Neutrophil Extracellular Traps Markers Research Articles

LL37/FPR2 regulates neutrophil mPTP promoting the development of neutrophil extracellular traps in diabetic retinopathy.

Diabetic retinopathy (DR) is characterized by chronic, low-grade inflammation. This state may be related to the heightened production of neutrophil extracellular traps (NETs) induced by high glucose (HG). Human cathelicidin antimicrobial peptide (LL37) is an endogenous ligand of G protein-coupled chemoattractant receptor formyl peptide receptor 2 (FPR2), expressed on neutrophils and facilitating the formation and stabilization of the structure of NETs. In this study, we detected neutrophils cultured under different conditions, the retinal tissue of diabetic mice, and fibrovascular epiretinal membranes (FVM) samples of patients with proliferative diabetic retinopathy (PDR) to explore the regulating effect of LL37/FPR2 on neutrophil in the development of NETs during the process of DR. Specifically, HG or NG with LL37 upregulates the expression of FPR2 in neutrophils, induces the opening of mitochondrial permeability transition pore (mPTP), promotes the increase of reactive oxygen species and mitochondrial ROS, and then leads to the rise of NET production, which is mainly manifested by the release of DNA reticular structure and the increased expression of NETs-related markers. The PI3K/AKT signaling pathway was activated in neutrophils, and the phosphorylation level was enhanced by FPR2 agonists invitro. Invivo, increased expression of NETs markers was detected in the retina of diabetic mice and in FVM, vitreous fluid, and serum of PDR patients. Transgenic FPR2 deletion led to decreased NETs in the retina of diabetic mice. Furthermore, invitro, inhibition of the LL37/FPR2/mPTP axis and PI3K/AKT signaling pathway decreased NET production induced by high glucose. These results suggested that FPR2 plays an essential role in regulating the production of NETs induced by HG, thus may be considered as one of the potential therapeutic targets.

The clinical characteristics of neutrophil extracellular trap in patients with severe cerebral venous sinus thrombosis and its prognostic value in acute and subacute patients

Objective: To explore the clinical characteristics of neutrophil extracellular trap (NET) in patients with severe cerebral venous sinus thrombosis (CVST) and to study their prognostic value in the acute and subacute phases. Methods: This study is a retrospective case series analysis. Clinical and pathological data of 52 patients with severe cerebral venous sinus thrombosis who underwent endovascular treatment in the Department of Neurosurgery, Tianjin Huanhu Hospital from June 2019 to June 2022 were retrospectively analyzed. There were 20 males and 32 females, with an age of (40.1±13.6) years(range:18 to 66 years). Forty-five healthy physical examinees were included in the control group. High-resolution MRI was used to stage the thrombus, with 11 cases in the acute group, 28 cases in the subacute group, and 13 cases in the chronic group. Thrombus specimens were obtained through endovascular treatment, and the fluorescence intensity of NET in peripheral blood at different time points was analyzed by immunofluorescence contrast,including the double-stranded DNA structure and adhesion protein components (citrolinated histone H3 (CitH3), myeloperoxidase-DNA complex(MPO-DNA), neutrophil elastase (NE)). The NET markers were determined by ELISA. Spearman rank correlation analysis was used to analyze the correlation between the NET markers in peripheral blood of patients with severe cerebral venous sinus thrombosis in the acute and subacute phases and the volume of venous sinus thrombus, the degree of venous sinus recanalization after treatment, and the discharge modified Rankin scale(mRS)score. The accuracy of NET markers in predicting the prognosis of patients with severe cerebral venous sinus thrombosis was analyzed by drawing receiver operating characteristic (ROC) curve and calculating the area under the curve (AUC). Results: The results of immunofluorescence staining and ELISA showed that no NET structure was formed in the peripheral blood of the control group, while CitH3, MPO-DNA and NE levels in the peripheral blood of CVST patients were increased, among which the acute stage group was the highest, followed by the subacute group, and the chronic group was the lowest. Spearman correlation analysis showed that CitH3, MPO-DNA and NE levels in peripheral blood of patients in acute group and subacute group were positively correlated with thrombus volume and mRS score at discharge (P<0.05). The levels of CitH3 and MPO-DNA in peripheral blood of patients with complete venous sinus recanalization were lower than those of patients with partial venous sinus recanalization (P<0.01). ROC curve analysis results showed that MPO-DNA and NE had no predictive ability for the prognosis of CVST patients (P values were 0.614 and 0.324, respectively), and the AUC of CitH3 was 0.800 (95%CI: 0.638~0.962, P=0.032), the best cut-off value was 13.5 μg/L, the sensitivity was 100%, and the specificity was 58.8%. Conclusions: A large number of NET are formed in patients with severe cerebral venous sinus thrombosis in acute stage. Patients with severe cerebral venous sinus thrombosis in acute stage and subacute stage with high peripheral blood NET content has a low rate of complete sinus revascularization and poor neurological function recovery after treatment.

