Macrophage Extracellular Traps Research Articles

Morphologic Analysis of M2 Macrophage in Glioblastoma: Involvement of Macrophage Extracellular Traps (METs).

Macrophages are classified into two phenotypes, M1 and M2, based on their roles. M2 macrophages suppress inflammation and increase in proportion to the malignancy of brain tumors. Recently, macrophage extracellular traps (METs), which change into a network, have been reported as a unique form of macrophage cell death. In this study, immunohistochemical analysis of macrophages in METs in human glioblastoma was performed. To distinguish between M1 and M2 macrophages, multiple immunostainings with Iba1 combined with CD163 or CD204 were performed. M2 macrophages were present in small amounts in normal and borderline areas but showed an increasing trend as they shifted to tumor areas, and most of them were the activated- or phagocytic-type. We also successfully detected METs coexisting with fibrin and lactoferrin near the border between the tumor and necrotic area. M2 macrophages not only suppressed inflammation but also were involved in the formation of METs. This study found that M2 macrophages play various roles in unstable situations.

Calcium phosphate-based biomaterials trigger human macrophages to release extracellular traps

As central part of the innate immune response, immune cells fight against invaders through various mechanisms, such as the release of extracellular traps (ETs). While this mechanism is mainly known for neutrophils in biomaterial contact, the release of macrophage extracellular traps (METs) in response to biomaterials has not yet been reported. An important application area for biomaterials is bone, where healing of defects of a critical size requires the implantation of grafts, which are often composed of calcium phosphates (CaPs). In this study, the response of human monocyte-derived macrophages in vitro to two different CaPs (α-tricalcium phosphate (α-TCP) and calcium deficient hydroxyapatite (CDHA)) as well as different pore structures was investigated. Scaffolds with anisotropic porosity were prepared by directional freezing, while samples with isotropic pore structure served as reference. It was revealed that ETs are released by human monocyte-derived macrophages in direct or indirect contact with CaP scaffolds. This was caused by mineral nanoparticles formed during incubation of α-TCP samples in culture medium supplemented with human platelet lysate, with an anisotropic pore structure attenuating MET formation. METs were significantly less pronounced or absent in association with CDHA samples. It was furthermore demonstrated that MET formation was accompanied by an increase in pro-inflammatory cytokines. Thus, this study provided the first evidence that macrophages are capable of releasing ETs in response to biomaterials.

Role of macrophage extracellular traps in innate immunity and inflammatory disease.

Macrophages play an integral role in initiating innate immune defences and regulating inflammation. They are also involved in maintaining homeostasis and the resolution of inflammation, by promoting tissue repair and wound healing. There is evidence that like neutrophils, macrophages can release extracellular traps following exposure to a range of pathogenic and pro-inflammatory stimuli. Extracellular traps are released by a specialised cell death pathway termed 'ETosis', and consist of a backbone of DNA and histones decorated with a range of other proteins. The composition of extracellular trap proteins can be influenced by both the cell type and the local environment in which the traps are released. In many cases, these proteins have an antimicrobial role and assist with pathogen killing. Therefore, the release of extracellular traps serves as a means to both immobilise and destroy invading pathogens. In addition to their protective role, extracellular traps are also implicated in disease pathology. The release of neutrophil extracellular traps (NETs) is causally linked to the development of wide range of human diseases. However, whether macrophage extracellular traps (METs) play a similar role in disease pathology is less well established. Moreover, macrophages are also involved in the clearance of extracellular traps, which could assist in the resolution of tissue damage associated with the presence of extracellular traps. In this review, we will provide an overview of the pathways responsible for macrophage extracellular trap release, and discuss the role of these structures in innate immunity and disease pathology and possible therapeutic strategies.

Enhanced Responsive Formation of Extracellular Traps in Macrophages Previously Exposed to Porphyromonas gingivalis.

