Citrullinated Histone H3, a Marker for Neutrophil Extracellular Traps, Is Associated with Poor Prognosis in Cutaneous Squamous Cell Carcinoma Developing in Patients with Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare severe hereditary skin disease characterized by skin and mucosa fragility, resulting in blister formation. The most severe complication in RDEB patients is the development of cutaneous squamous cell carcinoma (SCC), leading to premature death. There is a great deal of evidence suggesting a permissive tumor microenvironment (TME) as a driver of SCC development in RDEB patients. In a cohort of RDEB patients, we characterized the immune profiles of RDEB-SCCs and compared them with clinical, histopathological, and prognostic features. RDEB-SCCs were subdivided into four groups based on their occurrence (first onset or recurrences) and grading according to clinical, histopathological parameters of aggressiveness. Thirty-eight SCCs from 20 RDEB patients were analyzed. Five RDEB patients experienced an unfavorable course after the diagnosis of the first SCC, with early recurrence or metastasis, whereas 15 patients developed multiple SCCs without metastasis. High-risk primary RDEB-SCCs showed a higher neutrophil-to-lymphocyte ratio in the tumor microenvironment and an increased proportion of neutrophil extracellular traps (NETs). Additionally, citrullinated histone H3, a marker of NETs, was increased in the serum of RDEB patients with high-risk primary SCC, suggesting that this modified form of histone H3 may serve as a potential blood marker of unfavorable prognosis in RDEB-SCCs.

The Therapeutic Potential of Neutrophil Extracellular Traps and NLRP3 Inflammasomes in Mycoplasma pneumoniae Pneumonia.

INTRODUCTION: Mycoplasma pneumoniae (MP) is the most common pathogen of community-acquired pneumonia in children. However, the role of neutrophil extracellular traps (NETs) in the pathogenesis of MP is unclear. METHODS: Both the level of NETs were detected between the 60 MP pneumonia patients and 20 healthy controls, whose the clinical characteristics were compared. Additionally, NETs formation induced by community-acquired respiratory distress syndrome (CARDS) toxin was also analyzed through transcriptome sequencing. RESULTS: The levels of cell-free DNA, Cit-H3, and MPO-DNA complexes were significantly increased in the patients with MP pneumonia. Importantly, both cell-free DNA and LDH were higher in hospitalized patients with severity than those without severity. In addition, CARDS toxin induced the NETs formation in vitro and in vivo. Transcriptomics GO and KEGG pathway analysis indicate that NOD like receptor signaling pathway and Toll-like receptor signaling pathway are significantly enriched. Finally, we found that DNase I significantly attenuated the higher levels of Cit-H3, and up-regulation of interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) by down-regulating the expression of NLRP3 and Caspase1(p20) in the lung tissues. DISCUSSION: These results indicate that inhibiting excessive activation of NLRP3 inflammasomes, and NETs formation may alleviate MP pneumonia.

Neutrophil Extracellular Trap Formation and Deoxyribonuclease I Activity in Patients with Otitis Media with Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

INTRODUCTION: No previous studies have evaluated the levels of neutrophil extracellular trap (NET) remnants or the importance of deoxyribonuclease (DNase) I activity based on the disease activity of otitis media with antineutrophil cytoplasmic antibody-associated vasculitis (OMAAV). The aim of this study was to explore the formation of NETs in the middle ear of patients with OMAAV during the onset and remission phases of the disease, with a particular focus on the relationships between the quantifiable levels of NET remnants and DNase I activity. METHODS: OMAAV patients were eligible for inclusion. Patients with otitis media with effusion (OME) were examined as controls. The levels of cell-free deoxyribonucleic acid (DNA), citrullinated-histone H3 (cit-H3)-DNA complex, and myeloperoxidase (MPO)-DNA complex were quantified using an enzyme-linked immunosorbent assay. DNase I activity was measured using a fluorometric method. RESULTS: The quantifiable levels of cell-free DNA, cit-H3-DNA complex, and MPO-DNA complex in the middle ear lavage of patients with OMAAV at onset were significantly higher than those in patients with OMAAV at remission and in patients with OME. DNase I activity in the patients with OMAAV at onset was significantly lower than those in patients with OMAAV at remission and OME and was negatively correlated with the level of MPO-DNA complex. CONCLUSIONS: This study suggests that NET remnants and DNase I activity may be potentially useful biomarkers for the diagnosis and disease activity of OMAAV.

