Citrullinated Histone H3 Research Articles

Placental neutrophil reverse trans-migration and maternal serum neutrophil extracellular trap expression in HIV infection co-morbid pre-eclampsia in women of African ancestry

Neutrophil extracellular traps (NETs) and placental neutrophil reverse transmigration (r-TM) are implicated in the pathogenesis of pre-eclampsia (PE). However, the role of the comorbidity of PE and human immunodeficiency virus (HIV) infection in placental neutrophil r-TM and serum NETs remains unknown. Human placental tissue (n = 160) and serum (n = 80) samples were obtained post-ethical approval and divided by pregnancy type and HIV status and across the study population. Immunohistochemistry and morphometry were performed to localize and quantify junctional adhesion molecule-C (JAM-C) expression as an inverse marker of neutrophil r-TM within placental villi. An enzyme-linked immunosorbent assay (ELISA) was performed to quantify the concentration of citrullinated histone H3 (cit-H3) as a marker of NETs. GraphPad Prism (version 8.0.2) was used to compare the results, and a p value of p < 0.05 was considered statistically significant. The localization of JAM-C was observed on the syncytiotrophoblasts (STBs) and endothelial cells of placental villi. The immunoexpression of JAM-C was elevated in PE vs. normotensive (N) placentae. In the exchange villi, JAM-C immunoexpression was higher in the N+ve vs. N-ve group. However, in PE comorbid HIV infection, JAM-C expression was lower in the PE+ve vs. PE-ve group. Citrullinated histone-H3 concentration was lower in the N+ve vs. N-ve group but elevated in early-onset PE (EOPE)+ve vs. late-onset PE (LOPE)+ve group. These results indicate that PE and HIV-infected placentae individually express elevated JAM-C, manifesting in less neutrophil r-TM. However, in exchange villi of PE comorbid with HIV infection reduced JAM-C enhances neutrophil r-TM, thus supporting the synergistic effect of PE comorbid with HIV.

Xuefu Zhuyu decoction alleviates deep vein thrombosis through inhibiting the activation of platelets and neutrophils via sirtuin 1/nuclear factor kappa-B pathway

Ethnopharmacological relevanceXuefu Zhuyu Decoction (XZD), a renowned traditional Chinese medicine prescription, is widely employed for the management of conditions characterized by qi-stagnation and blood stasis. Although its anti-thrombotic effect on deep vein thrombosis (DVT) patients has been clinically observed, the underlying mechanism remains largely unexplored. Aim of the studyOur aim was to investigate the mechanisms by which XZD exerted its effect on DVT. Materials and methodsThe ultra performance liquid chromatography (UPLC) technique was employed to evaluate quality of XZD. To examine the effect of XZD on DVT, a DVT rat model with inferior vena cava (IVC) stenosis was established. The 4D-label-free proteomics approach was then utilized to uncover the possible mechanisms of XZD against DVT. Based on proteomics, citrullinated histone H3 (CitH3), along with serum levels of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) were observed the inhibitory activity of XZD on neutrophil activation. Subsequently, the marker of platelet activation, specifically glycoprotein IIb (CD41) and glycoprotein IIIa (CD61), were assessed along with the secretion of von Willebrand factor (vWF) to investigate the inhibitory activity of XZD on platelet activation. Finally, we explored the impact of XZD on the sirtuin 1 (SIRT1)/nuclear factor kappa-B (NF-κB) pathway, which was associated with the activation of platelets and neutrophils. ResultsEight distinct components were identified for the quality control of XZD. XZD effectively reduced thrombus weight and length in DVT rats, without affecting the coagulation function or hematological parameters in the systemic circulation. Proteomics analysis revealed that XZD alleviated DVT by inhibiting the activation of platelets and neutrophils. The protein expression of CitH3, along with serum levels of TNF-α and IL-1β, were reduced in XZD-treated DVT rats. Similarly, protein expressions of CD41 and CD61, along with the release of vWF, were markedly down-regulated in XZD-treated DVT rats. Finally, treatment with XZD resulted in an up-regulation of SIRT1 protein expression and a down-regulation of both acetylated NF-κB/p65 and phosphorylated NF-κB/p65 protein expressions in endothelium. ConclusionsXZD alleviates DVT by inhibiting the activation of platelets and neutrophils at the injured endothelium via the regulation of SIRT1/NF-κB pathway.

A New Approach for Assessment of Neutrophil Extracellular Traps Through Immunofluorescence Staining in Whole Blood Smears.

