Induction of neutrophil extracellular traps (NETs) by the pneumococcal toxin, pneumolysin (Ply) (original) (raw)
Pneumolysin activates neutrophil extracellular trap formation
The primary objective of the current study was to investigate the potential of the pneumococcal toxin, pneumolysin (Ply), to activate neutrophil extracellular trap (NET) formation in vitro. Isolated human blood neutrophils were exposed to recombinant Ply (5-20 ng ml21) for 30–90 min at 378C and NET formation measured using the following procedures to detect extracellular DNA: (i) flow cytometry using VybrantVR DyeCycleTM Ruby; (ii) spectrofluorimetry using the fluorophore, SytoxVR Orange (5 lM); and (iii) NanoDropVR technology. These procedures were complemented by fluorescence microscopy using 40, 6-diamino-2-phenylindole (DAPI) (nuclear stain) in combination with anti-citrullinated histone monoclonal antibodies to visualize nets. Exposure of neutrophils to Ply resulted in relatively rapid (detected within 30–60 min), statistically significant (P < 005) dose- and time-related increases in the release of cellular DNA impregnated with both citrullinated histone and myeloperoxidase. Microscopy revealed that NETosis appeared to be restricted to a subpopulation of neutrophils, the numbers of NET-forming cells in the control and Ply-treated systems (10 and 20 ng ml21) were 43 (42), 14.3 (99) and 165 (75), respectively (n 5 4, P < 00001 for comparison of the control with both Ply-treated systems). Ply-induced NETosis occurred in the setting of retention of cell viability, and apparent lack of involvement of reactive oxygen species and Toll-like receptor 4. In conclusion, Ply induces vital NETosis in human neutrophils, a process which may either contribute to host defence or worsen disease severity, depending on the intensity of the inflammatory response during pneumococcal infection.
Neutrophil extracellular traps (NETs) - formation and implications
Acta Biochimica Polonica, 2013
Neutrophils are cells of the immune system which freely circulate in blood vessels and are recruited to the inflammation sites when the human organism responds to microbial infections. One of the mechanisms of neutrophil action is the formation of neutrophil extracellular traps (NETs) The process of NET generation, called netosis, is a specific type of cell death, different from necrosis and apoptosis. NETs are formed by neutrophils upon contact with various bacteria or fungi as well as with activated platelets or under the influence of numerous inflammatory stimuli, and this process is associated with dramatic changes in the morphology of the cells. The main components of NETs, DNA and granular antimicrobial proteins, determine their antimicrobial properties. The pathogens trapped in NETs are killed by oxidative and non-oxidative mechanisms. On the other hand, it was also discovered that chromatin and proteases released into the circulatory system during NET formation can regulate ...
Microbial Pathogenesis, 2019
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Neutrophil Extracellular Traps in Infectious Human Diseases
Role of Neutrophils in Disease Pathogenesis, 2017
Neutrophils, as the main cells of the first line of host defense against microbial pathogens, are responsible for pathogen recognition, inhibition of pathogen spreading into the host tissue, and finally, killing the invader cells. Neutrophils carry out these functions via numerous mechanisms, including a relatively recently described activity based on a release of neutrophil extracellular traps (NETs), a process called netosis. NETs are structures composed of DNA backbone, decorated with antimicrobial factors, derived from neutrophil granules. The structure of NETs and their enzymatic and microbicidal inclusions enable efficient trapping and killing of microorganisms within the neutrophil extracellular space. However, the efficiency of NETs depends on neutrophil ability to recognize pathogen signals and to trigger rapid responses. In this chapter, we focus on possible pathways involved in the release of NETs and summarize the current knowledge on triggers of this process during bacterial, fungal, protozoan, and viral infections. We also consider the mechanisms used by microorganisms to evade NET-killing activity and analyze the harmful potential of NETs against the host cells and the contribution of NETs to noninfectious human diseases.
Microbial Pathogenesis, 2020
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
FEMS Immunology & Medical Microbiology, 2010
Epithelial cells act as the first line of host defense against microorganisms by producing a range of molecules for clearance. Proinflammatory cytokines facilitate the clearance of invaders by the recruitment and activation of leukocytes. Upregulation of cytokine expression thus represents an important host innate defense response against invading microorganisms such as Streptococcus pneumoniae. Histological analysis of the airway revealed less leukocyte infiltration during the early stage of pneumococcal infection, when compared with nontypable Haemophilus influenzae (NTHi) infection. Here, we report that S. pneumoniae is less potent in inducing proinflammatory cytokine expression compared with NTHi. Among numerous virulence factors, pneumococcal pneumolysin was found to be the major factor responsible for the induction of inflammation. Interestingly, pneumolysin induces cytokine expression to a lesser extent at the early stage of infection, but becomes more potent in inducing inflammation at the late stage. Thus, this study reveals that pneumolysin induces the proinflammatory cytokine expression in a time-dependent manner.
bioRxiv (Cold Spring Harbor Laboratory), 2023
Streptococcus pneumoniae is a global priority respiratory pathogen that kills over a million people annually and produces the pore-forming cytotoxin, pneumolysin (PLY). Host cells expel membrane assembled toxin by shedding microvesicles, but the composition and pathophysiological sequelae of the toxin-induced host extracellular vesicles (EVs) are unknown. Here, we found that EVs shed from PLY-challenged monocytes (PLY-EVs) harbor membrane-bound toxin that induced cytotoxicity upon fusion with recipient cells. EVs from human monocytes challenged with recombinant PLY as well as PLY-expressing pneumococcal strains, but not the isogenic PLY mutant, primed dendritic cells and evoked higher pro-inflammatory cytokines upon infection. Proteomic analysis revealed that PLY-EVs are enriched for key antimicrobial and inflammatory host proteins such as IFI16, NLRC4, PTX3 and MMP9. In vivo, zebrafish and mice administered with PLY-EVs showed mortality, pericardial edema, tissue damage and inflammation. Our findings show that host EVs bearing the cytotoxin PLY constitute a previously unexplored mechanism of pneumococcal pathogenesis.
Innate immunity, 2017
Neutrophils play an important role in the innate immune response to infection with Streptococcus pneumoniae, the pneumococcus. Pneumococci are phagocytosed by neutrophils and undergo killing after ingestion. Other cellular processes may also be induced, including autophagy and the formation of neutrophil extracellular traps (NETs), which may play a role in bacterial eradication. We set out to determine how these different processes interacted following pneumococcal infection of neutrophils, and the role of the major pneumococcal toxin pneumolysin in these various pathways. We found that pneumococci induced autophagy in neutrophils in a type III phosphatidylinositol-3 kinase dependent fashion that also required the autophagy gene Atg5. Pneumolysin did not affect this process. Phagocytosis was inhibited by pneumolysin but enhanced by autophagy, while killing was accelerated by pneumolysin but inhibited by autophagy. Pneumococci induced extensive NET formation in neutrophils that was n...