Delayed neutrophil apoptosis enhances NET formation in cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) lung disease is defined by large numbers of neutrophils and associated damaging products in the airway. Delayed neutrophil apoptosis is described in CF although it is unclear whether this is a primary neutrophil defect or a response to chronic inflammation. Increased levels of neutrophil extracellular traps (NETs) have been measured in CF and we aimed to investigate the causal relationship between these phenomena and their potential to serve as a driver of inflammation. We hypothesised that the delay in apoptosis in CF is a primary defect and preferentially allows CF neutrophils to form NETs, contributing to inflammation. METHODS: Blood neutrophils were isolated from patients with CF, CF pigs and appropriate controls. Neutrophils were also obtained from patients with CF before and after commencing ivacaftor. Apoptosis was assessed by morphology and flow cytometry. NET formation was determined by fluorescent microscopy and DNA release assays. NET interaction with macrophages was examined by measuring cytokine generation with ELISA and qRT-PCR. RESULTS: CF neutrophils live longer due to decreased apoptosis. This was observed in both cystic fibrosis transmembrane conductance regulator (CFTR) null piglets and patients with CF, and furthermore was reversed by ivacaftor (CFTR potentiator) in patients with gating (G551D) mutations. CF neutrophils formed more NETs and this was reversed by cyclin-dependent kinase inhibitor exposure. NETs provided a proinflammatory stimulus to macrophages, which was enhanced in CF. CONCLUSIONS: CF neutrophils have a prosurvival phenotype that is associated with an absence of CFTR function and allows increased NET production, which can in turn induce inflammation. Augmenting neutrophil apoptosis in CF may allow more appropriate neutrophil disposal, decreasing NET formation and thus inflammation.

C5a-mediated neutrophil dysfunction is RhoA-dependent and predicts infection in critically ill patients.

Critically ill patients are at heightened risk for nosocomial infections. The anaphylatoxin C5a impairs phagocytosis by neutrophils. However, the mechanisms by which this occurs and the relevance for acquisition of nosocomial infection remain undetermined. We aimed to characterize mechanisms by which C5a inhibits phagocytosis in vitro and in critically ill patients, and to define the relationship between C5a-mediated dysfunction and acquisition of nosocomial infection. In healthy human neutrophils, C5a significantly inhibited RhoA activation, preventing actin polymerization and phagocytosis. RhoA inhibition was mediated by PI3Kδ. The effects on RhoA, actin, and phagocytosis were fully reversed by GM-CSF. Parallel observations were made in neutrophils from critically ill patients, that is, impaired phagocytosis was associated with inhibition of RhoA and actin polymerization, and reversed by GM-CSF. Among a cohort of 60 critically ill patients, C5a-mediated neutrophil dysfunction (as determined by reduced CD88 expression) was a strong predictor for subsequent acquisition of nosocomial infection (relative risk, 5.8; 95% confidence interval, 1.5-22; P = .0007), and remained independent of time effects as assessed by survival analysis (hazard ratio, 5.0; 95% confidence interval, 1.3-8.3; P = .01). In conclusion, this study provides new insight into the mechanisms underlying immunocompromise in critical illness and suggests novel avenues for therapy and prevention of nosocomial infection.

The human cathelicidin LL-37 preferentially promotes apoptosis of infected airway epithelium.

Cationic host defense peptides are key, evolutionarily conserved components of the innate immune system. The human cathelicidin LL-37 is an important cationic host defense peptide up-regulated in infection and inflammation, specifically in the human lung, and was shown to enhance the pulmonary clearance of the opportunistic pathogen Pseudomonas aeruginosa in vivo by as yet undefined mechanisms. In addition to its direct microbicidal potential, LL-37 can modulate inflammation and immune mechanisms in host defense against infection, including the capacity to modulate cell death pathways. We demonstrate that at physiologically relevant concentrations of LL-37, this peptide preferentially promoted the apoptosis of infected airway epithelium, via enhanced LL-37-induced mitochondrial membrane depolarization and release of cytochrome c, with activation of caspase-9 and caspase-3 and induction of apoptosis, which only occurred in the presence of both peptide and bacteria, but not with either stimulus alone. This synergistic induction of apoptosis in infected cells was caspase-dependent, contrasting with the caspase-independent cell death induced by supraphysiologic levels of peptide alone. We demonstrate that the synergistic induction of apoptosis by LL-37 and Pseudomonas aeruginosa required specific bacteria-epithelial cell interactions with whole, live bacteria, and bacterial invasion of the epithelial cell. We propose that the LL-37-mediated apoptosis of infected, compromised airway epithelial cells may represent a novel inflammomodulatory role for this peptide in innate host defense, promoting the clearance of respiratory pathogens.

