Changes Of Spleen NK Cell Numbers And Their Subsets In Cecal Ligation And Puncture Murine Model

Sepsis,a life-threatening organ dysfunction caused by the host response to infection disorders,is a common disease in the intensive care unit(ICU)with a high incidence and mortality.Over the past decade,many studies have confirmed that immunosuppression in the late stage of sepsis is the main cause of death,and more and more researches have shown that NK cells play a key role in the immune response to sepsis.As a kind of large granular lymphocytes which are involved in both innate immune responses and adaptive immune responses,NK cells play a critical role in the immune response to sepsis.Clinical studies have confirmed that NK cells and NK cell subsets CD56 hiNK cells and CD56 lowNK cells are decreased significantly in the late phase of sepsis.However,CD56 is not present on NK cells of mouse,which limits the NK cells research in sepsis to some extent.In recent years,it has been found that NK cells can be divided into four subsets according to surface markers CD27 and CD11b(CD27-CD11b+,CD27+CD11b+,CD27+CD11b-,CD27-CD11b-).They also can be divided into three functional subsets(NKcytotoxic,NKregulatory,NKtolerant).NKcytotoxic subset is mainly CD27-CD11b+NK cells,NKregulatory subset is mainly CD27+CD11b+and CD27+CD11b-NK cells,NKtolerantsubset is mainly CD27-CD11 bNK cells.In this study,we analyzed the spleen NK cells in mice,the subsets marked by CD27 and CD11 b,the changes of the corresponding functional subsets in sepsis immunosuppression period,which not only provided a bridge for the basic and clinical research on NK cells in sepsis but also provided a theoretical basis for the further study on the mechanism and treatment of sepsis.Part1:The survival rate of sepsis mice,Pathological changes of lung tissue and intestinal tissue,changes of serum inflammatory factorsObjective:(1)Observation of changes in the survival rate of sepsis mice.(2)The pathological changes of lung tissues and intestinal tissues in sepsis model at different time points were evaluated by hematoxylin/eosin(HE)staining.(3)Changes of serum TNF-α and IL-10 at different time points were detected by ELISA kit.Methods:(1)60 Male C57bl/6 mice,with body weight ranging from 20 to 30 gand age ranging from 8 to 10 weeks,were randomly divided into two groups: sham operation group(Sham group)and sepsis group(CLP group).Thirty mice were used to prepare cecal ligation perforation(CLP)model and thirty mice were used to prepare sham operation mode.The survival rate was recorded for 8 days.(2)Male C57bl/6 mice,with body weight ranging from 20 to 30 g and age ranging from 8 to10 weeks,were used to prepare the celestial ligation and perforation(CLP)model.The small intestine tissues and lung tissues were taken at 24 h,48h and 72 h after operation(with 3 mice each group).The histopathological changes of small intestine and lung tissues in different groups were observed by HE staining.(3)Male C57bl/6mice,with body weight ranging from 20 to 30 g and age ranging from 8 to 10 weeks,were used to prepare the celestial ligation and perforation(CLP)model.Mice were sacrificed at 2h,4h,6h,24 h,48h and 72 h after operation(with 6 mice each group).The serum was prepared,followed by incubation at-80 ° C.Concentrations of the serum TNF-α and IL-10 were measured by ELISA kit.Results:(1)The survival rate of Sham group was 100% within 8 days after modeling,and the survival rate of the CLP group was lower than that of the Sham group at different time points.(2)HE staining showed that the small intestine mucosa of Sham group was normal,with clear and intact villi.In contrast,the structure of small intestine tissues of CLP mice was disordered,with obvious congestion and visible inflammatory cell infiltration.Moreover,the intestinal villi were fractured and exfoliated,which was most obvious in the CLP24 h group.The lung tissue structure in Sham group was normal,with intact alveolar,and no telangiectasia,alveolar wall widening and inflammatory cell infiltration were observed.In contrast,the lung tissues of CLP group showed alveolar wall thickening,congestion,edema,disordered alveolar structure,alveolar infiltration of a large number of inflammatory cells,and telangiectasia,which was most obvious in the CLP24 h group.(3)The serum concentration of TNF-α was dramatically increased from 2 hours and reached peak level at 6 hours(P<0.05).At 24 h and 48 h there is no significant difference in serum concentration of TNF-α between CLP group and Sham group.However,it was significantly decreased compared to CLP6 h group.The serum concentration of IL-10 was significant increased at 6 hours,24 hours and 72 hours compared to group Sham(P<0.05),but no significant change was observed at 48 h.Conclusions:The survival rate of cecal ligation model mice was low and the CLP model was established.The inflammatory pathological changes were observed in lung and small intestine,which was the earliest infected and the earliest be thansferred,respectively.The pro-inflammatory cytokine TNF-α played a key role within the first hours of sepsis.However,at a later stage,the anti-inflammatory cytokine IL-10 played a key role.Furthermore,the results provided time basis for the study of NK cells in the immunosuppression phase of sepsis.Part2:Changes of spleen NK cells and NK cell subsets in sepsis miceObjective:To investigate the changes of splenic NK cells and their subsets in sepsis mice,and to provide a reference for the further study of sepsis mechanism and treatment.Methods:Male C57bl/6 mice,with body weight ranging from 20 to 30 and age ranging from 8 to 10 weeks,were used to prepare the celestial ligation and perforation(CLP)model.The percentage of NK cells and their subsets(CD27-CD11b+,CD27+ CD11b+,CD27+ CD11b-,CD27-CD11b-)were detected by flow cytometry at 24 h,48 h and 72 h after operation(with 6 mice each group).Results Spleen NK cells and their subsets in mice were assessed by flow cytometry.A decreased NK cell numbers of was observed starting 24 hours of infection and maintained up to 72 hours(P<0.05).A decreased percent of NKcytotoxic(CD27-CD11b+NK)was found at 48 hours(P<0.05).However,an increased percentage of NKregulatory(CD27+CD11b+ NK and CD27+CD11b-NK)was observed at48 hours(P<0.05).In addition,the percentage of NKtolerant(CD27-CD11b-NK)was decreased at 24 hours and 48 hours but increased at 72 hours.Conclusion NK cells and their subsets are involved in the formation of sepsis immunosuppressive mechanisms and play an important role in sepsis immunosuppression.