Page 266 - Haematologica May 2020

J. Köhler et al.
Results
Plasma proteome from septic mice
In order to gain more information about contact factors in sepsis, we performed a quantitative proteome analysis of plasma from healthy or S. pyogenes-infected mice. The subcutaneous infection model has been shown to become septic and activate the contact system,9 which is similar to the situation encountered in human streptococcal toxic shock syndrome.15 Plasma was collected 24 hours (h) after infection and a total number of 137 proteins including PK, kininogen-1 and FXII could be quantified based on the abundance of at least two peptides (Online Supplementary Table S1). There was a rise in the concentration of 38 pro- teins due to infection (Online Supplementary Table S2). Within this set of elevated proteins, typical positive acute phase proteins such as serum amyloid, C-reactive protein or fibrinogen were detected. In addition, we also identi- fied 47 proteins with significantly decreased concentration due to infection (Table 1). Here, again, we found classical negative acute phase proteins such as retinol binding pro- tein or antithrombin III. PK concentrations were signifi- cantly reduced in infected animals (Table 1). Kininogen-1 and FXII levels were also reduced; however, this was not statistically significant (Online Supplementary Table S1).
F12 and Klkb1 mRNA levels decline in vitro and in vivo after infection with S. pyogenes
We next investigated in vitro how mRNA expression of contact factors is affected in liver cells in response to infec- tion. HepG2 cells were treated with IL6 or living bacteria and mRNA was analyzed by quantitative real-time PCR. In accordance with Citarella et al.,16 F12 mRNA levels were significantly decreased in cells treated with IL6 for 6 and 24 h (Figure 1A). The same was observed in cells infected with S. pyogenes (Figure 1A). Klkb1 mRNA levels also sig- nificantly declined upon treatment with either IL6 or S. pyogenes (Figure 1B).
To investigate the hepatic expression of Klkb1 and F12 mRNA in vivo, we used the streptococcal murine sepsis model. Mice were infected subcutaneously with 1.5-2x107 colony forming units (CFU) of S. pyogenes and samples col- lected 6 and 24 h after infection. Six h after infection, Klkb1 mRNA levels were reduced by approximately 50% compared to non-infected controls (Figure 1C). Twenty- four h after infection, Klkb1 mRNA levels dropped down to undetectable levels (Figure 1C), indicating that Klkb1 mRNA production was discontinued upon bacterial spreading. Relative expression of F12 was also significant- ly reduced at 6 and 24 h after infection (Figure 1D); how- ever, this effect was not as pronounced as for the Klkb1 gene. We also measured fibrinogen alpha (FGA) mRNA levels and found significantly increased FGA expression at 6 and 24 h after infection (Figure 1E), which is consistent with the data from proteome analysis. Ninety-percent of mice were bacteremic at 6 h after infection, containing bacteria in their liver and/or spleen (Figure 1F).
Plasma kallikrein concentration decline significantly in vivo after infection with S. pyogenes
In accordance with the quantitative proteome analysis and Klkb1 mRNA data, we detected a significant decrease in PK levels in mice 24 h after infection, and an even greater decrease 48 h after infection (Figure 2A). This was accompanied by a decreased proteolytic potential of
PK/FXIIa in plasma after activation with Dapptin reagent (Figure 2B), and a significantly prolonged activated partial thromboplastin time (aPTT) (Figure 2C) but not PT (Figure 2D). In accordance with mass spectrometry data, FXII plasma levels did not change significantly in infected mice (data not shown).
Knockdown of plasma prekallikrein diminishes streptococcal dissemination, dampens kidney damage, cyto- and chemokines, but raises RANTES
Such quick downregulation of protein expression during sepsis implicates that the protein is not required, or is even harmful, under these conditions. To investigate the func- tional contribution of PK and FXII during sepsis, we inhib- ited Klkb1 or F12 gene expression by antisense oligonu- cleotides (ASO), using established protocols,11,17 prior to infection with S. pyogenes. As expected, pre-treatment of female BALB/c mice with PPK ASO (Online Supplementary Figure S1A) or FXII ASO (Online Supplementary Figure S1B) reduces relative expression levels more than 90%, which is in accordance with previous studies in male BALB/c mice. As shown before,11,17 plasma protein level of PPK on FXII depletion, or FXII on PPK depletion, were increased (Online Supplementary Figure S1C-F). This indicates stabi- lization of FXII or PPK as response to decreased basal acti- vation. As a consequence of PPK or FXII knockdown, decreased proteolytic potential of PK/FXIIa (Online Supplementary Figure S1G) and prolongation of activated partial thromboplastin time (aPTT) (Online Supplementary Figure S1H) in plasma was demonstrated, compared to mice treated with control ASO. Moreover, as expected, PT was not affected by the treatment with either ASO (data not shown).
The PPK and FXII-depleted mice were challenged with S. pyogenes as described above. Bacterial dissemination and histopathology of the kidneys was determined 24 h after infection. PPK-depleted mice had significantly fewer bacteria in the spleen and blood compared to control-ASO mice (Figure 3A and B). Intriguingly, there was no differ- ence in bacterial loads in spleen, blood or kidneys between FXII-depleted mice and controls (data not shown).
Formation of microvascular fibrin deposition in kidneys was described in lethal human sepsis.18 In our animal model, fibrin was detected 24 h after subcutaneous (sc.) infection in kidneys from septic animals (Figure 3C). A quantification of fibrin areas (> 5 mm) on a scale from 0 to 3 (0: absent; 1: ≤ 20 fibrin areas; 2: 20-50 fibrin areas; 3: >50 fibrin areas) revealed that the mean score in PPK-ASO treated animals was lower (1.7) than that of the control- ASO treated group (2.0) or the FXII-ASO treated group (2.5). This was reflected by increased creatinine values in plasma from control-ASO (8±2.9 mmol/L) and FXII-ASO (8.25±1.9 mmol/L) mice, compared to values in PPK-ASO mice (6±1 mmol/L).
In addition, a panel of 20 cytokines, chemokines and growth factors was measured in healthy, infected control- ASO and PPK-ASO treated mice. Infection boosted the pro-inflammatory response yielding a robust increase of 17 cytokines/chemokines. In agreement with lower bac- terial loads, infected PPK-ASO treated mice had signifi- cantly lower levels of Gm-CsF (Figure 4A) MIP-1 beta (Figure 4B), and MIP-2 (Figure 4C) compared to infected control mice. Interestingly, CCL5 was significantly increased in infected PPK-ASO mice compared to infected controls (Figure 4D).
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