Abstract
Mycoplasma bovis (M. bovis) is a significant worldwide pathogen of cattle. Neutrophils have an important role in the innate immune response during infection with M. bovis. However, even though neutrophils accumulate in M. bovis infection, the interaction of M. bovis and neutrophils has not been fully elucidated. We attempted to elucidate the innate immune response of neutrophils stimulated with M. bovis and evaluate the transcriptome and functional analysis of bovine neutrophils stimulated with M. bovis. Proinflammatory cytokines, such as inducible nitric oxide (iNOS), which was the most increased gene in transcriptome analysis, were increased in quantitative polymerase chain reaction analysis of bovine neutrophils stimulated with live or heat-killed M. bovis. Nitric oxide and intracellular reactive oxygen species production of neutrophils stimulated with M. bovis was significantly increased. Neutrophils stimulated with M. bovis showed an increased ratio of nonapoptotic cell death compared to unstimulated controls. We demonstrated that neutrophil extracellular traps (NETs) formation was not recognized in neutrophils stimulated with live M. bovis. However, heat-killed M. bovis induced NETs formation. We also showed the interaction with M. bovis and bovine neutrophils regarding proinflammatory cytokine gene expression and functional expression related to NETs formation. Live and killed M. bovis induced innate immune responses in neutrophils and had the potential to induce NETs formation, but live M. bovis escaped NETs.
Highlights
Mycoplasmas are classified under the class Mollicutes, which do not have a cell wall and are cause widespread infections of eukaryotes in nature [1]
Detection of apoptotic cells and quantity of nitric oxide (NO) and reactive oxygen species (ROS) production To evaluate NO production and the ratio of apoptosis cells in neutrophils stimulated with M. bovis, neutrophils (2 × 105 cells/200 μL RPMI 1640 medium in 96-well tissue culture plates) were incubated in the presence of live M. bovis at an Multiplicity of infection (MOI) of 1000 (10 μL) for 1, 3, and 6 h at 37 °C and under 5% CO2
To measure the quantity of intracellular ROS in neutrophils stimulated with M. bovis, neutrophils (2 × 105 cells/1 mL RPMI 1640 medium in a 3 cm dish) were stimulated with 10 μL conteining live M. bovis and/or phorbol myristate acetate (PMA; SigmaAldrich Corp.) or phosphate-buffered saline (PBS) for control at an MOI of 1000 for 30 min at 37 °C and 5% CO2
Summary
Mycoplasmas are classified under the class Mollicutes, which do not have a cell wall and are cause widespread infections of eukaryotes in nature [1]. Mycoplasma bovis (M. bovis) is a significant worldwide pathogen of cattle [2, 3] and is known to cause pneumonia, arthritis, and mastitis [2, 4], resulting in calf mortality, weight loss in surviving calves, and decreased milk production in dairy cows [2, 5], which all contribute to significant economic losses [2, 6]. In M. bovis infection, neutrophils constitute the major accumulation of cells at an infection site [7]. M. bovis reportedly suppressed the production of reactive oxygen species (ROS) in the immune response of neutrophils [8]. ROS is the major innate immune factor of neutrophils to pathogens and is required for neutrophil extracellular traps (NETs) formation [9]. M. bovis was considered to escape the host immune response, and we previously reported that M. bovis escaped bovine NETs following the degradation of nucleic acid [12]
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