Biomarkers of peripheral blood neutrophil extracellular traps in the diagnosis and progression of malignant tumors.

BACKGROUND AND AIMS: The mortality rate associated with malignant tumors remains high and there is a lack of effective diagnostic and tumor progression markers. Neutrophil extracellular traps (NETs) can promote tumor-associated thrombosis, invasive metastasis, and inflammatory responses, but there is a lack of research on the value of measuring NETs in the peripheral blood of patients with malignancies. METHODS: We included 263 patients with malignancies (55 gliomas, 101 ovarian, 64 colorectal, and 43 lung cancers) and 75 healthy controls in this study. We compared the levels of citrullinated histone H3 (citH3), cell-free DNA (cfDNA), and systemic inflammation-related parameters, including neutrophils, lymphocytes, monocytes, platelets, neutrophil-to-lymphocyte ratio, monocyte-to-lymphocyte ratio, platelet-to-lymphocyte ratio, systemic immune inflammation index, and systemic inflammation response index. We assessed the value of changes in NETs in peripheral blood to determine the diagnosis, venous thromboembolism, clinical staging, and systemic inflammatory response in patients with malignancy. RESULTS: The levels of citH3 and cfDNA in peripheral blood can distinguish between healthy controls and tumor patients. The levels of citH3 and cfDNA before clinical intervention did not predict the risk of combined venous thromboembolism in oncology patients in the short-term after clinical intervention. The levels of citH3, cfDNA, and systemic inflammation-related parameters in the peripheral blood of tumor patients increased with the clinical stage. There was a correlation between cfDNA levels in peripheral blood and systemic inflammation-related parameters in tumor patients, and this correlation was more significant in patients with advanced tumors. CONCLUSIONS: Changes in NETs in the peripheral blood differ between healthy controls and patients with malignant tumors. NETs may be involved in tumor-induced systemic inflammatory responses through interaction with circulating inflammatory cells, thus promoting tumor progression. NETs may be used as markers to assist in the diagnosis and progression of tumor malignancy.

SARS-CoV-2 Spike Proteins and Cell-Cell Communication Induce P-Selectin and Markers of Endothelial Injury, NETosis, and Inflammation in Human Lung Microvascular Endothelial Cells and Neutrophils: Implications for the Pathogenesis of COVID-19 Coagulopathy.

COVID-19 progression often involves severe lung injury, inflammation, coagulopathy, and leukocyte infiltration into pulmonary tissues. The pathogenesis of these complications is unknown. Because vascular endothelium and neutrophils express angiotensin-converting enzyme-2 and spike (S)-proteins, which are present in bodily fluids and tissues of SARS-CoV-2-infected patients, we investigated the effect of S-proteins and cell-cell communication on human lung microvascular endothelial cells and neutrophils expression of P-selectin, markers of coagulopathy, NETosis, and inflammation. Exposure of endothelial cells or neutrophils to S-proteins and endothelial-neutrophils co-culture induced P-selectin transcription and expression, significantly increased expression/secretion of IL-6, von Willebrand factor (vWF, pro-coagulant), and citrullinated histone H3 (cit-H3, NETosis marker). Compared to the SARS-CoV-2 Wuhan variant, Delta variant S-proteins induced 1.4-15-fold higher P-selectin and higher IL-6 and vWF. Recombinant tissue factor pathway inhibitor (rTFPI), 5,5'-dithio-bis-(2-nitrobenzoic acid) (thiol blocker), and thrombomodulin (anticoagulant) blocked S-protein-induced vWF, IL-6, and cit-H3. This suggests that following SARS-CoV-2 contact with the pulmonary endothelium or neutrophils and endothelial-neutrophil interactions, S-proteins increase adhesion molecules, induce endothelial injury, inflammation, NETosis and coagulopathy via the tissue factor pathway, mechanisms involving functional thiol groups, and/or the fibrinolysis system. Using rTFPI, effectors of the fibrinolysis system and/or thiol-based drugs could be viable therapeutic strategies against SARS-CoV-2-induced endothelial injury, inflammation, NETosis, and coagulopathy.

Low serum level of citrullinated histone H3 in patients with dermatomyositis.

