Cryoelectron tomography reveals the multiplex anatomy of condensed native chromatin and its unfolding by histone citrullination.

Nucleosome chains fold and self-associate to form higher-order structures whose internal organization is unknown. Here, cryoelectron tomography (cryo-ET) of native human chromatin reveals intrinsic folding motifs such as (1) non-uniform nucleosome stacking, (2) intermittent parallel and perpendicular orientations of adjacent nucleosome planes, and (3) a regressive nucleosome chain path, which deviates from the direct zigzag topology seen in reconstituted nucleosomal arrays. By examining the self-associated structures, we observed prominent nucleosome stacking in cis and anti-parallel nucleosome interactions, which are consistent with partial nucleosome interdigitation in trans. Histone citrullination strongly inhibits nucleosome stacking and self-association with a modest effect on chromatin folding, whereas the reconstituted arrays undergo a dramatic unfolding into open zigzag chains induced by histone citrullination. This study sheds light on the internal structure of compact chromatin nanoparticles and suggests a mechanism for how epigenetic changes in chromatin folding are retained across both open and condensed forms.

Candida albicans induces neutrophil extracellular traps and leucotoxic hypercitrullination via candidalysin.

The peptide toxin candidalysin, secreted by Candida albicans hyphae, promotes stimulation of neutrophil extracellular traps (NETs). However, candidalysin alone triggers a distinct mechanism for NET-like structures (NLS), which are more compact and less fibrous than canonical NETs. Candidalysin activates NADPH oxidase and calcium influx, with both processes contributing to morphological changes in neutrophils resulting in NLS formation. NLS are induced by leucotoxic hypercitrullination, which is governed by calcium-induced protein arginine deaminase 4 activation and initiation of intracellular signalling events in a dose- and time-dependent manner. However, activation of signalling by candidalysin does not suffice to trigger downstream events essential for NET formation, as demonstrated by lack of lamin A/C phosphorylation, an event required for activation of cyclin-dependent kinases that are crucial for NET release. Candidalysin-triggered NLS demonstrate anti-Candida activity, which is resistant to nuclease treatment and dependent on the deprivation of Zn2+ . This study reveals that C. albicans hyphae releasing candidalysin concurrently trigger canonical NETs and NLS, which together form a fibrous sticky network that entangles C. albicans hyphae and efficiently inhibits their growth.

Inhibiting Neutrophil Extracellular Traps Protects against Ultraviolet B-Induced Skin Damage: Effects of Hochu-ekki-to and DNase I.

UV-B radiation induces sunburn, and neutrophils are pivotal in this inflammation. In this study, we examined the potential involvement of neutrophil extracellular traps (NETs) in ultraviolet B (UVB)-induced skin inflammation, correlating the skin inflammation-mitigating effects of Hochu-ekki-to on UV-B irradiation and NETs. To elucidate NET distribution in the dorsal skin, male ICR mice, exposed to UVB irradiation, were immunohistologically analyzed to detect citrullinated histone H3 (citH3) and peptidylarginine deiminase 4 (PAD4). Reactive oxygen species (ROS) production in the bloodstream was analyzed. To establish the involvement of NET-released DNA in this inflammatory response, mice were UV-B irradiated following the intraperitoneal administration of DNase I. In vitro experiments were performed to scrutinize the impact of Hochu-ekki-to on A23187-induced NETs in neutrophil-like HL-60 cells. UV-B irradiation induced dorsal skin inflammation, coinciding with a significant increase in citH3 and PAD4 expression. Administration of DNase I attenuated UV-B-induced skin inflammation, whereas Hochu-ekki-to administration considerably suppressed the inflammation, correlating with diminished levels of citH3 and PAD4 in the dorsal skin. UV-B irradiation conspicuously augmented ROS and hydrogen peroxide (H2O2) production in the blood. Hochu-ekki-to significantly inhibited ROS and H2O2 generation. In vitro experiments demonstrated that Hochu-ekki-to notably inhibited A23187-induced NETs in differentiated neutrophil-like cells. Hence, NETs have been implicated in UV-B-induced skin inflammation, and their inhibition reduces cutaneous inflammation. Additionally, Hochu-ekki-to mitigated skin inflammation by impeding neutrophil infiltration and NETs in the dorsal skin of mice.

