Citrullinated Histone H3 Research Articles

The role of protein arginine deiminase 4-dependent neutrophil extracellular traps formation in ulcerative colitis.

Neutrophil extracellular traps (NETs) play an important role in the development and progression of ulcerative colitis (UC). Peptidyl arginine deiminase 4 (PAD4) is essential for the formation of NETs via catalyzing histone citrullination. This study mainly to explore the role of PAD4-mediated NETs in intestinal inflammation of dextran sulfate sodium (DSS)-induced UC. Acute and chronic colitis mouse models were established by supplementing DSS in drinking water. Colon tissues from colitis mice were analyzed for the level of PAD4 expression, citrullinated histone H3(Cit-H3), intestinal histopathology, and inflammatory cytokines secretion. Serum samples were tested for systemic neutrophil activation biomarkers. Colitis mice administered with Cl-amidine, a PAD4 inhibitor, and PAD4 knockout mice were investigated to detect NETs formation, intestinal inflammation, and barrier function. We found the formation of NETs significantly increased in DSS-induced colitis mice and was correlated with disease markers. Blocking NETs formation by Cl-amidine or PAD4 genetic knockout could alleviate clinical colitis index, intestinal inflammation, and barrier dysfunction. This study provided a research basis for the role of PAD4-mediated NETs formation in the pathogenesis of UC and suggested that inhibition of PAD4 activity and the formation of NETs may be helpful for the prevention and treatment of UC.

Calprotectin in chronic rhinosinusitis eosinophil extracellular traps.

Calprotectin is an antimicrobial peptide primarily secreted by neutrophils. Furthermore, calprotectin secretion increases in patients with chronic rhinosinusitis (CRS) with polyps (CRSwNP) and positively correlates with neutrophil markers. However, CRSwNP is known to be associated with type 2 inflammation related to tissue eosinophilia. Therefore, the authors investigated calprotectin expression in eosinophils and eosinophil extracellular traps (EETs) and explored the associations between tissue calprotectin and the clinical findings of patients with CRS. A total of 63 patients participated, and patients diagnosed with CRS were classified based on the Japanese Epidemiological Survey of Refractory Eosinophilic Chronic Rhinosinusitis (JESREC) score. The authors performed hematoxylin and eosin staining, immunohistochemistry, immunofluorescence with calprotectin, myeloperoxidase (MPO), major basic protein (MBP), and citrullinated histone H3 with the participant's tissues. Finally, correlations between calprotectin and the clinical data were examined. Calprotectin-positive cells are co-localized not only in MPO-positive cells but also in MBP-positive cells in human tissues. Calprotectin was also involved in EETs and neutrophil extracellular traps. The number of calprotectin-positive cells in the tissue was positively correlated with the number of tissue and blood eosinophils. In addition, calprotectin in the tissue is associated with the olfactory function, Lund-Mackay computed tomography score, and JESREC score. Calprotectin, known to be secreted by neutrophils, in CRS was also expressed in eosinophils. In addition, calprotectin, which functions as an antimicrobial peptide, may play an important role in the innate immune response based on its EET involvement. Therefore, calprotectin expression could reflect as a disease severity biomarker for CRS.

Neutrophil Extracellular Traps and Platelet Activation for Identifying Severe Episodes and Clinical Trajectories in COVID-19

The role of NETs and platelet activation in COVID-19 is scarcely known. We aimed to evaluate the role of NETs (citrullinated histone H3 [CitH3], cell-free DNA [cfDNA]) and platelet activation markers (soluble CD40 ligand [CD40L] and P-selectin) in estimating the hazard of different clinical trajectories in patients with COVID-19. We performed a prospective study of 204 patients, categorized as outpatient, hospitalized and ICU-admitted. A multistate model was designed to estimate probabilities of clinical transitions across varying states, such as emergency department (ED) visit, discharge (outpatient), ward admission, ICU admission and death. Levels of cfDNA, CitH3 and P-selectin were associated with the severity of presentation and analytical parameters. The model showed an increased risk of higher levels of CitH3 and P-selectin for ED-to-ICU transitions (Hazard Ratio [HR]: 1.35 and 1.31, respectively), as well as an elevated risk of higher levels of P-selectin for ward-to-death transitions (HR: 1.09). Elevated levels of CitH3 (HR: 0.90), cfDNA (HR: 0.84) and P-selectin (HR: 0.91) decreased the probability of ward-to-discharge transitions. A similar trend existed for elevated levels of P-selectin and ICU-to-ward transitions (HR 0.40); In conclusion, increased NET and P-selectin levels are associated with more severe episodes and can prove useful in estimating different clinical trajectories.

