Peptidylarginine Deiminase Research Articles

481 Pathogenic variants affecting peptidyl arginine deiminase 3 and its major substrates underlie central centrifugal cicatricial alopecia

481 Pathogenic variants affecting peptidyl arginine deiminase 3 and its major substrates underlie central centrifugal cicatricial alopecia

Inhibition of PAD4-mediated neutrophil extracellular traps formation attenuates hypoxic–ischemic brain injury in neonatal mice

Neonatal hypoxic–ischemic encephalopathy (HIE) is the primary cause of neonatal mortality and severe neurological sequelae. The interaction of neuroinflammation with the immune system represents a significant pathological mechanism underlying the development of HIE. Neutrophil extracellular traps (NETs) are a recently identified antimicrobial mechanism utilized by neutrophils. NETs can act as damage-associated molecular patterns, thereby amplifying the immune response and exerting proinflammatory effects. However, further research is needed to elucidate their role in the pathogenesis of HIE. In this study, we investigated the role of NETs in a hypoxic–ischemic brain injury (HIBI) model. We first reported that a pharmacological intervention to inhibit peptidylarginine deiminase type IV (PAD4) may constitute an effective strategy for reducing HI insult-induced neuroinflammation, neuronal apoptosis, and brain tissue destruction while also enhancing long-term neurobehavioral function in mice. These results support a pathological role for NETs in HIBI, and targeting PAD4 is a potential direction for the treatment of HIE.

Ferroptosis Induces gut microbiota and metabolic dysbiosis in Collagen-Induced arthritis mice via PAD4 enzyme

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation and joint destruction, with emerging evidence implicating gut microbiota dysbiosis in its pathogenesis. The current study explores the role of ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation, in modulating gut microbiota and metabolic dysregulation through the enzyme peptidyl arginine deiminase 4 (PAD4) in collagen-induced arthritis (CIA) mouse model. Our findings demonstrate that ferroptosis exacerbates RA-related inflammatory responses and joint damage by upregulating PAD4 expression, which, in turn, influences the gut microbial composition and associated metabolite profiles. Erastin, a known ferroptosis agonist, significantly increased the relative abundance of pro-inflammatory bacteria such as Proteobacteria while reducing beneficial taxa like Firmicutes and Bacteroidetes. This microbial shift was associated with heightened oxidative stress and an imbalance in key metabolites, such as lysophosphatidyl ethanolamine 14:0 (LysoPE 14:0), further exacerbated by ferroptosis. Co-treatment with GSK484, a PAD4 inhibitor, reversed these effects, restoring microbial homeostasis and reducing joint inflammation. This study suggests that ferroptosis-mediated PAD4 activity contributes to RA pathogenesis by disrupting the gut-joint axis, providing novel insights into potential therapeutic targets for RA. Our results highlight the intricate interplay between immune-mediated cell death, gut microbiota, and systemic inflammation, emphasizing the importance of ferroptosis as a therapeutic target in mitigating RA progression.

Identification of novel PAD2 inhibitors using pharmacophore-based virtual screening, molecular docking, and MD simulation studies

In the realm of epigenetic regulation, Protein arginine deiminase 2 (PAD2) stands out as a therapeutic target due to its significant role in neurological disorders, rheumatoid arthritis (RA), multiple sclerosis (MS), and various cancers. To date, no in silico studies have focused on PAD2 for lead compound identification. Therefore, we conducted structure-based pharmacophore modeling, virtual screening, molecular docking, molecular dynamics (MD) simulations, and essential dynamics studies using PCA and free energy landscape analyses to identify repurposed drugs and selective inhibitors against PAD2. The best pharmacophore model, ‘Pharm_01,’ had a selectivity score of 10.485 and an excellent ROC curve quality of 0.972. Pharm1 consisted of three hydrogen bond donors (HBD) and two hydrophobic (Hy) features (DDDHH). A virtual screening of about 9.2 million compounds yielded 2575 hits using a fit value threshold of 2.5 and drug-likeness criteria. Molecular docking identified the top ten molecules, which were verified using MD simulations. Stability was verified using MM-PBSA studies, whereas conformational differences were investigated using PCA and free energy landscape analyses. Two hits (Leads 1 and 2) from the DrugBank dataset showed promise for repurposing as PAD2 inhibitors, while one hit compound (Lead 8) from the ZINC database emerged as a novel PAD2 inhibitor. These findings indicate that the discovered compounds may be potent PAD2 inhibitors, necessitating additional preclinical and clinical research to produce viable treatments for cancer and neurological disorders.

