Citrullinated Histone H3 Research Articles

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 < 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 < 0.01) and MPO:Ag (ρ = 0.87; p < 0.01); increase in MPO:Ag level was coupled to rise in vWF:Ag (ρ = 0.70; p< 0.01), but increase in vWF:Ag occurred along with decline in ADAMTS-13:Ac (ρ = –0.43; p < 0.01) and ADAMTS-13:Ag (ρ= –0.42; p < 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.

Activation of GPR81 aggravated intestinal ischemia/reperfusion injury-induced acute lung injury via HMGB1-mediated neutrophil extracellular traps formation.

Intestinal ischemia/reperfusion (II/R) injury is a life-threatening situation accompanied by severe organ injury, especially acute lung injury (ALI). A great body of evidence indicates that II/R injury is usually associated with hyperlactatemia. G-protein-coupled receptor 81 (GPR81), a receptor of lactate, has been recognized as a regulatory factor in inflammation, but whether it was involved in II/R injury-induced ALI is still unknown. To establish the II/R injury model, the superior mesenteric artery of the mice was occluded gently by a microvascular clamp for 45min to elicit intestinal ischemia and then a 90-min reperfusion was performed. Broncho-alveolar lavage fluid (BALF) and lung tissues were obtained to evaluate the lung injury after II/R. The pulmonary histopathological alteration was evaluated by H&E staining. The concentration of proteins, the number of infiltrated cells, and the level of IL-6 were measured in BALF. The formation of neutrophil extracellular traps (NETs) was evaluated by the level of double-stranded DNA (dsDNA) and myeloperoxidase- double-stranded DNA (MPO-dsDNA) complex in BALF, and the content of citrullinated histone H3 (Cit-H3) in lung tissue. The level of HMGB1 in the BALF and plasma was measured by enzyme linked immunosorbent assay (ELISA). Administration of the GPR81 agonist 3,5-dihydroxybenzoic acid (DHBA) aggravated II/R injury-induced lung histological abnormalities, upregulated the concentration of proteins, the number of infiltrated cells, and the level of IL-6 in BALF. In addition, DHBA treatment increased the level of dsDNA and MPO-dsDNA complex in BALF, and promoted the elevation of Cit-H3 in lung tissue and the release of HMGB1 in BALF and plasma. After induction of ALI by II/R, the administration of DHBA aggravated ALI through NETs formation in the lung.

IgD enhances the release of neutrophil extracellular traps (NETs) via FcδR in rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory disorder affecting primarily the joints. Neutrophils and the release of neutrophil extracellular traps (NETs) contribute to the pathogenesis of RA. However, IgD, which was abnormally higher in RA, has not been studied for its pathological role in neutrophil activation and NETs formation. To investigate the effects of IgD on neutrophil activation and NETs formation via IgD receptor (FcδR), we collect peripheral blood of RA patients and established adjuvant-induced arthritis (AA) rat model. We found that the expression of FcδR on neutrophils was significantly higher in RA patients compared with healthy controls. As a specific marker of NETs, the level of citrullinated histone H3 was positively correlated with sIgD and FcδR in RA patients. IgD enhances the release of NETs and promotes the proliferation of fibroblast-like synoviocytes (FLS) from RA patients by activating neutrophils. As a competitive FcδR blocker, IgD-Fc-Ig fusion protein could significantly reduce NETs formation and FcδR expression on neutrophils in vitro. In vivo, IgD-Fc-Ig could restrain IgD-induced neutrophil activation and NETs formation, thus inhibited FLS proliferation in AA rats. Data presented here demonstrate that neutrophils could be triggered by IgD to release NETs and take part in FLS proliferation in RA patients with excessive IgD. Blocking IgD-FcδR could inhibit neutrophil activation and NETs formation, and represent an additional attractive novel therapeutic strategy for the treatment of RA.