Neutrophils, constituting 50%-70% of circulating leukocytes, play crucial roles in host defense and exhibit anti-tumorigenic properties. An elevated peripheral blood neutrophil-to-lymphocyte ratio is associated with decreased survival rates in cancer patients. In response to exposure to various antigens, neutrophils release neutrophil granular proteins, which combine to form web-like structures known as neutrophil extracellular traps (NETs). Previously, the relative percentage of NETs was found to be increased in resected tumor tissue samples from patients with gastrointestinal malignancies. The presence of NETs in peripheral blood is indicative of underlying pathological conditions. Hence, employing a non-invasive method to detect NETs in peripheral blood, along with other diagnostic tests, shows potential as a valuable tool not just for identifying different inflammatory disorders but also for assessing disease severity and determining patient suitability for surgical resection. While reliable methods exist for identifying NETs in tissue, accurately quantifying them in whole blood remains challenging. Many previous methods are time-consuming and rely on a limited set of markers that are inadequate for fully characterizing NETs. Therefore, we established a unique sensitive smear immunofluorescence assay based on blood smears to identify NETs in only as little as 2 μL of whole blood. To identify the NET complexes that have enhanced specificities, this combines the use of various antibodies against neutrophil-specific CD15, NET-specific myeloperoxidase (MPO), citrullinated histone H3 (Cit H3), and nuclear DNA. This protocol offers an easy, affordable, rapid, and non-invasive method for identifying NETs; thus, it can be utilized as a diagnostic marker and targeted through various therapeutic approaches for treating human malignancies. Key features • Characterization of neutrophil extracellular traps in whole blood smears through immunofluorescence staining. • Affordable and quantitative approach to neutrophil extracellular trap detection.

Complement activation is associated with right ventricular dysfunction and the severity of pulmonary embolism: links with prothrombotic state.

Little is known about the role of complement activation in acute pulmonary embolism (PE). We investigated whether complement activation is associated with the severity of acute PE, along with the associated prothrombotic state, systemic inflammation and neutrophil extracellular traps (NETs) formation. We studied 109 normotensive, non-cancer PE patients (aged 58.1±15.0 years). On admission prior to initiation of anticoagulation, plasma soluble complement components, i.e., C3a and sC5b-9, were measured with enzyme-linked immunosorbent assay (ELISA), along with thrombin generation, fibrinolysis proteins (plasminogen, antiplasmin, plasminogen activator inhibitor-1), factor VIII (FVIII) activity, and fibrin clot properties, including clot permeability (Ks, a measure of clot density) and clot lysis time (CLT). Moreover, we determined inflammatory markers and citrullinated histone H3, a specific marker of NETs formation. Patients in the lower tertile of C3a (≤1.45 ng/mL, n=37) had lower simplified Pulmonary Embolism Severity Index (sPESI) values and were less likely to have right ventricular (RV) dysfunction compared to the remaining subjects. The former subgroup also had 13% lower FVIII activity, but not fibrinogen, interleukin-6, fibrinolysis proteins, and thrombin generation. Plasma C3a levels correlated inversely with Ks and positively with CLT indicating formation of denser and poorly lysable clots in subjects with elevated C3a. Despite a positive association between C3a and sC5b-9, the latter parameter was solely associated with higher FVIII, but not with other variables. We showed that in acute PE enhanced complement activation characterizes patients with poorer short-term prognosis who display prothrombotic fibrin clot properties and elevated FVIII, which supports the involvement of complement proteins in acute thromboembolism.

Da-yuan-yin decoction alleviates ulcerative colitis by inhibiting complement activation, LPS-TLR4/NF-κB signaling pathway and NET formation

Ethnopharmacological relevanceDa-yuan-yin decoction (DYY) is a classical traditional Chinese medicine prescription for ulcerative colitis (UC). Aim of studyThis study explored the protective effects and mechanisms of DYY on UC. Materials and methodsThe mice were fed 2.5% dextran sulfate sodium (DSS) for 7 days to establish UC. On the second day, DYY (0.4 g/kg, 0.8 g/kg, 1.6 g/kg) was orally administered daily for 7 consecutive days. The colon tissues and serum were measured by histopathological examination and biochemical analysis. ResultsDYY significantly reduced the disease activity index (DAI) and severity of colon shortening and alleviated pathological changes in the colon tissue. DYY restored the protein expression of intestinal tight junction (TJ) protein (ZO-1, occludin and claudin-3). DYY remarkably decreased the level of lipopolysaccharide (LPS), Lactic acid (LA), circulating free DNA (cfDNA), complement (C3, C3a, C3c, C3aR1, C5a and C5aR1) and regulated the levels of inflammatory cytokines in serum. DYY significantly inhibited the expressions of nuclear factor kappa-B p65 (NF-κB p65) and Toll-like receptor 4 (TLR4), citrullinated histone H3 (CitH3) and myeloperoxidase (MPO), reactive oxygen species (ROS) peptidylarginine deiminase 4 (PAD4) and CD 11b, the mRNA levels of PADI4, MPO and ELANE in colon tissues. ConclusionsDYY significantly attenuated DSS-induced UC, which was related with regulating the inflammatory response by the inhibition of complement activation, the LPS-TLR4/NF-κB signaling pathway and neutrophil extracellular traps (NETs) formation. DYY is a potential therapeutic agent for UC.