Tolerance is defined to be a hyporesponsive state following repeated stimulations with bacteria or their virulence factors and has potential impacts on the development of periodontitis. Recently, macrophages have been reported to release chromatin and antimicrobial peptides to form extracellular traps upon bacterial or chemical stimulations. Thus, we explored the roles and mechanisms of tolerance induced by Porphyromonas gingivalis (P. gingivalis) in macrophage extracellular traps (METs). Tolerance in peritoneal macrophages from mice was triggered by repeated P. gingivalis stimulation. METs were observed using fluorescence microscopy, and the levels of extracellular DNA were determined by microplate reader assays. The expression of p-RAF, p-MEK, and p-ERK was examined by Western blot, and reactive oxygen species (ROS) production was explored using flow cytometry. Moreover, the levels of intracellular Ca2+ were also determined by confocal microscopy to identify the possible mechanisms related to the changes in METs in P. gingivalis-pretreated macrophages. Repeated P. gingivalis stimulation contributed to the formation of METs and increased levels of extracellular DNA (p < 0.05). ROS generation and RAF/MEK/ERK phosphorylation were decreased in P. gingivalis-pretreated macrophages compared with non-pretreated cells (p < 0.05), which was inconsistent with the changes in METs. However, in P. gingivalis-pretreated macrophages, the levels of intracellular Ca2+ were significantly increased compared with the single stimulation group. Additionally, inhibition of intracellular Ca2+ resulted in a decrease in the levels of extracellular DNA in P. gingivalis-pretreated cells (p < 0.05). Taken together, P. gingivalis-pretreated macrophages released more METs, possibly related to the increased levels of intracellular Ca2+.

Macrophage Extracellular Traps: Current Opinions and the State of Research regarding Various Diseases.

Macrophages are an important component of the human immune system and play a key role in the immune response, which can protect the body against infection and regulate the development of tissue inflammation. Some studies found that macrophages can produce extracellular traps (ETs) under various conditions of stimulation. ETs are web-like structures that consist of proteins and DNA. ETs are thought to immobilize and kill microorganisms, as well as play an important role in tissue damage, inflammatory progression, and autoimmune diseases. In this review, the structure, identification, mechanism, and research progress of macrophage extracellular traps (METs) in related diseases are reviewed.

Extracellular CIRP Induces Macrophage Extracellular Trap Formation Via Gasdermin D Activation.

Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern promoting inflammation and tissue injury. During bacterial or viral infection, macrophages release DNA decorated with nuclear and cytoplasmic proteins known as macrophage extracellular traps (METs). Gasdermin D (GSDMD) is a pore-forming protein that has been involved in extracellular trap formation in neutrophils. We hypothesized that eCIRP induces MET formation by activating GSDMD. Human monocytic cell line THP-1 cells were differentiated with phorbol 12-myristate 13-acetate (PMA) and treated with recombinant murine (rm) CIRP. The MET formation was detected by three methods: time-lapse fluorescence microscopy (video imaging), colorimetry, and ELISA. Cleaved forms of GSDMD, and caspase-1 were detected by Western blotting. Treatment of THP-1 cells with rmCIRP increased MET formation as revealed by SYTOX Orange Staining assay in a time- and dose-dependent manner. METs formed by rmCIRP stimulation were further confirmed by extracellular DNA, citrullinated histone H3, and myeloperoxidase. Treatment of THP-1 cells with rmCIRP significantly increased the cleaved forms of caspase-1 and GSDMD compared to PBS-treated cells. Treatment of macrophages with caspase-1, and GSDMD inhibitors z-VAD-fmk, and disulfiram, separately, significantly decreased rmCIRP-induced MET formation. We also confirmed rmCIRP-induced MET formation using primary cells murine peritoneal macrophages. These data clearly show that eCIRP serves as a novel inducer of MET formation through the activation of GSDMD and caspase-1.

Interaction Between Macrophage Extracellular Traps and Colon Cancer Cells Promotes Colon Cancer Invasion and Correlates With Unfavorable Prognosis.

