Christine Schauer

Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines

Gout is characterized by an acute inflammatory reaction and the accumulation of neutrophils in response to monosodium urate (MSU) crystals. Inflammation resolves spontaneously within a few days, although MSU crystals can still be detected in the synovial fluid and affected tissues. Here we report that neutrophils recruited to sites of inflammation undergo oxidative burst and form neutrophil extracellular traps (NETs). Under high neutrophil densities, these NETs aggregate and degrade cytokines and chemokines via serine proteases. Tophi, the pathognomonic structures of chronic gout, share characteristics with aggregated NETs, and MSU crystals can induce NETosis and aggregation of NETs. In individuals with impaired NETosis, MSU crystals induce uncontrolled production of inflammatory mediators from neutrophils and persistent inflammation. Furthermore, in models of neutrophilic inflammation, NETosis-deficient mice develop exacerbated and chronic disease that can be reduced by adoptive transfer of aggregated NETs. These findings suggest that aggregated NETs promote the resolution of neutrophilic inflammation by degrading cytokines and chemokines and disrupting neutrophil recruitment and activation.

PMA and crystal‐induced neutrophil extracellular trap formation involves RIPK1‐RIPK3‐MLKL signaling

Neutrophil extracellular trap (NET) formation contributes to gout, autoimmune vasculitis, thrombosis, and atherosclerosis. The outside‐in signaling pathway triggering NET formation is unknown. Here, we show that the receptor‐interacting protein kinase (RIPK)‐1‐stabilizers necrostatin‐1 or necrostatin‐1s and the mixed lineage kinase domain‐like (MLKL)‐inhibitor necrosulfonamide prevent monosodium urate (MSU) crystal‐ or PMA‐induced NET formation in human and mouse neutrophils. These compounds do not affect PMA‐ or urate crystal‐induced production of ROS. Moreover, neutrophils of chronic granulomatous disease patients are shown to lack PMA‐induced MLKL phosphorylation. Genetic deficiency of RIPK3 in mice prevents MSU crystal‐induced NET formation in vitro and in vivo. Thus, neutrophil death and NET formation may involve the signaling pathway defining necroptosis downstream of ROS production. These data imply that RIPK1, RIPK3, and MLKL could represent molecular targets in gout or other crystallopathies.

Neutrophil extracellular trap formation can involve RIPK1-RIPK3-MLKL signalling.

Neutrophil extracellular trap (NET) formation contributes to gout, autoimmune vasculitis, thrombosis, and atherosclerosis. The outside‐in signaling pathway triggering NET formation is unknown. Here, we show that the receptor‐interacting protein kinase (RIPK)‐1‐stabilizers necrostatin‐1 or necrostatin‐1s and the mixed lineage kinase domain‐like (MLKL)‐inhibitor necrosulfonamide prevent monosodium urate (MSU) crystal‐ or PMA‐induced NET formation in human and mouse neutrophils. These compounds do not affect PMA‐ or urate crystal‐induced production of ROS. Moreover, neutrophils of chronic granulomatous disease patients are shown to lack PMA‐induced MLKL phosphorylation. Genetic deficiency of RIPK3 in mice prevents MSU crystal‐induced NET formation in vitro and in vivo. Thus, neutrophil death and NET formation may involve the signaling pathway defining necroptosis downstream of ROS production. These data imply that RIPK1, RIPK3, and MLKL could represent molecular targets in gout or other crystallopathies.

Why does the gout attack stop? A roadmap for the immune pathogenesis of gout

Gout is one of the most severe and frequent rheumatic diseases. Clinical manifestations of gout arise from uric acid crystal deposition in the musculoskeletal tissue. At high concentrations of uric acid in the body (hyperuricaemia), needle-shaped monosodium urate (MSU) crystals are formed. The structures are ingested by neutrophils and monocytes and thereby trigger robust activation of the inflammasome, an intracellular protein complex mounting an inflammatory response. Inflammasome activation builds interleukin-1, which acts as a proinflammatory mediator and induces vasodilation, recruitment of additional leucocytes and the expression of proinflammatory cytokines and chemokines. This process is associated with the clinical manifestation of an acute gout attack. Such attacks, however, stop rather rapidly and the process of resolution of inflammation in gout is now better defined. Neutrophils having ingested MSU crystals undergo a specific form of cell death called NETosis, which is characterised by the formation of neutrophil extracellular traps (NETs). During this process, DNA is extruded, allowing the dense packaging of MSU crystals as well as the degradation of proinflammatory cytokines, thereby allowing the stopping of the inflammatory process. Reactive oxygen species are essential for forming NETs and for allowing the resolution of inflammation in gout. This process of NETosis is critical for understanding tophaceous gout, since tophi are composed of NETs and densely packed MSU crystals.

