| Background:Gram negative?G-?bacterial infection is the leading cause of sepsis.The elevation of circulating endotoxin,also known as lipopolysaccharide?LPS?,released from G-bacteria,has a dominant role in systemic inflammatory response syndrome in sepsis.The host innate immune system is responsible for'sensing'and eliminating the invading G-negative bacteria.When bacterial infection cannot be controlled,bacteria and the released factors such as LPS provoke overwhelmed systemic inflammatory response,as a consequence,the host response becomes unbalanced and harmful,and fails to return to homeostasis.The sustained excessive inflammation is often associated with an increase in circulating LPS levels.Despite significant advances in supportive care and the availability of potent,broad-spectrum antibiotics,the overall mortality due to sepsis is still approximately 35%,and this increases to 60%if patients develop septic shock.Therefore,the mechanisms underlying the transition of the host response from local defense to dysregulated systemic inflammatory state causing multiple organ damage is still poorly understood.Further investigation of the host response to bacterial infection,as well as the uncontrolled inflammatory response to G-bacteria is very important,it will not only provide novel insight into the pathogenesis of sepsis,but will also identify new potential therapeutic targets.High molecular weight kininogen?HK?is a plasma protein and a major component of plasma kallikrein-kinin system?KKS?.Evolutionary analysis has demonstrated that the KKS and the innate immunity have co-evolved,and HK has a central role in modulation of immune response.Moreover,HK binds to a variety of G-bacteria and its cleavage is associated with bacterial infection and sepsis.However,the role of HK in host response to G-bacterial infection has never been characterized.Objective:To determine the role of HK in the host response to G-bacterial infection and the systemic inflammation initiated by LPS,as well as the underlying mechanism.Methods:1.Generation of Kng1-/-miceTo generate a Kng1 knockout strain,Cre/loxp technology was used to delete exon2-3 of the Kng1 gene,which lead to the loss of Kng1 mRNA and immunologically detectable plasma HK.2.The role of HK in host response to bacterial infectionPhenotypes of mice lacking HK(Kng1-/-)mice were characterized in two infection models,E.coli intraperitoneal infection and cecal ligation and puncture?CLP?.Survival rate,H&E staining of lung,bacteria load and circulating cytokine levels were compared between Kng1+/+mice and Kng1-/-mice.3.The effect of HK on E.coli outgrowthTo evaluate the effect of HK on bacterial proliferation,E.coli bacteria were incubated with normal or HK deficient plasma,the changes of OD value were recorded real time using a microplate reader to plot the growth curve of bacteria.The bacteria survival rate were determined by bacteria colony-forming units CFU on LB agar plates.4.The interaction between HK and E.coli?1?In vivo autoradiograpHy imaging System was applied to trace colocalization of125I-HK and E.coli.?2?Flow cytometry was used to analyze the binding capacity of HK to E.coli.5.The mechanism by which HK interact with G-bacteria?1?Using flow cytometry to determine whether HK binding to E.coli is dependent on LPS.?2?Analysis of LPS-associated HK was detected by LPS coated-beads pull-down assay and ELISA affinity test.Surface Plasmon Resonance?SPR?assay was used to quantify the binding affinity of HK for different LPS species from G-bacteria.HK-LPS complexes were further characterized by fast protein liquid chromatograpHy?FPLC?,and the HK and LPS content,as well as the stoichiometry,was subsequently determined.?3?Map the binding site for LPS in D5 of HK.Eight 30-mer peptides with 15 aa overlap within D5 were synthesized.After preincubation with these peptides,LPS coated-beads were incubated with HK,followed by pull-down.The beads-associated proteins were analyzed by anti-HK immunoblotting.?4?To identify whether HK bound to G-bacteria becomes activatedCirculating levels of BK in mice infection with E.coli was measured by ELISA.In plasma from both humans and mice with Gram-negative bacterial infection,the cleaved fragment of HK was detected by 6C9G4,a monoclonal antibody against DHG15peptide?DHGHKHKHGHGHGKH?,which is the binding region of HK in Domain 5 to LPS.6.The underlying mechanism of HK as host defense factor during G-bacterial infection?1?The ExPASy website software?Compute pI/Mw?was used to analyze the pHysicochemical properties of the DHG15 