Fig 2: PKK depletion results in enhanced expression of multiple innate immune pathways in lungs of uninfected mice. Mice were treated subcutaneously with PKK ASO or control ASO twice weekly for 3 weeks and euthanized 4 days after final dosing. RNA was purified from whole lungs and genome‐wide mRNA expression was analyzed. (A) Volcano plots [integrating adjusted P values and fold‐expression (log2 fold‐change)] depicting the global alteration in gene expression after treatment with PKK ASO relative to control ASO administration. Horizontal line indicates Benjamini–Hochberg (BH) adjusted p < 0.05. Red dots denote overexpressed genes; turquoise dots indicate underexpressed genes. (B) Ingenuity pathway analysis of elevated transcripts (red bars) with Benjamini–Hochberg (BH) adjusted Fisher's p < 0.01 demarcating significance; pathway analysis did not reveal significantly altered pathways for underexpressed genes. (C) Heatmap representation of transcript expression (rows) grouped as Ingenuity canonical signaling pathways in PKK ASO‐ and control ASO‐treated animals. Red, high expression; turquoise, low expression.

Fig 3: Klebsiella pneumoniae does not activate the contact system in vivo or in vitro. Mice were treated subcutaneously with PKK ASO (open bars) or control ASO (grey bars) twice weekly for 3 weeks; 4 days after final dose, mice were infected with K. pneumoniae via airways (t = 0) and euthanized at 12 or 36 h after infection. (A) Western blots showing full length PKK and the cleaved chain of α‐PKa in plasma obtained 36 h after infection using either reduced or non‐reduced samples; naive mouse plasmas pretreated with 100 μg/ml kaolin for the indicated time (15 min to 1 h) were used as positive controls. (B, C) K. pneumoniae, E. coli Ymel or E. coli Ymel‐1 was incubated in citrated plasma from healthy volunteers and bacterial bound PKK (B) and HK (C) proteins were detected by western blotting after reducing SDS‐PAGE. cHK, cleaved chains of HK; α‐PKahc, α‐PKa heavy chain.

Fig 4: Inhibition of PKK expression by PKK ASO treatment. Mice were treated subcutaneously with PKK ASO (open bars) or control (Ctrl) ASO (grey bars) twice weekly for 3 weeks; 4 days after final dose, mice were infected with K. pneumoniae via the airways (t = 0). (A) Western blot of mouse plasma PKK (t = 0). Plasma (0.2 μl diluted in buffer; total volume 10 μl) from four PKK ASO‐ or four Ctrl ASO‐treated mice was loaded; ‘standard’ indicates volumes (μl) of plasma in buffer (total volume 10 μl) from one uninfected mouse (treated with Ctrl ASO). The blot shown is representative of three independent blots. (B) Klkb1 mRNA expression in livers of mice treated with PKK or Ctrl ASO at corresponding time points. (C) Plasma PKK activity. (D) Activated partial thromboplastin time (aPTT). (E) Prothrombin time (PT). Data are mean and SD for bar charts in B and C or medians for box and whisker plots in D and E, eight mice per group. **p < 0.01, ***p < 0.001 (versus Ctrl ASO at the same time point); φ p < 0.05, φφ p < 0.01, φφφ p < 0.001 (versus t = 0 in the same treatment group).

Fig 5: PKK depletion attenuates distant organ injury. Mice were treated subcutaneously with PKK ASO (open bars) or control ASO (grey bars) twice weekly for 3 weeks; 4 days after final dose, mice were infected with K. pneumoniae via airways (t = 0) and euthanized at 12 or 36 h after infection (n = 8 per group at each time point). Data are shown as box and whisker plots with medians. ***p < 0.001.