Nanoparticles size-dependently initiate self-limiting NETosis-driven inflammation

Significance The current widespread exposure of humans to natural as well as man-made nanomaterials due to the deployment of nanoparticles (NPs) as food additives, as vaccine- or drug-delivery vehicles, and in diagnostic procedures encourages the evaluation of their interaction with the innate immune system. Understanding how organisms cope with hydrophobic and chemically inert particulate matter, which is excluded from metabolic processing, is of major importance for interpreting the responses associated with the use of NPs in the biosphere. The containment of NPs within neutrophil-derived aggregates locally orchestrates the resolution of inflammation. Overriding this mechanism bears the risk of inducing chronic inflammation and causing tissue damage. The critical size for strong interaction of hydrophobic particles with phospholipid bilayers has been predicted to be 10 nm. Because of the wide spreading of nonpolar nanoparticles (NPs) in the environment, we aimed to reveal the ability of living organisms to entrap NPs via formation of neutrophil extracellular traps (NETs). Upon interaction with various cell types and tissues, 10- to 40-nm-sized NPs induce fast (<20 min) damage of plasma membranes and instability of the lysosomal compartment, leading to the immediate formation of NETs. In contrast, particles sized 100–1,000 nm behaved rather inertly. Resulting NET formation (NETosis) was accompanied by an inflammatory reaction intrinsically endowed with its own resolution, demonstrated in lungs and air pouches of mice. Persistence of small NPs in joints caused unremitting arthritis and bone remodeling. Small NPs coinjected with antigen exerted adjuvant-like activity. This report demonstrates a cellular mechanism that explains how small NPs activate the NETosis pathway and drive their entrapping and resolution of the initial inflammatory response.

How neutrophil extracellular traps orchestrate the local immune response in gout

Neutrophil granulocytes possess a large arsenal of pro-inflammatory substances and mechanisms that empower them to drive local acute immune reactions to invading microorganisms or endogenous inflammatory triggers. The use of this armory needs to be tightly controlled to avoid chronic inflammation and collateral tissue damage. In gout, inflammation arises from precipitation of uric acid in the form of needle-shaped monosodium urate crystals. Inflammasome activation by these crystals in local immune cells results in a rapid and dramatic recruitment of neutrophils. This neutrophil influx is accompanied by the infamously intense clinical symptoms of inflammation during an acute gout attack. Neutrophilic inflammation however is equipped with a built-in safeguard; activated neutrophils form neutrophil extracellular traps (NETs). At the very high neutrophil densities that occur at the site of inflammation, NETs build aggregates that densely pack the monosodium urate (MSU) crystals and trap and degrade pro-inflammatory mediators by inherent proteases. Local removal of cytokines and chemokines by aggregated NETs explains how acute inflammation can stop in the consistent presence of the inflammatory trigger. Aggregated NETs resemble early stages of the typical large MSU deposits that constitute the pathognomonic structures of gout, tophi. Although tophi contribute to muscosceletal damage and mortality in patients with chronic gout, they can therefore be considered as a payoff that is necessary to silence the intense inflammatory response during acute gout.

Review: Neutrophils as Invigorated Targets in Rheumatic Diseases.

