The Protective Influence Of Triggering Receptor Expressed On Myeloid Cells-1 Binding Peptide On The Cytokine Release Of Monocytes And Septic Mice

Background: Sepsis is systemic inflammatory response syndrome that results from a harmful host response to infection. Clinically, features may include fever or hypothermia, leucocytosis or hypolekocytosis, tachycardia , tachypnea and so on. It correlates to physiological change of multisystem and multiorgan and has become an obstacle to improve the prognosis of sepsis. A recent epidemiological study from North America and Europe found that there were over 18 million severe sepsis cases per year ,and 14,000 patients died from severe sepsis per day with 1.5% increasing rate per year .After infection the activation of inflammatory cell induces production of several proinflammatory cytokines ,which results in more inflammatory cells activated.Then inflammatory cascade comes up and diffuses to cause sepsis.Recently we make use of supporting treatment such as fluid resuscitation ,antibiotherapy,mechanical ventilation and blood purification to treat sepsis. Even plenty of medical resource had been costed the mortality of sepsis still stayed high .So it is of great importance to figure out the pathogenesis of sepsis and find out new therapeutic target. The triggering receptor expressed on myeloid cells (TREM)-1 is a newly found member of the immunoglobulin superfamily expressed by neutrophils, macrophages and mature monocytes. The expression of TREM-1 of skin, body fluid and tissue dramatically increases after bacterium or fungi infection.TREM-1 and TLRs appear to cooperate to produce an inflammatory response and increase the expression of TREM-1 as a positive feedback. TREM-1 is able to enhance the pro-inflammatory effect of neutrophil,which is modulated by NF-κB.TREM-1 and its soluble form sTREM-1 can be a biologic marker as they both are up-regulated during sepsis .The natural ligand of TREM-1 stays unknown and may exist on the platelet of septic patient. The blockade of TREM-1 with mTREM-1/IgG1 (a murine TREM-1 extracellular domain and human IgG1 Fc fragment fusion protein) could protect mice against both LPS-induced shock and microbial sepsis caused by administration of live Escherichia coli or by CLP . A 17 peptide containing CDR-3 and'F'βchain of the TREM-1 extracellular region could compete with the natural ligand of TREM-1 binding to its receptor to modulate the inflammation,which had been confirmed by in vitro and in vivo studies. The reduction of NF-κB activation and cytokine production can protect the mice from excess inflammation through the modulation of TREM-1.With gene knock-out the inflammatory cell produces less myeloid differential protein 88, interleukin-1βafter LPS stimulation. Study of TREM-1 was considered as an important achievement because of its role as the key mediator in SIRS. In prophase study, We has isolated the monocytes from peripheral blood of patients with severe pneumonia whose monocytes expressed TREM-1 gene in a very high level,and constructed the vector of TREM-1 ,then expressed and purified the TREM-1 protein .With the recombinant protein of TREM-1 as the target molecule,the binding peptides will be screened from a random phage display peptide library. The binding peptides which bind specifically with TREM-1 and inhibit the TREM-1 pathway will be found.Purpose: to study the protective influence of triggering receptor expressed on myeloid cells-1 binding peptide on the cytokine release of monocytes and septic miceMethods:1. Monocytes were prepared from peripheral blood of healthy volunteer donor originating from laboratory staff and divided into①monocytes②monocytes + platelets③monocytes + LPS④monocytes + platelets + LPS⑤monocytes + LPS+ platelets + TREM-1 binding peptide (10ng/ml)⑥monocytes + LPS + platelets + TREM-1 binding peptide (50ng/ml)⑦monocytes + LPS+ platelets + TREM-1 binding peptide (100ng/ml).The cell supernatant was obtained after monocytes had been maintained in a 5% CO2 incubator at 37℃for 24h and the levels of TNF-α,IL-1βwere detected by ELISA.2. Mice sepsis models were established by LPS through peritoneal injection. Mice were divided into blank ,LPS-induced sepsis, binding peptide,LPS+TREM-1 monoclonal antibody ,LPS+ TREM-1 monoclonal antibody + binding peptide protection ,early binding peptide protection and delayed binding peptide protection. Peripheral blood,lung and liver were obtained at the set time .The level of serum TNF-α,IL-1βwere detected by ELISA .Also TREM-1,TNF-α,IL-1βmRNA were determined by quantitative reverse transcriptional polymerase chain reaction. Detection of Pathohistology for lung and liver was also carried out3. Mice sepsis models were established through cecum ligation and puncture. Mice were divided into blank,sham-operation, CLP-induced sepsis, early protection (peritoneal injection of 500μg/mouse immediately after CLP) and delayed protection (peritoneal injection of 500μg/mouse 4 hours after CLP) groups. Every 6 hours in the next 4 days we detected the survival rate of every group miceResults:1. Significant TNF-αand IL-1βproduction was observed in the supernatant of monocytes cultured with LPS and platelets.The inducible release of proinflammatory cytokines was significantly lower after LPS and platelets stimulation when the medium was supplemented with TREM-1 binding peptide.2.. TREM-1 mRNA increased while the mice were treated with LPS.The early binding peptide protection could decrease the liver TNF-α,IL-1βmRNA and serum TNF-α,IL-1β, depressing the inflammation of the LPS-induced injury on the lung and liver,which was inhibited by TREM-1 agonistic antibody and not detected in the delayed protection group.3. The 72 hours survival rate of CLP- induced sepsis mice was 10%,which was lower than blank and sham-operation groups mice(P<0.01,P<0.01). Compared with the sepsis group, early protection group had an significantly increased survival rate to 60%(P<0.05).But delayed protection group didn′t show survival advantage with 72 hours survival rate being 0%(P>0.05).Conclusions:1. LPS and platelets simultaneously increased the release of TNF-α,IL-1βfrom monocytes cultured with LPS and platelets, which was inhibited by TREM-1 binding peptide.2. TREM-1 binding peptide has obviously protective effect on LPS-induced sepsis in mice .The blockage of SIRS induced by TREM-1 maybe a new pathway for the treatment of sepsis3. TREM-1 binding peptide has obviously protective effect on CLP-induced sepsis in mice, but the delayed treatment of the binding peptide didn't show survival advantage.