were successfully established. RNA interfere technique were used to knock down the Tim3 expression in macrophage cell line Raw264.7. Roles of Tim3 in regulating the activity of macrophage were investigated by ELISA and Flow Cytometry respectively. (2) Tim-3 signaling pathway in macrophage was detected in vitrol by silencing or activating the Tim3 on Raw264.7 cells then detecting TNF-αand IL-6 production by ELISA. Then we examined the role of AKT and NF-kB pathway in Tim-3 mediated macrophage activation by Western Blot. The cross-talk between Tim3 and TLR4 pathway were analyzed by ELISA and Flow cytometry. The activity of NF-kB was detected by Dual-Luciferase reporter gene system. (3) We established sepsis model in C57BL/6 mice and investigated the different role of Tim3 in early and late stage of sepsis. (4) Finally, effects of Tim-3 on adaptive imunity were detected by examing Th1, Th17 differentiation and CD80/CD86 expression in vivo and in vitro.Results: (1) A RAW264.7 cell line silenced of Tim-3 expression was successfully established. (2) To study the regulation of Tim3 in innate immunity, we investigated the role of Tim3 in activiting of RAW264.7 macrophage cell line. The results suggested that Tim3 can promote TNF-αand IL-6 production from macrophage. It also suggested that there is an antagonistic effect between Tim3 and TLR4 pathway in promoting TNF-αproduction from macrophage. Western Blot data showed that the antagonistic effects between Tim3 and TLR4 associated with AKT,NF-kB signaling pathways. We deduced that greater phophorylation of AKT can significantly inhibit the activation of NF-kB, which decreased TNF-αproduction in response to LPS stimulation. (3) In sepsis mouse models, our data shown that the up-regulated expression of Tim3 in early stage of sepsis which mediated pro-inflammatory. However, the expression of Tim3 is deregulated in late sepsis. (4) Oue in vivo and vitro data showed that Tim3 also regulates the expression of the co-stimulatory molecules CD80 and CD86 on macrophage, and thus promotes Th1, Th17 polarization by improving IFN-γand IL-17 production.Conclusions: (1) ELISA data showed that activation of Tim3 pathway enhanced TNF-αand IL-6 secretion from macrophage, which suggest that Tim3 is especially important for regulating innate immune response. (2) Our data suggested that there is an antagonistic effect between Tim3 and TLR4 pathway. (3) The results showed that Tim-3, by increasing CD86 and decreasing CD80, promote Th1,Th17 polarization. (4) Our dates provide new information concerning the role of Tim3 in innate and adaptive immunity, especially provide valuable knowledge about the regulatory roles of Tim3 in macrophage mediated immune response.