OBJECTIVES: We aimed at analyzing the serum levels of citrullinated histone H3 (CitH3) in patients with dermatomyositis (DM) and their association with disease activity. METHODS: Serum CitH3 levels were measured using enzyme-linked immunosorbent assays in serum samples obtained from 93 DM patients and 56 healthy controls (HCs). Receiver operating characteristic (ROC) curve analysis was performed to evaluate the discriminant capacity of CitH3 and other disease variables. The association between CitH3 and disease variables was analyzed using Pearson's rank correlation. RESULTS: Serum CitH3 level was significantly lower in DM patients than in HCs (p < 0.001). The ROC curve analysis revealed that CitH3 strongly discriminated DM patients from HCs (area under the curve [AUC], 0.86), and a combination of CitH3 and the ratio of neutrophil to lymphocyte counts (NLR) showed a greater diagnostic value (AUC, 0.92). Serum CitH3 levels were markedly lower in DM patients with normal muscle enzyme levels than in HCs (all p < 0.001), and when compared to an elevated group, the CitH3 levels were comparable (all p > 0.05). The CitH3 levels showed no difference between DM in active and remission groups. However, in a paired test with 18 hospitalized DM patients, the CitH3 levels were higher in remission state than in active state. Moreover, the CitH3 levels showed no correlation with disease variables that were associated with the disease activity of DM. CONCLUSIONS: Serum CitH3 level may serve as a useful biochemical marker for screening patients with DM from HCs, while its role in monitoring DM disease activity requires further research.

Chloroquine reduces neutrophil extracellular trap (NET) formation through inhibition of peptidyl arginine deiminase 4 (PAD4).

Neutrophil extracellular traps (NETs) occur when chromatin is decondensed and extruded from the cell, generating a web-like structure. NETs have been implicated in the pathogenesis of several sterile disease states and thus are a potential therapeutic target. Various pathways have been shown to induce NETs, including autophagy, with several key enzymes being activated like peptidyl arginine deiminase 4 (PAD4), an enzyme responsible for citrullination of histones, allowing for DNA unwinding and subsequent release from the cell. Pre-clinical studies have already demonstrated that chloroquine (CQ) and hydroxychloroquine (HCQ) are able to reduce NETs and slow disease progression. The exact mechanism as to how these drugs reduce NETs has yet to be elucidated. CQ and HCQ decrease NET formation from various NET activators, independent of their autophagy inhibitory function. CQ and HCQ were found to inhibit PAD4 exclusively, in a dose-dependent manner, confirmed with reduced CitH3+ NETs after CQ or HCQ treatment. Circulating CitH3 levels were reduced in pancreatic cancer patients after HCQ treatment. In silico screening of PAD4 protein structure identified a likely binding site interaction at Arg639 for CQ and Trp347, Ser468, and Glu580 for HCQ. SPR analysis confirmed the binding of HCQ and CQ with PAD4 with KD values of 54.1 µM (CQ) and 88.1 µM (HCQ). This data provide evidence of direct PAD4 inhibition as a mechanism for CQ/HCQ inhibition of NETs. We propose that these drugs likely reduce NET formation through multiple mechanisms; the previously established TLR9 and autophagy inhibitory mechanism and the novel PAD4 inhibitory mechanism.

A prospective marker for the prediction of postoperative deep venous thrombosis: Neutrophil extracellular traps.

Deep venous thrombosis (DVT) is a common medical complication in patients with lumbar fractures. The current study aimed to investigate the predictive value of neutrophil extracellular traps (NETs) in postoperative DVT formation in patients with lumbar fractures and to develop a nomogram relating clinical admission information for prediction. Patients who underwent open reduction and pedicle screw internal fixation in the treatment of single-segment lumbar fracture in the Department of Spine Surgery, the First Affiliated Hospital of Nanjing Medical University, from December 2020 to June 2022 were enrolled in this study. Baseline data and laboratory results were collected from enrollees, and the primary study endpoint event was the occurrence of DVT in patients after surgery. Multivariable logistic regression analysis was used to identify risk factors associated with higher odds of DVT after surgery. A nomogram was constructed using the results of the multivariable model. The calibration plot and receiver operating characteristics (ROC) curve were used to show the satisfactory predictive capacity of the model. Of these 393 patients who did not have DVT preoperatively, 79 patients developed it postoperatively, and 314 did not, respectively. Multivariate analysis showed that higher body mass index (BMI) (BMI between 24 and 28: RR = 1.661, 95% CI = 0.891-3.094; BMI ≤28: RR = 5.625, 95% CI = 2.590-12.217; reference: BMI <24), neutrophils (RR = 1.157, 95% CI 1.042-1.285), D-dimer (RR = 1.098, 95% CI 1.000-1.206), and citrullinated histone H3 (CitH3) (RR = 1.043, 95% CI 1.026-1.060) were independent risk factors for postoperative DVT. Using the multivariable analysis, we then constructed a nomogram to predict DVT, which was found to have an area under the curve of 0.757 (95% CI = 0.693-0.820). Calibration plots also showed the satisfied discrimination and calibration of the nomogram. In conclusion, patients with lumbar fractures with postoperative DVT had higher levels of NETs in the circulation preoperatively compared to those without postoperative DVT. Furthermore, based on BMI, D-dimer, neutrophils, and CitH3, we developed a predictive model to predict postoperative DVT incidence in these patients.