Abstract PR010: DICER1 cancer predisposition tumor promotion mediated non-cell autonomously via neutrophils and NETosis

Abstract DICER1 cancer predisposition syndrome is a heritable condition where affected patients have increased risk of neoplasms including fusion-negative rhabdomyosarcoma (FN-RMS). The syndrome is defined by germline heterozygous loss of function mutations in DICER1, but it remains unclear exactly how these mutations predispose children to cancer. To gain insight into the role of Dicer1 loss in DICER1 cancer predisposition, we deleted the Dicer1 gene both in the germline or conditionally within the tumor cell in our previously established mouse model of FN-RMS. One would expect the germline loss of one allele to provide a sensitized background in the tumor cell to acquire a second hit mutation. However, we observed faster tumor onset and increased penetrance in mice with germline heterozygous Dicer1 loss (Dicer1 +/- ) but not in mice when Dicer1 loss was restricted to the tumor cells (Dicer1 Flox/+ ), demonstrating that haploinsufficient tumor suppressor activity of Dicer1 loss requires non-cell autonomous cell types. Single cell RNA sequencing (scRNA-Seq) revealed significant expansion of immature neutrophils in Dicer1 +/- tumors, which were enriched for proteases associated with neutrophil extracellular traps (NETs). NETs are webs of chromatin and proteases released when neutrophils in response to inflammatory stimuli in process termed NETosis. We show Dicer1 +/- murine and human tumors are enriched for neutrophils and tumor-bearing mice had abundant circulating NETs. NETs cause significant damage to the local extracellular matrix (ECM) and have been implicated in tumor promotion. In addition, neutrophil enrichment, we found that Dicer1 +/- tumors show significant ECM remodeling and are increased C5a, a potent neutrophil chemoattractant and NET-priming factor. These results suggest NETosis is upregulated in Dicer1 +/- FN-RMS tumors. Ligand-receptor pair analysis (iTALK) on the scRNA- Seq data revealed a putative interaction between a NET-derived ligand and EGFR and IGF1R on tumor cells, suggesting direct tumor promoting signaling from NET-to-tumor. We validated this novel FN-RMS promotion mechanism in vitro using human FN-RMS cell lines and observed that this NET-derived ligand promoted FN-RMS growth through both EGFR and IGF1R signaling. NET release requires citrullination of histone H3 mediated by peptidyl arginine deiminase 4 (PADI4) to facilitate decondensation of chromatin. We intercrossed the Padi4 -/- allele into our tumor model to genetically block NETosis and observed complete rescue of the accelerated tumor onset and increased penetrance observed in Dicer1 +/- mice. These results demonstrate that NETosis promotes the growth of Dicer1 +/- FN-RMS tumors and is required for Dicer1 +/- haploinsufficiency. These findings may be applicable to many DICER1 syndrome- associated cancers, as neutrophils which get converted to tumor promoters by heterozygous DICER1 loss are present in DICER1 syndrome patients regardless of tumor type and suggest targeting neutrophils/NET-release may reduce cancer risk in DICER1 +/- individuals. Citation Format: Randolph K Larsen, Jason A Hanna, Hongjian Jin, Kristen B Reed, Darden W Kimbrough, Kyna Voung, Myron K Evans, Casey G Langdon, Catherine J Drummond, Matthew R Garcia, David Finkelstein, Patrick A Schreiner, Jerold E Rehg, Kris Ann P Schultz, Mark E Hatley. DICER1 cancer predisposition tumor promotion mediated non-cell autonomously via neutrophils and NETosis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr PR010.

Helicobacter pylori upregulates PAD4 expression via stabilising HIF-1α to exacerbate rheumatoid arthritis

ObjectiveHelicobacter pylori infection has been reported to aggravate rheumatoid arthritis (RA), but the relevant mechanism remains unclear. This study aimed to investigate the underlying pathogenic mechanism of H. pylori infection...

Abstract 4122257: Inhibition of Neutrophil Extracellular Traps Prevents Heart Failure by Preserving Mitochondrial Function of Cardiomyocytes in Mice