Sodium Acetate Enhances Neutrophil Extracellular Trap Formation via Histone Acetylation Pathway in Neutrophil-like HL-60 Cells.

Neutrophil extracellular trap formation has been identified as a new cell death mediator, termed NETosis, which is distinct from apoptosis and necrosis. NETs capture foreign substances, such as bacteria, by releasing DNA into the extracellular environment, and have been associated with inflammatory diseases and altered immune responses. Short-chain fatty acids, such as acetate, are produced by the gut microbiota and reportedly enhance innate immune responses; however, the underlying molecular mechanisms remain unclear. Here, we investigated the effects of sodium acetate, which has the highest SCFA concentration in the blood and gastrointestinal tract, on NETosis by focusing on the mechanisms associated with histone acetylation in neutrophil-like HL-60 cells. Sodium acetate enhanced NETosis, as shown by fluorescence staining with SYTOX green, and the effect was directly proportional to the treatment duration (16-24 h). Moreover, the addition of sodium acetate significantly enhanced the acetylation of Ace-H3, H3K9ace, and H3K14ace. Sodium acetate-induced histone acetylation rapidly decreased upon stimulation with the calcium ionophore A23187, whereas histone citrullination markedly increased. These results demonstrate that sodium acetate induces NETosis via histone acetylation in neutrophil-like HL-60 cells, providing new insights into the therapeutic effects based on the innate immunity-enhancing effect of dietary fiber.

DNA demethylation increases NETosis.

Neutrophil extracellular traps (NETs) occur during the development of autoimmune diseases, cancer and diabetes. A novel form of cell death that is induced by NETs is called NETosis. Although these diseases are known to have an epigenetic component, epigenetic regulation of NETosis has not previously been explored. In the present study, we investigated the effects of epigenetic change, especially DNA demethylation, on NETosis in neutrophil-like cells differentiated from HL-60 cells, which were incubated for 72 h in the presence of 1.25% DMSO. DMSO-differentiated neutrophil-like cells tended to have increased methylation of genomic DNA. NETosis in the neutrophil-like cells was induced by the treatment with A23187, calcium ionophore, and increased by the addition of the DNMT inhibitor 5-azacytidine (Aza) during differentiation. Interestingly, Aza-stimulated neutrophil-like cell induced NETosis without treatment with A23187. Although reactive oxygen species (ROS), especially superoxide and hypochlorous acid, are important in NETosis induction, treatment with Aza decreased production of ROS, while mitochondria ROS scavenger tended to decrease Aza-induced NETosis. Moreover, the genomic DNA in Aza-stimulated neutrophil-like cell was demethylated, and the expression of peptidylarginine deiminase4 (PAD4) and citrullinated histone H3 (R2+R8+R17) was increased, but myeloperoxidase expression was unaffected. Additionally, PAD4 inhibition tended to decrease Aza-induced NETosis. The DNA demethylation induced by the DNMT inhibitor in neutrophil-like cells enhanced spontaneous NETosis through increasing PAD4 expression and histone citrullination. This study establishes a relationship between NETosis and epigenetics for the first time, and indicates that various diseases implicated to have an epigenetic component might be exacerbated by excessive NETosis also under epigenetic control.

Halogen Bonding Increases the Potency and Isozyme Selectivity of Protein Arginine Deiminase 1 Inhibitors.

Protein arginine deiminases (PADs) hydrolyze the side chain of arginine to form citrulline. Aberrant PAD activity is associated with rheumatoid arthritis, multiple sclerosis, lupus, and certain cancers. These pathologies established the PADs as therapeutic targets and multiple PAD inhibitors are known. Herein, we describe the first highly potent PAD1-selective inhibitors (1 and 19). Detailed structure-activity relationships indicate that their potency and selectivity is due to the formation of a halogen bond with PAD1. Importantly, these inhibitors inhibit histone H3 citrullination in HEK293TPAD1 cells and mouse zygotes with excellent potency. Based on this scaffold, we also developed a PAD1-selective activity-based probe that shows remarkable cellular efficacy and proteome selectivity. Based on their potency and selectivity we expect that 1 and 19 will be widely used chemical tools to understand PAD1 biology.

