Inhibition Of Neutrophil Extracellular Traps Formation Research Articles

Guizhi Shaoyao Zhimu decoction inhibits neutrophil extracellular traps formation to relieve rheumatoid arthritis via gut microbial outer membrane vesicles

BackgroundRheumatoid arthritis (RA) is a common autoimmune disease with a high disability rate. Accumulating studies suggest that neutrophil extracellular traps (NETs) play a crucial role in the pathogenesis of RA and targeting NETs has emerged as a potential therapeutic strategy for RA. As a traditional Chinese medicine, Guizhi-Shaoyao-Zhimu Decoction (GSZD) has exhibited good efficacy in the treatment of rheumatoid arthritis (RA), while the underly mechanism especially the possibility that GSZD alter NETs formation to relieve RA remains unknown. PurposeOur study aimed to investigate relationship between GSZD and NETs in RA treatment and revealed underlying mechanism. MethodsWe constructed collagen-induced arthritis (CIA) model and treated CIA mice with GZSY to validate therapeutic effects of GSZD and examine whether GZSD could inhibit NETs formation in RA. And 16S rRNA sequencing and Fecal microbiota transplantation (FMT) experiment were performed to determine whether GSZD could reduce NETs formation to alleviate RA in gut microbiota-associated manner and identify crucial bacterium in response to GSZD administration. CIA mice treated with effective bacteria and its outer membrane vesicles (OMVs) with oral administration to investigate protective effect against RA and NETs regulative efficiency. We utilized small interfering RNA in vivo and vitro to silence gene mediating effect of GZSD-gut microbiota-NETs. ResultsGSZD could inhibit NETs formation and relive arthritis in CIA mice. Additionally, GSZD alter gut microbiota composition and significantly increase intestinal Parabacteroides goldsteinii (P.goldsteinii) abundance. Mechanistically, P.goldsteinii enriched by GSZD secreted outer membrane vesicles (OMVs) to translocate into joints and activate Cav-1-Nrf2 axis, leading to reduced NETs formation and alleviate arthritis. In clinical, the abundance of P.goldsteinii exhibited negative correlation with NETs indexes and RA disease activities. ConclusionOur findings suggest that GSZD inhibits NETs formation to relieve RA in P.goldsteinii-Cav-1-Nrf2 associated manner, which could provide new sight of the prevention and treatment of RA.

The Glucagon-Like Peptide-1 (GLP-1) Receptor Agonist Liraglutide Regulates Sirtuin-1-Mediated Neutrophil Extracellular Traps to Improve Diabetes-Induced Bone Metabolism Imbalance

Background: Diabetes mellitus (DM) is a chronic metabolic disorder that disrupts normal bone remodeling. Objectives: This study aimed to investigate how the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide (LIR) addresses bone metabolism imbalances induced by type-II diabetes. Methods: Type-II diabetic rat models were established through a single intraperitoneal injection of streptozotocin (STZ). Blood glucose levels were measured using a blood glucose meter, and insulin levels were assessed using an assay kit. Bone formation markers [alkaline phosphatase (ALP), osteocalcin (OCN), and procollagen I N-terminal propeptide (PINP)] and bone resorption markers [tartrate-resistant acid phosphatase (TRACP) and CTX-1] were monitored using assay kits. Bone marrow mesenchymal stem cells (BMSCs) were cultured in vitro under high-fat and high-glucose (HFHS) conditions to mimic diabetic bone metabolism dysregulation. Neutrophil extracellular traps (NETs) formation was examined through immunofluorescent staining and Western blot analysis. Results: Liraglutide was found to reduce STZ-induced NETs formation, as indicated by decreased expression of cit-H3 by 36.90% - 53.57%, myeloperoxidase (MPO) by 55.81% - 65.12%, NE by 53.95% - 65.17%, and PAD4 by 46.81% - 63.83%, alongside increased Sirtuin-1 (SIRT1) expression in femur tissue (70.71% - 91.19%). In vitro, LIR enhanced osteogenesis and inhibited apoptosis, effects that were partially reversed by SIRT1 knockdown. Additionally, SIRT1 knockdown partially restored LIR-induced reductions in oxidative stress, inflammation, and NETs formation. Conclusions: LIR mitigates diabetes-induced bone metabolism imbalance by inhibiting NETs formation through SIRT1 mediation.

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.

