| Sepsis is defined as systemic inflammatory response syndrome(SIRS)caused by pathogenic microorganisms infection,the pathogens include bacteria,fungi,viruses and parasites.Severe sepsis?septic shock and multiple organ disfunction syndrome(MODS)are the sepsis progressive complications.At present,sepsis has been listed as one of the top ten cause of death that threatens human life.About 19 million people were diagnosed as sepsis every year.High morbidity and mortality make sepsis is a major stress in maintaining public health.Thus,identifing the pathogens and precise mechanism of sepsis are important for sepsis prevention and treatment.The response degree of innate immune system to pathogens plays a key role in sepsis.The imbalance of immunoinflammatory response is considered as the core performance of sepsis.Although excessive inflammatory response is beneficial to pathogens clearance,may lead to organ dysfunction or even death.Under normal circumstances,the pattern recognition receptors of innate immune system can recognize specifical pathogens,activate intracellular signaling cascades,promote immune cell release multiple inflammatory cytokines,then initiate innate and adaptive immune responses to clear the infection pathogens.It has been reported that sepsis has relationship with genetic background.The mutation of key genes in the immune response pathway can affect the response of the individual immune system to pathogens.Therefore,finding immune genes associated with the development of sepsis will be beneficial to developing new genetic diagnostic techniques,screening high-risk patients with clinical sepsis,finding new drugs and therapeutic targets to improve the prognosis of patients with sepsis.C-type lectin receptors(C LRs)can recognize specifical structures of fungi and bacteria,activate Syk-CARD9-NF-?B pathway,and release inflammatory cytokines to kill infection pathogens.For example,Dectin1 of CLRs recognizes ?-glucan on fungal surface,Dectin2 can recognize mannose of fungal surface and mannosylated lipopolysaccharide on the surface of certain bacteria,Dectin3 and Mincle can identify the mycobacterial cord factor trehalose-6,6'-dimycolate(TDM).In this study,Dectin1,Dectin3 and CARD9 knockout mice were used to investigate the role and mechanism of C LRs and their downstream adapter protein CARD9 in the development of sepsis.In this study,the mice model of sepsis is prepared by "cecal ligation and puncture"(CLP),a gold standard which is internationally accepted in sepsis research.The main operation is to ligature part of the cecum and puncture,resulting in local necrosis and intestinal contents leakage,causing abdominal infection,then developing to sepsis.The results showed that the severity of sepsis was determined by the length of cecum ligated and the needle puncture size.With the increase of the puncture needle size,the pathogens number of lung and peritoneal increased significantly,and the level of TNF-? were significantly increased,too.The Dectin1,Dectin3 and CARD9 knockout mice with sepsis were used to compare the survival rate with wild-type mice at the same sepsis severity.The results showed that the wild-type mice all survived after 7 days follow up,Dectin3 knockout mice all died within 48 hours,but the mortality rates and median survival time of the Dectin1 or CARD9 knockout mice were not significantly different from those of wild-type mice.These results indicate that Dectin3 is a key receptor in the development of sepsis.The lung homogenate and peritoneal lavage fluid were cultured to identify the bacteria and fungi respectively.The results showed that the bacterial culture was positive and the results of the fungal culture were negative,indicating that the mice model was the polymicrobial sepsis model.The number of bacteria in the lung and peritoneal lavage fluid of Dectin3 knockout mice increased sharply and was significantly higher than that of wild-type mice.The levels of different inflammation cytokines in lung,peritoneal lavage fluid and serum were detected by ELISA,the amount of TNF-? of Dectin3 knockout mice was significantly increased than that of wild-type mice,but other inflammatory cytokines such as IL-6,IL-12 and IL-1? were not notice significantly difference.Pathological analysis the lungs after HE staining,showed that the alveolar structure disappeared and inflammation was serious in the lung of Dectin3 knockout mice.Flow cytometry results showed that the number and proportion of neutrophils in Dectin3 knockout mice reduced significantly after sepsis,and was statistically significant with that of wild-type mice.These results suggest that Dectin3 plays an important role in the identification and clearance of bacteria and reduces sepsis damage to the organism,however,Dectin1 and CARD9 may not play a role in the immunoprotection of sepsis mice.Bacterial species from lung of Dectin3 knockout mice were identified by PCR amplification the 16 S r RNA,sequencing and blast with database in NCBI website.The results showed that 91% of the bacteria belonged to Escherichia coli and the remaining 9% was Shigella flexneri.According to the results of database,the Escherichia coli serotype is most likely to be O83:H1.Further,PCR amplification was used to amplification the Escherichia coli O83:H1 special virulent gene ibeA,fim H,fim A.The results of blast showed that the strain was Escherichia coli O83:H1 str.NRG 857 c.The above results suggest that Dectin3 likely recognize this serotype of Escherichia coli.The strain is referred to as "E.coli O83:H1" hereinafter.Bone marrow derived macrophages(BMDM)from mice were stimulated with E.coli O83:H1 in vitro and the level of cytokines produced by BMDM were detected by ELISA.It was found that the levels of TNF-? and IL-1? produced by Dectin3-deficient BMDM was significantly lower than that produced by wild-type mouse cells,indicating that Dectin3 is the specific pattern recognition receptor of E.coli O83:H1.The production of cytokine TNF-? from the wild-type BMDM co-stimulated by NF-?B and ERK signal pathway inhibitors and E.coli O83:H1 was detected by ELISA,the results showed that TNF-? production was significantly decreased when Syk?p65 inhibited,and the TNF-? expression was statistically lower than that produced by E.coli O83:H1 stimulates alone,indicating that Dectin3 can recognize E.coli O83:H1 and eventually though activate NF-?B pathway to play antibacterial effect.Lipopolysaccharides extracted from other serotypes of E.coli cannot stimulate BMDM cells to produce the same results.Intraperitoneal injection of lipopolysaccharide was used to simulation sepsis model.There was no significant difference in the reactivity between wild-type mice and Dectin3 knockout mice,and the survival rate was not statistically significant yet.The in vivo and in vitro results indicate that Dectin3 can only recognize the O83:H1 serotype in E.coli,but not other serotypes.Our laboratory previous studies have found that the Flos Rosae Chinensis extract has antifungal effect in vitro,oral administration could significantly prolonged the survival time of mice with systemic fungal infection.The fungl infection sepsis model was established.First,mice were treated with containing ceftriaxone sterile water to destroy the bacterial colonies in the intestine,then intragastric administration of Flos Rosae Chinensis extract and Candida albicans,successively.Finally,the fungl infection sepsis model was done by CLP.It was found that intragastric administration of Flos Rosae Chinensis extract could significantly decrease the mortality rate and prolong the survival time of fungal infection sepsis mice and polymicrobial sepsis,which indicates that the Flos Rosae Chinensis extract had protective effect on the fungal and polymicrobial sepsis.The results of in vitro drug sensitivity tests showed that E.coli O83:H1 was sensitive to the clinical use of antimicrobial agents,and resistance to the Flos Rosae Chinensis extract.The results showed that infections caused by E.coli O83:H1 can be treated with conventional antimicrobial agents.This study is the first time to illustrate that Dectin3 is associated with the severity of sepsis,Dectin3 deficiency reduces the survival rate of sepsis mice.It is also first found that Dectin3 can recognize the O83:H1 serotype in E.coli and activate NF-?B pathway to play an anti-bacterial infection effect.The mechanism of Dectin3 in anti-bacterial effect was not related to CARD9. |
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