Peptidylarginine Deiminase Research Articles

Neutrophil autophagy induced by monosodium urate crystals facilitates neutrophil extracellular traps formation and inflammation remission in gouty arthritis.

Neutrophil extracellular traps (NETs) are composed of chromatin filaments coated with granular and cytosolic proteins, which contribute to the pathogenesis and progression of immune-related diseases. NETs are frequently observed in gouty arthritis, but the related mechanisms remain poorly understood. The aim of our study was to systematically elucidate the molecular mechanisms of self-remitting effects in gouty arthritis, and the causative relationship between neutrophil autophagy and NETs. The air pouch and paw edema model were used to simulate gouty arthritis in mice. Neutrophil infiltration and the formation of NETs were found in gouty arthritis. Interestingly, monosodium urate (MSU) crystals could induce the formation of NETs, degrade inflammatory factors, and alleviate the inflammatory response in gouty arthritis. In addition, MSU crystals resulted in profound molecular alterations in neutrophils using RNA-seq analysis, including autophagy activation. MSU crystals could activate neutrophil autophagy in vitro, and autophagy activators and inhibitors could regulate the formation of NETs. Furthermore, we explored the mechanism of autophagy-induced NETs. Autophagy related protein 7 (ATG7) produced by neutrophils stimulated with MSU crystals worked synergistically with p53 to enter the nucleus, promoting peptidyl arginine deiminase 4 (PAD4) expression, and inducing the formation of NETs. Finally, we substantiated that neutrophil autophagy regulates the severity of gouty arthritis via the formation of NETs in PAD4 -/- mice. Our results indicated that the autophagy of neutrophils regulates the formation of NETs and degrades inflammatory factors. Regulating autophagy and interfering with the formation of NETs represents a potential therapeutic approach against gouty arthritis during clinical practice.

Smart Liposomal Nanocarrier Enhanced the Treatment of Ischemic Stroke through Neutrophil Extracellular Traps and Cyclic Guanosine Monophosphate-Adenosine Monophosphate Synthase-Stimulator of Interferon Genes (cGAS-STING) Pathway Inhibition of Ischemic Penumbra.

Brain inflammation is regarded as one of the leading causes that aggravates secondary brain injury and hinders the prognosis of ischemic stroke. After ischemic stroke, high quantities of peripheral neutrophils are recruited to brain lesions and release neutrophil extracellular traps (NETs), leading to the aggravation of blood-brain barrier (BBB) damage, activation of microglia, and ultimate neuronal death. Herein, a smart multifunctional delivery system has been developed to regulate immune disorders in the ischemic brain. Briefly, Cl-amidine, an inhibitor of peptidylarginine deiminase 4 (PAD4), is encapsulated into self-assembled liposomal nanocarriers (C-Lipo/CA) that are modified by reactive oxygen species (ROS)-responsive polymers and fibrin-binding peptide to achieve targeting ischemic lesions and stimuli-responsive release of a drug. In the mouse model of cerebral artery occlusion/reperfusion (MCAO), C-Lipo/CA can suppress the NETs release process (NETosis) and further inhibit the cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes (cGAS-STING) pathway in an ischemic brain. In addition, MCAO mice treated with C-Lipo/CA significantly mitigated ischemic and reperfusion injury, with a reduction in the area of cerebral infarction to 12.1%, compared with the saline group of about 46.7%. These results demonstrated that C-Lipo/CA, which integrated microglia regulation, BBB protection, and neuron survival, exerts a potential therapy strategy to maximize ameliorating the mortality of ischemic stroke.