Abstract 3134: Integrated Multi-Omics Profiling Reveals Neutrophil Extracellular Traps Promote Aortic Dissection

Background: Negative aortic remodeling increases the risk of aorta-related adverse events (AAEs) after thoracic endovascular aortic repair (TEVAR) for patients with aortic dissection (AD). Research Questions and Aims: It is worth investigating prognostic indicators to streamline the identification of individuals at elevated risk for postoperative AAEs, and therapeutic targets to optimize the efficacy of TEVAR. Methods: We performed single-cell transcriptomic and proteomic profiling of aortic lesions and peripheral blood samples, respectively. We then integrated single-cell with publicly accessible bulk data to identify phenotype-relevant subpopulations. We further confirmed the results in human plasma samples and in mouse models. The Cox regression analysis were used to identify independent risk factors. Results: Integrated multi-omics analysis identified a highly phenotype-associated and disease-relevant macrophage subset, which closely interacted with neutrophil via CXCL3/CXCR2 axis, and promoted neutrophil extracellular traps (NETs) within aortic lesion. Likewise, increased NETs formation is a defining feature of systemic immunity of AD, with an expansion of inflammatory cytokines in the peripheral blood. Furthermore, we demonstrated that in a prospective cohort of 115 patients with a median follow-up of 13 months, the level of citrullinated histone H3 (CitH3), a NETs marker, could serve as a risk factor for the occurrence of AAEs following TEVAR (HR 8.78, p &lt; .001). The CitH3 level, determined at a cut-off value of 6.9 ng/ml, predicted the postoperative AAEs with a sensitivity and specificity of 0.6 and 0.9, respectively, and an area under the curve of 0.75. In addition, inhibiting NETs formation through the blockade of CitH3 alleviated the progression and rupture of AD in mice. Conclusions: The multi-omics profiling reveals highly disease-relevant macrophage subpopulation, which promotes NETs formation and inflammation infiltration in the development of AD. Increased NETs formation is a defining feature of both aortic lesion and peripheral blood of patients with AD. CitH3 could facilitate the risk stratification and prognostic evaluation, and might serve as potential therapeutic targets of patients with AD.

Neutrophil extracellular traps promote acetaminophen-induced acute liver injury in mice via AIM2.

Excessive acetaminophen (APAP) can induce neutrophil activation and hepatocyte death. Along with hepatocyte dysfunction and death, NETosis (a form of neutrophil-associated inflammation) plays a vital role in the progression of acute liver injury (ALI) induced by APAP overdose. It has been shown that activated neutrophils tend to migrate towards the site of injury and participate in inflammatory processes via formation of neutrophil extracellular traps (NETs). In this study we investigated whether NETs were involved in hepatocyte injury and contributed to APAP-induced ALI progression. ALI mouse model was established by injecting overdose (350 mg/kg) of APAP. After 24 h, blood and livers were harvested for analyses. We showed that excessive APAP induced multiple programmed cell deaths of hepatocytes including pyroptosis, apoptosis and necroptosis, accompanied by significantly increased NETs markers (MPO, citH3) in the liver tissue and serum. Preinjection of DNase1 (10 U, i.p.) for two consecutive days significantly inhibited NETs formation, reduced PANoptosis and consequently alleviated excessive APAP-induced ALI. In order to clarify the communication between hepatocytes and neutrophils, we induced NETs formation in isolated neutrophils, and treated HepaRG cells with NETs. We found that NETs treatment markedly increased the activation of GSDMD, caspase-3 and MLKL, while pre-treatment with DNase1 down-regulated the expression of these proteins. Knockdown of AIM2 (a cytosolic innate immune receptor) abolished NETs-induced PANoptosis in HepaRG cells. Furthermore, excessive APAP-associated ALI was significantly attenuated in AIM2KO mice, and PANoptosis occurred less frequently. Upon restoring AIM2 expression in AIM2KO mice using AAV9 virus, both hepatic injury and PANoptosis was aggravated. In addition, we demonstrated that excessive APAP stimulated mtROS production and mitochondrial DNA (mtDNA) leakage, and mtDNA activated the TLR9 pathway to promote NETs formation. Our results uncover a novel mechanism of NETs and PANoptosis in APAP-associated ALI, which might serve as a therapeutic target.

Abstract 1026: Post-surgery sequelae unrelated to disease progression and chemotherapy revealed in follow-up of stage III colon cancer patients

Abstract A gap currently exists in our understanding of causes of elevated total circulating DNA (cirDNA) levels in cancer patients following tumor resection, and the influence of anesthesia or surgery on cirDNA release. Indeed, the post-surgery dynamics of cirDNA have been largely overlooked to date, despite their importance in determining the optimal timing for assessment of minimal residual disease (MRD). Since cirDNA levels were recently found to be associated with neutrophil extracellular traps (NETs) formation in various cancers, we investigated NETs and cirDNA post-surgery.We conducted two clinical studies to explore the dynamics of cirDNA quantity and of neutrophil extracellular traps (NETs) markers: (1) a peri-surgery study over a duration of up to 72 hours, which involved stage I-III colon (n=10), prostate (n=10), and breast (n=9) cancer patients; and (2) a post-surgery study that extended up to two years post-surgery, and which involved 74 stage III colon cancer patients. We assessed plasma levels of cirDNA using qPCR, and assessed two protein markers of NETs using ELISA, in both cancer patients and control group of healthy individuals (HI) (N=114). This second study is the first prospective, multicenter, blinded study of the dynamics of the total cirDNA post-surgery, for a period of up to two years.We observed that (i), NETs formation contributes to post-surgery conditions; (ii), peri- and post-surgery cirDNA levels were highly associated with NETs formation in colon cancer; (iii), each tumor type showed a specific pattern of the peri-surgery dynamics of cirDNA and NETs markers; peri-operative levels of these markers are significantly lower in breast cancer patients as compared to prostate and colorectal cancer patients; (iv), a significant proportion of patients showed pre- (58.1%) and post-surgery (80.4%) median marker values higher than in HI, even after 2 years following tumor resection, (v) these markers were either equal to or greater (23.2%) than their pre-surgery counterparts; and (vi), elevated values of these markers did not derive from chemotherapy toxicity.We provide evidence that, for cancer patients in the post-surgery period, cirDNA originates mainly from NETs. This finding calls into question the current method of assessing MRD according to the fraction of mutant cirDNA, given that the level of NETs formation appears to be patient dependant. The peri-surgery dynamics of NETs formation and cirDNA release vary according to the surgical procedure and cancer type. In a significant part of patients with colon cancer, NETs continue to persist for more than a year after surgery, irrespective of disease progression or chemotherapy use. Nevertheless, cirDNA and NETs marker levels vary in cases with post-surgery inflammatory/thrombotic adverse events. Our work highlights the existence of long-lasting “sequelae” effects of cancer, previously unreported. Citation Format: Andrei Kudriavtsev, Alexia Mirandola, Catalina I. Cofre Muñoz, Raquel Comas Navarro, Marco Macagno, Brice Pastor, Ekaterina Pisareva, Mireia Sanchis Marin, Javier Gonzalo Ruiz, Anna Sapino, Alice Bartolini, Massimo Di Maio, Cynthia Sanchez, Yann Gricourt, Xavier Capdevila, Gerald Lossaint, Evelyne Crapez, Marc Ychou, Ramon Salazar Soler, Elisabetta Fenocchio, Paula X. Fernandez Calotti, Philippe Cuvillon, Thibault Mazard, Cristina Santos Vivas, Elez M. Elez, Federica Di Nicolantonio, Alain R. Thierry. Post-surgery sequelae unrelated to disease progression and chemotherapy revealed in follow-up of stage III colon cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1026.