Assessment of the role of neutrophil extracellular traps in SARS-CoV-2 pneumonia.

BACKGROUND: Abnormal neutrophil extracellular traps are associated with lung diseases, thrombosis, increased mucosal secretion in the airways. The aim of this study is to evaluate the possible place of the most specific NETosis marker Cit-H3 protein in diagnostic algorithms by revealing its relationship with the severity, mortality and prognosis of SARS-CoV-2 pneumonia. PATIENTS AND METHODS: Patients (n = 78) who applied to the Emergency Department between March 11, 2020 and June 10, 2020, with positive SARS-CoV-2 polymerase chain reaction (PCR) test and lung involvement were included in the prospective study. Serum Cit-H3 levels and critical laboratory parameters were measured at baseline on the day of clinical deterioration and before recovery/discharge/death. Cit-C3 levels were determined by enzyme immunassay method. RESULTS: Cit-H3 levels in patients with SARS-CoV-2 pneumonia during their first admission to the hospital were significantly higher compared to the healthy control group (p < 0.05). Repeated measurements of Cit-H3 levels of the patients significantly correlated with D-dimer, procalcitonin, Neutrophil/ Lymphocyte ratio, lymphocyte, CRP, and oxygen saturation. Cit-H3 levels of the patients who died were significantly higher than that of those who survived (p < 0.05). Cit-H3 levels were found to be statistically significantly higher in patients who developed acute respiratory distress syndrome, were admitted to the intensive care unit, and had mortality (p < 0.05). CONCLUSIONS: Cit-H3 plays a role in inflammatory processes in SARS-CoV-2 pneumonia, and changes in serum Cit-H3 levels of these patients can be used to determine prognosis and mortality (Tab. 5, Fig. 1, Ref. 21).

High Circulating Levels of Neutrophil Extracellular Traps Parameters Predicting Poor Outcome in COVID-19.

OBJECTIVE: Exploration of biomarkers to predict the severity of COVID-19 is important to reduce mortality. Upon COVID-19 infection, neutrophil extracellular traps (NET) are formed, which leads to a cytokine storm and host damage. Hence, the extent of NET formation may reflect disease progression and predict mortality in COVID-19. METHODS: We measured 4 NET parameters - cell-free double stranded DNA (cell-free dsDNA), neutrophil elastase, citrullinated histone H3 (Cit-H3), and histone - DNA complex - in 188 COVID-19 patients and 20 healthy controls. Survivors (n=166) were hospitalized with or without oxygen supplementation, while non-survivors (n=22) expired during in-hospital treatment. RESULTS: Cell-free dsDNA was significantly elevated in non-survivors in comparison with survivors and controls. The survival rate of patients with high levels of cell-free dsDNA, neutrophil elastase, and Cit-H3 was significantly lower than that of patients with low levels. These three markers significantly correlated with inflammatory markers (absolute neutrophil count and C-reactive protein). CONCLUSION: Since the increase in NET parameters indicates the unfavourable course of COVID-19 infection, patients predisposed to poor outcome can be rapidly managed through risk stratification by using these NET parameters.

Increased serum citrullinated histone H3 levels in COVID-19 patients with acute ischemic stroke.

