Neutrophils engulf and kill bacteria when their antimicrobial granules fuse with the phagosome. Here, we describe that, upon activation, neutrophils release granule proteins and chromatin that together form extracellular fibers that bind Gram-positive and -negative bacteria. These neutrophil extracellular traps (NETs) degrade virulence factors and kill bacteria. NETs are abundant in vivo in experimental dysentery and spontaneous human appendicitis, two examples of acute inflammation. NETs appear to be a form of innate response that binds microorganisms, prevents them from spreading, and ensures a high local concentration of antimicrobial agents to degrade virulence factors and kill bacteria.

http://science.sciencemag.org/content/sci/303/5663/1532.full.pdf

Neutrophil extracellular traps kill bacteria

Systemic lupus erythematosus (SLE) is a clinically heterogeneous disease, which is autoimmune in origin and is characterized by the presence of autoantibodies directed against nuclear antigens. It is a multi-system disease, and patients can present in vastly different ways. Prevalence varies with ethnicity, but is estimated to be about 1 per 1000 overall with a female to male ratio of 10:1. The clinical heterogeneity of this disease mirrors its complex aetiopathogenesis, which highlights the importance of genetic factors and individual susceptibility to environmental factors. SLE can affect every organ in the body. The most common manifestations include rash, arthritis and fatigue. At the more severe end of the spectrum, SLE can cause nephritis, neurological problems, anaemia and thrombocytopaenia. Over 90% of patients with SLE have positive anti-nuclear antibodies (ANA). Significant titres are accepted to be of 1:80 or greater. SLE is a relapsing and remitting disease, and treatment aims are threefold: managing acute periods of potentially life-threatening ill health, minimizing the risk of flares during periods of relative stability, and controlling the less life-threatening, but often incapacitating day to day symptoms. Hydroxychloroquine and non-steroidal anti-inflammatory drugs are used for milder disease; corticosteroids and immunosuppressive therapies are generally reserved for major organ involvement; anti-CD20 monoclonal antibody is now used in patients with severe disease who has not responded to conventional treatments. Despite enormous improvements in prognosis since the introduction of corticosteroids and immunosuppressive drugs, SLE continues to have a significant impact on the mortality and morbidity of those affected.

/pdf/systemic-lupus-erythematosus-34b38trluu.pdf

Systemic lupus erythematosus

Neutrophils have traditionally been thought of as simple foot soldiers of the innate immune system with a restricted set of pro-inflammatory functions. More recently, it has become apparent that neutrophils are, in fact, complex cells capable of a vast array of specialized functions. Although neutrophils are undoubtedly major effectors of acute inflammation, several lines of evidence indicate that they also contribute to chronic inflammatory conditions and adaptive immune responses. Here, we discuss the key features of the life of a neutrophil, from its release from bone marrow to its death. We discuss the possible existence of different neutrophil subsets and their putative anti-inflammatory roles. We focus on how neutrophils are recruited to infected or injured tissues and describe differences in neutrophil recruitment between different tissues. Finally, we explain the mechanisms that are used by neutrophils to promote protective or pathological immune responses at different sites.

Neutrophil recruitment and function in health and inflammation

Neutrophil extracellular traps (NETs) are extracellular structures composed of chromatin and granule proteins that bind and kill microorganisms. We show that upon stimulation, the nuclei of neutrophils lose their shape, and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate, allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death process is distinct from apoptosis and necrosis and depends on the generation of reactive oxygen species (ROS) by NADPH oxidase. Patients with chronic granulomatous disease carry mutations in NADPH oxidase and cannot activate this cell-death pathway or make NETs. This novel ROS-dependent death allows neutrophils to fulfill their antimicrobial function, even beyond their lifespan.

