Induce Neutrophil Extracellular Traps Formation Research Articles

IL33-induced neutrophil extracellular traps (NETs) mediate a positive feedback loop for synovial inflammation and NET amplification in rheumatoid arthritis.

This study investigated the mechanisms driving the induction and sustained presence of neutrophil extracellular traps (NETs) in the synovial microenvironment of rheumatoid arthritis (RA). Synovial tissue and fluid samples were collected from patients with RA and osteoarthritis (OA), and NET levels and cytokine concentrations were measured using a cytometric bead array and enzyme-linked immunosorbent assay (ELISA). The ability of interleukin-33 (IL-33) to induce NET formation was evaluated using quantitative assays, immunofluorescence staining, live-cell imaging, and electron microscopy. Coincubation experiments of NETs with fibroblast-like synovial cells (FLSs) were conducted, and a modified Transwell migration assay was designed to assess neutrophil migration. The role of IL-33 and NETs in RA progression was further investigated using a collagen antibody-induced arthritis (CAIA) mouse model. The results revealed an increase in NETs and IL-33 levels in the synovial fluid of RA patients, with a significant positive correlation between them. NET formation assays confirmed that IL-33 activates neutrophils to produce NETs and that neutrophils from RA patients exhibit increased responsiveness to IL-33 stimulation. Both in vitro and in vivo evidence has demonstrated that NETs stimulate FLSs to secrete IL-33 and the chemokine CXCL8 via Toll-like receptor 9, promoting further neutrophil recruitment and increasing NET production within the RA synovium. This study reveals a novel positive feedback loop involving NETs and FLSs that is mediated by IL-33 that increases NET accumulation in RA. Targeting IL-33 or NET formation and amplification may offer new therapeutic strategies for managing RA.

Neutrophil extracellular DNA traps activate the TLR9 signaling pathway of pancreatic ductal epithelial cells in patients with type 2 autoimmune pancreatitis

The presence of neutrophil infiltration around the pancreatic ducts has been found to be associated with type 2 autoimmune pancreatitis (AIP). However, the functional role and clinical significance of neutrophil migration in the progression of pancreatitis is not fully understood. Here, we found that neutrophil extracellular traps (NETs) are abundant around the pancreatic duct in patients with type 2 AIP. We also observed an increased expression of toll-like receptor 9 (TLR9) in pancreatic ductal epithelial cells (HPDEC) in type 2 AIP patients compared to other pancreatic diseases. TLR9 acts as the DNA component of NETs (NET-DNA) receptor in HPDEC, which senses extracellular DNA and subsequently activates the NF-κB pathway to promote neutrophil recruitment and induce NET formation. In addition, our results indicated that the hydroxychloroquine (HCQ), acting as a TLR9 antagonist, could effectively inhibit the activation of inflammatory pathways, reduce neutrophil migration and block the positive feedback loop. The intervention positions HCQ acts as a potential target drug for the clinical treatment of type 2 AIP.

5-oxoETE promote thrombosis in antiphospholipid syndrome by triggering NETs formation through PLC/PKC/ERK pathway.

One mechanism by which antiphospholipid syndrome (APS) IgG contribute to thrombotic events in patients with APS is through the potentiation of neutrophil extracellular traps (NETs) release. However, the exact mechanism by which APS IgG induces NETs formation and thrombosis has not been fully elucidated. We conducted untargeted metabolomics on serum samples from thrombotic APS patients to identify metabolic changes. The effect of 5-oxoETE on NETs formation and oxidative stress was evaluated in vitro by treating neutrophils with various concentrations of 5-oxoETE. The involvement of the PLC/PKC/ERK signaling pathway in 5-oxoETE-induced NETs formation was examined using pharmacological inhibitors. In vivo, we assessed the effects of inhibiting 5-oxoETE synthesis or blocking its receptor (OXE-R) on NETs formation and thrombosis in APS mouse models. Serum metabolomics revealed significantly elevated levels of 5-oxoETE in APS patients. In vitro experiments demonstrated that 5-oxoETE, via OXE-R activation of the PLC/PKC/ERK signaling pathway, increased NETs formation and oxidative stress in a dose-dependent manner. In vivo, inhibiting 5-oxoETE synthesis or OXE-R reduced NETs formation and attenuated venous thrombosis in APS mice models. This study identifies 5-oxoETE as a critical mediator of NET formation and thrombosis in APS. Targeting 5-oxoETE or OXE-R may offer a promising therapeutic approach for thrombotic APS and other NET-associated autoimmune diseases.