CD177 Inhibits Neutrophil Extracellular Trap Formation and Protects against Acute Pancreatitis in Mice

The inflammatory immune response mediated by neutrophils is closely related to the progression of acute pancreatitis. Previous studies confirmed that CD177 is a neutrophil-specific marker involved in the pathogenesis of conditions such as systemic vasculitis, asthma, and polycythemia vera. Neutrophil extracellular trap (NET) formation is a specific death program by which neutrophils release nuclear DNA covered with histones, granule proteins, etc. It also plays an important role in host defense and various pathological reactions. However, the function of CD177 in regulating the generation of NETs and the development of acute pancreatitis (AP) is unclear. In our manuscript, CD177 was significantly elevated in blood neutrophils in patients and positively correlated with the AP disease severity. Then, recombinant human CD177 protein (rhCD177) could significantly improve pancreatic injury and the inflammatory response in AP mice, and reduce AP-related lung injury. Mechanistically, we found that rhCD177 could inhibit the formation of NETs by reducing reactive oxygen species (ROS) and myeloperoxidase (MPO)/citrullinated histone H3 (CitH3) release. For the first time, we discovered the potential of rhCD177 to protect AP in mice and inhibit the NET formation of AP. CD177 may be a potential treatment strategy for preventing or inhibiting the aggravation of AP.

Infection with SARS-CoV-2 Is Associated with Elevated Levels of IP-10, MCP-1, and IL-13 in Sepsis Patients.

Immunothrombosis, an excessive inflammatory response with simultaneous overactivation of the coagulation system, is a central pathomechanism in sepsis and COVID-19. It is associated with cellular activation, vascular damage, and microvascular thrombosis, which can lead to multiple organ failure and death. Here, we characterized factors related to immunothrombosis in plasma samples from 78 sepsis patients. In the course of routine clinical testing, SARS-CoV-2 was detected in 14 of these patients. Viral infection was associated with a higher mortality. Both, COVID-19 negative and COVID-19 positive sepsis patients showed increased levels of effectors of immunothrombosis, including platelet factor 4, D-dimer, nucleosomes, citrullinated histone H3, high mobility group box-1 protein, as well as phosphatidylserine-expressing platelet-derived extracellular vesicles, compared to healthy controls (n = 25). Using a 27-plex cytokine bead array, we found that Interleukin (IL)-1ra, IL-6, IL-8, IL-13, tumor necrosis factor (TNF)-α, interferon inducible protein (IP)-10, monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, and granulocyte-colony stimulating factor (G-CSF) were elevated in both, COVID-19 negative and COVID-19 positive sepsis patients, as compared to healthy controls. SARS-CoV-2 infection was associated with elevated levels of IP-10, MCP-1, and IL-13, while all other mediators widely overlapped between COVID-19 negative and COVID-19 positive patients.

Rat bronchoalveolar lavage proteome changes following e-cigarette aerosol exposures.

