Neutrophil Extracellular Traps Research Articles

Increased neutrophil extracellular traps caused by diet-induced obesity delay fracture healing.

Obesity, recognized as a risk factor for nonunion, detrimentally impacts bone health, with significant physical and economic repercussions for affected individuals. Nevertheless, the precise pathomechanisms by which obesity impairs fracture healing remain insufficiently understood. Multiple studies have identified neutrophil granulocytes as key players in the systemic immune response, being the predominant immune cells in early fracture hematomas. This study identified a previously unreported critical period for neutrophil infiltration into the callus. Invivo experiments demonstrated that diet-induced obesity (DIO) mice showed earlier neutrophil infiltration, along with increased formation of neutrophil extracellular traps (NETs), compared to control mice during the endochondral phase of fracture repair. Furthermore, Padi4 knockout was found to reduce NET formation and mitigate the fracture healing delays caused by high-fat diets. Mechanistically, invitro analyses revealed that NETs, by activating NLRP3 inflammasomes, inhibited the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and concurrently promoted M1-like macrophage polarization. These findings establish a connection between NET formation during the endochondral phase and delayed fracture healing, suggesting that targeting NETs could serve as a promising therapeutic approach for addressing obesity-induced delays in fracture recovery.

Neutrophil extracellular traps, deoxyribonuclease and fibrosis in thoracic aortic aneurysm

Abstract Introduction Thoracic aortic aneurysm (TAA) is a rare but severe cardiovascular disease. The condition often goes undiagnosed, increasing the risk of acute aortic events. It is characterized by medial degeneration, caused by neutrophils infiltrating aortic tissue. Neutrophils can form proinflammatory and prothrombotic neutrophil extracellular traps (NETs), promoting disease progression. Purpose Recently, NETs and abdominal aortic aneurysm (AAA) were linked, while their effects on TAA development and progression remain unclear. We aimed to characterize NETs and NET-specific markers in tissue and plasma samples of TAA patients. Methods We included 171 patients with either ascending TAA (n=153) or Type A dissection (n=18). Healthy subjects were recruited as controls (n=135). Sample fiber composition was analyzed using trichrome; NET burden was quantified using immunofluorescence. Plasma NET surrogate markers including double-stranded (ds)DNA, myeloperoxidase (MPO), DNase activity and matrixmetalloproteinases (MMP) were measured using a fluorescence-based assay and ELISA, respectively. mRNA expression was assessed using qPCR. Results Median patient age was 65 years, and most were male (68.4%). Cardiovascular risk factors were common, including hypertension (77.8%), hyperlipidemia (57.9%), coronary artery disease (26.3%), and diabetes (18.7%). TAA patients were older with high BMI and impaired kidney function. Trichrome staining revealed abundant fibrotic material (median 47.99%), while collagen and elastic fiber distributions were 27.67% and 20.17%, respectively. TAA patients had increased amounts of aortic fibrotic material (47.99% vs 38.94%, p=0.001). Using immunofluorescence, we found higher NET expression in diseased tissue (0.87 [0.3, 1.85] vs 0.15 [0.03, 0.22]%, p=0.0017). This increase was primarily driven by the dissection patients (1.76 [0.89, 2.83]%, p=0.0037). Plasma MPO levels were increased in TAA compared to controls (36.93 [23.46, 57.55] vs 26.8 [19.7, 36.2]ng/mL, p<0.001), while DNase activity was decreased in patients (4.16 [2.66, 6.24] vs 6.06 [4.25, 8.11]mU/mL, p<0.001). Moreover, circulating plasma levels of MMP2 were elevated in TAA (250.36 [190.24, 351.32] vs 218.85 [171.32, 263.87]ng/mL, p=0.004). qPCR analysis of mRNA expressions in TAA cryospecimens revealed upregulations of TNFα (p=0.033) and CD45 (p=0.003), when compared to aortic disease-free controls. Conclusion Aortic fibrosis at the aneurysm site may be caused by inflammatory mechanisms involving TNFα and leucocytes (CD45+). High local NET-burden is indicative of activated neutrophils in the aortic media. TAA patients have suppressed DNase activity and higher MPO levels in venous blood, leading to dsDNA accumulation and increased neutrophil turnover. Elevated MMP2 suggests its involvement in TAA formation. Our findings could enable further research targeting NETs and inflammation in TAA, possibly finding novel therapies capable of mitigating aneurysm development.

