Overexpression of activated protein C hampers bacterial dissemination during pneumococcal pneumonia

Abstract

Background: During pneumonia, inflammation and coagulation are activated as part of anti-bacterial host defense. Activated protein C (APC) has anticoagulant and anti-inflammatory properties and until recently was a registered drug for the treatment of severe sepsis. Streptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia.

Methods: We aimed to investigate the effect of high APC levels during experimental pneumococcal pneumonia. Wild type (WT) and APC overexpressing (APC(high))-mice were intranasally infected with S. pneumoniae and sacrificed after 6, 24 or 48 hours, or followed in a survival study.

Results: In comparison to WT mice, APC(high)-mice showed decreased bacterial dissemination to liver and spleen, while no differences in bacterial loads were detected at the primary site of infection. Although no differences in the extent of lung histopathology were seen, APC(high)-mice showed a significantly decreased recruitment of neutrophils into lung tissue and bronchoalveolar lavage fluid. Activation of coagulation was not altered in APC(high)-mice. No differences in survival were observed between WT and APC(high)-mice (P =0.06).

Conclusion: APC overexpression improves host defense during experimental pneumococcal pneumonia. This knowledge may add to a better understanding of the regulation of the inflammatory and procoagulant responses during severe Gram-positive pneumonia.

Figure 1

APC-overexpression is associated with reduced bacterial dissemination . Mice were intranasally inoculated with 5*10⁴ CFU of S. pneumoniae and sacrificed after 6, 24 or 48 hours. Bacterial loads were determined in lung homogenates (A), blood (B), liver homogenates (C) and spleen homogenates (D). Data are expressed as box and whisker plots showing the smallest observation, lower quartile, median, upper quartile and largest observation. Grey boxes represent WT mice, white boxes represent APC^high-mice (n =8 mice per group for each time point). *P <0.05 and **P <0.01 for the difference between WT and APC^high-mice (Mann–Whitney U test). BD below detection limits.

Figure 2

APC-overexpression does not impact on lung pathology but attenuates neutrophil influx into the lungs . Histology scores were similar in WT and APC^high-mice 6, 24 and 48 hours post-infection with 5*10⁴ CFU of S. pneumoniae (A). Representative photographs of WT (B) and APC^high-mice (C) (H&E staining, original magnification x100) 48 hours post-infection. Decreased granulocyte influx as measured by Ly-6G expression in APC^high-mice infected with S. pneumoniae, 24 hours after infection (D). Representative photographs of Ly-6G immunostaining (original magnification x100) for granulocytes of WT (E) and APC^high-mice (F), 24 hours post-infection. APC^high-mice showed decreased numbers of total cell counts in bronchoalveolar lavage fluid (BALF) compared to WT mice (G), which was caused by a decreased influx of neutrophils (H). No differences in influx of macrophages (I) and lymphocytes (J) were found. Data are expressed as box and whisker plots showing the smallest observation, lower quartile, median, upper quartile and largest observation. Numbers of Ly-6G + granulocytes are expressed as the percentage of the total lung surface area. Neutrophils, macrophages and lymphocytes are expressed as absolute numbers. Grey boxes represent WT mice, white boxes represent APC^high-mice (n =8 mice per group for each time point). *P <0.05 and **P <0.01 for WT versus APC^high-mice (Mann–Whitney U test).

Figure 3

APC ^high -mice show an unaltered procoagulant response. Mice were infected intranasally with 5*10⁴ CFU of S. pneumoniae. After 6 and 48 hours levels of TATc were measured in lung homogenates (A) and plasma (B) of WT and APC^high-mice. No differences were seen between WT and APC^high-mice. Grey boxes represent WT mice, white boxes represent APC^high-mice (n =8 mice per group for each time point). *P <0.05 for WT versus APC^high-mice (Mann–Whitney U test). NEM not enough material for analysis.

Figure 4

Survival. APC^high-mice show a trend towards a delayed mortality during pneumococcal pneumonia. Wild type (WT) (n =16) and APC^high-mice (n =12) were infected intranasally with 5*10⁴ CFU of S. pneumoniae and mortality was assessed every 6 hours, Comparison between groups was done by using Kaplan-Meier analysis followed by log rank tests.