BackgroundMacrophage extracellular traps (METs) and tumor-infiltrating macrophages contribute to the progression of several diseases. But the role of METs and tumor-infiltrating macrophages in colon cancer (CC) has not been illuminated. In this study, we aimed to clarify the prognostic value of METs for CC patients and to explore the interaction between CC cells and METs in vitro and in vivo.MethodsA training cohort consisting of 116 patients and a validation cohort of 94 patients were enrolled in this study. Immunofluorescence (IF) staining was conducted to determine METs formation in CC patients. Cox regression was used to perform prognostic analysis and screen out the best prognostic model. A nomogram was established to predict 5-year overall survival (OS). The correlation between METs with clinicopathological features and inflammatory markers was analyzed. The formation of METs in vitro was detected by SYTOX® green and IF staining, and the effect of METs on CC cells was detected by transwell assays. PAD2-IN-1, a selective inhibitor of peptidylarginine deiminase 2 (PAD2), was introduced to destroy the crosstalk between CC cells and METs in vitro and in vivo.ResultsMETs levels were higher in CC tissues and were an independent prognostic factor for CC patients. The prognostic model consisting of age, tumors local invasion, lymph node metastasis and METs were confirmed to be consistent and accurate for predicting the 5-year OS of CC patients. Besides, METs were correlated with distant metastasis and inflammation. Through in vitro experiments, we confirmed that there was a positive feedback loop between CC cells and METs, in that METs promoted the invasion of CC cells and CC cells enhanced the production of METs, in turn. This interaction could be blocked by PAD2-IN-1 inhibitors. More importantly, animal experiments revealed that PAD2-IN-1 inhibited METs formation and CC liver metastasis in vivo.ConclusionsMETs were the potential biomarker of CC patient prognosis. PAD2-IN-1 inhibited the crosstalk between CC cells and METs in vitro and in vivo, which should be emphasized in CC therapy.

Neutrophil Elastase Triggers the Release of Macrophage Extracellular Traps: Relevance to Cystic Fibrosis.

Neutrophil extracellular traps increase cystic fibrosis (CF) airway inflammation. We hypothesized that macrophage exposure to neutrophil elastase (NE) would trigger the release of macrophage extracellular traps (METs), a novel mechanism to augment NE-induced airway inflammation in CF. Experiments were performed using human blood monocyte derived macrophages (hBMDM) from patients with and without CF to test specific mechanisms associated with MET release, and MET release by NE was confirmed in alveolar macrophages from Cftr-null and wild-type littermate mice exposed to intratracheal NE in vivo. Human BMDM were exposed to FITC-NE, and intracellular FITC-NE was localized to cytoplasmic and nuclear domains. Intracellular NE was proteolytically active as indicated by DQ-Elastin substrate cleavage. NE (100 to 500 nM) significantly increased extracellular PicoGreen fluorescence consistent with DNA release/ MET release from hBMDM in the absence of cell death. MET release was further confirmed by confocal microscopy in hBMDM treated with NE, and in alveolar macrophages from Cftr-null and wild-type littermate mice that had been exposed to intratracheal NE. NE-triggered MET release was associated with H3 citrullination detected by immunofluorescence assays and with partial cleavage of histone H3 but not H4. Exposure to NE caused release of METs from both CF and non-CF hBMDM invitro and murine alveolar macrophages invivo. MET release was associated with NE-activated H3 clipping, a mechanism associated with chromatin decondensation, a prerequisite for METs.

Bacillus licheniformis and Bacillus subtilis, Probiotics That Induce the Formation of Macrophage Extracellular Traps.

Probiotics are considered living microorganisms that help preserve the health of the host who uses them. Bacillus are a genus of Gram-positive bacteria used as probiotics for animal and human consumption. They are currently distributed in various commercial forms. Two of the species used as probiotics are B. licheniformis and B. subtilis. Macrophages are central cells in the immune response, being fundamental in the elimination of microbial pathogens, for which they use various mechanisms, including the formation of extracellular traps (METs). There have been very few studies carried out on the participation of macrophages in response to the interaction of probiotics of the genus Bacillus with the host. In this work, we used macrophages from the J774A mouse cell line.1, and we found that they are susceptible to infection by the two Bacillus species. However, both species were eliminated as the infection progressed. Using confocal microscopy, we identified the formation of METs from the first hours of infection, which were characterized by the presence of myeloperoxidase (MPO) and citrullinated histone (Hit3Cit). Quantitative data on extracellular DNA release were also obtained; release was observed starting in the first hour of infection. The induction of METs by B. licheniformis caused a significant decrease in the colony-forming units (CFU) of Staphylococcus aureus. The induction of METS by bacteria of the Bacillus genus is a mechanism that participates in controlling the probiotic and potentially pathogenic bacteria such as S. aureus. The induction of METs to control pathogens may be a novel mechanism that could explain the beneficial effects of probiotics of the genus Bacillus.