/pdf/novel-cell-death-program-leads-to-neutrophil-extracellular-3y3jbyjspz.pdf

Novel cell death program leads to neutrophil extracellular traps

Macrophages are heterogeneous and their phenotype and functions are regulated by the surrounding micro-environment. Macrophages commonly exist in two distinct subsets: 1) Classically activated or M1 macrophages, which are pro-inflammatory and polarized by lipopolysaccharide (LPS) either alone or in association with Th1 cytokines such as IFN-γ, GM-CSF, and produce pro-inflammatory cytokines such as interleukin-1β (IL-1β), IL-6, IL-12, IL-23, and TNF-α; and 2) Alternatively activated or M2 macrophages, which are anti-inflammatory and immunoregulatory and polarized by Th2 cytokines such as IL-4 and IL-13 and produce anti-inflammatory cytokines such as IL-10 and TGF-β. M1 and M2 macrophages have different functions and transcriptional profiles. They have unique abilities by destroying pathogens or repair the inflammation-associated injury. It is known that M1/M2 macrophage balance polarization governs the fate of an organ in inflammation or injury. When the infection or inflammation is severe enough to affect an organ, macrophages first exhibit the M1 phenotype to release TNF-α, IL-1β, IL-12, and IL-23 against the stimulus. But, if M1 phase continues, it can cause tissue damage. Therefore, M2 macrophages secrete high amounts of IL-10 and TGF-β to suppress the inflammation, contribute to tissue repair, remodeling, vasculogenesis, and retain homeostasis. In this review, we first discuss the basic biology of macrophages including origin, differentiation and activation, tissue distribution, plasticity and polarization, migration, antigen presentation capacity, cytokine and chemokine production, metabolism, and involvement of microRNAs in macrophage polarization and function. Secondly, we discuss the protective and pathogenic role of the macrophage subsets in normal and pathological pregnancy, anti-microbial defense, anti-tumor immunity, metabolic disease and obesity, asthma and allergy, atherosclerosis, fibrosis, wound healing, and autoimmunity.

Macrophage plasticity, polarization, and function in health and disease.

Neutrophil extracellular traps (NETs) are part of the innate immune response to infections. NETs are a meshwork of DNA fibers comprising histones and antimicrobial proteins. Microbes are immobilized in NETs and encounter a locally high and lethal concentration of effector proteins. Recent studies show that NETs are formed inside the vasculature in infections and noninfectious diseases. Here we report that NETs provide a heretofore unrecognized scaffold and stimulus for thrombus formation. NETs perfused with blood caused platelet adhesion, activation, and aggregation. DNase or the anticoagulant heparin dismantled the NET scaffold and prevented thrombus formation. Stimulation of platelets with purified histones was sufficient for aggregation. NETs recruited red blood cells, promoted fibrin deposition, and induced a red thrombus, such as that found in veins. Markers of extracellular DNA traps were detected in a thrombus and plasma of baboons subjected to deep vein thrombosis, an example of inflammation-enhanced thrombosis. Our observations indicate that NETs are a previously unrecognized link between inflammation and thrombosis and may further explain the epidemiological association of infection with thrombosis.

https://www.pnas.org/content/pnas/107/36/15880.full.pdf

Extracellular DNA traps promote thrombosis

Hyperinflammatory responses can lead to a variety of diseases, including sepsis. We now report that extracellular histones released in response to inflammatory challenge contribute to endothelial dysfunction, organ failure and death during sepsis. They can be targeted pharmacologically by antibody to histone or by activated protein C (APC). Antibody to histone reduced the mortality of mice in lipopolysaccharide (LPS), tumor necrosis factor (TNF) or cecal ligation and puncture models of sepsis. Extracellular histones are cytotoxic toward endothelium in vitro and are lethal in mice. In vivo, histone administration resulted in neutrophil margination, vacuolated endothelium, intra-alveolar hemorrhage and macro- and microvascular thrombosis. We detected histone in the circulation of baboons challenged with Escherichia coli, and the increase in histone levels was accompanied by the onset of renal dysfunction. APC cleaves histones and reduces their cytotoxicity. Co-infusion of APC with E. coli in baboons or histones in mice prevented lethality. Blockade of protein C activation exacerbated sublethal LPS challenge into lethality, which was reversed by treatment with antibody to histone. We conclude that extracellular histones are potential molecular targets for therapeutics for sepsis and other inflammatory diseases.

