Unraveling NETosis: From Immune Mechanisms to Therapeutic Innovations

CML-271: Neutrophil Extracellular Traps Are Increased in Chronic Myeloid Leukemia and Are Differentially Affected by Tyrosine Kinase Inhibitors

Introduction Histone hypercitrullination by the enzyme peptidylarginine deiminase 4 (PAD4) leads to nuclear chromatin decondensation that is needed for neutrophil extracellular trap (NET) formation. NETs consist of chromatin and granule proteins that are released into the extracellular environment. NETs were shown to be involved in thrombosis by promoting coagulation and platelet adhesion and were identified in the thrombus scaffold in animal models of deep vein thrombosis (DVT). Objective Whether NETs are involved in the pathogenesis of DVT or whether they are merely a consequence of neutrophil recruitment to the thrombus is unknown. We hypothesized that NET formation would be impaired in PAD4-deficient mice during deep vein thrombosis and that this may affect thrombus formation and/or stability. Methods PAD4-deficient mice are incapable of citrullinating histones and therefore fail to decondense chromatin during NETosis. We performed the inferior vena cava stenosis model of DVT in wild-type or PAD4-/- mice. Intravital microscopy was done to assess leukocyte vessel wall interaction in PAD4 deficiency. Results We induced NET formation in isolated peripheral blood mouse neutrophils with ionomycin and found that PAD4-/- neutrophils had a complete inability to produce NETs (WT, 20.65±2.61% NETs; PAD4-/-, not detected. n=4). Leukocyte-endothelial interactions in PAD4-/- mice were not impaired upon induction of systemic Weibel-Palade body release (WT, 55.2±11.8; PAD4-/-, 62.0±17.5 cells/min, n=5-6). In the DVT model, while a majority (9/10) of wild-type mice formed a thrombus 48 hours after stenosis, only 1 of 11 PAD4-/- mice formed a thrombus. Thrombus formation could be rescued by infusions of isolated WT bone marrow neutrophils into PAD4-/- mice, and extracellular H3Cit+ areas were seen within these thrombi. This data suggests that neutrophil PAD4 was essential for thrombus formation in deep veins. Conclusion NETs comprise a crucial part of the pathologic thrombus scaffold, and here we report that the lack of NETs inhibits pathological thrombosis. Chromatin decondensation initiated by PAD4 in neutrophils is a key player in the formation of deep vein thrombi and targeting neutrophil histone modification could be a new way to prevent DVT.

Neutrophil extracellular traps (NETs) occur when chromatin is decondensed and extruded from the cell, generating a web-like structure. NETs have been implicated in the pathogenesis of several sterile disease states and thus are a potential therapeutic target. Various pathways have been shown to induce NETs, including autophagy, with several key enzymes being activated like peptidyl arginine deiminase 4 (PAD4), an enzyme responsible for citrullination of histones, allowing for DNA unwinding and subsequent release from the cell. Pre-clinical studies have already demonstrated that chloroquine (CQ) and hydroxychloroquine (HCQ) are able to reduce NETs and slow disease progression. The exact mechanism as to how these drugs reduce NETs has yet to be elucidated. CQ and HCQ decrease NET formation from various NET activators, independent of their autophagy inhibitory function. CQ and HCQ were found to inhibit PAD4 exclusively, in a dose-dependent manner, confirmed with reduced CitH3+ NETs after CQ or HCQ treatment. Circulating CitH3 levels were reduced in pancreatic cancer patients after HCQ treatment. In silico screening of PAD4 protein structure identified a likely binding site interaction at Arg639 for CQ and Trp347, Ser468, and Glu580 for HCQ. SPR analysis confirmed the binding of HCQ and CQ with PAD4 with KD values of 54.1 µM (CQ) and 88.1 µM (HCQ). This data provide evidence of direct PAD4 inhibition as a mechanism for CQ/HCQ inhibition of NETs. We propose that these drugs likely reduce NET formation through multiple mechanisms; the previously established TLR9 and autophagy inhibitory mechanism and the novel PAD4 inhibitory mechanism.

