The Role And Mechanism Of Triggering Receptor Expressed On Myeloid Cells2(TREM-2) In Sepsis

Part1Expression of TREM-2in sepsis and its relationship to sepsisObjective:TREM-2is a kind of cell-surface receptor primary expressing in activated macrophages and belonging to the immunoglobulin superfamily. Type Ⅱ cytokines (such as IL-4) can induce the expression of TREM-2. Studies have shown that TREM-2is a phagocytic receptor for microorganisms such as Gram+and Gram-bacteria as well as yeast. TREM-2mediated signaling pathways can decrease the ability of macrophages to generate pro-inflammatory mediators. Thus, TREM-2may play important roles in sepsis. This part of the study was designed to detect the expression level of TREM-2during sepsis and analysis the relationship between the TREM-2expression level and the development of sepsis.Methods:Sepsis was induced by CLP. Sham-operated mice were included as a control group. Samples such as liver, lung, spleen, abdominal cavity lavage fluid and peripheral blood were collected at0h,6h,12h,24h,72h,120h and168h after the surgery. TREM-2expression was detected by means of immunohistochemistry and flow cytometry. Abdominal bacterial infection was assessed using agar plating method. Plasma concentrations of IL-4, IL-13and IL-10were assayed using ELISA.Results:At Oh after the surgery, the expression of TREM-2was negative both in septic mice and control mice. In the septic mice, TREM-2expression level in the liver, lung and spleen began to increase at6h after CLP, and reached the peak at72h after the operation, and then decreased gradually. Double immunofluorescence labeling analysis showed that TREM-2expression was located in tissue macrophages. However, the expression of TREM-2was not detected at each time point in the control mice. In the peritoneal macrophages in mice with sepsis, the expression pattern of TREM-2was consistent with that in the other organs. The expression level of TREM-2had significant difference at72h after the operation between the two groups. In septic mice, plasma concentration of IL-4reached peak at24h after CLP. The dynamic change of IL-4level was earlier than that of TREM-2, while the pattern was similar to that of TREM-2. The dynamic change of bacterial infection in abdominal cavity was exactly same as that of TREM-2expression. There were no relationships between TREM-2expression pattern and the changes in plasma levels of IL-13and IL-10.Conclusions:In septic mice, the expression level of TREM-2increased first, and reached peak at72h after CLP, and decreased thereafter. The inducible expression of TREM-2may be related to IL-4. TREM-2may play an important role in immune defense against infection during sepsis. Part2The impact of blocking the effect of TREM-2on sepsisObjective:In view of the expression pattern of TREM-2in the pathogenesis of sepsis, this part of the study was designed to investigate the impact of blocking the effect of TREM-2on sepsis.Methods:At18h after CLP, the septic mice were randomly divided into two groups: one group was administrated with20μg recombinant mouse TREM-2/human IgGl protein (mTREM-2/IgG1group) by tail vein injection, and the other group was given with20μg recombinant human IgGl protein (IgGl group) by tail vein injection. The mice were observed for7days to access survival rate. On the other hand, at24h,72h and120h after giving the recombinant protein, peritoneal infection was evaluated using agarose plate method, and IL-10level was assayed using ELISA.Results:The7-day survival rate in the mTREM-2/IgGl group was8.3%, significantly lower than that in the IgGl group (50.0%; P<O.05). At24h after administration of recombinant protein, no significant difference in abdominal bacterial infection was observed between the two groups. At72h and120h after administration of recombinant protein, intraperitoneal bacterial infection in the mTREM-2/IgG1group was significantly higher than those in the IgGl group. The IL-10levels in both the peritoneal lavage fluid and the plasma were not significantly different.Conclusions:Blocking the effect of TREM-2reduced the7-day survival rate in septic mice, the mechanism of which was related to increased bacterial infection in the septic Part3Overexpression of TREM-2in the early stage of sepsis is protective against sepsisObjective:Inducible expression of TREM-2in sepsis is peaked lately after onset of sepsis, while blocking the effect of TREM-2increased the severity of sepsis. Therefore, this part of the study focuses on the effect of overexpression of TREM-2in the early stage of sepsis and its mechanism.Methods:Recombinant adenovirus expressing TREM-2and the control adenovirus expressing GFP were constructed and infected BMMC. The expression level of TREM-2protein was detected using Western-blot and flow cytometry analysis. Using an agonistic antibody to activate TREM-2combined with100ng/ml LPS stimulation, the effect of recombinant TREM-2on inflammatory response were analyzed. On the other hand, the effect of TREM-2on phagocytosis of fluorescent particles and bacteria was also investigated. In vivo, BMMCs overexpressing TREM-2or GFP were administrated to septic mice at different time points (2h,4h,6h) after CLP, and7-day survival rates were evaluated. In the groups given with BMMCs at2h after CLP, samples such as peripheral blood, peritoneal lavage fluid, liver, lung and spleen were harvested at24h and48h after the cell transfusion, respectively. The morphology and function of the organs were accessed. Systemic and local bacterial infections were detected, and the concentrations of IL-10were assayed.Results:TREM-2was successfully expressed in BMMC after infection by recombinant adenovirus. Cross-link of TREM-2with agonistic antibody can activate its downstream signaling molecule Akt, and significantly increased IL-10mRNA level after LPS stimulation. BMMCs overexpressing TREM-2showed significantly enhanced phagocytosis of fluorescent particles and bacteria. BMMCs overexpressing TREM-2 improved the7-day survival rate of septic mice after infusion at2h and4h after CLP. BMMCs overexpressing TREM-2also protected liver and lung injury, and apoptosis of splenic cells. The local and systemic bacterial loads were decreased after administration of BMMCs overexpressing TREM-2, while the levels of IL-10in peripheral blood and peritoneal lavage fluid showed no significant changes.Conclusions:In the early stage of sepsis, increased expression of TREM-2protects against sepsis via enhanced clearance of bacteria.