Macrophage Extracellular Trap Formation Research Articles

Toll-like Receptors as Pro-Thrombotic Drivers in Viral Infections: A Narrative Review.

Toll-like receptors (TLRs) have a critical role in the pathogenesis and disease course of viral infections. The induced pro-inflammatory responses result in the disturbance of the endovascular surface layer and impair vascular homeostasis. The injury of the vessel wall further promotes pro-thrombotic and pro-coagulatory processes, eventually leading to micro-vessel plugging and tissue necrosis. Moreover, TLRs have a direct role in the sensing of viruses and platelet activation. TLR-mediated upregulation of von Willebrand factor release and neutrophil, as well as macrophage extra-cellular trap formation, further contribute to (micro-) thrombotic processes during inflammation. The following review focuses on TLR signaling pathways of TLRs expressed in humans provoking pro-thrombotic responses, which determine patient outcome during viral infections, especially in those with cardiovascular diseases.

The escape of Candida albicans from macrophages is enabled by the fungal toxin candidalysin and two host cell death pathways.

The egress of Candida hyphae from macrophages facilitates immune evasion, but it also alerts macrophages to infection and triggers inflammation. To better define the mechanisms, here we develop an imaging assay to directly and dynamically quantify hyphal escape and correlate it to macrophage responses. The assay reveals that Candida escapes by using two pore-forming proteins to permeabilize macrophage membranes: the fungal toxin candidalysin and Nlrp3 inflammasome-activated Gasdermin D. Candidalysin plays a major role in escape, with Nlrp3 and Gasdermin D-dependent and -independent contributions. The inactivation of Nlrp3 does not reduce hyphal escape, and we identify ETosis via macrophage extracellular trap formation as an additional pathway facilitating hyphal escape. Suppressing hyphal escape does not reduce fungal loads, but it does reduce inflammatory activation. Our findings explain how Candida escapes from macrophages by using three strategies: permeabilizing macrophage membranes via candidalysin and engaging two host cell death pathways, Gasdermin D-mediated pyroptosis and ETosis.

Macrophage extracellular traps aggravate iron overload-related liver ischaemia/reperfusion injury.

Macrophages regulate iron homeostasis in the liver and play important role in hepatic ischaemia/reperfusion (I/R) injury. This study investigates the role of macrophages in iron overload-related hepatocyte damage during liver I/R. Liver biopsies from patients undergoing partial hepatectomy with or without hepatic portal occlusion were recruited and markers of hepatocyte cell death and macrophage extracellular traps (METs) were detected. A murine hepatic I/R model was also established in high-iron diet-fed mice. Ferrostatin-1 and deferoxamine were administered to investigate the role of ferroptosis in hepatic I/R injury. The macrophage inhibitor liposome-encapsulated clodronate was used to investigate the interaction between macrophages and ferroptosis. AML12 hepatocytes and RAW264.7 macrophages were co-cultured in vitro. An inhibitor of macrophage extracellular traps was used to evaluate the role and mechanism of these traps and ferroptosis in hepatic I/R injury. Hepatocyte macrophage extracellular trap formation and ferroptosis were greater in patients who underwent hepatectomy with hepatic portal occlusion and in mice subjected to hepatic I/R. Macrophage extracellular traps increased when macrophages were subjected to hypoxia/reoxygenation and when they were co-cultured with hepatocytes. Ferroptosis increased and post-hypoxic hepatocyte survival decreased, which were reversed by inhibition of macrophage extracellular traps. Ferroptosis inhibition attenuated post-ischaemic liver damage. Moreover, iron overload induced hepatic ferroptosis and exacerbated post-ischaemic liver damage, which were reversed by the iron chelator. Macrophage extracellular traps are in volved in regulating ferroptosis highlighting the therapeutic potential of macrophage extracellular traps and ferroptosis inhibition in reducing liver I/R injury.