The influence of agents differentiating HL-60 cells toward granulocyte-like cells on their ability to release neutrophil extracellular traps.

Studies on neutrophil extracellular traps (NETs) are challenging as neutrophils live shortly and easily become activated. Thus, availability of a cell line model closely resembling the functions of peripheral blood neutrophils would be advantageous. Our purpose was to find a compound that most effectively differentiates human promyelocytic leukemia (HL-60) cells toward granulocyte-like cells able to release NETs. HL-60 cells were differentiated with all-trans retinoic acid (ATRA), dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) and stimulated with phorbol 12-myristate 13-acetate (PMA) or calcium ionophore A23187 (CI). Cell differentiation, phagocytosis and calcium influx were analyzed by flow cytometry. Reactive oxygen species production and NETs release were measured fluorometrically and analyzed microscopically. LC3-II accumulation and histone 3 citrullination were analyzed by western blot. ATRA most effectively differentiated HL-60 cells toward granulocyte-like cells. ATRA-dHL-60 cells released NETs only upon PMA stimulation, DMSO-dHL-60 cells only post CI stimulation, while DMF-dHL-60 cells formed NETs in response to both stimuli. Oxidative burst was induced in ATRA-, DMSO- and DMF-dHL-60 cells post PMA stimulation and only in DMF-dHL-60 cells post CI stimulation. Increased histone 3 citrullination was observed in stimulated DMSO- and DMF-, but not in ATRA-dHL-60 cells. The calcium influx was diminished in ATRA-dHL-60 cells. Significant increase in autophagosomes formation was observed only in PMA-stimulated DMF-dHL-60 cells. Phagocytic index was higher in ATRA-dHL-60 cells than in control, DMSO- and DMF-dHL-60 cells. We conclude that ATRA, DMSO and DMF differentiate HL-60 in different mechanisms. DMF is the best stimulus for HL-60 cell differentiation for NETs studies.

Neutrophil extracellular traps are critical for pregnancy loss in sphingosine kinase-deficient mice on 129Sv/C57BL/6 background.

Exaggerated maternal immune responses must be strictly controlled to ensure a successful pregnancy. Neutrophil extracellular traps (NETs) have recently been implicated as a potential mechanism for promoting inflammation in pregnancy-related disorders. In this study, we demonstrated that NETs play a key role in the pathogenesis of sphingosine kinase (Sphk)-mediated pregnancy loss. Perturbing the sphingolipid pathway by disrupting Sphk genes during pregnancy led to excessive NET formation exclusively at the fetomaternal interface and early fetal death. Neutrophils that formed NETs were characterized by histone hypercitrullination and peptidylarginine deiminase 4 (PAD4) overexpression. In addition, thrombus formation was enhanced in the decidua, but not in the plasma, of Sphk-deficient mice. Blocking NET formation with a PAD4 inhibitor protected Sphk-deficient mice from pregnancy loss. The PAD4 inhibition significantly reduced the expression of hypercitrullinated histone in neutrophils and ameliorated vascular injury in the decidua of Sphk-deficient mice. Moreover, NET formation was induced in human neutrophils stimulated with Sphk-deficient human decidual cells. Together, these findings indicate that targeting NETs might be a novel therapeutic strategy to treat idiopathic pregnancy loss in humans.-Mizugishi, K., Yamashita, K. Neutrophil extracellular traps are critical for pregnancy loss in sphingosine kinase-deficient mice on 129Sv/C57BL/6 background.

Neutrophil histone modification by peptidylarginine deiminase 4 is critical for deep vein thrombosis in mice.