Background: We have previously demonstrated that neutrophil extracellular traps (NETs) in myocardial tissue are associated with cardiac dysfunction and adverse outcomes in patients with heart failure with reduced ejection fraction. Here, we aimed to elucidate the underlying mechanisms and to clarify whether inhibiting NETs could rescue the cardiac phenotype using ex vivo and in vivo models. Methods and Results: Given that peptidyl arginine deiminase 4 (PAD4) is essential for the formation of NETs, PAD4 knockout mice were used for this study. First, we assessed the mitochondrial function of cardiomyocytes by conducting real-time measurements of the oxygen consumption rate in ex vivo . Extracellular flux analysis revealed that NETs-containing conditioned medium from wild-type neutrophils or purified NET components led to impaired mitochondrial oxygen consumption of adult mouse cardiomyocytes, while these effects were abolished when PAD4 in neutrophils was genetically ablated (Figure 1). In a murine model of pressure overload-induced heart failure, transverse aortic constriction (TAC) operation was performed on wild-type and PAD4 knockout mice. Immunohistological analysis demonstrated that TAC induced the formation of NETs in the myocardium, with the greatest number of NETs occurring at 1 day, persisting into the failing stage at 4 weeks in wild-type mice, while PAD4-deficient hearts displayed no presence of NETs in the heart tissue (Figure 2). Four weeks after TAC, left ventricular ejection fraction was reduced in wild-type mice, whereas PAD4 knockout mice displayed preserved left ventricular ejection fraction (Figure 2). Finally, we investigated the bioenergetics of cardiomyocytes isolated from PAD4 knockout mice after TAC. Maximal mitochondrial oxygen consumption rate in cardiomyocytes isolated from TAC-operated PAD4 knockout mice was significantly higher than in those from TAC-operated wild-type mice (Figure 3). Conclusion: Our data suggest that NETs directly derived impairment of mitochondrial function in cardiomyocytes. Inhibiting NETs represents a potential therapeutic approach for heart failure, preventing mitochondrial dysfunction of cardiomyocytes.

The Coexistence of Klebsiella pneumoniae and Candida albicans Enhanced Biofilm Thickness but Induced Less Severe Neutrophil Responses and Less Inflammation in Pneumonia Mice Than K. pneumoniae Alone.

Due to the possible coexistence of Klebsiella pneumoniae (KP) and Candida albicans (CA), strains of KP and CA with biofilm production properties clinically isolated from patients were tested. The production of biofilms from the combined organisms (KP+CA) was higher than the biofilms from each organism alone, as indicated by crystal violet and z-stack immunofluorescence. In parallel, the bacterial abundance in KP + CA was similar to KP, but the fungal abundance was higher than CA (culture method), implying that CA grows better in the presence of KP. Proteomic analysis was performed to compare KP + CA biofilm to KP biofilm alone. With isolated mouse neutrophils (thioglycolate induction), KP + CA biofilms induced less prominent responses than KP biofilms, as determined by (i) neutrophilic supernatant cytokines (ELISA) and (ii) neutrophil extracellular traps (NETs), using immunofluorescent images (neutrophil elastase, myeloperoxidase, and citrullinated histone 3), peptidyl arginine deiminase 4 (PAD4) expression, and cell-free DNA. Likewise, intratracheal KP + CA in C57BL/6 mice induces less severe pneumonia than KP alone, as indicated by organ injury (serum creatinine and alanine transaminase) (colorimetric assays), cytokines (ELISA), bronchoalveolar lavage fluid parameters (bacterial culture and neutrophil abundances using a hemocytometer), histology score (H&E stains), and NETs (immunofluorescence on the lung tissue). In conclusion, the biofilm biomass of KP + CA was mostly produced from CA with less potent neutrophil activation and less severe pneumonia than KP alone. Hence, fungi in the respiratory tract might benefit the host in some situations, despite the well-known adverse effects of fungi.

A cyclic peptide toolkit reveals mechanistic principles of peptidylarginine deiminase IV regulation

Peptidylarginine deiminase IV (PADI4, PAD4) deregulation promotes the development of autoimmunity, cancer, atherosclerosis and age-related tissue fibrosis. PADI4 additionally mediates immune responses and cellular reprogramming, although the full extent of its physiological roles is unexplored. Despite detailed molecular knowledge of PADI4 activation in vitro, we lack understanding of its regulation within cells, largely due to a lack of appropriate systems and tools. Here, we develop and apply a set of potent and selective PADI4 modulators. Using the mRNA-display-based RaPID system, we screen >1012 cyclic peptides for high-affinity, conformation-selective binders. We report PADI4_3, a cell-active inhibitor specific for the active conformation of PADI4; PADI4_7, an inert binder, which we functionalise for the isolation and study of cellular PADI4; and PADI4_11, a cell-active PADI4 activator. Structural studies with PADI4_11 reveal an allosteric binding mode that may reflect the mechanism that promotes cellular PADI4 activation. This work contributes to our understanding of PADI4 regulation and provides a toolkit for the study and modulation of PADI4 across (patho)physiological contexts.

Association of PADI4 Gene Polymorphisms With Susceptibility to Rheumatoid Arthritis: Evidence From 24 Case-Control Studies.