Treatment of human cardiac fibroblasts with the protein arginine deiminase inhibitor BB-Cl-amidine activates the Nrf2/HO-1 signaling pathway

BackgroundCardiac fibrosis contributes to end-stage extracellular matrix remodeling and heart failure (HF). Cardiac fibroblasts (CFs) differentiate into myofibroblasts (myoFbs) to preserve the structural integrity of the heart; however, the molecular mechanisms regulating CF transdifferentiation remain poorly understood. Protein arginine deiminase (PAD), which converts arginine to citrulline, has been shown to play a role in myocardial infarction, fibrosis, and HF. This study aimed to investigate the role of PAD in CF differentiation to myoFbs and identify the citrullinated proteins that were associated with phenotypic changes in CFs. ResultsGene expression analysis showed that PAD1 and PAD2 isoforms, but not PAD4 isoforms, were abundant in both CFs and myoFbs, and PAD1 was significantly upregulated in myoFbs. The pan-PAD inhibitor BB-Cl-amidine (BB-Cl) downregulated the mRNA expression of PAD1 and PAD2 as well as the protein expression of the fibrosis marker COL1A1 in CFs and myoFbs. Interestingly, a proteomic approach pointed to the activation of the Nrf2/HO-1 signaling pathway upon BB-Cl treatment in CFs and myoFbs. BB-Cl administration resulted in the upregulation of HO-1 at both the gene and protein levels in CFs and myoFbs. Importantly, the protein citrullination landscape of CFs consisting of 86 novel citrullination sites associated with focal adhesion (FN1(R1054)), inflammation (TAGLN(R12)) and DNA replication (EEF2(R767)) pathways was identified. ConclusionsIn summary, we revealed that BB-Cl treatment resulted in increased HO-1 expression via the Nrf2 pathway, which could prevent excessive tissue damage, thereby leading to substantial clinical benefits for the treatment of cardiac fibrosis.

Thymopentin ameliorates experimental colitis via inhibiting neutrophil extracellular traps

BackgroundThe long-term prognosis of Crohn’s disease (CD) remains unsatisfactory. Therefore, we assessed the therapeutic effect of thymopentin (TP5) in a mouse model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis, which mimics CD, and analyzed its impact on neutrophil extracellular traps (NETs). MethodsNET markers, including myeloperoxidase (MPO), neutrophil elastase (NE), citrullinated histone H3 (CitH3), peptidyl arginine deiminase IV (PAD4), and double-stranded DNA (dsDNA) were assessed by immunostaining and enzyme-linked immunosorbent assay. NET formation was evaluated in vitro. Neoseptin 3, a specific NET agonist, was used to reverse the effect of TP5 on TNBS-induced colitis. The action mechanism of TP5 was investigated using RNA-seq. ResultsTP5 ameliorated weight loss (P < 0.001), disease activity index (DAI) (P = 0.05), colon shrinkage (P = 0.04), and elevated levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, and neutrophils in the TNBS group. The TNBS group exhibited increased MPO, NE, CitH3, PAD4, dsDNA and MPO-DNA levels (all P < 0.001), which decreased after TP5 administration (P = 0.01, P < 0.001, P < 0.001, P < 0.001, P = 0.02, and P = 0.02 respectively). Tissue CitH3 levels were positively correlated with DAI and TNF-α levels (P < 0.05). Furthermore, phorbol 12-myristate 13-acetate-stimulated NET formation increased by 1.8-, 2.8-, and 2.3-fold in vitro in the control, TNBS + saline, and TNBS + TP5 groups, respectively. Neoseptin 3 significantly reversed the effect of TP5. RNA-seq revealed potential pathways underlying the effect of TP5. ConclusionTP5 effectively ameliorated colitis by suppressing NETs in the experimental CD model.

Molecular Factors in PAD2 (PADI2) and PAD4 (PADI4) Are Associated with Interstitial Lung Disease Susceptibility in Rheumatoid Arthritis Patients.