Evaluation of Neutrophil Extracellular Trap Formation on Smokers and Non-Smokers with Periodontitis

Objevtive: In this study, we aimed to evaluate neutrophil extracellular trap (NET) in smoker and non-smoker periodontitis patients. Materials and Methods: 21 subjects [non-smokers and periodontally healthy (S-P-) 7 people, non-smokers with periodontitis (S-P+) 7 people, smokers with periodontitis (S+P+) 7 people] were included in this study. Plaque index (PI), gingival index (GI), probing depth (PD) and clinical attachment level (CAL) parameters were recorded. The peripheral neutrophils isolated from the subjects were activated by phorbol 12-myristate 13-acetate (PMA) to induce NET formation. NET formed after the activation was measured with the fluorometer. Results: In the S-P- group PI, GI, PD and CAL values were found statistically significantly low (P=0,001; P=

Poly(ADP-Ribose) Polymerase-1 Regulates Pyroptosis Independent Function of NLRP3 Inflammasome in Neutrophil Extracellular Trap Formation.

Neutrophil extracellular traps (NETs) function to control infectious agents as well as to propagate inflammatory response in a variety of disease conditions. DNA damage associated with chromatin decondensation and NACHT domain-leucine-rich repeat-and pyrin domain-containing protein 3 (NLRP3) inflammasome activation have emerged as crucial events in NET formation, but the link between the two processes is unknown. In this study, we demonstrate that poly(ADP-ribose) polymerase-1 (PARP-1), a key DNA repair enzyme, regulates NET formation triggered by NLRP3 inflammasome activation in neutrophils. Activation of mouse neutrophils with canonical NLRP3 stimulants LPS and nigericin induced NET formation, which was significantly abrogated by pharmacological inhibition of PARP-1. We found that PARP-1 is required for NLRP3 inflammasome assembly by regulating post-transcriptional levels of NLRP3 and ASC dimerization. Importantly, this PARP-1-regulated NLRP3 activation for NET formation was independent of inflammasome-mediated pyroptosis, because caspase-1 and gasdermin D processing as well as IL-1β transcription and secretion remained intact upon PARP-1 inhibition in neutrophils. Accordingly, pharmacological inhibition or genetic ablation of caspase-1 and gasdermin D had no effect on NLRP3-mediated NET formation. Mechanistically, PARP-1 inhibition increased p38 MAPK activity, which was required for downmodulation of NLRP3 and NETs, because concomitant inhibition of p38 MAPK with PARP-1 restored NLRP3 activation and NET formation. Finally, mice undergoing bacterial peritonitis exhibited increased survival upon treatment with PARP-1 inhibitor, which correlated with increased leukocyte influx and improved intracellular bacterial clearance. Our findings reveal a noncanonical pyroptosis-independent role of NLRP3 in NET formation regulated by PARP-1 via p38 MAPK, which can be targeted to control NETosis in inflammatory diseases.

Fibrin aggravates periodontitis through inducing NETs formation from mitochondrial DNA.

This study investigated the role of fibrin on neutrophil extracellular traps (NETs) formation from neutrophils and to elucidate the involvement of mitochondria in NETs formation during periodontitis. Plasminogen-deficient (Plg-/-) mice were employed to evaluate the effects of fibrin deposition on inflammation, bone resorption, and neutrophil infiltration in periodontal tissues. In addition, invitro tests evaluated fibrin's impact on neutrophil-driven inflammation. Mitochondrial reactive oxygen species (mtROS) levels within neutrophils were quantified utilizing flow cytometry and immunofluorescence invitro. Furthermore, the anti-inflammatory properties of the mtROS scavenger, Mito-TEMPO, were confirmed to regulate the NET formation invitro and invivo. Plasminogen deficiency resulted in increased fibrin deposition, neutrophil infiltration, inflammatory factors concentration, and alveolar bone resorption in periodontal tissues. After neutrophils were treated by fibrin invitro, the expression of inflammatory factors, the formation of mtROS, and NETs enriched in mitochondrial DNA (mtDNA) were upregulated, which were reversed by Mito-TEMPO invitro. Moreover, Mito-TEMPO alleviated inflammation in Plg-/- mice. This study showed that fibrin deposition in gingiva induced the NET formation in Plg-/- mice, in which the DNA in NETs was from mitochondria depending on increasing mtROS.

