Final Report Summary - NETSSEPSIS (Defining the mechanisms by which platelets regulate neutrophil extracellular traps in sepsis) The aim of the NETSSEPSIS project was to investigate the molecular mechanisms that regulated the formation of neutrophils extracellular traps (NETs) that trapped and killed bacteria in infectious diseases. Many patients with kidney failure opt for continuous ambulatory peritoneal dialysis (CAPD) at home in favour of haemodialysis with a machine. Bacterial peritonitis, an infection of the peritoneal cavity, is a frequent complication of CAPD and the most common cause of CAPD failure, resulting in switching to haemodialysis. Around 60 % of patients will have at least one episode of peritonitis within a year of starting CAPD. Dissemination of bacteria into the blood stream following the evasion of immune defence is known as sepsis and is a dangerous complication of peritonitis. In addition to traditional mechanisms for killing bacteria, such as phagocytosis, it has recently been observed that activated neutrophils release NETs. These comprise an extracellular scaffold of deoxyribonucleic acid (DNA) that harbours various anti-microbial proteins. Intra-vascular NET can catch circulating bacteria as they pass through the vessels, but are also associated with tissue injury. Levels of free circulating plasma DNA are elevated in sepsis and this correlates with patient outcome. 5-Lipoxygenase (5-LOX) and its free acid products are central to inflammation regulating adhesion, chemotaxis and bacterial killing. However, the known free acid products do not fully account for its bioactivity in vivo, suggesting the participation of further uncharacterised molecular species. This indicates that the identification of novel lipids derived from this pathway was an important and clinically relevant goal. Using lipidomic techniques we identified a new family of 5-LOX derived lipids comprising 5-hydroxyeicosatetraenoic acids (5-HETE) attached to membrane phospholipids (PL). Using precursor-scanning liquid chromatography tandem mass spectrometry (LC/MS/MS), these esterified eicosanoids were structurally characterised (18:0p/5-HETE-PE, 18:1p/5-HETE-PE, 16:0p/5-HETE-PE and 16:0a/5-HETE-PC). They were found in the peritoneal effluent of CAPD patient peritonitis and in an animal model of peritonitis and sepsis, i.e. in intraperitoneal live staphylococcus epidermidis (S epi) in mice. These novel lipid mediators were also formed in vitro by primary human neutrophils, following exposure serum-opsonised S epi or bacterial products. They were formed within two minutes of neutrophil activation in vitro by fMLP or serum-opsonised S epi. These novel esterified eicosanoids were retained by the neutrophils and were present in nuclear and extra-nuclear membrane fractions suggesting an autocrine mode of action. Furthermore, levels were elevated in both murine and human bacterial peritonitis, consistent with their generation in vivo in response to infection. Formation of NETs was significantly attenuated by addition of exogenous phospholipid-esterified HETEs (HETE-PLs) in vitro. Consistent with this, inhibition of 5-LOX enhanced NET formation. Interestingly, when examining other neutrophil responses they were either unaffected or, in the case of superoxide or interleukin-8 generation, modestly enhanced by exogenous 5-HETE-PLs. NETs were important in anti-microbial defence, but their formation occurred at the expense of tissue injury. Regulation of NETs might be an anti-inflammatory or protective activity of these novel lipids. We identified novel neutrophil 5-LOX products that had not been previously described and might be involved in regulatory pathways governing neutrophil function in peritonitis and sepsis. A full understanding of the actions of 5-LOX in regulating the immune system might lead to design of new anti-inflammatory therapies based on mimicking or interfering with the action of these new molecules. Identification of new products of this pathway represented an important step in this direction and was to be followed by a full characterisation of their roles in health and disease.
