A20 deficiency in myeloid cells protects mice from diet-induced obesity and insulin resistance due to increased fatty acid metabolism.

Obesity-induced inflammation is a major driving force in the development of insulin resistance, type 2 diabetes (T2D), and related metabolic disorders. During obesity, macrophages accumulate in the visceral adipose tissue, creating a low-grade inflammatory environment. Nuclear factor κB (NF-κB) signaling is a central coordinator of inflammatory responses and is tightly regulated by the anti-inflammatory protein A20. Here, we find that myeloid-specific A20-deficient mice are protected from diet-induced obesity and insulin resistance despite an inflammatory environment in their metabolic tissues. Macrophages lacking A20 show impaired mitochondrial respiratory function and metabolize more palmitate both in vitro and in vivo. We hypothesize that A20-deficient macrophages rely more on palmitate oxidation and metabolize the fat present in the diet, resulting in a lean phenotype and protection from metabolic disease. These findings reveal a role for A20 in regulating macrophage immunometabolism.

Patients with COVID-19: in the dark-NETs of neutrophils.

SARS-CoV-2 infection poses a major threat to the lungs and multiple other organs, occasionally causing death. Until effective vaccines are developed to curb the pandemic, it is paramount to define the mechanisms and develop protective therapies to prevent organ dysfunction in patients with COVID-19. Individuals that develop severe manifestations have signs of dysregulated innate and adaptive immune responses. Emerging evidence implicates neutrophils and the disbalance between neutrophil extracellular trap (NET) formation and degradation plays a central role in the pathophysiology of inflammation, coagulopathy, organ damage, and immunothrombosis that characterize severe cases of COVID-19. Here, we discuss the evidence supporting a role for NETs in COVID-19 manifestations and present putative mechanisms, by which NETs promote tissue injury and immunothrombosis. We present therapeutic strategies, which have been successful in the treatment of immunο-inflammatory disorders and which target dysregulated NET formation or degradation, as potential approaches that may benefit patients with severe COVID-19.

Epithelial HMGB1 Delays Skin Wound Healing and Drives Tumor Initiation by Priming Neutrophils for NET Formation.

Regenerative responses predispose tissues to tumor formation by largely unknown mechanisms. High-mobility group box 1 (HMGB1) is a danger-associated molecular pattern contributing to inflammatory pathologies. We show that HMGB1 derived from keratinocytes, but not myeloid cells, delays cutaneous wound healing and drives tumor formation. In wounds of mice lacking HMGB1 selectively in keratinocytes, a marked reduction in neutrophil extracellular trap (NET) formation is observed. Pharmacological targeting of HMGB1 or NETs prevents skin tumorigenesis and accelerates wound regeneration. HMGB1-dependent NET formation and skin tumorigenesis is orchestrated by tumor necrosis factor (TNF) and requires RIPK1 kinase activity. NETs are present in the microenvironment of keratinocyte-derived tumors in mice and lesional and tumor skin of patients suffering from recessive dystrophic epidermolysis bullosa, a disease in which skin blistering predisposes to tumorigenesis. We conclude that tumorigenicity of the wound microenvironment depends on epithelial-derived HMGB1 regulating NET formation, thereby establishing a mechanism linking reparative inflammation to tumor initiation.

Citrullination Licenses Calpain to Decondense Nuclei in Neutrophil Extracellular Trap Formation.

Neutrophils respond to various stimuli by decondensing and releasing nuclear chromatin characterized by citrullinated histones as neutrophil extracellular traps (NETs). This achieves pathogen immobilization or initiation of thrombosis, yet the molecular mechanisms of NET formation remain elusive. Peptidyl arginine deiminase-4 (PAD4) achieves protein citrullination and has been intricately linked to NET formation. Here we show that citrullination represents a major regulator of proteolysis in the course of NET formation. Elevated cytosolic calcium levels trigger both peptidylarginine deiminase-4 (PAD4) and calpain activity in neutrophils resulting in nuclear decondensation typical of NETs. Interestingly, PAD4 relies on proteolysis by calpain to achieve efficient nuclear lamina breakdown and chromatin decondensation. Pharmacological or genetic inhibition of PAD4 and calpain strongly inhibit chromatin decondensation of human and murine neutrophils in response to calcium ionophores as well as the proteolysis of nuclear proteins like lamin B1 and high mobility group box protein 1 (HMGB1). Taken together, the concerted action of PAD4 and calpain induces nuclear decondensation in the course of calcium-mediated NET formation.