This study aims to investigate the association of rs11203366, rs11203367, rs874881, rs2240340 and rs1748033 polymorphisms of protein-arginine deiminase type 4 (PADI4) gene with the risk of rheumatoid arthritis (RA) through a meta-analysis that was followed with a bioinformatics approach. The data were collected from reputable articles and underwent quantitative analysis, followed by in silico analysis using some bioinformatics tools. The results showed that rs874881 polymorphism in Latino (G vs. C: OR=1.35, 95% CI=1.11-1.65, p=0.003; GG+CG vs. CC: OR=2.02, 95% CI=1.41-2.89, p=0.0001; CG vs. CC+GG: OR=1.38, 95% CI=1.04-1.83, p=0.027; GG vs. CC: OR=2.09, 95% CI=1.35-3.23, p=0.001; CG vs. CC: OR=1.98, 95% CI=1.36-2.87, p=0.00033) and rs1748033 in Caucasian population (T vs. C: OR=1.25, 95% CI=1.07-1.45, p=0.005; TT vs. CT+CC: OR=1.34, 95% CI=1.09-1.64, p=0.005, TT+CT vs. CC: OR=1.26, 95% CI=1.09-1.44, p=0.001; TT vs. CC: OR=1.59, 95% CI=1.13-2.23, p=0.007; CT vs. CC: OR=1.20, 95% CI: 1.04-1.39, p=0.015) are associated with increased risk of RA. Moreover, rs11203366 (G vs. A: OR=1.46, 95% CI=1.19-1.78, p=0.0002, GG vs. AG+AA: OR=1.42, 95% CI=1.01-2.01, p=0.043; GG+AG vs. AA: OR=2.03, 95% CI=1.45-2.86, p=0.00004; GG vs. AA: OR=2.29, 95% CI=1.49-3.51, p=0.0002; AG vs. AA: OR=1.93, 95% CI=1.35-2.76, p=0.0003) and rs11203367 (T vs. C: OR=1.50, 95% CI=1.23-1.83, p=0.00007; TT vs. CT+CC: OR=1.56, 95% CI=1.12-2.18, p=0.009; TT+CT vs. CC: OR=2.02, 95% CI=1.43-2.84, p=0.00007, TT vs. CC: OR=2.43, 95% CI=1.59-3.71, p=0.0004; CT vs. CC: OR=1.86, 95% CI=1.30-2.68, p=0.0007) had an impact in the Latino population. Bioinformatics tools showed the effect of these polymorphisms on gene function. These findings suggest that rs11203366, rs11203367, rs874881 and rs1748033 polymorphisms may be genetic risk factors for RA. Moreover, differences between populations suggest that ethnicity may play an important role in the effect of these polymorphisms on RA risk.

Circulating levels of neutrophil extracellular traps reflects local neutrophil activity but not fibrinolytic activity in coronary thrombi from patients with ST-elevation myocardial infarction

Abstract Introduction Neutrophil extracellular traps (NETs) are associated with impaired fibrinolysis in ischemic stroke, while NETs degradation enhances lysis of coronary thrombi in ex vivo experiments. Whether circulating NET markers are associated with local fibrinolytic activity in patients with ST-elevation myocardial infarction (STEMI) has not been reported. Purpose We investigated associations between circulating NET markers and gene expression of fibrinolytic markers in aspirated coronary thrombi from patients with STEMI. Furthermore, we explored whether local upregulation of NET formation by peptidylarginine deiminase 4 (PAD4) was reflected in the circulation. Methods Coronary thrombi from 35 STEMI patients undergoing primary percutaneous coronary intervention (PCI) were aspirated and promptly snap-frozen to -80°C. Peripheral blood samples were collected simultaneously. Median time from start of symptoms to PCI was 152 min. Thrombus gene expression of tissue Plasminogen Activator (tPA), urinary-type Plasminogen Activator (uPA), plasminogen-activation inhibitor type 1 (PAI-1) and PAD4 were quantified with RT-PCR. Gene expression of PAD4 was also measured in circulating leukocytes. Circulating NET markers were measured by a fluorescent nucleic acid stain using fluorometry (dsDNA), an in-house ELISA technique (MPO-DNA) and commercial ELISA (H3Cit). Correlations were tested using Spearman’s rho. Results There were no correlations between any of the circulating NET markers and gene expression of the fibrinolytic markers tPA, uPA and PAI-1 in the thrombus. Of the circulating NET markers, dsDNA correlated with thrombus gene expression of PAD4 (rs=0.491 p=0.006), whereas none of the NET markers correlated with gene expression of PAD4 in circulating leukocytes. Conclusion Circulating NET markers were not associated with gene expression of fibrinolytic activity in thrombi from patients with STEMI. Local NET formation in the thrombi, measured as PAD4 expression, was associated with the circulating NET marker dsDNA. These findings suggest that circulating NET markers have limited utility for assessing local fibrinolytic activity, but might reflect PAD4-dependent NET formation in coronary thrombi.