Deep vein thrombosis and pulmonary embolism are major health problems associated with high mortality. Recently, DNA-based neutrophil extracellular traps (NETs) resulting from the release of decondensed chromatin, were found to be part of the thrombus scaffold and to promote coagulation. However, the significance of nuclear decondensation and NET generation in thrombosis is largely unknown. To address this, we adopted a stenosis model of deep vein thrombosis and analyzed venous thrombi in peptidylarginine deiminase 4 (PAD4)-deficient mice that cannot citrullinate histones, a process required for chromatin decondensation and NET formation. Intriguingly, less than 10% of PAD4(-/-) mice produced a thrombus 48 h after inferior vena cava stenosis whereas 90% of wild-type mice did. Neutrophils were abundantly present in thrombi formed in both groups, whereas extracellular citrullinated histones were seen only in thrombi from wild-type mice. Bone marrow chimera experiments indicated that PAD4 in hematopoietic cells was the source of the prothrombotic effect in deep vein thrombosis. Thrombosis could be rescued by infusion of wild-type neutrophils, suggesting that neutrophil PAD4 was important and sufficient. Endothelial activation and platelet aggregation were normal in PAD4(-/-) mice, as was hemostatic potential determined by bleeding time and platelet plug formation after venous injury. Our results show that PAD4-mediated chromatin decondensation in the neutrophil is crucial for pathological venous thrombosis and present neutrophil activation and PAD4 as potential drug targets for deep vein thrombosis.

PLA2G2E-mediated lipid metabolism triggers brain-autonomous neural repair after ischemic stroke.

The brain is generally resistant to regeneration after damage. The cerebral endogenous mechanisms triggering brain self-recovery have remained unclarified to date. We here discovered that the secreted phospholipase PLA2G2E from peri-infarct neurons generated dihomo-γ-linolenic acid (DGLA) as necessary for triggering brain-autonomous neural repair after ischemic brain injury. Pla2g2e deficiency diminished the expression of peptidyl arginine deiminase 4 (Padi4), a global transcriptional regulator in peri-infarct neurons. Single-cell RNA sequencing (scRNA-seq) and epigenetic analysis demonstrated that neuronal PADI4 had the potential for the transcriptional activation of genes associated with recovery processes after ischemic stroke through histone citrullination. Among various DGLA metabolites, we identified 15-hydroxy-eicosatrienoic acid (15-HETrE) as the cerebral metabolite that induced PADI4 in peri-infarct-surviving neurons. Administration of 15-HETrE enhanced functional recovery after ischemic stroke. Thus, our research clarifies the promising potential of brain-autonomous neural repair triggered by the specialized lipids that initiate self-recovery processes after brain injury.

Padi2/3 Deficiency Alters the Epigenomic Landscape and Causes Premature Differentiation of Mouse Trophoblast Stem Cells.

Histone citrullination is a relatively poorly studied epigenetic modification that involves the irreversible conversion of arginine residues into citrulline. It is conferred by a small family of enzymes known as protein arginine deiminases (PADIs). PADI function supports the pluripotent state of embryonic stem cells, but in other contexts, also promotes efficient cellular differentiation. In the current study, we sought to gain deeper insights into the possible roles of PADIs in mouse trophoblast stem cells (TSCs). We show that Padi2 and Padi3 are the most highly expressed PADI family members in TSCs and are rapidly down-regulated upon differentiation. Padi2/3 double knockout (DKO) TSCs express lower levels of stem cell transcription factors CDX2 and SOX2 and are prone to differentiate into extremely large trophoblast giant cells, an effect that may be mediated by centrosome duplication defects. Interestingly, Padi2/3 DKO TSCs display alterations to their epigenomic landscape, with fewer H3K9me3-marked chromocentric foci and globally reduced 5-methylcytosine levels. DNA methylation profiling identifies that this effect is specifically evident at CpG islands of critical trophoblast genes, such as Gata3, Peg3, Socs3 and Hand1. As a consequence of the hypomethylated state, these factors are up-regulated in Padi2/3 DKO TSCs, driving their premature differentiation. Our data uncover a critical epigenetic role for PADI2/3 in safeguarding the stem cell state of TSCs by modulating the DNA methylation landscape to restrict precocious trophoblast differentiation.

Insights into peptidylarginine deiminase expression and citrullination pathways.

