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.

Keywords: Cystic fibrosis; DNase; Inflammation; Macrophages; NETs; Neutrophil extracellular traps; Neutrophils.

Fig. 1

Overlay of immunofluorescence images demonstrating co-localisation of DNA and myeloperoxidase (MPO) on in vitro NETs stained with Hoechst antibody (blue, binds DNA) and anti-MPO antibody (green)

Fig. 2

Neutrophil Extracellular Trap (NET) production by a neutrophil. Neutrophils are stimulated to form NETs by several microbial, inflammatory and sterile endogenous triggers. These bind onto cell surface receptors including Toll-like Receptor 4, cytokine and complement receptors. Receptor binding leads to increased calcium release from the endoplasmic reticululm, activating Protein Kinase C (PKC). This leads to activation of NADPH Oxidase on the cell membrane and lysosomes, forming superoxide which reacts with water and chloride to form hypochlorite. Hypochlorite activates Protein Arginine Deiminase 4 (PAD4) which translocates to the nucleus where it catalyses hypercitrullination of histones 3 and 4 [26]. This causes the histones to lose their positive charge and in doing so weakens their binding to DNA, leading to decondensation of chromatin. There is loss of plasma membrane integrity then decondensed chromatin and histones are expelled into the extracellular space where they form complexes with granule/cytosolic proteins such as myeloperoxidase, neutrophil elastase and calprotectin. Recent research suggests NET production is an end-point of numerous cell signalling pathways – not all of which require each of the above steps – dependent upon the stimulant used to induce NETosis [25].

Fig. 3

Several mechanisms require investigation concerning the dysfunctional innate immune response and NETs in Cystic Fibrosis (CF). It may be that CFTR ^−/− neutrophils are hyper-stimulatory to macrophages due to increased NET production and CFTR ^−/− alveolar macrophages produce higher levels of pro-inflammatory cytokines, driving chemotaxis of more neutrophils in a self-perpetuating cycle of inflammation. Co-culture experiments with airway epithelial cells, NETs and macrophages would mimic in vivo conditions. Targeting this cycle, e.g. by reducing NET production, inhibiting NET-protein’s deleterious functions or promoting NET clearance would provide new targets for the treatment of CF