NLRP3-Induced NETosis: A Potential Therapeutic Target for Ischemic Thrombotic Diseases?

Ischemic thrombotic disease, characterized by the formation of obstructive blood clots within arteries or veins, is a condition associated with life-threatening events, such as stroke, myocardial infarction, deep vein thrombosis, and pulmonary embolism. The conventional therapeutic strategy relies on treatments with anticoagulants that unfortunately pose an inherent risk of bleeding complications. These anticoagulants primarily target clotting factors, often overlooking upstream events, including the release of neutrophil extracellular traps (NETs). Neutrophils are integral components of the innate immune system, traditionally known for their role in combating pathogens through NET formation. Emerging evidence has now revealed that NETs contribute to a prothrombotic milieu by promoting platelet activation, increasing thrombin generation, and providing a scaffold for clot formation. Additionally, NET components enhance clot stability and resistance to fibrinolysis. Clinical and preclinical studies have underscored the mechanistic involvement of NETs in the pathogenesis of thrombotic complications, since the clots obtained from patients and experimental models consistently exhibit the presence of NETs. Given these insights, the inhibition of NETs or NET formation is emerging as a promising therapeutic approach for ischemic thrombotic diseases. Recent investigations also implicate a role for the nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome as a mediator of NETosis and thrombosis, suggesting that NLRP3 inhibition may also hold potential for mitigating thrombotic events. Therefore, future preclinical and clinical studies aimed at identifying and validating NLRP3 inhibition as a novel therapeutic intervention for thrombotic disorders are imperative.

DEL-1 deficiency aggravates pressure overload-induced heart failure by promoting neutrophil infiltration and neutrophil extracellular traps formation.

Recent studies have shown that neutrophils play an important role in the development and progression of heart failure. Developmental endothelial locus-1 (DEL-1) is an anti-inflammatory glycoprotein that has been found to have protective effects in various cardiovascular diseases. However, the role of DEL-1 in chronic heart failure is not well understood. In a mouse model of pressure overload-induced non-ischemic cardiac failure, we found that neutrophil infiltration in the heart increased and DEL-1 levels decreased in the early stages of heart failure. DEL-1 deficiency worsened pressure overload-induced cardiac dysfunction and remodeling in mice. Mechanistically, DEL-1 deficiency promotes neutrophil infiltration and the formation of neutrophil extracellular traps (NETs) through the regulation of P38 signaling. In vitro experiments showed that DEL-1 can inhibit P38 signaling and NETs formation in mouse neutrophils in a MAC-1-dependent manner. Depleting neutrophils, inhibiting NETs formation, and inhibiting P38 signaling all reduced the exacerbation of heart failure caused by DEL-1 deletion. Overall, our findings suggest that DEL-1 deficiency worsens pressure overload-induced heart failure by promoting neutrophil infiltration and NETs formation.

Effect of different sperm populations on neutrophils extracellular traps (NETs) formation in cattle.

In cattle, clinical and subclinical inflammation in the bovine female reproductive tract (FRT) significantly reduces fertility. PMN participate in this FRT-associated inflammation by eliminating pathogens by eliciting various defense mechanisms, with the release of neutrophil extracellular traps NETs) being the latest process discovered. Consistently, human-, bovine- and porcine-derived spermatozoa induce release of NETs in exposed PMN of the same species origin, and thereby decreasing sperm motility through NETs-mediated entrapment. The release of NETs in the presence of different sperm sub-populations is evaluated in this work. Cryopreserved bovine sperm were selected and different sperm populations were used: viable sperm, sperm with oxidative stress, capacitated sperm, and sperm with loss of viability. Isolated PMN of dairy cows were co-incubated with these sperm populations for 4 h. Neutrophil elastase (NE) and DNA were detected by fluorescence microscopy analysis. It was noted that exposed bovine PMN released NETs in the presence of sperm. Moreover, sperm-triggered NETosis resulted different phenotypes of NETs, i. e. spread NETs (sprNETs), diffused NETs (diffNETs) and aggregated NETs (aggNETs). Viable/motile spermatozoa induced a higher proportion of NETotic cells at 15, 60 and 120 min in comparison to controls. In conclusion, all bovine sperm populations in co-culture with PMN generated NETs extrusion while viable sperm activated NETotic cells to a greater extent. With this being an early event in the activation of bovine PMN.