Malondialdehyde-specific natural IgM inhibit toll-like receptor 4 and peptidyl-arginine deiminase 4-dependent NETosis triggered by coronary extracellular vesicles of myocardial infarction patients

Abstract Background Neutrophil extracellular traps (NETs) are critical mediators of thromboinflammation during acute myocardial infarction (AMI). However, triggers and signalling pathways of NETosis in AMI remain incompletely understood. Levels of extracellular vesicles (EV) carrying oxidation-specific epitopes (OSE) originating from lipid peroxidation are increased at the culprit site in AMI. Importantly, natural IgM antibodies with specificity for OSE, such as malondialdehyde (MDA), have been shown to modulate functional effects of EV, and are associated with reduced cardiovascular risk. Purpose We investigated the stimulatory capacity of EV on neutrophil effector functions and specifically targeted key mediators of NET formation using pharmacological inhibitors and atheroprotective natural IgM to inhibit this process. Methods Patients were included after diagnosis of ST-segment elevation myocardial infarction and blood was aspirated from the culprit site and peripheral arterial site (n=28) during primary percutaneous coronary intervention (pPCI). Myocardial function was documented by cardiac magnetic resonance imaging 4±2 days and 195±15 days after pPCI. EV were isolated from cell culture supernatants and culprit site plasma for neutrophil stimulation in vitro. Pharmacological inhibitors were used to map NET signalling pathways of EV. Isolated EV were used for neutrophil stimulation in a murine injection model in the presence of the MDA-specific IgM LR04 or an isotype control. NET formation and other neutrophil functions were assessed by flow cytometry, ELISA and fluorescence microscopy. Results Levels of NET surrogate markers and CD45+ MDA+ EV were higher at the culprit site than in the peripheral circulation. EV generated by LPS-activated THP-1 monocytic cells induced in vitro NET formation, release of neutrophil elastase and IL-8, and degranulation of MPO and NGAL in primary human neutrophils, but not reactive oxygen species. Toll-like receptor 4 (TLR4) and peptidyl-arginine deiminase 4 (PAD4) were identified as key mediators of NET formation induced by in vitro-generated EV and EV isolated from the culprit site of AMI patients. Functionally, the MDA-specific monoclonal IgM antibody LR04, but not an isotype control, inhibited the ability of patient-derived and in vitro-generated EV to trigger release of NETs in primary human neutrophils and in mice. Titres of MDA-specific IgM antibodies were inversely associated with the NET marker citH3 in blood of AMI patients. Finally, we found that the concentration of CD45+ MDA+ EV per protective MDA-specific IgM at the culprit site showed an inverse association with left ventricular ejection fraction 72 hours and 6 months after AMI. Conclusions We show that EV can trigger several neutrophil effector functions. EV-induced NET formation is TLR4- and PAD4-dependent and can be inhibited by OSE-specific natural IgM potentially influencing cardiovascular outcomes after an acute event.

Circulating levels of neutrophil extracellular traps reflects local neutrophil activity but not fibrinolytic activity in coronary thrombi from patients with ST-elevation myocardial infarction

Abstract Introduction Neutrophil extracellular traps (NETs) are associated with impaired fibrinolysis in ischemic stroke, while NETs degradation enhances lysis of coronary thrombi in ex vivo experiments. Whether circulating NET markers are associated with local fibrinolytic activity in patients with ST-elevation myocardial infarction (STEMI) has not been reported. Purpose We investigated associations between circulating NET markers and gene expression of fibrinolytic markers in aspirated coronary thrombi from patients with STEMI. Furthermore, we explored whether local upregulation of NET formation by peptidylarginine deiminase 4 (PAD4) was reflected in the circulation. Methods Coronary thrombi from 35 STEMI patients undergoing primary percutaneous coronary intervention (PCI) were aspirated and promptly snap-frozen to -80°C. Peripheral blood samples were collected simultaneously. Median time from start of symptoms to PCI was 152 min. Thrombus gene expression of tissue Plasminogen Activator (tPA), urinary-type Plasminogen Activator (uPA), plasminogen-activation inhibitor type 1 (PAI-1) and PAD4 were quantified with RT-PCR. Gene expression of PAD4 was also measured in circulating leukocytes. Circulating NET markers were measured by a fluorescent nucleic acid stain using fluorometry (dsDNA), an in-house ELISA technique (MPO-DNA) and commercial ELISA (H3Cit). Correlations were tested using Spearman’s rho. Results There were no correlations between any of the circulating NET markers and gene expression of the fibrinolytic markers tPA, uPA and PAI-1 in the thrombus. Of the circulating NET markers, dsDNA correlated with thrombus gene expression of PAD4 (rs=0.491 p=0.006), whereas none of the NET markers correlated with gene expression of PAD4 in circulating leukocytes. Conclusion Circulating NET markers were not associated with gene expression of fibrinolytic activity in thrombi from patients with STEMI. Local NET formation in the thrombi, measured as PAD4 expression, was associated with the circulating NET marker dsDNA. These findings suggest that circulating NET markers have limited utility for assessing local fibrinolytic activity, but might reflect PAD4-dependent NET formation in coronary thrombi.

