Induced Neutrophil Extracellular Trap Research Articles

Quercetin Mitigates Lysophosphatidylcholine (LPC)-Induced Neutrophil Extracellular Traps (NETs) Formation through Inhibiting the P2X7R/P38MAPK/NOX2 Pathway.

Neutrophil extracellular traps (NETs) are three-dimensional reticular structures that release chromatin and cellular contents extracellularly upon neutrophil activation. As a novel effector mechanism of neutrophils, NETs possess the capacity to amplify localized inflammation and have been demonstrated to contribute to the exacerbation of various inflammatory diseases, including cardiovascular diseases and tumors. It is suggested that lysophosphatidylcholine (LPC), as the primary active component of oxidized low-density lipoprotein, represents a significant risk factor for various inflammatory diseases, such as cardiovascular diseases and neurodegenerative diseases. However, the specific mechanism of NETs formation induced by LPC remains unclear. Quercetin has garnered considerable attention due to its anti-inflammatory properties, serving as a prevalent flavonoid in daily diet. However, little is currently known about the underlying mechanisms by which quercetin inhibits NETs formation and alleviates associated diseases. In our study, we utilized LPC-treated primary rat neutrophils to establish an in vitro model of NETs formation, which was subsequently subjected to treatment with a combination of quercetin or relevant inhibitors/activators. Compared to the control group, the markers of NETs and the expression of P2X7R/P38MAPK/NOX2 pathway-associated proteins were significantly increased in cells treated with LPC alone. Quercetin intervention decreased the LPC-induced upregulation of the P2X7R/P38MAPK/NOX2 pathway and effectively reduced the expression of NETs markers. The results obtained using a P2X7R antagonist/activator and P38MAPK inhibitor/activator support these findings. In summary, quercetin reversed the upregulation of the LPC-induced P2X7R/P38MAPK/NOX2 pathway, further mitigating NETs formation. Our study investigated the potential mechanism of LPC-induced NETs formation, elucidated the inhibitory effect of quercetin on NETs formation, and offered new insights into the anti-inflammatory properties of quercetin.

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.

Stroke and myocardial infarction induce neutrophil extracellular trap release disrupting lymphoid organ structure and immunoglobulin secretion

Post-injury dysfunction of humoral immunity accounts for infections and poor outcomes in cardiovascular diseases. Among immunoglobulins (Ig), IgA, the most abundant mucosal antibody, is produced by plasma B cells in intestinal Peyer’s patches (PP) and lamina propria. Here we show that patients with stroke and myocardial ischemia (MI) had strongly reduced IgA blood levels. This was phenocopied in experimental mouse models where decreased plasma and fecal IgA were accompanied by rapid loss of IgA-producing plasma cells in PP and lamina propria. Reduced plasma IgG was detectable in patients and experimental mice 3–10 d after injury. Stroke/MI triggered the release of neutrophil extracellular traps (NETs). Depletion of neutrophils, NET degradation or blockade of NET release inhibited the loss of IgA+ cells and circulating IgA in experimental stroke and MI and in patients with stroke. Our results unveil how tissue-injury-triggered systemic NET release disrupts physiological Ig secretion and how this can be inhibited in patients.

Resveratrol Attenuates Rheumatoid Arthritis Induce Neutrophil Extracellular Traps via TLR-4 Mediated Inflammation in C57BL/6 Mice.