Background and purpose: Prevalence of acute ische-mic stroke (AIS) is increased in patients with coronavirus disease 2019 (COVID-19). A proposed hypothesis is increased virus-induced propensity to hypercoagulation resulting in arterial thrombosis. Our aim was to provide evidence regarding the involvement of neutrophil extracellular trap (NET) formation (NETosis) in COVID-19 related AIS. Methods: Twenty-six consecutively enrolled COVID-19+ pneumonia patients with AIS, 32 COVID-19+ pneumonia patients without AIS and 24 AIS patients without COVID-19 infection were included to the study. Clinical characteristics of recruited patients were collected. Serum levels of citrullinated histone H3 (H3Cit; a factor of NETosis), IL-8 and C5a (mediators associated with NETosis) were measured by ELISA (enzyme-linked immunosorbent assay). Results: H3Cit levels were significantly higher in COVID-19+ AIS patients, whereas all study groups showed comparable IL-8 and C5a levels. There were no significant differences among etiological subgroups of AIS patients with or without COVID-19. AIS patients with COVID-19 showed relatively increased white blood cell, lymphocyte, neutrophil, D-dimer, C-reactive protein and procalcitonin levels than control groups. H3Cit levels did not correlate with clinical/prognostic features and inflammation parameters. H3Cit and IL-8 levels were correlated in COVID-19 patients without stroke but not in COVID-19 positive or negative AIS patients. Conclusion: Increased levels of inflammation parameters and H3Cit in COVID-19 related AIS suggest that NETosis may cause susceptibility to arterial thrombosis. However, H3Cit levels do not correlate with clinical severity measures and inflammation parameters diminishing the prognostic biomarker value of NETosis factors. Moreover, the link between IL-8 and NETosis appears to be abolished in AIS.

Detection of Extracellular Traps in Canine Steroid-Responsive Meningitis-Arteritis.

Extracellular traps (ETs) are DNA networks formed by immune cells to fight infectious diseases by catching and attacking pathogenic microorganisms. Uncontrolled ET formation or impaired ET clearance can cause tissue and organ damage. Steroid-responsive meningitis-arteritis (SRMA) represents an immune-mediated, presumably non-infectious, purulent leptomeningitis and fibrinoid-necrotizing arteritis and periarteritis of young-adult dogs. Chronic and recurrent cases of SRMA are characterized by lymphohistiocytic inflammatory cell infiltration in the meninges and perivascular tissue. This study aimed to identify extracellular traps in dogs with SRMA, a model for immune-mediated diseases in the central nervous system (CNS). Hematoxylin and eosin-stained samples of two young dogs with chronic, recurrent SRMA were examined by light microscopy for characteristic lesions and consecutive slices of affected tissues were stained for detection of ETs by immunofluorescence microscopy using antibodies against DNA-histone-1 complexes, myeloperoxidase, and citrullinated histone H3. Histology revealed purulent and lymphohistiocytic leptomeningitis (n = 2/2) with meningeal periarteritis (n = 2/2) and periadrenal located lymphohistiocytic periarteritis (n = 1). Extracellular DNA networks and inflammatory cell infiltrates of macrophages, neutrophil granulocytes, and lymphocytes were detected in the subarachnoid space of the leptomeninx (n = 2/2) and perivascularly in meningeal (n = 2/2) as well as periadrenal vessels (n = 1/1). In summary, extracellular DNA fibers and attached ET markers are detectable in affected perivascular and meningeal tissues of dogs suffering from SRMA. The proof of principle could be confirmed that ETs are present in canine, inflammatory, and non-infectious CNS diseases and possibly play a role in the pathogenesis of SRMA.

Circulating cell free DNA and citrullinated histone H3 as useful biomarkers of NETosis in endometrial cancer.