Peptidylarginine deiminases (PADs) are calcium-dependent enzymes that mediate citrullination, an irreversible post-translational modification (PTM). PAD enzymes have received increasing attention in (patho-)physiology since multi-omics analysis accelerated their expression profiling. Here, we provide a comprehensive overview of PAD expression at the RNA and protein levels, and a list of annotated substrates per PAD isozyme. We discuss novel roles of citrullination in cellular growth, epigenetic regulation, tissue remodeling, inflammation, and cancer in mouse models and humans. Additionally, we cluster similar effects of protein deimination to offer a different perspective and improve our understanding of citrullination in health and disease. Citrullination should no longer be considered as a rare PTM, but as an important regulatory mechanism in physiology and pathology.

Differentiation of HL-60 cells in serum-free hematopoietic cell media enhances the production of neutrophil extracellular traps.

Neutrophil extracellular traps (NETs) are web-like structures made of chromatin and have been identified to have a role in the host's immune defense. Differentiated human promyelocytic leukemia HL-60 cells (dHL-60) have been used to study the mechanisms of NETs formation, as neutrophils have a short lifespan that limits their use. However, dHL-60 cells are inefficient at generating NETs and therefore are not ideal replacements for neutrophils in studying of NET formation. In the present study, the optimal cell culture conditions and differentiation time that result in the most effective release of NETs from dHL-60 cells upon stimulation were determined. HL-60 cells were cultured in serum (FBS) or serum-free (X-VIVO) medium and differentiated using all-trans retinoic acid (ATRA) or dimethyl sulfoxide (DMSO). dHL-60 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) or Ca2+ ionophore (CI). Cell differentiation and apoptosis, as well as the formation of reactive oxygen species (ROS) and citrullinated histone H3 (citH3) were analyzed using flow cytometry. NETs were visualized using fluorescence microscopy and NET quantification was performed using PicoGreen. Induction of HL-60 cells for five days produced the best results in terms of differentiation markers and cell viability. Both ATRA- and DMSO-induced dHL-60 cells were able to release NETs upon PMA and CI stimulation; dHL-60 cells in serum-free medium produced more NETs than those in serum-containing medium. DMSO-dHL-60 (X-VIVO) cells were most efficient at producing NETs and ROS upon stimulation with PMA, while ATRA-dHL-60 (X-VIVO) cells were most efficient at producing NETs and citH3 upon stimulation with CI. It was concluded that DMSO-dHL-60 (X-VIVO) may be a model for the study of ROS-high NETosis and ATRA-dHL-60 (X-VIVO) may be suitable for ROS-low NETosis.

Sex-dependent mechanisms involved in renal tolerance to ischemia-reperfusion: Role of inflammation and histone H3 citrullination.

Ischemia-reperfusion (I/R) injury, an inevitable result of kidney transplantation, triggers early inflammatory events that affect graft viability. Evidence from human transplantation and preclinical models of I/R suggests that a female hormonal environment positively influences the ability to recover from ischemic injury. However, the mechanisms behind these effects remain mostly unexplored. Here, we studied the influence of sex on pro-inflammatory mediators involved in the pathophysiology of acute I/R injury in male, female, and female ovariectomized (OVX) Wistar rats that underwent unilateral renal ischemia for 45 min, followed by 24 h of reperfusion. We found improved renal function, reduced cytokine expression, and decreased infiltration of myeloperoxidase-positive cells in females after I/R, when compared to their male and female OVX counterparts. Remarkably, citrullination of histone H3 was exacerbated in serum and renal tubules of females after I/R. In contrast, we observed lower levels of citrullinated histone H3 in male and female OVX rats in response to I/R, mostly in neutrophil extracellular traps. Our results demonstrate that female sex promotes renal I/R tolerance by attenuating pro-inflammatory mediators involved in I/R-induced damage.

Post-Translational Modifications in NETosis and NETs-Mediated Diseases.