E-cigarette liquids are complex mixtures of chemicals consisting of humectants, such as propylene glycol (PG) and vegetable glycerin (VG), with nicotine or flavorings added. Published literature emphasizes the toxicity of e-cigarette aerosols with flavorings whereas much less attention has been given to the biologic effects of humectants. The purpose of the current study was to provide a comprehensive view of the acute biologic effects of e-cigarette aerosols on rat bronchoalveolar lavage (BAL) using mass spectrometry-based global proteomics. Sprague-Dawley rats were exposed to e-cigarette aerosol for 3 h/day for three consecutive days. Groups included: PG/VG alone, PG/VG + 2.5% nicotine (N), or PG/VG + N + 3.3% vanillin (V). Right lung lobes were lavaged for BAL and supernatants prepared for proteomics. Extracellular BAL S100A9 concentrations and BAL cell staining for citrullinated histone H3 (citH3) were also performed. From global proteomics, ∼2,100 proteins were identified from rat BAL. The greatest change in number of BAL proteins occurred with PG/VG exposures alone compared with controls with biological pathways enriched for acute phase responses, extracellular trap formation, and coagulation. Extracellular BAL S100A9 concentrations and the number of citH3 + BAL cells also increased significantly in PG/VG and PG/VG + 2.5% N. In contrast to PG/VG or PG/VG + N, the addition of vanillin to PG/VG + N increased BAL neutrophilia and downregulated lipid transport proteins. In summary, global proteomics support e-cigarette aerosol exposures to PG/VG alone as having a significant biologic effect on the lung independent of nicotine or flavoring with increased markers of extracellular trap formation.

An impact of neutrophil extracellular traps to the prothrombotic state and tumor progression in gynecological cancer patients

Introduction. One of the leading causes in the mortality pattern of cancer patients is accounted for by thrombotic complications. Recent studies have shown that neutrophil extracellular traps (NETs) are involved in the activation of coagulation, contribute to the initiation and progression of thrombosis. In addition, NET-related effect on tumor progression and metastasis has been actively studied.Aim: to evaluate NET-related procoagulant activity in gynecological cancer patients.Materials and Methods. From April 2020 to October 2022, a prospective controlled interventional non-randomized study was conducted with 120 women. The main group included 87 patients aged 32 to 72 years with malignant neoplasms of the female genital organs and mammary glands who were hospitalized for elective surgical treatment or chemotherapy: uterine body cancer (subgroup 1; n = 18), ovarian cancer (subgroup 2; n = 26), cervical cancer – adenocarcinoma of the cervical canal (subgroup 3; n = 13), breast cancer (subgroup 4; n = 30). The control group consisted of 33 healthy women aged 32 to 68 years. In all women, plasma concentrations of citrullinated histone H3 (citH3), myeloperoxidase antigen (MPO:Ag), D-dimer, and thrombin–antithrombin (TAT) complexes were evaluated.Results. The magnitude of NETosis in cancer patients, assessed by level of citH3 (2.5 ± 0.7; 1.9 ± 0.8; 2.5 ± 0.7; 0.7 ± 0.5 ng/ml in four subgroups, respectively) and MPO:Ag (29.5 ± 13.1; 12.8 ± 3.7; 22.8 ± 8.7; 6.6 ± 2.5 ng/ml in four subgroups, respectively) was significantly higher compared to women in the control group (0.3 ± 0.1 ng/ml; p = 0.0001 and 2.5 ± 0.2 ng/ml; p = 0.0001). In parallel with increased NETosis markers in accordance with the disease stage, there was an increase in the concentration of hemostasis activation markers – D-dimer (1.7 ± 0.6; 2.0 ± 0.7; 1.4 ± 0.5; 1.5 ± 0.7 µg/ml in four subgroups, respectively) and TAT complexes (729.8 ± 43.9; 794.1 ± 164.8; 636.2 ± 149.5; 699.6 ± 165.7 pg/ml in four subgroups, respectively) exceeding their level in the control group (respectively, 0.4 ± 0.1 μg/ml; p = 0.0001 and 362.3 ± 0.1 pg/ml; p = 0.0001). The maximum values of parameters occurred at later stages according to the Classification of Malignant Tumours (tumor, nodus, metastasis, TNM). A significant correlation between TAT level and the concentrations of citH3 (r = 0.586; р = 0.04) and MPO:Ag was revealed (r = 0.631; р = 0.04).Conclusion. Tumor tissue creates milieu that stimulates NETs release, which, in turn, not only contribute to the creating a procoagulant state, but also might act as one of the factors that ensure tumor progression and metastasis. The development of targeted therapies acting on NETs has a potential to affect hemostasis in cancer patients and reduce rate of tumor growth and metastasis.

Circulating neutrophil extracellular traps in cats with hypertrophic cardiomyopathy and cardiogenic arterial thromboembolism.

