Investigating The Mechanism Of Macrophage TREM-1Pathway And Lymphocyte Tim-3Pathway In The Development Of Sepsis

Part I. Investigating the expression and the mechanism of TREM-1pathway in sepsis[Objective] To investigate the expression of TREM-1molecule on sepsis relatedinflammatory cells and the mechanism of TREM-1pathway in sepsis.[Methods] The model of peritoneal sepsis was constructed in BALB/c mice. Peritonealcells were harvested and the expression of TREM-1molecule on different kinds ofinflammatory cells was detected by Flow cytometry; the peritoneal macrophages fromnormal mice were purified and were co-cultured with Pseudomonas aeruginosa, and theTREM-1fusion protein was added to block TREM-1/DAP12pathway. The phagocytosis ofmacrophages was determined by Wright-Giemsa’s staining. The secretion of cytokines bymacrophages was detected by ELISA. The expression of co-stimulation molecule onmacrophages was detected by RT-PCR; Pseudomonas aeruginosa-induced sepsis wasconstructed and TREM-1blocking protein or control IgG was administrated. The survivalof mice with bacterial-induced sepsis was analyzed. In other experiment groups, the micewere sacrificed and the levels of serum IL-1、TNF-α、MCP-1were detected, and theexpression of CD40and CD86on macrophages was also investigated by using Flowcytometry.[Results] Our results showed that TREM-1was not expressed on lymphocytes butemerged on the cell surface of neutrophils and peritoneal macrophages. Blockade ofTREM-1signaling significantly prolonged survival of mice with P. aeruginosa-inducedperitonitis. However, blocking TREM-1signaling had no effect on macrophagephagocytosis in vitro. Interestingly, the expression of the costimulatory molecules CD40and CD86on macrophages was significantly decreased after blocking TREM-1signaling.Furthermore, interfering with TREM-1engagement led to significant reduction ofpro-inflammatory mediators such as IL-1, TNF-α and MCP-1.[Conclusion] TREM-1molecule was expressed on macrophages and neutrophils. Blocking TREM-1pathway significantly decreased the levels of IL-1and TNF-α secretedby macrophages and simultaneously reduced the activation of macrophages, resulting inobvious prolonged survival time in mice with bacterial induced sepsis. Part II. Investigating the relationship between Tim-3pathway andTh17cells and the role of Tim-3pathway in Klebsiellapneumonia-induced pulmonary infection[Objective] To investigate the relationship between Tim-3pathway and Th17cells, and toanalyze the mechanism of Tim-3pathway in acute pneumonia.[Methods] The splenic lymphocytes of normal mice were purified and Th17-skewed cellswere stimulated by adding some specific cytokines to culture medium. The expression ofTim-3on Th17-skewed cells was analyzed by Flow cytometry after four days of culture.The apoptosis of Th17-skewed cells treated with10μg/ml Galectin-9was also analyzed byFlow cytometry; The model of Klebsiella pneumonia-induced pulmonary infection with thetreatment of Galectin-9or control PBS was constructed. For each treatment, the meansurvival time (MST) was indicated. In some other experimental groups, the infected micewith the treatment of Galectin-9or control PBS were sacrificed and the levels of serumIL-17, IFN-γ, IL-4, G-CSF and MIP-2were detected. Furthermore, the absolute numbers ofCD4~+T cells, CD8+T cells and peripheral neutrophils were counted and the number ofKlebsiella pneumonia in lung was also detected.[Results] We demonstrated that T cells were differentiated to Th17cells after thestimulation of anti-CD3, anti-CD28, TGF-β and IL-6and Tim-3was expressed on most ofthe Th17-skewed cells. Th17-skewed cells were sensitive to Galectin-9-induced apoptosis.In vitro administration of Galectin-9decreased stimulated Th17cells and inhibited theproduction of IL-17. Interestingly, Klebsiella pneumoniae infection led to enhanced IL-17levels. Recombinant Galectin-9significantly decreased IL-17and IFN-γ in vivo, leading tolow levels of G-CSF and MIP-2, which resulted in not enough of neutrophils migrating to inflammatory sites.[Conclusion] Tim-3was expressed on Th17cells and Th17cells were also sensitive toGalectin-9-induced apoptosis. Using Galectin-9significantly decreased the secretion ofIL-17and the number of neutrophils, which resulted in reduced bacterial clearance and highmortality. Part III. Tim-3pathway involves with the function of CD4~+T cells,CD8~+T cells and CD11c~+DC cells and plays important role inPseudomonas aeruginosa-induced sepsis[Objective] To investigate the expression of Tim-3molecule on sepsis relatedinflammatory cells and the mechanism of Tim-3pathway in sepsis.[Methods] Pseudomonas aeruginosa-induced sepsis was constructed. The total peritonealcells were harvested and the expression of Tim-3molecule on inflammatory related cellswas analyzed by Flow cytometry; Pseudomonas aeruginosa-induced sepsis with thetreatment of Tim-3blocking protein or control IgG was constructed and the survival time ofeach group was recorded; The experimental animals were sacrificed and serum levels ofIL-1, TNF-α, IFN-γ and IL-4were detected by ELISA; The splenic lymphocytes wereisolated and LPS was added to culture medium in the present of Tim-3blocking protein orcontrol IgG. The levels of inflammatory cytokines were detected and the proliferation andapoptosis of lymphocytes was also analyzed by Flow cytometry. Furthermore, the absolutenumbers of inflammatory macrophages and peritoneal Pseudomonas aeruginosa werecounted. The proliferation and activation of lymphocytes was detected and the phagocytosisand apoptosis of macrophages was also analyzed in vivo.[Results] Tim-3was expressed on peritoneal CD4~+T cells, CD8~+T cells and CD11c+DCcells, but not emerged on neutrophils and macrophages. The percentage of Tim-3positivecells in CD4~+T cells, CD8~+T cells and CD11c~+DC cells was significantly increased afterPseudomonas aeruginosa infection, up to28%,38%and23%in respectively;Administration of Tim-3blocking protein significantly decreased the levels of IL-1, TNF-α and IFN-γ, resulting in obviously prolonged survival time of infected mice. Usageof Tim-3blocking protein in vitro significantly decreased the proliferation and activation oflymphocytes, but the apoptosis of lymphocytes was increased. Administration of Tim-3blocking protein in vivo also decreased the activation of T cells and DC cells.Administration of Tim-3blocking protein in vivo had no obvious effect on the phagocytosisof macrophages, but significantly increased the migration of macrophages to peritonealcavity, which resulted in increased bacterial clearance.[Conclusion] Tim-3molecule was expressed on peritoneal CD4~+T cells, CD8~+T cells andCD11c~+DC cells, but not on neutrophils and macrophages in sepsis. Blocking Tim-3pathway decreased the levels of IL-1, TNF-α and IFN-γ, resulting in prolonged survivaltime of infected mice. The roles of Tim-3pathway in sepsis involved with the proliferation,activation, migration and apoptosis of lymphocytes and macrophages.