Association of Neutrophil extracellular traps burden with clinical and angiographic characteristics in patients with ST-elevation myocardial infarction

Abstract Background The precise triggers for arterial plaque rupture and the underlying pathophysiology of thrombogenesis remain elusive. Polymorphonuclear neutrophils (PMN), particularly their formation of Neutrophil Extracellular Traps (NETs), have garnered attention in the context of coronary atherothrombosis. This study sought to explore the association of NETs burden with clinical and angiographic characteristics in ST-elevation myocardial infarction (STEMI) patients undergoing primary percutaneous coronary intervention (pPCI) and thrombus aspiration (TA). Methods This study included 336 consecutive STEMI patients undergoing pPCI and TA. Aspirated thrombi underwent histological analysis and NETs quantification via immunohistochemistry. Potential associations of clinical variables and angiographic outcomes with NETs burden were assessed. Results Manual TA was selectively performed in 72 cases with increased thrombotic burden and 60 thrombi were suitable for analysis. Most thrombi specimens displayed lytic features (63%) and almost three out of four specimens were identified as white thrombi. Increased NETs burden was significantly associated with prolonged pain-to-balloon time (>300 minutes) OR=10.29 (95% CI 2.11-42.22, p=0.001) and stress-induced hyperglycemia (SIH) OR=6.58 (95% CI 1.23-52.63, p=0.04) after multivariate regression analysis. Additionally, distal embolization (DE), a predictor of adverse outcomes, was more frequent among patients with an elevated NETs burden OR=16.9 (95% CI 4.23-44.52, p<0.001). Conversely, no significant association between NETs burden and final TIMI flow=3 was observed. Conclusion Elevated NETs burden in STEMI thrombi is linked to delayed reperfusion, SIH and increased risk of DE. Further research is needed to elucidate the role of NETs as a potential therapeutic target in acute atherothrombosis.

TLR7 deficiency alleviated myocardial ischemia reperfusion injury via regulating macrophage-neutrophil interaction

Abstract Background Toll-like receptor 7 (TLR7) is an intracellular innate immune receptor activated by single-stranded RNA. We previously reported that TLR7 could recognize RNA from dying cardiac cells and thereby deteriorating post myocardial infarction left ventricular remodeling. However, its effect on myocardial ischemia reperfusion (IR) injury is still unknown. Purpose In this study, we aimed to investigate the role of TLR7 in myocardial IR injury and the underlying mechanisms. Methods C57BL/6J wild type (WT) and TLR7 deficient (TLR7-/-) mice underwent left anterior descending artery occlusion for 30min followed by reperfusion. Myocardial infarct size was measured by Evan’s blue and TTC staining. mRNA expression levels of cytokines and phagocytosis-related receptors were detected by qPCR. TUNEL assay was conducted to determine apoptosis in the heart. Influx of neutrophils and macrophages in ischemic myocardium was analyzed by flow cytometry and Histone Cit3 was imaged to examine formation of neutrophils extracellular traps (NETs), cardiac function was evaluated by echocardiography during post-IR injury follow-up. Results TLR7 expression was up-regulated after IR injury in ischemic myocardium of WT mice. Compared to WT mice, fewer apoptotic cells and smaller infarct size (34±2% vs. 23±3% of area at risk, p<0.01) were observed in TLR7-/- mice at 24 hours post-IR, which was accompanied by reduced inflammatory cytokines (IL-1β: 110.5±14.1 vs. 54.8±13.7, p<0.05; TNF-α: 4.6±1.0 vs. 2.0±0.4, p<0.05) and increased anti-inflammatory factors (TNF-β: 2.9±0.3 vs. 8.6±2.6, p<0.05; IL-10: 14.2±2.1 vs. 32.3±8.8, p<0.05). Surprisingly, although the influx of neutrophils and macrophages increased to a similar extent in WT and TLR7-/- mice, the latter exhibited remarkably less NETs formation and enhanced macrophage phagocytosis in the infarct region. Moreover, TLR7 deficiency improved cardiac function, as indicated by increased left ventricular ejection fraction (LVEF) and fraction shortening (FS) at 56d post-IR. Conclusions TLR7 deficiency alleviates myocardial IR injury and improves cardiac function probably via enhancing macrophage clearance of NETs in the injured myocardium.TLR7 deficiency in cardiac functionTLR7 deficiency decreased NETs formation