Expression level of neutrophil extracellular traps in peripheral blood of patients with chronic heart failure complicated with venous thrombosis and its clinical significance

ObjectivePrevious studies have reported that neutrophil extracellular traps (NETs) have been identified to be involved in thrombosis, but the clinical value in chronic heart failure (CHF) patients with venous thrombosis is unclear. This study focused on the expression level of NETs in the peripheral blood of patients with CHF complicated with venous thrombosis and its clinical value.Methods80 patients with CHF were included and divided into 2 groups according to the occurrence of venous thrombosis, and the expression levels of NETs in peripheral venous blood and lesion veins of the patients were detected through fluorescent staining. Myeloperoxidase-DNA (MPO-DNA) and citrullinated histone H3 (CitH3), markers of NETs, were detected by enzyme linked immunosorbent assay kit. The receiver operating characteristic (ROC) curve was used to analyze the value of peripheral venous blood NETs in the diagnosis of venous thrombosis in CHF patients, while the relationship between NETs in peripheral and lesion veins was analyzed by a unitary linear regression model.ResultsThe results showed that the concentration of NETs, MPO-DNA, and CitH3 in CHF patients combined with venous thrombosis was markedly higher than that in patients without venous thrombosis, and the concentration of NETs, MPO-DNA, and CitH3 in lesion venous blood was notably higher than that in peripheral venous blood. Binary logistics regression analysis showed that NETs in peripheral venous blood were an independent risk factor for venous thrombosis in patients with heart failure. The unitary linear regression model fitted well, indicating a notable positive correlation between NETs concentrations in peripheral and lesion veins. The area under the ROC curve for diagnosing venous thrombosis was 0.85, indicating that peripheral blood NETs concentration levels could effectively predict venous thrombosis in CHF patients.ConclusionThe expression level of NETs was high in the peripheral blood of CHF patients combined with venous thrombosis and was the highest in lesion venous blood. NETs levels in peripheral blood had the value of diagnosing venous thrombosis in CHF patients, and the concentrations of NETs in peripheral and lesion veins are markedly positively correlated.

Impaired balance between neutrophil extracellular trap formation and degradation by DNases in COVID-19 disease

BackgroundThrombo-inflammation and neutrophil extracellular traps (NETs) are exacerbated in severe cases of COVID-19, potentially contributing to disease exacerbation. However, the mechanisms underpinning this dysregulation remain elusive. We hypothesised that lower DNase activity may be associated with higher NETosis and clinical worsening in patients with COVID-19.MethodsBiological samples were obtained from hospitalized patients (15 severe, 37 critical at sampling) and 93 non-severe ambulatory cases. Our aims were to compare NET biomarkers, functional DNase levels, and explore mechanisms driving any imbalance concerning disease severity.ResultsFunctional DNase levels were diminished in the most severe patients, paralleling an imbalance between NET markers and DNase activity. DNase1 antigen levels were higher in ambulatory cases but lower in severe patients. DNase1L3 antigen levels remained consistent across subgroups, not rising alongside NET markers. DNASE1 polymorphisms correlated with reduced DNase1 antigen levels. Moreover, a quantitative deficiency in plasmacytoid dendritic cells (pDCs), which primarily express DNase1L3, was observed in critical patients. Analysis of public single-cell RNAseq data revealed reduced DNase1L3 expression in pDCs from severe COVID-19 patient.ConclusionSevere and critical COVID-19 cases exhibited an imbalance between NET and DNase functional activity and quantity. Early identification of NETosis imbalance could guide targeted therapies against thrombo-inflammation in COVID-19-related sepsis, such as DNase administration, to avert clinical deterioration.Trial registration: COVERAGE trial (NCT04356495) and COLCOV19-BX study (NCT04332016).Graphical

The mechanism of intravenous immunoglobulin (IVIG) in vascular endothelial injury in kawasaki disease based on neutrophil extracellular traps