: Neutrophils undergo a unique form of cell death that generates neutrophil extracellular traps (NETs) that may help to neutralize invading pathogens and restore homeostasis. However, uncontrolled NET formation (NETosis) can result in numerous diseases that adversely affect health. Recent studies further elucidate the mechanistic details of the different forms of NETosis and their common end structure, as NETs were constantly found to contain DNA, modified histones and cytotoxic enzymes. In fact, emerging evidence reveal that the post translational modifications (PTMs) of histones in neutrophils have a critical role in regulating neutrophil death. Histone citrullination is shown to promote a rapid form of NET formation independent of NADPH oxidase (NOX), which relies on calcium influx. Interestingly, few studies suggest an association between histone citrullination and other types of PTMs to control cell survival and death, such as histone methylation. Even more exciting is the finding that histone acetylation has a biphasic effect upon NETosis, where histone deacetylase (HDAC) inhibitors promote baseline, NOX-dependent and -independent NETosis. However, increasing levels of histone acetylation suppresses NETosis, and to switch neutrophil death to apoptosis. Interestingly, in the presence of NETosis-promoting stimuli, high levels of HDACis limit both NETosis and apoptosis, and promote neutrophil survival. Recent studies also reveal the importance of the PTMs of neutrophils in influencing numerous pathologies. Histone modifications in NETs can act as a double-edged sword, as they are capable of altering multiple types of neutrophil death, and influencing numerous NET-mediated diseases, such as acute lung injury (ALI), thrombosis, sepsis, systemic lupus erythematosus, and cancer progression. A clear understanding of the role of different PTMs in neutrophils would be important for an understanding of the molecular mechanisms of NETosis, and to appropriately treat NETs-mediated diseases.

Identification of macrophage extracellular trap-like structures in mammary gland adipose tissue: a preliminary study.

PAD4-mediated hypercitrullination of histone H4 arginine 3 (H4R3) has been previously found to promote the formation of Neutrophil Extracellular Traps in inflamed tissues and the resulting histone H4 citrulline 3 (H4Cit3) modification is thought to play a key role in extracellular trap (ET) formation by promoting chromatin decondensation. In addition to neutrophils, macrophages have also recently been found to generate functional extracellular traps (METs). However, a role for PADs in ET formation in macrophages has not been previously described. Transcripts for PAD2 and PAD4 are found in mature macrophages and these cells can be induced to citrullinate proteins, thus raising the possibility that PADs may play a direct role in ET formation in macrophages via histone hypercitrullination. In breast and visceral white adipose tissue from obese patients, infiltrating macrophages are often seen to surround dead adipocytes forming characteristic "crown-like structures" (CLS) and the presence of these lesions is associated with increased levels of inflammatory mediators. In light of these observations, we have initiated studies to test whether PADs are expressed in CLS macrophages and whether these macrophages might form METs. Our preliminary findings show that PAD2 (and to a lesser extent, PAD4) is expressed in both in the macrophage cell line (RAW 264.7) and in CLS lesions. Additionally, we provide evidence that macrophage-derived extracellular histones are seen around presumptive macrophages within CLS lesions and that these histones contain the H4Cit3 modification. These initial findings support our hypothesis that obesity-induced adipose tissue inflammation promotes the formation of METs within CLS lesions via PAD-mediated histone hypercitrullination. Subsequent studies are underway to further validate these findings and to investigate the role in PAD-mediated MET formation in CLS function in the mammary gland.

Abundant neutrophil extracellular traps in thrombus of patient with microscopic polyangiitis.

This is a case study of a patient diagnosed with microscopic polyangiitis (MPA) and complicated with deep vein thrombosis (DVT), who died of respiratory failure despite treatment. Autopsy revealed severe crescentic glomerulonephritis and massive alveolar hemorrhage. The thrombus contained abundant neutrophils. Although it is reported that patients with ANCA-associated vasculitis (AAV) have an increased risk of DVT, it remains elusive why they are prone to thrombosis. A recent study has demonstrated the presence of neutrophil extracellular traps (NETs), a newly recognized mode of neutrophil cell-death, in glomerular crescents of MPA patients. Interestingly, NETs were identified in the thrombus as well as in the glomerular crescents in the present case. When compared to other thrombi unrelated to MPA, the amount of NETs was significantly greater in the MPA patient. On the other hand, NETs are critically involved in thrombogenesis because histones within NETs can bind platelets and blood coagulants. Although this is important in regard to containment of microbes within NETs, excessive NETs could cause thrombosis. The collective findings suggest the possibility that thrombosis could be critically associated with MPA via NETs, and that NETs could be a therapeutic target in MPA patients.