Bacteriophages isolated from mouse feces attenuates pneumonia mice caused by Pseudomonas aeruginosa.

Most of the current bacteriophages (phages) are mostly isolated from environments. However, phages isolated from feces might be more specific to the bacteria that are harmful to the host. Meanwhile, some phages from the environment might affect non-pathogenic bacteria for the host. Here, bacteriophages isolated from mouse feces were intratracheally (IT) or intravenously (IV) administered in pneumonia mice caused by Pseudomonas aeruginosa at 2 hours post-intratracheal bacterial administration. As such, the mice with phage treatment, using either IT or IV administration, demonstrated less severe pneumonia as indicated by mortality, serum cytokines, bacteremia, bacterial abundance in bronchoalveolar lavage fluid (BALF), and neutrophil extracellular traps (NETs) in lung tissue (immunofluorescence of neutrophil elastase and myeloperoxidase). Interestingly, the abundance of phages in BALF from the IT and IV injections was similar, supporting a flexible route of phage administration. With the incubation of bacteria with neutrophils, the presence of bacteriophages significantly improved bactericidal activity, but not NETs formation, with the elevated supernatant IL-6 and TNF-α, but not IL-1β. In conclusion, our findings suggest that bacteriophages against Pseudomonas aeruginosa can be discovered from feces of the host. The phages attenuate pneumonia partly through an enhanced neutrophil bactericidal activity, but not via inducing NETs formation. The isolation of phages from the infected hosts themselves might be practically useful for future treatment. More studies are warranted.

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.

IL-8 Induces Neutrophil Extracellular Trap Formation in Severe Thermal Injury.

Neutrophil extracellular traps (NETs) have a dual role in the innate immune response to thermal injuries. NETs provide an early line of defence against infection. However, excessive NETosis can mediate the pathogenesis of immunothrombosis, disseminated intravascular coagulation (DIC) and multiple organ failure (MOF) in sepsis. Recent studies suggest that high interleukin-8 (IL-8) levels in intensive care unit (ICU) patients significantly contribute to excessive NET generation. This study aimed to determine whether IL-8 also mediates NET generation in patients with severe thermal injuries. IL-8 levels were measured in serum samples from thermally injured patients with ≥15% of the total body surface area (TBSA) and healthy controls (HC). Ex vivo NET generation was also investigated by treating isolated neutrophils with serum from thermal injured patients or normal serum with and without IL-8 and anti-IL-8 antibodies. IL-8 levels were significantly increased compared to HC on days 3 and 5 (p < 0.05) following thermal injury. IL-8 levels were also significantly increased at day 5 in septic versus non-septic patients (p < 0.001). IL-8 levels were also increased in patients who developed sepsis compared to HC at days 3, 5 and 7 (p < 0.001), day 10 (p < 0.05) and days 12 and 14 (p < 0.01). Serum containing either low, medium or high levels of IL-8 was shown to induce ex vivo NETosis in an IL-8-dependent manner. Furthermore, the inhibition of DNase activity in serum increased the NET-inducing activity of IL-8 in vitro by preventing NET degradation. IL-8 is a major contributor to NET formation in severe thermal injury and is increased in patients who develop sepsis. We confirmed that DNase is an important regulator of NET degradation but also a potential confounder within assays that measure serum-induced ex vivo NETosis.

Neutrophil Extracellular Traps in Myocardial Tissue Drive Cardiac Dysfunction and Adverse Outcomes in Patients With Heart Failure With Dilated Cardiomyopathy.