PAD4 Inhibitor-Loaded Layered Double Hydroxide Nanosheets as a Multifunctional Nanoplatform for Photodynamic Therapy-Mediated Tumor Metastasis Treatment.

Photodynamic therapy (PDT) is demonstrated to be effective in inducing antitumor immune responses for tumor metastasis treatment. However, tumor hypoxia, inferior tissue penetration of light, and low singlet oxygen (1O2) quantum yield significantly hamper the efficacy of PDT, thus weakening its immune function. Moreover, PDT-mediated neutrophil extracellular traps (NETs) formation can further reduce the therapeutic effectiveness. Herein, the use of defect-rich CoMo-layered double hydroxide (DR-CoMo-LDH) nanosheets as a carrier to load a typical peptidyl arginine deiminase 4 inhibitor, i.e., YW4-03, to construct a multifunctional nanoagent (403@DR-LDH) for PDT/immunotherapy, is reported. Specifically, 403@DR-LDH inherits excellent 1O2 generation activity under 1550nm laser irradiation and improves the half-life of YW4-03. Meanwhile, 403@DR-LDH plus 1550nm laser irradiation can stimulate immunogenic cell death to promote the maturation of dendric cells and activation/infiltration of T cells and significantly downregulate H3cit protein expression to inhibit NETs formation, synergistically promoting the antitumor metastasis effect. Taken together, 403@DR-LDH can kill cancer cells and inhibit tumor growth/metastasis under 1550nm laser irradiation. Single-cell analysis indicates that 403@DR-LDH can regulate the ratio of immune cells and immune-related proteins to improve the tumor immune microenvironment, showing strong efficacy to inhibit the tumor growth, metastasis, and recurrence.

Aloin alleviates corneal injury in alkali burn via inhibiting neutrophil extracellular traps and promoting Nrf2.

Ocular chemical burns are a leading cause of blindness. The cornea is injured by alkali-induced oxidative disturbances and an inflammatory response. The aim of this study was to evaluate the protective effects of aloin, an antioxidant, and anti-inflammatory compound, on corneal alkali burn. Mice eyes were injured by NaOH and subsequently treated with aloin eye drop and intraperitoneal injection. Pathological characteristics of the eyes were examined, and corneal samples were collected for further analysis. Aloin diminished neutrophil infiltration and the production of proinflammatory cytokines. Aloin also attenuated apoptosis in human corneal epithelial cells (HCEs) by reducing oxidative stress through the activation of the Nrf2 pathway. Additionally, aloin suppressed the formation of neutrophil extracellular traps (NETs) and inhibited their deposition on the cornea. Moreover, aloin mitigated alkali-induced apoptosis in HCEs caused by NETs. These findings suggest that aloin has potential as an antioxidant and anti-inflammatory compound for treating corneal alkali burn by inhibiting NETs formation and promoting Nrf2.

Bone morphogenetic protein 4 inhibits corneal neovascularization by blocking NETs-induced disruption to corneal epithelial barrier

Corneal neovascularization (CoNV) is the second leading cause of visual impairment worldwide, and current drugs have certain limitations. Inflammatory response is the core pathological process of CoNV. Neutrophil extracellular traps (NETs) are generated after neutrophil activation, which promotes neovascularization. Prior studies demonstrated that bone morphogenetic protein 4 (BMP4) could significantly reduce inflammation and CoNV formation, its exact molecular mechanism remains unclear. Therefore, we stimulated human peripheral blood neutrophils with phorbol myristate acetate (PMA) or deoxyribonuclease I (DNase I) to induce or inhibit NETs formation. By using corneal sutures and subconjunctival injections of NETs or DNase I, rat CoNV models were established. Compared with the suture group, NETs formation and inflammatory cell infiltration in the corneal stroma were significantly increased, corneal edema was aggravated, and the length, area and diameter of CoNV were significantly enhanced in the NETs group. Furthermore, by curetting the corneal epithelial apical junctional complexes (AJCs), a crucial component in preserving the function of the corneal epithelial barrier, we discovered that the damage of AJCs had a significant role in inducing CoNV formation. NETs could induce CoNV formation by injuring corneal epithelial AJCs. Finally, by comparing the aforementioned indicators after the intervention of BMP4, BMP4 inhibitor Noggin and NADPH oxidase (NOX) inhibitor, we finally demonstrated that BMP4 could inhibit NETs-induced inflammation and corneal epithelial AJC injury, repair corneal epithelial barrier function and eventually inhibit CoNV formation by blocking NOX-2-dependent NETs formation.