Astilbin inhibited neutrophil extracellular traps in gouty arthritis through suppression of purinergic P2Y6 receptor

BackgroundGouty arthritis (GA), a common inflammatory condition triggered by monosodium urate crystal accumulation, often necessitates safer treatment alternatives due to the limitations of current therapies. Astilbin, a flavonoid from Smilax glabra Roxb, has demonstrated potential in traditional Chinese medicine for its anti-inflammatory properties. However, the anti-GA effect and its underlying mechanism have not been fully elucidated. PurposeThis study aimed to investigate the therapeutic potential of astilbin in GA, focusing on its effects on neutrophil extracellular traps (NETs), as well as the potential molecular target of GA both in vitro and in vivo. Study designFirstly, astilbin inhibited the citrullinated histone H3 (Cit h3) protein levels and reduced the NETs formation in neutrophils stimulated by monosodium urate (MSU). Secondly, we wondered the effect of astilbin on migration of neutrophils and dimethyl-sulfoxide (DMSO)-differentiated HL-60 (dHL-60) cells under the stimulation of MSU. Then, the effect of astilbin on suppressing NETs through purinergic P2Y6 receptor (P2Y6R) and Interlukin-8 (IL-8)/ CXC chemokine receptor 2 (CXCR2) pathway was investigated. Also, the relationship between P2Y6R and IL-8/CXCR2 was explored in dHL-60 cells under stimulation of MSU. Finally, we testified the effect of astilbin on reducing NETs in GA through suppressing P2Y6R and then down-regulating IL-8/CXCR2 pathway. MethodsMSU was used to induce NETs in neutrophils and dHL-60 cells. Real-time formation of NETs and migration of neutrophils were monitored by cell living imaging with or without MSU. Then, the effect of astilbin on NETs formation, P2Y6R and IL-8/CXCR2 pathway were detected by immunofluorescence (IF) and western blotting. P2Y6R knockdown dHL-60 cells were established by small interfering RNA to investigate the association between P2Y6R and IL-8/CXCR2 pathway. Also, plasmid of P2Y6R was used to overexpress P2Y6R in dHL-60 cells, which was employed to explore the role of P2Y6R in astilbin inhibiting NETs. Within the conditions of knockdown and overexpression of P2Y6R, migration and NETs formation were assessed by transmigration assay and IF staining, respectively. In vivo, MSU-induced GA mice model was established to assess the effect of astilbin on inflammation by haematoxylin-eosin and ELISA. Additionally, the effects of astilbin on neutrophils infiltration, NETs, P2Y6R and IL-8/CXCR2 pathway were analyzed by IF, ELISA, immunohistochemistry (IHC) and western blotting. ResultsUnder MSU stimulation, astilbin significantly suppressed the level of Cit h3 and NETs formation including the fluorescent expressions of Cit h3, neutrophils elastase, myeloperoxidase, and intra/extracellular DNA. Also, results showed that MSU caused NETs release in neutrophils as well as a trend towards recruitment of dHL-60 cells to MSU. Astilbin could markedly decrease expressions of P2Y6R and IL-8/CXCR2 pathway which were upregulated by MSU. By silencing P2Y6R, the expression of IL-8/CXCR2 pathway and migration of dHL-60 cells were inhibited, leading to the suppression of NETs. These findings indicated the upstream role of P2Y6R in the IL-8/CXCR2 pathway. Moreover, overexpression of P2Y6R was evidently inhibited by astilbin, causing a downregulation in IL-8/CXCR2 pathway, migration of dHL-60 cells and NETs formation. These results emphasized that astilbin inhibited the IL-8/CXCR2 pathway primarily through P2Y6R. In vivo, astilbin administration led to marked reductions in ankle swelling, inflammatory infiltration as well as neutrophils infiltration. Expressions of P2Y6R and IL-8/CXCR2 pathway were evidently decreased by astilbin and P2Y6R inhibitor MRS2578 either alone or in combination. Also, astilbin and MRS2578 showed notable effect on reducing MSU-induced NETs formation and IL-8/CXCR2 pathway whether used alone or in combination, parallelly demonstrating that astilbin decreased NETs formation mainly through P2Y6R. ConclusionThis study revealed that astilbin suppressed NETs formation via downregulating P2Y6R and subsequently the IL-8/CXCR2 pathway, which evidently mitigated GA induced by MSU. It also highlighted the potential of astilbin as a promising natural therapeutic for GA.