The immune systems and chronic inflammation are implicated in the pathogenesis of dilated cardiomyopathy (DCM) and heart failure. However, the significance of neutrophil extracellular traps (NETs) in heart failure remains to be elucidated. We enrolled consecutive 62 patients with heart failure with idiopathic DCM who underwent endomyocardial biopsy. Biopsy specimens were subjected to fluorescent immunostaining to detect NETs, and clinical and outcome data were collected. Ex vivo and in vivo experiments were conducted. The numbers of NETs per myocardial tissue area and the proportion of NETs per neutrophil were significantly higher in patients with DCM compared with non-DCM control subjects without heart failure, and the numbers of NETs were negatively correlated with left ventricular ejection fraction. Patients with DCM with NETs (n=32) showed lower left ventricular ejection fraction and higher BNP (B-type natriuretic peptide) than those without NETs (n=30). In a multivariable Cox proportional hazard model, the presence of NETs was independently associated with an increased risk of adverse cardiac events in patients with DCM. To understand specific underlying mechanisms, extracellular flux analysis in ex vivo revealed that NETs-containing conditioned medium from wild-type neutrophils or purified NET components led to impaired mitochondrial oxygen consumption of cardiomyocytes, while these effects were abolished when PAD4 (peptidyl arginine deiminase 4) in neutrophils was genetically ablated. In a murine model of pressure overload, NETs in myocardial tissue were predominantly detected in the acute phase and persisted throughout the ongoing stress. Four weeks after transverse aortic constriction, left ventricular ejection fraction was reduced in wild-type mice, whereas PAD4-deficient mice displayed preserved left ventricular ejection fraction without inducing NET formation. NETs in myocardial tissue contribute to cardiac dysfunction and adverse outcomes in patients with heart failure with DCM, potentially through mitochondrial dysfunction of cardiomyocytes.

Low density granulocytes and neutrophil extracellular trap formation are increased in incomplete systemic lupus erythematosus.

To investigate the proportion of low-density granulocytes (LDGs), circulating plasma neutrophil extracellular traps (NETs), and serum-induced NET formation in patients with incomplete systemic lupus erythematosus (iSLE) and systemic lupus erythematosus (SLE). LDGs were measured cross-sectionally in 18 iSLE patients, 11 SLE patients and 14 healthy controls (HCs), whereas circulating NETs and serum-induced NET formation were assessed in 35 iSLE patients, 41 SLE patients and 16 HCs. LDGs (CD14lowCD15+) were measured in PBMCs using flow cytometry and circulating plasma NETs were measured using anti-myeloperoxidase-DNA, anti-citrullinated histone H3 and anti-elastase-DNA complex ELISAs. Serum-induced NET formation was assessed by incubating healthy neutrophils with serum from iSLE patients, SLE patients or HCs and visualizing NETs with fluorescence microscopy. Proportions of LDGs and circulating plasma NETs were similarly elevated in iSLE and SLE patients compared with those in HCs. Furthermore, patients under hydroxychloroquine (HCQ) treatment had lower proportions of LDGs than those without. Serum from iSLE and SLE patients similarly induced NET formation in healthy neutrophils. In iSLE patients, myeloperoxidase-DNA complexes were correlated with proportions of age-associated B-cells, memory B-cells and negatively with naïve B-cells, while we did not find associations between measures of NETs or serum-induced NET formation and interferon score or clinical parameters. These results show that neutrophil dysfunction, including higher proportions of LDGs, and increased NET formation, already occur in iSLE, similar to SLE, despite differences in disease manifestations. Thereby, neutrophil dysfunction may contribute to sustained exposure to autoantigens and autoreactivity in early stages of SLE.

NETosis and SARS-COV-2 Infection-Related Autoimmunity: A Narrative Review

Background: Neutrophil extracellular traps (NETs) are crucial for antimicrobial defense in sepsis, trapping and neutralizing pathogens while minimizing damage to host cells. Neutrophil extracellular traps, comprising nuclear chromatin, histones, and antimicrobial cytoplasmic granules, are significant. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) triggers multiple complications in patients. Decondensed NETs and associated proteins can become a critical source of autoantigens, presented to the immune system, thereby inducing hyperinflammation and autoimmunity in sepsis and viral diseases. Objectives: This study aimed to explore NETosis and its role in autoimmunity in coronavirus disease 2019 (COVID-19). Methods: We reviewed the PubMed database using keywords (autoimmune disease OR autoimmunity AND SARS-CoV-2 OR COVID-19 AND NETosis OR NETs) to enhance understanding of NETosis in autoimmune diseases and COVID-19, ultimately analyzing 55 articles. Results: Neutrophil extracellular traps play a pivotal role in host defense against pathogens. However, their overproduction can lead to self-tolerance failure and immune system activation, contributing to autoimmunity. Autoimmune phenomena have been observed in many COVID-19 patients, with a pathophysiology similar to other viral infections that induce NET formation. Conclusions: SARS-CoV-2 infections may cause immune system hyperactivation and subsequent autoimmunity. Like in other viral infections, NETosis could play a significant role in the autoimmunity associated with SARS-CoV-2. Modulating NET formation and degradation could potentially reduce the severity of the disease.