This study explores the role of Neutrophil extracellular traps (NETs) in kawasaki disease (KD)-induced vascular inflammatory injury and the protective effect and mechanism of IVIG on vascular endothelial damage. A total of 37 children diagnosed with KD and admitted to Dongguan maternal and Child Health Care Hospital between March 2020 and June 2022 were included in the study. The children were divided into different groups based on their treatment and the presence or absence of coronary artery damage: IVIG treatment group (KDIVIG group), subgroup with coronary artery damage (KDCAL group), and subgroup without coronary artery damage (KDNCAL group), and a Control group consisting of 9 children who underwent surgical treatment. Flow cytometry was used to detect the proportion of neutrophils and the number of NETs in peripheral blood. It was found that the proportion of neutrophils in the peripheral blood of the acute KD group significantly increased with the presence of NETs. RT-PCR and ELISA detection showed that the levels of inflammatory factors TNF-α, IL-6 and CitH3 were abnormally elevated in this acute KD group, and the CAL group exhibited higher proportions of neutrophils and NETs-related markers compared to the NCAL group, while the IVIG group had significantly decreased proportions of neutrophils. PMA culture of neutrophils induced an increase expression of NETs marker protein, the content of NETs cfDNA increased. NETs culture could promote the secretion of TNF-α, whereas IVIG cultured cells inhibited the secretion of TNF-α. Finally, HCAEC cells were cultured with different levels of TNF-α, and the function of HCAEC cells was assessed using CCK8, scratch assay and flow cytometry. The high expression of TNF-α in the NETs group inhibited the proliferation and migration of HUVEC cells and enhanced their apoptosis. In contrast, the IVIG culture group exhibited similar effects to the TNF-α monoclonal antibody, as it inhibited HUVEC cell apoptosis and improved their viability by reducing TNF-α expression. Total protein was extracted from the cells using nano-magnetic beads, and RT-PCR and western blot detection indicated that the increase of TNF-α expression could increase the phosphorylation of NF-κB and and the expression of MMP-9. However, when TNF-α was inhibited by IVIG and TNF-α monoclonal antibody culture, the activity of NF-κB/MMP-9 athway was decreased. Therefore, IVIG may inhibit the production of NETs in KD children, thereby reducing TNF-α/NF-NF-κB/MMP-9 mediated inflammatory response process and protecting the function of vascular endothelial cells.

Expression of neutrophil extracellular traps and phagocytic functions among patients with hepatic alveolar echinococcosis

To investigate the expression of neutrophil extracellular traps (NETs) and phagocytic function in the peripheral blood of patients with hepatic alveolar echinococcosis (HAE), and to examine their correlations with clinical inflamma tory indicators and liver functions. A total of 50 patients with HAE admitted to Department of Hepatobiliary and Pancreatic Surgery, The Affiliated Hospital of Qinghai University from August 2022 to June 2023 were enrolled, while 50 age- and gender-matched healthy individuals from the Centre for Healthy Examinations of the hospital during the same period served as controls. The levels of NETs markers neutrophil myeloperoxidase (MPO) and neutrophil elastase (NE) were measured using enzyme-linked immunosorbent assay (ELISA). Peripheral blood neutrophils were isolated using density gradient centrifugation, stimulated in vitro using phorbol 12-myristate 13 acetate (PMA), and the levels of MPO and citrullination histone H3 (CitH3) released by neutrophils were quantified using flow cytometry. The phagocytic functions of neutrophils were examined using flow cytometry. In addition, the correlations of MPO and NE levels with clinical inflammatory indicators and liver biochemical indicators were examined using Spearman correlation analysis among HAE patients. The peripheral blood plasma MPO［(417.15 ± 76.08) ng/mL vs. (255.70 ± 80.84) ng/mL; t = 10.28, P < 0.05］, NE［(23.16 ± 6.75) ng/mL vs. (11.92 ± 3.17) ng/mL; t = 10.65, P < 0.05］and CitH3 levels［(33.93 ± 18.93) ng/mL vs. (19.52 ± 13.89) ng/mL; t = 4.34, P < 0.05］were all significantly higher among HAE patients than among healthy controls, and a lower phagocytosis rate of neutrophils was detected among HAE patients than among healthy controls［(70.85 ± 7.32)% vs. (94.04 ± 3.90)%; t = 20.18, P < 0.05］, and the ability to produce NETs by neutrophils was higher among HAE patients than among healthy controls following in vitro PMA stimulation. Pearson correlation analysis showed that the phagocytosis rate of neutrophils correlated negatively with platelet-to-lymphocyte ratio (PLR), neutrophil-to-lymphocyte ratio (NLR), interleukin-6 (IL-6) level and C-reactive protein (CRP) level (rs = -0.515 to -0.392, all P values < 0.05), and the MPO and NE levels positively correlated with inflammatory markers NLR, PLR, CRP and IL-6 (rs = 0.333 to 0.445, all P values < 0.05) and clinical liver biochemical indicators aspartic transaminase, alanine aminotransferase, direct bilirubin and total bilirubin among HAE patients (rs = 0.290 to 0.628, all P values < 0.001). Excessive formation of NETs is found among HAE patients, which affects the phagocytic ability of neutrophils and results in elevated levels of inflammatory indicators. NETs markers may be promising novel biomarkers for early diagnosis, monitoring, and severity assessment of liver disease.

Impact of bronchoalveolar lavage from influenza A virus diseased pigs on neutrophil functions and growth of co-infecting pathogenic bacteria.