Cats with hypertrophic cardiomyopathy (HCM) are at risk of cardiogenic arterial thromboembolism (CATE). Neutrophil extracellular traps (NETs) may be a potential biomarker and therapeutic target for cardiomyopathy in cats. Characterize NETs in cats with HCM or CATE. We hypothesized that circulating NETs assessed in the form of cell-free DNA (cfDNA) and citrullinated histone H3 (citH3) are increasedin cats with HCM and CATE and associated with reported predisposing factors for thrombus formation. Eighty-five cats including client-owned cats with HCM and CATE and staff- and student-owned clinically healthy cats without HCM. After echocardiographic evaluations, NETs were measured as cfDNA and citH3. Cats with CATE had significant increases in cfDNA (11.2ng/μL; interquartile range [IQR],8.1 to 29.6) compared to those without HCM (8.2ng/μL; IQR,5.7 to 11.7μL; P=.01) and were responsible for 75% to 83% of cases with cfDNA fragments sized 100 to 2000 base pairs. Citrullinated histone 3, detected in 52% of cats with HCM (31.1ng/mL; IQR, 16.9 to 29.8), was significantly lower than in those with CATE (48.2ng/mL; IQR, 34.2 to 60.2; P=.007). The citH3 concentrations correlated significantly with reported risk factors of CATE, such as left atrial auricular velocity. Neutrophil extracellualr traps, especially citH3, are increased in cats with HCM and CATE. They may serve as a novel therapeutic target and biomarker of thrombosis in cats with HCM.

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

We aimed at analyzing the serum levels of citrullinated histone H3 (CitH3) in patients with dermatomyositis (DM) and their association with disease activity. 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. 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. 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.

Anti-DNA-IgM Favors the Detection of NET-Associated Extracellular DNA.

During inflammatory responses, neutrophils enter the sites of attack where they execute various defense mechanisms. They (I) phagocytose microorganisms, (II) degranulate to release cytokines, (III) recruit various immune cells by cell-type specific chemokines, (IV) secrete anti-microbials including lactoferrin, lysozyme, defensins and reactive oxygen species, and (V) release DNA as neutrophil extracellular traps (NETs). The latter originates from mitochondria as well as from decondensed nuclei. This is easily detected in cultured cells by staining of DNA with specific dyes. However, in tissues sections the very high fluorescence signals emitted from the condensed nuclear DNA hamper the detection of the widespread, extranuclear DNA of the NETs. In contrast, when we employ anti-DNA-IgM antibodies, they are unable to penetrate deep into the tightly packed DNA of the nucleus, and we observe a robust signal for the extended DNA patches of the NETs. To validate anti-DNA-IgM, we additionally stained the sections for the NET-markers histone H2B, myeloperoxidase, citrullinated histone H3, and neutrophil elastase. Altogether, we have described a fast one-step procedure for the detection of NETs in tissue sections, which provides new perspectives to characterize neutrophil-associated immune reactions in disease.

Simvastatin Reduces NETosis to Attenuate Severe Asthma by Inhibiting PAD4 Expression.