The objective of this study was to evaluate whether RSV inhibits neutrophil extracellular traps (NETs) that induce joint hyperalgesia in C57BL/6 mice after adjuvant-induced arthritis. A subplantar injection of Freund's complete adjuvant was administered to C57BL/6 mice on day 0 for immunization in the AIA model. Resveratrol (RSV, 25 mg/kg) was administered intraperitoneally once daily starting on day 22 and continuing for two weeks. The effects of mechanical hyperalgesia and edema formation have been assessed in addition to histopathological scoring. Mice were sacrificed on day 35 to determine cytokine levels and PADI4 and COX-2 expression levels. ELISA was used to quantify neutrophil extracellular traps (NETs) along with neutrophil elastase-DNA and myeloperoxidase-DNA complexes in neutrophils. An immunohistochemical stain was performed on knee joints to determine the presence of nuclear factor kappa B p65 (NF-kappaB p65). AIA mice were found to have higher levels of NET in joints and their joint cells demonstrated an increased expression of the PADI4 gene. Treatment with RSV in AIA mice (25 mg/kg, i.p.) significantly (P<0.05) inhibited joint hyperalgesia, resulting in a significant increase in mechanical threshold, a decrease in articular edema, a decrease in the production of inflammatory cytokines, increased COX-2 expression, and a decrease in the immunostaining of NF-kappaB. Furthermore, treatment with RSV significantly reduced the amount of neutrophil elastase (NE)-DNA and MPO-DNA complexes, which were used as indicators of NET formation (P<0.05). This study indicates that RSV reduces NET production and hyperalgesia by reducing inflammation mediated by PADI4 and COX-2. According to these data, NETs contribute to joint pain and resveratrol can be used to treat pain in RA through this pathway.

Interleukin-8 and neutrophil extracellular traps in children with lupus nephritis and vitamin C deficiency.

Vitamin C is a potent scavenger of reactive oxygen species, which induce neutrophil extracellular trap (NET) formation. NETs are a major source of autoantigens and are involved in systemic lupus erythematosus (SLE) pathogenesis. We determined vitamin C status and evaluated NET formation and inflammatory cytokines in children with lupus nephritis. Serum vitamin C was measured in 46 patients (82.6% females, mean age 14.5 ± 0.3years). Vitamin C levels < 0.3mg/dL indicated vitamin C deficiency. Patients were divided into two groups according to serum vitamin C levels: normal and low (< 0.3mg/dL). We compared NET formation and levels of SLE-related cytokines, including interleukin (IL)-8, IL-10, and tumor necrosis factor-α (TNF-α), between groups. NET formation was determined through measurement of serum citrullinated histone 3 levels and mRNA expression of peptidyl arginine deiminase-4 and assessment of the percentage of neutrophils with NETs by immunofluorescence. Nine patients (19.6%) had vitamin C deficiency. Kidney pathology assessment at disease onset revealed that histological activity index and number of kidney biopsies containing crescentic glomeruli were higher in vitamin C-deficient patients, but chronicity index was not. NET formation and serum IL-8 were more prominent in vitamin C-deficient patients. Serum IL-8 levels were 12.9 ± 5.2pg/mL in low vitamin C group and 5.2 ± 0.9pg/mL in normal vitamin C group (p = 0.03). Serum IL-10 and TNF-α were similar between groups. Our study demonstrated correlation among vitamin C deficiency, increased NET formation, and IL-8 upregulation in children with lupus nephritis. A prospective study is required to evaluate cause‒effect relationships of vitamin C status, NET formation and IL-8 expression.

Biophysical Changes of Leukocyte Activation (and NETosis) in the Cellular Host Response to Sepsis.

Sepsis, the leading cause of mortality in hospitals, currently lacks effective early diagnostics. A new cellular host response test, the IntelliSep test, may provide an indicator of the immune dysregulation characterizing sepsis. The objective of this study was to examine the correlation between the measurements performed using this test and biological markers and processes associated with sepsis. Phorbol myristate acetate (PMA), an agonist of neutrophils known to induce neutrophil extracellular trap (NET) formation, was added to whole blood of healthy volunteers at concentrations of 0, 200, and 400 nM and then evaluated using the IntelliSep test. Separately, plasma from a cohort of subjects was segregated into Control and Diseased populations and tested for levels of NET components (citrullinated histone (cit-H3) DNA and neutrophil elastase (NE) DNA) using customized ELISA assays and correlated with ISI scores from the same patient samples. Significant increases in IntelliSep Index (ISI) scores were observed with increasing concentrations of PMA in healthy blood (0 and 200: p < 10-10; 0 and 400: p < 10-10). Linear correlation was observed between the ISI and quantities of NE DNA and Cit-H3 DNA in patient samples. Together these experiments demonstrate that the IntelliSep test is associated with the biological processes of leukocyte activation and NETosis and may indicate changes consistent with sepsis.

