Neutrophils are innate immune phagocytes that have a central role in immune defence. Our understanding of the role of neutrophils in pathogen clearance, immune regulation and disease pathology has advanced dramatically in recent years. Web-like chromatin structures known as neutrophil extracellular traps (NETs) have been at the forefront of this renewed interest in neutrophil biology. The identification of molecules that modulate the release of NETs has helped to refine our view of the role of NETs in immune protection, inflammatory and autoimmune diseases and cancer. Here, I discuss the key findings and concepts that have thus far shaped the field of NET biology.

Neutrophil elastase escapes azurophilic granules, translocates to the nucleus, and degrades histones to promote chromatin decondensation necessary for NET formation.

Secretion of the cytokine interleukin-1β (IL-1β) by macrophages, a major driver of pathogenesis in atherosclerosis, requires two steps. First, priming signals promote transcription of immature IL-1β and then, endogenous "danger" signals activate innate immune signaling complexes called inflammasomes, to process IL-1β processing for secretion. While cholesterol crystals act as danger signals in atherosclerosis, what primes IL-1β transcription remains elusive. Using a murine model of atherosclerosis, we show that cholesterol crystals acted both as priming and danger signals for IL-1β production. Cholesterol crystals triggered neutrophils to release neutrophil extracellular traps (NETs). NETs primed macrophages for cytokine release, activating Th-17 cells that amplify immune cell recruitment in atherosclerotic plaques. Therefore, danger signals may drive sterile inflammation, such as that seen in atherosclerosis, through their interactions with neutrophils.

Neutrophils are critical for antifungal defense, but the mechanisms that clear hyphae and other pathogens that are too large to be phagocytosed remain unknown. We show that neutrophils sense microbial size and selectively release neutrophil extracellular traps (NETs) in response to large pathogens, such as Candida albicans hyphae and extracellular Mycobacterium bovis aggregates, but not small yeast and single bacteria. NETs are fundamental in countering large pathogens in vivo. Phagocytosis via dectin-1, acts as a sensor for microbial size preventing NETosis by downregulating neutrophil elastase (NE) translocation to the nucleus. Dectin-1 deficiency leads to aberrant NETosis and NET-mediated tissue damage during infection. Size-tailored neutrophil responses clear large microbes and minimize pathology when microbes are small enough to be phagocytosed.

IntroductionMyeloperoxidase (MPO) is one of the most abundant proteins in neutrophils, accounting for 5% of the dry weight of the cell. 1 Stored in the azurophilic granules and released when neutrophils are stimulated, MPO catalyzes the oxidation of chloride and other halide ions in the presence of hydrogen peroxide 2,3 to generate hypochlorous acid and other highly reactive products that mediate efficient antimicrobial action. 4,5 Several inherited mutations and deletions in the gene encoding MPO result in decreased enzyme production and activity. 6,7 Using automated hematological devices, clinicians can distinguish between partial and complete MPO deficiencies. 8 MPO deficiency is reported to have an incidence of 1 in 2000-4000 in the United States and Europe and 1 in 55 000 in Japan. 9-13 Candida infections are common in MPO-deficient patients, especially in those that also develop diabetes. 9,14-18 Occasionally, serious infectious or inflammatory complications have been observed in completely MPOdeficient patients as well. 8 Consistently, MPO knockout mice are susceptible to particular bacterial and fungal infections. 19 Neutrophil extracellular traps (NETs) are part of the neutrophil response to microbes. Activated neutrophils die and release these structures composed of decondensed chromatin and antimicrobial proteins 20,21 that trap and inhibit a broad range of microbes. 22 Little is known about the molecular mechanism that regulates NET formation, making the antimicrobial role of NETs in vivo difficult to assess.Interestingly, neutrophils from chronic granulomatous disease (CGD) patients fail to make NETs. 20 CGD is caused by mutations that disrupt the ability of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to generate superoxide, which dismutates to hydrogen peroxide, the substrate of MPO. CGD patients are prone to recurrent and severe infections, as well as to persistent inflammation that can occur independently of infection. [23][24][25] NET formation by CGD neutrophils is restored by the addition of exogenous hydrogen peroxide, indicating that reactive oxygen species are required for NET formation. 20 Here we show that MPO is necessary for making NETs and suggest that defective NET formation may undermine host defense in patients lacking MPO. Methods Donor consentAll donors gave consent to blood drawing in accordance with the Declaration of Helsinki and to functional and genetic analysis. Samples were collected with approval from the ethical committees at each institution. Neutrophil isolationNeutrophils were isolated by centrifuging heparinized venous blood over Histopaque 1119 (Sigma-Aldrich) and subsequently over a discontinuous Percoll (Amersham Biosciences) gradient as described previously. 20 Cells were stored in Hank buffered salt solution (-) or Dulbecco phosphatebuffered saline (-), without calcium or magnesium, before experiments. NET formation and visualizationNeutrophils (5 ϫ 10 4 ) were seeded per well in 24-well tissue culture plates, in Hanks buffered salt solution (...