Abstract PO2-25-01: Tumor Derived Chitinase-3 like 1 Induces Neutrophil Extracellular Traps that Promote T cell Exclusion and Resistance to Immunotherapies in Breast Cancer

Abstract Breast cancer remains the most prevalent cancer and a major cause of mortality affecting women worldwide. Chemotherapy along with radiation and surgery have long been the standard of care for breast cancer patients. However, devastating side effects, resistance to treatments and relapse are major concerns for patients and physicians. With the rise of immunotherapies as a treatment option for various cancers, there is a critical need to understand the mechanisms through which cancers evade the immune system. Targeting such mechanisms holds the key to improve patient response to immunotherapy, especially in breast cancer where efficacy rates remain low. In a subset of poor outcome Triple Negative Breast Cancers, cytotoxic T cells are excluded from the tumor nest and restricted to the surrounding stroma, a phenomenon known as stromal restriction. Our data from genetically engineered mouse models and human tumors identified that the secreted cytokine Chitinase-3 like 1 (Chi3l1) promotes stromal restriction of T cells in several tumor types including breast, lung and colon. Chi3l1 ablation in pre-clinical models of breast cancer results in increased T cell infiltration into the tumor nest which delayed mammary tumorigenesis and improved response to immunotherapy. We further demonstrate that Chi3l1 promotes T cell exclusion through the direct induction and deposition of neutrophil extracellular traps (NETs) which form a barrier that restricts T cell entry. Neutrophil depletion or pharmacological disruption of NET formation abrogates Chi3l1-induced T cell exclusion and histologically phenocopies Chi3l1 ablation. Thus, our results indicate that Chi3l1 is a major immunosuppressive cytokine that promotes breast cancer progression by inducing NET formation and inhibiting T cell infiltration. Given the importance of T cell infiltration in immune elimination of nascent tumors, the future targeting of Chi3l1 should improve immunotherapy efficacy and outcomes in patients with tumors characterized by a T cell excluded microenvironment. Citation Format: Tarek Taifour, Sherif Samer Attalla, Dongmei Zuo, Yu Gu, Virginie Sanguin-Gendreau, Hailey Dall-Proud, Emilie Solymoss, Tung Bui, Hellen Kuasne, Vasilios Papavasiliou, Chun Geun Lee, Suchitra Kamle, Peter Siegel, Jack Elias, Morag Park, William Muller. Tumor Derived Chitinase-3 like 1 Induces Neutrophil Extracellular Traps that Promote T cell Exclusion and Resistance to Immunotherapies in Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-25-01.

Saikosaponin a promotes neutrophil extracellular trap formation and bactericidal activity

This study aimed to investigate the effects of SSa, one of the major triterpenoid saponins extracted from Radix bupleuri, on neutrophil extracellular trap (NET) formation and the mechanism associated with this process. Using Sytox green and immunofluorescence assays, we found SSa rapidly induced NET formation, which depended on NADPH oxidase (NOX)-independent ROS production and autophagy. Pharmacologic inhibitor studies indicated that ERK and PI3K/AKT signalling were also required for SSa-induced NET formation, whereas protein arginine deiminase 4 (PAD4) was not required. Furthermore, we found that SSa promoted neutrophil bactericidal activity mainly through NET formation. Based on flow cytometry and the Cell Counting Kit-8 (CCK-8) assays, the results demonstrated that SSa-induced NET formation occurred without neutrophil death. Taken together, these findings indicated that SSa could be a potential natural product to boost innate immune defense against pathogen attack via NET formation.

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

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

The In Vitro Effects of Carprofen on Lipopolysaccharide-Induced Neutrophil Extracellular Trap Formation in Dairy Cows.

