Induce Neutrophil Extracellular Traps Formation Research Articles

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 IL-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 CFU E. coli intraperitoneally (i.p.). After 4 and 20 hours, 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 i.p. 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 overall survival rate in B-1a cell-treated septic mice. To understand the mechanism, we co-cultured bone marrow-derived neutrophils (BMDNs) with peritoneal B-1a cells in a contact or non-contact condition using an insert and stimulated them with eCIRP. After 4 hours, we found that eCIRP significantly increased NET formation in BMDNs. 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 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.

Neuron Derived Cold-Inducible RNA-Binding Protein Promotes NETs Formation to Exacerbate Brain Endothelial Barrier Disruption after Ischemic Stroke.

In ischemic stroke, neutrophils are the first-line peripheral immune cells infiltrating the brain tissue to form neutrophil extracellular traps (NETs). The present study aimed to investigate the role of neuronal cold-inducible RNA-binding protein (CIRP) in promoting NETs-induced brain endothelial barrier destruction and cerebral edema after ischemic stroke. We found that the expression of NETs and neuronal CIRP in the penumbra increased at 6 hours after transient middle cerebral artery occlusion (tMCAO) and increased significantly at 24 hours, reaching a peak at 3 days. NETs degradation or CIRP inhibition can alleviate the leakage of brain endothelial barrier and reverse the decreased expression of tight junction proteins (zonula occludens-1, claudin-5 and occludin) in tMCAO mice. Oxygen-glucose deprivation/reperfusion treated primary neurons or recombinant CIRP could induce NETs formation via TLR4/p38 signaling pathway in vitro. Transcription factor specificity protein 1 (sp1) was responsible for the increased neuronal CIRP expression and the inhibition of sp1 could suppress the increased CIRP expression, reduce NETs formation, and diminish brain endothelial barrier leakage in tMCAO mice. We also found the upregulated CIRP level was associated with severe cerebral edema in patients with acute ischemic stroke. In conclusion, the increased expression of transcription factor sp1 after ischemic stroke can lead to elevated CIRP expression and release from the neurons, which subsequently interacts with neutrophils and promotes NETs formation, resulting in brain endothelial barrier destruction and cerebral edema.

Pyroptotic Macrophage-Derived Microvesicles Accelerate Formation of Neutrophil Extracellular Traps via GSDMD-N-expressing Mitochondrial Transfer during Sepsis.

Macrophage pyroptosis and neutrophil extracellular traps (NETs) play a critical role in sepsis pathophysiology; however, the role of macrophage pyroptosis in the regulation of NETs formation during sepsis is unknown. Here, we showed that macrophages transfer mitochondria to neutrophils through microvesicles following pyroptosis; this process induces mitochondrial dysfunction and triggers the induction of NETs formation through mitochondrial reactive oxygen species (mtROS)/Gasdermin D (GSDMD) axis. These pyroptotic macrophage-derived microvesicles can induce tissues damage, coagulation, and NETs formation in vivo. Disulfiram partly inhibits these effects in a mouse model of sepsis. Pyroptotic macrophage-derived microvesicles induce NETs formation through mitochondrial transfer, both in vitro and in vivo. Microvesicles-mediated NETs formation depends on the presence of GSDMD-N-expressing mitochondria in the microvesicles. This study elucidates a microvesicles-based pathway for NETs formation during sepsis and proposes a microvesicles-based intervention measure for sepsis management.

Grass carp Il-2 promotes neutrophil extracellular traps formation via inducing ROS production and autophagy in vitro