Advancing osteoarthritis therapy with GMOCS hydrogel-loaded BMSCs-exos

This study investigated the mechanism of the extracellular matrix-mimicking hydrogel-mediated TGFB1/Nrf2 signaling pathway in osteoarthritis using bone marrow mesenchymal stem cell-derived exosomes (BMSCs-Exos). A GMOCS-Exos hydrogel was synthesized and evaluated for its impact on chondrocyte viability and neutrophil extracellular traps (NETs) formation. In an OA rat model, GMOCS-Exos promoted cartilage regeneration and inhibited NETs formation. Transcriptome sequencing identified TGFB1 as a key gene, with GMOCS-Exos activating Nrf2 signaling through TGFB1. Depletion of TGFB1 hindered the cartilage-protective effect of GMOCS-Exos. This study sheds light on a promising therapeutic strategy for osteoarthritis through GMOCS-Exos-mediated TGFB1/Nrf2 pathway modulation.

Neutrophil extracellular traps mediate the crosstalk between plaque microenvironment and unstable carotid plaque formation

The development of unstable carotid atherosclerotic plaques is associated with the induction of neutrophil extracellular traps (NETs) via the activation of diverse inflammatory mediators in the circulating bloodstream. However, the underlying mechanisms through which NETs influence the microenvironment of atherosclerotic plaques and contribute to the development of unstable carotid plaques remain largely elusive. The objective of this study was to elucidate the role of myeloid differentiation protein 1 (MD-1, LY86)-induced NETs underlying the crosstalk between unstable plaque formation and the plaque microenvironment. We employed bioinformatics analysis to identify key genes associated with carotid-unstable plaque, followed by comprehensive validation using various experimental approaches on tissue specimens and plasma samples classified based on pathological characteristics. Patients with carotid-unstable plaques exhibited elevated plasma concentrations of MD-1 (LY86), while patients with stable plaques demonstrated comparatively lower levels. Furthermore, soluble MD-1 was found to induce the formation of NETs through activation of Toll-like receptor signaling pathway. The proliferative and immature vascularization effects of NETs on endothelial cells, as well as their inhibitory impact on cell migration, are directly correlated with the concentration of NETs. Additionally, NETs were found to activate the NF-κB signaling pathway, thereby upregulating ICAM1, VCAM1, MMP14, VEGFA, and IL6 expression in both Human umbilical vein endothelial cells (HUVECs) and HAECs. Subsequently, a significant increase in intraplaque neovascularization by NETs results in poor carotid plaque stability, and NETs in turn stimulate macrophages to produce more MD-1, generating a harmful positive feedback loop. Our findings suggest that soluble MD-1 in the bloodstream triggers the production of NETs through activation of the Toll-like receptor signaling pathway and further indicate NETs mediate a crosstalk between the microenvironment of the carotid plaque and the neovascularization of the intraplaque region. Inhibiting NETs formation or MD-1 secretion may represent a promising strategy to effectively suppress the development of unstable carotid plaques.

G-CSF induces neutrophil extracellular traps formation and promotes ovarian cancer peritoneal dissemination.

Epithelial ovarian cancer is characterized by aggressive peritoneal dissemination. Neutrophils are mobilized to peritoneal cavity in some patients with ovarian cancer dissemination; however, its pathological significance remains unknown. This study aimed to investigate the role of neutrophil extracellular traps (NETs) in ovarian cancer dissemination. We conducted a retrospective analysis of clinical data and samples from 340 patients with ovarian cancer who underwent primary surgery between 2007 and 2016 at the Osaka University Hospital. In vitro, NETs formation was induced by stimulating human peripheral neutrophils. The human ovarian cancer cell line, OVCAR8, was cocultured with NETs. For an ovarian cancer dissemination mouse model, we performed an intraperitoneal injection of OVCAR8 cells into nude mice. The association between NETs and peritoneal dissemination was explored, and model mice were treated with the PAD4 inhibitor GSK484 to assess antitumor efficacy. Neutrophilia (neutrophil count >7000/mm3) correlated with shorter survival, advanced peritoneal dissemination, elevated granulocyte colony-stimulating factor (G-CSF) levels, increased neutrophil count in ascites, and augmented NETs foci in peritoneal dissemination sites. In vitro assays revealed that G-CSF stimulated neutrophils to form NETs, promoting cancer cell adhesion. In vivo investigations revealed that G-CSF-producing tumor-bearing mice had accelerated peritoneal dissemination and poor prognosis. NETs formation was pathologically observed at the peritoneal dissemination sites. Inhibition of NETs formation by GSK484 significantly delayed peritoneal dissemination in vivo. In conclusion, G-CSF was associated with intra-abdominal NETs formation and increased peritoneal dissemination. NETs represent potential therapeutic targets for ovarian cancer, particularly in patients with neutrophilia.

