Study On The Mechanism Of Immune Dysfunction In Pediatric Sepsis And Its Therapeutic Strategy

Background: Sepsis is a clinical syndrome caused by infection and is characterized by the systemic inflammatory response syndrome(SIRS).Sepsis remains a major cause of mortality and morbidity in children,especially in neonates and infants,which can also cause serious financial difficulties for families and communities.Unfortunately,sepsis-specific treatment options are lacking,which the physicians still rely on administration of antibiotics and organ support.With the increased prevalence of resistant pathogens and opportunistic infection in recent years,the worldwide incidence of pediatric sepsis appears to be rising.Therefore,the pathogenesis and treatment of sepsis require more study.The pathogenesis of sepsis is extremely complex and remains to be fully elucidated.Previous studies have shown that,in the early phase of sepsis,innate immune cells are stimulated by pathogen-associated molecular pattern(PAMP)and damage-associated molecular pattern(DAMP),which lead to an abnormal and amplified inflammatory response referring as "cytokine storm".This severe inflammation may cause septic shock and death.The late phase of sepsis is characterized by adaptive immune suppression in patients,which have been associated with an increased risk of secondary infections.This study will focus on the mechanism of immune dysfunction during the early and late phase of sepsis as well as the strategy in the treatment to sepsis.Part I Expression characteristics of myeloid-related proteins inpediatric acute appendicitis and its diagnostic valueObjective: To investigate the expression characteristics of myeloid-related protein(MRP)8,MRP14 and MRP8/14 in peripheral blood of children with acute appendicitis(AA),and to analyze the relationship between DAMP and tissue injury in pediatric AA.Methods: This study enrolled a total of 89 patients who were diagnosed with AA and underwent appendectomy in Children's Hospital of Soochow University from June 2013 to January 2015.The peripheral blood samples were collected and divided into simple groups and complicated group,which can be further divided into two groups as phlegmonous and gangrenous,based on the pathology diagnosis.At the same time,52 children who were scheduled for elective surgery of inguinal hernia repair were selected as the control group.The plasma levels of MRP8,MRP14,MRP8/14,IL-6 and TNF-a were measured and analyzed with clinical markers.Results:(1)The levels of MRP8,MRP14,MRP8/14 and IL-6 in AA group was significantly higher than that in control group(P < 0.001,P < 0.001,P < 0.001,P < 0.001),and no difference was found in the production of TNF-a between the two groups.(2)There was a positive correlation between MRP8 and MRP8/14(P < 0.001),while MRP8 was positively correlated with MRP14(P < 0.001),but there was no correlation between MRP14 and MRP8/14.MRP8/14 was positively correlated with LDH(P < 0.05),and negatively correlated with platelet count(P < 0.001).(3)The level of MRP8 in phlegmonous group and gangrenous group was significantly higher than that in control group(P < 0.05,P < 0.05),but there was no significant difference found in the level of MRP8/14 among the three groups.(4)For the prediction of AA,the area under ROC curve(AUC)of CRP,IL-6,MRP8,MRP14,and MRP8/14 was 0.976,0.947,0.836,0.823,and 0.763.For the prediction of complicated AA,AUC of CRP,MRP8,IL-6,MRP14,and MRP8/14 was 0.809,0.763,0.756,0.754,and 0.635.Conclusion: MRP8,MRP14,MRP8/14 were significantly increased in AA children,and their expression levels may be closely related to tissue injury.In addition,MRP8 and MRP14 in the complicated AA was higher than simple AA,MRP8 remains of great value in the diagnosis of pediatric AA.Part II Analysis of T helper lymphocyte-specific cytokines in pediatric childrenObjective: To study the characteristics of T helper lymphocyte(Th)-specific cytokines in pediatric sepsis,and to provide experimental evidence for clinical individualized therapy.Methods: We enrolled 53 children with sepsis and 13 children with SIRS in the PICU at Children's Hospital of Soochow University from July 2015 to December 2016.Peripheral blood samples were collected.Meanwhile,41 children with recurrent inguinal hernia were selected as the control group.The levels of IL-2,IL-4,IL-5,IL-6,IL-9,IL-10,IL-13,IL-17 A,IL-17 F,IL-21,IL-22,TNF-a and IFN-g in plasma were detected by flow cytometry.Cluster analysis was performed in sepsis and control group,sepsis group and SIRS group.Results:(1)Compared with the control group,the levels of IFN-?(P < 0.001),IL-2(P < 0.001),IL-4(P < 0.001),IL-5(P < 0.01),IL-13(P < 0.05),IL-17A(P < 0.001),IL-17F(P < 0.001),IL-9(P < 0.01),IL-21(P < 0.001),and TNF-a(P < 0.001)were lower in the sepsis group.And the level of IL-10(P < 0.05)was higher in the sepsis group,while no difference between the two groups was found in the levels of IL-6 and IL-22.(2)According to the expression levels of Th cell-specific cytokines in the sepsis group and the control group,there were two subgroups found by clustered analysis.(3)Compared with the SIRS group,the levels of IL-4(P < 0.001),IL-5(P < 0.01),IL-17A(P < 0.001),and IL-9(P < 0.01)were lower in the sepsis group.The levels of IL-10(P < 0.05)were higher in the sepsis group.The other cytokines were similar between the two groups.(4)According to the expression levels of Th cell-specific cytokines in the sepsis group and the SIRS group,no subgroup was found by clustered analysis.Conclusion: The general characteristics in pediatric sepsis were Th cell suppression and an increased heterogeneity of immune status.Compared with SIRS children,no difference was found in the profile of Th cell-specific cytokines,but IL-4,IL-5,IL-17 A,IL-9,IL-10 may be useful in the differential diagnosis.Part III Effects of small molecule drug C646 on macrophage function and its therapeutic potential in sepsisObjective: To study the effect and mechanism of small molecule drug C646 on macrophage and to evaluate the effects in murine model of endotoxic shock.Methods: The effect of C646 on the expression of inflammatory cytokines in vitro or in vivo was detected by flow cytometry and Real-time PCR.The effect of C646 on intracellular signaling pathway was detected by Western Blot.The effect of C646 on the binding of LPS to macrophages,phagocytic receptor expression,and the ability of bacterial uptake and phagocytosis was monitored by flow cytometry.The effect of C646 on phagosomal maturation was observed by confocal fluorescence microscopy.The effect of C646 on macrophage bactericidal ability was detected by in vitro assay.To study the effect of C646 on survival rate and tissue inflammatory injury in endotoxic shock model.Results:(1)The expression levels of TNF-a,MCP-1 and IL-6 in macrophages were inhibited by C646.The levels of CD14 and TLR4/MD2 were inhibited,but no obvious effect was found in the binding ability of LPS to macrophages.C646 also inhibits JNK,ERK1/2,and p65 phosphorylation,but not p38 phosphorylation.(2)The expression levels of Fc?RIII/II and CR3 on macrophages were inhibited by C646,but no significant effect on the level of Fc?RI.C646 can also significantly inhibit macrophage uptake and phagocytosis of E.coli,and can inhibit the fusion of the phagosome with the lysosomes.Moreover,macrophage bactericidal activity was inhibited by C646.(3)The plasma levels of TNF-a and IL-6 in endotoxin-shock mice were inhibited by C646,but the survival of mice with endotoxic shock was not improved by the pretreatment with C646.LPS induced lung and liver injury in mice was not reduced by C646 as well.Conclusion: Macrophage-mediated inflammatory response could be inhibited by C646,but the severity of endotoxic shock in mice was not reduced,which may be related to the inhibition of macrophage phagocytosis and bactericidal function.