Influenza A viruses (IAVs) infect the respiratory tract of mainly humans, poultry, and pigs. Co-infections with pathogenic lung bacteria are a common event and contribute to the severity of disease progression. Neutrophils are a major cell type of the innate immune system and are rapidly recruited to the site of infection. They have several effector functions to fight invading pathogens such as the secretion of reactive oxygen species (ROS) or the release of neutrophil extracellular traps (NETs). NETs are known to promote the growth of Pasteurellaceae bacteria, especially if degraded by nucleases. In this study, bronchoalveolar lavage fluid (BALF) from 45 field-infected pigs was analyzed for 1) NET markers, 2) influence on growth of lung bacteria, and 3) impact on neutrophil functions. BALF samples from 21 IAV-positive pigs and 24 lung diseased but IAV-negative pigs were compared. Here, we show that neutrophils in the lungs of IAV-positive pigs release vesicular NETs. Several NET markers were increased in the BALF of IAV-positive pigs compared with the BALF from IAV-negative pigs. The amount of NET markers positively correlated with the viral load of the IAV infection. Interestingly, the BALF of IAV-positive pigs enhanced the growth of bacteria belonging to the family of Pasteurellaceae as potential coinfecting bacteria. These effects were weaker with the BALF derived from IAV-negative pigs with other lung infections. The intensity of oxidative burst in neutrophils was significantly decreased by BALF from IAVpositive pigs, indicating impaired antimicrobial activity of neutrophils. Finally, the lung milieu reflected by IAV-positive BALF does not enable neutrophils to kill Actinobacillus pleuropneumoniae but rather enhances its growth. In summary, our data show that an IAV infection is affecting neutrophil functions, in particular the release of NETs and ROS. Furthermore, IAV infection seems to provide growth-enhancing factors for especially coinfecting Pasteurellaceae and reduces the killing efficiency of neutrophils.

Neutrophils and Neutrophil Extracellular Traps Cause Vascular Occlusion and Delayed Cerebral Ischemia After Subarachnoid Hemorrhage in Mice.

After subarachnoid hemorrhage (SAH), neutrophils are deleterious and contribute to poor outcomes. Neutrophils can produce neutrophil extracellular traps (NETs) after ischemic stroke. Our hypothesis was that, after SAH, neutrophils contribute to delayed cerebral ischemia (DCI) and worse outcomes via cerebrovascular occlusion by NETs. SAH was induced via endovascular perforation, and SAH mice were given either a neutrophil-depleting antibody, a PAD4 (peptidylarginine deiminase 4) inhibitor (to prevent NETosis), DNAse-I (to degrade NETs), or a vehicle control. Mice underwent daily neurological assessment until day 7 and then euthanized for quantification of intravascular brain NETs (iNETs). Subsets of mice were used to quantify neutrophil infiltration, NETosis potential, iNETs, cerebral perfusion, and infarction. In addition, NET markers were assessed in the blood of aneurysmal SAH patients. In mice, SAH led to brain neutrophil infiltration within 24 hours, induced a pro-NETosis phenotype selectively in skull neutrophils, and caused a significant increase in iNETs by day 1, which persisted until at least day 7. Neutrophil depletion significantly reduced iNETs, improving cerebral perfusion, leading to less neurological deficits and less incidence of DCI (16% versus 51.9%). Similarly, PAD4 inhibition reduced iNETs, improved neurological outcome, and reduced incidence of DCI (5% versus 30%), whereas degrading NETs marginally improved outcomes. Patients with aneurysmal SAH who developed DCI had elevated markers of NETs compared with non-DCI patients. After SAH, skull-derived neutrophils are primed for NETosis, and there are persistent brain iNETs, which correlated with delayed deficits. The findings from this study suggest that, after SAH, neutrophils and NETosis are therapeutic targets, which can prevent vascular occlusion by NETs in the brain, thereby lessening the risk of DCI. Finally, NET markers may be biomarkers, which can predict which patients with aneurysmal SAH are at risk for developing DCI.

Abstract WMP112: Neutrophil Extracellular Traps Occlude the Cerebrovasculature Causing Delayed Cerebral Ischemia After Subarachnoid Hemorrhage in Mice

Neutrophils are reported to be critical mediators of to poor outcome after subarachnoid hemorrhage (SAH). Following ischemic stroke, neutrophils cause vascular occlusion via neutrophil extracellular traps (NETs). In this study, our hypothesis was that NETs cause vascular occlusion leading to delayed cerebral ischemia (DCI) and worse outcome after SAH. SAH mice (male and female, young and aged) were administered one of the following: a neutrophil depleting antibody, a PAD4 inhibitor (to prevent NETosis), DNAse-I (to degrade NETs), or a vehicle (control). Mice had neurological assessment daily until day 7 and then were euthanized to quantify the amount of brain vascular NETs. Various subcohorts of mice were used to assess neutrophil propensity to NETose, in vivo formation of NETs via intravital microscopy, and MRI assessment of cerebral perfusion and infarct development. We also examined if NETs were present after SAH in 128 humans by measuring NETs markers via ELISA at 1, 2, 4, 7, and 10 days post-SAH. In mice, SAH induced a pro-NETosis phenotype selectively in skull neutrophils and caused intravascular NETs to be present by day 1 which persisted until at least day 7. Neutrophil depletion reduced NETs, improved cerebral perfusion and neurological outcome, and attenuated the incidence of DCI. Similar findings were observed for PAD4 inhibition. However, degrading NETs only marginally improved outcomes. Aneurysmal SAH patients who developed DCI had elevated markers of NETs compared to non-DCI patients. In conclusion, after SAH, skull-derived neutrophils are primed for NETosis, and neutrophils and NETosis are therapeutic targets to reduce DCI by preventing vascular occlusion by NETs.