Alpha-1 antitrypsin targeted neutrophil elastase protects against sepsis-induced inflammation and coagulation in mice via inhibiting neutrophil extracellular trap formation

AimsSepsis pathophysiology is complex and identifying effective treatments for sepsis remains challenging. The study aims to identify effective drugs and targets for sepsis through transcriptomic analysis of sepsis patients, repositioning analysis of compounds, and validation by animal models. Main methodsGSE185263 obtained from the GEO database that includes gene expression profiles of 44 healthy controls and 348 sepsis patients categorized by severity. Bioinformatic algorithms revealed the molecular, function, and immune characteristics of the sepsis, and constructed sepsis-related protein-protein interaction networks. Subsequently, Random Walk with Restart analysis was applied to identify candidate drugs for sepsis, which were tested in animal models for survival, inflammation, coagulation, and multi-organ damage. Key findingsOur analysis found 1862 genes linked to sepsis development, enriched in functions like neutrophil extracellular trap formation (NETs) and complement/coagulation cascades. With disease progression, immune activation-associated cells were inhibited, while immune suppression-associated cells were activated. Next, the drug repositioning method identified candidate drugs, such as alpha-1 antitrypsin, that may play a therapeutic role by targeting neutrophil elastase (NE) to inhibit NETs. Animal experiments proved that alpha-1 antitrypsin treatment can improve the survival rate, reduce sepsis score, reduce the levels of inflammation markers in serum, and alleviate muti-organ morphological damage in mice with sepsis. The further results showed that α-1 antitrypsin can inhibit the NETs by suppressing the NE for the treatment of sepsis. SignificanceAlpha-1 antitrypsin acted on the NE to inhibit NETs thereby protecting mice from sepsis-induced inflammation and coagulation.

Exogenous serpin B1 restricts immune complex-mediated NET formation via inhibition of a chymotrypsin-like protease and enhances microbial phagocytosis

Immune complex (IC)-driven formation of neutrophil extracellular traps (NETs) is a major contributing factor to the pathogenesis of autoimmune diseases including systemic lupus erythematosus (SLE). Exogenous recombinant human serpin B1 (rhsB1) can regulate NET formation; however, its mechanism(s) of action is currently unknown as is its ability to regulate IC-mediated NET formation and other neutrophil effector functions. To investigate this, we engineered or post-translationally modified rhsB1 proteins that possessed specific neutrophil protease inhibitory activities and pretreated isolated neutrophils with them prior to inducing NET formation with ICs derived from patients with SLE, PMA, or the calcium ionophore A23187. Neutrophil activation and phagocytosis assays were also performed with rhsB1 pretreated and IC-activated neutrophils. rhsB1 dose-dependently inhibited NET formation by all three agents in a process dependent on its chymotrypsin-like inhibitory activity, most likely cathepsin G. Only one variant (rhsB1 C344A) increased surface levels of neutrophil adhesion/activation markers on IC-activated neutrophils and boosted intracellular ROS production. Further, rhsB1 enhanced complement-mediated neutrophil phagocytosis of opsonized bacteria but not ICs. In conclusion, we have identified a novel mechanism of action by which exogenously administered rhsB1 inhibits IC, PMA, and A2138-mediated NET formation. Cathepsin G is a well-known contributor to autoimmune disease but to our knowledge, this is the first report implicating it as a potential driver of NET formation. We identified the rhsB1 C334A variant as a candidate protein that can suppress IC-mediated NET formation, boost microbial phagocytosis, and potentially impact additional neutrophil effector functions including ROS-mediated microbial killing in phagolysosomes.

Gene Regulation of Neutrophils Mediated Liver and Lung Injury through NETosis in Acute Pancreatitis.