BACKGROUND: Cancer mortality is mainly caused by organ failure and thrombotic events. It has been demonstrated that NETosis, a chromatin release mechanism implemented by neutrophils, may contribute to these lethal systemic effects. Our aim was to investigate NETosis biomarkers in endometrial cancer (EC). METHODS: The experiments were conducted on 21 healthy subjects (HS) with no gynecological conditions, and on 63 EC patients. To assess the presence of NETosis features, IHC and IF was performed using antibodies against citrullinated histone H3 (citH3), neutrophil elastase (NE) and histone 2B. Serum levels of cell free DNA (cfDNA), cell free mitochondrial DNA (cfmtDNA) and citH3 were measured by qPCR using one microliter of deactivated serum, and by ELISA assay respectively. Fragmentation pattern of serum cfDNA was analyzed using the Agilent 2100 Bioanalyzer and High Sensitivity DNA Chips. Receiver operating characteristic (ROC) analysis was used to identify a cut off for cfDNA and cfmtDNA values able to discriminate between ECs and HSs. Correlation analysis and multiple correspondence analysis (MCA) between cfDNA, mtcfDNA, citH3 and blood parameters were used to identify the potential association among serum parameters in EC grades. RESULTS: We demonstrated the presence of NETosis features in tissues from all EC grades. Serum cfDNA and cfmtDNA levels discriminate ECs from HSs and a direct correlation between citH3 and cfDNA content and an inverse correlation between cfmtDNA and citH3 in EC sera was observed, not detectable in HSs. MCA indicates cfDNA, cfmtDNA and citH3 as features associated to G1 and G2 grades. A correlation between increased levels of cfDNA, citH3 and inflammation features was found. Finally, serum nucleosomal cfDNA fragmentation pattern varies in EC sera and correlates with increased levels of cfDNA, citH3, lymphocytes and fibrinogen. CONCLUSION: Our data highlight the occurrence of NETosis in EC and indicate serum cfDNA and citH3 as noninvasive biomarkers of tumor-induced systemic effects in endometrial cancer.

H2 Inhibits the Formation of Neutrophil Extracellular Traps.

Neutrophil extracellular traps (NETs) contribute to inflammatory pathogenesis in numerous conditions, including infectious and cardiovascular diseases, and have attracted attention as potential therapeutic targets. H2 acts as an antioxidant and has been clinically and experimentally proven to ameliorate inflammation. This study was performed to investigate whether H2 could inhibit NET formation and excessive neutrophil activation. Neutrophils isolated from the blood of healthy volunteers were stimulated with phorbol-12-myristate-13-acetate (PMA) or the calcium ionophore A23187 in H2-exposed or control media. Compared with control neutrophils, PMA- or A23187-stimulated human neutrophils exposed to H2 exhibited reduced neutrophil aggregation, citrullination of histones, membrane disruption by chromatin complexes, and release of NET components. CXCR4high neutrophils are highly prone to NETs, and H2 suppressed Ser-139 phosphorylation in H2AX, a marker of DNA damage, thereby suppressing the induction of CXCR4 expression. H2 suppressed both myeloperoxidase chlorination activity and production of reactive oxygen species to the same degree as N-acetylcysteine and ascorbic acid, while showing a more potent ability to inhibit NET formation than these antioxidants do in PMA-stimulated neutrophils. Although A23187 formed NETs in a reactive oxygen species-independent manner, H2 inhibited A23187-induced NET formation, probably via direct inhibition of peptidyl arginine deiminase 4-mediated histone citrullination. Inhalation of H2 inhibited the formation and release of NET components in the blood and bronchoalveolar lavage fluid in animal models of lipopolysaccharide-induced sepsis (mice and aged mini pigs). Thus, H2 therapy can be a novel therapeutic strategy for NETs associated with excessive neutrophil activation.

Neutrophil extracellular traps in feline cardiogenic arterial thrombi: a pilot study.

OBJECTIVES: The aim of this study was to investigate the spatial distribution of neutrophil extracellular traps (NETs) in cardiogenic arterial thromboembolism (CATE). Specifically, we aimed to examine the related structural features of NETs in feline arterial thrombi in relation to their arterial locations. METHODS: Paraffin-embedded aortic bifurcations from nine cats with hypertrophic cardiomyopathy (four with CATE and five without) were deparaffinized, and NETs were identified by immunodetection based on colocalization of cell-free DNA, citrullinated histone H3 and neutrophil elastase. The distribution of NETs in thrombi within the aortic bifurcations and common iliac arteries (CIAs) was compared based on their proximity to the descending aorta (proximal, mid, distal). Ten random fields per section were captured at × 10 and × 20 magnification for each section of the clot and analyzed. RESULTS: The distributions of NETs in thrombi within the aortic bifurcation and CIAs were found to differ in relation to their assigned zones (proximal, mid, distal; P = 0.04); NETs were concentrated mostly in the proximal region in the aortic bifurcations (47.56%, interquartile range [IQR] 14.07-77.95) and CIAs (44.69%, IQR 24.65-85.28), compared with the distal regions (2.69%, IQR 0.10-50.04 [P = 0.027]; 7.08%, IQR 1.27-59.33 [P = 0.02]). CONCLUSIONS AND RELEVANCE: The variation in NET distribution within arterial thrombi may shed light on the pathogenesis of thrombus growth. This may be due to possible neutrophil entrapment or variations in shear stress.