P108 Response to vedolizumab in ulcerative colitis is associated with reduced neutrophil extracellular traps formation and IL-1β production: insights from NEUTROVED study

Abstract Background Vedolizumab (VDZ) is a humanized mAb against integrin α4β7. VDZ inhibits gut inflammation by impeding intestinal T-cell homing, however, recent data suggest that effects of α4β7 blocking extend beyond T-cells to alterations in innate immunity. Neutrophils are key components of innate immune response and inflammation in ulcerative colitis (UC). Studies have provided evidence linking neutrophils with UC pathogenesis, through autophagy induction and production of IL-1β-enriched neutrophil extracellular traps (NETs). NEUTROVED aimed to investigate the effects of VDZ treatment on neutrophils that may be associated with clinical and endoscopic response. Methods Patients with moderate-to-severe UC who received VDZ in standard medical care were evaluated in two timepoints, 24 hours before treatment initiation (T1) and at week 14 of treatment (T2). Clinical/endoscopic scores and common laboratory indexes were evaluated. In parallel, RNA-sequencing in peripheral neutrophils was performed, and markers of NETs in plasma and intestinal biopsies were assessed. Results In total 5 patients completed the study until today (Table 1). In T2, 4 patients demonstrated complete remission, and one patient had mild disease. No adverse events were recorded. RNA-sequencing analysis of paired samples revealed 248 significantly downregulated and 109 upregulated genes in peripheral neutrophils after VDZ treatment. Bioinformatics analysis using the GeneCodis4 web-based tool revealed that downregulated DEGs clustered mainly in immune-related pathways, including neutrophil degranulation, Toll-like receptor cascades, signaling by interleukins, NETs formation, and HIF-1 signaling pathway, while upregulated DEGs were involved in RNA metabolism-related processes, and the ribosome biogenesis pathway. A comparative analysis between these differentially expressed genes (DEGs) and our previous RNA-sequencing data of peripheral neutrophils isolated from active UC patients (n=24) highlighted a set of 168 DEGs as restored targets after VDZ therapy. Of note, several unique genes implicated in neutrophil activation, including IL-1 signaling components, were found significantly downregulated after VDZ therapy. In line with the abovementioned transcriptome alterations, markers of NETs and IL1β-bearing NETs in plasma and colonic tissue were significantly reduced, recapitalizing clinical and endoscopic response to VDZ. Conclusion Response to VDZ treatment in UC is related to anti-inflammatory actions on neutrophils characterized by reduced NETosis and IL-1β production through NETs. Further mechanistic studies investigating the possible effects of VDZ on innate immunity and neutrophil-mediated responses are warranted.

What is the actual relationship between neutrophil extracellular traps and COVID-19 severity? A longitudinal study

BackgroundNeutrophil extracellular traps (NETs) have repeatedly been related to COVID-19 severity and mortality. However, there is no consensus on their quantification, and there are scarce data on their evolution during the disease. We studied circulating NET markers in patients with COVID-19 throughout their hospitalization.MethodsWe prospectively included 93 patients (201 blood samples), evaluating the disease severity in 3 evolutionary phases (viral, early, and late inflammation). Of these, 72 had 180 samples in various phases. We also evaluated 55 controls with similar age, sex and comorbidities. We measured 4 NET markers in serum: cfDNA, CitH3, and MPO-DNA and NE-DNA complexes; as well as neutrophil-related cytokines IL-8 and G-CSF.ResultsThe COVID-19 group had higher CitH3 (28.29 vs 20.29 pg/mL, p = 0.022), and cfDNA, MPO-DNA, and NE-DNA (7.87 vs 2.56 ng/mL; 0.80 vs 0.52 and 1.04 vs 0.72, respectively, p < 0.001 for all) than the controls throughout hospitalisation. cfDNA was the only NET marker clearly related to severity, and it remained higher in non-survivors during the 3 phases. Only cfDNA was an independent risk factor for mortality and need for intensive care. Neutrophil count, IL-8, and G-CSF were significantly related to severity. MPO-DNA and NE-DNA showed significant correlations (r: 0.483, p < 0.001), including all 3 phases and across all severity grades, and they only remained significantly higher on days 10–16 of evolution in those who died. Correlations among the other NET markers were lower than expected.ConclusionsThe circulating biomarkers of NETs were present in patients with COVID-19 throughout hospitalization. cfDNA was associated with severity and mortality, but the three other markers showed little or no association with these outcomes. Neutrophil activity and neutrophil count were also associated with severity. MPO-DNA and NE-DNA better reflected NET formation. cfDNA appeared to be more associated with overall tissue damage; previous widespread use of this marker could have overestimated the relationship between NETs and severity. Currently, there are limitations to accurate NET markers measurement that make it difficult to assess its true role in COVID-19 pathogenesis.

Pancreatectomy Induces Cancer-Promoting Neutrophil Extracellular Traps.

