Study On Pre-exposure To CpG DNA Protect Sepsis Model Mice

Objective Sepsis, a common complication in patients with extensive deep burns and severe traumas, has a high mortality. It is urgent and important to look for methods to prevent and treat sepsis. Unmethylated CpG-containing oligodeoxynucleotides (CpG ODN) can mimic the immunostimulatory effects of bacterial DNA (bDNA). They are potent immunomodulators. Recently, it was shown that CpG DNA could increase resistance against acute nonspecific polymicrobial sepsis in mice and chicken. The mechanism is probably associated with its inhibition TNF-a and IL-6 release from monocytes or macrophages.With these considerations in mind, we undertook the current study to investigate whether pre-exposure with low amount of CpG DNA could protect on mice challenged with heat-killed E.coli. And possible molecular mechanisms related to the protection were investigated, too.Methods ① In vitro, RAW264.7 cells were pretreated with saline, CpG DNA, and then cells were stimulated by CpG DNA. TNF-a in the supernant was tested by ELISA. ② In vivo, murine sepsis models was made by intravenously LD90 heat-killed E.Coli. Then protection of low-amount of CpG ODN in mice sepsis model was observed. ③ Effect of pretreatment with low-amount of CpG DNA on cell surface binding and internalization large-amount of CpG DNA was observed in RAW264.7 cells using flow cytometry. ④ Effect of pretreatment with low-amount of CpG DNA on cell-surface and intracellular TLR9 in RAW264.7 cells was observed using flow cytometry, immunocytochemical method and confocal laser scanning microscope. ⑤ Inhibition of pretreatment with low-amount of CpG DNA on NF-kB activation was observed using electrophoretic mobility shift assay (EMSA).Results We found that pretreatment with 4 μg/ml of CpG DNA inhibited TNF-α release from RAW264.7 cells induced by CpG ODN in a dose- and time-dependent manners. In vivo, pre-exposure of 2.5 mg/kg of CpG DNA could protect mice from lethal challenge by inactivated E. coli in a dose- and time-dependent manner. FACS analysisdemonstrated that both cell surface binding and low-dose CpG DNA at same concentrations that blocked cytokine release significantly reduced interaalization of large-dose of 6-FAM CpG DNA. FACS analysis and immunocytochemical method analysis demonstrated that Pretreatment of low-amount of CpG DNA also down-regulated intracellular TLR9 expression, but increased cell-surface TLR9 expression. Furthermore, pretreatment of low-amount of CpG DNA inhibited NF-kB activation induced by large -amount of CpG DNA.Conclusions Our results demonstrated that pre-exposure to low-dose of CpG DNA protectd mice challenged with lethal heat-killed E.coli was correlating with reduction of TNF-a release, lower binding and intemalization of CpG DNA, down-regulation of intracellular TLR9, and inhibition of activation of NF-kB.