Regulation Of Macrophages Phagocytosis And Cytokine Expressions By TRIM59 And Its Effect On Sepsis

Sepsis is a life-threatening syndrome that affects the health of millions of patients,especially hospitalized patients.The mortality rate of sepsis is as high as 25%.Sepsis certainly imposes significant global health costs due to its high incidence and mortality rates.Therefore,it is importance to determine the pathogenesis of sepsis and its mitigating factors.Patients with sepsis mainly died of microvascular dysfunction,which is characterized by impaired vascular barrier function,excessive inflammatory immune cell infiltration,excessive coagulation,impaired vascular regulation and ischemia,and eventually leads to multiple organ failure.Sepsis is characterized by a systemic maladjusted response of the host to the infection.When the pattern recognition receptors(including TLRs)on the cells of the innate immune system recognize pathogens,the innate immune cells are activated and trigger pro-inflammatory and anti-inflammatory cytokines through the transcription factor NF-?B,such as TNF-?,IL-1,IL-2,IL-6,IL-8,etc.,which can cause neutrophil endothelial cell adhesion,activate complement and coagulation,and lead to microthrombosis.When pathogens invade,some innate immune cells,such as neutrophils and macrophages,can also reduce pathogen invasion by phagocytizing pathogens,thus reducing sepsis.Therefore,innate immune response plays an important role in sepsis.Macrophages are an integral part of the innate immune response and play a key role in many diseases.Macrophages have multiple functions,including:(1)production of cytokines,like IL-1,IL-6,and TNF-?;(2)phagocytosis of pathogens;and(3)presentation of antigens.TNF-?,IL-1?,and IL-6 are important cytokines that mediate initial response of innate immune system to infections.TNF-?and IL-1?both activate endothelial cells and attract circulating polymorphonuclear white blood cells to the infection site.However,these cytokines also enter the bloodstream and cause fever and other systemic symptoms.IL-6 stimulates the liver to produce acute phase reactants to stimulate the inflammatory response and aggravate the inflammation.Therefore,in the early stage of sepsis,a large number of pro-inflammatory cytokines produced by macrophages can aggravate the progress of the disease.The surface receptors of macrophages can recognize and phagocytize apoptotic cells and PAMPs,thus reduced the release of contents from apoptotic cells and PAMPs,and controled the aggravation of inflammation.Previous studies have demonstrated that reduced expression of MHCII proteins on the surface of macrophages in patients with sepsis,which resulted in T cells activating uneffectively and aggravating the sepsis.Therefore,Macrophages play an important role in the development of sepsis through antigen presentation,phagocytosis and production of various cytokines.TRIM59 is a member of TRIM family,which containing a RING finger domain,a B-Box motif(B-Box2),a coiled-coil region and a transmembrane domain.The TRIM protein family is also referred to as the RBCC protein family due to its highly conservative structure.TRIM proteins are involved in the innate immune response,recent studies have confirmed that TRIM proteins could restrict viral infections and influenced various signaling pathways such as IFN signaling pathway and NF-?B signaling pathway.Additionally,TRIM family proteins participated in the regulation of gene transcription.TRIM59 was upregulated in various cancers and promotes the development of tumors,down regulated in LPS stimulated macrophage cell lines,and affected the activation level of LPS stimulated macrophages.Other studies found that TRIM59 could interact with ECSIT and regulate innate immune response through NF-?B and IRF3/7-mediated signaling pathways.Our previous study also found that TRIM59 could promote phagocytosis of macrophages.Therefore,TRIM59 may play an important role in inflammation related diseases through macrophages.TRIM59 regulated NF-?B and IRF3/7-mediated signaling pathways by interacting with ECSIT,thus affecting the innate immune response.TRIM59 also affected the activation of LPS stimulated macrophages and enhanced the phagocytosis of macrophages.Macrophages play an important role in the development of sepsis through antigen presentation,phagocytosis and production of various cytokines.However,the role of TRIM59 in sepsis has not been explored.Therefore,in order to explore the role of TRIM59 in macrophages and sepsis,we carried out research from the following three parts:1.The effects of TRIM59 protein deletion in macrophages on septic miceBMDMs were induced from bone marrow stem cells of C57BL/6N mice in order to investigate the expression of TRIM59 in LPS stimulated macrophages.BMDMs and RAW264.7 cells were stimulated with LPS in time and concentration gradients.The results showed that the expression of TRIM59 was significantly decreased after LPS stimulation,and the expression of TRIM59 could be reduced by low concentration of LPS in a short stimulation time.In order to investigate the effects of TRIM59 protein deletion in macrophages on septic mice,we successfully constructed Trim59-c KO mice with conditional knockout of BMDMs by Loxp-Cre system.The classic sepsis model was established by CLP surgery on Trim59^flox/flox and Trim59-c KO mice.The survival time of mice was observed for 7 days,and 24 hours after CLP surgery,the heart,liver,spleen,lung,kidney and brain of Trim59^flox/flox and Trim59-c KO mice were taken to detect the damage and the infiltration of inflammatory cells.Besides,macropahges,neutrophils,dendritic cells,B cells,CD4⁺T and CD8⁺T cells in peripheral blood,spleen and m LNs were detected by flow cytometry.Results showed that the survival time of Trim59-c KO mice was shortened,liver bleeding was increased,and liver