Patients with severe asthma respond poorly to corticosteroids, and their care accounts for more than 60% of the total costs attributed to asthma. Neutrophils form neutrophil extracellular traps (NETs), which play a crucial role in severe asthma. Statins have shown anti-inflammatory effects by reducing NETosis. In this study, we investigate if simvastatin can attenuate severe asthma by reducing NETosis and the underlying mechanism. Mice were concomitantly sensitized with ovalbumin (OVA), house dust mite (HDM), and lipopolysaccharide (LPS) during sensitization to establish a mouse model of severe asthma with neutrophil predominant inflammation (OVA+LPS mice) and treated with or without simvastatin. In inflammatory response, proportions of Th2, Th17, and Treg cells in lung tissue were detected by flow cytometry, and the levels of cytokines, dsDNA, and MPO-DNA in bronchoalveolar lavage fluid (BALF) were analyzed by ELISA. Citrullinated histone H3 (CitH3) and peptidyl arginine deiminase 4 (PAD4) in lung tissue were determined by Western blot and immunofluorescence imaging. PAD4 mRNA was determined by quantitative PCR (qPCR). HL-60 cells were differentiated into neutrophil-like cells by 1.25% DMSO. The neutrophil-like cells were treated with or without LPS, and simvastatin was then stimulated with PMA. CitH3 and PAD4 expressions were determined. Sensitization with OVA, HDM, and LPS resulted in neutrophilic inflammation and the formation of NETs in the lungs. Simvastatin treatment reduced the inflammation score, cytokine levels, total cells, and neutrophil counts in the BALF and reduced proportions of Th2 and Th17 but increased Treg cells in lungs of OVA+LPS mice. Simvastatin-treated OVA+LPS mice show reduced NET formation in BALF and lung tissue compared to control mice. Adoptive transfer of neutrophils was sufficient to restore NETosis and neutrophilic inflammation in simvastatin-treated OVA+LPS mice. Simvastatin reduced PAD4 mRNA and protein expression in lung tissues and neutrophils isolated from lungs of OVA+LPS mice and consequent NET formation. In vitro, simvastatin reduced LPS-induced PAD4 upregulation and NETosis in HL-60-differentiated neutrophil-like cells. Furthermore, PAD4-overexpressed lentiviral transduction was sufficient to restore PAD4 protein expression and NETosis in simvastatin-treated HL-60-differentiated neutrophil-like cells. Simvastatin reduces Th17-mediated neutrophilic inflammation and airway hyperreactivity by reducing PAD4 expression and inhibiting NETosis in a mouse model of severe asthma. Severe asthmatic patients with high levels of circulating NETs or sputum NETs may show improved responses to statin treatment.

Homeostatic, Non-Canonical Role of Macrophage Elastase in Vascular Integrity.

Matrix metalloproteinase (MMP)-12 is highly expressed in abdominal aortic aneurysms and its elastolytic function has been implicated in the pathogenesis. This concept is challenged, however, by conflicting data. Here, we sought to revisit the role of MMP-12 in abdominal aortic aneurysm. Apoe-/- and Mmp12-/-/Apoe-/- mice were infused with Ang II (angiotensin). Expression of neutrophil extracellular traps (NETs) markers and complement component 3 (C3) levels were evaluated by immunostaining in aortas of surviving animals. Plasma complement components were analyzed by immunoassay. The effects of a complement inhibitor, IgG-FH1-5 (factor H-immunoglobulin G), and macrophage-specific MMP-12 deficiency on adverse aortic remodeling and death from rupture in Ang II-infused mice were determined. Unexpectedly, death from aortic rupture was significantly higher in Mmp12-/-/Apoe-/- mice. This associated with more neutrophils, citrullinated histone H3 and neutrophil elastase, markers of NETs, and C3 levels in Mmp12-/- aortas. These findings were recapitulated in additional models of abdominal aortic aneurysm. MMP-12 deficiency also led to more pronounced elastic laminae degradation and reduced collagen integrity. Higher plasma C5a in Mmp12-/- mice pointed to complement overactivation. Treatment with IgG-FH1-5 decreased aortic wall NETosis and reduced adverse aortic remodeling and death from rupture in Ang II-infused Mmp12-/- mice. Finally, macrophage-specific MMP-12 deficiency recapitulated the effects of global MMP-12 deficiency on complement deposition and NETosis, as well as adverse aortic remodeling and death from rupture in Ang II-infused mice. An MMP-12 deficiency/complement activation/NETosis pathway compromises aortic integrity, which predisposes to adverse vascular remodeling and abdominal aortic aneurysm rupture. Considering these new findings, the role of macrophage MMP-12 in vascular homeostasis demands re-evaluation of MMP-12 function in diverse settings.