The role of neutrophil extracellular traps in sepsis and sepsis-related acute lung injury.

Neutrophils release neutrophil extracellular traps (NETs) to trap pathogenic microorganisms. NETs are involved in the inflammatory response and bacterial killing and clearance. However, their excessive activation can lead to an inflammatory storm in the body, which may damage tissues and cause organ dysfunction. Organ dysfunction is the main pathophysiological cause of sepsis and also a cause of the high mortality rate in sepsis. Acute lung injury caused by sepsis accounts for the highest proportion of organ damage in sepsis. NET formation can lead to the development of sepsis because by promoting the release of interleukin-1 beta, interleukin-8, and tumor necrosis factor-alpha, thereby accelerating acute lung injury. In this review, we describe the critical role of NETs in sepsis-associated acute lung injury and review the current knowledge and novel therapeutic approaches.

NET-targeted therapy: effects, limitations, and potential strategies to enhance treatment efficacy.

Neutrophil extracellular traps (NETs) are complex structures released by activated neutrophils during inflammatory responses. Due to their unique potential for causing tissue damage and modulating immune responses, there is increasing interest in studying these structures as potential targets for the treatment of infectious diseases, autoimmune diseases, and cancer. However, therapeutic targeting of NETs might trigger deleterious effects that may limit treatment efficacy. NET disruption may increase the microbial load in infection; in autoimmunity, NET targeting might impair peripheral tolerance, but it might reduce adaptive immune responses in cancer. In this review, we explore the therapeutic and deleterious effects of NET-targeted therapy while shedding light on novel strategies to overcome treatment-related limitations and enhance treatment efficacy.

A Tandem-Locked Fluorescent NETosis Reporter for the Prognosis Assessment of Cancer Immunotherapy.

NETosis, the peculiar type of neutrophil death, plays important roles in pro-tumorigenic functions and inhibits cancer immunotherapy. Non-invasive real-time imaging is thus imperative for prognosis of cancer immunotherapy yet remains challenging. Herein, we report a Tandem-locked NETosis Reporter 1 (TNR1 ) that activates fluorescence signals only in the presence of both neutrophil elastase (NE) and cathepsin G (CTSG) for the specific imaging of NETosis. In the aspect of molecular design, the sequence of biomarker-specific tandem peptide blocks can largely affect the detection specificity towards NETosis. In live cell imaging, the tandem-locked design allows TNR1 to differentiate NETosis from neutrophil activation, while single-locked reporters fail to do so. The near-infrared signals from activated TNR1 in tumor from living mice were consistent with the intratumoral NETosis levels from histological results. Moreover, the near-infrared signals from activated TNR1 negatively correlated with tumor inhibition effect after immunotherapy, thereby providing prognosis for cancer immunotherapy. Thus, our study not only demonstrates the first sensitive optical reporter for noninvasive monitoring of NETosis levels and evaluation of cancer immunotherapeutic efficacy in tumor-bearing living mice, but also proposes a generic approach for tandem-locked probe design.

NETs-Induced Thrombosis Impacts on Cardiovascular and Chronic Kidney Disease.

Arterial and venous thrombosis constitute a major source of morbidity and mortality worldwide. Association between thrombotic complications and cardiovascular and other chronic inflammatory diseases are well described. Inflammation and subsequent initiation of thrombotic events, termed immunothrombosis, also receive growing attention but are still incompletely understood. Nevertheless, the clinical relevance of aberrant immunothrombosis, referred to as thromboinflammation, is evident by an increased risk of thrombosis and cardiovascular events in patients with inflammatory or infectious diseases. Proinflammatory mediators released from platelets, complement activation, and the formation of NETs (neutrophil extracellular traps) initiate and foster immunothrombosis. In this review, we highlight and discuss prominent and emerging interrelationships and functions between NETs and other mediators in immunothrombosis in cardiovascular disease. Also, with patients with chronic kidney disease suffering from increased cardiovascular and thrombotic risk, we summarize current knowledge on neutrophil phenotype, function, and NET formation in chronic kidney disease. In addition, we elaborate on therapeutic targeting of NETs-induced immunothrombosis. A better understanding of the functional relevance of antithrombotic mediators which do not increase bleeding risk may provide opportunities for successful therapeutic interventions to reduce thrombotic risk beyond current treatment options.