injury was aggravated.In addition,serum ALT and AST were increased in Trim59-c KO mice,indicating that the liver damage was more serious.In Trim59-c KO mice,the lung structure was destroyed more seriously,inflammatory cells were infiltrated more,and neutrophils in peripheral blood were also increased.These results suggested that when TRIM59 protein was deleted in macrophages,sepsis was aggravated in mice.2.TRIM59 regulated macrophage phagocytosis in sepsisTo explore whether TRIM59 regulates macrophage phagocytosis in sepsis,we collected peripheral blood and peritoneal fluid of Trim59^flox/flox and Trim59-c KO mice24 hours after CLP for bacterial culture,stimulated BMDMs of Trim59^flox/flox and Trim59-c KO mice with LPS,then added appropriate amount of Ig G pretreated with E.coli expressing GFP.The changes of phagocytic capacity were detected by immunofluorescence and fluorescence microscopy.The results showed that the bacterial content in blood and peritoneal fluid of Trim59-c KO mice increased.After Trim59 knockout,the phagocytic capacity of BMDMs decreased significantly,and the Ig G mediated opsonic phagocytosis also decreased.The opsonic phagocytosis of macrophages is an important part of macrophage phagocytosis.Previous results suggested that TRIM59 regulated the phagocytosis mediated by Ig G in macrophages.To explore the underlying mechanism,we detected the expression of Fc?receptors(CD16/32 and CD64)in BMDMs of Trim59^flox/flox and Trim59-c KO mice by flow cytometry.When stimulated by LPS,the expression of Fc?receptors in TRIM59 deficient BMDMs were significantly down regulated.These results suggested that the loss of TRIM59 in BMDMs was associated with decreased expression of Fc?receptors in inflammatory environment.The antigen presenting function of macrophages also plays a role in sepsis.In order to explore the expression of antigen-presenting related receptors in BMDMs after TRIM59 deletion,we detected the expression of MHCII,CD80 and CD86 in Trim59^flox/flox and Trim59-c KO mice by flow cytometry.Results showed that there was no significant change in MHCII,CD80 and CD86 when stimulated by LPS,indicated the loss of TRIM59 did not affect the expression of antigen presentation related receptors in BMDMs,TRIM59 may not regulate macrophage antigen-presenting function in sepsis.3.TRIM59 regulates the expression of cytokines in macrophages in sepsisIn addition to phagocytosis,macrophages can also secrete a large number of cytokines to affect the process of sepsis.In the above results,we observed that the neutrophil infiltration in peripheral blood of Trim59-c KO septic mice increased,and the injury of important organs and inflammatory cell infiltration also increased,indicating that TRIM59 may affect the expression of inflammatory cytokines in macrophages in sepsis.Therefore,we collected peripheral blood and BALFs of Trim59^flox/flox and Trim59-c KO mice 24 hours after CLP surgery.It was found that the secretions of TNF-?and IL-6 in serum of Trim59-c KO sepsis mice were significantly increased,and the expressions of TNF-?,IL-6 and IL-1?in BALFs were up-regulated,besides,in the sham group,the levels of TNF-?and IL-6 in serum and the expression of IL-1?in BALFs of Trim59-c KO mice were increased.The expressions of Tnf-?,Il-6,Il-1?and Nos2 in BMDMs from Trim59-c KO mice were increased at the m RNA level,and TNF-?,IL-1?were increased at the protein level.The expression of IL-1?in BMDMs of Trim59-c KO mice without LPS stimulation was also increased.When stimulated Trim59 knockdown RAW264.7 cell line and its control cell line with LPS,resultes showed that the secretions of TNF-?,IL-6 and IL-1?of Trim59 knockdown cells were increased.These results indicated that the deletion of TRIM59 in macrophages increased the secretion of pro-inflammatory cytokines both in vivo and in vitro,and under normal conditions,TRIM59 also has a certain anti-inflammatory effect.Activation of NF-?B signal pathway is important in LPS induced inflammatory response of macrophages.In order to investigate the effect of TRIM59 on NF-?B signal pathway,we stimulated BMDMs of Trim59^flox/flox and Trim59-c KO mice with LPS.We found that p-IKK?/?,p-I?B?and p-p65 were up-regulated after LPS stimulation in TRIM59 deficient BMDMs.The expression of p65 in necleus in TRIM59 deficient BMDMs was also increased.These results indicated that the activation of NF-?B signaling pathway was enhanced after TRIM59 deletion in macrophages,and TRIM59enhanced LPS induced inflammatory response through NF-?B pathway.Previous studies have shown that TRIM59 could regulate NF-?B signaling pathway through ECSIT,and regulate the proliferation of a variety of cells through PI3K/AKT signaling pathway.PI3K/AKT signaling pathway can activate NF-?B signaling pathway through IKKs.To determine whether TRIM59 regulates LPS activated NF-?B signaling pathway through ECSIT/MAP3K1 and PI3K/AKT signaling pathways,we stimulated BMDMs of Trim59^flox/flox and Trim59-c KO mice with the above experimental conditions.The expressions of ECSIT,MAP3K1 and PI3K/AKT signaling pathway were not significantly changed after LPS stimulation,suggesting that TRIM59 can directly regulate inflammatory response through NF-?B signaling pathway,but its specific binding sites and related molecules need further study.Our study is the first to demonstrate that the deletion of Trim59 in macrophages promotes the progression of sepsis in mice.In Trim59-c KO mice,the mortality,liver and lung injury,and bacterial load were all increased after CLP surgery.In TRIM59knockout BMDMs,the expressions of Fc?receptors and phagocytic function decreased,and the secretions of pro-inflammatory cytokines increased as well as the activation of NF-?B.This study broadens the way of sepsis related research and provides new evidence for the role of TRIM protein in inflammation related diseases.