Berbamine ameliorates DSS-induced colitis by inhibiting peptidyl-arginine deiminase 4-dependent neutrophil extracellular traps formation

Ulcerative colitis (UC) is a chronic inflammatory bowel disease with immune dysregulation affecting colon inflammatory response. Recent studies have highlighted that neutrophil extracellular traps (NETs) play an important role in the pathogenesis of UC. Berbamine (BBM), one of the bioactive ingredients extracted from Chinese herbal medicine Berberis vulgaris L, has attracted intensive attentions due to its significant anti-inflammatory activity and a marketing drug for treating leukemia in China. However, the exact role and potential molecular mechanism of BBM against UC remains elusive. In the present study, our results showed that BBM could markedly improve the pathological phenotype and the colon inflammation in mice with dextran sulfate sodium (DSS)-induced colitis. Then, comprehensive approaches combining network pharmacology and molecular docking analyses were employed to predict the therapeutic potential of BBM in treating UC by peptidyl-arginine deiminase 4 (PAD4), a crucial molecule involved in NETs formation. The molecular docking results showed BBM had a high affinity for PAD4 with a binding energy of −9.3 kcal/mol Moreover, PAD4 expression and NETs productions, including citrullination of histone H3 (Cit-H3), neutrophil elastase (NE), myeloperoxidase (MPO) in both neutrophils and colonic tissue were reduced after BBM administration. However, in the mice with DSS-induced colitis pretreated with GSK484, a PAD4-specific inhibitor, BBM could not further reduce disease related indexes, expression of PAD4 and NETs productions. Above all, the identification of PAD4 as a potential target for BBM to inhibit NETs formation in colitis provides novel insights into the development of BBM-derived drugs for the clinical management of UC.

Optimization and verification of conditions for induction of neutrophil extracellular traps in vitro

This study aims to optimize the conditions for the formation of neutrophil extracellular traps(NETs) in vitro, so as to establish a relatively stable experimental research platform. Different conditions were compared, including commonly used laboratory animals(rats and mice) and a variety of cell sources(bone marrow neutrophils and peripheral blood neutrophils separated by percoll density gradient centrifugation). Different inducers like lipopolysaccharide(LPS) and phorbol 12-myristate 13-acetate(PMA) were used for induction in vitro. Myeloperoxidase(MPO)/citrullinated histone H3(CitH3)/DAPI immunofluorescence and cell free DNA(cf-DNA) content determination were used for comprehensive evaluation to screen the optimal conditions for the formation of NETs induced in vitro. Furthermore, the stability of the selected conditions for inducing the formation of NETs in vitro was evaluated by tetramethylpyrazine(TMP), an active component in Chinese herbal medicines. The results showed that coated poly-D-lysine(PDL) induced the formation of NETs in bone marrow neutrophils of mice to a certain extent. Both LPS and PMA significantly up-regulated the protein levels of MPO and CitH3 in mouse bone marrow neutrophils and elevated the cfDNA level in the supernatant of rat peripheral blood neutrophils. The cfDNA level in the PMA-induced group increased more significantly than that in the LPS-induced group(P&lt;0.05). The results of immunofluorescence staining showed that the expression of MPO and CitH3 in mouse bone marrow neutrophils, rat bone marrow neutrophils, and rat peripheral blood neutrophils were significantly increased after PMA induction, especially in rat peripheral blood neutrophils. TMP significantly down-regulated the protein levels of MPO, CitH3, and neutrophil elastase(NE) in rat peripheral blood neutrophils induced by PMA. In conclusion, treating the peripheral blood neutrophils of rats with PMA is the optimal condition for inducing the formation of NETs in vitro. This study provides an optimal platform for in vitro studies based on NETs and a basis for studying the effects of traditional Chinese medicines targeting NETs.

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.