Neutrophil extracellular traps are an important component of chronic myocardial inflammation in patients with heart failure

Abstract Background and aim We have previously shown that heart failure (HF) is characterized by chronic inflammation. However, the role of neutrophils (N) and extracellular neutrophil traps (NETs) in the myocardium of patients with HF is largely unknown. The present study investigated the number of neutrophils (N) and their proportion that develop NETs in HF. Methods We used quantitative confocal microscopy and NETs markers in the left ventricular biopsies obtained from 5 controls and from patients with HF due to dilated (DCM, n=7), ischemic (ICM, n=7) and inflammatory (infCMP, n=7) cardiomyopathy. We used immunolabeling for CD45 (N) and for NETs (citrullinated histone 3 (CitH3), peptidylarginine deiminase-4 (PAD-4), neutrophil elastase (NE) and myeoloperoxidase (MPO). Results Compared to the control, the number of N was 4.3-5.7 times higher in HF. Using single labeling for NETs marker revealed that 41.1±4.6% of N in DCM, 42.9±4.1% in ICM and 47.6±2.9% in infCMP developed NETs. The use of dual labeling (MPO with CitH3, MPO with PAD-4 and CitH3 with NE) resulted in 54.5±4.7% of N-developing NETs in DCM, 59.5±4.1% in ICM and 77.6%±4.2 in infCMP. The difference between N-undergoing NETs was statistically different in infCMP than in DCM.and ICM. The proportion of N-forming NETs in control tissue was less than 5% and was significantly different from that in HF patients regardless of etiology. Conclusions This is the first study to show the presence of NETs in human hearts in situ and demonstrates that NETs are an important component of chronic inflammation in HF due to cardiomyopathies.

Malondialdehyde-specific natural IgM inhibit toll-like receptor 4 and peptidyl-arginine deiminase 4-dependent NETosis triggered by coronary extracellular vesicles of myocardial infarction patients

Abstract Background Neutrophil extracellular traps (NETs) are critical mediators of thromboinflammation during acute myocardial infarction (AMI). However, triggers and signalling pathways of NETosis in AMI remain incompletely understood. Levels of extracellular vesicles (EV) carrying oxidation-specific epitopes (OSE) originating from lipid peroxidation are increased at the culprit site in AMI. Importantly, natural IgM antibodies with specificity for OSE, such as malondialdehyde (MDA), have been shown to modulate functional effects of EV, and are associated with reduced cardiovascular risk. Purpose We investigated the stimulatory capacity of EV on neutrophil effector functions and specifically targeted key mediators of NET formation using pharmacological inhibitors and atheroprotective natural IgM to inhibit this process. Methods Patients were included after diagnosis of ST-segment elevation myocardial infarction and blood was aspirated from the culprit site and peripheral arterial site (n=28) during primary percutaneous coronary intervention (pPCI). Myocardial function was documented by cardiac magnetic resonance imaging 4±2 days and 195±15 days after pPCI. EV were isolated from cell culture supernatants and culprit site plasma for neutrophil stimulation in vitro. Pharmacological inhibitors were used to map NET signalling pathways of EV. Isolated EV were used for neutrophil stimulation in a murine injection model in the presence of the MDA-specific IgM LR04 or an isotype control. NET formation and other neutrophil functions were assessed by flow cytometry, ELISA and fluorescence microscopy. Results Levels of NET surrogate markers and CD45+ MDA+ EV were higher at the culprit site than in the peripheral circulation. EV generated by LPS-activated THP-1 monocytic cells induced in vitro NET formation, release of neutrophil elastase and IL-8, and degranulation of MPO and NGAL in primary human neutrophils, but not reactive oxygen species. Toll-like receptor 4 (TLR4) and peptidyl-arginine deiminase 4 (PAD4) were identified as key mediators of NET formation induced by in vitro-generated EV and EV isolated from the culprit site of AMI patients. Functionally, the MDA-specific monoclonal IgM antibody LR04, but not an isotype control, inhibited the ability of patient-derived and in vitro-generated EV to trigger release of NETs in primary human neutrophils and in mice. Titres of MDA-specific IgM antibodies were inversely associated with the NET marker citH3 in blood of AMI patients. Finally, we found that the concentration of CD45+ MDA+ EV per protective MDA-specific IgM at the culprit site showed an inverse association with left ventricular ejection fraction 72 hours and 6 months after AMI. Conclusions We show that EV can trigger several neutrophil effector functions. EV-induced NET formation is TLR4- and PAD4-dependent and can be inhibited by OSE-specific natural IgM potentially influencing cardiovascular outcomes after an acute event.

