Emerging insights into macrophage extracellular traps in bacterial infections.

Macrophages possess a diverse range of well-defined capabilities and roles as phagocytes, encompassing the regulation of inflammation, facilitation of wound healing, maintenance of tissue homeostasis, and serving as a crucial element in the innate immune response against microbial pathogens. The emergence of extracellular traps is a novel strategy of defense that has been observed in several types of innate immune cells. In response to infection, macrophages are stimulated and produce macrophage extracellular traps (METs), which take the form of net-like structures, filled with strands of DNA and adorned with histones and other cellular proteins. METs not only capture and eliminate microorganisms but also play a role in the development of certain diseases such as inflammation and autoimmune disorders. The primary objective of this study is to examine the latest advancements in METs for tackling bacterial infections. We also delve into the current knowledge and tactics utilized by bacteria to elude or endure the effects of METs. Through this investigation, we hope to shed light on the intricate interactions between bacteria and the host's immune system, particularly in the context of microbicidal effector mechanisms of METs. The continued exploration of METs and their impact on host defense against various pathogens opens up new avenues for understanding and potentially manipulating the immune system's response to infections.

Sperm induce macrophage extracellular trap formation via phagocytosis-dependent mechanism.

Infertility is a public health concern worldwide. Asthenozoospermia is a common cause of male infertility and is characterized by decreased motility. Sperm motility ensures that sperm migrate to complete fertilization. Macrophages are an essential component of innate immunity in the female reproductive tract. Macrophage extracellular traps are induced by various microorganisms to capture and mediate the clearance of microorganisms. The relationship between sperm and macrophage extracellular traps is unclear. The human monocyte leukemia (THP-1) cells differentiated by phorbol myristate acetate (PMA) are widely used as surrogate of human macrophages. This study investigated sperm-induced macrophage extracellular trap formation and clarified some of the mechanisms affecting macrophage extracellular trap production. Sperm-induced macrophage extracellular traps were visualized and components of macrophage extracellular traps were identified by immunofluorescence analyses and scanning electron microscopy. By inhibiting macrophage extracellular trap production and macrophage phagocytosis, the relationship between macrophage phagocytosis and macrophage extracellular trap production was analyzed. Sperm could trigger PMA-differentiated THP-1 macrophages to produce extracellular traps. Sperm-triggered macrophage extracellular traps are dependent on phagocytosis and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Sperm from asthenozoospermia donors are more likely to be phagocytosed by macrophages than sperm from healthy donors, which induce more macrophage extracellular trap release. These data confirm the phenomenon and partial mechanism of sperm-induced macrophage extracellular trap formation in vitro. These may partly provide evidence to explain the mechanisms of clearing abnormally morphological or hypomotile sperm in the female reproductive tract and the rationale for the decreased probability of successful fertilization in asthenozoospermia.

Impact of endogenous and exogenous nitrogen species on macrophage extracellular trap (MET) formation by bone marrow-derived macrophages.

Macrophage extracellular traps (METs) represent a novel defense mechanism in the antimicrobial arsenal of macrophages. However, mechanisms of MET formation are still poorly understood and this is at least partially due to the lack of reliable and reproducible models. Thus, we aimed at establishing a protocol of MET induction by bone marrow-derived macrophages (BMDMs) obtained from cryopreserved and then thawed bone marrow (BM) mouse cells. We report that BMDMs obtained in this way were morphologically (F4/80+) and functionally (expression of inducible nitric oxide (NO) synthase and NO production) differentiated and responded to various stimuli of bacterial (lipopolysaccharide, LPS), fungal (zymosan) and chemical (PMA) origin. Importantly, BMDMs were successfully casting METs composed of extracellular DNA (extDNA) serving as their backbone to which proteins such as H2A.X histones and matrix metalloproteinase 9 (MMP-9) were attached. In rendered 3D structure of METs, extDNA and protein components were embedded in each other. Since studies had shown the involvement of oxygen species in MET release, we aimed at studying if reactive nitrogen species (RNS) such as NO are also involved in MET formation. By application of NOS inhibitor - L-NAME or nitric oxide donor (SNAP), we studied the involvement of endogenous and exogenous RNS in traps release. We demonstrated that L-NAME halted MET formation upon stimulation with LPS while SNAP alone induced it. The latter phenomenon was further enhanced in the presence of LPS. Taken together, our findings demonstrate that BMDMs obtained from cryopreserved BM cells are capable of forming METs in an RNS-dependent manner.

