The Role Of Complement System In Regulation Of Adaptive Immunity In Severe Intra-abdominal Infection

Severe sepsis is a multifactorial,progressive,and life-threatening clinical syndrome that results from the innate immune response to infection and can emerge as a complication in conditions like trauma,intra-abdominal infection,surgery,and cancer.Despite advanced progresses made in discovering its pathophysiology,the current therapy,such as source control,fluid resuscitation,and anti-microbial treatment,is insufficient for sepsis management.The sepsis-associated mortality and morbidity rates remain unacceptably high,with poorer outcomes for septic shock with multiple organ dysfunctionsThe complement system consists of about 30 soluble and membrane-bound proteins,and can be activated by various activation cascades(the classic,alternative,lectin pathway,and the fourth way).This system is generally regarded as a first line of fighting against heterologous cells and bacteria,with a firm band to innate immunity.The major functions of complement,mainly through classic or alternative pathway during sepsis,consist of killing target cells with the membrane attack complex,promoting pathogenic phagocytosis by leukocytes and macrophages,and amplifying pro-inflammatory attributes by the release of anaphylatoxins(C3a,C4a,C5a,etc.).However,during sepsis,when complement is excessively activated,complement exhaustion would emerge at last and the original beneficial functions can rapidly threaten to the host.The effects of complement system on adaptive immunity,particularly T-cell-dependent responses,were diverse in various invasive pathogens.For instance,C3a has been reported to favor Thl differentiation by up-regulating the secretion of IL-12 in antigen presenting cells(APCs).Although several hypotheses have been proposed,there is not yet to be a consensus on the precise mechanism by which complement regulates T-cell immunity.In the study,we first explored the complement C3 depletion in late stage of sepsis,and then we evaluated the efficacy of exogenous C3 protein applied in different time on treatment of such sepsis.Afterward,we further investigated the relationship between C3 depletion and inflammatory process during sepsis.At last,we found the potential mechanism for C3 in regulation of adaptive immunity.This thesis can be divided into the following three parts:PART ?:The effect of human complement C3 protein applied in different time on treatment of polymicrobial sepsis Objective:This study was designed to investigate the effect of exogenous complement C3 administration on outcomes of sepsis and identify an optimal time for this therapy.Materials&Methods:Colon ascendens stent peritonitis(CASP)was performed to induce sepsis in C57BL/6 mice,with sham-operated mice as control.Human purified C3(HuC3,1 mg)was administered via intraperitoneal injection,with 200 ?l phosphate-buffered saline as control.Mice were categorized as the initiation time of HuC3 treatment.Survival,bacterial burden,vital organ damages,histology changes,and expression of C3 were compared among the groups.Results:CASP-induced sepsis caused rapid complement C3 depletion and severe organ damages.Vital organs suffered from substantial bacterial loads.Exogenous C3 applied in early stage of sepsis was associated with attenuated organ injuries,enhanced bacterial clearance,and improved survival.Exogenous C3 application promoted the synthesis of C3 in early stage of sepsis.Maybe 6 h post CASP surgery is the optimal time for HuC3 therapy.Conclusions:The study confirmed the positive effect of exogenous C3 on treatment of polymicrobial sepsis.C3 supplementation prior to the appearance of complement depletion could protect vital organs and should be encouraged to apply in early stage of sepsis.PART II:Exogenous C3 postpones complement exhaustion and confers organ protection in murine sepsisObjective:Sepsis in human being is a challenging and life-threatening problem.Complement activation is an essential event in sepsis.This present study observed the dynamic levels of complement components in sepsis and evaluated the role of exogenous complement protein in outcomes.The relationship between complement and inflammatory cytokines was also investigated.Materials&Methods:Colon ascendens stent peritonitis(CASP)surgery was performed in wild-type C57BL/6 mice to induce sepsis.After 6 hours of CASP,a single intraperitoneal injection of human purified C3(HuC3,lmg)was performed,with 200 ?1 phosphate-buffered saline injection for control purpose.Plasma levels of C3,complement factor H(CFH and inflammatory cytokines at different time points were detected.Bacterial burden and organ damages were evaluated after 24 hours of surgical procedure.Results:The plasma C3 levels began to fall at 6 h post CASP,followed by an irreversible process of consumption.A single injection of HuC3 stabilized C3 levels for about 6 hours,decreasing the 24 h mortality from 60%to 20%.Administration of exogenous C3 reduced bacterial burden and attenuated organ injury in sepsis.Plasma levels of CFH and TNF-a were correlated with the depletion of C3.Conclusions:We demonstrated a consumptive depletion of complement components toward the septic peritonitis.Exogenous C3 supplementation in early stage of sepsis is helpful to sustain C3 levels,with enhanced bacterial clearance and improved outcomes.PART III:Exogenous C3 protein enhances the adaptive immune response to sepsis through down-regulation of regulatory T cellsObjective:The role of complement system in bridging innate and adaptive immunity has been confirmed in various invasive pathogens.It is still obscure how complement proteins promote T cell-mediated immune during sepsis.This study was designed to investigate the role of exogenous C3 protein in the T-cell responses to sepsis.Materials&Methods:Sepsis was induced by colon ascendens stent peritonitis(CASP)in wild-type C57BL/6 mice,sham-operated mice for control.Human purified C3 protein(HuC3,1 mg)was intraperitoneally injected at 6 h post-surgery,with 200 ?l phosphate-buffered saline as control.The levels of C3 and cytokines,the expression of FOXP3 and NF-?B,and the percentages of CD4+ T-cell subsets were compared among the groups at given time points.Results:The polymicrobial sepsis produced considerable release of TNF-a and IL-10,and caused complement C3 exhaustion.Exogenous C3 administration markedly improved the 48 h survival rate,as compared with nontreatment(40%vs.5%,P<0.01).The expression of FOXP3 protein was increased during sepsis,but can be suppressed by HuC3 administration.A single injection of HuC3 postponed the decline of differentiated Thl cells,and depressed the activation of Th2/Th17 cells.Besides,the Thl-Th2 shift in late stage of sepsis can be controlled under C3 supplementation.The suppression of NF-?B pathway might be related to the appearance of immunocompromise.Conclusions:The study confirmed the important role of exogenous C3 in up-regulation of adaptive immune response to sepsis.The complement pathway would be a pivotal target for severe sepsis management.