Extracellular histones: a unifying mechanism driving platelet-dependent extracellular vesicle release and thrombus formation in COVID-19

BackgroundCOVID-19 can cause profound inflammation and coagulopathy, and while many mechanisms have been proposed, there is no known common pathway leading to a prothrombotic state. ObjectivesFrom the beginning of the COVID-19 pandemic, elevated levels of extracellular histones have been found in plasma of patients infected with SARS-CoV-2. We hypothesized that platelet activation triggered by extracellular histones might represent a unifying mechanism leading to increased thrombin generation and thrombosis. MethodsWe utilized blood samples collected from an early clinical trial of hospitalized COVID-19 patients (NCT04360824) and recruited healthy subjects as controls. Using plasma samples, we measured the procoagulant and prothrombotic potential of circulating extracellular histones and extracellular vesicles (EVs). Platelet prothrombotic activity was assessed via thrombin generation potential and platelet thrombus growth. Circulating EVs were assessed for thrombin generation potential in vitro in plasma and enhancement of thrombotic susceptibility in vivo in mice. ResultsCompared with controls, COVID-19 patients had elevated plasma levels of citrullinated histone H3, cell-free DNA, nucleosomes, and EVs. Plasma from COVID-19 patients promoted platelet activation, platelet-dependent thrombin generation, thrombus growth under venous shear stress, and release of platelet-derived EVs. These prothrombotic effects of COVID-19 plasma were inhibited by an RNA aptamer that neutralizes both free and DNA-bound histones. EVs isolated from COVID-19 plasma enhanced thrombin generation in vitro and potentiated venous thrombosis in mice in vivo. ConclusionWe conclude that extracellular histones and procoagulant EVs drive the prothrombotic state in COVID-19 and that histone-targeted therapy may prove beneficial.

Lidocaine effects on neutrophil extracellular trapping and angiogenesis biomarkers in postoperative breast cancer patients with different anesthesia methods: a prospective, randomized trial

BackgroundAnesthesia techniques and drug selection may influence tumor recurrence and metastasis. Neutrophil extracellular trapping (NETosis), an immunological process, has been linked to an increased susceptibility to metastasis in individuals with tumors. Furthermore, recurrence may be associated with vascular endothelial growth factor A (VEGF-A), a mediator of angiogenesis. This study investigates the impact of lidocaine (combined with sevoflurane or propofol anesthesia ) during breast cancer surgery inhibits the expression of biomarkers associated with metastasis and recurrence (specifically H3Cit, NE, MPO, MMP-9 and VEGF-A).MethodsWe randomly assigned 120 women undergoing primary or invasive breast tumor resection to receive one of four anesthetics: sevoflurane (S), sevoflurane plus i.v. lidocaine (SL), propofol (P), and propofol plus i.v. lidocaine (PL). Blood samples were collected before induction and 3 h after the operation. Biomarkers associated with NETosis (citrullinated histone H3 [H3Cit], myeloperoxidase [MPO], and neutrophil elastase [NE]) and angiogenesis were quantified using enzyme-linked immunosorbent assays.ResultsPatient and breast tumor characteristics, along with perioperative management, did not differ between study groups. In intra-group comparisons, S and P groups demonstrated a statistically significant increase in post-operative MPO (S group: 10.39[6.89–17.22] vs. 14.31[8.55–20.87] ng ml-1, P = 0.032; P group: 9.45[6.73–17.37] vs. 14.34[9.87–19.75] ng ml-1, P = 0.035)and NE(S group: 182.70[85.66-285.85] vs. 226.20[91.85-391.65] ng ml-1, P = 0.045; P group: 154.22[97.31–325.30] vs. 308.66[132.36-483.57] ng ml-1, P = 0.037) concentrations compared to pre-operative measurements, whereas SL and PL groups did not display a similar increase. H3Cit, MMP-9, and VEGF-A concentrations were not significantly influenced by the anesthesia techniques and drugs.ConclusionsRegardless of the specific technique employed for general anesthesia, there was no increase in the postoperative serum concentrations of MPO and NE after perioperative lidocaine infusion compared to preoperative serum concentrations. This supports the hypothesis that intravenous lidocaine during cancer surgery aimed at achieving a cure may potentially decrease the likelihood of recurrence. Further interpretation and discussion of clinical implications are warranted, emphasizing the significance of these findings in the context of cancer surgery and recurrence prevention.Clinical trial registrationChiCTR2300068563.

NETosis Drives Blood Pressure Elevation and Vascular Dysfunction in Hypertension.