Interleukin (IL)-2 has been reported to regulate neutrophil functions in humans, mice, pigs and chicken although it is a key regulator of T cells. Consistently, we found that grass carp (Ctenopharyngodon idellus) interleukin-2 (gcIl-2) is capable of modulating the antimicrobial activities of neutrophils via regulating granzyme B- and perforin-like gene expression in our previous study. In the present study, stimulation of gcIl-2 on neutrophil extracellular traps (NETs) formation in grass carp neutrophils was demonstrated by detecting free DNA release, histone H3 citrullination and morphological changes of the cells. Further investigation revealed that reactive oxygen species (ROS) production from NADPH oxidase but not mitochondria was involved in NETosis induced by gcIl-2. Aside from ROS, autophagy was disclosed to be indispensable for NETosis induced by gcIl-2. These converging lines of evidence suggested that fish Il-2 could induce NETs formation via NADPH oxidase-derived ROS- and autophagy-dependent pathways in fish species which is evolutionarily conserved with that in mammals. It is noteworthy that these two pathways did not interplay with each other in Il-2-stimulated NETosis. The mechanisms governing autophagy induced by Il-2 were also explored in the present study, showing that Il-2 modulated the action of high mobility group box 1 (HMGB1) protein to stimulate autophagy, leading to NETs formation in fish neutrophils. These results provided a new insight to the function of Il-2 in fish neutrophils, and a clue about the regulation of NETosis in the lower vertebrates.

Tacrolimus Maintains the Balance of Neutrophil Extracellular Traps by Inducing DNA Methylation of Neutrophils to Reduce Immune Rejection.

Immune rejection is a significant concern in organ transplantation, as it can lead to damage to and failure of the transplanted organ. To prevent or treat immune rejection, transplant recipients are commonly administered immunosuppressive drugs. Tacrolimus (FK506) is a widely used immunosuppressive drug in organ transplantation. The excessive formation of neutrophil extracellular traps (NETs) can contribute to inflammation and tissue damage. Although NETs play an antimicrobial role, their overproduction can be harmful. To investigate the mechanism by which FK506 suppresses immune rejection, we utilized HL-60 cells, which were differentiated into neutrophils using DMSO and induced to form NETs with phorbol myristate acetate (PMA), a very efficient and frequently used drug for inducing NET formation. By comparing pre- and post-treatment with FK506, we examined whether FK506 affects the formation of NETs. Various experimental techniques were employed, including confocal imaging for visualizing cell NETs, qPCR and Western blotting for gene and protein expression analyses, ELISAs for protein content detection, and LC-MS/MS for methylation detection. In our study, we discovered that FK506 can enhance DNA methylation, which likely contributes to the reduction in NETs. Genes and proteins related to methylation, namely, DNMT3B and TET3, exhibited significant correlations with methylation. Consistent changes in both genes and proteins suggest that DNMT3B and TET3 are key factors that are influenced by FK506, resulting in enhanced DNA methylation and the potential inhibition of PMA-induced NET production. In summary, we have identified a novel mechanism by which FK506 inhibits NET production through the enhancement of DNA methylation. This finding highlights a new aspect of FK506's immunosuppressive effect. Our results provide valuable insights for clinical research, immunosuppression, and organ preservation strategies.

Aging dysregulates neutrophil extracellular trap formation in response to HIV in blood and genital tissues.

Women acquire HIV through sexual transmission, with increasing incidence in women >50 years old. Identifying protective mechanisms in the female genital tract (FGT) is important to prevent HIV-acquisition in women as they age. Human genital and blood neutrophils inactivate HIV by releasing neutrophil extracellular traps (NETs), an innate protective mechanism against HIV-infection. However, how NET formation is triggered by HIV in different tissues and whether this mechanism is affected by aging remain unknown. We demonstrate that the mechanisms that trigger NET release in response to HIV are different in blood and genital tissues, and that NET release decreases with aging. In blood neutrophils, HIV stimulation independently activated calcium pathways and endosomal TLR8, but aging reduced calcium responses, resulting in delayed NET release. In contrast, calcium responses were absent in genital neutrophils and NET release was triggered preferentially through TLR8 activation, but aging impaired this pathway. HIV induced NET formation through non-lytic pathways in blood and FGT neutrophils, except for a small subset of NETs that incorporated annexin V and lactoferrin predominantly in blood, suggesting proinflammatory and lytic NET release. Our findings demonstrate that blood neutrophils cannot model genital neutrophil responses which has important implications to understanding protection against HIV acquisition.