sequence such as pI values and cationicity.?2?The binding ability of DHG15 peptide to E.coli was determined by Flow cytometry.?3?Antibacterial activity of DHG15 was evaluated by Propidium Iodide positive bacteria and CFU counts analyzed by Flow cytometry and viable count assay respectively.?4?Cytotoxicity effect and hemolytic activity of DHG15 peptide was measured by CCK-8 kit and hemolysis assay respectively.?5?The effect of DHG15 peptide on bacterial growth was evaluated in vivo by i.v injection of DHG15 peptide to mice with E.coli infection.7.To determine the role of HK in the host response to LPS induced endotoxemiaKng1+/+and Kng1-/-mice were challenged with a lethal dose of LPS?50 mg/kg?.Survival rate,H&E staining of lung and circulating cytokine levels were compared between Kng1+/+and Kng1-/-mice.8.Effects of HK on LPS clearance and metabolism?1?Kng1-/-mice and their littermate control mice were challenged with LPS?5mg/kg,i.p.?,and blood was collected at the indicated time points.The plasma LPS levels were measured using the chromogenic LAL detection method.The quantitation of 3-hydroxytetradecanoic acid?or 3HM?by HPLC/MS/MS was also used to determined LPS concentration.?2?125I-LPS 0.125ug/g weight was i.v injected into Kng1+/+and Kng1-/-mice.At30 min after injection,the distribution of125I-LPS in liver and plasma was determined by Gamma radioimmunoassay counter.?3?To further determine whether the LPS-binding region DHG15 of HK is important for supporting endotoxemia,a recombinant mutant HK lacking the DHG15?HK/?492-506?was generated.Kng1+/+mice,Kng1-/-mice,and Kng1-/-mice reconstituted with HK/?492-506?n=6?were challenged by LPS?5 mg/kg,i.p.?,followed by measurement of circulating LPS levels.9.The effects of HKa and HK light chain?LC?on LPS activity?1?Dynamic binding of HK and its derivatives to LPS and their capacity in disaggregating LPS was investigated by using a BODIPY FL-LPS fluorescence assay.?2?To determine the role of HKa and LC in LPS-induced inflammatory responses,Raw 264.7 cells were incubated with,or without human HKa or LC in the presence of different species of LPS,the concentration of TNF-?in the culture supernatants was determined using ELISA.10.The therapeutic potential of targeting HK in treatment of LPS induced endotoxemia8-weeks-old WT C57BL/6 mice challenged with LPS were treated with 6C9G4antibody to block the HK-LPS binding or mIgG respectively.Survival rate,circulation LPS levels,H&E staining of lung,BALF's total protein concentration and white blood cell content and circulating cytokine levels were compared.11.The effects of hHKa and mHKa on LPS activity?1?The capacity of hHKa and mHKa in disaggregating LPS was investigated by using a BODIPY FL-LPS fluorescence assay.?2?The role of hHKa and mHKa in LPS-induced inflammatory responses of Raw264.7 cells was determined using ELISA.?3?The concentration of single chain HK?SCHK?which is the selective for binding LPS was measured by Western blotting.Results:1.Generation of Kng1-/-miceGenotyping,using PCR analysis of tail DNA snips,indicated successful excision of the exon 2-3 of Kng1.Kng1 mRNA was absent in the liver of Kng1-/-mice,although Kng2 mRNA remained expressed normally.Moreover,kininogen protein was absent in Kng1-/-mice.Thus,the Kng1-/-mice lack Kng1 mRNA expression in liver tissue and HK antigens in plasma.2.HK improves host defense against G-bacterial infection.Compared with Kng1+/+mice,Kng1-/-mice showed a significantly decrease in survival rate in two infection models,accompanied with enhanced plasma proinflammatory cytokines levels and tissue injury of lung,as well as increased bacterial outgrowth.3.HK deficient plasma increases bacterial survivalWhen incubated E.coli with normal or HK deficient plasma,the proliferation and survival rate of bacteria was significantly higher in HK deficient plasma than normal plasma,indicating that HK has the antibacterial effect.4.HK directly binds to E.coli?1?When 125I-HK was injected into Kng1-/-mice bearing E.coli infection,it was rapidly recruited into the site of infection.?2?As detected by Flow cytometry,HK directly bound to E.coli in a concentration dependent manner,which is saturated at about 100nM HK.5.HK identifies invading G-bacteria by recognizing LPS and possesses antibacterial activity through its DHG15 region.?1?As detected by flow cytometry,the binding of HK to E.coli was almost abolished when EDTA was used to release LPS from E.coli,indicated that HK binds to E.coli dependently on LPS.?2?Plasma HK was found to be specifically bound to LPS beads but not