The Contribution of Neutrophil Extracellular Traps to Coagulopathy in Patients with COVID-19-Related Thrombosis

COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is associated with hypercoagulability and increased incidence of thrombotic events. In this study, we investigated the levels of neutrophil extracellular trap biomarkers and von Willebrand factor to assess if these could predict the occurrence of a thrombotic event in COVID-19 patients. We enrolled 202 patients hospitalized with symptomatic COVID-19 infection. Of those, 104 patients did not experience any type of thrombotic events before or during their hospitalization. These patients were compared to the other cohort of 98, who experienced thrombotic events before or during their hospitalization. In total, 61 patients who experienced thrombotic events had the event after initial blood collection, so the predictive capacity of biomarkers in these patients was evaluated. Citrullinated histone H3 was the best predictive biomarker for thrombotic events in COVID-19 regardless of age, sex, and race; disease severity was also a significant predictor in most thrombotic event groups. These results may better inform treatment and prophylaxis of thrombotic events in COVID-19 and similar viral illnesses in the future to improve outcomes and reduce mortality.

Neutrophil Extracellular Traps: Potential Thrombotic Markers and Therapeutic Targets in Colorectal Cancer.

Neutrophil extracellular traps (NETs) are promising promoters in venous thromboembolism (VTE). In the present study, we have investigated the potential thrombogenic role of NETs in colorectal cancer (CRC). A total of 583 patients with gastrointestinal malignancies who were diagnosed with or without VTE by extremities arteriovenous ultrasound and computed tomography were enrolled. The incidence of VTE in CRC was as high as 17.53%. In serological ELISA experiments, Cit-H3, MPO and cfDNA were significantly overexpressed in CRC patients with VTE compared to CRC patients without VTE and healthy individuals. Neutrophils from CRC patients with VTE produced appreciable amounts of NETs after stimulation with phorbol-12-myristate-13-acetate, which were lacking in CRC patients without VTE and healthy individuals. CfDNA was positively correlated with plasmin-α2-antiplasmin complex and tissue plasmin activator inhibitor-1 complex, and Cit-H3 was positively correlated with plasmin-α2-antiplasmin complex, suggesting that NETs are associated with increased fibrinolytic activity. We screened some NETs-related genes by analysing several high-throughput sequencing datasets of VTE and NETs. FCGR1A was identified as the optimal target gene by pan-cancer expression analysis and survival analysis. FCGR1A was significantly overexpressed in the peripheral blood of CRC patients without VTE compared to healthy individuals and showed a positive correlation with cfDNA. Neutrophil-derived NETs were significantly reduced by FCGR1A inhibitor exposure. These findings indicate that NETs are actively involved in VTE in CRC. NETs are promising thrombotic marker and therapeutic target in CRC to prevent the thrombotic consequences of cancer.

TNF inhibitors target a mevalonate metabolite/TRPM2/calcium signaling axis in neutrophils to dampen vasculitis in Behçet’s disease

TNF inhibitors have been used to treat autoimmune and autoinflammatory diseases. Here we report an unexpected mechanism underlying the therapeutic effects of TNF inhibitors in Behçet’s disease (BD), an autoimmune inflammatory disorder. Using serum metabolomics and peripheral immunocyte transcriptomics, we find that polymorphonuclear neutrophil (PMN) from patients with BD (BD-PMN) has dysregulated mevalonate pathway and subsequently increased farnesyl pyrophosphate (FPP) levels. Mechanistically, FPP induces TRPM2-calcium signaling for neutrophil extracellular trap (NET) and proinflammatory cytokine productions, leading to vascular endothelial inflammation and damage. TNF, but not IL-1β, IL-6, IL-18, or IFN-γ, upregulates TRPM2 expression on BD-PMN, while TNF inhibitors have opposite effects. Results from mice with PMN-specific FPP synthetase or TRPM2 deficiency show reduced experimental vasculitis. Meanwhile, analyses of public datasets correlate increased TRPM2 expressions with the clinical benefits of TNF inhibitors. Our results thus implicate FPP-TRPM2-TNF/NETs feedback loops for inflammation aggravation, and novel insights for TNF inhibitor therapies on BD.