Around 50% of rheumatoid arthritis (RA) patients show some extra-articular manifestation, with the lung a usually affected organ; in addition, the presence of anti-citrullinated protein antibodies (ACPA) is a common feature, which is caused by protein citrullination modifications, catalyzed by the peptidyl arginine deiminases (PAD) enzymes. We aimed to identify single nucleotide variants (SNV) in PADI2 and PADI4 genes (PAD2 and PAD4 proteins, respectively) associated with susceptibility to interstitial lung disease (ILD) in RA patients and the PAD2 and PAD4 levels. Material and methods: 867 subjects were included: 118 RA-ILD patients, 133 RA patients, and 616 clinically healthy subjects (CHS). Allelic discrimination was performed in eight SNVs using qPCR, four in PADI2 and four in PADI4. The ELISA technique determined PAD2 and PAD4 levels in serum and bronchoalveolar lavage (BAL) samples, and the population structure was evaluated using 14 informative ancestry markers. Results: The rs1005753-GG (OR = 4.9) in PADI2 and rs11203366-AA (OR = 3.08), rs11203367-GG (OR = 2.4) in PADI4 are associated with genetic susceptibility to RA-ILD as well as the ACTC haplotype (OR = 2.64). In addition, the PAD4 protein is increased in RA-ILD individuals harboring the minor allele homozygous genotype in PADI4 SNVs. Moreover, rs1748033 in PADI4, rs2057094, and rs2076615 in PADI2 are associated with RA susceptibility. In conclusion, in RA patients, single nucleotide variants in PADI4 and PADI2 are associated with ILD susceptibility. The rs1748033 in PADI4 and two different SNVs in PADI2 are associated with RA development but not ILD. PAD4 serum levels are increased in RA-ILD patients.

Abstract P182: Neutrophil Extracellular Traps Are Formed In Response To Hypertensive Stretch And Contribute To Vascular Dysfunction In Hypertension

Background: Neutrophils play a crucial role in the innate immune response, in part by the release of neutrophil extracellular traps (NETs), which consist of decondensed chromatin and proteins including citrullinated histone 3 (H3-Cit). The process of NETosis involves the disassociation of DNA from the nucleosome catalyzed by the enzyme peptidyl arginine deiminase 4 (PAD4). TRPV4 is a mechanosensitive calcium channel expressed in neutrophils. Studies of human subjects and rodent models have described the accumulation of NETs in renal and vascular tissue in hypertension (HTN), however the causative role of NETs has not been described. We hypothesized that NETs form in response to hypertensive stretch and contribute to endothelial cell (EC) dysfunction and HTN. Methods: Mice deficient for Padi4 lack the PAD4 enzyme and cannot efficiently form NETs. Padi4 -/- and C57Bl/6J mice were treated with 490 ng/kg/min angiotensin (Ang) II for 4 weeks. Blood pressure was measured by radiotelemetry. Vascular reactivity studies were performed on mesenteric arteries. Vascular infiltration was measured by flow cytometry. Murine neutrophils and aortic ECs were co-cultured and subjected to uniaxial stretch under normotensive (5%) and hypertensive (10%) conditions. Neutrophils were treated with a TRPV4 agonist (GSK1016790A) and NETs were quantified by immunofluorescence. Results: Padi4 -/- mice exhibit attenuated HTN and improved vascular reactivity following Ang II treatment (systolic BP 159.4 ± 1.4 mmHg in C57Bl/6J mice vs 147.9 ± 2.6 mmHg in Padi4 -/- mice, n= 7-8, p= 0.007). Flow cytometry revealed a marked reduction in aortic infiltration of immune cells in Padi4 -/- mice compared to C57Bl/6J mice. Co-culture of murine neutrophils with ECs improves neutrophil survival and augments NETosis. Hypertensive stretch of co-cultured neutrophils and ECs augmented NETosis compared to normotensive stretch. Immunoblot revealed increased H3-Cit in neutrophils exposed to hypertensive stretch. Neutrophils treated with GSK1016790A exhibit augmented NETosis. Conclusions: These findings suggest a vital role of stretch-induced NETosis as a mediator of EC dysfunction and HTN. Mechanistically, both agonism of neutrophil TRPV4 and hypertensive stretch of ECs augment NETosis.