Neutrophil extracellular traps (NETs) are composed of DNA, enzymes, and citrullinated histones that are expelled by neutrophils in the process of NETosis. NETs accumulate in the aorta and kidneys in hypertension. PAD4 (protein-arginine deiminase-4) is a calcium-dependent enzyme that is essential for NETosis. TRPV4 (transient receptor potential cation channel subfamily V member 4) is a mechanosensitive calcium channel expressed in neutrophils. Thus, we hypothesize that NETosis contributes to hypertension via NET-mediated endothelial cell (EC) dysfunction. NETosis-deficient Padi4-/- mice were treated with Ang II (angiotensin II). Blood pressure was measured by radiotelemetry, and vascular reactivity was measured with wire myography. Neutrophils were cultured with or without ECs and exposed to normotensive or hypertensive uniaxial stretch. NETosis was measured by flow cytometry. ECs were treated with citrullinated histone H3, and gene expression was measured by quantitative reverse transcription PCR. Aortic rings were incubated with citrullinated histone H3, and wire myography was performed to evaluate EC function. Neutrophils were treated with the TRPV4 agonist GSK1016790A. Calcium influx was measured using Fluo-4 dye, and NETosis was measured by immunofluorescence. Padi4-/- mice exhibited attenuated hypertension, reduced aortic inflammation, and improved EC-dependent vascular relaxation in response to Ang II. Coculture of neutrophils with ECs and exposure to hypertensive uniaxial stretch increased NETosis and accumulation of neutrophil citrullinated histone H3. Histone H3 and citrullinated histone H3 exposure attenuates EC-dependent vascular relaxation. Treatment of neutrophils with the TRPV4 agonist GSK1016790A increases intracellular calcium and NETosis. These observations identify a role of NETosis in the pathogenesis of hypertension. Moreover, they define an important role of EC stretch and TRPV4 as initiators of NETosis. Finally, they define a role of citrullinated histones as drivers of EC dysfunction in hypertension.

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

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

Neutrophil extracellular trap formation and its implications in nonsteroidal anti-inflammatory drug-induced small intestinal injury.

Nonsteroidal anti-inflammatory drugs (NSAIDs) damage the small intestine via neutrophil infiltration driven by the mucosal invasion of enterobacteria. The antimicrobial function of neutrophils is partially dependent on neutrophil extracellular traps (NETs). Excessive NET formation has been associated with several inflammatory diseases. Here, we aimed to investigate the role of NETs in NSAID-induced small intestinal damage using human samples and an experimental mouse model. Human small intestine specimens were obtained from NSAID users during double-balloon enteroscopy. Wild-type, protein arginine deiminase 4 (PAD4) knockout, and antibiotic-treated mice were administered indomethacin to induce small intestinal injury. The expression of NET-associated proteins, including PAD4, citrullinated histone H3 (CitH3), cell-free DNA, and myeloperoxidase (MPO), was evaluated. The double-positive stained area with CitH3 and MPO, which is specific for neutrophil-derived extracellular traps, was significantly high in the injured small intestinal mucosa of NSAID users. In a mouse model, small intestinal damage developed at 6h after indomethacin administration, accompanied by increased mRNA levels of interleukin-1β and keratinocyte chemoattractant and elevated NET-associated protein levels of PAD4, CitH3, and MPO in small intestine and serum levels of cell-free DNA. Both genetic deletion and pharmacological inhibition of PAD4 attenuated this damage by reducing the mRNA expression of inflammatory cytokines and NET-associated proteins. Furthermore, mice pretreated with antibiotics showed resistance to indomethacin-induced small intestinal damage, with less NET formation. These results suggest that NETs aggravate NSAID-induced small intestinal injury. Therefore, NET inhibition could be a potential treatment for NSAID-induced small intestinal injury.

Interactions between integrin α9β1 and VCAM-1 promote neutrophil hyperactivation and mediate poststroke DVT