Understanding the complex chromatin dynamics in primary human neutrophils during PMA-induced NET formation

BackgroundPrimary human neutrophils play a pivotal role in innate immunity, mainly through the formation of neutrophil extracellular traps (NETs) in a process known as NETosis. This cell-death pathway is crucial for combating infections but is also implicated in many inflammatory diseases, such as sepsis, systemic lupus erythematosus, and rheumatoid arthritis.MethodsThe study presented here investigates chromatin dynamics during NET formation by stimulating primary human neutrophils with phorbol 12-myristate 13-acetate (PMA). We adapt the ATAC-Seq (assay for transposase-accessible chromatin using sequencing) method to isolated neutrophils and characterize a time-dependent chromatin response.ResultsWe found that chromatin accessibility patterns are consistent across individual donors and most chromatin changes occur within 30 min, with many continuing across the 90 min assessed in this study. Regulatory regions gaining accessibility were associated with the activity of pathways that have been implicated in NOX-dependent NET formation.ConclusionsOur findings increase the understanding of the chromatin changes underlying NET formation and also identify potential early-acting targets for modulating this process in inflammatory diseases.

Neutrophil extracellular traps as immunofibrotic mediators in RA-ILD; pilot evaluation of the nintedanib therapy

ObjectiveRheumatoid arthritis-associated interstitial lung disease (RA-ILD) is a significant pulmonary complication of RA. This study tried to elucidate the mechanisms enhancing inflammation and causing lung injury in RA-ILD, focusing on the role of neutrophil extracellular traps (NETs). The study also investigated the potential benefits of nintedanib in advanced disease.MethodsNine RA-ILD patients and nine healthy controls were included in the study. Inflammatory markers in patients’ circulation were evaluated with immunoassays. The formation of NETs was examined using a citrullinated histone H3 (CitH3) ELISA and cell immunofluorescence. Inflammatory proteins expressed in neutrophils/NETs were studied with real-time qPCR and NET ELISA. To assess the effect of nintedanib, an intracellular tyrosine kinase inhibitor with antifibrotic properties, in RA-ILD a paired study was conducted in five patients before treatment administration and 16 weeks later.ResultsThe soluble terminal complement complex sC5b-9 and the levels of CitH3 were significantly elevated in patients with RA-ILD, compared to healthy controls. In addition, neutrophils isolated from RA-ILD patients released NETs enriched with tissue factor and interleukin-17A. Inflammatory NETs had a dynamic role, increasing the fibrotic potential of human pulmonary fibroblasts (HPFs). On the other hand, nintedanib treatment decreased NETs and sC5b-9 levels in RA-ILD patients.ConclusionThe findings propose an interplay between circulating NETs and HPFs, establishing the immunofibrotic aspects of RA-ILD. They also support the effectiveness of nintedanib in reducing key pathological processes of the disease. Further research is needed to fully understand these mechanisms and optimize treatment strategies for RA-ILD.

Microbial and Transcriptomic Landscape Associated With Neutrophil Extracellular Traps in Perianal Fistulizing Crohn's Disease.

Perianal fistulizing Crohn's disease (pfCD) poses significant healing challenges, closely associated with neutrophil extracellular traps (NETs). This study aimed to investigate the microbe-host interactions influencing NETs in pfCD. From January 2019 to July 2022, patients with pfCD were screened at Ren Ji Hospital. Patients in remission following comprehensive treatment were recruited. We documented clinical characteristics, medication regimens, healing outcomes, and infliximab levels in fistula tissues. NET positivity was confirmed by positive results in citrullinated histone H3 (CitH3) enzyme-linked immunosorbent assay (ELISA) and dual immunofluorescence staining for myeloperoxidase and CitH3. Microbial and transcriptomic profiles from fistula tissues, obtained during surgery, were analyzed using 16S rRNA gene sequencing and RNA sequencing. Differences in microbiome and transcriptomic profiles were evaluated, and their relationships were assessed using Mantel's and Spearman's coefficients. Significant differences in microbial communities were found between groups (P = .007). Representatively differential microbes such as Prevotella bivia, Streptococcus gordonii, and Bacteroides dorei were enriched in NETs-positive fistulas (P < .05). Functional analysis of microbes revealed reduced ubiquinol biosynthesis and butanoate production in NETs-negative fistulas (P < .05). Transcriptomic analysis indicated increased neutrophil and monocyte infiltration in NETs-positive fistulas, associated with pathways involving bacterial response, neutrophil chemotaxis, secretory processes, and peptidase activity (P < .05). Species prevalent in NETs-positive fistulas correlated positively with immune responses and wound healing pathways, whereas bacteria in NETs-negative fistulas correlated negatively. NETs were negatively associated with tissue infliximab levels (P = .001) and healing outcomes (P = .025). Our findings reveal unique microbial and transcriptomic signatures associated with NETs in pfCD, highlighting their profound influence on clinical outcomes.