Extracellular traps and PAD4 released by macrophages induce citrullination and auto-antibody production in autoimmune arthritis

The mechanisms underlying the transition of rheumatoid arthritis (RA) systemic autoimmunity to the joints remain largely unknown. Here, we demonstrate that macrophages in the secondary lymphoid organs (SLOs) and synovial ectopic lymphoid-like structures (ELSs) express peptidylarginine deiminase 4 (PAD4) in murine collagen induced arthritis (CIA) and synovial biopsies from RA patients. Moreover, peptidyl citrulline colocalized with macrophages in SLOs and ELSs, and depletion of macrophages in CIA decreased lymphoid tissue citrullination and serum anti-citrullinated protein/peptide antibody (ACPA) levels. Furthermore, PAD was released from activated murine and RA synovial tissue and fluid (SF) macrophages which functionally deiminated extracellular proteins/peptides in vitro. Additionally, activated murine and SF macrophages displayed macrophage extracellular trap formation (METosis) and release of intracellular citrullinated histones. Moreover, presentation of citrullinated proteins induced ACPA production in vitro. Thus, lymphoid tissue macrophages contribute to self-antigen citrullination and ACPA production, indicating that their selective targeting would potentially ameliorate citrullination-dependent autoimmune disorders.

Role of hypochlorous acid (HOCl) and other inflammatory mediators in the induction of macrophage extracellular trap formation

The infiltration of activated leukocytes, including macrophages, at sites of inflammation and the formation and presence of hypochlorous acid (HOCl) are interlinked hallmarks of many debilitating disease processes, including atherosclerosis, arthritis, neurological and renal disease, diabetes and obesity. The production of extracellular traps by activated leukocytes in response to a range of inflammatory stimuli is increasingly recognised as an important process within a range of disease settings. We show that exposure of human monocyte-derived macrophages to pathophysiological levels of HOCl results in the dose-dependent extrusion of DNA and histones into the cellular supernatant, consistent with extracellular trap formation. Concurrent with, but independent of these findings, macrophage exposure to HOCl also resulted in an immediate and sustained cytosolic accumulation of Ca2+, culminating in the increased production of cytokines and chemokines. Polarisation of the macrophages prior to HOCl exposure revealed a greater propensity for inflammatory M1 macrophages to produce extracellular traps, whereas alternatively-activated M2 macrophages were less susceptible to HOCl insult. M1 macrophages also produced extracellular traps on exposure to phorbol myristate acetate (PMA), interleukin-8 (IL-8) and tumour necrosis factor α (TNFα). Taken together, these data indicate a potential role for macrophages in mediating extracellular trap formation, which may be relevant in pathological conditions characterised by chronic inflammation or excessive HOCl formation.

Macrophage extracellular trap formation promoted by platelet activation is a key mediator of rhabdomyolysis-induced acute kidney injury.

Rhabdomyolysis is a serious syndrome caused by skeletal muscle injury and the subsequent release of breakdown products from damaged muscle cells into systemic circulation. The muscle damage most often results from strenuous exercise, muscle hypoxia, medications, or drug abuse and can lead to life-threatening complications, such as acute kidney injury (AKI). Rhabdomyolysis and the AKI complication can also occur during crush syndrome, an emergency condition that commonly occurs in victims of natural disasters, such as earthquakes, and man-made disasters, such as wars and terrorism. Myoglobin released from damaged muscle is believed to trigger renal dysfunction in this form of AKI. Recently, macrophages were implicated in the disease pathogenesis of rhabdomyolysis-induced AKI, but the precise molecular mechanism remains unclear. In the present study, we show that macrophages released extracellular traps (ETs) comprising DNA fibers and granule proteins in a mouse model of rhabdomyolysis. Heme-activated platelets released from necrotic muscle cells during rhabdomyolysis enhanced the production of macrophage extracellular traps (METs) through increasing intracellular reactive oxygen species generation and histone citrullination. Here we report, for the first time to our knowledge, this unanticipated role for METs and platelets as a sensor of myoglobin-derived heme in rhabdomyolysis-induced AKI. This previously unknown mechanism might be targeted for treatment of the disease. Finally, we found a new therapeutic tool for prevention of AKI after rhabdomyolysis, which might rescue some sufferers of this pathology.

Nontypeable Haemophilus influenzae induces sustained lung oxidative stress and protease expression.