Simple SummaryNeutrophil extracellular traps (NETs) are a recently described form of neutrophil cellular death that has been associated with a thrombotic tendency in many diseases. We studied NET formation in neutrophils derived from patients with chronic myeloid leukemia (CML) and in CML neutrophil cell lines and demonstrated that NETs are increased in CML and that certain drugs used to treat CML (tyrosine kinase inhibitors—TKIs) increase NET formation. These findings may shed light on a novel mechanism linking CML, TKIs and vascular toxicity.Cardiovascular complications are increasingly reported with the use of certain tyrosine kinase inhibitors (TKIs) to treat chronic myeloid leukemia (CML). We studied neutrophil extracellular trap (NET) formation in CML and evaluated the effect of TKIs on NET formation. Neutrophils isolated from treatment-naïve patients with CML showed a significant increase in NET formation compared to matched controls at baseline and after stimulation with ionomycin (IO) and phorbol 12-myristate 13-acetate (PMA). Expression of citrullinated histone H3 (H3cit), peptidyl arginine deiminase 4 (PAD4) and reactive oxygen species (ROS) was significantly higher in CML samples compared to controls. Pre-treatment of neutrophils with TKIs was associated with a differential effect on NET formation, and ponatinib significantly augmented NET-associated elastase and ROS levels as compared to controls and other TKIs. BCR-ABL1 retroviral transduced HoxB8-immortalized mouse hematopoietic progenitors, which differentiate into neutrophils in-vitro, demonstrated increased H3cit & myeloperoxidase (MPO) expression consistent with excess NET formation. This was inhibited by Cl-amidine, a PAD4 inhibitor, but not by the NADPH inhibitor diphenyleneiodonium (DPI). Ponatinib pre-exposure significantly increased H3cit expression in HoxB8-BCR-ABL1 cells after stimulation with IO. In summary, CML is associated with increased NET formation, which is augmented by ponatinib, suggesting a possible role for NETs in promoting vascular toxicity in CML.

Neutrophil Extracellular Traps Accelerate Cholestatic Liver Injury through Bile Acids in Bile Duct Ligation Mice

Aberrant neutrophil extracellular trap (NET) formation has been implicated as a mechanism to induce autoreactivity in individuals at risk of autoimmune diseases. The objective of this study was to assess whether medications implicated in cases of drug-induced autoimmunity (hydralazine and procainamide) and medications less commonly associated with drug-induced autoimmunity (minocycline and clozapine) induce NET formation and/or prevent NET degradation. Human neutrophils were incubated with the drugs of interest and resultant NET formation was quantified by fluorescent microscopy. The ability of these drugs to interfere with NET degradation by serum nuclei was assessed. Pathways of drug-induced NET formation were studied with pharmacologic inhibitors of reactive oxygen species (ROS), peptidylarginine deiminases (PADs), and muscarinic receptors, and by assessment of intracellular calcium levels by flow cytometry. To determine if NET protein cargo varies by drug stimulus and/or neutrophil source, proteomic analysis of NET lysates induced by specific medications was compared using neutrophils from healthy donors and from patients with autoimmune diseases. Hydralazine and procainamide significantly induced NET formation while minocycline and clozapine did not. None of the medications significantly impaired NET degradation. NETosis induced by these drugs required NADPH oxidase and PAD4 activation. Procainamide triggered NETs via muscarinic receptor engagement on neutrophils, while hydralazine modulated calcium release from intracellular stores. Differences in protein cargo, particularly histone content, were observed in NETs induced by hydralazine and procainamide. Medications commonly implicated in drug-induced autoimmunity trigger NET formation displaying distinct protein cargo, via common and specific pathways. NETosis may play a role in the pathogenesis of drug-induced autoimmunity.