Immunohistochemical study of chicken fat clots: Investigation of the formation mechanism

In forensic practice, the presence of chicken fat clots (CFCs) in the heart and/or large blood vessels of cadavers has been empirically used to estimate the time from the onset of fatal events to death. However, little scientific evidence of its significance exists, and the mechanism of its formation has not been elucidated. CFCs contain large amounts of leukocytes; thus, we hypothesized that leukocytes might contribute to their formation. Since leukocytes, especially neutrophils, are considered to be involved in blood coagulation through the formation of neutrophil extracellular traps (NETs), we aimed to investigate whether NETs are related to the formation of CFCs through immunohistochemistry. Most cells in the CFCs were myeloperoxidase- and neutrophil elastase-positive, strongly suggesting that they were neutrophils. Since chromatin is released extracellularly during NET formation, immunostaining was performed against some types of histones in CFCs. A certain number of neutrophils in CFCs showed positive extra-nuclear and extracellular signals of histones. In addition, citrullination of histone H3, which is considered important for histone release, was immunohistochemically detected in some neutrophils. These results suggest that neutrophils may affect the formation of CFCs through histone release. Although it was not clear how and when citrullination and extracellular release of histones in CFCs occur in this study, our findings provide insights into the events occurring at the time of death in a human body.

Immunothrombosis in cancer patients: contribution of neutrophil extracellular traps, ADAMTS-13 and von Willebrand factor

Introduction. Neutrophil extracellular traps (NETs) and von Willebrand factor (vWF) are integral players in thrombosis and inflammation in cancer patients. It has been increasingly evident that an active interplay exists between NETs and vWF. Some studies suggest that NETs cause decrease in ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13) activity, being an arm in the pathogenesis of both thrombotic microangiopathies (TMA) and other thrombotic complications during oncological process.Aim: to assess a crosstalk between NETs, vWF, and ADAMTS-13 in uterine, ovarian, breast malignant neoplasms as well as cervical canal adenocarcinoma.Materials and Methods. From September 2019 to July 2022, a prospective controlled interventional non-randomized study was carried out with 106 patients hospitalized for planned surgical treatment aged 30 to 72 years. The main group included 73 patients with malignant neoplasms of the female genital organs and mammary glands, stage I–III: uterine cancer (subgroup 1; n = 18), ovarian cancer (subgroup 2; n = 21), cervical cancer – adenocarcinoma of cervical canal (subgroup 3; n = 9) and breast cancer (subgroup 4; n = 25). The control group consisted of 33 women with female genital tract and breast benign neoplasms. In all patients, serum levels of vWF, citrullinated histone H3 (citH3), MPO (myeloperoxidase) antigen, ADAMTS-13 activity, ADAMTS-13 antigen, and D-dimer were evaluated.Results. The study revealed significant differences in the concentration of NETosis markers between the main and control groups. Patients with uterine cancer and adenocarcinoma of the cervical canal peaked at NETosis markers. At the same time, there were significant differences in citH3 concentration among patients with «early» (stage I) and «not early» (stage II–III) disease forms. While assessing level of von Willebrand factor (vWF:Ag), antigen (ADAMTS-13:Ag), and ADAMTS-13 activity (ADAMTS-13:Ac), significant differences were found between the main and control groups (p &lt; 0.0001). The vWF in the main groups was sharply increasedwhereas ADAMTS-13 antigen concentration and activity were decreased. A сorrelation analysis among oncological patients in main group showed that while citH3 level increased, it was also paralleled with rise in vWF:Ag (ρ = 0.80; p &lt; 0.01) and MPO:Ag (ρ = 0.87; p &lt; 0.01); increase in MPO:Ag level was coupled to rise in vWF:Ag (ρ = 0.70; p&lt; 0.01), but increase in vWF:Ag occurred along with decline in ADAMTS-13:Ac (ρ = –0.43; p &lt; 0.01) and ADAMTS-13:Ag (ρ= –0.42; p &lt; 0.01).Conclusion. The interplay between NET, vWF, and ADAMTS-13 leads to a vicious circle, reduces ADAMTS-13 activity by increasing serum vWF concentration, which positively correlates with severity and mortality in TMA, acute ischemic infarction, and COVID-19. Targeting the NETs-vWF axis may pave the way for therapeutic strategies for immunothrombosis in various diseases, including cancer.

Neutrophil Extracellular Traps Promote Metastases of Colorectal Cancers through Activation of ERK Signaling by Releasing Neutrophil Elastase.