Investigation of the Effectiveness of Cl-Amidine on Wound Healing: An In Vitro Study

Objective: Peptidylarginine deiminases (PADs) are enzymes converting the arginine to citrulline. They play a role in embryogenesis and cell signaling activities. But excessive or dysregulated PAD levels were determined to be associated with disorders and to increase in many diseases. It has been shown that Chloramidine (Cl-amidine) used as a PAD inhibitor suppresses increased PAD activity and shows anti-cancer, anti-inflammatory and antioxidant activities. Anti-inflammatory and antioxidant properties play an important role in wound healing. In this study, the possible efficacy of Cl-amidine on wound healing in the keratinocyte cell line was investigated by considering these parameters. Methods: Cell proliferation evaluations of Cl-amidine concentrations (500, 125, 31.25 and 7.81 µM) determined according to the results of MTT method on HaCaT keratinocyte cells were performed using Real-Time Cell Analysis System (RTCA DP). COL1A1 mRNA expression levels were analyzed by RT (Real Time)-PCR (Polymerase Chain Reaction) method at the concentrations where proliferation was achieved (125, 31.25 µM). Migration effects of Cl-amidine on cells were evaluated by performing scratch analysis. MTT results were statistically analyzed with one-way ANOVA and Tukey test, and p&amp;lt;0.05 was accepted as significant. RTCA DP and RT-PCR results were evaluated using device software programs. Results: In the study, it was found that certain concentrations of Cl-amidine had a proliferative effect on HaCaT keratinocyte cells. It was determined that Cl-amidine increased the amount of type 1 collagen, which is an important parameter for wound healing, by RT-PCR method. In addition, according to scratch analysis, it was detected that it positively affected cell migration in relation to wound closure. Conclusion: This research shows that Cl-amidine may have a significant potential for wound healing.

Peptidylarginine deiminase 2 plays a key role in osteogenesis by enhancing RUNX2 stability through citrullination

Peptidylarginine deiminase (PADI) 2 catalyzes the post-translational conversion of peptidyl-arginine to peptidyl-citrulline in a process called citrullination. However, the precise functions of PADI2 in bone formation and homeostasis remain unknown. In this study, our objective was to elucidate the function and regulatory mechanisms of PADI2 in bone formation employing global and osteoblast-specific Padi2 knockout mice. Our findings demonstrate that Padi2 deficiency leads to the loss of bone mass and results in a cleidocranial dysplasia (CCD) phenotype with delayed calvarial ossification and clavicular hypoplasia, due to impaired osteoblast differentiation. Mechanistically, Padi2 depletion significantly reduces RUNX2 levels, as PADI2-dependent stabilization of RUNX2 protected it from ubiquitin-proteasomal degradation. Furthermore, we discovered that PADI2 binds to RUNX2 and citrullinates it, and identified ten PADI2-induced citrullination sites on RUNX2 through high-resolution LC-MS/MS analysis. Among these ten citrullination sites, the R381 mutation in mouse RUNX2 isoform 1 considerably reduces RUNX2 levels, underscoring the critical role of citrullination at this residue in maintaining RUNX2 protein stability. In conclusion, these results indicate that PADI2 plays a distinct role in bone formation and osteoblast differentiation by safeguarding RUNX2 against proteasomal degradation. In addition, we demonstrate that the loss-of-function of PADI2 is associated with CCD, thereby providing a new target for the treatment of bone diseases.

A novel homozygous variant in PADI6 is associate with human cleavage-stage embryonic arrest.

Repeated absence of useable embryos is a difficult problem for infertility patients. Among them, embryonic developmental arrest is more common, but the genetic cause is not known. The embryos of a patient who came to our hospital three times could not develop beyond the four-cell stage. In addition to recording the developmental details of the embryos by daily photo-taking, the PADI6 R132C homozygous variants was further confirmed by whole-exome sequencing. Subsequently, PADI6 R132C was analyzed by bioinformatics methods for conservativeness across species. In addition, the possible impact of the pathogenic mutation on the structure of the protein PADI6 were also assessed. Generally, we identified a homozygous variants [NM_207421.4, c.394C>T(p.R132C] in the middle protein-arginine deiminase domain in PADI6 gene. The homozygous variant is highly conserved across species. Homozygous variant in PADI6 R132C could cause a human cleavage-stage embryonic arrest in female patients. These findings provide further evidence for the important roles of the homozygous PADI6R132C variant in embryonic development. Our findings contribute to a deeper understanding of the molecular genetic basis of female infertility.

