The Effect And Mechanism Of Trigeering Receptor Expressed On Myeloid Cells-1on Monocyte Apoptosis

Part1The relationship between triggering receptor expressed on myeloid cells-1and monocyte apoptosis in sepsisObjective:Triggering receptor expressed on myeloid cells-1(TREM-1) is a cell surface receptor selectively expressed on myeloid cells, which can amplify pro-inflammatory response initiated by Toll-like receptors and Nod-like receptors, and may contribute to the pathogenesis of inflammatory disease such as sepsis. However, the role of TREM-1in monocyte fate and the detailed molecular mechanisms evoked by TREM-1are unknown. Thus, the expression levels of TREM-1in monocyte and the magnitude of monocyte apoptosis were measured.Methods:From July1st2012, to September4th2012, patients who were newly hospitalized in the surgical Intensive Care Unit in the University Hospitals were screened for eligibility. The diagnosis of sepsis met the criteria recommended by American College of Chest Physicians and Society of Critical Care Medicine Consensus Conference. The Acute Physiology and Chronic Health Evaluation Ⅱ (APACHE Ⅱ) score, and the Sequential Organ Failure Assessment (SOFA) score were recorded. Results of microbiological culture were also recorded. Notable exclusion criteria included:human immunodeficiency virus (HIV) positive, treatment with long-term corticosteroids within6months or short-term corticosteroids within4weeks, chemotherapy or radiation therapy within4weeks, or a history of organ transplantation. Ten milliliters of EDTA-anticoagulation peripheral whole blood were drawn from each patient within24hours after diagnosis of sepsis. Monocytes and neutrophils were isolated and extracted for detection of TREM-1by real-time PCR; meanwhile, the magnitude of apoptosis in monocytes and neutrophils in the septic patients were measured by flow cytometry.Results:During the study period, twenty-six patients who were diagnosed as sepsis were enrolled in the study. Among them, peritonitis (26.9%) and pneumonia (26.9%) were the most frequent initial diagnosis for the patients. Other diagnosis included obstructive cholecystitis, severe acute pancreatitis, and so on. Microbiological inspection evidenced that16patients experienced a documented infection. The other10patients showed clinically suspected infections stated by senior intensivists. The expression levels of TREM-1and the magnitude of apoptosis in monocyte were successfully detected in all of the patients. Correlation analysis showed that the expression levels of TREM-1were reversely correlated with the magnitude of apoptosis in monocyte (r=-0.49, P=0.01). In addition, the expression levels of TREM-1and the magnitude of apoptosis in neutrophil were measured in22out of26septic patients. Although the expression levels of TREM-1were not significantly correlated with the magnitude of apoptosis (r=-0.28, P=0.21), a trend that the neutrophil with higher expression levels of TREM-1have less magnitude of apoptosis was observed.Conclusion:In septic patients, TREM-1levels were reversely correlated to the magnitude of apoptosis in monocyte. This study demonstrated that TREM-1played an important role in monocyte apoptosis. Part2The effect and mechanism of TREM-1on monocyte apoptosisObjective:To investigate the effect and mechanism of TREM-1on monocyte apoptosis.Method:Adenoviruses overexpressing TREM-1were constructed and transfected into monocytic THP-1cells. After activation of TREM-1by agonist antibody with/without lipopolysaccharide, apoptosis was induced by staurosporine and assayed using flow cytometry. The signaling pathways downstream of TREM-1were illustrated by inhibitory experiments. Pro-/anti-apoptotic protein levels were measured using immunoblot.Results:Activation of TREM-1protected monocytes from staurosporine induced apoptosis (41.8%vs.63%, p<0.05). This characteristic was also obtained under lipopolysaccharide stimulation (33.7%vs.54.3%, p<0.05), an in vitro inflammatory model. The protection of TREM-1against monocyte apoptosis was abrogated after inhibition of extracellular-signal-regulated kinase (Erk)(77.7%vs.29.8%, p<0.05) or PI3K/Akt (69.3%vs.29.8%, p<0.05) signaling. Western blot analysis showed that cross-linking of TREM-1remarkably upregulated myeloid cell leukemia-1(Mcl-1) protein level and reversed staurosporine-suppressed Mcl-1expression (p<0.05).Conclusion:This study demonstrated that activation of TREM-1protected monocytic cells from apoptosis through activation of both Erk and Akt pathways and increased expression of Mcl-1protein. Part3The importance of myeloid cell leukemia-1in TREM-1mediated protection against apoptosisObjective:To illustrate the importance of myeloid cell leukemia-1(Mcl-1) in TREM-1mediated protection against apoptosis.Method:The extracellular-signal-regulated kinase (Erk) and/or PI3K/Akt, down stream of TREM-1signaling, were inhibited to detect Mcl-1expression level. Then a cyclin dependent kinase inhibitor, Roscovitine, which could specifically downregulate Mcl-1, was added into the transfected cells simultaneously with STS in a final concentration of20μM.Results:Inhibition of Erk or Akt resulted in the reduction of Mcl-1expression (p<0.05). Treatment of the agonist-activated Ad.TREM-1-transfected cells with20μM Roscovitine for4hours did suppress the protein levels of Mcl-1(p<0.05). Roscovitine abrogated the protective effect of TREM-1against apoptosis (68.1%vs.39.5%, p<0.05).Conclusion:This study confirming that Mcl-1was a critical molecule accounting for TREM-1mediated signaling pathway against apoptosis.