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A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide
Published on: December 15, 2014
Pneumolysin Induces 12-Lipoxygenase-Dependent Neutrophil Migration during Streptococcus pneumoniae Infection
Walter Adams1,2, Rudra Bhowmick1, Elsa N Bou Ghanem1
1Department of Molecular Biology and Microbiology, Tufts University, Boston, MA 02111.
Abstract:
Streptococcus pneumoniae is a major cause of pneumonia, wherein infection of respiratory mucosa drives a robust influx of neutrophils. We have previously shown that S. pneumoniae infection of the respiratory epithelium induces the production of the 12-lipoxygenase (12-LOX)-dependent lipid inflammatory mediator hepoxilin A3, which promotes recruitment of neutrophils into the airways, tissue damage, and lethal septicemia. Pneumolysin (PLY), a member of the cholesterol-dependent cytolysin (CDC) family, is a major S. pneumoniae virulence factor that generates ∼25-nm diameter pores in eukaryotic membranes and promotes acute inflammation, tissue damage, and bacteremia. We show that a PLY-deficient S. pneumoniae mutant was impaired in triggering human neutrophil transepithelial migration in vitro. Ectopic production of PLY endowed the nonpathogenic Bacillus subtilis with the ability to trigger neutrophil recruitment across human-cultured monolayers. Purified PLY, several other CDC family members, and the α-toxin of Clostridium septicum, which generates pores with cross-sectional areas nearly 300 times smaller than CDCs, reproduced this robust neutrophil transmigration. PLY non-pore-forming point mutants that are trapped at various stages of pore assembly did not recruit neutrophils. PLY triggered neutrophil recruitment in a 12-LOX-dependent manner in vitro. Instillation of wild-type PLY but not inactive derivatives into the lungs of mice induced robust 12-LOX-dependent neutrophil migration into the airways, although residual inflammation induced by PLY in 12-LOX-deficient mice indicates that 12-LOX-independent pathways also contribute to PLY-triggered pulmonary inflammation. These data indicate that PLY is an important factor in promoting hepoxilin A3-dependent neutrophil recruitment across pulmonary epithelium in a pore-dependent fashion.
Insights
Pneumolysin (PLY), a toxin from Streptococcus pneumoniae, triggers neutrophil recruitment via pore formation and 12-lipoxygenase (12-LOX) signaling. This mechanism is crucial for pneumonia pathogenesis and airway inflammation.
Area of Science:
- Microbiology
- Immunology
- Molecular Biology
Background:
- Streptococcus pneumoniae causes pneumonia, characterized by neutrophil influx into the respiratory mucosa.
- Pneumolysin (PLY), a cholesterol-dependent cytolysin (CDC) from S. pneumoniae, is a key virulence factor that forms pores in cell membranes, driving inflammation and tissue damage.
- Previous research identified a 12-lipoxygenase (12-LOX)-dependent pathway involving hepoxilin A3 in S. pneumoniae-induced neutrophil recruitment.
Purpose of the Study:
- To investigate the role of Pneumolysin (PLY) in neutrophil recruitment during S. pneumoniae infection.
- To determine if PLY's pore-forming activity is essential for triggering neutrophil migration.
- To elucidate the involvement of the 12-lipoxygenase (12-LOX) pathway in PLY-mediated neutrophil recruitment.
Main Methods:
- Assessed neutrophil transepithelial migration in vitro using PLY-deficient S. pneumoniae mutants and wild-type PLY.
- Transferred PLY-expressing genes into non-pathogenic Bacillus subtilis to evaluate its effect on neutrophil recruitment.
- Utilized purified PLY, other CDC members, and alpha-toxin, along with non-pore-forming PLY mutants, to study pore-dependent effects.
- Administered wild-type PLY and its derivatives to mouse lungs to assess in vivo neutrophil migration and inflammation, including in 12-LOX-deficient mice.
Main Results:
- A PLY-deficient S. pneumoniae mutant showed impaired ability to trigger human neutrophil transepithelial migration in vitro.
- Ectopic expression of PLY in Bacillus subtilis induced neutrophil recruitment across human cell monolayers.
- Purified PLY and other pore-forming toxins, but not non-pore-forming PLY mutants, robustly promoted neutrophil transmigration.
- In vivo, wild-type PLY induced significant 12-LOX-dependent neutrophil migration into mouse airways, though 12-LOX-independent pathways also contributed to inflammation.
Conclusions:
- Pneumolysin (PLY) is a critical virulence factor that promotes neutrophil recruitment across pulmonary epithelium.
- PLY's pore-forming activity is essential for triggering this neutrophil migration, acting in a pore-dependent manner.
- The PLY-induced neutrophil recruitment is largely dependent on the 12-lipoxygenase (12-LOX) pathway and hepoxilin A3 production.
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