Signal Pathway Of TRIM59 Expression And The Regulation Of TRIM59 On Biological Activities Of Macrophages

Macrophages are one of the most important natural immune cells,expressing multiple patterns of pattern recognition receptors,modality receptors,cytokines receptors.Macrophages are activated by various factors such as LPS,IFN,BCG,etc.,and participate in the scavenging of pathogens and target cells,which are closely related to inflammation.Macrophages are also specialized antigen presenting cells,and are involved in immune regulation by secreting a variety of cytokines.TRIM59 is the protein that is expressed in the BCG activation of macrophages,and the protein is part of the family of the TRIM protein.The TRIM structure is highly conserved,and it is classified as E3 ubiquitin ligase due to the RING structure.It participates in the ubiquitination process and has a positive or negative effect on the intrinsic immune response.It is shown that TRIM59 is expressed on The endoplasmic reticulum and is highly expressed on a variety of tumors,and the previous work of our team found that the protein is a membrane protein of BCG activated macrophage,and the direct contact killing effect of macrophage is significantly reduced after blocking TRIM59 with antibody,so the protein plays an important role in the antitumor effect of BCG activated macrophage.It is also found that the protein can regulate the innate immune response and play a negative regulatory role in combination with ECSIT.However,it is not clear what roles the expression of TRIM59 on macrophage membrane by BCG regulation plays on the biological activity of macrophages such as proliferation,apoptosis,phenotype,phagocytosis,and how BCG activates macrophages so as to increase the expression TRIM59.Based on the above research background,cell models of this study were established by stimulating RAW264.7 cells with BCG,and transfection technology was used to produce highly TRIM59 expression RAW264.7 cells(TRIM59 cells)and their control cells(Vector cells),as well as the low TRIM59 expression cells(si-TRIM59 cells)and their control cells(Scramble cells).This study is carried out in two aspects as follows:1.Signal pathways that participate in the BCG regulation of TRIM59 expression.As to explore the expression mechanism and levels of TRIM59 in BCG stimulate RAW264.7 cells,we used the BCG stimulated RAW264.7 cells,and used TLR2 and TLR4 neutralizing antibody to block these two receptors,and then used siRNA to interfere the expression of IRF5,then found that TRIM59 was highly expressed on the membrane of BCG activated RAW264.7 cells,this is consistent with the experiment results in vivo,and found that high expression of TRIM59 is through TLR2/4/IRF5 signaling pathways.2.Research on TRIM59 regulates macrophage biological activities.In order to define the TRIM59 regulation function of macrophages,we used TRIM59 highly and lowly expression RAW264.7 cells as models,detected changes in cell surface molecules,cytokine secretion,phagocytosis,proliferation and apoptosis.We found that the antigen presenting related membrane molecular MHCII,CD86 is reduced,and the non-essential phagocytosis related molecular CD172 a,CD206 is increased,the receptor mediated phagocytosis and IgG CD16,CD32,CD64 increases,and the complement mediated phagocytosis CD11 b related receptors is reduced,Moreover,TRIM59 can significantly promote the expression of IL-10,inhibit the expression of IFN-γ,promote the expression of TNF-α,has time phase effect on the expression of IL-1β,while no obvious effect was found on IL-12 expression.In terms of phagocytosis,TRIM59 can promote the non-essential phagocytosis and IgG inducing essential phagocytosis of macrophages,and inhibit the complement mediated essential phagocytosis.Moreover,after the high expression of TRIM59,the G1 phase cells decreased,the S-phase cells increased,and the apoptotic cells did not change significantly.These results indicated that TRIM59 could have a broad effect on macrophages,promoting the secretion of macrophages secreting inhibitory factors,promoting phagocytosis,and promoting the proliferation of macrophages themselves.This study explored the mechanism of BCG regulation of TRIM59 expression,elaborated the regulation effect of TRIM59 on macrophages’ own proliferation and apoptosis,and explored the regulation of TRIM59 on the membrane molecules,cytokines and phagocytic function of macrophages,clarified the expression significance of TRIM59 in macrophages,and provided new experimental methods and thoughts for studying the regulation TRIM family protein in macrophages.