S-07-6: NEUTROPHIL EXTRACELLULAR TRAPS AS MEDIATORS OF ENDOTHELIAL DAMAGE IN HYPERTENSION AND DIABETES

Objective: Endothelial dysfunction (ED) plays a key role in the pathogenesis of hypertension and diabetes, yet its molecular determinants are poorly understood. Concomitant hypertension and diabetes can synergistically increase damage to the vasculature, with increasing evidence suggesting that resulting immune dysregulation may contribute to endothelial injury. Neutrophil extracellular traps (NETs), networks of chromatin and protein-containing extracellular fibers released to fight infection, are generated from neutrophils following activation of the citrullinating enzyme peptidylarginine deiminase 4 (PAD4). PAD4 has been shown to be upregulated in atherosclerosis, vasculitis, and various inflammatory autoimmune diseases, leading to NET-induced endothelial injury. Whether NETs could contribute to hypertension- and diabetes-induced ED has not been established. The objective of the present study is to investigate the impact of hypertension and diabetes on PAD4 activity and the contribution of NETs to resulting vascular injury. Design and Methods: Promyelocytic HL-60 cells were differentiated to neutrophil-like cells and treated with angiotensin II (10−7 M) and/or high glucose (25 mM) for 24 hours, with or without subsequent NET-inducing PMA stimulation (500 nM). NETs were isolated from the culture medium by differential centrifugation and assessed by Western blot analysis of citrullinated histone H3 (citH3, a primary NET component). Isolated NETs were used to treat Human Umbilical Vein Endothelial Cells (HUVECs) at 0.5–500 ng/ml for 24 hours, after which cell viability (XTT cell assay) and proliferation (BrdU assay) were measured. We also assessed NETosis in mice expressing human renin in the liver (TTRhRen) intercrossed with OVE26 diabetic mice [i.e. hypertensive-diabetic, HD mice], compared with wild-type (WT) FVBN/J mice. We examined kidney levels of citH3, neutrophil elastase (NE), and PAD4 expression by immunohistochemistry. Results: Treatment with high glucose, but not angiotensin II, increased citH3 levels in neutrophil-like cells. NETs isolated from PMA-stimulated cells decreased HUVEC viability by ∼2-fold at 5 ng/ml (P = < 0.0001), and dose dependently decreased proliferation from 0.5–500 ng/ml, with a ∼80% decrease at 500 ng/ml. In HD mice, we observed a significant increase in kidney levels of citH3 (P < 0.0001), NE (P < 0.0001), and PAD4 (P < 0.0001), which was associated with the presence of albuminuria and increased blood pressure levels. Conclusion: These results suggest that NETs are upregulated in hypertension and diabetes and may contribute to endothelial damage and microvascular injury.

Neutrophil extracellular traps (NETs) in aortic stenosis: Comparison of methods for assessment of NETs formation

Abstract Introduction We previously showed increased neutrophil extracellular traps (NETs) formation (NETosis) in patients with severe aortic stenosis (AS). Our aim was to comprehensively assess NETosis in AS using a relatively simple and appropriate technique. We investigated circulating NETosis markers by ELISA and assessed the potential of blood neutrophils to release NETs by flow cytometry. Materials and Methods We enrolled 13 patients aged 66 [Q1–Q3, 60–70] years with severe isolated AS without diabetes, chronic kidney disease, and atrial fibrillation. Nine apparently healthy volunteers of similar sex and age served as controls. Serum concentrations of citrullinated histone H3 (citH3), circulating nucleosomes, myeloperoxidase (MPO), and deoxyribonuclease-1 (DNASE1) were measured using ELISAs. Peripheral blood NET-releasing neutrophils were detected by flow cytometry as MPO/citH3-positive cells. Results AS patients compared to controls presented 174% higher concentrations of citH3 (p<0.001), 456% higher nucleosomes (p<0.001), 136% higher MPO (p=0.021) and 19% higher DNASE1 levels (p=0.039), together with 101% elevated percentage of NET-releasing neutrophils assessed by flow cytometry (p=0.003). In AS patients, the proportion of blood NET-releasing neutrophils positively correlated with citH3 (r=0.86, p<0.001) and nucleosome (r=0.58, p=0.041) concentrations but not with MPO or DNASE1. Moreover, we observed a strong association between AS severity, measured as aortic valve area (AVA), and serum citH3 concentrations (r=−0.75, p=0.003), but not the number of NET-releasing neutrophils. Conclusions Our study showed increased blood neutrophil potential to release NETs together with increased levels of serum markers of NETos is in severe AS patients. However, we recommend ELISA to assess NETosis in vivo as simpler technique giving more unequivocal results compared to flow cytometry.

Phthalide derivative CD21 regulates the platelet- neutrophil extracellular trap-thrombin axis and protects against ischemic brain injury in rodents.

