The Research About TREM-1 Regulation On The Activation Of Macrophages Induced By Escherichia Coli. Or Double-stranded RNA

IntroductionAfter the pathogen enters organism, its pathogen-associated molecular patterns(PAMPs) can be combined with the pattern recognition receptors(PRRs) on the membrane or in the cytoplasm of the innate immune cells. Then the pathogen can be internalized and/or killed by the phagocytes, meanwhile, the inflammation response arises. Uncontrolled inflammation and severe infection can lead to multiple organ failure and even death. Because the fatality rate in those patients is very high and few treatments are effective to them, the further investigation on the mechanism about subsequent inflammation signal cascade and how pathogen is recognized and eliminated by innate immune system need to be done. In recent years, it has been confirmed that triggering receptors expressed on myeloid cells-1(TREM-1) has important and extensive roles in adjusting the immune cells functions. After this receptor ligation, the production of multiple proinflammatory cytokines can significantly increase and the signaling pathways related with infection can be amplified when the polymorphonuclear leukocytes are chanllenged with gram-negative bacteria/LPS. So TREM-1 plays an important role in the development of sepsis induced by infection and may be an important therapeutic target. Myeloid differentiation protein-2(MD-2) is the ligand for TLR4 and the receptor for LPS. MD-2 binds tightly to the LPS on the surface of Gram-negative bacteria and transmits an activating signal to TLR4. Then the inflammation arises. And uncontrolled inflammation is the development foundation of sepsis. But most of the anti-sepsis treatment strategies targeting TLR4/MD-2 receptor complex can not achieve a satisfactory curative effect. In our knowledge, Gram-negative bacterias/LPS can be internalized into the phagocytes and killed or cleaved after Gram-negative bacterias/LPS bind to TLR4/MD-2 receptor complex, which has important roles of anti-infection and tissue repair. TLR4/MD-2 receptor complex intervention may distinctly damage the internalization and removal of gram-negative bacterias/LPS, which lead to serious complications. But it is unknow whether TREM-1 can affect internalization and removal of gram-negative bacteria/LPS, aithough this receptor activation can amplify the inflammation signal induced by Gram-negative bacterias/LPS and accelerate sepsis. So an Escherichia coli(E. coli) which can express intracellular Enhanced Green Fluorescent Protein(EGFP) protein, was produced by gene engineering methods in our research. We confirmed that TREM-1 ligation could promote internalization and intercellular killing of this E. coli by macrophages.TREM-1 is a receptor on the membrane of the myeloid cells and can not directly combine with LPS, so it is impossible that TREM-1 recognize the LPS/bacteria and make them be internalized. For answering the question why TREM-1 ligation can enhance the internalization, RAW264.7 cells were also infected with different lentivirus which could interfere with the TREM-1 expression. Then the macrophage-like cell lines with different TREM-1 expression were successfully established and their differences in genome were compared using gene chip technology.We also had a new finding when the RAW264.7 cells were infected with the control lentivirus. TREM-1 expression is notably up-regulated on the control lentivirus-infected cells. When the cells are infected with the lentivirus, a significant amount of viral RNA(target sequence) can be delivered into the DNA of the host cells, and new progeny virus can not be packaged and produced. The target sequence, integrated into host genome, can continuously transcribe new double-stranded RNA(ds RNA). Our result shows the exogenous scramble ds RNA can induce the TREM-1 expression by macrophages. Double-stranded RNA is one of the PAMPs of double-stranded RNA virus. After ds RNA is combined by the PRRs in cytoplasm or inner body of the immune cells, the type Ⅰ interferon and inflammatory medium can be secreted to protect the host from the virus infection.It has been confirmed that the TREM- 1 is involved in the innate immunity against gram positive bacteria, fungi and virus in several researches. TREM-1 activation, synergizing with many kinds of PAMPs, can regulation the subsequent signal pathway. But the roles and mechanisms of TREM-1 in the anti-virus immunity induced by ds RNA is lack of clear understanding. In this research, we found that the expression of TREM-1 raised by ds RNA, and the TREM-1 activation enhanced the anti-viral immunity. TREM-1 activation can synergistically increase the MAPK signal pathway activity and virus-related PRRs expression.The main experimental methods and results:1. The BMDMs were infected with 2% serum opsonized