Phillyrin inhibits oxidative stress and neutrophil extracellular trap formation through the KEAP1/NRF2 pathway in gouty arthritis.

Gouty arthritis (GA) is an inflammatory disorder characterized by deposition of monosodium urate (MSU) crystal in joints. Phillyrin, a natural compound with anti-inflammatory properties, shows promise in mitigating inflammatory responses. This study investigates the therapeutic potential of phillyrin in GA and explores its mechanisms of action. GA was induced in mice via intraarticular MSU injection, and joint inflammation, inflammatory cell infiltration, and their level in serum/tissue were assessed. Key proteins in the NF-κB and NLRP3 pathways were examined using western blot analysis. The impact of phillyrin on oxidative stress, neutrophil extracellular trap (NET) formation, and neutrophil accumulation was evaluated by measuring CD11b + Ly6G + cells, MPO, CitH3, extracellular DNA ratio, and oxidative stress markers. In vitro studies assessed the effects of phillyrin on oxidative stress, cell viability, cytokine production, and NET formation in MSU-treated neutrophils. The KEAP1/NRF2 pathway's role was analyzed using ML385, an NRF2 inhibitor. Phillyrin significantly reversed MSU-induced ankle swelling and inflammatory cell infiltration in joint tissues. It suppressed pro-inflammatory cytokines and proteins in the NF-κB and NLRP3 pathways. Phillyrin reduced neutrophil infiltration, evidenced by lower MPO activity and NET formation, marked by reduced CitH3 expression. In vitro, phillyrin inhibited inflammatory marker expression and NET formation without affecting cell viability. It also restored antioxidant enzyme levels and reduced ROS production, regulating the KEAP1/NRF2 pathway, enhancing NRF2 expression and stability. These effects were reversed by NRF2 inhibition with ML385. Phillyrin alleviates GA by reducing joint inflammation, inhibiting NET formation, and suppressing oxidative stress through NRF2 modulation.

The impact of trauma relevant concentrations of prostaglandin E2 on the anti-microbial activity of the innate immune system

BackgroundThe mechanisms underlying the state of systemic immune suppression that develops following major trauma are poorly understood. A post-injury increase in circulating levels of prostaglandin E2 (PGE2) has been proposed as a contributory factor, yet few studies have addressed how trauma influences PGE2 biology.MethodsBlood samples from 95 traumatically-injured patients (injury severity score ≥8) were collected across the pre-hospital (≤2 hours), acute (4-12 hours) and subacute (48-72 hours) post-injury settings. Alongside ex vivo assessments of lipopolysaccharide (LPS)-induced cytokine production by monocytes, neutrophil reactive oxygen species production and phagocytosis, serum concentrations of PGE2 and its scavenger albumin were measured, and the expression of enzymes and receptors involved in PGE2 synthesis and signalling analysed. Leukocytes from trauma patients were treated with cyclooxygenase (COX) inhibitors (indomethacin or NS-398), or the protein kinase A inhibitor H89, to determine whether injury-induced immune suppression could be reversed by targeting the PGE2 pathway. The effect that trauma relevant concentrations of PGE2 had on the anti-microbial functions of neutrophils, monocytes and monocyte-derived macrophages (MDMs) from healthy controls (HC) was examined, as was the effect of PGE2 on efferocytosis. To identify factors that may trigger PGE2 production post-trauma, leukocytes from HC were treated with mitochondrial-derived damage associated molecular patterns (mtDAMPs) and COX-2 expression and PGE2 generation measured.ResultsPGE2 concentrations peaked in blood samples acquired ≤2 hours post-injury and coincided with significantly reduced levels of albumin and impaired LPS-induced cytokine production by monocytes. Significantly higher COX-2 and phospholipase A2 expression was detected in neutrophils and/or peripheral blood mononuclear cells isolated from trauma patients. Treatment of patient leukocytes with indomethacin, NS-398 or H89 enhanced LPS-induced cytokine production and neutrophil extracellular trap generation. Exposure to physiological concentrations of PGE2 suppressed the anti-microbial activity of monocytes, neutrophils and MDMs of HC, but did not influence efferocytosis. In a formyl-peptide receptor-1 dependent manner, mtDAMP treatment significantly increased COX-2 protein expression in neutrophils and monocytes, which resulted in increased PGE2 production.ConclusionsPhysiological concentrations of PGE2 suppress the anti-microbial activities of neutrophils, monocytes and MDMs. Targeting the PGE2 pathway could be a therapeutic approach by which to enhance innate immune function post-injury.