Acute pancreatitis (AP) is one of the most common gastrointestinal emergencies, often resulting in self-digestion, edema, hemorrhage, and even necrosis of pancreatic tissue. When AP progresses to severe acute pancreatitis (SAP), it often causes multi-organ damage, leading to a high mortality rate. However, the molecular mechanisms underlying SAP-mediated organ damage remain unclear. This study aims to systematically mine SAP data from public databases and combine experimental validation to identify key molecules involved in multi-organ damage caused by SAP. Retrieve transcriptomic data of mice pancreatic tissue for AP, lung and liver tissue for SAP, and corresponding normal tissue from the Gene Expression Omnibus (GEO) database. Conduct gene differential analysis using Limma and DEseq2 methods. Perform enrichment analysis using the clusterProfiler package in R software. Score immune cells and immune status in various organs using single-sample gene set enrichment analysis (ssGSEA). Evaluate mRNA expression levels of core genes using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Validate serum amylase, TNF-α, IL-1β, and IL-6 levels in peripheral blood using enzyme-linked immunosorbent assay (ELISA), and detect the formation of neutrophil extracellular traps (NETs) in mice pancreatic, liver, and lung tissues using immunofluorescence. Differential analysis reveals that 46 genes exhibit expression dysregulation in mice pancreatic tissue for AP, liver and lung tissue for SAP, as well as peripheral blood in humans. Functional enrichment analysis indicates that these genes are primarily associated with neutrophil-related biological processes. ROC curve analysis indicates that 12 neutrophil-related genes have diagnostic potential for SAP. Immune infiltration analysis reveals high neutrophil infiltration in various organs affected by SAP. Single-cell sequencing analysis shows that these genes are predominantly expressed in neutrophils and macrophages. FPR1, ITGAM, and C5AR1 are identified as key genes involved in the formation of NETs and activation of neutrophils. qPCR and IHC results demonstrate upregulation of FPR1, ITGAM, and C5AR1 expression in pancreatic, liver, and lung tissues of mice with SAP. Immunofluorescence staining shows increased levels of neutrophils and NETs in SAP mice. Inhibition of NETs formation can alleviate the severity of SAP as well as the levels of inflammation in the liver and lung tissues. This study identified key genes involved in the formation of NETs, namely FPR1, ITGAM, and C5AR1, which are upregulated during multi-organ damage in SAP. Inhibition of NETs release effectively reduces the systemic inflammatory response and liver-lung damage in SAP. This research provides new therapeutic targets for the multi-organ damage associated with SAP.

Zixue Powder attenuates septic thrombosis via reducing neutrophil extracellular trap through blocking platelet STING activation

Ethnopharmacological relevanceMicrothrombosis is commonly seen in sepsis and COVID-19. Zixue Powder (ZXP) is a traditional Chinese herbal formula with the potential to treat microvascular and infectious diseases. However, the role and mechanism of ZXP in sepsis-associated thrombosis remain unclear. Aim of the studyInvestigating the therapeutic effectiveness and underlying mechanisms of ZXP in septic thrombosis. Materials and methodsZXP's compositions were examined with UPLC-QTOF-MS. The efficacy of ZXP on sepsis-induced thrombosis was assessed through various methods: liver tissue pathology was examined using hematoxylin-eosin staining, platelet count was determined by a blood cell analyzer, and an enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of serum tissue factor (TF), thromboxane B2 (TXB2), D-Dimer, and plasminogen activator inhibitor-1 (PAI-1). Neutrophil extracellular traps (NETs) were localized and expressed in liver tissues by immunofluorescence, and the number of NETs in peripheral blood was evaluated by ELISA, which measured the quantity of cf-DNA and MPO-DNA in serum. Platelet P-selectin expression and platelet-neutrophil aggregation were measured by flow cytometry, and plasma P-selectin expression was measured by ELISA. Furthermore, the mechanism of the stimulator of interferon genes (STING) signaling pathway in ZXP's anti-sepsis thrombosis effect was investigated using the STING agonist, Western blot experiments, and immunoprecipitation experiments. ResultsUPLC-QTOF-MS identified 40 chemical compositions of ZXP. Administration of ZXP resulted in significant improvements in liver thrombosis, platelet counts, and levels of TXB2, TF, PAI-1, and D-Dimer in septic rats. Moreover, ZXP inhibited NETs formation in both liver tissue and peripheral blood. Additionally, ZXP decreased the levels of P-selectin in both platelets and plasma, as well as the formation of platelet-neutrophil aggregates, thereby suppressing P-selectin-mediated NETs release. Immunoprecipitation and immunofluorescence staining experiments revealed that ZXP attenuated P-selectin secretion by inhibiting STING-mediated assembly of platelet soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) complex, ultimately preventing inhibition of NETs formation. ConclusionOur study showed that ZXP effectively mitigates platelet granule secretion primarily through modulation of the STING pathway, consequently impeding NET-associated thrombosis in sepsis. These findings offer valuable insights for future research on the development and application of ZXP.

