Synergistic Effects Of Endotoxin, Bacterial Lipoprootein And Bacterial DNA

Gram-negative bacterial infection still remains one of the commonest infections in clinic, which is one of major death causes for the critically ill patients. It has been found recently that besides endotoxin or lipoplysaccharide (LPS), other G- bacterial components, such as Bacterial lipoprotein (BLP) and bacterial DNA also have markedly toxic effects to host. However, there are little reports about the synergistic effects among LPS, BLP and bacterial DNA. Based on the current researches, the present study was designed to investigate the synergistic effects of bacterial components through the following four aspects: (1) To investigate the expression of LPS receptors (CD14, Toll-like receptor 4 (TLR4), scavenger receptor(SR)), BLP receptor (TLR2) and bacterial DNA receptor (TLR9) and their relationship with pulmonary injury in abdominal infection-induced sepsis; (2) To examine the synergistic effects of LPS, BLP and bacterial DNA in mice; (3) To further determine the synergistic effects of LPS, BLP and bacterial DNA on the expression of pattern recognition receptors(PRR) on the cell surface of mouse alveolar macrophages and cellular activation; and (4) To examine the effects of TNFαand IFNγ on the expression of the above PRRs on the cell surface of mouse alveolar macrophages. The purposes of this study is to further elucidate the toxic effects of other bacterial components and their synergistic effects with LPS, and then provide more evidence for deepening the understanding of pathogenesis of sepsis and finding effective therapeutic measures for sepsis treatment. The main results and conclusions are shown as follows: 1. The expression of LPS receptors(CD14,TLR4,SR), BLP receptor(TLR2) and bacterial DNA receptor(TLR9) and their relationship with pulmonary injury were systemically investigated in sepsis induced by cecal ligation puncture and ligation (CLP), which is most close to clinical situtation of G- bacterial infection. It was found that the expression of receptors for LPS, BLP and bacterial DNA in pulmonary tissues was markedly changed in CLP-induced sepsis, showing upregulation of CD14,TLR2,TLR4 and TLR9 at different degrees, among which the expression of TLR9 kept increasing. The expression of SR showed continuous down-regulation. There was a marked correlation between the changes of PRR expression and the increases of MPO and TNFαlevels in pulmonary tissues. 2. The synergistic effects of LPS, BLP and bacterial DNA were investigated in vivo for the first time. The results showed that LPS, BLP or bacterial DNA in itself has toxic insult to mice, but they had significant synergistic effects against mice, showing significant increases in both serum TNFαlevels and mortality rate when injected by combination of LPS with BLP or bacterial DNA, and the strongest when given with all of them together. 3. The synergistic effects of LPS, BLP and bacterial DNA were further examined in vitro at the level of PRRs on the cell surface of effector cells using cultured primary alveolar macrophages. The results indicated that LPS,BLP and CpG-ODN not only synergistically stimulated alveolar macrophages to produce TNFα, but also synergistically enhanced the expression of CD14, TLR4, TLR2 and TLR9. The synergistic effects were strongest when they were present together. 4. The effects of TNFαand IFNγ on the expression of PRRs on the surface of alveolar macrophages were further investigated. The results revealed that besides the direct effects of LPS, BLP and bacterial DNA, the cytokines such as TNF-α and IFNγ could also produce feedback regulation on the expression of PRRs at the levels of genes and proteins, showing upregulation of CD14, TLR2, TLR4 and TLR9, and downregulation of SR. Such regulation on the PRR expression would be of significance for further amplification of inflammation cascade and eventually leading to uncontrolled inflammation.