SAT0010 Excessive formation of neutrophil extracellular traps have a different role in the pathogenesis of anca-associated vasculitis and systemic lupus erythematosus

Simple SummaryIn the present study, white blood cells (neutrophils) were isolated from the blood of postpartum dairy cows and subjected in vitro to conditions designed to mimic an inflammatory reaction induced by the bacterial toxin named lipopolysaccharide (LPS). The production of neutrophil extracellular traps (NETs), reactive oxygen species (ROS), gene expression of NETs-related proteins and inflammatory factors were quantified. The results indicate that LPS stimulation resulted in a concentration-dependent release of NETs, while ROS exhibited rather minor changes. Nevertheless, the presence of the anti-inflammatory drug Carprofen (CA) resulted in a decrease in NETs formation, a downregulation of gene expression of related proteins as well as pro-inflammatory genes. These results indicate that CA has the potential to serve as a therapeutic for alleviating an excessive inflammatory reaction characterized by an overabundance of NETs. Furthermore, this study illuminates the potential of CA in regulating the immune response and its potential benefits in the treatment of inflammatory conditions in (transition) 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.

PAD4 takes charge during neutrophil activation: Impact of PAD4 mediated NET formation on immune‐mediated disease

Neutrophil extracellular traps promote M1 macrophage polarization in gouty inflammation via targeting hexokinase-2

Neutrophil extracellular traps in inflammatory disorders

Psoriasis is a common inflammatory skin disease with chronic manifestation in which the role of neutrophil extracellular traps (NETs) and alarmins are increasingly recognized as contributors to systemic and cutaneous inflammation. However, the interaction between alarmins and NET-driven immune responses remains poorly defined. The main aim of this study is to define the role of target alarmins (i.e., IL-33 and TSLP) in NETs induction and its subsequent impact on oxidative stress and inflammation in the peripheral blood. In the present study, we recruited active psoriasis patients (n = 56) and control (n = 56) subjects. The frequency of circulating neutrophils, the levels of NET-associated markers (MPO (myeloperoxidase)-DNA complex, CitH3 (citrullinated histone H3), PAD4 (peptidyl arginine deiminase4), NADPH oxidase, and NE (neutrophil elastase)), and alarmin transcripts (IL (interleukin)-33, TSLP (thymic stromal lymphopoietin), S100A7, S100B, HSP (heat shock protein) 60/70 were quantified using flow cytometry, ELISA (Enzyme-linked immunosorbent assay), and qPCR (quantitative polymerase chain reaction), respectively, in each group. The NET formation potential of isolated neutrophils was assessed in the presence or absence of rhIL-33 and rhTSLP by immunocytofluorescence. The effect of rhIL-33- and rhTSLP-primed NETs in augmenting oxidative stress and inflammation was evaluated on peripheral blood mononuclear cells (PBMCs) by ELISA. Significantly higher circulating neutrophils (p < 0.001) and levels of NET-associated markers (i.e., MPO-DNA complex, CitH3, PAD4, NADPH oxidase, and NE) were observed in active psoriasis patients compared to controls. Lesional skin exhibited strong expression of MPO (p < 0.001) compared to normal skin. The alarmins, IL-33 and TSLP, were markedly upregulated in the blood and skin (p < 0.05). The rhIL-33 and rhTSLP treated neutrophils demonstrated enhanced NETosis in patients (p < 0.001). Increased expression of inflammatory cytokines and oxidative stress markers were reported in PBMCs when incubated with rhIL-33- and rhTSLP-primed NETs. Taken together, our investigation demonstrated the novel mechanism wherein the alarmins IL-33 and TSLP exacerbate NET formation that may drive enhanced inflammation and oxidative stress in psoriasis.

Heme-Induced Neutrophil Extracellular Traps (NETs) Formation Contributes To Sickle Cell Disease Pathogenesis

Peptidylarginine deiminase inhibition disrupts NET formation and protects against kidney, skin and vascular disease in lupus-prone MRL/lpr mice

Uric acid induces NADPH oxidase-independent neutrophil extracellular trap formation