Treatment with DNases rescues hidden neutrophil elastase from aggregated NETs.

The release of neutrophil extracellular traps (NETs) is one of the weapons neutrophils have in their armory. NETs consist of extracellular chromatin fibers decorated with a plethora of cytoplasmic and granular proteins, such as the antimicrobial serine protease neutrophil elastase (NE). Because the first description of NETs as beneficial to the host, reports on their double-faced role in health and disease have considerably increased recently. On one hand, NETs reportedly trap and kill bacteria and also participate in the resolution of the acute inflammation associated with infection and with tissue damage. On the other hand, numerous negative aspects of NETs contribute to the etiopathogenesis of autoimmune disorders. Employing soluble and solid fluorescent substrates, we demonstrate the interaction of NE with aggregated NETs (aggNETs), the limitation of its enzymatic activity and the containment of the enzyme from surrounding tissues. These events prevent the spread of inflammation and tissue damage. The detection of DNase 1-dependent elevation of NE activity attests the continuous presence of patrolling neutrophils forming NETs and aggNETs even under conditions physiologic conditions.

Neutrophil Extracellular Traps Initiate Gallstone Formation.

The presence of gallstones (cholelithiasis) is a highly prevalent and severe disease and one of the leading causes of hospital admissions worldwide. Due to its substantial health impact, we investigated the biological mechanisms that lead to the formation and growth of gallstones. We show that gallstone assembly essentially requires neutrophil extracellular traps (NETs). We found consistent evidence for the presence of NETs in human and murine gallstones and describe an immune-mediated process requiring activation of the innate immune system for the formation and growth of gallstones. Targeting NET formation via inhibition of peptidyl arginine deiminase type 4 or abrogation of reactive oxygen species (ROS) production, as well as damping of neutrophils by metoprolol, effectively inhibit gallstone formation in vivo. Our results show that after the physicochemical process of crystal formation, NETs foster their assembly into larger aggregates and finally gallstones. These insights provide a feasible therapeutic concept to prevent cholelithiasis in patients at risk.

Living on the Edge: Efferocytosis at the Interface of Homeostasis and Pathology.

Nearly every tissue in the body undergoes routine turnover of cells as part of normal healthy living. The majority of these cells undergoing turnover die via apoptosis, and then are rapidly removed by phagocytes by the process of efferocytosis that is anti-inflammatory. However, a number of pathologies have recently been linked to defective clearance of apoptotic cells. Perturbed clearance arises for many reasons, including overwhelming of the clearance machinery, disruptions at different stages of efferocytosis, and responses of phagocytes during efferocytosis, all of which can alter the homeostatic tissue environment. This review covers linkages of molecules involved in the different phases of efferocytosis to disease pathologies that can arise due to their loss or altered function.

Frontline Science: Aggregated neutrophil extracellular traps prevent inflammation on the neutrophil-rich ocular surface.

Eye rheum is a physiological discharge, which accumulates at the medial angle of the healthy eye soon after opening in the morning. Microscopic evaluation of eye rheum revealed the presence of viable neutrophils, bacteria, epithelial cells, and particles, aggregated by neutrophil extracellular traps. We observed that in the evening, during eye closure, high C5a recruited neutrophils to the tear film and activated them. In this hypoxic area rich in CO2 , neutrophils fight microbial aggressors by degranulation. Immediately after eye opening, the microenvironment of the ocular surface changes, the milieu gets normoxic, and loss of CO2 induces subtle alkalinization of tear film. These conditions favored the formation of neutrophil extracellular traps (NETs) that initially covers the ocular surface and tend to aggregate by eyelid blinking. These aggregated neutrophil extracellular traps (aggNETs) are known as eye rheum and contain several viable neutrophils, epithelial cells, dust particles, and crystals packed together by NETs. Similar to aggNETs induced by monosodium urate crystals, the eye rheum shows a robust proteolytic activity that degraded inflammatory mediators before clinically overt inflammation occur. Finally, the eye rheum passively floats with the tear flow to the medial angle of the eye for disposal. We conclude that the aggNETs-based eye rheum promotes cleaning of the ocular surface and ameliorates the inflammation on the neutrophil-rich ocular surfaces.