Neutrophil biology. Neutrophils are terminally differentiated innate immune cells that play fundamental roles in host defense against microbes. The bone marrow is the primary site of neutrophil production, and under conditions of homeostasis, these cells can also be found in the lung, spleen, and liver. Characteristically, neutrophils have a short half-life, and constant production in the bone marrow is needed to maintain a steady state (1). However, there is still significant uncertainty regarding neutrophil turnover in the bone marrow and circulation under homeostatic and nonhomeostatic conditions (for review, see ref. 2). Neutrophil production is tightly controlled through synthesis of granulocyte-colony-stimulating factor (G-CSF) in response to interleukin-17 (IL-17). Neutrophils express 4 categories of intracytoplasmic granules: primary (azurophilic), which contain among many proteins myeloperoxidase (MPO), defensins, neutrophil elastase, and membrane-bound CD63, secondary, which contain various molecules, such as lactoferrin, LL-37 peptide, and membrane-bound CD66b, tertiary, which are loaded with gelatinase, cathepsin, and various toxic proteins, and secretory vesicles, which are considered an important store of membrane-bound receptors and which express alkaline phosphatase and CD35 on their membranes (3) (Figure 1). In the circulation, neutrophils remain in a resting state, ensuring that their toxic intracellular material is not accidentally released. Significant decreases in neutrophil numbers or their function can lead to severe infections, while very tight regulation of these cells is crucial to preventing tissue injury (4). Under inflammatory conditions, circulating neutrophils are recruited to tissues according to a gradient, and their half-life may increase to support proper response to noxious stimuli (1,3). Inflammatory tissue neutrophils share many of the functions of macrophages, including synthesis of cytokines and chemokines, phagocytosis, and presentation of major histocompatibility complex (MHC) class II–dependent antigen. In tissues, neutrophils use different mechanisms to destroy pathogens, including phagocytosis, degranulation, and a distinct form of cell death characterized by the extracellular extrusion of structures formed by intracellular proteins bound to a meshwork of chromatin and other nuclear material, called neutrophil extracellular traps (NETs) (5) (Figure 1). Neutrophils are now recognized as being major orchestrators of inflammation and as having a strong influence on the phenotype and function of other innate and adaptive immune cells (4) (Figure 1). Uptake of apoptotic neutrophils by conventional myeloid dendritic cells (DCs) can enhance their antigen-presenting capabilities (6). Neutrophils synthesize cytokines that are fundamental to B cell ontogeny, such as BAFF (7) and APRIL (8). Splenic neutrophils can display T cell–independent B cell helper capabilities (9). Furthermore, neutrophils can both suppress and stimulate T cell responses, cross-prime CD81 T cells in an MHC class I– dependent manner, and activate g/d T cells (10). Neutrophils promote T cell apoptosis through programmed death ligand 1 (PDL-1)/PD-1 interactions (11), while their proteases can inactivate T cell–stimulating cytokines, including IL-6 and IL-2 (12). Neutrophils can down-regulate the T cell receptor z chain, thereby promoting cell cycle arrest, and this appears to be triggered by reactive oxygen species (ROS) production and argiSupported in part by the NIH (National Institute of Arthritis and Musculoskeletal and Skin Diseases Intramural Research Program, project ZIA-AR-041199). Peter C. Grayson, MD, Mariana J. Kaplan, MD: National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, Bethesda, Maryland; Christine Schauer, PhD, Martin Herrmann, PhD: Friedrich Alexander University Erlangen–Nuremberg and Universit€atsklinikum Erlangen, Erlangen, Germany. Address correspondence to Mariana J. Kaplan, MD, Systemic Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, 10 Center Drive, Building 10, Room 6D/47C, Bethesda, MD 20892-1560. E-mail: mariana.kaplan@nih.gov. Submitted for publication February 11, 2016; accepted in revised form May 3, 2016.

Blood-borne phagocytes internalize urate microaggregates and prevent intravascular NETosis by urate crystals

Hyperuricemia is strongly linked to cardiovascular complications including atherosclerosis and thrombosis. In individuals with hyperuricemia, needle-shaped monosodium urate crystals (nsMSU) frequently form within joints or urine, giving rise to gouty arthritis or renal calculi, respectively. These nsMSU are potent instigators of neutrophil extracellular trap (NET) formation. Little is known on the mechanism(s) that prevent nsMSU formation within hyperuricemic blood, which would potentially cause detrimental consequences for the host. Here, we report that complement proteins and fetuins facilitate the continuous clearance by blood-borne phagocytes and resident macrophages of small urate microaggregates (UMA; <1 μm in size) that initially form in hyperuricemic blood. If this clearance fails, UMA exhibit bipolar growth to form typical full-sized nsMSU with a size up to 100 μm. In contrast to UMA, nsMSU stimulated neutrophils to release NETs. Under conditions of flow, nsMSU and NETs formed densely packed DNase I-resistant tophus-like structures with a high obstructive potential, highlighting the importance of an adequate and rapid removal of UMA from the circulation. Under pathological conditions, intravascularly formed nsMSU may hold the key to the incompletely understood association between NET-driven cardiovascular disease and hyperuricemia.