Nicotine and Cotinine Induce Neutrophil Extracellular Trap Formation-Potential Risk for Impaired Wound Healing in Smokers.

Smoking undoubtedly affects human health. Investigating 2318 representative patients at a level 1 trauma center identified delayed wound healing, tissue infections, and/or sepsis as main complications in smokers following trauma and orthopedic surgery. Therefore, smoking cessation is strongly advised to improve the clinical outcome in these patients, although smoking cessation often fails despite nicotine replacement therapy raising the need for specific interventions that may reduce the complication rate. However, the underlying mechanisms are still unknown. In diabetics, delayed wound healing and infections/sepsis are associated with increased neutrophilic PADI4 expression and formation of neutrophil extracellular traps (NETs). The aim was to investigate if similar mechanisms hold for smokers. Indeed, our results show higher PADI4 expression in active and heavy smokers than non-smokers, which is associated with an increased complication rate. However, in vitro stimulation of neutrophils with cigarette smoke extract (CSE) only moderately induced NET formation despite accumulation of reactive oxygen species (ROS). Physiological levels of nicotine and its main metabolite cotinine more effectively induced NET formation, although they did not actively induce the formation of ROS, but interfered with the activity of enzymes involved in anti-oxidative defense and NET formation. In summary, we propose increased formation of NETs as possible triggers for delayed wound healing, tissue infections, and/or sepsis in smokers after a major trauma and orthopedic surgery. Smoking cessation might reduce this effect. However, our data show that smoking cessation supported by nicotine replacement therapy should be carefully considered as nicotine and its metabolite cotinine effectively induced NET formation in vitro, even without active formation of ROS.

Interleukin-33 Induces Neutrophil Extracellular Trap (NET) Formation and Macrophage Necroptosis via Enhancing Oxidative Stress and Secretion of Proatherogenic Factors in Advanced Atherosclerosis.

Interleukin-33 (IL-33) acts as an 'alarmin', and its role has been demonstrated in driving immune regulation and inflammation in many human diseases. However, the precise mechanism of action of IL-33 in regulating neutrophil and macrophage functioning is not defined in advanced atherosclerosis (aAT) patients. Further, the role of IL-33 in neutrophil extracellular trap (NET) formation in aAT and its consequent effect on macrophage function is not known. In the present study, we recruited n = 52 aAT patients and n = 52 control subjects. The neutrophils were isolated from both groups via ficoll/percoll-based density gradient centrifugation. The effect of IL-33 on the NET formation ability of the neutrophils was determined in both groups. Monocytes, isolated via a positive selection method, were used to differentiate them into macrophages from each of the study subjects and were challenged by IL-33-primed NETs, followed by the measurement of oxidative stress by calorimetric assay and the expression of the proinflammatory molecules by quantitative PCR (qPCR). Transcript and protein expression was determined by qPCR and immunofluorescence/ELISA, respectively. The increased expression of IL-33R (ST-2) was observed in the neutrophils, along with an increased serum concentration of IL-33 in aAT compared to the controls. IL-33 exacerbates NET formation via specifically upregulating CD16 expression in aAT. IL-33-primed NETs/neutrophils increased the cellular oxidative stress levels in the macrophages, leading to enhanced macrophage necroptosis and the release of atherogenic factors and matrix metalloproteinases (MMPs) in aAT compared to the controls. These findings suggested a pathogenic effect of the IL-33/ST-2 pathway in aAT patients by exacerbating NET formation and macrophage necroptosis, thereby facilitating the release of inflammatory factors and the release of MMPs that may be critical for the destabilization/rupture of atherosclerotic plaques in aAT. Targeting the IL-33/ST-2-NETs axis may be a promising therapeutic target for preventing plaque instability/rupture and its adverse complications in aAT.