To NET or not to NET:current opinions and state of the science regarding the formation of neutrophil extracellular traps.

Since the discovery and definition of neutrophil extracellular traps (NETs) 14 years ago, numerous characteristics and physiological functions of NETs have been uncovered. Nowadays, the field continues to expand and novel mechanisms that orchestrate formation of NETs, their previously unknown properties, and novel implications in disease continue to emerge. The abundance of available data has also led to some confusion in the NET research community due to contradictory results and divergent scientific concepts, such as pro- and anti-inflammatory roles in pathologic conditions, demarcation from other forms of cell death, or the origin of the DNA that forms the NET scaffold. Here, we present prevailing concepts and state of the science in NET-related research and elaborate on open questions and areas of dispute.

Chemical Tools for Targeted Amplification of Reactive Oxygen Species in Neutrophils.

A number of chemical compounds are known, which amplify the availability of reactive oxygen species (ROS) in neutrophils both in vitro and in vivo. They can be roughly classified into NADPH oxidase 2 (NOX2)-dependent and NOX2-independent reagents. NOX2 activation is triggered by protein kinase C agonists (e.g., phorbol esters, transition metal ions), redox mediators (e.g., paraquat) or formyl peptide receptor (FPR) agonists (e.g., aromatic hydrazine derivatives). NOX2-independent mechanisms are realized by reagents affecting glutathione homeostasis (e.g., l-buthionine sulfoximine), modulators of the mitochondrial respiratory chain (e.g., ionophores, inositol mimics, and agonists of peroxisome proliferator-activated receptor γ) and chemical ROS amplifiers [e.g., aminoferrocene-based prodrugs (ABPs)]. Since a number of inflammatory and autoimmune diseases, as well as cancer and bacterial infections, are triggered or enhanced by aberrant ROS production in neutrophils, it is tempting to use ROS amplifiers as drugs for the treatment of these diseases. However, since the known reagents are not cell specific, their application for treatment likely causes systemic enhancement of oxidative stress, leading to severe side effects. Cell-targeted ROS enhancement can be achieved either by using conjugates of ROS amplifiers with ligands binding to receptors expressed on neutrophils (e.g., the GPI-anchored myeloid differentiation marker Ly6G or FPR) or by designing reagents activated by neutrophil function [e.g., phagocytic activity or enzymatic activity of neutrophil elastase (NE)]. Since binding of an artificial ligand to a receptor may trigger or inhibit priming of neutrophils the latter approach has a smaller potential for severe side effects and is probably better suitable for therapy. Here, we review current approaches for the use of ROS amplifiers and discuss their applicability for treatment. As an example, we suggest a possible design of neutrophil-specific ROS amplifiers, which are based on NE-activated ABPs.

Experimental lupus is aggravated in mouse strains with impaired induction of neutrophil extracellular traps.

Many effector mechanisms of neutrophils have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). Neutrophil extracellular traps (NETs) have been assigned a particularly detrimental role. Here we investigated the functional impact of neutrophils and NETs on a mouse model of lupus triggered by intraperitoneal injection of the cell death-inducing alkane pristane. Pristane-induced lupus (PIL) was aggravated in 2 mouse strains with impaired induction of NET formation, i.e., NOX2-deficient (Ncf1-mutated) and peptidyl arginine deiminase 4-deficient (PAD4-deficient) mice, as seen from elevated levels of antinuclear autoantibodies (ANAs) and exacerbated glomerulonephritis. We observed a dramatically reduced ability to form pristane-induced NETs in vivo in both Ncf1-mutated and PAD4-deficient mice, accompanied by higher levels of inflammatory mediators in the peritoneum. Similarly, neutropenic Mcl-1ΔMyelo mice exhibited higher levels of ANAs, which indicates a regulatory function in lupus of NETs and neutrophils. Blood neutrophils from Ncf1-mutated and human individuals with SLE exhibited exuberant spontaneous NET formation. Treatment with specific chemical NOX2 activators induced NET formation and ameliorated PIL. Our findings suggest that aberrant NET is one of the factors promoting experimental lupus-like autoimmunity by uncontrolled release of inflammatory mediators.