SARS-CoV-2 primed platelets–derived microRNAs enhance NETs formation by extracellular vesicle transmission and TLR7/8 activation

BackgroundHyperactive neutrophil extracellular traps (NETs) formation plays a key role in the pathogenesis of severe COVID-19. Extracellular vesicles (EVs) are vehicles which carry cellular components for intercellular communication. The association between COVID-19 patients-derived EVs and NETs formation remains elusive.MethodsWe explored the roles of EVs in NETs formation from 40 COVID-19 patients with different disease severities as well as 30 healthy subjects. The EVs-carried microRNAs profile was analyzed using next generation sequencing approach which was validated by quantitative reverse transcription PCR. The regulatory mechanism of EVs on NETs formation was investigated by using an in vitro cell-based assay, including immunofluorescence assay, flow cytometry, and immunoblotting.ResultsCOVID-19 patient–derived EVs induced NETs formation by endocytosis uptake. SARS-CoV-2 spike protein-triggered NETs formation was significantly enhanced in the presence of platelet–derived EVs (pEVs) and this effect was Toll-like receptor (TLR) 7/8- and NADPH oxidase-dependent. Increased levels of miR-21/let-7b were revealed in EVs from COVID-19 patients and were associated with disease severity. We demonstrated that the spike protein activated platelets directly, followed by the subsequent intracellular miR-21/let-7b upregulation and then were loaded into pEVs. The pEVs-carried miR-21 interacted with TLR7/8 to prime p47phox phosphorylation in neutrophils, resulting in NADPH oxidase activation to promote ROS production and NETs enhancement. In addition, miR-21 modulates NF-κB activation and IL-1β/TNFα/IL-8 upregulation in neutrophils upon TLR7/8 engagement. The miR-21 inhibitor and TLR8 antagonist could suppress efficiently spike protein-induced NETs formation and pEVs primed NETs enhancement.ConclusionsWe identified SARS-CoV-2 triggered platelets–derived GU-enriched miRNAs (e.g., miR-21/let-7b) as a TLR7/8 ligand that could activate neutrophils through EVs transmission. The miR-21-TLR8 axis could be used as a potential predisposing factor or therapeutic target for severe COVID-19.

Neutrophil extracellular traps are involved in the occurrence of interstitial lung disease in a murine experimental autoimmune myositis model.

The excessive formation of neutrophil extracellular traps (NETs) has been demonstrated to be a pathogenic mechanism of idiopathic inflammatory myopathy (IIM)-associated interstitial lung disease (ILD). This study aimed to answer whether an experimental autoimmune myositis (EAM) model can be used to study IIM-ILD and whether NETs participate in the development of EAM-ILD. An EAM mouse model was established using skeletal muscle homogenate and pertussis toxin (PTX). The relationship between NETs and the ILD phenotype was determined via histopathological analysis. As NETs markers, serum cell-free DNA (cfDNA) and serum citrullinated histone 3 (Cit-H3)-DNA were tested. The healthy mouse was injected with PTX intraperitoneally to determine whether PTX intervention could induce NETs formation in vivo. Neutrophils isolated from the peripheral blood of healthy individuals were given different interventions to determine whether PTX and skeletal muscle homogenate can induce neutrophils to form NETs in vitro. EAM-ILD had three pathological phenotypes similar to IIM-ILD. Cit-H3, neutrophil myeloperoxidase, and neutrophil elastase were overexpressed in the lungs of EAM model mice. The serum cfDNA level and Cit-H3-DNA complex level were significantly increased in EAM model mice. Serum cfDNA levels were increased significantly in vivo intervention with PTX in mice. Both PTX and skeletal muscle homogenate-induced neutrophils to form NETs in vitro. EAM-ILD pathological phenotypes are similar to IIM-ILD, and NETs are involved in the development of ILD in a murine model of EAM. Thus, the EAM mouse model can be used as an ideal model targeting NETs to prevent and treat IIM-ILD.