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.

Neutrophil Extracellular Traps and Macrophage Extracellular Traps Predict Postoperative Recurrence in Resectable Nonfunctional Pancreatic Neuroendocrine Tumors.

BackgroundExtracellular traps (ETs) and tumor-infiltrating immune cells can contribute to disease progression. The clinical significance of tumor-infiltrating neutrophils and macrophages and related extracellular traps in pancreatic neuroendocrine tumors (pNETs) has not been fully elucidated. This study aimed to explore the prognostic value of tumor infiltration and ET formation by neutrophils and macrophages in pNETs.MethodsA total of 135 patients with radical resection of nonfunctional pNETs were analyzed retrospectively. Immunohistochemistry and immunofluorescence were utilized to stain tumor tissue sections. The recurrence-free survival (RFS) of subgroups determined by Kaplan-Meier analysis was compared with the log-rank test. Univariate and multivariate Cox regression analyses were used to identify independent prognostic factors. A nomogram was established to predict 3-year RFS.ResultsPatients with high tumor-infiltrating neutrophils or macrophages or positive expression of neutrophils ETs or macrophage ETs displayed worse RFS (all p<0.05). Moreover, univariate and multivariate Cox regression analyses showed that neutrophil and macrophage infiltration and ETs were independent prognostic factors for RFS (all p<0.05). A combined parameter including WHO grade, TNM stage, tumor-infiltrating neutrophils and macrophages, and neutrophil and macrophage ETs had the highest C-index (0.866) and lowest Akaike information criteria (326.557). The calibration plot of nomogram composed of the combined parameter exhibited excellent prognostic values for 3-year RFS.ConclusionsInfiltration and ETs by neutrophils and macrophages can be used as biological indicators of patient prognosis, suggesting the treatment potential for targeting those in nonfunctional pNETs.

Salmonella Typhimurium Triggers Extracellular Traps Release in Murine Macrophages.

Salmonella comprises two species and more than 2500 serovars with marked differences in host specificity, and is responsible for a wide spectrum of diseases, ranging from localized gastroenteritis to severe life-threatening invasive disease. The initiation of the host inflammatory response, triggered by many Pathogen-Associated Molecular Patterns (PAMPs) that Salmonella possesses, recruits innate immune cells in order to restrain the infection at the local site. Neutrophils are known for killing bacteria through oxidative burst, amid other mechanisms. Amongst those mechanisms for controlling bacteria, the release of Extracellular Traps (ETs) represents a newly described pathway of programmed cell death known as ETosis. Particularly, Neutrophil Extracellular Traps (NETs) were first described in 2004 and since then, a number of reports have demonstrated their role as a novel defense mechanism against different pathogens. This released net-like material is composed of cellular DNA decorated with histones and cellular proteins. These structures have shown ability to trap, neutralize and kill different kinds of microorganisms, ranging from viruses and bacteria to fungi and parasites. Salmonella was one of the first microorganisms that were reported to be killed by NETs and several studies have confirmed the observation and deepened into its variants. Nevertheless, much less is known about their counterparts in other immune cells, e.g. Macrophage Extracellular Traps (METs) and Salmonella-induced MET release has never been reported so far. In this work, we observed the production of METs induced by Salmonella enterica serovar Typhimurium and recorded their effect on bacteria, showing for the first time that macrophages can also release extracellular DNA traps upon encounter with Salmonella Typhimurium. Additionally we show that METs effectively immobilize and reduce Salmonella survival in a few minutes, suggesting METs as a novel immune-mediated defense mechanism against Salmonella infection. Of note, this phenomenon was confirmed in primary macrophages, since MET release was also observed in bone marrow-derived macrophages infected with Salmonella. The evidence of this peculiar mechanism provides new incipient insights into macrophages´ role against Salmonella infection and can help to design new strategies for the clinical control of this transcendental pathogen.