Elevation of circulating neutrophil extracellular traps, interleukin (IL)-8, IL-22, and vascular endothelial growth factor in patients with venomous snake mamushi (Gloydius blomhoffii) bites.

Mamushi bites cause swelling and pain that extend from the bitten site. The coagulopathic, anti-coagulopathic, and vasculopathic actions of mamushi venom result in various laboratory abnormalities, occasionally with muscular, renal, and other organ damage. We investigated the serum biomarkers that were associated with the pathogenesis of mamushi bites, focusing on markers related to tissue-damage and neutrophil activation. Twenty patients (one case of grade 2, 13 cases of grade 3, and six cases of grade 4 of severity) seen by us in one summer season were enrolled. Peripheral blood samples were taken from the patients on day 0, day 2, and day 7 after mamushi bites. In addition to routine blood examination, serum samples were subjected to enzyme-linked immunosorbent assay for citrullinated histone H3 (CitH3), interleukin (IL)-8, IL-17A, IL-22, vascular endothelial growth factor (VEGF), high mobility group box protein 1 (HMGB1), tumor necrosis factor (TNF)-α, and IL-33. Creatinine kinase (CK) values significantly correlated with prothrombin time (PT) levels, suggesting that muscular damage is associated with exaggerated coagulation and fibrinolysis. In the vast majority of patients, HMGB1, TNF-α, and IL-33 were under detection levels. Neutrophil counts did not correlate with PT or CK, indicating that the coagulation disorder and muscular damage were virtually independent of the neutrophil activation. The neutrophil number significantly correlated with CitH3, a representative marker of neutrophil extracellular traps. Moreover, there were significant correlations between neutrophil number, CitH3, IL-8, IL-22, and VEGF. Our study suggests that there are two major cascades in mamushi bites. One is an already characterized venom effect on coagulation, vessels, and muscles. In the other novel cascade, we propose that neutrophil activation with IL-8 leads to the production of IL-22 and VEGF. This sequential event may contribute to both vascular damage and repair.

Circulating CitH3 Is a Reliable Diagnostic and Prognostic Biomarker of Septic Patients in Acute Pancreatitis.

Purpose: Acute pancreatitis (AP) is an inflammatory disease. AP starts with sterile inflammation and is often complicated with critical local or systemic infection or sepsis in severe cases. Septic AP activates peptidyl arginine deiminase (PAD) and citrullinates histone H3 (CitH3), leading to neutrophil extracellular trap (NET) formation. Investigating the role of NETs and underlying mechanisms in septic AP may facilitate developing diagnostic and therapeutic approaches. In this study, we sought to identify the expression of CitH3 in septic AP patients and to analyze the correlation of CitH3 concentration with NET components as well as clinical outcomes. Methods: Seventy AP patients with or without sepsis (40 septic cases, 30 nonseptic cases) and 30 healthy volunteers were recruited in this study. Concentration of NET components (CitH3 and double-strain DNA) and key enzymes (PAD2/4) were measured. Clinical and laboratory characteristics of patients were recorded and analyzed. Results: Levels of CitH3 were elevated significantly in septic AP patients compared with those in nonseptic AP and healthy volunteers. The area under the curve (AUC, 95% confidence interval) for diagnosing septic AP was 0.93 (0.86-1.003), and the cutoff was 43.05 pg/ml. Among septic AP cases (n = 40), the concentration of CitH3 was significantly increased in those who did not survive or were admitted to the intensive care unit, when compared with that in those who survived or did not require intensive care unit. Association analysis revealed that CitH3 concentration was positively correlated with PAD2, PAD4, dsDNA concentration, and Sequential Organ Failure Assessment scores. Conclusion: CitH3 concentration increased in septic AP patients and was closely correlated with disease severity and clinical outcomes. CitH3 may potentially be a diagnostic and prognostic biomarker of septic AP.

Citrullinated Histone H3 Mediates Sepsis-Induced Lung Injury Through Activating Caspase-1 Dependent Inflammasome Pathway.