Extracellular vesicles and citrullination signatures are novel biomarkers in sturgeon (Acipenser gueldenstaedtii) during chronic stress due to seasonal temperature challenge

Acipenser gueldenstaedtii is one of the most cultured sturgeon species worldwide and of considerable economic value for caviar production. There are though considerable challenges around chronic stress responses due to increased summer temperatures, impacting sturgeons’ immune responses and their susceptibility to opportunistic infections. The identification of molecular and cellular pathways involved in stress responses may contribute to identifying novel biomarkers reflective of fish health status, crucial for successful sturgeon aquaculture. Protein citrullination is a calcium-catalysed post-translational modification caused by peptidylarginine deiminases (PADs), altering target protein function and affecting protein interactions in physiological and pathobiological processes. PADs can also modulate extracellular vesicle (EVs) profiles, which play critical roles in cellular communication, via transport of their cargoes (proteins, including post-translationally modified proteins, genetic material and micro-RNAs).This study identified differences in EV signatures, and citrullinated proteins in sera from winter and summer farmed sturegeons. EVs were significantly elevated in sera of the summer chronically stressed group. The citrullinated proteins and associated gene ontology (GO) pathways in sera and serum-EVs of chronically heat stressed A. gueldenstaedtii, showed some changes, with specific citrullinated serum protein targets including alpa-2-macroglobulin, alpha globin, calcium-dependent secretion activator, ceruloplasmin, chemokine XC receptor, complement C3 isoforms, complement C9, plectin, selenoprotein and vitellogenin. In serum-EVs, citrullinated protein cargoes identified only in the chronically stressed summer group included alpha-1-antiproteinase, apolipoprotein B-100, microtubule actin crosslinking factor and histone H3. Biological gene ontology (GO) pathways related to citrullinated serum proteins in the chronically stressed group were associated with innate and adaptive immune responses, stress responses and metabolic processes. In serum-EVs of the heat-stressed group the citrullinome associated with various metabolic GO pathways.In addition to modified citrullinated protein content, Serum-EVs from the stressed summer group showed significantly increased levels of the inflammatory associated miR-155 and the hypoxia-associated miR-210, but significantly reduced levels of the growth-associated miR-206.Our findings highlight roles for protein citrullination and EV signatures in response to chronic heat stress in A. gueldenstaedtii, indicating a trade-off in immunity versus growth and may be of value for sturgeon aquaculture.

The vacuolar anti-Pseudomonal activity of neutrophil primary granule peptidyl-arginine deiminase enzymes.

The role of neutrophils in host defense involves several cell processes including phagocytosis, degranulation of antimicrobial proteins, and the release of neutrophil extracellular traps (NETs). In turn, dysregulated cell activity is associated with the pathogenesis of airway and rheumatic diseases, in which neutrophil-derived enzymes including peptidyl-arginine deiminases (PADs) play a role. Known physiological functions of PADs in neutrophils are limited to the activity of PAD isotype 4 in histone citrullination in NET formation. The aim of this study was to extend our knowledge on the role of PADs in neutrophils and, specifically, bacterial killing within the confines of the phagocytic vacuole. Human neutrophils were fractionated by sucrose gradient ultracentrifuge and PADs localized in subcellular compartments by Western blot analysis. Direct interaction of PADs with Pseudomonas aeruginosa (P. aeruginosa) was assessed by flow cytometry and Western blot overlay. The participation of neutrophil PAD2 and PAD4 in killing of P. aeruginosa was assessed by inclusion of PAD-specific inhibitors. In vitro, bactericidal activity of recombinant human PAD2 or PAD4 enzymes against P. aeruginosa was determined by enumeration of colony-forming units (CFU). Together with neutrophil elastase (NE), PAD2 and PAD4 were localized to primary granules and, following activation with particulate stimuli, were degranulated in to the phagocytic vacuole. In vitro, PAD2 and PAD4 bound P. aeruginosa (p = 0.04) and significantly reduced bacterial survival to 49.1 ± 17.0 (p < 0.0001) and 48.5 ± 13.9% (p < 0.0001), respectively. Higher antibacterial activity was observed at neutral pH levels with the maximum toxicity at pH 6.5 and pH 7.5, comparable to the effects of neutrophil bactericidal permeability increasing protein. In phagosomal killing assays, inclusion of the PAD2 inhibitor, AFM-30a, or PAD4 inhibitor, GSK484, significantly increased survival of P. aeruginosa (AFM-30a, p = 0.05; and GSK484, p = 0.0079). Results indicate that PAD2 and PAD4 possess antimicrobial activity and are directly involved in the neutrophil antimicrobial processes. This study supports further research into the development of PAD-based antimicrobials.