Heme activates platelets and exacerbates rhabdomyolysis-induced acute kidney injury via CLEC-2 and GPVI/FcRγ

There is increasing evidence that platelets participate in multiple pathophysiological processes other than thrombosis and hemostasis, such as immunity, inflammation, embryonic development, and cancer progression. A recent study revealed that heme (hemin)-activated platelets induce macrophage extracellular traps (METs) and exacerbate rhabdomyolysis-induced acute kidney injury (RAKI); however, how hemin activates platelets remains unclear. Here, we report that both C-type lectin-like receptor-2 (CLEC-2) and glycoprotein VI (GPVI) are platelet hemin receptors and are involved in the exacerbation of RAKI. We investigated hemin-induced platelet aggregation in humans and mice, binding of hemin to CLEC-2 and GPVI, the RAKI-associated phenotype in a mouse model, and in vitro MET formation. Using western blotting and surface plasmon resonance, we showed that hemin activates human platelets by stimulating the phosphorylation of SYK and PLCγ2 and directly binding to both CLEC-2 and GPVI. Furthermore, hemin-induced murine platelet aggregation was partially reduced in CLEC-2–depleted and FcRγ-deficient (equivalent to GPVI-deficient) platelets and almost completely inhibited in CLEC-2–depleted FcRγ-deficient (double-knockout) platelets. In addition, hemin-induced murine platelet aggregation was inhibited by the CLEC-2 inhibitor cobalt hematoporphyrin or GPVI antibody (JAQ-1). Renal dysfunction, tubular injury, and MET formation were attenuated in double-knockout RAKI mice. Furthermore, in vitro MET formation assay showed that the downstream signaling pathway of CLEC-2 and GPVI is involved in MET formation. We propose that both CLEC-2 and GPVI in platelets play an important role in RAKI development.

Protective Role of Activated Protein C against Viral Mimetic Poly(I:C)-Induced Inflammation.

Activated protein C (APC) is an anticoagulant plasma serine protease which exhibits potent cytoprotective and anti-inflammatory activities. Here, we studied protective effects of APC on the proinflammatory function of polyinosinic:polycytidylic acid [poly(I:C)], a synthetic analog of viral double-stranded RNA, in cellular and animal models. Poly(I:C) induced histone H3 extranuclear translocation via interaction with toll-like receptor 3 in two established endothelial cell lines. Furthermore, poly(I:C) induced histone H3 extranuclear translocation in J774A.1 macrophages and human neutrophils and formation of macrophage and neutrophil extracellular traps (ETs). Mechanistically, poly(I:C) was found to upregulate expression of peptidylarginine deiminase 4 and enhance its interaction with histone H3, thereby leading to increased histone citrullination and neutrophil ET formation. Poly(I:C) elicited proinflammatory signaling responses by inducing nuclear factor kappa B activation and disrupting endothelial cell permeability. In vivo, poly(I:C) enhanced cell surface expression of Mac-1 on neutrophils in mice and facilitated their infiltration to lung tissues. Poly(I:C) also downregulated thrombomodulin expression in mouse tissues and reduced its circulating soluble level in plasma. We demonstrate in this study that APC and a signaling-selective mutant of APC effectively inhibit proinflammatory signaling effects of poly(I:C) in both cellular and animal models. We further demonstrate that unlike the requirement for endothelial protein C receptor on endothelial cells, the integrin Mac-1 is involved in the protease-activated receptor 1-dependent APC inhibition of macrophage ET formation in J774A.1 cells. Taken together, these results support a key role for APC signaling in inhibiting the viral mimetic-induced proinflammatory signaling responses and histone translocation-associated formation of ETs by innate immune cells.