Porphyromonas gingivalis Peptidyl Arginine Deiminase (PPAD) in the Context of the Feed-Forward Loop of Inflammation in Periodontitis.

Periodontitis is a widespread chronic inflammatory disease caused by a changed dysbiotic oral microbiome. Although multiple species and risk factors are associated with periodontitis, Porphyromonas gingivalis has been identified as a keystone pathogen. The immune-modulatory function of P. gingivalis is well characterized, but the mechanism by which this bacterium secretes peptidyl arginine deiminase (PPAD), a protein/peptide citrullinating enzyme, thus contributing to the infinite feed-forward loop of inflammation, is not fully understood. To determine the functional role of citrullination in periodontitis, neutrophils were stimulated by P. gingivalis bearing wild-type PPAD and by a PPAD mutant strain lacking an active enzyme. Flow cytometry showed that PPAD contributed to prolonged neutrophil survival upon bacterial stimulation, accompanied by the secretion of aberrant IL-6 and TNF-α. To further assess the complex mechanism by which citrullination sustains a chronic inflammatory state, the ROS production and phagocytic activity of neutrophils were evaluated. Flow cytometry and colony formation assays showed that PPAD obstructs the resolution of inflammation by promoting neutrophil survival and the release of pro-inflammatory cytokines, while enhancing the resilience of the bacteria to phagocytosis.

Peptidylarginine Deiminase Type 2 Predicts Tumor Progression and Poor Prognosis in Patients with Curatively Resected Biliary Tract Cancer.

(1) Background: PADI2 is a post-translational modification (PTM) enzyme that catalyzes citrullination, which then triggers autoimmune disease and cancer. This study aimed to evaluate the prognostic value of peptidylarginine deiminase 2 (PADI2) protein expression in biliary tract cancer (BTC) patients. (2) Methods: Using immunohistochemistry, the PADI2 protein expression in BTC tissues was analyzed. The correlations between PADI2 protein expression and clinicopathologic characteristics were analyzed using Chi-square tests. The Kaplan-Meier procedure was used for comparing survival distributions. We used Cox proportional hazards regression for univariate and multivariate analyses. From 2014 to 2020, 30 resected BTC patients were enrolled in this study. (3) Results: Patients with high PADI2 protein expression were associated with shorter progress-free survival (PFS; p = 0.041), disease-specific survival (DSS; p = 0.025), and overall survival (OS; p = 0.017) than patients with low PADI2 protein expression. (4) Conclusions: The results indicated that PADI2 protein expression was an independent poor prognostic factor for BTC patients regarding PFS, DSS, and OS.

Cannabidiol alleviates perfluorooctane sulfonate-induced macrophage extracellular trap mediate inflammation and fibrosis in mice liver

As a new type of persistent organic pollutant, perfluorooctane sulphonate (PFOS) has received extensive attention worldwide. Cannabidiol (CBD) is a non-psychoactive natural cannabinoid extract that has been proved to have antioxidation, regulation of inflammation and other functions. However, the effects of PFOS on liver injury and whether CBD can alleviate PFOS-induced liver injury are still unclear. Therefore, in this study, we used CBD (10 mg/kg) and/or PFOS (5 mg/kg) to intraperitoneally inject mice for 30 days. We found that PFOS exposure led to inflammatory infiltration in the liver of mice, increased the formation of macrophage extracellular trap (MET), and promoted fibrosis. In vitro, we established a coculture system of RAW264.7, AML12 and LX-2 cells, and treated them with CBD (10 μM) and/or PFOS (200 μM). The results showed that PFOS could also induce the expression of MET, inflammation and fibrosis marker genes in vitro. Coiled-coil domain containing protein 25 (CCD25), as a MET-DNA sensor, was used to investigate its ability to regulate inflammation and fibrosis, we knocked down CCDC25 and its downstream proteins (integrin-linked kinase, ILK) by siRNA technology, and used QNZ to inhibit NF-κB pathway. The results showed that the knockdown of CCDC25 and ILK and the inhibition of NF-κB pathway could inhibit MET-induced inflammation and fibrosis marker gene expression. In summary, we found that PFOS-induced MET can promote inflammation and fibrosis through the CCDC25-ILK-NF-κB signaling axis, while the treatment of CBD showed a protective effect, and it is proved by Macromolecular docking that this protective effect is achieved by combining CBD with peptidylarginine deiminase 4 (PAD4) to alleviate the release of MET. Therefore, regulating the formation of MET and the CCDC25-ILK-NF-κB signaling axis is an innovative treatment option that can effectively reduce hepatotoxicity. Our study reveals the mechanism of PFOS-induced hepatotoxicity and provides promising insights into the protective role of CBD in this process.

