Role Of The Contact System In Sepsis

Sepsis is defined as organ dysfunction caused by dysregulated host response to infection.Even though great improvement has been achieved in the management of sepsis,sepsis is still a major public health concern and its mortality remains high.Research on its pathogenesis and investigating potential new target for treatment may bring novel therapy for sepsis.During the course of sepsis,a bunch of host response will be initiated including pro-inflammatory response,anti-inflammatory response,activation of coagulation pathways and complement system as well as some immune cells.These host response acts like a double-edged sword,they could help to eliminate the invading pathogens to protect the host while the persistent exacerbated host response could also injure tissues/organs of the host.The contact system is regarded as part of coagulation system,it consists of three zymogen factors(i.e factor ?,factor ? and prekallikrein)and a cofactor highmolecular-weight kininogen(HK).Activation of the contact system is reported in sepsis patients,which may be involved in coagulation fibrinolysis,and inflammation.The role of contact system in the pathogenesis of sepsis remains elusory.The excessive activation of contact system and production of bradykinin could induce hypotension and inflammation,while the cleavage of HK could exert some immune effects by releasing some anti-microbial peptides.The present project aims to study the role of bradykinin during sepsis(Part ?),and investigate the effect of depleting HK or PKK on the host response during gram-negative sepsis(Part ?,?).To this end,we will use corresponding genetic deficient mice or make use of anti-sense oligonucleotide for gene knockdown,and we will set up the sepsis disease model by inoculating alive gramnegative bacteria(Klebsiella pneumoniae)intranasally.We will obtain more knowledge on the role of contact system during sepsis and the mechanism it may involve.Part ? Role of bradykinin in the host response in gram-negative sepsisAim:As an endogenous vasoactive peptide and inflammation mediator,bradykinin(BK)is one of the main end-product by contact activation.We aim to investigate the role of BK in the host response during gram-negative sepsis.Methods:Wild-type C57BL/6J mice treated with BK B2 and/or B1 receptor antagonist(R-715,HOE-140,B-9430)or BK receptor knockout(Bdkrbl/b2-/-)mice were infected by inhaling alive Klebsiella pneumoniae intranasally.Mice were euthanized at 12h or 36h after infection,CFUs in the lung,liver,spleen and blood,and inflammatory cytokine/chemokine levels in the lung were determined.Results:The genetic deficiency of BK receptors or using BK antagonists did not affect the bacterial outgrowth or dissemination both at early and late timepoints.The pulmonary cytokine/chemokine levels were also comparable with corresponding control groups.Conclusion:BK exerts limited effect on the host response during gram-negative sepsis.Key words:bradykinin;bacterial infection;sepsis;B1/B2 receptor;cytokinePart ? Limited role of kininogen in the host response during gramPart ? Limited role of kininogen in the host response during gramnegative pneumonia derived sepsisBackground/Objective:High molecular weight kininogen(HK),together with Factor ?,Factor ? and prekallikrein,is part of the contact system,which has proinflammatory,prothrombotic and vasoactive properties.We hypothesized that HK plays a role in the host response during pneumonia derived sepsis.Methods:Wildtype C57BL/6J mice were depleted of kininogen(KNG)by repeated treatment with a specific antisense oligonucleotide(KNG ASO)for three weeks prior to infection with the common human sepsis pathogen Klebsiella pneumoniae via the airways,resulting in a gradually evolving infection of the lungs followed by sepsis with distant organ injury.Mice were sacrificed at 12/36 hours after infection,blood and organs were collected for measurements.Results:Plasma HK levels increased during infection in mice treated with a scrambled control ASO(Ctrl ASO),while HK level in KNG ASO treated group was reduced to around 30%of that in corresponding Ctrl ASO group both before and after infection.KNG depletion did not influence bacterial growth in lungs or dissemination to distant body sites.KNG depletion was associated with lower lung CXC chemokine and myeloperoxidase levels,but did not impact neutrophil influx,lung pathology,activation of the vascular endothelium,activation of the coagulation system or the extent of distant organ injury.These results were corroborated by studies in mice with a genetic deficiency of KNG,which were indistinguishable from wild-type mice during Klebsiella induced sepsis.Both KNG depletion and KNG deficiency were associated with strongly reduced plasma prekallikrein levels,indicating the carrier function of HK for this zymogen.Conclusion:This study suggests that KNG does not significantly contribute to the host defense during gram-negative pneumonia derived sepsis.Part ? Depletion of prekallikrein improves host defense during gram-negative pneumonia derived sepsisBackground/Objective:Prekallikrein(PKK)is an essential zymogen component of the contact system.Knowledge of the role of PKK in local host defense during bacterial infection and the subsequent development of sepsis is limited.We aim to determine the role of PKK in the host response to gram-negative pneumonia derived sepsis.Methods:We used a common pathogen for pneumosepsis in humans,Klebsiella pneumonia(K.p),to infect C57BL/6J mice via the airways.Mice were pretreated with a PKK antisense oligonucleotide(PKK ASO)to deplete PKK,or a control(Ctrl)ASO.Mice were followed for 10 days after infection,or sacrificed at an early time point(12 h after infection)or during late-stage sepsis(36 h).Activation of contact system was detected in vivo and vitro.RNA was extracted from lung and liver homogenates for qPCR and transcriptome profiling.Results:PKK ASO pretreatment reduced PKK mRNA levels in the liver and PKK protein levels in plasma by over 85%compared with Ctrl ASO.PKK depleted mice had a strongly prolonged survival compared with mice treated with Ctrl ASO,which was accompanied by significantly lower bacterial loads in the lungs,blood and distant organs.PKK depletion reduced pulmonary levels of proinflammatory cytokines and chemokines in both early and late stage of sepsis,and attenuated distal organ damage as reflected by lower plasma levels of transaminases and LDH.Protective effect of PKK ASO was also observed in F? knockout mice.K.p did not activate contact system in vivo or in vitro.Mild increase of cytokine/chemokines were observed in PKK depleted mice before infection,and whole genome transcriptome profiling revealed upregulation of genes enriched in pathways including pathogen recognition,phagocytosis in macrophage,granulocyte adhesion and diapedesis.Conclusion:PKK inhibition improves host defense during Klebsiella induced pneumosepsis as indicated by prolonged survival,diminished bacterial growth and dissemination,and attenuated local and systemic inflammation.PKK may exert its detrimental effect by regulating specific pathways involved in innate/adaptive immunity,rather than by contact pathway since K.p can hardly activate contact system.