to the control beads.Coating of microplates with HK significantly increased FITC-LPS binding.The sensorgrams showed that HK bound to different LPS species through the carbohydrate region with a high affinity,reflected by dissociation constants?KD?in the picomolar range.FPLC results indicated that HK shifted from an unbound state toward a LPS-bound state in the presence of LPS.The molar ratio of LPS to HK is 1:1.76±0.45,suggesting that on average,one LPS molecule binds one or two HK molecules in the complex.suggesting that HK binds directly to LPS.?3?As indicated by immunoblotting,peptide 6 and peptide 7 blocked the binding of HK to LPS.The overlapping sequence between peptide 6 and peptide 7 was 15 aa in length,namely,DHG HKH KHG HGH GKH?referred to here as DHG15?,suggesting that DHG15 amino acid region in D5 is the binding site of HK for LPS.?4?In plasma from both humans and mice with G-bacterial infection,the cleaved fragment of HK detected by a monoclonal antibody against DHG15 peptide were observed.6.DHG15 peptide is a host defense peptide.DHG15 sequence shares many features with antibacterial peptides?such as cationicity?.Flow cytometry showed that DHG15 binds to E.coli surface and efficiently killed E.coli,the antimicrobial effects of DHG15 is comparable with LL37.Furthermore,no discernible hemolysis or cytotoxic effects on eukaryotic cells were noted.In wild-type mice with E.coli infection,DHG15 significantly decreased bacterial outgrowth in circulation and liver.7.Mice with a deficiency in HK are resistant to LPS-induced mortalityAlthough 90%of the Kng1+/+mice died within 50 h,all of the Kng1-/-mice were still alive when challenged with LPS.Consistently,HK deficiency significantly ameliorated systemic inflammation and lung injury.These results demonstrated that HK deficiency protected mice from LPS-induced mortality.8.?1?HK deficiency decreases circulating endotoxin levelHK deficiency dramatically decreased plasma endotoxin levels in both mice and rats and promoted LPS clearance by liver.?2?LPS-binding region of DHG15 is responsible for supporting endotoxemiaReconstitution of Kng1-/-mice with HK/?492-506 did not lead to a recovery in circulating LPS to the levels of WT mice,supporting the notion that the LPS binding region of DHG15 is responsible for supporting endotoxemia.9.HK amplifies the activity of LPS by its light chain.The two-chain form of HK?cleaved HK,HKa?and its LC directly mediated LPS disaggregation and potentiated TNF-?production in Raw264.7 monocytes challenged with LPS.10.mAb against the DHG15?6C9G4?ameliorates LPS-induced endotoxemiaTreatment with 6C9G4,the mAb against DHG15 of HK significantly increased the survival rate of WT mice challenged with a lethal dose of LPS.Consistent with this,6C9G4 significantly reduced circulating LPS levels and LPS-induced systemic inflammation.In addition,6C9G4 attenuated the lung injury and reduced the total leukocyte number and protein levels in BALF,as well as the MPO levels in lung homogenates.These data suggest that blockade of the HK-LPS binding protects mice against LPS-induced sepsis.11.hHKa and mHKa display similar activities in their interaction with LPSIn disaggregation assay,human HKa and mouse HKa had a similar ability to disaggregate LPS.When incubated with Raw264.7 monocytes,both human HKa and mouse HKa enhanced LPS-stimulated TNF-?to comparable extents.As detected by immunoblots using the anti-DHG15 antibody 6C9G4,the concentration of single chain HK?SCHK?which is the selective for binding LPS in human plasma is about 2 times higher than that in mouse plasma.Conclusion:1.HK is a novel pattern recognition molecules?PRMs?.HK identifies invading G-bacteria by recognizing LPS and possesses antibacterial activity through its DHG15region,therefore contributing to the host defense against G-bacterial infection.2.HK,as the crucial LPS carrier to support LPS levels in circulation,which is likely the mechanism for the pathogenesis of endotoxemia and dysregulated systemic inflammatory response at the late stage of G-bacterial infection.3.The role of HK in host response to G-bacterial infection is a‘double-edged sword'.At the early stage of G-bacterial infection,HK senses G-bacteria and exerts bactericidal effect;When G-bacterial infection cannot be controlled and the released LPS is abundant in circulation,HK serve as the LPS carrier elevating the LPS levels in circulation and promote systemic inflammation.4.HK is an novel therapeutic target for anti-endotoxin treatment. |
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