Nontypeable Haemophilus influenzae (NTHi) is a prevalent bacterium found in a variety of chronic respiratory diseases. The role of this bacterium in the pathogenesis of lung inflammation is not well defined. In this study we examined the effect of NTHi on two important lung inflammatory processes 1), oxidative stress and 2), protease expression. Bronchoalveolar macrophages were obtained from 121 human subjects, blood neutrophils from 15 subjects, and human-lung fibroblast and epithelial cell lines from 16 subjects. Cells were stimulated with NTHi to measure the effect on reactive oxygen species (ROS) production and extracellular trap formation. We also measured the production of the oxidant, 3-nitrotyrosine (3-NT) in the lungs of mice infected with this bacterium. NTHi induced widespread production of 3-NT in mouse lungs. This bacterium induced significantly increased ROS production in human fibroblasts, epithelial cells, macrophages and neutrophils; with the highest levels in the phagocytic cells. In human macrophages NTHi caused a sustained, extracellular production of ROS that increased over time. The production of ROS was associated with the formation of macrophage extracellular trap-like structures which co-expressed the protease metalloproteinase-12. The formation of the macrophage extracellular trap-like structures was markedly inhibited by the addition of DNase. In this study we have demonstrated that NTHi induces lung oxidative stress with macrophage extracellular trap formation and associated protease expression. DNase inhibited the formation of extracellular traps.

Activation of the stress response in macrophages alters the M1/M2 balance by enhancing bacterial killing and IL-10 expression.

Macrophages (Mϕs) play an important role in the inflammatory response during injury by participating in the removal of injurious stimuli, such as bacteria, and promoting tissue healing to restore homeostasis. Mϕs can acquire distinct functional phenotypes along a spectrum between two opposite stages (M1/M2) during activation. In the present study, we induced a stress response in Mϕs via heat shock (HS) and found that it incurred an increase in phagocytosis (1.6-fold, P < 0.05) and bacterial killing (2.8-fold, P < 0.01). Upon heat stress activation, Mϕs respond to group B Streptococcus (GBS) infection with lower levels of pro-inflammatory cytokines, TNF-α (2.25-fold, P < 0.01), IL-6 (7-fold, P < 0.001), and inducible nitric oxide synthase (iNOS) (2.22-fold, P < 0.05), but higher levels of the anti-inflammatory cytokine IL-10 (3.9-fold, P < 0.01). Stressed Mϕs exposed to GBS display rapid phagosome maturation, increased extracellular trap (ET) formation and elevated cathelicidin antimicrobial peptide expression (2.5-fold, P < 0.001). These findings are consistent with a heretofore uncharacterized Mϕ activation state formed in response to stress, associated with secretion of large quantities of anti-inflammatory mediators and redirection of antimicrobial mechanisms to NADPH-oxidase-independent pathways. This "friendly activation" of Mϕs is characterized by increased bactericidal activity and more rapid and controlled resolution of the inflammatory response. Macrophages form a dual pro-bactericidal and anti-inflammatory state. Stress in the setting of infection triggers friendly activation in macrophages. Heat shock plus infection increases macrophage bactericidal activity. Heat shock plus infection increases macrophage extracellular trap formation. Heat shock plus infection increases macrophage production of cathelicidin and IL-10.

Mechanistic Studies of Protein Arginine Deiminase 2: Evidence for a Substrate-Assisted Mechanism

Citrullination, which is catalyzed by protein arginine deiminases (PADs 1–4 and 6), is a post-translational modification (PTM) that effectively neutralizes the positive charge of a guanidinium group by its replacement with a neutral urea. Given the sequence similarity of PAD2 across mammalian species and the genomic organization of the PAD2 gene, PAD2 is predicted to be the ancestral homologue of the PADs. Although PAD2 has long been known to play a role in myelination, it has only recently been linked to other cellular processes, including gene transcription and macrophage extracellular trap formation. For example, PAD2 deiminates histone H3 at R26, and this PTM leads to the increased transcription of more than 200 genes under the control of the estrogen receptor. Given that our understanding of PAD2 biology remains incomplete, we initiated mechanistic studies on this enzyme to aid the development of PAD2-specific inhibitors. Herein, we report that the substrate specificity and calcium dependence of PAD2 are similar to those of PADs 1, 3, and 4. However, unlike those isozymes, PAD2 appears to use a substrate-assisted mechanism of catalysis in which the positively charged substrate guanidinium depresses the pKa of the nucleophilic cysteine. By contrast, PADs 1, 3, and 4 use a reverse-protonation mechanism. These mechanistic differences will aid the development of isozyme-specific inhibitors.