Neutrophil activation markers can predict rheumatoid arthritis treatment response to the Janus Kinase 1/2 inhibitor baricitinib.

Neutrophils play an important role in regulating immune and inflammatory responses in rheumatoid arthritis (RA). We assessed whether baricitinib, a JAK1/JAK2 inhibitor, could reduce neutrophil activation, and whether a neutrophil activation score could predict treatment response. Markers of neutrophil activation, calprotectin, and neutrophil extracellular traps (NETs) were analyzed using ELISA in RA plasma (n=271) and healthy controls (n=39). For RA patients, neutrophil activation markers were measured at baseline, 12 weeks, and 24 weeks after treatment with placebo, 2 mg, and 4 mg baricitinib. Whole blood RNA analyses from multiple randomized baricitinib RA trials were performed to study neutrophil-related transcripts (n=1651). Baseline levels of plasma neutrophil markers were elevated in RA patients compared to healthy controls (p<0.001). Baricitinib reduced levels of soluble calprotectin at 12 and 24 weeks, especially in RA patients responding to treatment, as determined by ACR20. Whole blood RNA analysis revealed similar changes in the predominant neutrophil markers calprotectin and FcαRI upon treatment with baricitinib in three randomized clinical trials involving RA patients at various stages of disease modifying therapy. Clustering analysis of plasma activation markers showed elevated levels of calprotectin and NETs, e.g., a neutrophil activation score, at baseline, could predict treatment response to baricitinib. In contrast, CRP levels could not distinguish between responders and non-responders. Neutrophil activation markers may add clinical value in predicting treatment response to baricitinib and other drugs targeting RA. This study supports personalized medicine, in treating RA patients, not only based on symptoms, but also based on immunophenotyping.

From inflammation to invasion: Neutrophils in cervical cancer pathogenesis

Cervical cancer, predominantly caused by high-risk human papillomavirus (HPV) infections, continues to be a major health challenge globally. Recent research has highlighted the significant role of neutrophils, a type of white blood cell integral to the immune system, in the pathogenesis of cervical cancer. This review examines the dualistic role of neutrophils in cervical cancer, emphasizing their contribution to both inflammation and tumor progression. Understanding the intricate relationship between neutrophils and cervical cancer could unveil new therapeutic targets for better disease management. Neutrophils are key mediators of the immune response and inflammation. In the context of cervical cancer, these cells are recruited to the tumor microenvironment where they can adopt tumor-promoting phenotypes. Tumor-associated neutrophils (TANs) facilitate various processes that aid tumor growth and metastasis, such as producing reactive oxygen species (ROS) and proteases that induce DNA damage, releasing cytokines and chemokines that promote angiogenesis, and forming neutrophil extracellular traps (NETs) that enhance metastatic potential. Furthermore, TANs contribute to immune suppression by inhibiting the activity of cytotoxic T lymphocytes and natural killer cells, allowing cancer cells to evade immune surveillance. Given their pivotal role in cervical cancer progression, neutrophils represent a promising target for novel therapeutic strategies. Approaches such as inhibiting neutrophil recruitment to the tumor site, blocking NET formation, and modulating TAN phenotypes from pro-tumor to anti-tumor are being explored. These strategies aim to disrupt the supportive role of neutrophils in tumor development and progression, potentially leading to improved outcomes for patients with cervical cancer.

Juvenile Dermatomyositis: Updates in Pathogenesis and Biomarkers, Current Treatment, and Emerging Targeted Therapies.

Juvenile dermatomyositis is a rare systemic inflammatory autoimmune disease involving muscle, skin, and vessels. Most patients do not fully respond to initial therapy, instead having a chronic refractory or polycyclic disease course. Pathogenesis is not completely understood, but immune cell dysregulation, particularly of B cells, mitochondrial dysfunction, changes in neutrophils and neutrophil extracellular traps (NETs), and increased type I and type II interferon (IFN) signaling have been described. There are limited randomized controlled trials of drugs in juvenile dermatomyositis (JDM), and treatment is largely based on lower-quality data such as case series, retrospective studies, and open-label prospective studies. These data have been compiled into expert recommendations or consensus treatment plans, which help guide therapy. While initial therapy is more standard with most including corticosteroids (high-dose oral and/or pulse intravenous methylprednisolone) and methotrexate, for refractory patients, guidelines are more varied with multiple options or combinations, including biologic therapies. There is a clear need for more efficacious and personalized therapy in JDM. Emerging treatment options worthy of further study in JDMinclude targetingIFN-signaling (JAK, IFNAR1, IFN beta),B-cells (CD20, CD19, BAFF, TACI, CD38, BCMA)including Chimeric Antigen Receptor (CAR)-T cell therapy, mitochondrial dysfunction, and NETs.