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.

Atorvastatin combined with imipenem alleviates lung injury in sepsis by inhibiting neutrophil extracellular trap formation via the ERK/NOX2 signaling pathway

Sepsis is a systemic inflammatory response syndrome caused by the invasion of pathogenic microorganisms. Despite major advances in diagnosis and technology, morbidity and mortality remain high. The level of neutrophil extracellular traps (NETs) is closely associated with the progression and prognosis of sepsis, suggesting the regulation of NET formation as a new strategy in sepsis treatment. Owing to its pleiotropic effects, atorvastatin, a clinical lipid-lowering drug, affects various aspects of sepsis-related inflammation and immune responses. To align closely with clinical practice, we combined it with imipenem for the treatment of sepsis. In this study, we used a cecum ligation and puncture-induced lung injury mouse model and employed techniques including western blot, immunofluorescence, and enzyme-linked immunosorbent assay to measure the levels of NETs and other sepsis-related lung injury indicators. Our findings indicate that atorvastatin effectively inhibited the formation of NETs. When combined with imipenem, it significantly alleviated lung injury, reduced systemic inflammation, and improved the 7-day survival rate of septic mice. Additionally, we explored the inhibitory mechanism of atorvastatin on NET formation in vitro, revealing its potential action through the ERK/NOX2 pathway. Therefore, atorvastatin is a potential immunomodulatory agent that may offer new treatment strategies for patients with sepsis in clinical settings.

Salidroside ameliorates acute liver transplantation rejection in rats by inhibiting neutrophil extracellular trap formation.

Acute rejection is an important factor affecting the survival of recipients after liver transplantation. Salidroside has various properties, including anti-inflammatory, antioxidant, and hepatoprotective properties. This study aims to investigate whether salidroside can prevent acute rejection after liver transplantation and to examine the underlying mechanisms involved. An in vivo acute rejection model is established in rats that are pretreated with tacrolimus (1 mg/kg/d) or salidroside (10 or 20 mg/kg/d) for seven days after liver transplantation. In addition, an in vitro experiment is performed using neutrophils incubated with salidroside (1, 10, 50 or 100 μM). Hematoxylin-eosin staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, immunosorbent assays, immunofluorescence analysis, Evans blue staining, and western blot analysis are performed to examine the impact of salidroside on NET formation and acute rejection in vitro and in vivo. We find that Salidroside treatment reduces pathological liver damage, serum aminotransferase level, and serum levels of IL-1β, IL-6, and TNF-α in vivo. The expressions of proteins associated with the HMGB1/TLR-4/MAPK signaling pathway (HMGB1, TLR-4, p-ERK1/2, p-JNK, p-P38, cleaved caspase-3, cleaved caspase-9, Bcl-2, Bax, IL-1β, TNF-α, and IL-6) are also decreased after salidroside treatment. In vitro experiments show that the release of HMGB1/TLR-4/MAPK signaling pathway-associated proteins from neutrophils treated with lipopolysaccharide is decreased by salidroside. Moreover, salidroside inhibits NETosis and protects against acute rejection by regulating the HMGB1/TLR-4/MAPK signaling pathway. Furthermore, salidroside combined with tacrolimus has a better effect than either of the other treatments alone. In summary, salidroside can prevent acute liver rejection after liver transplantation by reducing neutrophil extracellular trap development through the HMGB1/TLR-4/MAPK signaling pathway.

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 < .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.

