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 extracellular traps in immunity and disease

The production of neutrophil extracellular traps (NETs) is a process that enables neutrophils to help catch and kill bacteria. However, increasing evidence suggests that this process might also occur in noninfectious, sterile inflammation. In this Review, we describe the role of NETosis in autoimmunity, coagulation, acute injuries and cancer, and discuss NETs as potential therapeutic targets. Furthermore, we consider whether extracellular DNA is always detrimental in sterile inflammation and whether the source is always NETs.

An emerging role for neutrophil extracellular traps in noninfectious disease

Social networking of human neutrophils within the immune system.

Objectives An imbalance between neutrophil extracellular trap (NET) formation and degradation has been described in systemic lupus erythematosus (SLE), potentially contributing to autoantigen externalisation, type I interferon synthesis and endothelial damage. We have demonstrated that peptidylarginine deiminase (PAD) inhibition reduces NET formation and protects against lupus-related vascular damage in the New Zealand Mixed model of lupus. However, another strategy for inhibiting NETs—knockout of NOX2 —accelerates lupus in a different murine model, MRL/ lpr . Here, we test the effects of PAD inhibition on MRL/ lpr mice in order to clarify whether some NET inhibitory pathways may be consistently therapeutic across models of SLE. Methods NET formation and autoantibodies to NETs were characterised in lupus-prone MRL/ lpr mice. MRL/ lpr mice were also treated with two different PAD inhibitors, Cl-amidine and the newly described BB-Cl-amidine. NET formation, endothelial function, interferon signature, nephritis and skin disease were examined in treated mice. Results Neutrophils from MRL/ lpr mice demonstrate accelerated NET formation compared with controls. MRL/ lpr mice also form autoantibodies to NETs and have evidence of endothelial dysfunction. PAD inhibition markedly improves endothelial function, while downregulating the expression of type I interferon-regulated genes. PAD inhibition also reduces proteinuria and immune complex deposition in the kidneys, while protecting against skin disease. Conclusions PAD inhibition reduces NET formation, while protecting against lupus-related damage to the vasculature, kidneys and skin in various lupus models. The strategy by which NETs are inhibited will have to be carefully considered if human studies are to be undertaken.

https://ard.bmj.com/content/annrheumdis/early/2014/08/07/annrheumdis-2014-205365.full.pdf

Peptidylarginine deiminase inhibition disrupts NET formation and protects against kidney, skin and vascular disease in lupus-prone MRL/lpr mice

Neutrophil Diversity in Health and Disease

Neutrophil extracellular traps (NETs) have been implicated in the pathogenesis of systemic Lupus erythematosus (SLE), since netting neutrophils release potentially immunogenic autoantigens including histones, LL37, human neutrophil peptide (HNP), and self-DNA. In turn, these NETs activate plasmacytoid dendritic cells resulting in aggravation of inflammation and disease. How suppression of NET formation can be targeted for treatment has not been reported yet. Signal Inhibitory Receptor on Leukocytes-1 (SIRL-1) is a surface molecule exclusively expressed on phagocytes. We recently identified SIRL-1 as a negative regulator of human neutrophil function. Here, we determine whether ligation of SIRL-1 prevents the pathogenic release of NETs in SLE. Peripheral blood neutrophils from SLE patients with mild to moderate disease activity and healthy donors were freshly isolated. NET release was assessed spontaneously or after exposure to anti-neutrophil antibodies or plasma obtained from SLE patients. The formation of NETs was determined by microscopic evaluation using DNA dyes and immunostaining of NET components, as well as by live cell imaging. We show that SLE neutrophils spontaneously release NETs. NET formation is enhanced by stimulation with antibodies against LL37. Inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and MEK-ERK signaling prevents NET release in response to these antibodies. Signaling via the inhibitory receptor SIRL-1 was induced by ligation with anti-SIRL-1 specific antibodies. Both spontaneous and anti-neutrophil antibody-induced NET formation is suppressed by engagement of SIRL-1. Furthermore, NET release by healthy neutrophils exposed to SLE plasma is inhibited by SIRL-1 ligation. Thus, SIRL-1 engagement can dampen spontaneous and anti-neutrophil antibody-induced NET formation in SLE, likely by suppressing NAPDH oxidase and MEK-ERK activity. Together, these findings reveal a regulatory role for SIRL-1 in NET formation, potentially providing a novel therapeutic target to break the pathogenic loop in SLE.

/pdf/ligation-of-signal-inhibitory-receptor-on-leukocytes-1-1ogpqjkb1v.pdf

Ligation of signal inhibitory receptor on leukocytes-1 suppresses the release of neutrophil extracellular traps in systemic lupus erythematosus.