The vacuolar anti-Pseudomonal activity of neutrophil primary granule peptidyl-arginine deiminase enzymes

The role of neutrophils in host defense involves several cell processes including phagocytosis, degranulation of antimicrobial proteins, and the release of neutrophil extracellular traps (NETs). In turn, dysregulated cell activity is associated with the pathogenesis of airway and rheumatic diseases, in which neutrophil-derived enzymes including peptidyl-arginine deiminases (PADs) play a role. Known physiological functions of PADs in neutrophils are limited to the activity of PAD isotype 4 in histone citrullination in NET formation. The aim of this study was to extend our knowledge on the role of PADs in neutrophils and, specifically, bacterial killing within the confines of the phagocytic vacuole. Human neutrophils were fractionated by sucrose gradient ultracentrifuge and PADs localized in subcellular compartments by Western blot analysis. Direct interaction of PADs with Pseudomonas aeruginosa (P. aeruginosa) was assessed by flow cytometry and Western blot overlay. The participation of neutrophil PAD2 and PAD4 in killing of P. aeruginosa was assessed by inclusion of PAD-specific inhibitors. In vitro, bactericidal activity of recombinant human PAD2 or PAD4 enzymes against P. aeruginosa was determined by enumeration of colony-forming units (CFU). Together with neutrophil elastase (NE), PAD2 and PAD4 were localized to primary granules and, following activation with particulate stimuli, were degranulated in to the phagocytic vacuole. In vitro, PAD2 and PAD4 bound P. aeruginosa (p = 0.04) and significantly reduced bacterial survival to 49.1 ± 17.0 (p &amp;lt; 0.0001) and 48.5 ± 13.9% (p &amp;lt; 0.0001), respectively. Higher antibacterial activity was observed at neutral pH levels with the maximum toxicity at pH 6.5 and pH 7.5, comparable to the effects of neutrophil bactericidal permeability increasing protein. In phagosomal killing assays, inclusion of the PAD2 inhibitor, AFM-30a, or PAD4 inhibitor, GSK484, significantly increased survival of P. aeruginosa (AFM-30a, p = 0.05; and GSK484, p = 0.0079). Results indicate that PAD2 and PAD4 possess antimicrobial activity and are directly involved in the neutrophil antimicrobial processes. This study supports further research into the development of PAD-based antimicrobials.

Real-Time, High-Throughput Microscopic Quantification of Human Neutrophil Extracellular Trap Release and Assessing the Pharmacology of Antagonists

Real-Time, High-Throughput Microscopic Quantification of Human Neutrophil Extracellular Trap Release and Assessing the Pharmacology of Antagonists

Exploring the role of aggrephagy-related signatures in immune microenvironment, prognosis, and therapeutic strategies of breast cancer.

Breast cancer (BC) is 1 of the most common malignant tumors among women globally. This study aimed to develop a prognostic signature based on aggrephagy-related genes (ARGs). Transcriptomic and clinical data for BC patients were downloaded from the cancer genome atlas and GEO databases. Differential expression analysis, univariate Cox proportional hazards regression and least absolute shrinkage and selection operator Cox regression were employed to construct a prognostic signature. Consensus clustering, evaluation of immune infiltration and drug sensitivity, and gene set enrichment analysis, and development of nomogram were performed. The expression of ARGs was validated using data from the Cancer Cell Line Encyclopedia and clinical samples. Eleven ARGs were abnormally expressed in BC, with 5 showing significant correlations with BC prognosis. Consensus clustering identified 2 molecular subtypes with distinct prognoses. A prognostic signature including 5 ARGs (VIM, TUBB1, TUBA3E, TUBA3D, TUBA1C) was developed, which showed high performance in predicting BC prognosis. The low-risk group showed enrichment in extracellular matrix organization and cell migration processes while chromosome separation was suppressed. Additionally, patients in this group also show activation in several signaling pathways including MAPK, PI3K-AKT, and cAMP pathways, whereas cell cycle and neutrophil extracellular trap formation were significantly inhibited. The signature was also associated with immune infiltration and drug sensitivity. A nomogram incorporating the risk signature, clinical stage and chemotherapy was constructed, demonstrating excellent performance in predicting prognosis. The expression of signature-related genes were validated in patients with BC. This study successfully constructed molecular subtypes and a prognostic signature based on ARGs in BC, and developed a nomogram.