Neutrophil extracellular traps (NETs) play important roles in host immunity, as there is increasing evidence of their contribution to the progression of several types of cancers even though their role in colorectal cancers (CRCs) remains unclear. To investigate the clinical relevance of NETs in CRCs, we examined the expression of citrullinated histone H3 using immunohistochemistry and preoperative serum myeloperoxidase-DNA complexes in CRC patients using an enzyme-linked immunosorbent assay. High expression of intratumoral or systemic NETs was found to correlate with poor relapse-free survival (RFS), for which it is an independent prognostic factor. In vitro investigations of CRC cells (HCT116, HT29) revealed that NETs did not affect their proliferation but did promote the migration of CRC cells mediated by neutrophil elastase (NE) released during NETosis to increase extracellular signal-regulated kinase (ERK) activity. In vivo experiments using nude mice (KSN/slc) revealed that NE inhibition suppressed liver metastases in CRC cells, although it did not affect the growth of subcutaneously implanted tumors. Taken together, these results suggest that NET formation correlates with poor prognoses of patients with CRC and that the inhibition of NE could be a potential therapy for CRC metastases.

Взаимодействие внеклеточных ловушек нейтрофилов и антифосфолипидных антител у онкологических больных

Activation of NETosis processes has been detected in numerous autoimmune and non-autoimmune diseases with increasing concentrations of antiphospholipid antibodies (APA). Neutrophil extracellular traps (NETs) are an important activator of the coagulation cascade and an integral component of arterial and venous clots in cancer patients. Therefore, APA and NETs may be the links in the pathogenesis of thrombosis in cancer patients. Objective. To evaluate the severity of NETosis, hemostatic system activation and APA circulation (anti-β2-glycoprotein 1 (anti-β2-GPI)) in cancer patients. Design. A prospective controlled interventional non-randomized study. Patients and methods. This study included 144 patients who were divided into two groups: the study group – 111 patients aged 34–72 years with malignant breast and gynecological diseases of stages I–III (endometrial cancer, n = 25; ovarian cancer, n = 36; cervical cancer, n = 17; breast cancer, n = 33), and the control group – 33 healthy women. Prior to therapy, serum concentrations of NETosis markers – citrullinated histone H3 (citH3) and human myeloperoxidase antigen (MPO:Ag), markers of hemostatic activation (D-dimer and thrombin-antithrombin complex), and anti-β2-GPI antibodies were assessed. Results. Anti-β2-GPI antibodies were significantly more common in patients with ovarian cancer compared to controls. A significant correlation between the circulation of anti-β2-GPI and the concentration of MPO:Ag was also detected in these patients. All patients in the study group showed a significant increase in the markers of NETosis and hemostatic activation compared to controls. The revealed correlation between the concentrations of NETosis markers and anti-β2-GPI antibodies in ovarian cancer suggests a possible pathogenetic role of APA in the progression of ovarian cancer and NETs-mediated prothrombotic state against the background of APA circulation. It can be assumed that APA formed in cancer patients may be one of the triggers of NETosis. NETosis itself contributes to the development of thrombotic events and stimulates further synthesis of APA. Perhaps it is the activation of NETosis that explains the worse prognosis in cancer patients with APA circulation. Conclusion. The results of this study improve the understanding of interactions between the immune and hemostatic systems. Further research is needed to determine the role of APA in NETosis, as well as the role of NETosis and APA circulation in tumor progression, metastasis, and cancer-associated thrombosis. Key words: neutrophil extracellular traps, NETosis, antiphospholipid antibodies, cancer, neutrophils, anti-β2-GPI

A New Methodology for the Quantification of Neutrophil Extracellular Traps in Patient Plasma.

Neutrophil extracellular traps (NETs) are web-like structures made up of decondensed chromatin fibers along with neutrophil granular proteins that are extruded by neutrophils after activation or in response to foreign microorganisms. NETs have been associated with autoimmune and inflammatory diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, coronavirus disease 2019 (COVID-19), and others. There are reliable methods available to quantitate NETs from neutrophils, but their accurate quantification in patient plasma or serum remains a challenge. We developed a highly sensitive ELISA to detect NETs in serum/plasma and designed a novel smear immunofluorescence assay to detect NETs in as little as 1 μL of serum/plasma. We further validated our technology on plasma samples from SLE patients and healthy donors that carry interferon regulatory factor 5 genetic risk. The multiplex ELISA combines the use of three antibodies against myeloperoxidase (MPO), citrullinated histone H3 (CitH3), and DNA to detect the NET complexes with higher specificities. The immunofluorescence smear assay can visually detect intact structures of NETs in 1 μL of serum/plasma and provide similar results that correlate with findings from the multiplex ELISA. Furthermore, the smear assay is a relatively simple, inexpensive, and quantifiable method of NET detection for small volumes.