Sepsis is a life-threatening organ dysfunction caused by dysregulated host response to infection that often results in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). An emerging mechanism of sepsis-induced ARDS involves neutrophils/macrophages undergoing cell death, releasing nuclear histones to cause tissue damage that exacerbates pulmonary injury. While published studies focus on unmodified histones, little is known about the role of citrullinated histone H3 (CitH3) in the pathogenesis of sepsis and ALI. In this study, we found that levels of CitH3 were elevated in the patients with sepsis-induced ARDS and correlated to PaO2/FiO2 in septic patients. Systematic administration of CitH3 peptide in mice provoked Caspase-1 activation in the lung tissue and caused ALI. Neutralization of CitH3 with monoclonal antibody improved survival and attenuated ALI in a mouse sepsis model. Furthermore, we demonstrated that CitH3 induces ALI through activating Caspase-1 dependent inflammasome in bone marrow derived macrophages and bone marrow derived dendritic cells. Our study suggests that CitH3 is an important mediator of inflammation and mortality during sepsis-induced ALI.

Involvement of Neutrophil Extracellular Traps in Cerebral Arteriovenous Malformations.

BACKGROUND: Cerebral arteriovenous malformations (cAVMs) represent tangles of abnormal vasculature without intervening capillaries. High-pressure vascular channels due to abnormal arterial and venous shunts can lead to rupture. Multiple pathways are involved in the pathobiology of cAVMs including inflammation and genetic factors such as KRAS mutations. Neutrophil release of nuclear chromatin, known as neutrophil extracellular traps (NETs), plays a multifunctional role in infection, inflammation, thrombosis, intracranial aneurysms, and tumor progression. However, the relationship between NETs and the pathobiology of cAVMs remains unknown. We tested whether NETs play a role in the pathobiology of cAVMs. METHODS: We analyzed samples from patients who had undergone surgery for cAVM and immunohistochemically investigated expression of citrullinated histone H3 (CitH3) as a marker of NETs. CitH3 expression was compared among samples from cAVM patients, epilepsy patients, and normal human brain tissue. Expressions of thrombotic and inflammatory markers were also examined immunohistochemically in samples from cAVM patients. RESULTS: Expression of CitH3 derived from neutrophils was observed intravascularly in all cAVM samples but not other samples. Nidi of AVMs showed migration of many Iba-I-positive cells adjacent to the endothelium and endothelial COX2 expression, accompanied by expression of IL-6 and IL-8 in the endothelium and intravascular neutrophils. Unexpectedly, expression of CitH3 was not necessarily localized to the vascular wall and thrombus. CONCLUSIONS: Our results offer the first evidence of intravascular expression of NETs, which might be associated with vascular inflammation in cAVMs.

The association between circulating neutrophil extracellular trap related biomarkers and retinal vein occlusion incidence: A case-control pilot study.

Retinal vein occlusion (RVO) is the second most common retinal vascular disorders and causes visual damage in a large population. Neutrophil extracellular traps (NETs) formation (NETosis) is an important cause of vascular diseases, however, the association between NETs related biomarkers and RVO development remained unclear. In this pilot study, a total of 77 RVO cases and 48 controls were included between Jan 2020 and July 2020. Besides, the circulating levels of three NETs related markers, cell-free DNA (cfDNA), myeloperoxidase (MPO)-DNA and citrullinated histone H3 (H3Cit), were detected in all the participants and thus the association between NETosis and RVO incidence was analyzed. Advanced assays were conducted to investigate the inflammation and thrombosis related biomarkers in RVO cases with higher or lower NETs biomarkers. When the results were considered, it was found that NETs biomarkers, including cfDNA, MPO-DNA and H3Cit, were increased in the RVO cases comparing with the controls (P < 0.05). Through the receiver operating characteristic (ROC) analyses, we found that circulating NETs related biomarkers demonstrated potential diagnostic effects for RVO and the AUCs of plasma cfDNA, MPO-DNA and H3Cit were 0.859, 0.871 and 0.928, respectively (P < 0.001). Through analyzing the correlations between circulating NETs markers and RVO stages and durations, inflammatory markers as well as thrombotic indexes, it was found that NETs were related with the RVO subtypes, inflammatory status and thrombus formation. In conclusion, the plasma NETs remnants are significantly increased in RVO cases. Besides, advanced studies demonstrate that inflammation as well as thrombus formation might be involved in this association.