Constraint-induced movement therapy alleviates motor impairment by inhibiting the accumulation of neutrophil extracellular traps in ischemic cortex.

Stroke is a major cause of mortality and morbidity and most acute strokes are ischemic. Evidence-based medicine has demonstrated the effectiveness of constraint-induced movement therapy (CIMT) in the recovery of motor function in patients after ischemic stroke, but the specific treatment mechanism remains unclear. Herein, our integrated transcriptomics and multiple enrichment analysis studies, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) studies show that CIMT conduction broadly curtails immune response, neutrophil chemotaxis, and chemokine-mediated signaling pathway, CCR chemokine receptor binding. Those suggest the potential effect of CIMT on neutrophils in ischemic mice brain parenchyma. Recent studies have found that accumulating granulocytes release extracellular web-like structures composed of DNA and proteins called neutrophil extracellular traps (NETs), which destruct neurological function primarily by disrupting the blood-brain barrier and promoting thrombosis. However, the temporal and spatial distribution of neutrophils and their released NETs in parenchyma and their damaging effects on nerve cells remain unclear. Thus, utilizing immunofluorescence and flow cytometry, our analyses uncovered that NETs erode multiple regions such as primary motor cortex (M1), striatum (Str), nucleus of the vertical limb of the diagonal band (VDB), nucleus of the horizontal limb of the diagonal band (HDB) and medial septal nucleus (MS), and persist in the brain parenchyma for at least 14 days, while CIMT can reduce the content of NETs and chemokines CCL2 and CCL5 in M1. Intriguingly, CIMT failed to further reduce neurological deficits after inhibiting the NET formation by pharmacologic inhibition of peptidylarginine deiminase 4 (PAD4). Collectively, these results demonstrate that CIMT could alleviate cerebral ischemic injury induced locomotor deficits by modulating the activation of neutrophils. These data are expected to provide direct evidence for the expression of NETs in ischemic brain parenchyma and novel insights into the mechanisms of CIMT protecting against ischemic brain injury.

Neutrophil Extracellular Traps in Cerebral Ischemia/Reperfusion Injury: Friend and Foe.

Cerebral ischemic injury, one of the leading causes of morbidity and mortality worldwide, triggers various central nervous system (CNS) diseases, including acute ischemic stroke (AIS) and chronic ischemia-induced Alzheimer's disease (AD). Currently, targeted therapies are urgently needed to address neurological disorders caused by cerebral ischemia/reperfusion injury (CI/RI), and the emergence of neutrophil extracellular traps (NETs) may be able to relieve the pressure. Neutrophils are precursors to brain injury following ischemic stroke and exert complicated functions. NETs extracellularly release reticular complexes of neutrophils, i.e., double-stranded DNA (dsDNA), histones, and granulins. Paradoxically, NETs play a dual role, friend and foe, under different conditions, for example, physiological circumstances, infection, neurodegeneration, and ischemia/reperfusion. Increasing evidence indicates that NETs exert anti-inflammatory effects by degrading cytokines and chemokines through protease at a relatively stable and moderate level under physiological conditions, while excessive amounts of NETs release (NETosis) irritated by CI/RI exacerbate the inflammatory response and aggravate thrombosis, disrupt the blood-brain barrier (BBB), and initiates sequential neuron injury and tissue damage. This review provides a comprehensive overview of the machinery of NETs formation and the role of an abnormal cascade of NETs in CI/RI, as well as other ischemia-induced neurological diseases. Herein, we highlight the potential of NETs as a therapeutic target against ischemic stroke that may inspire translational research and innovative clinical approaches.

Neutrophil Extracellular Traps (NETs): A New Therapeutic Target for Neuroinflammation and Microthrombosis After Subarachnoid Hemorrhage?

Neutrophil extracellular traps (NETs) play a major role in intrinsic immunity by limiting and killing pathogens. Recently, a series of studies have confirmed that NETs are closely associated with vascular injury and microthrombosis. Furthermore, NETs play an important role in neuroinflammation after ischemic and hemorrhagic stroke. Neuroinflammation and microthrombosis after subarachnoid hemorrhage are key pathophysiological processes associated with poor prognosis, but their crucial formation mechanisms and interventions remain to be elucidated. Could NETs, as an emerging and important pathogenesis, be a new therapeutic target after subarachnoid hemorrhage?