AbstractVenous thromboembolic events are significant contributors to morbidity and mortality in patients with stroke. Neutrophils are among the first cells in the blood to respond to stroke and are known to promote deep vein thrombosis (DVT). Integrin α9 is a transmembrane glycoprotein highly expressed on neutrophils and stabilizes neutrophil adhesion to activated endothelium via vascular cell adhesion molecule 1 (VCAM-1). Nevertheless, the causative role of neutrophil integrin α9 in poststroke DVT remains unknown. Here, we found higher neutrophil integrin α9 and plasma VCAM-1 levels in humans and mice with stroke. Using mice with embolic stroke, we observed enhanced DVT severity in a novel model of poststroke DVT. Neutrophil-specific integrin α9–deficient mice (α9fl/flMrp8Cre+/−) exhibited a significant reduction in poststroke DVT severity along with decreased neutrophils and citrullinated histone H3 in thrombi. Unbiased transcriptomics indicated that α9/VCAM-1 interactions induced pathways related to neutrophil inflammation, exocytosis, NF-κB signaling, and chemotaxis. Mechanistic studies revealed that integrin α9/VCAM-1 interactions mediate neutrophil adhesion at the venous shear rate, promote neutrophil hyperactivation, increase phosphorylation of extracellular signal-regulated kinase, and induce endothelial cell apoptosis. Using pharmacogenomic profiling, virtual screening, and in vitro assays, we identified macitentan as a potent inhibitor of integrin α9/VCAM-1 interactions and neutrophil adhesion to activated endothelial cells. Macitentan reduced DVT severity in control mice with and without stroke, but not in α9fl/flMrp8Cre+/− mice, suggesting that macitentan improves DVT outcomes by inhibiting neutrophil integrin α9. Collectively, we uncovered a previously unrecognized and critical pathway involving the α9/VCAM-1 axis in neutrophil hyperactivation and DVT.

Abstract 1392: Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling

Abstract Neutrophil extracellular traps (NETs) are an important contributor to the immunosuppressive tumor microenvironment and facilitate metastasis of solid cancers. NETs have been shown to stimulate breast and colon cancer cell metastasis through a mechanism potentially involving an interaction between integrin-linked kinase (ILK) and the cell surface receptor coiled-coil domain containing protein 25 (CCDC25). Here, we have interrogated whether ILK is required for NET-induced metastasis in pancreatic ductal adenocarcinoma (PDAC), an inflammation-driven tumor that is difficult to treat. Evaluation of the circulating neutrophil to lymphocyte ratio (NLR) in the blood samples of patients presenting with metastatic PDAC showed that a higher baseline NLR is associated with significantly shorter overall patient survival. Western blot and immunofluorescence analyses demonstrated that human PDAC cell lines, patient-derived xenograft cells, cells derived from the KPCY genetic model of PDAC and patient-derived organoids co-express CCDC25 and ILK. Co-immunoprecipitation analyses showed that CCDC25 and ILK associate in both mouse and human PDAC cells, and that exposure of the cells to NETs results in an increased amount of ILK associated with CCDC25. Exposure of human and KPCY mouse PDAC cells to NETs increases phosphorylation of GSK3-β and ERK, increases activation of Rac1 and Cdc42 and upregulates markers of epithelial-to-mesenchymal transition (EMT), including ZEB1 and Snail, in a time and concentration-dependent manner. Furthermore, time-lapse imaging using Incucyte-based wounding assays showed that exposure to NETs induces pseudopodia-like protrusions, migration and invasion by these cells. RNAi-mediated suppression of ILK expression or inhibition of ILK activity using a highly selective inhibitor of ILK, QLT-0267, significantly inhibits the NET-induced increase in GSK3-β phosphorylation, Rac1/Cdc42 activation, migration and invasion of PDAC cells. In vivo, co-localization of CCDC25 and ILK is observed in tumors and metastases from KPCY mouse and MIA PaCa-2 human PDAC xenograft models stained by multispectral immunofluorescence and analyzed by confocal microscopy. Tumors and metastases show the presence of infiltrating neutrophils and NETs as indicated by staining for myeloperoxidase and citrullinated histone H3. Importantly, doxycycline-inducible shRNA-mediated suppression of ILK expression by luciferase-positive PDAC cells following injection of these cells through the tail vein of mice administered LPS intranasally results in a significant reduction in metastasis as measured by bioluminescence-based imaging, compared to control animals. Collectively, these data identify NET-induced ILK signaling as an important contributor to PDAC progression and suggest the potential for targeting this axis to prevent PDAC progression and metastasis. Citation Format: Paul C. McDonald, Shannon J. Awrey, Hossein Tavakoli, Rebekah Carroll, Zachary J. Gerbec, Joanna M. Karasinska, David F. Schaeffer, Daniel J. Renouf, Shoukat Dedhar. Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling [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 1392.

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.

