Trends in Immunology
ReviewSpecial issue: novel functions of neutrophilsNETs: a new strategy for using old weapons
Introduction
The innate immune system provides a generic and immediate defense against invading pathogens, and is absolutely essential for the survival of multicellular organisms [1]. Neutrophils play a central role in the innate immune system [2]. Armed and dangerous to both friend and foe alike, they circulate within the blood stream waiting to be called into action. The presence of microbes is detected by resident macrophages and other local sentinel cells, which signal to neutrophils. Neutrophils leave the blood stream and are rapidly recruited to the site of infection. They are typically the first immune cells to appear, whereupon they employ several strategies to contain and clear the infection. Neutrophils have evolved to fulfill a key role in the innate immune system through rapid deployment and effective antimicrobial action against a broad range of pathogens. Hence they are armed with a wide variety of weapons that can be deployed by different microbicidal strategies.
Section snippets
Neutrophil strategies: phagocytosis, degranulation, and NETs
Until a few years ago, neutrophils were thought to employ essentially only two major antimicrobial strategies: 1) phagocytosis which involves the engulfment and subsequent elimination of microbes in specialized phagolysosome compartments and 2) degranulation, which releases antimicrobial molecules in the vicinity of infection. Recently, a third strategy was uncovered i.e. the formation of Neutrophil Extracellular Traps (NETs). NETs arise from the release of the neutrophiĺs nuclear contents into
Laying the traps
Two models describing the release of NETs have been proposed: a novel cell death mechanism, and a DNA extrusion mechanism from intact cells. Fuchs at al. have addressed the question of NET formation by monitoring individual cells via live video microscopy [11]. These experiments demonstrated that ex vivo, activated neutrophils enter a cell-death program where the nuclear and granular membranes dissolve, and the nuclear contents decondense into the cytoplasm. Finally, the plasma membrane
Reactive oxygen species (ROS)
The molecular basis of NET formation is still poorly understood. It is clear however, that ROS play a central role in initiating the program. It was demonstrated that neutrophils derived from patients with the severe immune deficiency Chronic Granulomatous Disease (CGD) are defective in NET formation 11, 13, 15. This defect is caused by mutations in genes that encode the NADPH oxidase and disrupt the ability of the complex to generate ROS [16]. These mutations hamper both the killing of
Decision making
Equally interesting is the question of how the neutrophil decides which of the three microbial killing strategies to pursue. Unlike phagocytosis, NET formation via cell death is final therefore the decision process must be tightly regulated. An appealing hypothesis can be formulated from the timing of these events. Ex vivo, neutrophils engage microbes by phagocytosis within minutes of exposure. The rate of degranulation varies depending on their content, with secretory vesicles being released
Microbial binding, killing and NET antimicrobial factors
NETs appear to be effective against both Gram-positive and Gram-negative bacteria, fungi, and parasites. For instance, NETs have been shown to bind and kill the bacteria S. aureus, Shigella flexeneri, Streptococcus pyogenes, and Bacillus anthracis, the fungus C. albicans, and the protozoan parasite Leishmania amazonensis 11, 13, 19, 30, 31. As mentioned earlier, NETs may play a vital role in combating pathogens which are too large to be phagocytosed such as fungal hyphae and perhaps helminths
NETs as a scaffold: containment of microbes and microbicidal synergy
Since NETs employ most of the same antimicrobials released via degranulation, why would the cell undergo NET formation other than for the release of histones? As a novel strategy, NETs may offer certain advantages: 1) to promote the physical containment of bacteria, 2) to allow synergy between antimicrobial agents and increasing their effective concentration by minimising diffusion, 3) to minimize damage to surrounding tissues by antimicrobials, and 4) to modulate the inflammatory response.
Microbial strategies for evading NETs, and the in vivo role of NETs
NETs have been found in vivo at the sites of infection, in human appendicitis, and associated with pre-eclampsia 3, 33, 68. Furthermore, NET formation has been observed in the bloodstream of animals undergoing septic shock and may be a significant factor in organ failure [12]. Thus far, the molecules that regulate NET formation, downstream of the NADPH oxidase, remain unknown. Therefore, at present, it is not possible to probe the role of NETs exclusively, in genetically modified animals.
NETs as modulators of inflammation
Another interesting facet of NETs is their potential for acting as signaling cues to modulate the inflammatory response and alert the immune system to infection. Dying cells have been shown to trigger neutrophil and monocyte responses [79]. NETs are a product of cell death and promote the co-localization of bacterial adjuvants such as LPS and BLP with extracellular host DNA. Recently, the effect of NETs on human macrophage activation by Mycobacterium tuberculosis was tested. In these
NETs and autoimmunity
Autoimmunity is a defect in the adaptive arm of the immune system whereby antibodies and cytotoxic T cells attack the host leading to tissue and/or organ damage. Antibodies against double stranded DNA (dsDNA), histones and MPO are a hallmark of Systemic Lupus Erythematosus (SLE), and Systemic Vasculitis [87]. Since these molecules are abundant in NETs, it has been postulated that NETs could be contributing to some autoimmune diseases either through the initiation or propagation of disease.
In closing
NETs are still shrouded in a degree of mystery. While we have few answers to some of the more burning questions of NET behavior, there has been plenty of insight into where they may play significant roles. Clearly they are of importance to microbial clearance and containment but equally they may be involved in the initiation and/or pathology of autoimmune disease. Are ETs going to be discovered in other cell types, or are they going to remain the privilege of granulocytes? They certainly seem
Acknowledgements
We thank C. Chaput, F. Meisner, and V. Brinkmann for useful comments on the manuscript and D. Schad for assistance with polishing the figures.
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