Cyclic guanosine monophosphate-adenosine monophosphate synthetase/stimulator of interferon genes signaling aggravated corneal allograft rejection through neutrophil extracellular traps

The activation of innate immunity following transplantation has been identified as a crucial factor in allograft inflammation and rejection. However, the role of cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)/stimulator of interferon genes (STING) signaling-mediated innate immunity in the pathogenesis of allograft rejection remains unclear. Utilizing a well-established murine model of corneal transplantation, we demonstrated increased expression of cGAS and STING in rejected-corneal allografts compared with syngeneic (Syn) and normal (Nor) corneas, along with significant activation of the cGAS/STING pathway, as evidenced by the enhanced phosphorylation of TANK-binding kinase 1and interferon regulatory factor 3. Pharmacological and genetic inhibition of cGAS/STING signaling markedly delayed corneal transplantation rejection, resulting in prolonged survival time and reduced inflammatory infiltration. Furthermore, we observed an increase in the formation of neutrophil extracellular traps (NETs) in rejected allografts, and the inhibition of NET formation through targeting peptidylarginine deiminase 4 and DNase I treatment significantly alleviated immune rejection and reduced cGAS/STING signaling activity. Conversely, subconjunctival injection of NETs accelerated corneal transplantation rejection and enhanced the activation of the cGAS/STING pathway. Collectively, these findings demonstrate that NETs contribute to the exacerbation of allograft rejection via cGAS/STING signaling, highlighting the targeting of the NETs/cGAS/STING signaling pathway as a potential strategy for prolonging allograft survival.

Corilagin Inhibits Neutrophil Extracellular Trap Formation and Protects against Hydrochloric Acid/Lipopolysaccharide-Induced Acute Lung Injury in Mice by Suppressing the STAT3 and NOX2 Signaling Pathways.

Acute lung injury (ALI) and its severe manifestation, acute respiratory distress syndrome (ARDS), are characterized by uncontrolled inflammatory responses, neutrophil activation and infiltration, damage to the alveolar capillary membrane, and diffuse alveolar injury. Neutrophil extracellular traps (NETs), formed by activated neutrophils, contribute significantly to various inflammatory disorders and can lead to tissue damage and organ dysfunction. Corilagin, a compound found in Phyllanthus urinaria, possesses antioxidative and anti-inflammatory properties. In this study, we investigated the protective effects and underlying mechanisms of corilagin in hydrochloric acid (HCl)/lipopolysaccharide (LPS)-induced lung injury. Mice received intraperitoneal administration of corilagin (2.5, 5, or 10 mg/kg) or an equal volume of saline 30 min after intratracheal HCl/LPS administration. After 20 h, lung tissues were collected for analysis. Corilagin treatment significantly mitigated lung injury, as evidenced by reduced inflammatory cell infiltration, decreased production of proinflammatory cytokines, and alleviated oxidative stress. Furthermore, corilagin treatment suppressed neutrophil elastase expression, reduced NET formation, and inhibited the expression of ERK, p38, AKT, STAT3, and NOX2. Our findings suggest that corilagin inhibits NET formation and protects against HCl/LPS-induced ALI in mice by modulating the STAT3 and NOX2 signaling pathways.

Neutrophil Extracellular Traps Regulate Surgical Brain Injury by Activating the cGAS-STING Pathway

Surgical brain injury (SBI), induced by neurosurgical procedures or instruments, has not attracted adequate attention. The pathophysiological process of SBI remains sparse compared to that of other central nervous system diseases thus far. Therefore, novel and effective therapies for SBI are urgently needed. In this study, we found that neutrophil extracellular traps (NETs) were present in the circulation and brain tissues of rats after SBI, which promoted neuroinflammation, cerebral edema, neuronal cell death, and aggravated neurological dysfunction. Inhibition of NETs formation by peptidylarginine deiminase (PAD) inhibitor or disruption of NETs with deoxyribonuclease I (DNase I) attenuated SBI-induced damages and improved the recovery of neurological function. We show that SBI triggered the activation of cyclic guanosine monophosphate–adenosine monophosphate synthase stimulator of interferon genes (cGAS-STING), and that inhibition of the cGAS-STING pathway could be beneficial. It is worth noting that DNase I markedly suppressed the activation of cGAS-STING, which was reversed by the cGAS product cyclic guanosine monophosphate–adenosine monophosphate (cGMP-AMP, cGAMP). Furthermore, the neuroprotective effect of DNase I in SBI was also abolished by cGAMP. NETs may participate in the pathophysiological regulation of SBI by acting through the cGAS-STING pathway. We also found that high-dose vitamin C administration could effectively inhibit the formation of NETs post-SBI. Thus, targeting NETs may provide a novel therapeutic strategy for SBI treatment, and high-dose vitamin C intervention may be a promising translational therapy with an excellent safety profile and low cost.Graphical The schematic diagram shows the formation of NETs activated cGAS-STING pathway after SBI, leading to increased microglia activation, accompanied with elevation of inflammatory factors, which in turn aggravated brain injury.