Neutrophils delay repair process in Wallerian degeneration by releasing NETs outside the parenchyma.

Although inflammation is indispensable for the repair process in Wallerian degeneration (WD), the role of neutrophils in the WD repair process remains unclear. After peripheral nerve injury, neutrophils accumulate at the epineurium but not the parenchyma in the WD region because of the blood-nerve barrier. An increase or decrease in the number of neutrophils delayed or promoted macrophage infiltration from the epineurium into the parenchyma and the repair process in WD. Abundant neutrophil extracellular traps (NETs) were formed around neutrophils, and its inhibition dramatically increased macrophage infiltration into the parenchyma. Furthermore, inhibition of either MIF or its receptor, CXCR4, in neutrophils decreased NET formation, resulting in enhanced macrophage infiltration into the parenchyma. Moreover, inhibiting MIF for just 2 h after peripheral nerve injury promoted the repair process. These findings indicate that neutrophils delay the repair process in WD from outside the parenchyma by inhibiting macrophage infiltration via NET formation and that neutrophils, NETs, MIF, and CXCR4 are therapeutic targets for peripheral nerve regeneration.

Aging hampers neutrophil extracellular traps (NETs) efficacy.

BACKGROUND: NETosis is a neutrophil-mediated defense mechanism during which DNA and enzymes are extruded forming a network (NETs) trapping and killing different pathogens. NETosis is reduced in both mice and humans during aging. AIMS: We explored the difference in the efficacy of NETs released in elderly (> 65 years) versus adults (20-50 years) subjects in inhibiting Staphylococcus aureus growth and activating the growth of keratinocytes. METHODS: Neutrophil granulocytes, obtained from venous blood both in healthy elderly and adult subjects, were stimulated by LPS (0-250 µg/ml) to induce the formation of NET. NETs were quantified by SYBR Green staining and growth inhibition of S. aureus was evaluated by disk diffusion test. Furthermore, NETs (0-500 ng/ml) were added to immortalized human keratinocytes (HaCaT cells), and their proliferation was evaluated by MTT assay after 24 h. Finally, the DNA size of NETs was evaluated by flow cytometry after SYBR Green staining. RESULTS: Greater production of NETs was observed in elderly subjects than in adults, but these NETs showed reduced bactericidal capacity and HaCaT cells' proliferation stimulation. The activities of the NETs are related to the size of the extruded DNA threads, and when NETs size was analyzed, DNA from elderly showed a higher size compared to that obtained by adults. DISCUSSION: Unexpected results showed aging-related NETs structural modification resulting in both a lower antimicrobial activity and keratinocyte proliferation stimulation compared to NETs obtained from adults. CONCLUSIONS: The NETs DNA size observed in elderly subjects has not been previously reported and could be part of other pathogenic mechanisms observed in aging.

Neutrophil Extracellular Traps, Local IL-8 Expression, and Cytotoxic T-Lymphocyte Response in the Lungs of Patients With Fatal COVID-19.

BACKGROUND: Excessive inflammation is pathogenic in the pneumonitis associated with severe COVID-19. Neutrophils are among the most abundantly present leukocytes in the inflammatory infiltrates and may form neutrophil extracellular traps (NETs) under the local influence of cytokines. NETs constitute a defense mechanism against bacteria, but have also been shown to mediate tissue damage in a number of diseases. RESEARCH QUESTION: Could NETs and their tissue-damaging properties inherent to neutrophil-associated functions play a role in the respiratory failure seen in patients with severe COVID-19, and how does this relate to the SARS-CoV-2 viral loads, IL-8 (CXCL8) chemokine expression, and cytotoxic T-lymphocyte infiltrates? STUDY DESIGN AND METHODS: Sixteen lung biopsy samples obtained immediately after death were analyzed methodically as exploratory and validation cohorts. NETs were analyzed quantitatively by multiplexed immunofluorescence and were correlated with local levels of IL-8 messenger RNA (mRNA) and the density of CD8+ T-cell infiltration. SARS-CoV-2 presence in tissue was quantified by reverse-transcriptase polymerase chain reaction and immunohistochemistry analysis. RESULTS: NETs were found in the lung interstitium and surrounding the bronchiolar epithelium with interindividual and spatial heterogeneity. NET density did not correlate with SARS-CoV-2 tissue viral load. NETs were associated with local IL-8 mRNA levels. NETs were also detected in pulmonary thrombi and in only one of eight liver tissues. NET focal presence correlated negatively with CD8+ T-cell infiltration in the lungs. INTERPRETATION: Abundant neutrophils undergoing NETosis are found in the lungs of patients with fatal COVID-19, but no correlation was found with viral loads. The strong association between NETs and IL-8 points to this chemokine as a potentially causative factor. The function of cytotoxic T-lymphocytes in the immune responses against SARS-CoV-2 may be interfered with by the presence of NETs.