Cover Story: Eur. J. Immunol. 1'23

European Journal of ImmunologyVolume 53, Issue 1 2370011 Cover PictureFree Access Cover Story: Eur. J. Immunol. 1'23 First published: 11 January 2023 https://doi.org/10.1002/eji.202370011AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract Our colorful cover features an immunofluorescence staining of neutrophil extracellular traps (NETs) in the lung tissues of mice with specifically deleted GSDMD gene and treated with purified NETs. NETs were stained with MPO (green), citrullinated histone H3 (red) and DAPI (blue). Extrinsic NET injection partially reverses the protective effect of GSDMD deletion in neutrophils during acute respiratory distress syndrome. The image is taken from Xie et al., where the authors show that GSDMD cleavage in neutrophils is essential for the NET release and may be a new target for treating acute respiratory distress syndrome. Volume53, Issue1January 20232370011 RelatedInformation

Effects of recombinant human soluble thrombomodulin on neutrophil extracellular traps in the kidney of a mouse model of endotoxin shock

Sepsis is a life-threatening condition characterized by multi-organ dysfunction due to host immune system dysregulation in response to an infection. During sepsis, neutrophils release neutrophil extracellular traps (NETs) as part of the innate immune response. However, excessive NETs play a critical role in the development of organ failure during sepsis. Although recombinant human soluble thrombomodulin (rTM) can inhibit NET formation in the lungs and liver of a mouse model of endotoxin shock, its effects on the kidneys are unclear. The specific effects of NETs and rTM on the renal cortex and renal medulla were examined in a mouse model of endotoxin shock generated by intraperitoneal (i.p.) injection of lipopolysaccharide (LPS), followed by i.p. injection of rTM or an identical volume of saline 1h later. LPS injection increased serum creatinine, blood urea nitrogen, and histone H3 levels. However, rTM administration significantly decreased histone H3 and citrullinated histone H3 (citH3) levels. Immunohistochemical analysis revealed no significant changes in citH3 quantity in the renal cortex of any group. However, in the renal medulla, the increase in citH3 induced by LPS was abolished in the LPS+rTM group. Our findings demonstrate that rTM can suppress NETs in the renal medulla of mice with endotoxin-induced acute kidney injury.

Visualizing NETosis Using a Novel Neutrophil Extracellular Trap-Specific Marker.

As the most abundant leukocyte in circulation, the neutrophil plays a far-reaching role in maintaining homeostasis. Within the context of disease, however, neutrophils can potentiate various pathophysiological mechanisms with disastrous consequences for patients. The role of the neutrophil in disease is complex with mechanisms like NETosis driving the progression of several pathologies. NETosis involves neutrophils extruding protein-decorated DNA webs called neutrophil extracellular traps (NETs), which facilitate the progression of inflammatory, non-infectious, and neoplastic pathologies. The need to visualize NETs has thus never been greater. Current approaches for visualizing NETs are limited in specificity and sensitivity, involving non-specific fluorescent DNA dyes or co-stains of neutrophil and DNA markers. Improved methodologies are needed to robustly distinguish NETs from other cell-free DNA. Excitingly, a novel NET-specific posttranslational modification involving cleavage on the N-terminus of histone H3 has recently been identified. Here, we demonstrate that this single marker is superior to the conventional use of the co-stain of the neutrophil marker, myeloperoxidase, and, the DNA marker, histone H3 citrullination in visualizing neutrophil NETosis. This is due to this single marker's unparalleled ability to identify, not only more NETs but also their formation at earlier stages of NETosis. Moreover, we additionally propose a stepwise mechanism of neutrophil NETosis in which a histone H3 cleavage event precedes histone H3 citrullination. Taken together, these results demonstrate a novel method for visualizing NETs, allowing for continued exploration of their multifaceted roles in immunity and disease.