Trends in Immunology
Volume 30, Issue 11, November 2009, Pages 513-521
Journal home page for Trends in Immunology

Review
Special issue: novel functions of neutrophils
NETs: a new strategy for using old weapons

https://doi.org/10.1016/j.it.2009.07.011Get rights and content

As key players in the host innate immune response, neutrophils are recruited to sites of infection and constitute the first line of defense. They employ three strategies to eliminate invading microbes: microbial uptake, the secretion of antimicrobials, and the recently described release of Neutrophil Extracellular Traps (NETs). Composed of decondensed chromatin and antimicrobial proteins, NETs bind and kill a variety of microbes including bacteria, fungi, and parasites. In addition to using a repertoire of known antimicrobials, NETs incorporate histones into the antimicrobial arsenal. Furthermore, NETs may contribute to microbial containment by forming a physical barrier and a scaffold, to enhance antimicrobial synergy while minimizing damage to host tissues. Their role in innate immunity is only now being uncovered.

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.

References (93)

  • V. Ramos-Kichik

    Neutrophil extracellular traps are induced by Mycobacterium tuberculosis

    Tuberculosis (Edinburgh, Scotland)

    (2009)
  • K. Beiter

    An endonuclease allows Streptococcus pneumoniae to escape from neutrophil extracellular traps

    Curr Biol

    (2006)
  • J.T. Buchanan

    DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps

    Curr Biol

    (2006)
  • A. Coxon

    A novel role for the beta 2 integrin CD11b/CD18 in neutrophil apoptosis: a homeostatic mechanism in inflammation

    Immunity

    (1996)
  • B. Zhang

    Elucidation of molecular events leading to neutrophil apoptosis following phagocytosis: cross-talk between caspase 8, reactive oxygen species, and MAPK/ERK activation

    The Journal of biological chemistry

    (2003)
  • K. Fujie

    Release of neutrophil elastase and its role in tissue injury in acute inflammation: effect of the elastase inhibitor, FR134043

    European journal of pharmacology

    (1999)
  • O.H. Ryu

    Proteolysis of macrophage inflammatory protein-1alpha isoforms LD78beta and LD78alpha by neutrophil-derived serine proteases

    The Journal of biological chemistry

    (2005)
  • J. Zhu

    Conversion of proepithelin to epithelins: roles of SLPI and elastase in host defense and wound repair

    Cell

    (2002)
  • F.Y. Jin

    Secretory leukocyte protease inhibitor: a macrophage product induced by and antagonistic to bacterial lipopolysaccharide

    Cell

    (1997)
  • S. Lobov

    Structural bases of the redox-dependent conformational switch in the serpin PAI-2

    Journal of molecular biology

    (2004)
  • P.E. Desrochers

    Proteolytic inactivation of alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin by oxidatively activated human neutrophil metalloproteinases

    The Journal of biological chemistry

    (1992)
  • T.W. Stief

    Oxidative inactivation of purified human alpha-2-antiplasmin, antithrombin III, and C1-inhibitor

    Thrombosis research

    (1988)
  • K. Beatty

    Kinetics of association of serine proteinases with native and oxidized alpha-1-proteinase inhibitor and alpha-1-antichymotrypsin

    The Journal of biological chemistry

    (1980)
  • D. Belorgey et al.

    DNA binds neutrophil elastase and mucus proteinase inhibitor and impairs their functional activity

    FEBS letters

    (1995)
  • H.W. Huang

    Molecular mechanism of antimicrobial peptides: the origin of cooperativity

    Biochimica et biophysica acta

    (2006)
  • A.K. Gupta

    Induction of neutrophil extracellular DNA lattices by placental microparticles and IL-8 and their presence in preeclampsia

    Hum Immunol

    (2005)
  • C.G. Messina

    Catalase negative Staphylococcus aureus retain virulence in mouse model of chronic granulomatous disease

    FEBS letters

    (2002)
  • J.A. Lekstrom-Himes et al.

    Immunodeficiency diseases caused by defects in phagocytes

    The New England journal of medicine

    (2000)
  • C. Nathan

    Neutrophils and immunity: challenges and opportunities

    Nat Rev Immunol

    (2006)
  • V. Brinkmann

    Neutrophil extracellular traps kill bacteria

    Science (New York, N.Y

    (2004)
  • P.M. Henson et al.

    Tissue injury in inflammation. Oxidants, proteinases, and cationic proteins

    The Journal of clinical investigation

    (1987)
  • S.J. Weiss

    Tissue destruction by neutrophils

    The New England journal of medicine

    (1989)
  • T.A. Fuchs

    Novel cell death program leads to neutrophil extracellular traps

    J Cell Biol

    (2007)
  • S.R. Clark

    Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood

    Nature medicine

    (2007)
  • S. Yousefi

    Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense

    Nature medicine

    (2008)
  • F.A. Clark et al.

    Chronic granulomatous disease: studies of a family with impaired neutrophil chemotactic, metabolic and bactericidal function

    The American journal of medicine

    (1978)
  • M. Bianchi

    Restoration of NET formation by gene therapy in CGD controls aspergillosis

    Blood

    (2009)
  • B.H. Segal

    Aspergillus nidulans infection in chronic granulomatous disease

    Medicine

    (1998)
  • C.F. Urban

    Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms

    Cell Microbiol

    (2006)
  • S. Bozza

    Dendritic cells transport conidia and hyphae of Aspergillus fumigatus from the airways to the draining lymph nodes and initiate disparate Th responses to the fungus

    J Immunol

    (2002)
  • G.R. Tintinger

    Accelerated calcium influx and hyperactivation of neutrophils in chronic granulomatous disease

    Clinical and experimental immunology

    (2001)
  • E.P. Reeves

    Killing activity of neutrophils is mediated through activation of proteases by K+ flux

    Nature

    (2002)
  • T.E. DeCoursey

    Electrophysiology of the phagocyte respiratory burst. Focus on “Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity”

    American journal of physiology

    (2007)
  • Y. Wang

    Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation

    J Cell Biol

    (2009)
  • I. Neeli

    Histone deimination as a response to inflammatory stimuli in neutrophils

    J Immunol

    (2008)
  • B.J. Bentwood et al.

    The sequential release of granule constitutents from human neutrophils

    J Immunol

    (1980)
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