Low-level ambient ozone exposure associated with neutrophil extracellular traps and pro-atherothrombotic biomarkers in healthy adults

Background and aimsUncertainty of the causality determinations for ambient ozone (O3) on cardiovascular events is heightened by the limited understanding of the mechanisms involved in humans. We aimed to examine the pro-atherothrombotic impacts of O3 exposure and to explore the potential mediating roles of dysfunctional neutrophils, focusing on neutrophil extracellular traps (NETs). MethodsA longitudinal panel study of 152 healthy adults was conducted in the cool to cold months with relatively low levels of O3 between September 2019 and January 2020 in Beijing, China. Four repeated measurements of indicators reflecting atherothrombotic balance and NETs were performed for each participant. ResultsDaily average exposure levels of ambient O3 were 16.6 μg/m3 throughout the study period. Per interquartile range increase in average concentrations of O3 exposure at prior up to 7 days, we observed elevations of 200.1–276.3% in D-dimer, 27.2–36.8% in thrombin-antithrombin complex, 10.8–60.3% in plasminogen activator inhibitor 1, 13.9–21.8% in soluble P-selectin, 16.5–45.1% in matrix metalloproteinase-8, and 2.4–12.4% in lipoprotein-associated phospholipase A2. These pro-atherothrombotic changes were accompanied by endothelial activation, lung injury, and immune inflammation. O3 exposure was also positively associated with circulating NETs indicators, including citrullinated histone H3, neutrophil elastase, myeloperoxidase, and double-stranded DNA. Mediation analyses indicated that NETs could mediate O3-associated pro-atherothrombotic responses. The observational associations remained significant and robust after controlling for other pollutants, and were generally greater in participants with low levels of physical activity. ConclusionsAmbient O3 exposure was associated with significant increases in NETs and pro-atherothrombotic potential, even at exposure levels well below current air quality guidelines of the World Health Organization.

Peptidylarginine Deiminases Inhibitors Decrease Endothelial Cells Angiogenic Potential by Affecting Akt Signaling and the Expression and Secretion of Angiogenic Factors.

Endothelial cells (ECs) play a crucial role in various physiological processes, particularly those related to the cardiovascular system, but also those affecting the entire organism. The biology of ECs is regulated by multiple biochemical stimuli and epigenetic drivers that govern gene expression. We investigated the angiogenic potential of ECs from a protein citrullination perspective, regulated by peptidyl-arginine deiminases (PADs) that modify histone and non-histone proteins. Although the involvement of PADs has been demonstrated in several physiological processes, inflammation-related disorders and cancer, their role in angiogenesis remains unclear. To elucidate the role of PADs in endothelial angiogenesis, we used two human EC models: primary vein (HUVECs) and microvascular endothelial cells (HMEC-1). PADs activity was inhibited using irreversible inhibitors: BB-Cl-amidine, Cl-amidine and F-amidine. We analyzed all three steps of angiogenesis in vitro : proliferation, migration, and capillary-like tube formation, as well as secretory activities, gene expression and signaling in ECs. All used PAD inhibitors reduced the histone H3 citrullination (H3cit) mark, inhibited endothelial cell migration and capillary-like tube formation, and favored an angiostatic activity in HMEC-1 cells, by increasing PEDF (pigment epithelium-derived factor) and reducing VEGF (vascular endothelial growth factor) mRNA expression and protein secretion. Additionally, BB-Cl-amidine reduced the total activity of MMPs (Matrix metalloproteinases). The observed effects were underlined by the inhibition of Akt phosphorylation.>. Our findings suggest that pharmacological inhibitors of citrullination are promising therapeutic agents to target angiogenesis.

Vitamin B12 inhibits peptidylarginine deiminases and ameliorates rheumatoid arthritis in CAIA mice

Rheumatoid arthritis is an autoimmune disease whose early onset correlates with dysregulated citrullination, a process catalyzed by peptidylarginine deiminase isoform 4 (PADI-4). Here, we report that PADI-4 is a novel target of vitamin B12, a water-soluble vitamin that serves as a cofactor in DNA synthesis and the metabolism of fatty and amino acids. Vitamin B12 preferentially inhibited PADI-4 over PADI-2 with comparable inhibitory activity to the reference compound Cl-amidine in enzymatic inhibition assays, and reduced total cellular citrullination levels including that of histone H3 citrullination mediated by PADI-4. We also demonstrated that hydroxocobalamin, a manufactured form of vitamin B12, significantly ameliorated the severity of collagen type II antibody induced arthritis (CAIA) in mice and diminished gene expression of the rheumatoid inflammatory factors and cytokines IL17A, TNFα, IL-6, COX-II and ANXA2, as well PADI-4. Therefore, the use of vitamin B12 to treat rheumatoid arthritis merits further study.