Prothrombotic and proinflammatory properties of neutrophil extracellular traps (NETs) contribute to brain damage after ischemic stroke. CD21 is a novel phthalide neuroprotectant against cerebral ischemia in rodents. This study investigated effects of CD21 on the platelet-NET-thrombin axis and ischemic brain injury and the underlying mechanism. CD21 exerteddose-dependent neuroprotectionin rats that were subjected to2h middle cerebral artery occlusion,dose-dependentlyinhibited adenosine diphosphate-mediatedplatelet aggregationin rats, and dose-dependentlyexertedanti-thrombotic activityin rodents that received a collagen-epinephrine combination, ferric chloride, or an arteriovenous shunt. Equimolar CD21 doses exerted stronger efficacy than 3-N-butylphthalide (NBP, natural phthalide for the treatment of ischemic stroke). CD21 dose-dependently improved regional cerebral blood flow, neurobehavioral deficits, and infarct volume in mice that were subjected to photothrombotic stroke (PTS). CD21 (13.79mg/kg, i.v.) significantly decreased NET components (plasma dsDNA concentrations; mRNA levels of elastase, myeloperoxidase, and neutrophil gelatinase-associated lipocalin and protein level of citrullinated histone H3 in ischemic brain tissues), mRNA and protein levels of peptidyl-arginine deiminase 4 (PDA4, NET formation enzyme), and mRNA levels of NET-related inflammatory mediators (interleukin-1β, interleukin-17A, matrix metalloproteinase 8, and matrix metalloproteinase 9) in ischemic brain tissues, despite no effect on mRNA levels of deoxyribonuclease I (NET elimination enzyme). Pretreatment with compound C (inhibitor of adenosine monophosphate-activated protein kinase [AMPK]) significantly reversed the inhibitory effects of CD21 on NETs, PDA4, and inflammatory mediators in PTS mice. These results suggest that CD21 might regulate the platelet-NET-thrombin axis and protect against ischemic brain injury partly through the induction of AMPK activation.

Иммунотромбоз, прогрессия опухоли и метастазирование. Роль интерлейкина-8 и внеклеточных ловушек нейтрофилов

Immunothrombosis, tumor progression and metastasis are inextricably linked. These close interactions are carried out by several actors. Neutrophils are considered as players in the antimicrobial host defense. Activated neutrophils release extracellular traps (NETs) involved in antimicrobial immune response and pathogenesis of various conditions including tumor growth. Neutrophils and interleukin-8 (IL-8) play a key role in the formation of NETs. Objective. To evaluate the interaction between NETs and IL-8, their contribution to the development of immunothrombosis and tumor progression in patients with reproductive system cancers and breast cancer of different stages. Patients and methods. This study included 77 patients with malignant tumors of the endometrium, ovaries, cervix, and breast, and 33 healthy controls. Concentrations of citrullinated histone H3 (citH3), human myeloperoxidase antigen (MPO:Ag), D-dimer, thrombin-antithrombin complexes (TAT), and IL-8 were assessed in all patients before surgical treatment or chemotherapy upon admission to the hospital. Results. It was shown that the concentration of NETosis markers was significantly higher in cancer patients compared to healthy controls, with an increase in indicators against the background of disease progression in stages. Naturally, markers of hemostatic activation were elevated in gynecologic cancer patients, also with increasing indicators against disease progression. The concentration of IL-8 was used as a marker of inflammation in this study, which was significantly elevated in cancer patients. The correlation analysis showed that IL-8 concentrations increased with increasing levels of NETosis markers, demonstrating maximum values at late stages of the disease, which indicates an important role of immunothrombosis in cancer progression and metastasis. Conclusion. The study showed that the concentrations of NETosis markers and IL-8 are elevated in cancer patients, and the degree of this elevation correlates with the stage of the disease. A possible mechanism is capturing of tumor cells by networks, their shielding from immune system, subsequent expression of IL-8 by tumor cells with further involvement of neutrophils and initiation of NETosis. Key words: neutrophil extracellular traps, interleukin-8, immunothrombosis, metastasis, tumor progression, neutrophils

Clinicopathological features and prognostic impact of dirty necrosis in metastatic lung cancers from the colon and rectum.