PAD4-Induced NETosis Via Cathepsin G-Mediated Platelet-Neutrophil Interaction in ChAdOx1 Vaccine-Induced Thrombosis.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare syndrome characterized by platelet anti-PF4 (platelet-activating antiplatelet factor 4)-related thrombosis. Platelet-neutrophil interaction has been suggested to play a role, but the underlying mechanism has not been fully elucidated. The study included 10 patients with VITT after ChAdOx1 nCoV-19 (Oxford-AstraZeneca) vaccine administration, 10 patients with ischemic stroke (IS), 10 patients with acute deep vein thrombosis, and 10 control subjects in whom blood levels of neutrophil extracellular traps (NETs), soluble TF (tissue factor), and thrombin generation were examined. Furthermore, we performed in vitro studies comparing the effect of serum from patients and controls on NETs formation. Finally, immunohistochemistry was performed in cerebral thrombi retrieved from a patients with VITT and 3 patients with IS. Compared with patients with IS, patients with deep vein thrombosis, controls, and patients with VITT had significantly higher blood values of CitH3 (citrullinated histone H3), soluble TF, D-dimer, and prothrombin fragment 1+2 (P<0.0001). Blood CitH3 significantly correlated with blood soluble TF (Spearman rank correlation coefficient=0.7295; P=0.0206) and prothrombin fragment 1+2 (Spearman rank correlation coefficient=0.6809; P<0.0350) in patients with VITT. Platelet-neutrophil mixture added with VITT plasma resulted in higher NETs formation, soluble TF and thrombin generation, and platelet-dependent thrombus growth under laminar flow compared with IS and deep vein thrombosis plasma; these effects were blunted by PAD4 (protein arginine deiminase 4) and cathepsin G inhibitors, anti-FcγRIIa, and high doses of heparin. Immunohistochemistry analysis showed a more marked expression of PAD4 along with more diffuse neutrophil infiltration and NETs formation as well as TF and cathepsin expression in VITT thrombus compared with thrombi from patients with IS. Patients with VITT display enhanced thrombogenesis by PAD4-mediated NETs formation via cathepsin G-mediated platelet/neutrophil interaction.

Voluntary Exercise-Mediated Protection in TNBS-Induced Rat Colitis: The Involvement of NETosis and Prdx Antioxidants.

Inflammatory bowel diseases (IBDs) are autoimmune disorders of the gut. It is increasingly clear that voluntary exercise (VE) may exert protection against IBDs, but the exact background mechanism needs to be elucidated. In the present study, we aimed to investigate the possible role of NETosis and the antioxidant peroxiredoxin (Prdx) enzyme family in VE-induced protection. Wistar Han rats were randomly divided into two groups: sedentary (SED) and VE. After the 6-week voluntary wheel running, animals were treated with 2,4,6-trinitrobenzene sulphonic acid (TNBS) as a model of colitis. Here, we found that VE significantly decreased inflammation and ulceration of the colon in the VE TNBS group compared with SED TNBS. We also found that VE significantly decreased the expression of protein arginine deiminase 4 (PAD4) and myeloperoxidase (MPO), and markedly reduced citrullinated histone H3 (citH3) compared with SED TNBS. Furthermore, VE caused a significant increase in the levels of Prdx6 in the control and TNBS groups. Taken together, we found that a prior 6-week VE effectively reduces inflammation in TNBS-induced colitis, and we suggest that the protective effect of VE may be mediated via the inhibition of NETosis and upregulation of Prdx6 antioxidant.