Mycobacterium tuberculosis Exploits Human Interferon γ to Stimulate Macrophage Extracellular Trap Formation and Necrosis

Human neutrophils form extracellular traps during M. tuberculosis infection, but a similar phenomenon has not been reported in human macrophages. Here we demonstrate that M. tuberculosis induces release of extracellular traps from human macrophages. This process is regulated by elastase activity, previously shown to regulate formation of extracellular traps by neutrophils. Interestingly, formation of extracellular traps by macrophages during M. tuberculosis infection is inducible by interferon γ (IFN-γ). These traps are mainly produced by heavily infected macrophages. Accordingly, IFN-γ is found to stimulate M. tuberculosis aggregation in macrophages. Both IFN-γ–inducible events, extracellular trap formation and mycobacterial aggregation, require the ESX-1 secretion system. In addition, IFN-γ is found to enhance ESX-1–mediated macrophage necrosis. In the absence of ESX-1, IFN-γ does not restore any extracellular trap formation, mycobacterial aggregation, or macrophage necrosis. Thus, initial characterization of macrophage extracellular trap formation due to M. tuberculosis infection led to the uncovering of a novel role for IFN-γ in amplifying multiple effects of the mycobacterial ESX-1.

Histophilus somni causes extracellular trap formation by bovine neutrophils and macrophages

Histophilus somni (formerly Haemophilus somnus) is a Gram-negative pleomorphic coccobacillus that causes respiratory, reproductive, cardiac and neuronal diseases in cattle. H. somni is a member of the bovine respiratory disease complex that causes severe bronchopneumonia in cattle. Previously, it has been reported that bovine neutrophils and macrophages have limited ability to phagocytose and kill H. somni. Recently, it was discovered that bovine neutrophils and macrophages produce extracellular traps in response to Mannheimia haemolytica, another member of the bovine respiratory disease complex. In this study, we demonstrate that H. somni also causes extracellular trap production by bovine neutrophils in a dose- and time-dependent manner, which did not coincide with the release of lactate dehydrogenase, a marker for necrosis. Neutrophil extracellular traps were produced in response to outer membrane vesicles, but not lipooligosacchride alone. Using scanning electron microscopy and confocal microscopy, we observed H. somni cells trapped within a web-like structure. Further analyses demonstrated that bovine neutrophils trapped and killed H. somni in a DNA-dependent manner. Treatment of DNA extracellular traps with DNase I freed H. somni cells and diminished bacterial death. Treatment of bovine monocyte-derived macrophages with H. somni cells also caused macrophage extracellular trap formation. These findings suggest that extracellular traps may play a role in the host response to H. somni infection in cattle.

Mannheimia haemolytica and Its Leukotoxin Cause Macrophage Extracellular Trap Formation by Bovine Macrophages

Human and bovine neutrophils release neutrophil extracellular traps (NETs), which are protein-studded DNA matrices capable of extracellular trapping and killing of pathogens. Recently, we reported that bovine neutrophils release NETs in response to the important respiratory pathogen Mannheimia haemolytica and its leukotoxin (LKT). Here, we demonstrate macrophage extracellular trap (MET) formation by bovine monocyte-derived macrophages exposed to M. haemolytica or its LKT. Both native fully active LKT and noncytolytic pro-LKT (produced by an lktC mutant of M. haemolytica) stimulated MET formation. Confocal and scanning electron microscopy revealed a network of DNA fibrils with colocalized histones in extracellular traps released from bovine macrophages. Formation of METs required NADPH oxidase activity, as previously demonstrated for NET formation. METs formed in response to LKT trapped and killed a portion of the M. haemolytica cells. Bovine alveolar macrophages, but not peripheral blood monocytes, also formed METs in response to M. haemolytica cells. MET formation was not restricted to bovine macrophages. We also observed MET formation by the mouse macrophage cell line RAW 264.7 and by human THP-1 cell-derived macrophages, in response to Escherichia coli hemolysin. The latter is a member of the repeats-in-toxin (RTX) toxin family related to the M. haemolytica leukotoxin. This study demonstrates that macrophages, like neutrophils, can form extracellular traps in response to bacterial pathogens and their exotoxins.