5'-Nucleotidase is dispensable for the growth of Salmonella Typhimurium but inhibits the bactericidal activity of macrophage extracellular traps.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic pathogen that causes severe gastroenteritis. The 5'-nucleotidases of pathogens can dephosphorylate adenosine phosphates, boost adenosine levels and suppress the pro-inflammatory immune response. In our previous study, an extracellular nuclease, 5'-nucleotidase, was identified in the extracellular proteins of S. Typhimurium. However, the nuclease activity and the function of the 5'-nucleotidase of S. Typhimurium have not been explored. In the present study, deletion of the 5'-nucleotidase gene is dispensable for S. Typhimurium growth, even under environmental stress. Fluorescence microscopy revealed that the 5'-nucleotidase mutant induced more macrophage extracellular traps (METs) than the wild type did. Furthermore, recombinant 5'-nucleotidase protein (r5Nuc) could degrade λDNA, and the nuclease activity of r5Nuc was optimum at 37 °C and pH 6.0-7.0. The Mg2+ enhanced the nuclease activity of r5Nuc, whereas Zn2+ inhibited it. Meanwhile, deletion of the 5'-nucleotidase gene increased the bactericidal activity of METs, and r5Nuc could degrade METs and inhibit the bactericidal activity of METs. In conclusion, S. Typhimurium growth was independent of 5'-nucleotidase, but the nuclease activity of 5'-nucleotidase assisted S. Typhimurium to evade macrophage-mediated extracellular killing through degrading METs.

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.

Leishmania donovani mevalonate kinase regulates host actin for inducing phagocytosis.

Despite the well-established role of macrophages in phagocytosing Leishmania, the contribution of the parasite to this process is not well understood. Present study provides insights into the mechanism underlying the MVK-induced entry of L. donovani and improve our knowledge of host-pathogen interactions. We have discussed Mevalonate kinase (MVK)-induced actin reorganization, modulation of signaling pathways and host cell functions. Our results show that LdMVK gains access to macrophage cytosol and induces actin assembly modulation through the activation of actin-related proteins: VASP, Src and ERM. We have also demonstrated that LdMVK induces Ca2+ signaling and Akt pathway in macrophages, which are critical components of Leishmania survival and proliferation. Interestingly, we found that antibodies against LdMVK can kill Leishmania-infected macrophages in culture by forming extracellular traps, highlighting the potential of LdMVK in inhibiting parasite death. Overall, LdMVK is a virulent factor in Leishmania that mediates parasite internalization and host modulation by targeting host proteins phosphorylation and calcium homeostasis having significant implications in disease progression.

Polystyrene microplastics promote liver inflammation by inducing the formation of macrophages extracellular traps.

Microplastics (MPs), a new and increasing environmental pollutant, can cause ongoing damage to organisms. Although recent studies have revealed mechanisms of action for some of the hepatotoxicity caused by MPs, the role-played by cellular interactions, particularly immune cells, in the process of liver injury has not been elucidated. In the present study, 5-µm polystyrene microplastics (PS-MPs) induced liver inflammation as well as the formation of Macrophage extracellular traps (METs). Macrophage and LMH cell co-culture systems confirmed that PS-MPs-induced METs promote inflammation in hepatocytes. Mechanistically, macrophages actively phagocytose particles after 4 h of exposure to PS-MPs. Subsequently PS-MPs elevated ROS levels and disrupt mitochondrial kinetic homeostasis. Further activation of mitochondrial autophagy and lysosomes. After phagocytosis of PS-MPs by macrophages for 12 h, continued autophagy and lysosome activation eventually lead to lysosome rupture and release of calcium ions to induce the formation of METs. Blocking ROS (NAC) and autophagy (3MA) partially alleviated mitochondrial and lysosomal damage and thus inhibited the formation of METs induced by PS-MPs. NAC also delayed the onset of respiratory burst to alleviate METs formation. In conclusion, our study reveals the mechanism of METs formation in liver inflammation induced by PS-MPs exposure and suggests that lysosomal damage may be one of the key players in the formation of METs induced by PS-MPs.

Polystyrene microplastics-induced macrophage extracellular traps contributes to liver fibrotic injury by activating ROS/TGF-ß/Smad2/3 signaling axis.

Microplastics (MPs) are a type of emerging pollutant, posing a great threat to human and animal health. While recent studies have revealed the link between MPs exposure and liver injury of organisms, the effect of particle size on the level of MPs-induced hepatotoxicity and the intrinsic mechanism remain to be explored. Here, we established a mouse model exposed to two-diameter polystyrene MPs (PS-MPs, 1-10 µm or 50-100 µm) for 30 days. The in vivo results revealed that PS-MPs caused liver fibrotic injury in mice, accompanied with macrophages recruitment and macrophage extracellular traps (METs) formation, which were negatively correlated with particle size. The data in vitro showed that PS-MPs treatment could induce macrophages to release METs in a reactive oxygen species (ROS)-independent manner, and the METs formation level caused by large-size particles was higher than small-size particles. Further mechanistic analysis of a cell co-culture system revealed that PS-MPs-induced METs release led to a hepatocellular inflammatory response and epithelial-mesenchymal transition (EMT) via activating the ROS/TGF-ß/Smad2/3 signaling axis, and this biological crosstalk could be relieved by DNase I. Overall, this findings demonstrates the key role of the action mechanism of METs in aggravating MPs-caused liver injury.