Neutrophils as Invigorated Targets in Rheumatic Diseases

Neutrophil biology. Neutrophils are terminally differentiated innate immune cells that play fundamental roles in host defense against microbes. The bone marrow is the primary site of neutrophil production, and under conditions of homeostasis, these cells can also be found in the lung, spleen, and liver. Characteristically, neutrophils have a short half-life, and constant production in the bone marrow is needed to maintain a steady state (1). However, there is still significant uncertainty regarding neutrophil turnover in the bone marrow and circulation under homeostatic and nonhomeostatic conditions (for review, see ref. 2). Neutrophil production is tightly controlled through synthesis of granulocyte-colony-stimulating factor (G-CSF) in response to interleukin-17 (IL-17). Neutrophils express 4 categories of intracytoplasmic granules: primary (azurophilic), which contain among many proteins myeloperoxidase (MPO), defensins, neutrophil elastase, and membrane-bound CD63, secondary, which contain various molecules, such as lactoferrin, LL-37 peptide, and membrane-bound CD66b, tertiary, which are loaded with gelatinase, cathepsin, and various toxic proteins, and secretory vesicles, which are considered an important store of membrane-bound receptors and which express alkaline phosphatase and CD35 on their membranes (3) (Figure 1). In the circulation, neutrophils remain in a resting state, ensuring that their toxic intracellular material is not accidentally released. Significant decreases in neutrophil numbers or their function can lead to severe infections, while very tight regulation of these cells is crucial to preventing tissue injury (4). Under inflammatory conditions, circulating neutrophils are recruited to tissues according to a gradient, and their half-life may increase to support proper response to noxious stimuli (1,3). Inflammatory tissue neutrophils share many of the functions of macrophages, including synthesis of cytokines and chemokines, phagocytosis, and presentation of major histocompatibility complex (MHC) class II–dependent antigen. In tissues, neutrophils use different mechanisms to destroy pathogens, including phagocytosis, degranulation, and a distinct form of cell death characterized by the extracellular extrusion of structures formed by intracellular proteins bound to a meshwork of chromatin and other nuclear material, called neutrophil extracellular traps (NETs) (5) (Figure 1). Neutrophils are now recognized as being major orchestrators of inflammation and as having a strong influence on the phenotype and function of other innate and adaptive immune cells (4) (Figure 1). Uptake of apoptotic neutrophils by conventional myeloid dendritic cells (DCs) can enhance their antigen-presenting capabilities (6). Neutrophils synthesize cytokines that are fundamental to B cell ontogeny, such as BAFF (7) and APRIL (8). Splenic neutrophils can display T cell–independent B cell helper capabilities (9). Furthermore, neutrophils can both suppress and stimulate T cell responses, cross-prime CD81 T cells in an MHC class I– dependent manner, and activate g/d T cells (10). Neutrophils promote T cell apoptosis through programmed death ligand 1 (PDL-1)/PD-1 interactions (11), while their proteases can inactivate T cell–stimulating cytokines, including IL-6 and IL-2 (12). Neutrophils can down-regulate the T cell receptor z chain, thereby promoting cell cycle arrest, and this appears to be triggered by reactive oxygen species (ROS) production and argiSupported in part by the NIH (National Institute of Arthritis and Musculoskeletal and Skin Diseases Intramural Research Program, project ZIA-AR-041199). Peter C. Grayson, MD, Mariana J. Kaplan, MD: National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, Bethesda, Maryland; Christine Schauer, PhD, Martin Herrmann, PhD: Friedrich Alexander University Erlangen–Nuremberg and Universit€atsklinikum Erlangen, Erlangen, Germany. Address correspondence to Mariana J. Kaplan, MD, Systemic Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, 10 Center Drive, Building 10, Room 6D/47C, Bethesda, MD 20892-1560. E-mail: mariana.kaplan@nih.gov. Submitted for publication February 11, 2016; accepted in revised form May 3, 2016.

Erratum: Reply to “Neutrophils are not required for resolution of acute gouty arthritis in mice”

Nat. Med. 22, 1384–1386 (2016); published online 06 December 2016; corrected after print 19 January 2017 In the version of this article initially published, the units (ml) for values reported in the methods are incorrect. The correct unit should be μl. The error has been corrected in the HTML and PDF versions of the article.