The survival factor was calculated based on the CFU at time point 0 h (see Fig. e, f A.pp was grown for 1 or 3 h at 37 ☌ in the absence or presence of neutrophils (PMN) and external added DNase as indicated. Representative pictures are shown (scale bar = 15 µm). Afterwards staining for NETs was conducted (blue = DNA (Hoechst), green = DNA/histone‐1‐complexes (NETs), red = A.pp). d Neutrophils were incubated with A.pp for 3 h 37 ☌, 5% CO 2 and the cells were fixed. Compared results of the mean values are presented with ±SD ( n = 3, one-way ANOVA P = 0.0031, followed by Tukey’s multiple comparison test). c NET induction was analyzed after treatment of neutrophils with BALF. Compared results of the mean values are presented with ±SD ( n = 3, one-way ANOVA P = 0.0083, followed by Tukey’s multiple comparison test). Per sample, six pictures were taken on two slides at predefined positions and the number of NET-releasing cells were determined. Treated cells were incubated as described in a. b Statistical analysis of NET induction assays was conducted from three independent experiments. Afterwards NET staining for immunofluorescence microscopy was conducted (blue = DNA (Hoechst), green = DNA/histone‐1‐complexes (NETs)). After 3 h incubation at 37 ☌ and 5% CO 2 the cells were fixed. In contrast, A.pp is able to use NETs, which have been degraded by host nucleases or nucleases from other co-infecting bacteria as a source for NAD needed for efficient growth.Ī Primary fresh blood-derived porcine neutrophils were isolated and treated with RPMI as negative control (RPMI, ctr), methyl-β-cyclodextrin (CD) as positive control, A.pp, BALF of uninfected or A.pp-infected pigs. In this study, we show that A.pp induces NETs but does not produce its own secreted NET-degrading nuclease. Furthermore, some bacteria like Streptococcus pneumoniae use NETs for a better spreading in the body 10. Shortly after the discovery of NETs, several authors demonstrated the ability of pathogenic bacteria to avoid entrapment and killing by NETs by extracellular secretion of DNA-degrading DNases 6, 7, 8, 9. The role of NETs during bacterial pneumonia in pigs has not been studied so far. NETs are released by activated neutrophils as response to infections and mediate entrapment and partial killing by released DNA fibers decorated with histones and antimicrobial granule components 4, 5. Neutrophil extracellular traps (NETs) have been characterized as additional extracellular protective defense mechanism against various pathogens 4. Neutrophilic granulocytes are considered as the prominent innate immune cells in this infection 2, but since this pathogen is producing high amount of repeats-in-toxin (RTX)-toxins that prevent phagocytosis, infiltrating neutrophils are not able to eliminate the pathogen 3. Additionally, also acute and chronic infections often occur 1. Peracute diseased pigs that suffer from an Actinobacillus pleuropneumoniae (A.pp) infection often die in <24 h. These data shed light on the detrimental effects of NETs during host immune response against certain bacterial species that require and/or efficiently take advantage of degraded DNA material, which has been provided by host nuclease or nucleases of other co-infecting bacteria, as growth source. influenzae benefits from host nucleases in the presence of neutrophils. A similar phenotype was found for the human pathogen Haemophilus (H.) influenzae and its growth in the presence of human neutrophils. This effect can be diminished by inhibiting the bacterial adenosine synthase indicating that degraded NETs serve as a source for NAD, which is required by A.pp for its growth. Thus, A.pp is not only evading the antimicrobial activity of NETs, A.pp is rather additionally using parts of NETs as growth factor thereby taking advantage of host nucleases as DNase1 or nucleases of co-infecting bacteria, which degrade NETs. Furthermore, the nucleases of co-infecting pathogens like Streptococcus suis increase growth of A.pp in presence of porcine NETs. A.pp itself releases no nuclease, but we identified host nucleases as sources that degrade NETs after A.pp infection. Importantly, A.pp is able to enhance its growth rate in the presence of NETs that have been degraded by nucleases efficiently. Numerous NET-markers were identified in bronchoalveolar lavage fluid (BALF) of A.pp-infected piglets in vivo, however, most NET fibers are degraded. Here we show that neutrophils release neutrophil extracellular traps (NETs) as response to A.pp infection. Neutrophils are the prominent innate immune cell in this infection that massively infiltrate the infected lung. Peracute diseased pigs die in <24 h with pneumonia. Actinobacillus pleuropneumoniae (A.pp) causes severe pneumonia associated with enormous economic loss in pigs.
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