Neutrophil extracellular traps contribute to liver damage and increase defective low-density neutrophils in alcohol-associated hepatitis

In alcohol-associated hepatitis (AH), inflammation and neutrophil counts correlate with poor clinical outcomes. Here, we investigated how neutrophils contribute to liver damage in AH. We isolated blood neutrophils from individuals with AH to examine neutrophil extracellular traps (NETs) and performed RNA sequencing to explore their unique characteristics. We observed a significant increase in NET production in AH. We also observed a unique low-density neutrophil (LDN) population in individuals with AH and alcohol-fed mice that was not present in healthy controls. Transcriptome analysis of peripheral LDNs and high-density neutrophils (HDNs) from individuals with AH revealed that LDNs exhibit a functionally exhausted phenotype, while HDNs are activated. Indeed, AH HDNs exhibited increased resting reactive oxygen species (ROS) production and produced more ROS upon lipopolysaccharide stimulation than control HDNs, whereas AH LDNs failed to respond to lipopolysaccharide. We show that LDNs are generated from HDNs after alcohol-induced NET release invitro, and this LDN subset has decreased functionality, including reduced phagocytic capacity. Moreover, LDNs showed reduced homing capacity and clearance by macrophage efferocytosis; therefore, dysfunctional neutrophils could remain in the circulation and liver. Depletion of both HDNs and LDNs invivo prevented alcohol-induced NET production and liver damage in mice. Granulocyte-colony stimulating factor treatment also ameliorated alcohol-induced liver injury in mice. Neutrophils contribute to liver damage through increased NET formation which increases defective LDNs in AH. Alcohol induces phenotypic changes in neutrophils; HDNs are activated whereas LDNs are defective. Our findings provide mechanistic insights that could guide the development of therapeutic interventions for AH. In this study we discovered heterogeneity of neutrophils in alcohol-associated hepatitis, including high-density and low-density neutrophils that show hyper-activated or exhausted transcriptomic profiles, respectively. We found that alcohol induces neutrophil extracellular trap (NET) formation, which contributes to liver damage. NET release by high-density neutrophils resulted in low-density neutrophils that reside in the liver and escape clean-up by macrophages. Our findings help to understand the opposing neutrophil phenotypes observed in individuals with alcohol-associated hepatitis and provide mechanistic insights that could guide therapeutic strategies targeting neutrophils.

HAO1-mediated oxalate metabolism promotes lung pre-metastatic niche formation by inducing neutrophil extracellular traps

Metabolic reprogramming has been shown to be involved in cancer-induced pre-metastatic niche (PMN) formation, but the underlying mechanisms have been insufficiently explored. Here, we showed that hydroxyacid oxidase 1 (HAO1), a rate-limiting enzyme of oxalate synthesis, was upregulated in the alveolar epithelial cells of mice bearing metastatic breast cancer cells at the pre-metastatic stage, leading to oxalate accumulation in lung tissue. Lung oxalate accumulation induced neutrophil extracellular trap (NET) formation by activating NADPH oxidase, which facilitated the formation of pre-metastatic niche. In addition, lung oxalate accumulation promoted the proliferation of metastatic cancer cells by activating the MAPK signaling pathway. Pharmacologic inhibition of HAO1 could effectively suppress the lung oxalate accumulation induced by primary cancer, consequently dampening lung metastasis of breast cancer. Breast cancer cells induced HAO1 expression and oxalate accumulation in alveolar epithelial cells by activating TLR3-IRF3 signaling. Collectively, these findings underscore the role of HAO1-mediated oxalate metabolism in cancer-induced lung PMN formation and metastasis. HAO1 could be an appealing therapeutic target for preventing lung metastasis of cancer.

Polystyrene Nanoplastics Induce Neutrophil Extracellular Traps in Mice Neutrophils.