In response to microbial invasion, neutrophils release neutrophil extracellular traps (NETs) to trap and kill extracellular microbes. Alternatively, NET formation can result in tissue damage in inflammatory conditions and may perpetuate autoimmune disease. Intervention strategies that are aimed at modifying pathogenic NET formation should ideally preserve other neutrophil antimicrobial functions. We now show that signal inhibitory receptor on leukocytes-1 (SIRL-1) attenuates NET release by human neutrophils in response to distinct triggers, including opsonized Staphylococcus aureus and inflammatory danger signals. NET release has different kinetics depending on the stimulus, and rapid NET formation is independent of NADPH oxidase activity. In line with this, we show that NET release and reactive oxygen species production upon challenge with opsonized S. aureus require different signaling events. Importantly, engagement of SIRL-1 does not affect bacterially induced production of reactive oxygen species, and intracellular bacterial killing by neutrophils remains intact. Thus, our studies define SIRL-1 as an intervention point of benefit to suppress NET formation in disease while preserving intracellular antimicrobial defense.

Signal Inhibitory Receptor on Leukocytes-1 Limits the Formation of Neutrophil Extracellular Traps, but Preserves Intracellular Bacterial Killing

ROS production is an important effector mechanism mediating intracellular killing of microbes by phagocytes. Inappropriate or untimely ROS production can lead to tissue damage, thus tight regulation is essential. We recently characterized signal inhibitory receptor on leukocytes-1 (SIRL-1) as an inhibitory receptor expressed by human phagocytes. Here, we demonstrate that ligation of SIRL-1 dampens Fc receptor-induced ROS production in primary human phagocytes. In accordance, SIRL-1 engagement on these cells impairs the microbicidal activity of neutrophils, without affecting phagocytosis. The inhibition of ROS production may result from reduced ERK activation, since co-ligation of Fc receptors and SIRL-1 on phagocytes inhibited phosphorylation of ERK. Importantly, we demonstrate that microbial and inflammatory stimuli cause rapid downregulation of SIRL-1 expression on the surface of primary neutrophils and monocytes. In accordance, SIRL-1 expression levels on neutrophils in bronchoalveolar lavage fluid from patients with neutrophilic airway inflammation are greatly reduced. We propose that SIRL-1 on phagocytes sets an activation threshold to prevent inappropriate production of oxygen radicals. Upon infection, SIRL-1 expression is downregulated, allowing microbial killing and clearance of the pathogen.

Signal inhibitory receptor on leukocytes-1 (SIRL-1) negatively regulates the oxidative burst in human phagocytes.

An innate immune response is essential for survival of the host upon infection, yet excessive inflammation can result in harmful complications [1]. Inhibitory signaling evolved to limit host responses and prevent inflammatory pathology [2,3]. Given the significance of inhibitory pathways for immunity and homeostasis, they provide ideal targets for manipulation by bacterial pathogens. Recent evidence highlights that bacteria have developed diverse strategies to exploit these inhibitory pathways to avoid host defense for their own benefit. In this review, we cover these different immune evasion strategies for the first time. The recent literature discussed emphasizes that bacteria subvert host immune responses not only by direct engagement of inhibitory receptors (i.e., often through "molecular mimicry" of host ligands [4,5]) but also through virulence factors that resemble intermediates of host inhibitory signaling and interfere with defense functions [6-8]. Understanding how bacteria manipulate inhibitory signaling affords promising opportunities to counteract these escape strategies and tip the balance in favor of the host. In addition, these understandings may provide useful insights on the functional roles of inhibitory pathways in limiting host responses and preventing pathology.

/pdf/bacterial-immune-evasion-through-manipulation-of-host-3du58x4k56.pdf

Bacterial immune evasion through manipulation of host inhibitory immune signaling

Over the past millennia, life expectancy has drastically increased. While a mere 25 years during Bronze and Iron ages, life expectancy in many European countries and in Japan is currently above 80 years. Such an increase in life expectancy is a result of improved diet, life style, and medical care. Yet, increased life span and aging also represent the most important non‐modifiable risk factors for several pathologies including cardiovascular disease, neurodegenerative diseases, and cancer. In recent years, neutrophils have been implicated in all of these pathologies. Hence, this review provides an overview of how aging impacts neutrophil production and function and conversely how neutrophils drive aging‐associated pathologies. Finally, we provide a perspective on how processes of neutrophil‐driven pathologies in the context of aging can be targeted therapeutically.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1111/imr.13153

Kristof Van Avondt

Papers

Ligation of signal inhibitory receptor on leukocytes-1 suppresses the release of neutrophil extracellular traps in systemic lupus erythematosus.

Signal Inhibitory Receptor on Leukocytes-1 Limits the Formation of Neutrophil Extracellular Traps, but Preserves Intracellular Bacterial Killing

Signal inhibitory receptor on leukocytes-1 (SIRL-1) negatively regulates the oxidative burst in human phagocytes.

Bacterial immune evasion through manipulation of host inhibitory immune signaling

Neutrophils in aging and aging‐related pathologies