Inosine Released from Dying or Dead Cells Stimulates Cell Proliferation via Adenosine Receptors.

INTRODUCTION: Many antitumor therapies induce apoptotic cell death in order to cause tumor regression. Paradoxically, apoptotic cells are also known to promote wound healing, cell proliferation, and tumor cell repopulation in multicellular organisms. We aimed to characterize the nature of the regenerative signals concentrated in the micromilieu of dead and dying cells. METHODS: Cultures of viable melanoma B16F10 cells, mouse fibroblasts, and primary human fibroblast-like synoviocytes (FLS) in the presence of dead and dying cells, their supernatants (SNs), or purified agonists and antagonists were used to evaluate the stimulation of proliferation. Viable cell quantification was performed by either flow cytometry of harvested cells or by crystal violet staining of adherent cells. High-performance liquid chromatography and liquid chromatography coupled with mass spectrometry of cell SNs were deployed to identify the nature of growth-promoting factors. Coimplantation of living cells in the presence of SNs collected from dead and dying cells and specific agonists was used to evaluate tumor growth in vivo. RESULTS: The stimulation of proliferation of few surviving cells by bystander dead cells was confirmed for melanoma cells, mouse fibroblasts, and primary FLS. We found that small soluble molecules present in the protein-free fraction of SNs of dead and dying cells were responsible for the promotion of proliferation. The nucleoside inosine released by dead and dying cells acting via adenosine receptors was identified as putative inducer of proliferation of surviving tumor cells after irradiation and heat treatment. CONCLUSION: Inosine released by dead and dying cells mediates tumor cell proliferation via purinergic receptors. Therapeutic strategies surmounting this pathway may help to reduce the rate of recurrence after radio- and chemotherapy.

Capability of Neutrophils to Form NETs Is Not Directly Influenced by a CMA-Targeting Peptide.

During inflammatory reaction, neutrophils exhibit numerous cellular and immunological functions, notably the formation of neutrophil extracellular traps (NETs) and autophagy. NETs are composed of decondensed chromatin fibers coated with various antimicrobial molecules derived from neutrophil granules. NETs participate in antimicrobial defense and can also display detrimental roles and notably trigger some of the immune features of systemic lupus erythematosus (SLE) and other autoimmune diseases. Autophagy is a complex and finely regulated mechanism involved in the cell survival/death balance that may be connected to NET formation. To shed some light on the connection between autophagy and NET formation, we designed a number of experiments in human neutrophils and both in normal and lupus-prone MRL/lpr mice to determine whether the synthetic peptide P140, which is capable of selectively modulating chaperone-mediated autophagy (CMA) in lymphocytes, could alter NET formation. P140/Lupuzor™ is currently being evaluated in phase III clinical trials involving SLE patients. Overall our in vitro and in vivo studies established that P140 does not influence NET formation, cytokine/chemokine production, or CMA in neutrophils. Thus, the beneficial effect of P140/Lupuzor™ in SLE is apparently not directly related to modulation of neutrophil function.

Ménage-à-Trois: The Ratio of Bicarbonate to CO2 and the pH Regulate the Capacity of Neutrophils to Form NETs.

In this study, we identified and characterized the potential of a high ratio of bicarbonate to CO2 and a moderately alkaline pH to render neutrophils prone to undergo neutrophil extracellular trap (NET) formation. Both experimental settings increased the rate of spontaneous NET release and potentiated the NET-inducing capacity of phorbol esters (phorbol-2-myristate-13-acetate), ionomycin, monosodium urate, and LPS. In contrast, an acidic environment impaired NET formation both spontaneous and induced. Our findings indicate that intracellular alkalinization of neutrophils in response to an alkaline environment leads to an increase of intracellular calcium and neutrophil activation. We further found that the anion channel blocker DIDS strongly reduced NET formation induced by bicarbonate. This finding suggests that the effects observed are due to a molecular program that renders neutrophils susceptible to NET formation. Inflammatory foci may be characterized by an acidic environment. Our data indicate that NET formation is favored by the higher pH at the border regions of inflamed areas. Moreover, our findings highlight the necessity for strict pH control during assays of NET formation.

