Role Of Plasma High Molecular Weight Kininogen In Host Defense Against Bacterial Infection And The Underlying Mechanism

Background and significance: High-molecular-weight kininogen(HK) is a major component of plasma kallikrein-kinin system(KKS). Previous work has demonstrated that domain 3 and domain 5 of HK are antibacterial in vitro. However, the physiological role of HK in host defense against bacterial infection has never been characterized using a genetic approach.Objective: To determine the role of HK in host defense against bacterial infection and the underlying mechanism.Methods: We generated Kng1-/- mice by disrupting exons 2 and 3 of murine kininogen-1 gene. Two bacterial infection models were employed in this study. One was E.coli intraperitoneal infection model and the other was cecal ligation and puncture(CLP) model. The phenotype of bacteria-induced lethality, the H&E staining of lung and liver, the bacterial growth in peritoneal fluid, blood, liver and spleen, the white blood cell cytokine m RNA expression profiles were compared between Kng1+/+ mice and Kng1-/- mice. Western blotting was used to detect plasma HK binding to E.coli and the degradation of HK. Kng1+/+ mouse and Kng1-/- mouse plasma and plasma supplemented with Kal and FXII inhibitors(DX-2930 and CTI) were used to determine the role of HK in bacteria clearance and the underlying mechanism.Results:(1) In Kng1-/- mice, the deficiency of kininogen protein in plasma and kininogen m RNA in liver tissue were confirmed by Western blotting and RT-PCR, suggesting that the Kng1-/- mouse model were generated successfully.(2) Kng1-/- mice displayed a significant decreased survival rate in host defense against bacterial infection compared with Kng1+/+ mice(67.5% vs. 11.111%, p=0.0290 in E.coli i.p model; 75% vs. 11.667%, p=0.0087 in CLP model; Kng1+/+ vs. Kng1-/-), suggesting that the presence of HK decreases bacteria-induced lethality.(3) Tissue injury in CLP model was evaluated by histological staining. The H&E staining indicated that the lung and liver of Kng1+/+ mice exhibited mild leukocyte infiltration and damage, suggesting that bacterial infection can cause tissue damage. However, the Kng1-/- mice exhibited significant inflammatory cells infiltration and pulmonary interstitial proliferation in the lung, and obvious ballooning degeneration in the liver.(4) Eighteen hours after intraperitoneal injection of E.coli(108 CFU, 200 ?l), compared with control Kng1+/+ mice, Kng1-/- mice had a decreased bacterial clearance in peritoneal lavage fluid, blood, liver and spleen(peritoneal lavage fluid: 235000±59690 vs. 944400±156400 CFU, p=0.0002; blood: 0.5000±0.1581 vs. 3.938±1.142 CFU, p=0.0056; liver: 90630±32260 vs. 845600±156200 CFU, p<0.0001; spleen: 338100±71100 vs. 1033000±154700 CFU, p= 0.0003; Kng1+/+ vs. Kng1-/-).(5) Six hours after intraperitoneal injection of E.coli(108 CFU, 200 ?l), m RNA expression profiles in white blood cells were determined by Quantitative RT-PCR. Compared with those of Kng1+/+ mice, the expression of TNF-α and IL-6 in white blood cells from Kng1-/- mice challenged by E.coli were significantly increased(TNF-α: 17.00±5.110 vs. 83.20±9.640, p=0.0001; IL-6: 70.61±38.39 vs. 1374±348.4, p=0.0059; Kng1+/+ vs. Kng1-/-), suggesting that mice with deficiency of HK display enhanced inflammatory response to bacterial infection.(6) In the in vitro assay, after plasma incubated with E.coli, HK bound to bacteria and became cleaved. When incubated with E.coli, Kng1-/- mouse plasma had lower clearance of bacteria than Kng1+/+ mouse plasma(21110±4231 vs. 47000±8825 CFU, p=0.0207, after 1-hour incubation; 4020±613.9 vs. 6910±122.74 CFU, p=0.0494, after 2-hour incubation). However, HK protein alone did not affect growth of E.coli in the bactericidal assay, suggesting that the integration of KKS is required for bacterial clearance.(7) The cleavage of HK was blocked when E.coli was incubated with human plasma supplemented with Kal inhibitor DX-2930 or FXII inhibitor CTI. In consistence, the clearance of bacteria in plasma supplemented with PK inhibitor and FXII inhibitor was significantly decreased compared with the control normal human plasma(39600±5913 vs. 68600±7305 CFU, p=0.0128, after 10-hour incubation; 22400±3473 vs. 36800±4236 CFU, p=0.0302, after 20-hour incubation; normal plasma vs. normal plasma supplemented with inhibitors). The results indicate that clearance of bacteria in plasma depends on KKS activation.Conclusion: Our results demonstrate for the first time a novel function of plasma HK in host defense against bacterial infection, and its bactericidal activity depends on the activation of plasma KKS.