Brain-Region-Specific Differences in Protein Citrullination/Deimination in a Pre-Motor Parkinson's Disease Rat Model.

The detection of early molecular mechanisms and potential biomarkers in Parkinson's disease (PD) remains a challenge. Recent research has pointed to novel roles for post-translational citrullination/deimination caused by peptidylarginine deiminases (PADs), a family of calcium-activated enzymes, in the early stages of the disease. The current study assessed brain-region-specific citrullinated protein targets and their associated protein-protein interaction networks alongside PAD isozymes in the 6-hydroxydopamine (6-OHDA) induced rat model of pre-motor PD. Six brain regions (cortex, hippocampus, striatum, midbrain, cerebellum and olfactory bulb) were compared between controls/shams and the pre-motor PD model. For all brain regions, there was a significant difference in citrullinated protein IDs between the PD model and the controls. Citrullinated protein hits were most abundant in cortex and hippocampus, followed by cerebellum, midbrain, olfactory bulb and striatum. Citrullinome-associated pathway enrichment analysis showed correspondingly considerable differences between the six brain regions; some were overlapping for controls and PD, some were identified for the PD model only, and some were identified in control brains only. The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways identified in PD brains only were associated with neurological, metabolic, immune and hormonal functions and included the following: "Axon guidance"; "Spinocerebellar ataxia"; "Hippo signalling pathway"; "NOD-like receptor signalling pathway"; "Phosphatidylinositol signalling system"; "Rap1 signalling pathway"; "Platelet activation"; "Yersinia infection"; "Fc gamma R-mediated phagocytosis"; "Human cytomegalovirus infection"; "Inositol phosphate metabolism"; "Thyroid hormone signalling pathway"; "Progesterone-mediated oocyte maturation"; "Oocyte meiosis"; and "Choline metabolism in cancer". Some brain-region-specific differences were furthermore observed for the five PAD isozymes (PADs 1, 2, 3, 4 and 6), with most changes in PAD 2, 3 and 4 when comparing control and PD brain regions. Our findings indicate that PAD-mediated protein citrullination plays roles in metabolic, immune, cell signalling and neurodegenerative disease-related pathways across brain regions in early pre-motor stages of PD, highlighting PADs as targets for future therapeutic avenues.

Citrullination Accompanies the Development of Carotid Atherosclerotic Plaques.

Citrullination represents a post-translational modification primarily mediated by peptidylarginine deiminase (PADI) 2 and 4 and resulting in the conversion of positively charged peptidylarginine to neutrally charged peptidylcitrulline. Molecular consequences of citrullination include the generation of neoepitopes which provoke the production of autoantibodies implicated in the development of autoimmune diseases. As citrullination initiates, promotes, and is enhanced by aseptic inflammation which plays a pivotal role in atherosclerosis, we proposed that citrullination might accompany the development of atherosclerotic vascular disease. To investigate features and patterns of citrullination in atherosclerotic plaques. We collected carotid atherosclerotic plaques (n = 14) and adjacent arterial segments (n = 14) which were pairwise excised during the carotid endarterectomy. The tissues were examined employing proteomic profiling (ultra-high performance liquid chromatography-tandem mass spectrometry analysis), haematoxylin and eosin staining, Western blotting and immunofluorescence staining for peptidylcitrulline, PADI2, and PADI4, and gene expression analysis. To better explore the mechanisms of citrullination in the neointima, we have also stained excised plaques for the extracellular vesicle markers (CD9 and CD81) and assessed co-localisation of PADI2 (a citrullination marker) with CD81 (an extracellular vesicle marker). In order to study the systemic response to citrullination in an atherosclerotic vascular disease setting, we measured the level of anti-citrullinated protein antibodies in the serum of patients with ischaemic stroke and healthy volunteers. Proteomic profiling found 213 plaque-specific and 111 intact arteria-specific proteins, as well as 46 proteins and 13 proteins which have been respectively upregulated or downregulated in plaques as compared with the adjacent intact segments. Among the top 20 upregulated proteins were atherogenic apolipoprotein B-100, iron-associated protein haptoglobin, and matrix metal-loproteinase-9, together indicating the advanced stage of plaque progression. In comparison with the intact arterial segments, plaques demonstrated protein signatures of innate immune response and oxidative stress, suggesting aseptic inflammation as a driver of atherosclerotic vascular disease. Both peptidylcitrulline and PADI2 have been abundant in the neointima but negligible in tunica media; further, the levels of peptidylcitrulline, PADI2, and PADI4 were elevated in plaque lysates in comparison with those from adjacent arterial segments (p = 0.025, 0.025, and 0.010, respectively). Notably, PADI2 and peptidylcitrulline were co-localised with the cells in the neointima and a considerable proportion of PADI2 was co-localised with CD81-positive extracellular vesicles (p = 0.003). Albeit citrullinated histone H3 and myeloperoxidase showed higher signal in the neointima than in tunica media (p = 0.048 and 0.023, respectively), we did not observe any signs of neutrophil extracellular traps (e.g., unwound chromatin or co-localisation of citrullinated histone H3 with neutrophil elastase) in the plaque tissue. Serum anti-citrullinated protein antibodies were not elevated in patients with ischaemic stroke (p = 0.71), suggesting that vascular citrullination likely does not trigger a generalised immune response. The development of carotid atherosclerosis is associated with citrullination, although it represents a local rather than systemic phenomenon in this clinical scenario.