Hepcidin gene silencing ameliorated inflammation and insulin resistance in adipose tissue of db/db mice via inhibiting METs formation

As a major feature of diabetes, inflammation is closely related to macrophage extracellular traps and the expression of hepcidin upregulated by diabetes is reportedly involved in chronic inflammation. Therefore, we aimed to explore whether hepcidin could be implicated in inflammation and macrophage extracellular traps (METs) formation. The diabetic db/db mouse model was established exhibiting insulin resistance (IR), inflammation, macrophages infiltration and higher expression of hepcidin, where samples were obtained from epididymal adipose tissue. We observed that inflammation and IR improved in adipose tissue of mice treated with hepcidin gene silencing. Furthermore, METs formation could be markedly inhibited via hepcidin gene silencing followed by attenuated inflammatory response due to METs, indicating hepcidin gene silencing played a key role in anti‐inflammation by inhibiting METs formation. So, we concluded that hepcidin gene silencing has a potential for treatment of diabetes due to its ability to ameliorate inflammation via inhibiting METs formation.

Hydrogen sulfide of air induces macrophage extracellular traps to aggravate inflammatory injury via the regulation of miR-15b-5p on MAPK and insulin signals in trachea of chickens

Hydrogen sulfide (H2S) is an environmental contaminant to cause the airway damage. The release of macrophage extracellular traps (METs) is the mechanism of immune protection to harmful stimulation via microRNAs, but excessive METs cause the injury. However, few studies have attempted to interpret the mechanism of an organism injury due to H2S via METs in chickens. Here, we investigated the transcriptome profiles, pathological morphologic changes and METs release from chicken trachea after H2S exposure. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that 10 differentially expressed genes were related to the METs release, the MAPK and insulin signaling pathways. Morphological and immunofluorescence analysis showed that H2S caused airway injury and MET release. H2S activated the targeting effect of miRNA-15b-5p on activating transcription factor 2 (ATF2). Western blotting and real time quantitative PCR results showed that H2S down-regulated the levels of dual specificity protein phosophatase1 (DUSP1) but up-regulated p38 MAP Kinase (p38) in the MAPK signal pathway. And the expression of phosphoinositide-dependent protein kinase 1 (PDK1), serine/threonine kinase (Akt), and protein kinase ζ subtypes (PKCζ) in the insulin signal pathway were increased after H2S exposure. These promoted the release of myeloperoxidase (MPO) and degradation histone 4 (H4) to induce the release of METs. Taken together, miR-15b-5p targeted ATF2 to mediate METs release, which triggered trachea inflammatory injury via MAPK and insulin signals after H2S exposure. These results will provide new insights into the toxicological mechanisms of H2S and environmental ecotoxicology.

Relevance of Macrophage Extracellular Traps in C. albicans Killing.

Candida albicans causes systemic life-threatening infections, particularly in immunocompromised individuals, such as patients in intensive care units, patients undergoing chemotherapy, and post-surgical and neutropenic patients. The proliferation of invading Candida cells is mainly limited by the action of the human innate immune system, in which phagocytic cells play a fundamental role. This function is, however, limited in neutropenic patients, who rely mainly on the protective immunity mediated by macrophages. Macrophages have been shown to release extracellular DNA fibers, known as macrophage extracellular traps (METs), which can entrap and kill various microbes by a process called ETosis. In this study, we observed that, upon contact with C. albicans, macrophages became active in phagocyting and engulfing yeast cells. ETosis was induced in 6% of macrophages within the first 30 min of contact, and this percentage increased with the multiplicity of infection until a plateau was reached. After 2.5 h incubation, the presence of extracellular macrophage DNA was observed in approximately half of the cells. This study suggests that the formation of METs occurs before pyroptosis (first 6–8 h) and macrophage cell death (up to 24 h), and thus, METs could be included in models describing C. albicans–macrophage interactions. We also observed that macrophage ETosis and phagocytosis can occur simultaneously and that, in the first hours of infection, both processes are similarly important in controlling the proliferation of yeast cells, this being critical in neutropenic patients. Finally, it can also be concluded that, since C. albicans can degrade DNA, the structural component of METs, yeast extracellular DNase activity can be considered as an important virulence factor.

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages.