Endothelial Dysfunction Induced by Extracellular Neutrophil Traps Plays Important Role in the Occurrence and Treatment of Extracellular Neutrophil Traps-Related Disease.

Many articles have demonstrated that extracellular neutrophil traps (NETs) are often described as part of the antibacterial function. However, since the components of NETs are non-specific, excessive NETs usually cause inflammation and tissue damage. Endothelial dysfunction (ED) caused by NETs is the major focus of tissue damage, which is highly related to many inflammatory diseases. Therefore, this review summarizes the latest advances in the primary and secondary mechanisms between NETs and ED regarding inflammation as a mediator. Moreover, the detailed molecular mechanisms with emphasis on the disadvantages from NETs are elaborated: NETs can use its own enzymes, release particles as damage-associated molecular patterns (DAMPs) and activate the complement system to interact with endothelial cells (ECs), drive ECs damage and eventually aggravate inflammation. In view of the role of NETs-induced ED in different diseases, we also discussed possible molecular mechanisms and the treatments of NETs-related diseases.

Hypoxia Promotes Neutrophil Survival After Acute Myocardial Infarction.

Phagocytosis, degranulation, and neutrophil extracellular traps (NETs) formation build the armory of neutrophils for the first line of defense against invading pathogens. All these processes are modulated by the microenvironment including tonicity, pH and oxygen levels. Here we investigated the neutrophil infiltration in cardiac tissue autopsy samples of patients with acute myocardial infarction (AMI) and compared these with tissues from patients with sepsis, endocarditis, dermal inflammation, abscesses and diseases with prominent neutrophil infiltration. We observed many neutrophils infiltrating the heart muscle after myocardial infarction. Most of these had viable morphology and only few showed signs of nuclear de-condensation, a hallmark of early NET formation. The abundance of NETs was the lowest in acute myocardial infarction when compared to other examined diseases. Since cardiac oxygen supply is abruptly abrogated in acute myocardial infarction, we hypothesized that the resulting tissue hypoxia increased the longevity of the neutrophils. Indeed, the viable cells showed increased nuclear hypoxia inducible factor-1α (HIF-1α) content, and only neutrophils with low HIF-1α started the process of NET formation (chromatin de-condensation and nuclear swelling). Prolonged neutrophil survival, increased oxidative burst and reduced NETs formation were reproduced under low oxygen tensions and by HIF-1α stabilization in vitro. We conclude that nuclear HIF-1α is associated with prolonged neutrophil survival and enhanced oxidative stress in hypoxic areas of AMI.

Auricular Vagus Nerve Stimulation Attenuates Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting Neutrophil Infiltration and Neutrophil Extracellular Traps Formation.

ABSTRACT: Vagus nerve stimulation has been shown to exert anti-inflammation activities in sepsis. However, surgical implantation of stimulation devices is performed under general anesthesia, which limits its clinical application. Auricular vagus nerve stimulation (AVNS) is a minimal invasive technique that delivers electrical currents to the auricular branch of the vagus nerve. The purpose of this study was to determine the effects of AVNS on systemic inflammation, lung injury, neutrophil infiltration, and neutrophil extracellular traps (NETs) formation in the lung. In a LPS challenge lung-injury mice model, AVNS was applied to bilateral ears. Twelve hours after LPS administration, samples of blood, bronchoalveolar lavage fluid (BALF), and lung tissues were processed for investigations. We found that the treatment with AVNS significantly attenuated histopathological changes and neutrophil infiltration in the lung tissue, inhibited inflammatory cytokine elevations in serum and BALF, and decreased protein concentrations in BALF. Besides, AVNS decreased leukocyte and neutrophil accounts in BALF. Furthermore, colocalization of citrullination of histone H3 and myeloperoxidase expressions (highly specific marker of NETs) was reduced in AVNS mice. In conclusion, AVNS reduced systemic inflammation, attenuated lung edema, and inhibited neutrophil infiltration and NETs formation in the lung in LPS mice.

