Impact of bronchoalveolar lavage from influenza A virus diseased pigs on neutrophil functions and growth of co-infecting pathogenic bacteria.

Introduction: Influenza A viruses (IAVs) infect the respiratory tract of mainly humans, poultry, and pigs. Co-infections with pathogenic lung bacteria are a common event and contribute to the severity of disease progression. Neutrophils are a major cell type of the innate immune system and are rapidly recruited to the site of infection. They have several effector functions to fight invading pathogens such as the secretion of reactive oxygen species (ROS) or the release of neutrophil extracellular traps (NETs). NETs are known to promote the growth of Pasteurellaceae bacteria, especially if degraded by nucleases. Methods: In this study, bronchoalveolar lavage fluid (BALF) from 45 field-infected pigs was analyzed for 1) NET markers, 2) influence on growth of lung bacteria, and 3) impact on neutrophil functions. BALF samples from 21 IAV-positive pigs and 24 lung diseased but IAV-negative pigs were compared. Results: Here, we show that neutrophils in the lungs of IAV-positive pigs release vesicular NETs. Several NET markers were increased in the BALF of IAV-positive pigs compared with the BALF from IAV-negative pigs. The amount of NET markers positively correlated with the viral load of the IAV infection. Interestingly, the BALF of IAV-positive pigs enhanced the growth of bacteria belonging to the family of Pasteurellaceae as potential coinfecting bacteria. These effects were weaker with the BALF derived from IAV-negative pigs with other lung infections. The intensity of oxidative burst in neutrophils was significantly decreased by BALF from IAVpositive pigs, indicating impaired antimicrobial activity of neutrophils. Finally, the lung milieu reflected by IAV-positive BALF does not enable neutrophils to kill Actinobacillus pleuropneumoniae but rather enhances its growth. Discussion: In summary, our data show that an IAV infection is affecting neutrophil functions, in particular the release of NETs and ROS. Furthermore, IAV infection seems to provide growth-enhancing factors for especially coinfecting Pasteurellaceae and reduces the killing efficiency of neutrophils.

The effect of doxycycline on canine neutrophil functions.

Doxycycline is a broad-spectrum tetracycline-class antibiotic that is frequently used to treat bacterial infections. Its use has also been described in immune-mediated diseases due to its immunomodulatory properties. The aim of this study was to evaluate the immunomodulatory effect of doxycycline on canine neutrophil functions. Therefore, the release of reactive oxygen species (ROS) and the formation of neutrophil extracellular traps (NETs) were determined after incubation of canine PMNs with doxycycline in three different concentrations (4 µg/mL, 20 µg/mL and 200 µg/mL) for one and three hours, respectively. Additionally, a neutrophil killing assay with a doxycycline-resistant Staphylococcus aureus was performed to determine the bactericidal effect of doxycycline treated PMNs in presence of plasma. Doxycycline significantly diminished the production of ROS. However, doxycycline concentrations of 4 µg/mL and 20 µg/mL significantly induced NETs. A synergistic bacteriostatic effect of PMNs and doxycycline on a doxycycline-resistant Staphylococcus aureus isolate was detectable. However, already PMNs and especially doxycycline alone inhibited the growth. In summary, doxycycline showed a concentration-dependent immunomodulatory property in canine PMNs with a reduced ROS production and increased NET-induction. This immunomodulatory effect resulted in a slightly increased elimination of a doxycycline-resistant Staphylococcus aureus by the doxycycline plasma concentrations achieved in dogs.

Studying the Interaction of Neutrophils and Glaesserella Parasuis Indicates a Serotype Independent Benefit from Degradation of NETs.

Glaesserella (G.) parasuis is one of the most important porcine pathogens causing Glaesser's disease. Neutrophil granulocytes are the major counteracting cell type of the innate immune system, which contribute to the host defense by phagocytosis or the formation of neutrophil extracellular traps (NETs). Recently, NET-formation has been shown to facilitate the survival of bacteria from the Pasteurellaceae family. However, the interaction of NETs and G. parasuis is unclear so far. In this study, we investigated the interplay of three G. parasuis serotypes with porcine neutrophils. The production of reactive oxygen species by neutrophils after G. parasuis infection varied slightly among the serotypes but was generally low and not significantly influenced by the serotypes. Interestingly, we detected that independent of the serotype of G. parasuis, NET formation in neutrophils was induced to a small but significant extent. This phenomenon occurred despite the ability of G. parasuis to release nucleases, which can degrade NETs. Furthermore, the growth of Glaesserella was enhanced by external DNases and degraded NETs. This indicates that Glaesserella takes up degraded NET components, supplying them with nicotinamide adenine dinucleotide (NAD), as this benefit was diminished by inhibiting the 5'-nucleotidase, which metabolizes NAD. Our results indicate a serotype-independent interaction of Glaesserella with neutrophils by inducing NET-formation and benefiting from DNA degradation.

Methylprednisolone Induces Extracellular Trap Formation and Enhances Bactericidal Effect of Canine Neutrophils.

Methylprednisolone is a glucocorticoid and can negatively influence immune defense mechanisms. During bacterial infections in the dog, neutrophils infiltrate infected tissue and mediate antimicrobial effects with different mechanisms such as phagocytosis and neutrophil extracellular trap (NET) formation. Here, we investigated the influence of methylprednisolone on canine NET formation and neutrophil killing efficiency of Gram positive and Gram negative bacteria. Therefore, canine blood derived neutrophils were treated with different concentrations of methylprednisolone over time. The survival factor of Staphylococcus pseudintermedius, Streptococcus canis or Escherichia coli was determined in presence of stimulated neutrophils. Additionally, free DNA and nucleosomes as NET marker were analyzed in supernatants and neutrophils were assessed for NET formation by immunofluorescence microscopy. Methylprednisolone concentrations of 62.5 and 625 µg/mL enhanced the neutrophil killing of Gram positive bacteria, whereas no significant influence was detected for the Gram negative Escherichia coli. Interestingly, higher amounts of free DNA were detected under methylprednisolone stimulation in a concentration dependency and in the presence of Streptococcus canis and Escherichia coli. The nucleosome release by neutrophils is induced by bacterial infection and differs depending on the concentration of methylprednisolone. Furthermore, immunofluorescence microscopy analysis identified methylprednisolone at a concentration of 62.5 µg/mL as a NET inducer. In summary, methylprednisolone enhances NET-formation and time-dependent and concentration-dependent the bactericidal effect of canine neutrophils on Gram positive bacteria.