Inhibition of protein arginine deiminase 4 prevents inflammation-mediated heart failure in arthritis.

Rheumatoid arthritis is a prototypic inflammatory condition with affected patients being at greater risk of incident heart failure (HF). Targeting innate immune cell function in the pathogenesis of HF bears the potential to guide the development of future therapies. A collagen-induced arthritis (CIA) model in DBA/1 J mice was used to generate arthritis. Mice with CIA developed concentric hypertrophic myocardial remodeling, left ventricular (LV) diastolic dysfunction, and HF with elevated plasma B-type natriuretic peptide levels but preserved LV ejection fraction. Key features of HF in CIA were increased infiltration of activated neutrophils, deposition of neutrophil extracellular traps in the myocardium, and increased tissue levels of the proinflammatory cytokine IL-1β. Specific inhibition of protein arginine deiminase 4 (PAD4) by an orally available inhibitor (JBI-589), administered after the onset of clinical arthritis, prevented HF with reduced neutrophil infiltration. We identify PAD4-mediated neutrophil activation and recruitment as the key thromboinflammatory pathway driving HF development in arthritis. Targeting PAD4 may be a viable therapeutic approach for the prevention of HF secondary to chronic inflammation.

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.

Role of Peptidylarginine Deiminase 4 in Central Nervous System Diseases.

The deimination or citrullination of arginine residues in the polypeptide chain by peptidylarginine deiminase 4 alters the charge state of the polypeptide chain and affects the function of proteins. It is one of the main ways of protein post-translational modifications to regulate its function. Peptidylarginine deiminase 4 is widely expressed in multiple tissues and organs of the body, especially the central nervous system, and regulates the normal development of organisms. The abnormal expression and activation of peptidylarginine deiminase 4 is an important pathological mechanism for the occurrence and development of central nervous system diseases such as multiple sclerosis, Alzheimer's disease, cerebral ischemia reperfusion injury, and glioblastoma.

The release of zearalenone-induced heterophil extracellular traps in chickens is associated with autophagy, glycolysis, PAD enzyme, and P2X1 receptor

Zearalenone (ZEA) is produced mainly by fungi belonging to genus Fusarium in foods and feeds. Heterophil extracellular traps (HETs) are a novel defense mechanism of chicken innate immunity involving activated heterophils. However, the conditions and requirements for ZEA-triggered HET release remain unknown. In this study, immunostaining analysis demonstrated that ZEA-triggered extracellular fibers were composed of histone and elastase assembled on DNA skeleton, showing that ZEA can induce the formation of HETs. Further experiments indicated that ZEA-induced HET release was concentration-dependent (ranging from 20 to 80 μM ZEA) and time-dependent (ranging from 30 to 180 min). Moreover, in 80 μM ZEA-exposed chicken heterophils, reactive oxygen species (ROS) level, catalase (CAT), superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and glutathione (GSH) content were increased. Simultaneously, ZEA at 80 μM activated ERK and p38 MAPK signaling pathways by increasing the phosphorylation level of ERK and p38 proteins. Pharmacological inhibition assays revealed that blocking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, ERK, and p38 mitogen-activated protein kinase (MAPK) reduced ZEA-induced ROS levels but had no impact on HET formation. Furthermore, immunostaining analysis indicated that the heterophil underwent the formation of autophagosome based on being stained with LC3B. The pharmacological inhibition assays demonstrated that rapamycin-, wortmannin-, and 3-methyladenine (3-MA)-treatments modulated ZEA-triggered HET formation, indicating that heterophil autophagy played a key role in ZEA-induced HET formation. Further studies on energy metabolism showed that inhibition of lactate/glucose transport, hexokinase-2 (HK-2), fructose-2,6-biphosphatase 3 (PFKFB3) in glycolysis abated ZEA-induced HETs, implying that glycolysis was one of the factors influencing the ZEA-induced HET formation. Besides, inhibition of the peptidylarginine deiminase (PAD) enzyme and P2X1 significantly reduced the ZEA-induced HET formation. In conclusion, we demonstrated that ZEA-triggered HET formation, which was associated with glycolysis, autophagy, PAD enzyme, and P2X1 receptor activation, providing valuable insight into the negative effect of ZEA on chicken innate immunity.