The objective of this study was to develop an in vitro model that mimics inflammatory reactions and neutrophil extracellular traps (NETs) formation by polymorphonuclear leukocytes (PMNs) in dairy cows. This model was used to examine the effect of carprofen (CA) on lipopolysaccharide (LPS)-induced NETs formation and expression of inflammatory factors. Peripheral blood samples were collected from 24 Holstein cows (3-11 days postpartum) and PMNs were isolated. In three replicates, PMNs were exposed to various treatments to establish an appropriate in vitro model, including 80 μg/mL of LPS for 2 h, followed by co-incubation for 1 h with 60 μmol/L CA and 80 μg/mL LPS. The effects of these treatments were evaluated by assessing NETs formation by extracellular DNA release, gene expression of pro-inflammatory cytokines, reactive oxygen species (ROS) production, and the expression of NETs-related proteins, including histone3 (H3), citrullinated histone (Cit-H3), cathepsin G (CG), and peptidyl arginine deiminase 4 (PAD4). The assessment of these parameters would elucidate the specific mechanism by which CA inhibits the formation of NETs through the PAD4 pathway instead of modulating the Nox2 pathway. This highlights CA's effect on chromatin decondensation during NETs formation. Statistical analyses were performed utilizing one-way ANOVA with Bonferroni correction. The results demonstrated that LPS led to an elevated formation of NETs, while CA mitigated most of these effects, concurrent the PAD4 protein level increased with LPS stimulating and decreased after CA administration. Nevertheless, the intracellular levels of ROS did not change under the presence of LPS. LPS supplementation resulted in an upregulation of H3 and Cit-H3 protein expression levels. Conversely, the CA administration inhibited their expression. Additionally, there was no change in the expression of CG with either LPS or LPS + CA co-stimulation. The gene expression of pro-inflammatory cytokines (tumor necrosis factor -α, interleukin (IL)-18, IL-1β, and IL-6) upregulated with LPS stimulation, while the treatment with CA inhibited this phenomenon. In conclusion, CA demonstrated a pronounced inhibitory effect on both LPS-induced NETs formation as well as the associated inflammatory response.

B-1a cells scavenge NETs to attenuate sepsis.

B-1a cells, a regulatory subset of B lymphocytes, produce natural IgM and interleukin-10. Neutrophil extracellular traps (NETs) play a crucial role in pathogen defense, but their excessive formation during sepsis can cause further inflammation and tissue damage. In sepsis, extracellular cold-inducible RNA-binding protein (eCIRP), a damage-associated molecular pattern, is released to induce NET formation. We hypothesize that B-1a cells clear NETs to prevent sepsis-induced injury. Sepsis in mice was induced by injecting 1 × 107 and 5 × 107 colony-forming units of Escherichia coli intraperitoneally. After 4 and 20 h, we assessed the number of B-1a cells in the peritoneal cavity using flow cytometry. Our results showed that the number of peritoneal B-1a cells was significantly decreased in E. coli sepsis mice. Importantly, replenishing B-1a cells via intraperitoneal injection in sepsis mice significantly decreased NETs in peritoneal neutrophils. We also observed a decrease in serum inflammation and injury markers and a significant increase in the overall survival rate in B-1a cell-treated septic mice. To understand the mechanism, we cocultured bone marrow-derived neutrophils with peritoneal B-1a cells in a contact or noncontact condition using an insert and stimulated them with eCIRP. After 4 h, we found that eCIRP significantly increased NET formation in bone marrow-derived neutrophils. Interestingly, we observed that B-1a cells inhibited NETs by 67% in a contact-dependent manner. Surprisingly, when B-1a cells were cultured in inserts, there was no significant decrease in NET formation, suggesting that direct cell-to-cell contact is crucial for this inhibitory effect. We further determined that B-1a cells promoted NET phagocytosis, and this was mediated through natural IgM, as blocking the IgM receptor attenuated the engulfment of NETs by B-1a cells. Finally, we identified that following their engulfment, NETs were localized into the lysosomal compartment for lysis. Thus, our study suggests that B-1a cells decrease NET content in eCIRP-treated neutrophils and E. coli sepsis mice.

PSTPIP2 ameliorates aristolochic acid nephropathy by suppressing interleukin-19-mediated neutrophil extracellular trap formation.