With growing applications of plastic products, there is a growing threat to human health. Therefore, it is important to understand their toxicological behaviors. The release of neutrophil extracellular traps (NETs) was clarified as a new immune defense mechanism against intruders. Here, we discovered that polystyrene nanoplastics (PS NPs) induce NET formation, with involvement of reactive oxygen species, peptidyl arginine deiminase 4 (PAD4), and neutrophil elastase. Moreover, overexpression of PAD4 induced by PS NPs further mediates histone citrullination and chromatin depolymerization. These results provide important information and promising strategies for safety and immunotoxicity assessment of NPs.

Activated Platelets Autocrine 5-Hydroxytryptophan Aggravates Sepsis-Induced Acute Lung Injury by Promoting Neutrophils Extracellular Traps Formation.

Excessive neutrophil extracellular trap (NET) formation is an important contributor to sepsis-induced acute lung injury (ALI). Recent reports indicate that platelets can induce neutrophil extracellular trap formation. However, the specific mechanism remains unclear. Tph1 gene, which encodes the rate-limiting enzyme for peripheral 5-hydroxytryptophan (5-HT) synthesis, was knocked out in mice to simulate peripheral 5-HT deficiency. Cecal ligation and puncture (CLP) surgery was performed to induce sepsis. We found that peripheral 5-HT deficiency reduced NET formation in lung tissues, alleviated sepsis-induced lung inflammatory injury, and reduced the mortality rate of CLP mice. In addition, peripheral 5-HT deficiency was shown to reduce the accumulation of platelets and NETs in the lung of septic mice. We found that platelets from wild-type (WT), but not Tph1 knockout (Tph1 −/− ), mice promote lipopolysaccharide (LPS)-induced NET formation. Exogenous 5-HT intervention increased LPS-induced NET formation when Tph1 −/− platelets were co-cultured with WT neutrophils. Therefore, our study uncovers a mechanism by which peripheral 5-HT aggravated sepsis-induced ALI by promoting NET formation in the lung of septic mice.

IgA Complexes Induce Neutrophil Extracellular Trap Formation More Potently Than IgG Complexes.

Neutrophil extracellular trap (NET) formation is a powerful instrument to fight pathogens, but may induce collateral damage in the affected tissues. Besides pathogen-derived factors, immune complexes are potent inducers of NET formation. Neutrophils express IgA and IgG specific Fc receptors (FcRs) and therefore respond to complexed IgA and IgG. Especially in the context of autoimmune diseases, IgA and IgG immune complexes have been shown to trigger NET formation, a process that putatively contributes to disease severity. However, it is of question if both antibody classes stimulate neutrophils to the same extent. In this study, we compared the capability of IgA and IgG complexes formed by heat aggregation to induce NET formation. While stimulation of neutrophils with IgA complexes robustly induced NET formation, complexed IgG only marginally increased the amount of NETs compared to the unstimulated control. Mixing IgA with IgG before heat aggregation did not increase the effect of complexed IgA on neutrophils. By contrast, the presence of IgG complexes seemed to disturb neutrophil stimulation by IgA complexes. The capacity of complexed IgG to induce NET formation could not be increased by the addition of autologous serum or the removal of terminal sialic acid in the Fc glycan. Together, our data show that IgA is a much more potent inducer of NET formation than IgG. IgA may thus be the main driving force in (auto)immune complex-mediated NET formation.

Peripheral 5-hydroxytryptophan aggravates lung injury in septic mice by inducing the formation of neutrophils extracellular trap