Neutrophil extracellular traps (NETs) occur when neutrophil chromatin is decondensed and extruded into the extracellular space in a web-like structure. Originally described as an anti-microbial function, this process has been implicated in the pathogenesis of pancreatic disease. In addition, NETs are upregulated during physiologic wound-healing and coagulation. This study evaluated how the inflammatory response to pancreatic surgery influences NET formation. For this study, 126 patients undergoing pancreatectomy gave consent before participation. Plasma was collected at several time points (preoperatively and through the postoperative outpatient visit). Plasma levels of NET markers, including cell-free DNA (cfDNA), citrullinated histone H3 (CitH3), interleukin (IL)-8, IL-6, and granulocyte colony-stimulating factor (G-CSF) were measured using enzyme-linked immunosorbent assay (ELISA). Patient clinical data were retrospectively collected from a prospectively maintained database. After pancreatic resection, NET markers (cfDNA and CitH3) were elevated, peaking on postoperative days 3 and 4. This increase in NETs was due to an inherent change in neutrophil biology. Postoperatively, NET-inducing cytokines (IL-8, IL-6, and G-CSF) were increased, peaking early in the postoperative course. The patients undergoing the robotic approach had a reduction in NETs during the postoperative period compared with those who underwent the open approach. The patients who experienced a pancreatic leak had an increase in NET markers during the postoperative period. Pancreatectomy induces cancer-promoting NET formation. The minimally invasive robotic approach may induce fewer NETs, although the current analysis was limited by selection bias. Pancreatic leak resulted in increased NETs. Further study into the potential for NET inhibition during the perioperative period is warranted.

Extracellular DNA and Markers of Neutrophil Extracellular Traps in Saliva from Patients with Periodontitis-A Case-Control Study.

Periodontitis is a chronic inflammatory disease. We have previously shown that salivary DNA is higher in patients with periodontitis. Neutrophil extracellular traps (NETs) are involved in the pathogenesis of chronic inflammatory diseases. The objective of this case-control study was to compare patients with periodontitis and healthy controls regarding the salivary concentrations of extracellular DNA and NET components. Unstimulated saliva samples were collected from 49 patients with periodontitis and 71 controls before an oral examination. Salivary extracellular DNA was isolated and quantified fluorometrically and using PCR. NET-associated markers were assessed using ELISA. We have found significantly higher concentrations of salivary extracellular DNA in samples from periodontitis patients (five-times higher for supernatant and three times for pellet). Our results show that patients also have three-times-higher salivary nucleosomes and NET-associated enzymes-myeloperoxidase and neutrophil elastase (both two-times higher). Neutrophil elastase and salivary DNA in the pellet correlated positively with the pocket depth/clinical attachment level in periodontitis patients (r = 0.31-weak correlation; p = 0.03 and r = 0.41-moderate correlation, p = 0.004). Correlations between salivary extracellular DNA and NET enzymes were positive and significant. Based on our results, the higher salivary extracellular DNA in periodontitis seems to be related to components of NETs, albeit with weak to moderate correlations indicating that NETs are produced in periodontitis and can play a role in its pathogenesis similarly to other inflammatory diseases. Further studies should prove this assumption with potential diagnostic and therapeutic consequences.

Characterization of young and aged ferrets as animal models for SARS-CoV-2 infection with focus on neutrophil extracellular traps.

Neutrophil extracellular traps (NETs) are net-like structures released by activated neutrophils upon infection [e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)] as part of the innate immune response that have protective effects by pathogen entrapment and immobilization or result in detrimental consequences for the host due to the massive release of NETs and their impaired degradation by nucleases like DNase-1. Higher amounts of NETs are associated with coronavirus disease 2019 (COVID-19) severity and are a risk factor for severe disease outcome. The objective of our study was to investigate NET formation in young versus aged ferrets to evaluate their value as translational model for SARS-CoV-2-infection and to correlate different NET markers and virological parameters. In each of the two groups (young and aged), nine female ferrets were intratracheally infected with 1 mL of 106 TCID50/mL SARS-CoV-2 (BavPat1/2020) and euthanized at 4, 7, or 21 days post-infection. Three animals per group served as negative controls. Significantly more infectious virus and viral RNA was found in the upper respiratory tract of aged ferrets. Interestingly, cell-free DNA and DNase-1 activity was generally higher in bronchoalveolar lavage fluid (BALF) but significantly lower in serum of aged compared to young ferrets. In accordance with these data, immunofluorescence microscopy revealed significantly more NETs in lungs of aged compared to young infected ferrets. The association of SARS-CoV-2-antigen in the respiratory mucosa and NET markers in the nasal conchae, but the absence of virus antigen in the lungs, confirms the nasal epithelium as the major location for virus replication as described for young ferrets. Furthermore, a strong positive correlation was found between virus shedding and cell-free DNA or the level of DNAse-1 activity in aged ferrets. Despite the increased NET formation in infected lungs of aged ferrets, the animals did not show a strong NET phenotype and correlation among tested NET markers. Therefore, ferrets are of limited use to study SARS-CoV-2 pathogenesis associated with NET formation. Nevertheless, the mild to moderate clinical signs, virus shedding pattern, and the lung pathology of aged ferrets confirm those animals as a relevant model to study age-dependent COVID-19 pathogenesis.

PCSK9-inhibitor therapy leads to changes in immune cell gene expression and function: A subset analysis of the PACMAN-AMI trial