Loss of PADI2 and PADI4 ameliorates sepsis-induced acute lung injury by suppressing NLRP3+ macrophages.

Sepsis-induced acute lung injury (ALI) is prevalent in septic patients and has a high mortality rate. Peptidyl arginine deiminase (PADI) 2 and PADI4 play crucial roles in mediating the host's immune response in sepsis, but their specific functions remain unclear. Our study shows that Padi2-/-Padi4-/- double knockout (DKO) improved survival, reduced lung injury, decreased bacterial load in Pseudomonas aeruginosa (PA) pneumonia-induced sepsis mice. Using single-cell RNA sequencing (scRNA-seq), we found that the deletion of Padi2 and Padi4 reduced the Nlrp3+ pro-inflammatory macrophages and fostered Chil3+ myeloid cell differentiation into anti-inflammatory macrophages. Additionally, we observed the regulatory role of NLRP3-Ym1 axis upon DKO, confirmed by Chil3 knockdown and Nlrp3 KO experiments. Thus, eliminating Padi2 and Padi4 enhances the polarization of Ym1+ M2 macrophages by suppressing NLRP3, aiding in inflammation resolution and lung tissue repair. study unveils the PADI2/PADI4-NLRP3-Ym1 pathway as a potential target in treatment of sepsis-induced ALI.

Neutrophil extracellular traps induce barrier dysfunction in DSS-induced ulcerative colitis via the cGAS-STING pathway

Peptidyl arginine deiminase 4 (PAD4)-mediated neutrophil extracellular traps (NETs) play a crucial role in the pathogenesis of ulcerative colitis (UC). The cGAS-STING intracellular DNA-sensing pathway has been recognized as a pivotal mediator of inflammation. This study aimed to explore how NETs contribute to intestinal inflammation and barrier dysfunction in UC, focusing on the cGAS-STING pathway. We observed a significant increase of STING expression in UC mouse colons, which was mitigated by blocking NET formation through PAD4 genetic knockout. Moreover, NETs were discovered to activate the cGAS-STING pathway in MC38 cells in a dose and time-dependent manner, leading to the secretion of inflammatory cytokines and impaired barrier function. Additionally, STING deficiency ameliorated the clinical colitis index, intestinal inflammation, and barrier dysfunction. These findings underscore the involvement of cGAS-STING in regulating NET-mediated intestinal inflammation, suggesting its potential as a novel therapeutic target for UC.

APOE Protects Against Severe Infection with Mycobacterium tuberculosis by Restraining Production of Neutrophil Extracellular Traps.

While neutrophils are the predominant cell type in the lungs of humans with active tuberculosis (TB), they are relatively scarce in the lungs of most strains of mice that are used to study the disease. However, similar to humans, neutrophils account for approximately 45% of CD45+ cells in the lungs of Apoe -/- mice on a high-cholesterol (HC) diet following infection with Mycobacterium tuberculosis (Mtb). We hypothesized that the susceptibility of Apoe -/- HC mice might arise from an unrestrained feed-forward loop in which production of neutrophil extracellular traps (NETs) stimulates production of type I interferons by pDCs which in turn leads to the recruitment and activation of more neutrophils, and demonstrated that depleting neutrophils, depleting plasmacytoid dendritic cells (pDCs), or blocking type I interferon signaling, improved the outcome of infection. In concordance with these results, we found that Mtb-infected in Apoe -/- HC mice produce high levels of LTB4 and 12-HETE, two eicosanoids known to act as neutrophil chemoattractants and showed that blocking leukotriene B4 (LTB4) receptor signaling also improved the outcome of tuberculosis. While production of NETs has been associated with severe tuberculosis in other mouse models and in humans, a causative role for NETs in the pathology has not been directly established. We demonstrate that blocking the activation of peptidylarginine deiminase 4 (PAD4), an enzyme critical to NET formation, leads to fewer NETs in the lungs and, strikingly, completely reverses the hypersusceptibility of Apoe -/- HC mice to tuberculosis.