To observe the effect of peripheral 5-hydroxytryptophan (5-HT)-induced neutrophil extracellular trap (NET) on lung injury in septic mice. Wild-type (WT type) and Tph1 knockout (KO) C57 mice (6-8 weeks) were selected and divided into WT mice sham group, WT mice sepsis group, Tph1KO mice sham group and Tph1KO mice sepsis group according to the random number table method. Mice in the sham group received sham surgery (only open the abdominal cavity to flip the cecum without ligation and puncture, and then close the abdominal cavity); the mice in the sepsis group received cecal ligation and perforature (CLP) to establish sepsis model. The mice were sacrificed 12 hours after the operation, and the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in bronchialalveolar lavage fluid (BALF) were detected by enzyme linked immunoadsordent assay (ELISA); at the same time, the lung tissues were collected, and the pathological changes of lung tissues were observed under light microscope, and the production of NET in lung tissues was observed by immunofluorescence microscope. The pathological results suggested that the lung tissue structure in sham groups was intact without exudation, while the alveolar structures of mice in the sepsis groups were damaged, with obvious exudation in the alveolar cavity and thickened alveolar walls accompanied by a large number of inflammatory cell infiltration, and the degree of lung injury in the sepsis group of WT mice was more severe than that of the sepsis group of Tph1KO mice. ELISA results showed that there was no statistically significant difference in the contents of TNF-α and IL-6 in mice BALF from different strains of the sham group; while the contents of TNF-α and IL-6 in BALF of septic mice group were significantly higher than those in sham group [WT mice: TNF-α (μg/L) was 158.20±28.46 vs. 14.00±3.28, IL-6 (μg/L) was 304.98±21.78 vs. 57.70±12.30; Tph1KO mice: TNF-α (μg/L) was 85.88±20.13 vs. 14.95±1.53, IL-6 (μg/L) was 169.50±45.61 vs. 55.05±12.68, all P < 0.01], and the above index levels in the sepsis group of WT mice were significantly higher than the sepsis group of Tph1KO mice [TNF-α (μg/L): 158.20±28.46 vs. 85.88±20.13, IL-6 (μg/L): 304.98±21.78 vs. 169.50±45.61, both P < 0.01]. Immunofluorescence staining showed that a very small amount of NET formation was detected in the mice lungs from the sham group; a large amount of NET formation was detected in the lung tissues in the sepsis group, which were significantly higher than those in sham group [WT mice: (34.75±7.27)% vs. (1.75±0.96)%, Tph1KO mice: (14.25±5.74)% vs. (2.50±1.29)%, both P < 0.01], and the amount of NET produced in the lung tissues of the WT mice sepsis group was significantly higher than that of the Tph1KO mice sepsis group [(34.75±7.27)% vs. (14.25±5.74)%, P < 0.01]. In sepsis, the increased production of inflammatory factors in the mice lung tissues induces to lung injury. The mechanism may relate to the increased production of NET in the lung tissues mediated by peripheral 5-HT synthesized by enterochromaffin cells and released into the blood; inhibiting the production of 5-HT in the peripheral blood can effectively reduce the production of NET in the lung tissues, thereby reducing lung injury.

Virus-induced senescence is a driver and therapeutic target in COVID-19.

Derailed cytokine and immune cell networks account for the organ damage and the clinical severity of COVID-19 (refs. 1-4). Here we show that SARS-CoV-2, like other viruses, evokes cellular senescence as a primary stress response in infected cells. Virus-induced senescence (VIS) is indistinguishable from other forms of cellular senescence and is accompanied by a senescence-associated secretory phenotype (SASP), which comprises pro-inflammatory cytokines, extracellular-matrix-active factors and pro-coagulatory mediators5-7. Patients with COVID-19 displayed markers of senescence in their airway mucosa in situ and increased serum levels of SASP factors. In vitro assays demonstrated macrophage activation with SASP-reminiscent secretion, complement lysis and SASP-amplifying secondary senescence of endothelial cells, which mirrored hallmark features of COVID-19 such as macrophage and neutrophil infiltration, endothelial damage and widespread thrombosis in affected lung tissue1,8,9. Moreover, supernatant from VIS cells, including SARS-CoV-2-induced senescence, induced neutrophil extracellular trap formation and activation of platelets and the clotting cascade. Senolytics such as navitoclax and a combination of dasatinib plus quercetin selectively eliminated VIS cells, mitigated COVID-19-reminiscent lung disease and reduced inflammation in SARS-CoV-2-infected hamsters and mice. Our findings mark VIS as a pathogenic trigger of COVID-19-related cytokine escalation and organ damage, and suggest that senolytic targeting of virus-infected cells is a treatment option against SARS-CoV-2 and perhaps other viral infections.