Dirty necrosis (DN) is a form of tumor necrosis (TN) with prominent neutrophil infiltration and cell detritus in the necrotic foci. This study aimed to characterize the clinicopathological features of DN in metastatic lung cancers of the colon and rectum (MLCRs). A total of 227 patients who underwent pulmonary metastasectomy and complete resection for MLCR were included in this study. TN was evaluated using digitally scanned resection specimens. These slides were immunostained for biomarkers of NETosis (citrullinated histone H3 [citH3] and myeloperoxidase [MPO]), and the area positive for citH3 and MPO was further quantified. TN was observed in 216 cases (95.2%), and 54 (25.0%) of these cases had DN. The presence of TN was not associated with a worse prognosis; however, patients with DN had a significantly shorter overall survival than those without DN (p < 0.01). Furthermore, the presence of DN was a poor prognostic factor in both the univariate and multivariate analyses. Immunohistochemical analysis revealed that the percentage of citH3-positive and MPO-positive areas in the DN-positive cases was significantly higher than that in the DN-negative cases (p < 0.01 and p < 0.01, respectively). In surgically resected MLCR, DN is the characteristic TN subtype associated with poor prognosis and neutrophil extracellular traps (NETs).

Tanshinone IIA Improves Acute Gouty Arthritis in Rats through Regulating Neutrophil Activation and the NLRP3 Inflammasome.

To investigate the prevention and treatment effect of tanshinone IIA (TIIA) on acute gouty arthritis (AGA) and its mechanism. The anti-AGA effect of TIIA was observed in vivo and in vitro. Neutrophils were isolated from the abdominal cavity of mice, and the anti-AGA effect of TIIA was investigated in a rat model of MSU-induced AGA. The pathological changes of the ankle joint tissues were assessed by H&E. Cytokine and chemokine expression were determined by ELISA and RT-qPCR. The NLRP3 inflammasome pathway protein levels in the ankle joint tissues were evaluated via western blotting. Neutrophil migration was evaluated in air pouch and transwell assays. Immunohistochemistry and immunofluorescence analysis evaluate the release of myeloperoxidase (MPO), neutrophil elastase (NE), and citrullination of histone H3 (CitH3). Beclin-1 and LC3B expressions were determined using western blotting and immunofluorescence. Key Findings. Treatment with TIIA alleviated synovial hyperplasia and neutrophil infiltration, regulated cytokine and chemokine expressions, and inhibited NLRP3 activation in AGA rats, neutrophil migration, MPO, NE, and CitH3 expression, and LC3B and Beclin-1 protein expression. These results demonstrate that TIIA can effectively enhance AGA by focusing on the neutrophils and NLRP3 inflammasome, demonstrating that TIIA may act as a potential helpful agent for AGA.

478. Lung Hybrid Neutrophils and Subsequent Extracellular Traps Are Protective In COVID-19-Associated Pulmonary Aspergillosis