Extracellular histones are major mediators of death in sepsis

Mer tyrosine kinase (MerTK) is a major macrophage apoptotic cell (AC) receptor. Its functional impairment promotes autoimmunity and atherosclerosis, whereas overexpression correlates with poor prognosis in cancer. However, little is known about mechanisms regulating MerTK expression in humans. We found that MerTK expression is heterogenous among macrophage subsets, being mostly restricted to anti-inflammatory M2c (CD14(+)CD16(+)CD163(+)CD204(+)CD206(+)CD209(-)) cells, differentiated by M-CSF or glucocorticoids. Small numbers of MerTK(+) "M2c-like" cells are also detectable among circulating CD14(bright)CD16(+) monocytes. MerTK expression levels adapt to changing immunologic environment, being suppressed in M1 and M2a macrophages and in dendritic cells. Remarkably, although glucocorticoid-induced differentiation is IL-10 independent, M-CSF-driven M2c polarization and related MerTK upregulation require IL-10. However, neither IL-10 alone nor TGF-β are sufficient to fully differentiate M2c (CD16(+)CD163(+)MerTK(+)) macrophages. M-CSF and IL-10, both released by T lymphocytes, may thus be required together to promote regulatory T cell-mediated induction of anti-inflammatory monocytes-macrophages. MerTK enables M2c macrophages to clear early ACs more efficiently than other macrophage subsets, and it mediates AC clearance by CD14(bright)CD16(+) monocytes. Moreover, M2c cells release Gas6, which in turn amplifies IL-10 secretion via MerTK. IL-10-dependent induction of the Gas6/MerTK pathway may, therefore, constitute a positive loop for M2c macrophage homeostasis and a critical checkpoint for maintenance of anti-inflammatory conditions. Our findings give new insight into human macrophage polarization and favor a central role for MerTK in regulation of macrophage functions. Eliciting M2c polarization can have therapeutic utility for diseases such as lupus, in which a defective AC clearance contributes to initiate and perpetuate the pathological process.

/pdf/efficient-clearance-of-early-apoptotic-cells-by-human-31w0qfd1d5.pdf

Efficient Clearance of Early Apoptotic Cells by Human Macrophages Requires M2c Polarization and MerTK Induction

We previously reported that extracellular histones are major mediators of death in sepsis. Infusion of extracellular histones leads to increased cytokine levels. Histones activate TLR2 and TLR4 in a process that is enhanced by binding to DNA. Activation of TLR4 is responsible for the histone-dependent increase in cytokine levels. To study the impact of histone release on pathology we used two models: a Con A-triggered activation of T cells to mimic sterile inflammation, and acetaminophen to model drug-induced tissue toxicity. Histones were released in both models and anti-histone Abs were protective. TLR2- or TLR4-null mice were also protected. These studies imply that histone release contributes to death in inflammatory injury and in chemical-induced cellular injury, both of which are mediated in part through the TLRs.

/pdf/extracellular-histones-are-mediators-of-death-through-tlr2-4qwevmraj2.pdf

Extracellular Histones Are Mediators of Death through TLR2 and TLR4 in Mouse Fatal Liver Injury

Neutrophils are key effectors of the host innate immune response against bacterial infection. Staphylococcus aureus is a preeminent human pathogen, with an ability to produce systemic infections even in previously healthy individuals, thereby reflecting a resistance to effective neutrophil clearance. The recent discovery of neutrophil extracellular traps (NETs) has opened a novel dimension in our understanding of how these specialized leukocytes kill pathogens. NETs consist of a nuclear DNA backbone associated with antimicrobial peptides, histones and proteases that provide a matrix to entrap and kill various microbes. Here, we used targeted mutagenesis to examine a potential role of S. aureus nuclease in NET degradation and virulence in a murine respiratory tract infection model. In vitro assays using fluorescence microscopy showed the isogenic nuclease-deficient (nuc-deficient) mutant to be significantly impaired in its ability to degrade NETs compared with the wild-type parent strain USA 300 LAC. Consequently, the nuc-deficient mutant strain was significantly more susceptible to extracellular killing by activated neutrophils. Moreover, S. aureus nuclease production was associated with delayed bacterial clearance in the lung and increased mortality after intranasal infection. In conclusion, this study shows that S. aureus nuclease promotes resistance against NET-mediated antimicrobial activity of neutrophils and contributes to disease pathogenesis in vivo.

/pdf/nuclease-expression-by-staphylococcus-aureus-facilitates-ezybjtlk3a.pdf

Marc Monestier

Papers

Extracellular DNA traps promote thrombosis

Extracellular histones are major mediators of death in sepsis

Efficient Clearance of Early Apoptotic Cells by Human Macrophages Requires M2c Polarization and MerTK Induction

Extracellular Histones Are Mediators of Death through TLR2 and TLR4 in Mouse Fatal Liver Injury

Nuclease Expression by Staphylococcus aureus Facilitates Escape from Neutrophil Extracellular Traps