Limited role of kininogen in the host response during gram-negative pneumonia-derived sepsis

Abstract

High-molecular-weight kininogen (HK), together with factor XI, factor XII and prekallikrein, is part of the contact system that has proinflammatory, prothrombotic, and vasoactive properties. We hypothesized that HK plays a role in the host response during pneumonia-derived sepsis. To this end mice were depleted of kininogen (KNG) to plasma HK levels of 28% of normal by repeated treatment with a specific antisense oligonucleotide (KNG ASO) for 3 wk before infection with the common human sepsis pathogen Klebsiella pneumoniae via the airways. Whereas plasma HK levels increased during infection in mice treated with a scrambled control ASO (Ctrl ASO), HK level in the KNG ASO-treated group remained reduced to 25-30% of that in the 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. This study suggests that KNG does not significantly contribute to the host defense during gram-negative pneumonia-derived sepsis.

Keywords: contact system; high-molecular weight kininogen; kallikrein-kinin system.

Fig. 1.

High-molecular-weight kininogen (HK) levels before and after induction of pneumonia-derived sepsis in mice pretreated with control (Ctrl) antisense oligonucleotide (ASO) or kininogen (KNG) ASO. Target gene (Kng1) expression level indicated as its mRNA by quantitative RT-PCR (A) and plasma HK level by Western blot after SDS-PAGE under reducing conditions (B); plasma HK levels before and during Klebsiella pneumonia in mice treated with Ctrl ASO (n = 8) or KNG ASO (n = 8) (D and E). Quantification of HK levels in naïve and infected mice (C) determined by densitometry of the HK bands at 120 kDa in B, D, and E and compared with a serial dilution of plasma as shown in B. Serial data were analyzed by Kruskal-Wallis test followed by Mann-Whitney U-tests. ^ΦP < 0.05 vs. corresponding time (t) = 0 group; *P < 0.05 and ***P < 0.001, Mann-Whitney U-tests between KNG ASO vs. Ctrl ASO.

Fig. 7.

Effect of kininogen depletion or kininogen genetic deficiency on prekallikrein (PKK) plasma level and factor XI (FXI) activity level. Plasma PKK levels in noninfected and 36-h-infected mice were determined by Western blot (nonreduced, A); full-length PKK (85- and 88-kDa doublet) was quantified by densitometry of PKK band with ImageJ (B). The mean values from Ctrl ASO mice or WT mice are presented as 100%. Kaolin-activated mouse plasma was used as a negative control. C: plasma FXI activity of 36-h-infected WT and Kng1^−/− mice. Data in B and C are presented as box-and-whisker plots showing interquartile ranges and medians. *P < 0.05 and **P < 0.01, Mann-Whitney U-test.

Fig. 2.

Kininogen depletion does not influence bacterial growth or dissemination during pneumonia-derived sepsis. Bacterial loads [colony-forming units (CFUs)] in the lung, blood, liver, and spleen 12 and 36 h after infection with K. pneumoniae via the airways in mice pretreated with Ctrl ASO or KNG ASO. Data were expressed as box-and-whisker diagrams (10-Log) of 8 mice/group at each time point. At 12 h the vast majority of cultures from blood, liver, and spleen were sterile, reflecting the initially localized infection. Differences between groups were not significant with Mann-Whitney U-test.

Fig. 3.

Kininogen depletion does not influence neutrophil influx but lowers myeloperoxidase (MPO) levels in lungs during pneumonia-derived sepsis. Ly6G (neutrophil) stainings of lung sections from mice before (A and B) and 12 (C and D) or 36 (E and F) h after infection with K. pneumoniae via the airways. A, C, and E are representative images from Ctrl ASO-treated mice; B, D, and F are from KNG ASO-treated mice. Original magnification ×4. G: percentage of Ly6G-positive area in lung analyzed with Image J. H: MPO levels in lung homogenates. Data are expressed as box-and-whisker diagrams showing interquartile ranges with medians of 8 mice/group at each time point. *P < 0.05 and **P < 0.01, Mann-Whitney U-test.

Fig. 4.

Histopathological change in the lung during pneumonia-derived sepsis. Hematoxylin and eosin (H&E) staining of lung sections from mice before (A and B) and 12 (C and D) or 36 (E and F) h after infection with K. pneumoniae via the airways. A, C, and E are representative images from Ctrl ASO-treated mice; B, D, and F are from KNG ASO-treated mice. Original magnification ×4. G: severity of inflammation in the lung was evaluated and scored as described in materials and methods.

Fig. 5.

Effect of kininogen depletion on plasma biomarkers for endothelial activation, coagulation activation, and organ injury during pneumonia-derived sepsis. Endothelial (A and B) and coagulation activation (C) were investigated both before and 12 and 36 h after infection, whereas the biomarkers for liver (D and E) and general organ injury (F) were measured at 12 and 36 h after infection. Data are from mice pretreated with Ctrl or KNG ASO (8 mice/group at each time point) and presented as box-and-whisker plots showing interquartile ranges and medians. sE-selectin, soluble E-selectin; cVCAM-1, soluble vascular cell adhesion molecule-1; TATc, thrombin-antithrombin complexes; ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase; LDH, lactate dehydrogenase. *P < 0.05, Mann-Whitney U-test.

Fig. 6.

Kininogen deficiency does not influence neutrophil influx in lungs or bacterial growth or dissemination during pneumonia-derived sepsis. Data are from wild-type (WT) and Kng1^−/− mice (8 mice/group) at 36 h after infection and presented as box-and-whisker plots showing interquartile ranges and medians. A: plasma HK level of WT and Kng1^−/− mice indicated by Western blot after SDS-PAGE under reducing conditions. B: percentage of Ly6G-positive area in the lung. C: bacterial loads (CFUs) in the lung, blood, liver, and spleen at 36 h after infection with K. pneumoniae in WT and Kng1^−/− mice. Differences between groups were not significant with Mann-Whitney U-test.