Abstract Introduction The PACMAN-AMI trial showed a significant reduction of plaque atheroma volume by alirocumab on top of high-intensity statin therapy as compared to placebo in 265 patients undergoing serial multi-vessel imaging after acute myocardial infarction (AMI). Neutrophils and the formation of neutrophil extracellular traps (NETs) have been implicated in the development and progression of atherosclerosis. High levels of low-density lipoprotein (LDL) have been associated with neutrophil activation. Objectives This study aimed to determine the influence of aggressive LDL-lowering on neutrophil activation, and immune cell gene expression of pro-inflammatory markers in a subset of the PACMAN-AMI cohort. Methods Neutrophils and peripheral blood mononuclear cells (PBMC) of 38 patients were isolated by serial density-gradient centrifugation to assess relative gene expression of IL-1b, IL-6, TNFa, X-box binding protein (XBP) 1, caspase 1 (CASP1), and NLRP3 by quantitative PCR. Neutrophils were stimulated to undergo NET formation using a calcium ionophore in the presence or absence of pro-protein convertase subtilisin/kexin type 9 (PCSK9), detecting the release of double-stranded (ds)DNA. Plasma samples were screened for dsDNA, citrullinated histone H3, neutrophil elastase, and myeloperoxidase. Healthy controls (n=30) were recruited during routine check-ups in a health and prevention center. Results After one year of double-blind injections, the probability to undergo NET formation in vitro was still higher in AMI patients than in healthy controls. Considering randomized treatment, only patients injected with alirocumab showed significantly higher NET release than controls. Additional stimulation with the alirocumab target PCSK9 could reduce NET formation in all cohorts. This effect was significantly higher in patients treated with alirocumab. Relative gene expression of CASP1, IL-1b, IL-6, and XBP 1 was higher in patients treated with alirocumab. There was no difference in plasma NET markers comparing healthy controls and both treatment arms. Conclusion Our data indicate that PCSK9 might have effects beyond its role in cholesterol metabolism influencing immune cell activation, which could mask effects of LDL lowering. Treatment does not directly translate to higher plasma NET levels without an additional inflammatory stimulus disrupting the steady state.

Fusobacterium nucleatum-triggered neutrophil extracellular traps facilitate colorectal carcinoma progression

BackgroundFusobacterium nucleatum (Fn) acts as a procarcinogenic bacterium in colorectal carcinoma (CRC) by regulating the inflammatory tumor microenvironment (TME). Neutrophil extracellular traps (NETs), which can be generated by persistent inflammation, have been recently considered to be significant contributors in promoting cancer progression. However, whether NETs are implicated in Fn-related carcinogenesis is still poorly characterized. Here, we explored the role of NETs in Fn-related CRC as well as their potential clinical significance.MethodsFn was measured in tissue specimens and feces samples from CRC patients. The expression of NET markers were also detected in tissue specimens, freshly isolated neutrophils and blood serum from CRC patients, and the correlation of circulating NETs levels with Fn was evaluated. Cell-based experiments were conducted to investigate the mechanism by which Fn modulates NETs formation. In addition, we clarified the functional mechanism of Fn-induced NETs on the growth and metastasis of CRC in vitro and in vivo experiments.ResultsTissue and blood samples from CRC patients, particularly those from Fn-infected CRC patients, exhibited greater neutrophil infiltration and higher NETs levels. Fn infection induced abundant NETs production in in vitro studies. Subsequently, we demonstrated that Fn-induced NETs indirectly accelerated malignant tumor growth through angiopoiesis, and facilitated tumor metastasis, as manifested by epithelial-mesenchymal transition (EMT)-related cell migration, matrix metalloproteinase (MMP)-mediated basement membrane protein degradation, and trapping of CRC cells. Mechanistically, the Toll-like receptor (TLR4)-reactive oxygen species (ROS) signaling pathway and NOD-like receptor (NOD1/2)-dependent signaling were responsible for Fn-stimulated NETs formation. More importantly, circulating NETs combined with carcinoembryonic antigen (CEA) could predict CRC occurrence and metastasis, with areas under the ROC curves (AUCs) of 0.92 and 0.85, respectively.ConclusionsOur findings indicated that Fn-induced NETs abundance by activating TLR4-ROS and NOD1/2 signalings in neutrophils facilitated CRC progression. The combination of circulating NETs and CEA was identified as a novel screening strategy for predicting CRC occurrence and metastasis.

Platelets and neutrophils cooperate to induce increased neutrophil extracellular trap formation in JAK2V617F myeloproliferative neoplasms

BackgroundNeutrophils participate in the pathogenesis of thrombosis through the formation of neutrophil extracellular traps (NETs). Thrombosis is the main cause of morbidity and mortality in patients with myeloproliferative neoplasms (MPNs). Recent studies have shown an increase in NET formation (NETosis) both in patients with JAK2V617F neutrophils and in mouse models, and reported the participation of NETosis in the pathophysiology of thrombosis in mice. ObjectivesThis study investigated whether JAK2V617F neutrophils are sufficient to promote thrombosis or whether their cooperation with other blood cell types is necessary. MethodsNETosis was studied in PF4iCre;Jak2V617F/WT mice expressing JAK2V617F in all hematopoietic lineages, as occurs in MPNs, and in MRP8Cre;Jak2V617F/WT mice in which JAK2V617F is expressed only in leukocytes. ResultsIn PF4iCre;Jak2V617F/WT mice, an increase in NETosis and spontaneous lung thrombosis abrogated by DNAse administration were observed. The absence of spontaneous NETosis or lung thrombosis in MRP8Cre;Jak2V617F/WT mice suggested that mutated neutrophils alone are not sufficient to induce thrombosis. Ex vivo experiments demonstrated that JAK2V617F-mutated platelets trigger NETosis by JAK2V617F-mutated neutrophils. Aspirin treatment in PF4iCre;Jak2V617F/WT mice reduced NETosis and reduced lung thrombosis. In cytoreductive-therapy-free patients with MPN treated with aspirin, plasma NET marker concentrations were lower than that in patients with MPN not treated with aspirin. ConclusionOur study demonstrates that JAK2V617F neutrophils alone are not sufficient to promote thrombosis; rather, platelets cooperate with neutrophils to promote NETosis in vivo. A new role for aspirin in thrombosis prevention in MPNs was also identified.