Oxidative Burst-Dependent NETosis Is Implicated in the Resolution of Necrosis-Associated Sterile Inflammation.

Necrosis is associated with a profound inflammatory response. The regulation of necrosis-associated inflammation, particularly the mechanisms responsible for resolution of inflammation is incompletely characterized. Nanoparticles are known to induce plasma membrane damage and necrosis followed by sterile inflammation. We observed that injection of metabolically inert nanodiamonds resulted in paw edema in WT and Ncf1** mice. However, while inflammation quickly resolved in WT mice, it persisted over several weeks in Ncf1** mice indicating failure of resolution of inflammation. Mechanistically, NOX2-dependent reactive oxygen species (ROS) production and formation of neutrophil extracellular traps were essential for the resolution of necrosis-induced inflammation: hence, by evaluating the fate of the particles at the site of inflammation, we observed that Ncf1** mice deficient in NADPH-dependent ROS failed to generate granulation tissue therefore being unable to trap the nanodiamonds. These data suggest that NOX2-dependent NETosis is crucial for preventing the chronification of the inflammatory response to tissue necrosis by forming NETosis-dependent barriers between the necrotic and healthy surrounding tissue.

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 and neutrophil extracellular traps orchestrate initiation and resolution of inflammation.

Neutrophils, the most abundant leukocytes in the human body, are considered to be the first line of defense in the fight against microorganisms. In this fight neutrophils employ weaponry such as reactive oxygen species produced via the NADPH oxidase complex 2 together with the release of intracellular granules containing antimicrobial agents. The discovery that activated neutrophils release decondensed chromatin as DNase-sensitive neutrophil extracellular traps (NETs) lead to a renewed interest in these leukocytes and the function of NETs in vivo. In this review, we will focus on desirable as well as detrimental features of NETs by the example of gout and pancreatitis. In our models we observed that neutrophils drive the initiation of inflammation and are required for the resolution of inflammation.

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

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.

Externalized decondensed neutrophil chromatin occludes pancreatic ducts and drives pancreatitis.

Ductal occlusion has been postulated to precipitate focal pancreatic inflammation, while the nature of the primary occluding agents has remained elusive. Neutrophils make use of histone citrullination by peptidyl arginine deiminase-4 (PADI4) in contact to particulate agents to extrude decondensed chromatin as neutrophil extracellular traps (NETs). In high cellular density, NETs form macroscopically visible aggregates. Here we show that such aggregates form inside pancreatic ducts in humans and mice occluding pancreatic ducts and thereby driving pancreatic inflammation. Experimental models indicate that PADI4 is critical for intraductal aggregate formation and that PADI4-deficiency abrogates disease progression. Mechanistically, we identify the pancreatic juice as a strong instigator of neutrophil chromatin extrusion. Characteristic single components of pancreatic juice, such as bicarbonate ions and calcium carbonate crystals, induce aggregated NET formation. Ductal occlusion by aggregated NETs emerges as a pathomechanism with relevance in a plethora of inflammatory conditions involving secretory ducts.

Molecular and Translational Classifications of DAMPs in Immunogenic Cell Death.

The immunogenicity of malignant cells has recently been acknowledged as a critical determinant of efficacy in cancer therapy. Thus, besides developing direct immunostimulatory regimens, including dendritic cell-based vaccines, checkpoint-blocking therapies, and adoptive T-cell transfer, researchers have started to focus on the overall immunobiology of neoplastic cells. It is now clear that cancer cells can succumb to some anticancer therapies by undergoing a peculiar form of cell death that is characterized by an increased immunogenic potential, owing to the emission of the so-called "damage-associated molecular patterns" (DAMPs). The emission of DAMPs and other immunostimulatory factors by cells succumbing to immunogenic cell death (ICD) favors the establishment of a productive interface with the immune system. This results in the elicitation of tumor-targeting immune responses associated with the elimination of residual, treatment-resistant cancer cells, as well as with the establishment of immunological memory. Although ICD has been characterized with increased precision since its discovery, several questions remain to be addressed. Here, we summarize and tabulate the main molecular, immunological, preclinical, and clinical aspects of ICD, in an attempt to capture the essence of this phenomenon, and identify future challenges for this rapidly expanding field of investigation.