Abstract Background COVID-19-associated pulmonary aspergillosis (CAPA) is a severe superinfection with the fungus Aspergillus frequently affecting critically ill COVID-19 patients. Pathophysiological insight, key to improve diagnostic and immunomodulatory therapeutic options, is lacking. Methods We performed single-cell RNA sequencing (scRNA-seq) on 37 bronchoalveolar lavage (BAL) samples from 37 critically ill COVID-19 patients. Three groups were defined: patients who did not develop aspergillosis (COVID-19-only, n=22), CAPA patients with sampling &amp;lt; 5 days after CAPA diagnosis (early CAPA, n=6) and CAPA patients with sampling 5-11 days after CAPA diagnosis (late CAPA, n=9). All CAPA patients had probable/proven CAPA according to the 2020 ECMM/ISHAM consensus criteria. Additionally, we assessed neutrophil extracellular trap (NET) levels in a separate cohort of 33 biobanked COVID-19-only BAL samples and 24 early CAPA samples. Results A total of 69008 cells passed quality filtering. CAPA patients had significantly lower BAL neutrophil proportions than COVID-19-only patients, particularly in early CAPA (Fig. 1A). Pseudotime inference revealed two neutrophil trajectories: a regular maturation trajectory, and a trajectory giving rise to “hybrid” neutrophils which express genes encoding proteins with antigen-presenting functions (Fig. 1B). The latter trajectory was dominant in CAPA patients (Fig. 1C). NETosis analyses revealed significantly higher levels of citrullinated histone H3 DNA complexes (H3Cit-DNA) in CAPA patients (Fig. 2A). This explains the low CAPA BAL neutrophil proportions, as neutrophils that underwent NETosis are no longer detected via scRNA-seq. CAPA patients with the lowest H3Cit-DNA levels had significantly decreased survival rates (Fig. 2B). Figure 1:Bronchoalveolar lavage fluid (BALF) neutrophil proportions are significantly decreased in CAPA patients, accompanied by a shift to hybrid neutrophil formation. Panel (A): BALF neutrophil proportions as analyzed by single-cell RNA sequencing using the Seurat R package are significantly lower in CAPA patients compared to COVID-19-only patients. Patients with early CAPA have significantly lower BALF neutrophil proportions than patients with late CAPA. Macrophage/monocyte and epithelial cell proportions are reciprocally increased in CAPA patients compared to COVID-19-only patients. P-values shown for differences between the pooled CAPA patients and the COVID-19-only patients. P-values were calculated using a generalized linear model correcting for age, Charlson Comorbidity Index at hospital admission, and administration of corticosteroids (prednisone equivalent dose 20 mg or higher) within 48 hours of BALF sampling. Panel (B): Two trajectories are defined using pseudotime inference calculated using the Slingshot R package: a trajectory dominant in COVID-19-only patients with regular maturation of progenitor neutrophils, and a trajectory dominant in CAPA patients with maturation towards a ‘hybrid neutrophil’ state, with neutrophils expression genes encoding proteins with functions in antigen presentation. Subsequently, the hybrid neutrophil proportion is significantly higher in CAPA patients compared to COVID-19-only patients, and is significantly higher in patients with early CAPA than those with late CAPA. The mature neutrophil proportion is reciprocally reduced in CAPA patients. P-values shown for differences between the pooled CAPA patients and the COVID-19-only patients. P-values were calculated using a generalized linear model correcting for age, Charlson Comorbidity Index at hospital admission, and administration of corticosteroids (prednisone equivalent dose 20 mg or higher) within 48 hours of BALF sampling. Figure 2:Neutrophil extracellular trap (NET) levels are increased in early CAPA and are associated with increased survival in CAPA patients specifically. Panel (A): Myeloperoxidase (MPO) DNA levels were analyzed as measure for general NET-formation, while citrullinated histone H3 bound DNA (H3Cit-DNA) levels were analyzed as more specific PAD4-dependent NET-formation, in BALF samples from early CAPA and COVID-19-only patients. A trend towards higher MPO-DNA levels was found in early CAPA patients, while H3Cit-DNA levels were significantly higher in early CAPA compared to COVID-19-only patients. P-values calculated using Mann-Whitney U test. Panel (B): Kaplan-Meier analysis of patients with NETosis analyses, divided in early CAPA and COVID-19-only patients and subdivided according to H3Cit-DNA levels (cut-off at 20000 ng/mL for early CAPA and at 8000 ng/mL for COVID-19-only). Log-rank test was used to compare survival distributions. For the comparison early CAPA (low H3Cit-DNA) versus early CAPA (high H3Cit-DNA), the log-rank p-value was 0.033. Conclusion CAPA patients display extremely high levels of released NETs in the lower respiratory tract, associated with a shift from the normal neutrophil maturation process towards “hybrid neutrophil” formation, probably upon encountering the fungus. In contrast to high NETosis contributing to mortality in severe COVID-19, CAPA patients likely require these NETs to survive aspergillosis. BAL NET levels hold promise as a tool to guide diagnosis, prognosis and treatment in these patients. Disclosures Simon Feys, MD, Pfizer: Travel support Katrien Lagrou, PharmD, PhD, FUJIFILM Wako: Speaker fee|Gilead: Advisor/Consultant|Gilead: Speaker fee|MRM Health: Advisor/Consultant|MSD: Advisor/Consultant|Pfizer: Speaker fee|Thermo fisher Scientific: Service fee Joost Wauters, MD, PhD, Gilead: Grant/Research Support|Gilead: Speaker's fee, travel support, advisory board|MSD: Grant/Research Support|MSD: Speaker's fee, travel support, posaconazole for interventional trial|Pfizer: Grant/Research Support|Pfizer: Speaker's fee, travel support, advisory board.