Role of phagocyte extracellular traps during Mycobacterium tuberculosis infections and tuberculosis disease processes.

Mycobacterium tuberculosis (M.tb) infections remain one of the most significant causes of mortality worldwide. The current situation shows an emergence of new antibiotic-resistant strains making it difficult to control the tuberculosis (TB) disease. A large part of its success as a pathogen is due to its ability to persist for years or even decades without causing evident clinical manifestations. M.tb is highly successful in evading the host-defense by manipulating host-signalling pathways. Although macrophages are generally viewed as the key cell type involved in harboring M.tb, growing evidence shows that neutrophils also play a fundamental role. Both cells are known to act in multiple ways when encountering an invading pathogen, including phagocytosis, release of cytokines and chemokines, and oxidative burst. In addition, the formation of neutrophil extracellular traps (NETs) and macrophage extracellular traps (METs) has been described to contribute to M.tb infections. NETs/METs are extracellular DNA fibers with associated granule components, which are released upon activation of the cells by the pathogen or by pro-inflammatory mediators. On one hand, they can lead to a protective immune response by entrapment and killing of pathogens. However, on the other hand, they can also play a severe pathological role by inducing tissue damage. Extracellular traps (ETs) produced in the pulmonary alveoli can expand easily and expose tissue-damaging factors with detrimental effects. Since host-directed therapies offer a complementary strategy in TB, the knowledge of NET/MET formation is important for understanding potential protective versus detrimental pathways during innate immune signaling. In this review, we summarize the progress made in understanding the role of NETs/METs in the pathogenesis of TB.

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+.

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.

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.

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

Background: Macrophage 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. Methods: A 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. Results: METs 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. Conclusions: METs 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.

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.

The Cording Phenotype of Mycobacterium tuberculosis Induces the Formation of Extracellular Traps in Human Macrophages.

The causative agent of tuberculosis, Mycobacterium tuberculosis, shares several characteristics with organisms that produce biofilms during infections. One of these is the ability to form tight bundles also known as cords. However, little is known of the physiological relevance of the cording phenotype. In this study, we investigated whether cord-forming M. tuberculosis induce the formation of macrophage extracellular traps (METs) in human monocyte-derived macrophages. Macrophages have previously been shown to produce extracellular traps in response to various stimuli. We optimized bacterial culturing conditions that favored the formation of the cord-forming phenotype as verified by scanning electron microscopy. Microscopy analysis of METs formation during experimental infection of macrophages with M. tuberculosis revealed that cord-forming M. tuberculosis induced significantly more METs compared to the non-cording phenotype. Deletion of early secreted antigenic target-6 which is an important virulence factor of M. tuberculosis, abrogated the ability of the bacteria to induce METs. The release of extracellular DNA from host cells during infection may represent a defense mechanism against pathogens that are difficult to internalize, including cord-forming M. tuberculosis.

Identification of macrophage extracellular trap-like structures in mammary gland adipose tissue: a preliminary study.

PAD4-mediated hypercitrullination of histone H4 arginine 3 (H4R3) has been previously found to promote the formation of Neutrophil Extracellular Traps in inflamed tissues and the resulting histone H4 citrulline 3 (H4Cit3) modification is thought to play a key role in extracellular trap (ET) formation by promoting chromatin decondensation. In addition to neutrophils, macrophages have also recently been found to generate functional extracellular traps (METs). However, a role for PADs in ET formation in macrophages has not been previously described. Transcripts for PAD2 and PAD4 are found in mature macrophages and these cells can be induced to citrullinate proteins, thus raising the possibility that PADs may play a direct role in ET formation in macrophages via histone hypercitrullination. In breast and visceral white adipose tissue from obese patients, infiltrating macrophages are often seen to surround dead adipocytes forming characteristic "crown-like structures" (CLS) and the presence of these lesions is associated with increased levels of inflammatory mediators. In light of these observations, we have initiated studies to test whether PADs are expressed in CLS macrophages and whether these macrophages might form METs. Our preliminary findings show that PAD2 (and to a lesser extent, PAD4) is expressed in both in the macrophage cell line (RAW 264.7) and in CLS lesions. Additionally, we provide evidence that macrophage-derived extracellular histones are seen around presumptive macrophages within CLS lesions and that these histones contain the H4Cit3 modification. These initial findings support our hypothesis that obesity-induced adipose tissue inflammation promotes the formation of METs within CLS lesions via PAD-mediated histone hypercitrullination. Subsequent studies are underway to further validate these findings and to investigate the role in PAD-mediated MET formation in CLS function in the mammary gland.