Viral mimetic triggers cerebral arteriopathy in juvenile brain via neutrophil elastase and NETosis

Stroke is among the top ten causes of death in children but has received disproportionally little attention. Cerebral arteriopathies account for up to 80% of childhood arterial ischemic stroke (CAIS) cases and are strongly predictive of CAIS recurrence and poorer outcomes. The underlying mechanisms of sensitization of neurovasculature by viral infection are undefined. In the first age-appropriate model for childhood arteriopathy—by administration of viral mimetic TLR3-agonist Polyinosinic:polycytidylic acid (Poly-IC) in juvenile mice—we identified a key role of the TLR3-neutrophil axis in disrupting the structural-functional integrity of the blood-brain barrier (BBB) and distorting the developing neurovascular architecture and vascular networks. First, using an array of in-vivo/post-vivo vascular imaging, genetic, enzymatic and pharmacological approaches, we report marked Poly-IC-mediated extravascular leakage of albumin (66kDa) and of a small molecule DiI (∼934Da) and disrupted tight junctions. Poly-IC also enhanced the neuroinflammatory milieu, promoted neutrophil recruitment, profoundly upregulated neutrophil elastase (NE), and induced neutrophil extracellular trap formation (NETosis). Finally, we show that functional BBB disturbances, NETosis and neuroinflammation are markedly attenuated by pharmacological inhibition of NE (Sivelestat). Altogether, these data reveal NE/NETosis as a novel therapeutic target for viral-induced cerebral arteriopathies in children.

Adjuvants and Vaccines Used in Allergen-Specific Immunotherapy Induce Neutrophil Extracellular Traps.

Aluminum hydroxide (alum) and monophosphoryl-lipid A (MPLA) are conventional adjuvants in vaccines for allergen-specific immunotherapy (AIT). Alum triggers the release of neutrophil extracellular traps (NETs) by neutrophils. NETs contain expelled decondensed chromatin associated with granular material and may act as danger-associated molecular patterns and activate antigen-presenting cells. We investigated whether adjuvant-induced NETs contribute to innate responses to AIT-vaccines. Human neutrophils were incubated with alum, MPLA and adjuvant-containing AIT-vaccine preparations. NETs were verified by time-lapse and confocal fluorescence microscopy and quantitatively assessed by DNA and elastase release and ROS production. In contrast to MPLA, alum represented a potent trigger for NET release. Vaccine formulations containing alum resulted in less NET release than alum alone, whereas the vaccine containing MPLA induced stronger NET responses than MPLA alone. NETs and alum alone and synergistically increased the expression of molecules involved in antigen presentation, i.e., CD80, CD86 and CD83, by peripheral blood monocytes. Monocyte priming with NETs resulted in individually differing IL-1β- and IL-6-responses. Thus, NETs induced by adjuvants in AIT-vaccines can provide autonomous and cooperative effects on early innate responses. The high diversity of individual innate responses to adjuvants and AIT-vaccines may affect their therapeutic efficacy.

Silver Nanoparticles Induce Neutrophil Extracellular Traps Via Activation of PAD and Neutrophil Elastase.