Recent progress in the mechanistic understanding of NET formation in neutrophils.

Neutrophils are the most abundant circulating white blood cells and one of the major cell types of the innate immune system. Neutrophil extracellular traps (NETs) are a result of the extracellular release of nuclear chromatin from the ruptured nuclear envelope and plasma membrane. The externalized chromatin is an ancient defense weapon for animals to entrap and kill microorganisms in the extracellular milieu, thus protecting animals ranging from lower invertebrates to higher vertebrates. Although the externalized chromatin has the advantage of acting as anti-infective to protect against infections, extracellular chromatin might be problematic in higher vertebrate animals as they have an adaptive immune system that can trigger further immune or autoimmune responses. NETs and their associated nuclear and/or cytoplasmic components may induce sterile inflammation, immune, and autoimmune responses, leading to various human diseases. Though important in human pathophysiology, the cellular and molecular mechanisms of NET formation (also called NETosis) are not well understood. Given that nuclear chromatin forms the backbone of NETs, the nucleus is the root of the nuclear DNA extracellular traps. Thus, nuclear chromatin decondensation, along with the rupture of nuclear envelope and plasma membrane, is required for nuclear chromatin extracellular release and NET formation. So far, most of the literature focuses on certain signaling pathways, which are involved in NET formation but without explanation of cellular events and morphological changes described above. Here, we have summarized emerging evidence and discuss new mechanistic understanding, with our perspectives, in NET formation in neutrophils.

NETosis in ischemic/reperfusion injuries: An organ-based review.

Neutrophil extracellular trap (NETosis), the web-like structures induced by neutrophil death, is an important inflammatory mechanism of the immune system leading to reactive oxygen species production/coagulopathy, endothelial dysfunction, atherosclerosis, and ischemia. NETosis exerts its role through different mechanisms such as triggering Toll-like receptors, inflammatory cytokines, platelet aggregation, neutrophil activation/infiltration, and vascular impairment. NETosis plays a key role in the prognosis of coronary artery disease, ischemic injury of kidney, lung, gastrointestinal tract and skeletal muscles. In this review, we explored the molecular mechanisms involved in NETosis, and ischemic/reperfusion injuries in body organs.

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.

Neutrophil extracellular traps modulate inflammatory markers and uptake of oxidized LDL by human and murine macrophages.

Neutrophil extracellular traps (NETs) are web-like structures, which are released upon neutrophil activation. It has previously been demonstrated that NETs are present in atherosclerotic lesions of both humans and animal models thus playing a decisive role in atherosclerosis. Besides, macrophages have a crucial role in disease progression, whereby classically activated M1 macrophages sustain inflammation and alternatively activated M2 macrophages display anti-inflammatory effects. Although NETs and macrophages were found to colocalize in atherosclerotic lesions, the impact of NETs on macrophage function is not fully understood. In the present study, we aimed to investigate the effect of NETs on human and murine macrophages in respect to the expression of pro-inflammatory cytokines, matrix metalloproteinases (MMPs) and uptake of oxidized LDL (oxLDL) in vitro. Human THP-1 and murine bone marrow-derived macrophages were cultured under M1 (LPS + IFN-γ)- and M2a (IL-4)-polarizing culture conditions and treated with NETs. To mimic intraplaque regions, cells were additionally cultured under hypoxic conditions. NETs significantly increased the expression of IL-1ß, TNF-α and IL-6 in THP-M1 macrophages under normoxia but suppressed their expression in murine M1 macrophages under hypoxic conditions. Notably, NETs increased the number of oxLDL-positive M1 and M2 human and murine macrophages under normoxia, but did not influence formation of murine foam cells under hypoxia. However, oxLDL uptake did not strongly correlate with the expression of the LDL receptor CD36. Besides, upregulated MMP-9 expression and secretion by macrophages was detected in the presence of NETs. Again, hypoxic culture conditions dampened NETs effects. These results suggest that NETs may favor foam cell formation and plaque vulnerability, but exert opposite effects in respect to the inflammatory response of human and murine M1 macrophages. Moreover, effects of NETs on macrophages' phenotype are altered under hypoxia.