Cathelicidin host defence peptide augments clearance of pulmonary Pseudomonas aeruginosa infection by its influence on neutrophil function in vivo.

Cathelicidins are multifunctional cationic host-defence peptides (CHDP; also known as antimicrobial peptides) and an important component of innate host defence against infection. In addition to microbicidal potential, these peptides have properties with the capacity to modulate inflammation and immunity. However, the extent to which such properties play a significant role during infection in vivo has remained unclear. A murine model of acute P. aeruginosa lung infection was utilised, demonstrating cathelicidin-mediated enhancement of bacterial clearance in vivo. The delivery of exogenous synthetic human cathelicidin LL-37 was found to enhance a protective pro-inflammatory response to infection, effectively promoting bacterial clearance from the lung in the absence of direct microbicidal activity, with an enhanced early neutrophil response that required both infection and peptide exposure and was independent of native cathelicidin production. Furthermore, although cathelicidin-deficient mice had an intact early cellular inflammatory response, later phase neutrophil response to infection was absent in these animals, with significantly impaired clearance of P. aeruginosa. These findings demonstrate the importance of the modulatory properties of cathelicidins in pulmonary infection in vivo and highlight a key role for cathelicidins in the induction of protective pulmonary neutrophil responses, specific to the infectious milieu. In additional to their physiological roles, CHDP have been proposed as future antimicrobial therapeutics. Elucidating and utilising the modulatory properties of cathelicidins has the potential to inform the development of synthetic peptide analogues and novel therapeutic approaches based on enhancing innate host defence against infection with or without direct microbicidal targeting of pathogens.

Activation of conventional protein kinase C (PKC) is critical in the generation of human neutrophil extracellular traps.

BACKGROUND: Activation of NADPH oxidase is required for neutrophil extracellular trap (NET) formation. Protein kinase C (PKC) is an upstream mediator of NADPH oxidase activation and thus likely to have a role in NET formation. METHODS: Pharmacological inhibitors were used to block PKC activity in neutrophils harvested from healthy donor blood. RESULTS: Pan PKC inhibition with Ro-31-8220 (p<0.001), conventional PKC inhibition with Go 6976 (p<0.001) and specific PKCß inhibition with LY333531 (p<0.01) blocked NET formation in response to PMA. Inhibition of novel and atypical PKC had no effect. LY333531 blocked NET induction by the diacylglycerol analogue OAG (conventional PKC activator) (p<0.001). CONCLUSIONS: Conventional PKCs have a prominent role in NET formation. Furthermore PKCß is the major isoform implicated in NET formation.

The human cathelicidin LL-37 has antiviral activity against respiratory syncytial virus.

Respiratory syncytial virus is a leading cause of lower respiratory tract illness among infants, the elderly and immunocompromised individuals. Currently, there is no effective vaccine or disease modifying treatment available and novel interventions are urgently required. Cathelicidins are cationic host defence peptides expressed in the inflamed lung, with key roles in innate host defence against infection. We demonstrate that the human cathelicidin LL-37 has effective antiviral activity against RSV in vitro, retained by a truncated central peptide fragment. LL-37 prevented virus-induced cell death in epithelial cultures, significantly inhibited the production of new infectious particles and diminished the spread of infection, with antiviral effects directed both against the viral particles and the epithelial cells. LL-37 may represent an important targetable component of innate host defence against RSV infection. Prophylactic modulation of LL-37 expression and/or use of synthetic analogues post-infection may represent future novel strategies against RSV infection.

Antiviral activity and increased host defense against influenza infection elicited by the human cathelicidin LL-37.

