Adenosine-A3 receptors in neutrophil microdomains promote the formation of bacteria-tethering cytonemes

The A3‐adenosine receptor (A3AR) has recently emerged as a key regulator of neutrophil behaviour. Using a fluorescent A3AR ligand, we show that A3ARs aggregate in highly polarized immunomodulatory microdomains on human neutrophil membranes. In addition to regulating chemotaxis, A3ARs promote the formation of filipodia‐like projections (cytonemes) that can extend up to 100 μm to tether and ‘reel in’ pathogens. Exposure to bacteria or an A3AR agonist stimulates the formation of these projections and bacterial phagocytosis, whereas an A3AR‐selective antagonist inhibits cytoneme formation. Our results shed new light on the behaviour of neutrophils and identify the A3AR as a potential target for modulating their function

Neutrophil extracellular traps and the dysfunctional innate immune response of cystic fibrosis lung disease:a review

Abstract Background Cystic Fibrosis (CF) is a devastating genetic disease characterised primarily by unrelenting lung inflammation and infection resulting in premature death and significant morbidity. Neutrophil Extracellular Traps (NETs) are possibly key to inflammation in the disease. This review aims to draw together existing research investigating NETs in the context of a dysfunctional innate immune system in CF. Main body NETs have a limited anti-microbial role in CF and studies have shown they are present in higher numbers in CF airways and their protein constituents correlate with lung function decline. Innate immune system cells express CFTR and myeloid-specific CFTR KO mice have greater neutrophil recruitment and higher pro-inflammatory cytokine production to both sterile and bacterial inflammatory challenges. CFTR KO neutrophils have impaired anti-microbial capacity and intrinsic abnormalities in the pH of their cytoplasm, abnormal protein trafficking, increased neutrophil elastase and myeloperoxidase function, and decreased hypochlorite concentrations in their phagolysosomes. Furthermore, neutrophils from CF patients have less intrinsic apoptosis and may be therefore more likely to make NETs. CFTR KO macrophages have high intraphagolysosomal pH and increased toll-like receptor 4 on their cell surface membranes, which inhibit their anti-microbial capacity and render them hyper-responsive to inflammatory stimuli, respectively. Pharmacological treatments for CF target these intrinsic abnormalities of immune dysfunction. Emerging evidence suggests that the absence of CFTR from neutrophils affects NETosis and the interaction of NETs with macrophages. Conclusion Current evidence suggests that NETs contribute to inflammation and lung destruction rather than working effectively in their anti-microbial capacity. Further studies focussing on the pro-inflammatory nature of NET constituents are required to identify the exact mechanistic role of NETs in CF and potential therapeutic interventions

Influences of Chloride and Hypochlorite on Neutrophil Extracellular Trap Formation

Background: The release by neutrophils of DNA-based extracellular traps (NETs) is a recently recognized innate immune phenomenon that contributes significantly to control of bacterial pathogens at tissue foci of infection. NETs have also been implicated in the pathogenesis of non-infectious diseases such as small vessel vasculitis, lupus and cystic fibrosis lung disease. Reactive oxygen species (ROS) are important mediators of NET generation (NETosis). Neutrophils with reduced ROS production, such as those from patients with chronic granulomatous disease or myeloperoxidase (MPO) deficiency, produce fewer NETs in response to inflammatory stimuli. To better understand the roles of various ROS in NETosis, we explore the role of MPO, its substrates chloride ion (Cl 2) and hydrogen peroxide (H2O2), and its product hypochlorite (HOCl) in NETosis. Findings: In human peripheral blood neutrophils, pharmacologic inhibition of MPO decreased NETosis. Absence of extracellular Cl 2, a substrate for MPO, also reduced NETosis. While exogenous addition of H2O2 and HOCl stimulated NETosis, only exogenous HOCl could rescue NETosis in the setting of MPO inhibition. Neither pharmacological inhibition nor genetic deletion of MPO in murine neutrophils blocked NETosis, in contrast to findings in human neutrophils. Conclusions: Our results pinpoint HOCl as the key ROS involved in human NETosis. This finding has implications for understanding innate immune function in diseases in which Cl 2 homeostasis is disturbed, such as cystic fibrosis. Our results also reveal an example of significant species-specific differences in NET phenotypes, and the need for caution i

N-Terminal ArgD Peptides from the Classical Staphylococcus aureus Agr System Have Cytotoxic and Proinflammatory Activities

SummaryAgrD is the precursor for the autoinducing peptide in a quorum-sensing system regulating virulence phenotypes of the preeminent pathogen Staphylococcus aureus. Mass spectrometry-based methods, including molecular networking, identified formylated and nonformylated peptide variants derived from the AgrD N-terminal leader domain in S. aureus cell-free culture supernatants. Functional assessment of these peptides revealed unexpected bioactivities, including human cell-line cytotoxicity, modulation of neutrophil chemotaxis, neutrophil extracellular trap formation, and the aggravation of skin lesions in vivo

NET formation requires extracellular chloride (Cl⁻).

<p>Human peripheral neutrophils (2×10⁶ cells/well) were incubated in minimal media with or without extracellular Cl⁻, and stimulated to form NETs with PMA for 3 h. NETosis was completely inhibited in the absence of extracellular Cl⁻, as seen by (A) immunofluorescence using DAPI to counterstain DNA (blue) and anti-MPO antibody (green) to visualize NETs, and (B) quantification of released extracellular DNA (each condition done in triplicate).</p

Human neutrophils form NETs in response to exogenous oxidants.

<p>(A) Hypochlorite (HOCl) release by activated neutrophils incubated in minimal media with or without Cl⁻ was measured using a colorimetric assay for chlorination of extracellular taurine. Production of HOCl by activated neutrophils requires extracellular Cl⁻. Each condition was done in triplicate. (B,C) NETosis increased in dose-dependent manner in response to exogenous hydrogen peroxide and sodium HOCl, as visualized using immunofluorescence and microscopy (blue: DAPI, green: anti-MPO antibody) (B), and using quantification of released extracellular DNA (C) as assays of NETosis. Neutrophils were plated at 2×10⁶ cells/well.</p