Silver nanoparticles (AgNPs) are widely used in various fields because of their antimicrobial properties. However, many studies have reported that AgNPs can be harmful to both microorganisms and humans. Reactive oxygen species (ROS) are a key factor of cytotoxicity of AgNPs in mammalian cells and an important factor in the immune reaction of neutrophils. The immune reactions of neutrophils include the expulsion of webs of DNA surrounded by histones and granular proteins. These webs of DNA are termed neutrophil extracellular traps (NETs). NETs allow neutrophils to catch and destroy pathogens in extracellular spaces. In this study, we investigated how AgNPs stimulate neutrophils, specifically focusing on NETs. Freshly isolated human neutrophils were treated with 5 or 100 nm AgNPs. The 5 nm AgNPs induced NET formation, but the 100 nm AgNPs did not. Subsequently, we investigated the mechanism of AgNP-induced NETs using known inhibitors related to NET formation. AgNP-induced NETs were dependent on ROS, peptidyl arginine deiminase, and neutrophil elastase. The result in this study indicates that treatment of 5 nm AgNPs induce NET formation through histone citrullination by peptidyl arginine deiminase and histone cleavage by neutrophil elastase.

Inhibition of Efferocytosis by Extracellular CIRP-Induced Neutrophil Extracellular Traps.

Phagocytic clearance of apoptotic cells by the macrophages (efferocytosis) is impaired in sepsis, but its mechanism is poorly understood. Extracellular cold-inducible RNA-binding protein (eCIRP) is a novel damage-associated molecular pattern that fuels inflammation. We identify that eCIRP-induced neutrophil extracellular traps (NETs) impair efferocytosis through a novel mechanism. Coculture of macrophages and apoptotic thymocytes in the presence of recombinant murine CIRP (rmCIRP)-induced NETs significantly inhibited efferocytosis. Efferocytosis was significantly inhibited in the presence of rmCIRP-treated wild-type (WT), but not PAD4-/- neutrophils. Efferocytosis in the peritoneal cavity of rmCIRP-injected PAD4-/- mice was higher than WT mice. Milk fat globule-EGF-factor VIII (MFG-E8), an opsonin, increased macrophage efferocytosis, whereas the inhibition of efferocytosis by NETs was not rescued upon addition of MFG-E8, indicating disruption of MFG-E8's receptor(s) αvβ3 or αvβ5 integrin by the NETs. We identified neutrophil elastase in the NETs significantly inhibited efferocytosis by cleaving macrophage surface integrins αvβ3 and αvβ5 Using a preclinical model of sepsis, we found that CIRP-/- mice exhibited significantly increased rate of efferocytosis in the peritoneal cavity compared with WT mice. We discovered a novel role of eCIRP-induced NETs to inhibit efferocytosis by the neutrophil elastase-dependent decrease of αvβ3/αvβ5 integrins in macrophages. Targeting eCIRP ameliorates sepsis by enhancing efferocytosis.

Substrate stiffness induces neutrophil extracellular trap (NET) formation through focal adhesion kinase activation

Neutrophils predominate the early inflammatory response to tissue injury and implantation of biomaterials. Recent studies have shown that neutrophil activation can be regulated by mechanical cues such as stiffness or surface wettability; however, it is not known how neutrophils sense and respond to physical cues, particularly how they form neutrophil extracellular traps (NET formation). To examine this, we used polydimethylsiloxane (PDMS) substrates of varying physiologically relevant stiffness (0.2–32 kPa) and examined the response of murine neutrophils to untreated surfaces or to surfaces coated with various extracellular matrix proteins recognized by integrin heterodimers (collagen, fibronectin, laminin, vitronectin, synthetic RGD). Neutrophils on higher stiffness PDMS substrates had increased NET formation and higher secretion of pro-inflammatory cytokines and chemokines. Extracellular matrix protein coatings showed that fibronectin induced the most NET formation and this effect was stiffness dependent. Synthetic RGD peptides induced similar levels of NET formation and pro-inflammatory cytokine release than the full-length fibronectin protein. To determine if the observed NET formation in response to substrate stiffness required focal adhesion kinase (FAK) activity, which is down stream of integrin activation, FAK inhibitor PF-573228 was used. Inhibition of FAK using PF-573228 ablated the stiffness-dependent increase in NET formation and pro-inflammatory molecule secretion. These findings demonstrate that neutrophils regulate NET formation in response to physical and mechanical biomaterial cues and this process is regulated through integrin/FAK signaling.