The extensive world-wide morbidity and mortality caused by influenza A viruses highlights the need for new insights into the host immune response and novel treatment approaches. Cationic Host Defense Peptides (CHDP, also known as antimicrobial peptides), which include cathelicidins and defensins, are key components of the innate immune system that are upregulated during infection and inflammation. Cathelicidins have immunomodulatory and anti-viral effects, but their impact on influenza virus infection has not been previously assessed. We therefore evaluated the effect of cathelicidin peptides on disease caused by influenza A virus in mice. The human cathelicidin, LL-37, and the murine cathelicidin, mCRAMP, demonstrated significant anti-viral activity in vivo, reducing disease severity and viral replication in infected mice to a similar extent as the well-characterized influenza virus-specific antiviral drug zanamivir. In vitro and in vivo experiments suggested that the peptides may act directly on the influenza virion rather than via receptor-based mechanisms. Influenza virus-infected mice treated with LL-37 had lower concentrations of pro-inflammatory cytokines in the lung than did infected animals that had not been treated with cathelicidin peptides. These data suggest that treatment of influenza-infected individuals with cathelicidin-derived therapeutics, or modulation of endogenous cathelicidin production may provide significant protection against disease.

Secondary necrosis of apoptotic neutrophils induced by the human cathelicidin LL-37 is not proinflammatory to phagocytosing macrophages.

Cathelicidins are CHDP with essential roles in innate host defense but also more recently associated with the pathogenesis of certain chronic diseases. These peptides have microbicidal potential and the capacity to modulate innate immunity and inflammatory processes. PMN are key innate immune effector cells with pivotal roles in defense against infection. The appropriate regulation of PMN function, death, and clearance is critical to innate immunity, and dysregulation is implicated in disease pathogenesis. The efferocytosis of apoptotic PMN, in contrast to necrotic cells, is proposed to promote the resolution of inflammation. We demonstrate that the human cathelicidin LL-37 induced rapid secondary necrosis of apoptotic human PMN and identify an essential minimal region of LL-37 required for this activity. Using these LL-37-induced secondary necrotic PMN, we characterize the consequence for macrophage inflammatory responses. LL-37-induced secondary necrosis did not inhibit PMN ingestion by monocyte-derived macrophages and in contrast to expectation, was not proinflammatory. Furthermore, the anti-inflammatory effects of apoptotic PMN on activated macrophages were retained and even potentiated after LL-37-induced secondary necrosis. However, this process of secondary necrosis did induce the release of potentially harmful PMN granule contents. Thus, we suggest that LL-37 can be a potent inducer of PMN secondary necrosis during inflammation without promoting macrophage inflammation but may mediate host damage through PMN granule content release under chronic or dysregulated conditions.

Proteomic approach to identify candidate effector molecules during the in vitro immune exclusion of infective Teladorsagia circumcincta in the abomasum of sheep.

In the present study we have employed an in vitro organ challenge model to study the post-challenge responses in parasite naïve and immune gastric tissue of sheep, in an attempt to identify the host derived factors involved in immune exclusion of Teladorsagia circumcincta larvae. Proteins present in the epithelial cells and mucus from ovine abomasa following parasite challenge in previously naïve and immune animals were analysed through Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-Tof)-MS and shotgun proteomics. MALDI-ToF analysis of epithelial cell lysates revealed that a number of proteins identified were differentially expressed in naïve and immune cells. These included intelectin and lysozymes, which were present at higher levels in epithelial cell lysates derived from immune samples. A large number of proteins were identified in the mucosal wash from immune tissue which were not present in the mucosal wash of the naïve tissue. Some of these proteins were present in washes of immune tissue prior to the parasite challenge including immunoglobulin A, galectin 14 and 15 and sheep mast cell protease 1. However, other proteins, such as calcium activated chloride channel and intelectin were only detected in the washings from the challenged tissue. The latter may be related to an enhanced mucus release, which may result in entrapment of infective larvae and thus reduced establishment in tissue that has been previously challenged with the parasite. In conclusion, several proteins have been identified which may be involved, either directly or indirectly, in the exclusion and immune elimination of incoming infective larvae. In the present study, the usefulness of the in vitro model has been confirmed, and the global proteomic approach has identified proteins that had not previously been associated with parasite exclusion from abomasal mucosa, such as the calcium activated chloride channel.