In vitro induction of neutrophil extracellular traps by SARS-CoV-2 is biased by extracellular mitochondria

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has a highly variable course that is dependent on the host immune system reaction. Lung tissue damage, endothelial dysfunction, and microthrombosis in severe COVID-19 is linked to neutrophilia and the production of neutrophil extracellular traps (NETs). Previous studies have shown that NETs are involved in the pathology of COVID-19 and that the virus itself induces NET formation, although the underlying mechanisms are not clear. In this study, we aimed to investigate the induction of NETs by SARS-CoV-2 in vitro. We have found that both, infectious and heat-inactivated virus induce NETs formation. Surprisingly, cell culture media derived from uninfected Vero cells exhibit similar potency. This suggests that NET inducers other than the virus might be involved. Mitochondria released from dying cells during SARS-CoV-2 infection acting as damage-associated molecular patterns (DAMPs) were identified as potential contributors to neutrophil activation and NET formation. Our findings point to an important source of bias when analyzing NETs induction by SARS-CoV-2 in vitro, but also the immune reaction to viruses in general. Further implications for the understanding of COVID-19 pathogenesis remain to be elucidated.

Mitochondria-induced formation of neutrophil extracellular traps is enhanced in the elderly via Toll-like receptor 9.

Neutrophil extracellular traps are potent antimicrobial weapons; however, their formation during sterile inflammation is detrimental, and the mechanism of induction is still unclear. Since advanced age is the primary clinical risk factor for poor outcomes in inflammatory diseases, we hypothesized that sterile stimuli, represented by mitochondria, would induce neutrophil extracellular trap formation in an age-dependent manner. Therefore, we analyzed induction of neutrophil extracellular traps in patients grouped according to age or immune status and observed that neutrophils from elderly patients responded to the presence of mitochondria with enhanced neutrophil extracellular trap formation. These neutrophil extracellular traps were also found to be more oxidized and exhibited higher resistance to DNase I degradation. Additionally, a higher concentration of residual neutrophil extracellular traps was detected in the plasma of the elderly. This plasma was capable of priming neutrophils through TLR9-mediated signaling, leading to further neutrophil extracellular trap formation, which was successfully inhibited with chloroquine. Finally, in a mouse model of mitochondria-induced acute lung injury, we observed that neutrophils from aged mice displayed impaired chemotactic activity but exhibited a trend of higher neutrophil extracellular trap formation. Thus, we propose that residual neutrophil extracellular traps circulating in the elderly preactivate neutrophils, making them more prone to enhanced neutrophil extracellular trap formation when exposed to mitochondria during sterile inflammation. Further investigation is needed to determine whether this vicious circle could be a suitable therapeutic target.

Peripheral nervous system is injured by neutrophil extracellular traps (NETs) elicited by nonstructural (NS) protein-1 from Zika virus.

The involvement of innate immune mediators to the Zika virus (ZIKV)-induced neuroinflammation is not yet well known. Here, we investigated whether neutrophil extracellular traps (NETs), which are scaffolds of DNA associated with proteins, have the potential to injure peripheral nervous. The tissue lesions were evaluated after adding NETs to dorsal root ganglia (DRG) explants and to DRG constituent cells or injecting them into mouse sciatic nerves. Identification of NET harmful components was achieved by pharmacological inhibition of NET constituents. We found that ZIKV inoculation into sciatic nerves recruited neutrophils and elicited the production of the cytokines CXCL1 and IL-1β, classical NET inducers, but did not trigger NET formation. ZIKV blocked PMA- and CXCL8-induced NET release, but, in contrast, the ZIKV nonstructural protein (NS)-1 induced NET formation. NET-enriched supernatants were toxic to DRG explants, decreasing neurite area, length, and arborization. NETs were toxic to DRG constituent cells and affected myelinating cells. Myeloperoxidase (MPO) and histones were identified as the harmful component of NETs. NS1 injection into mouse sciatic nerves recruited neutrophils and triggered NET release and caspase-3 activation, events that were also elicited by the injection of purified MPO. In summary, we found that ZIKV NS1 protein induces NET formation, which causes nervous tissue damages. Our findings reveal new mechanisms leading to neuroinflammation by ZIKV.