BL21(DE3) plys S or nonopsonized BL21(DE3) plys S, then the cell culture supernatant were collected after 12 hrs. TNF-α and IL-6 in the cell culture supernatant were detected through ELISA method. The BMDMs infected by E. coli could heavily secrete TNF-α and IL-6(P < 0.01), which could be significantly amplified by TREM-1 ligation(P<0.05). And compared with non-opsonized BL21(DE3) plys S, the 2% serum opsonized BL21(DE3) plys S possessed the stronger effect for inducing TNF-α and IL-6 secretion by the BMDMs(P < 0.05).2. An EGFP prokaryotic expression vector was constructed using genetic engineering method, and transfected into an E. coli. Then EGFP protein was induced by IPTG in the E. coli, which mad the bacteria possess stable green fluorescence. This is a powerful tool in visually observing the internalization of bacteria and quantitatively measuring the phagocytosis of phagocytes by flow cytometry.3. TREM-1 was activated by incubated with its agonist antibody for half an hour. Then the bacteria with green fluorescence was added into the supernatant. Half an hour later, the fluorescence in the cells was determined by flow cytometry. And we found that phagocytosis of the macrophage pre-incubated with agonist anti-TREM-1 antibody significantly raised. The average fluorescence intensity of cell and phagocytosis index(P < 0.01) significantly increased. We also detected the phagocytosis of RAW264.7 cell using the same method. The phagocytosis of RAW264.7 cell was weaker than that of BMDMs, and the regulation of TREM-1 activation was faint.4. After the macrophage have been pre-incubated with agonist anti-TREM-1 antibody for half an hour, fluorescent labeled microspheres particles or neutral red were added into the culture supernatant. Then the pinocytosis of cells was detected. Our results found that pinocytosis of cells did not raised, compared with the untreated cells. But the pinocytosis of the macrophages stimulated with LPS was markedly enhanced(P < 0.01).5. After E. coli was internalized by BMDMs with activated TREM-1, the antibiotic was added to the supernatant to kill the extracellular bacteria. Five hours later, cells were lysed with sterilized water and 1/40 of the lysate was plated for colony count(CFU). With activated TREM-1, intracellular killing of the E. coli by BMDMs was significantly increased(P < 0.05).6. TREM-1 expression raised by the RAW264.7 cells infected with control Lentivirus. TREM-1 knock-down did not affect the TLR4 and MD-2 expression.7. After RAW264.7 cells were challenged with Poly IC, the level of TREM-1 expression could up-regulate markedly in a dose-dependent manner(P < 0.001). Inhibitor studies disclosed mitogen-activated protein kinase(MAPK) p38 and PI3 K pathways were involved in ds RNA-induced up-regulation of TREM-1(P < 0.01). Poly IC also could continuously up-regulate TREM-1 expression by the BMDMs within 36 hrs(P < 0.001), but the TREM-1 peaked at 24 hrs on the cells challenged by LPS. At the same time, we found that short ds RNA could also activate TREM-1 signal.8. After the BMDMs have been incubated with the agonist anti-TREM-1 antibody for 15 min, the effects of Poly IC on JNK, ERK1/2 and p38 phosphorylation were evaluated. We found the phosphorylation of all three MAPKs was enhanced and prolonged.9. After ligation with the agonist antibody, TREM-1 can significantly potentiate type I interferon(IFN), TNF-α and IL-6 production(P < 0.01), and the m RNA transcription of IL-1β, IL-10 and MCP-1 significantly enhanced. And we also found some differences in TREM-1 function between RAW264.7 cells and BMDMs.10. The TREM-1 activation could synergistically up-regulate the expression of MDA5, TLR3 and TLR7 by the BMDMs stimulated with Poly IC. After the cells were stimulated with short ds RNA, the receptor affected by TREM-1 was RIG-I. The expressions of those receptors were abnormal in RAW264.7 cells.Conclusion:1. TREM-1 activation amplifies the production of pro-inflammatory cytokine, such as TNF-α and IL-6. Meanwhile, internalization and intracellular killing of E. coli by macrophages can selectively promoted. But TREM-1 activation does not enhance the nonspecific pinocytosis of macrophages. This partly answer the question why TREM-1 activation can amplify the inflammatory signal arised by E. coli infection.2. TREM-1 knock-down does not change TLR4 and MD-2 expression. So the amplification of TREM-1 might relate with the function of TLR4/MD-2 and the change of MAPK signal pathway after TREM-1 is combined with its agonist antibody.3. The results of microarray detection and in vitro experiment confirm that ds RNA can raise the expression of TREM-1 in dose- and time-dependent manners.4. TREM-1 can regulate the anti-viral immune response, because TREM-1 activation synergistically raised RLRs and TLRs receptors expression and MAPKs activation in BMDMs challenged with ds RNA.