Aristolochic acid nephropathy (AAN) is a progressive kidney disease caused by herbal medicines. Proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2) and neutrophil extracellular traps (NETs) play important roles in kidney injury and immune defense, respectively, but the mechanism underlying AAN regulation by PSTPIP2 and NETs remains unclear. We found that renal tubular epithelial cell (RTEC) apoptosis, neutrophil infiltration, inflammatory factor, and NET production were increased in a mouse model of AAN, while PSTPIP2 expression was low. Conditional knock-in of Pstpip2 in mouse kidneys inhibited cell apoptosis, reduced neutrophil infiltration, suppressed the production of inflammatory factors and NETs, and ameliorated renal dysfunction. Conversely, downregulation of Pstpip2 expression promoted kidney injury. In vivo, the use of Ly6G-neutralizing antibody to remove neutrophils and peptidyl arginine deiminase 4 (PAD4) inhibitors to prevent NET formation reduced apoptosis, alleviating kidney injury. In vitro, damaged RTECs released interleukin-19 (IL-19) via the PSTPIP2/nuclear factor (NF)-κB pathway and induced NET formation via the IL-20Rβ receptor. Concurrently, NETs promoted apoptosis of damaged RTECs. PSTPIP2 affected NET formation by regulating IL-19 expression via inhibition of NF-κB pathway activation in RTECs, inhibiting RTEC apoptosis, and reducing kidney damage. Our findings indicated that neutrophils and NETs play a key role in AAN and therapeutic targeting of PSTPIP2/NF-κB/IL-19/IL-20Rβ might extend novel strategies to minimize Aristolochic acid I-mediated acute kidney injury and apoptosis.

Ferritin-mediated neutrophil extracellular traps formation and cytokine storm via macrophage scavenger receptor in sepsis-associated lung injury

BackgroundSepsis is a severe systemic inflammatory disorder manifested by a dysregulated immune response to infection and multi-organ failure. Numerous studies have shown that elevated ferritin levels exist as an essential feature during sepsis and are able to suggest patients’ prognoses. At the same time, the specific mechanism of ferritin-induced inflammatory injury remains unclear.MethodsHyper-ferritin state during inflammation was performed by injecting ferritin into a mouse model and demonstrated that injection of ferritin could induce a systemic inflammatory response and increase neutrophil extracellular trap (NET) formation.Padi4−/−, Elane−/− and Cybb−/− mice were used for the NETs formation experiment. Western blot, immunofluorescence, ELISA, and flow cytometry examined the changes in NETs, inflammation, and related signaling pathways.ResultsFerritin induces NET formation in a peptidylarginine deiminase 4 (PAD4), neutrophil elastase (NE), and reactive oxygen species (ROS)-dependent manner, thereby exacerbating the inflammatory response. Mechanistically, ferritin induces the expression of neutrophil macrophage scavenger receptor (MSR), which promotes the formation of NETs. Clinically, high levels of ferritin in patients with severe sepsis correlate with NETs-mediated cytokines storm and are proportional to the severity of sepsis-induced lung injury.ConclusionsIn conclusion, we demonstrated that hyper-ferritin can induce systemic inflammation and increase NET formation in an MSR-dependent manner. This process relies on PAD4, NE, and ROS, further aggravating acute lung injury. In the clinic, high serum ferritin levels are associated with elevated NETs and worse lung injury, which suggests a poor prognosis for patients with sepsis. Our study indicated that targeting NETs or MSR could be a potential treatment to alleviate lung damage and systemic inflammation during sepsis.CG19NQAB-3s1ZkbgUbzmoXVideo

Nanoparticles for stimulation of neutrophil extracellular trap-mediated immunity.

Neutrophil extracellular traps (NETs) have been identified as triggers for a self-limited inflammatory reaction upon contact with nanoparticles within our bodies. This typically results in entrapping potentially harmful nano- or micro-objects following an immune burst. The demand for potent adjuvants has led to research on particulate-based adjuvants, particularly those that act via NET formation. Various particles, including hydrophobic nanoparticles, needle-like microparticles, and other natural and artificial crystals, have been shown to induce NET formation, eliciting a robust humoral and cellular immune response toward co-injected antigens. The NET formation was found to be the basis of the efficient use of alum as a vaccine adjuvant. Thus, nanoparticles with specific surface properties serve as NET-stimulating adjuvants. In this mini-review, we aim to summarize the current knowledge about the surface properties of particulate objects and the molecular pathways involved in inducing NET formation by neutrophils. Additionally, we discuss the potential use of nanoparticles for activating neutrophils in the tissues and the exploitation of such activation for enhancing vaccine adjuvants.