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic inflammation. Neutrophil ETs (NETs) consist of a mesh of DNA, histone proteins, and various granule proteins (i.e., myeloperoxidase, elastase, and cathepsin G). Other immune cells, including macrophages, can also produce ETs; however, to what extent this occurs in vivo and whether macrophage extracellular traps (METs) play a role in pathological mechanisms has not been examined in detail. To better understand the role of METs in inflammatory pathologies, a protocol was developed for visualizing MET release from primary human macrophages in vitro, which can also be exploited in immunofluorescence experiments. This allows further characterization of these structures and their comparison to ETs released from neutrophils. Human monocyte-derived macrophages (HMDM) produce METs upon exposure to different inflammatory stimuli following differentiation to the M1 pro-inflammatory phenotype. The release of METs can be visualized by microscopy using a green fluorescent nucleic acid stain that is impermeant to live cells (e.g., SYTOX green). Use of freshly isolated primary macrophages, such as HMDM, is advantageous in modeling in vivo inflammatory events that are relevant to potential clinical applications. This protocol can also be used to study MET release from human monocyte cell lines (e.g., THP-1) following differentiation into macrophages with phorbol myristate acetate or other macrophage cell lines (e.g., the murine macrophage-like J774A.1 cells).

Deletion of MCP-1 enhances susceptibility to carbapenem-resistant Klebsiella pneumoniae via modulating neutrophil and macrophage function

Abstract Monocyte chemoattractant protein-1 (MCP-1) is important for monocyte recruitment to the lungs in response to bacterial infection. MCP-1 is also essential for neutrophil-dependent host defense during Escherichia coli infection. Carbapenem-Resistant Klebsiella pneumoniae (CRKP) infection has been rapidly growing as a life-threatening nosocomial disease in the U.S. and world. However, the role of chemokines and cellular responses in protective immunity to pulmonary infection with CRKP is largely unknown. Here, we investigated the role of MCP-1 in pulmonary host defense against CRKP infection using MCP-1 knockout (KO) mice. We determined survival, bacterial burden, and neutrophil influx, cytokine/chemokine expression as well as cellular functions, such as formation of neutrophil extracellular traps (NETs) and macrophage extracellular traps (METs). MCP-1 is essential for survival and restricting bacterial outgrowth organs. Compared to the C57Bl/6 (control) mice, KO mice showed impaired influx of neutrophils in the airways as assessed by nuclear cell morphology in BALF and by myeloperoxidase activity (MPO) in lung parenchyma. Moreover, the NETs and METs induced by CRKP were reduced in KO mice in both in vitro or in vivo. Intriguingly, subsequent to CRKP infection, KO mice demonstrated increased pyroptosis through enhanced activation of caspase-1, higher expression of IL-1β and cleaved gasdermin-D in the lungs. These findings enhance our understanding of the critical role of MCP-1 in modulating neutrophil function, such as NETosis, METosis and pyroptosis and suggest that therapies targeting modulation of NETs, METs and pyroptosis during CRKP infection can be explored through clinical trials.

Utilization of macrophage extracellular trap nucleotides by Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia in swine, an important disease worldwide. It has finite biosynthetic capabilities, including a deficit in de novo nucleotide synthesis. The source(s) for nucleotides in vivo are unknown, but mycoplasmas are known to carry membrane-bound nucleases thought to participate in the acquisition of nucleotides from host genomic DNA. Recent research has demonstrated that neutrophils can produce extracellular traps (NETs), chromatin NETs decorated with granular proteins to interact with and eliminate pathogens. We hypothesized that M. hyopneumoniae could utilize its membrane nuclease to obtain nucleotides from extracellular traps to construct its own DNA. Using the human monocytic cell line THP-1, we induced macrophage extracellular traps (METs), which are structurally similar to NETs. The thymidine analogue ethynyl deoxyuridine (EdU) was incorporated into THP-1 DNA and METs were induced. When incubated with M. hyopneumoniae, METs were degraded and the modified nucleotide label could be co-localized within M. hyopneumoniae DNA. When the nucleases were inhibited, MET degradation and nucleotide transfer were also inhibited. Controls confirmed that the EdU originated directly from the METs and not from free nucleotides arising from intracellular pools released during extrusion of the chromosomal DNA. M. hyopneumoniae incorporated labelled nucleotides more efficiently when 'fed' on METs than from free nucleotides in the medium, suggesting a tight linkage between nuclease degradation of DNA and nucleotide transport. These results strongly suggest that M. hyopneumoniae could degrade extracellular traps formed in vivo during infection and incorporate those host nucleotides into its own DNA.