The Extracellular Vesicle Citrullinome and Signature in a Piglet Model of Neonatal Seizures.

Neonatal seizures are commonly associated with acute perinatal brain injury, while understanding regarding the downstream molecular pathways related to seizures remains unclear. Furthermore, effective treatment and reliable biomarkers are still lacking. Post-translational modifications can contribute to changes in protein function, and post-translational citrullination, which is caused by modification of arginine to citrulline via the calcium-mediated activation of the peptidylarginine deiminase (PAD) enzyme family, is being increasingly linked to neurological injury. Extracellular vesicles (EVs) are lipid-bilayer structures released from cells; they can be isolated from most body fluids and act as potential liquid biomarkers for disease conditions and response to treatment. As EVs carry a range of genetic and protein cargo that can be characteristic of pathological processes, the current study assessed modified citrullinated protein cargo in EVs isolated from plasma and CSF in a piglet neonatal seizure model, also following phenobarbitone treatment. Our findings provide novel insights into roles for PAD-mediated changes on EV signatures in neonatal seizures and highlight the potential of plasma- and CSF-EVs to monitor responses to treatment.

Targeting Neutrophil α9 Improves Functional Outcomes After Stroke in Mice With Obesity-Induced Hyperglycemia.

Obesity-induced hyperglycemia is a significant risk factor for stroke. Integrin α9β1 is expressed on neutrophils and stabilizes adhesion to the endothelium via ligands, including Fn-EDA (fibronectin containing extra domain A) and tenascin C. Although myeloid deletion of α9 reduces susceptibility to ischemic stroke, it is unclear whether this is mediated by neutrophil-derived α9. We determined the role of neutrophil-specific α9 in stroke outcomes in a mice model with obesity-induced hyperglycemia. α9Neu-KO (α9fl/flMRP8Cre+) and littermate control α9WT (α9fl/flMRP8Cre-) mice were fed on a 60% high-fat diet for 20 weeks to induce obesity-induced hyperglycemia. Functional outcomes were evaluated up to 28 days after stroke onset in mice of both sexes using a transient (30 minutes) middle cerebral artery ischemia. Infarct volume (magnetic resonance imaging) and postreperfusion thrombo-inflammation (thrombi, fibrin, neutrophil, phospho-nuclear factor kappa B [p-NFκB], TNF [tumor necrosis factor]-α, and IL [interleukin]-1β levels, markers of neutrophil extracellular traps) were measured post 6 or 48 hours of reperfusion. In addition, functional outcomes (modified Neurological Severity Score, rota-rod, corner, and wire-hanging test) were measured for up to 4 weeks. Stroke upregulated neutrophil α9 expression more in obese mice (P<0.05 versus lean mice). Irrespective of sex, deletion of neutrophil α9 improved functional outcomes up to 4 weeks, concomitant with reduced infarct, improved cerebral blood flow, decreased postreperfusion thrombo-inflammation, and neutrophil extracellular traps formation (NETosis) (P<0.05 versus α9WT obese mice). Obese α9Neu-KO mice were less susceptible to thrombosis in FeCl3 injury-induced carotid thrombosis model. Mechanistically, we found that α9/cellular fibronectin axis contributes to NETosis via ERK (extracellular signal-regulated kinase) and PAD4 (peptidyl arginine deiminase 4), and neutrophil α9 worsens stroke outcomes via cellular fibronectin-EDA but not tenascin C. Obese wild-type mice infused with anti-integrin α9 exhibited improved functional outcomes up to 4 weeks (P<0.05 versus vehicle). Genetic ablation of neutrophil-specific α9 or pharmacological inhibition improves long-term functional outcomes after stroke in mice with obesity-induced hyperglycemia, most likely by limiting thrombo-inflammation.