Peptidylarginine Deiminase Enzymes Research Articles

Neutrophil PAD4 negatively influences cardiac function and remodeling with increasing age

Abstract Background Aging can be viewed as a status of chronic inflammation, in which neutrophils have a lower threshold for activation. The enzyme peptidylarginine deiminase 4 (PAD4), which catalyzes the conversion of arginine to citrulline, will be activated in a certain population of neutrophils. When this conversion takes place on the histones, neutrophils can form neutrophil extracellular traps (NETs), which are both prothrombotic and proinflammatory. Mice lacking this enzyme systemically were previously reported to be protected from age-related fibrosis. Purpose We aimed to study the long-term effect of neutrophils on cardiac health during the process of natural aging. We hypothesized that neutrophil PAD4, and in consequence NETs, are involved in cardiac fibrosis development, which in turn will result in impaired cardiac function. Methods We generated a mouse model of impaired NET release capability via deletion of PAD4, a NET-essential gene, under the neutrophil-specific promoter (PAD4fl/flMRP8Cre+). In order to study heart failure (HF) development, these specific deletion mice and their littermate controls were aged for a period of two years (coinciding with approximately 70 years of age in the human population; the age at which HF is the number one cause of hospitalization), after which cardiac function and remodeling were evaluated by echocardiography and histology, respectively. A separate set of young mice (12 weeks) were evaluated in parallel. Results We performed a comprehensive echocardiography analysis including both structural and functional parameters. As for systolic function, we could see that in old wild type (WT) mice, ejection fraction (EF) significantly decreased as compared to EF in young and healthy (YH) mice (YH - 67±6%, WT - 53±10%; p<0.0001) (Figure 1B). However, this decrease in systolic function was absent in the old PAD4fl/flMRP8Cre+ mice, with EF being comparable to the YH group (PAD4fl/flMRP8Cre+ - 67±7%; p=0.9169) (figure 1 A,B). As for diastolic function, again we could see a marked decrease in E/A ratio in the WT as compared to the YH population (YH- 1.50±0.23, WT – 1.21±0.17; p<0.0001), while this functional deterioration was absent in aged PAD4fl/flMRP8Cre+ animals (PAD4fl/flMRP8Cre+ - 1.38±0.21; p=0.0837) (Figure 1 C,D). To link this decline in heart function to tissue remodeling, we quantified collagen deposition in the heart. We saw that natural aging resulted in an increase in cardiac collagen deposition in the WT population as compared to YH mice (YH – 0.86±0.63%, WT – 4.02±1.71%). This increased collagen deposition was absent in the neutrophil deletion mice (PAD4fl/flMRP8Cre+ - 1.7±0.76%). Additionally, when comparing WT to PAD4 deletion-mice, we saw that the increase in collagen deposition is significantly higher in the WT mice (p<0.0001). Conclusion Our data confirms neutrophil PAD4 involvement in heart failure progression by promoting cardiac fibrosis, resulting in cardiac dysfunction. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Fonds Wetenschappelijk Onderzoek (FWO) - Vlaanderen

Zinc-dependent histone deacetylases drive neutrophil extracellular trap formation and potentiate local and systemic inflammation

SummaryNeutrophil extracellular traps (NETs) have been implicated in the pathogenesis of acute respiratory distress syndrome (ARDS) driven by viruses or bacteria, as well as in numerous immune-mediated disorders. Histone citrullination by the enzyme peptidylarginine deiminase 4 (PAD4) and the consequent decondensation of chromatin are hallmarks in the induction of NETs. Nevertheless, additional histone modifications that may govern NETosis are largely overlooked. Herein, we show that histone deacetylases (HDACs) play critical roles in driving NET formation in human and mouse neutrophils. HDACs belonging to the zinc-dependent lysine deacetylases family are necessary to deacetylate histone H3, thus allowing the activity of PAD4 and NETosis. Of note, HDAC inhibition in mice protects against microbial-induced pneumonia and septic shock, decreasing NETosis and inflammation. Collectively, our findings illustrate a new fundamental step that governs the release of NETs and points to HDAC inhibitors as therapeutic agents that may be used to protect against ARDS and sepsis.

CD8+ T Cells Variably Recognize Native Versus Citrullinated GRP78 Epitopes in Type 1 Diabetes.

In type 1 diabetes, autoimmune β-cell destruction may be favored by neoantigens harboring posttranslational modifications (PTMs) such as citrullination. We studied the recognition of native and citrullinated glucose-regulated protein (GRP)78 peptides by CD8+ T cells. Citrullination modulated T-cell recognition and, to a lesser extent, HLA-A2 binding. GRP78-reactive CD8+ T cells circulated at similar frequencies in healthy donors and donors with type 1 diabetes and preferentially recognized either native or citrullinated versions, without cross-reactivity. Rather, the preference for native GRP78 epitopes was associated with CD8+ T cells cross-reactive with bacterial mimotopes. In the pancreas, a dominant GRP78 peptide was instead preferentially recognized when citrullinated. To further clarify these recognition patterns, we considered the possibility of citrullination in the thymus. Citrullinating peptidylarginine deiminase (Padi) enzymes were expressed in murine and human medullary epithelial cells (mTECs), with citrullinated proteins detected in murine mTECs. However, Padi2 and Padi4 expression was diminished in mature mTECs from NOD mice versus C57BL/6 mice. We conclude that, on one hand, the CD8+ T cell preference for native GRP78 peptides may be shaped by cross-reactivity with bacterial mimotopes. On the other hand, PTMs may not invariably favor loss of tolerance because thymic citrullination, although impaired in NOD mice, may drive deletion of citrulline-reactive T cells.

Structural characterization of human peptidyl-arginine deiminase type III by X-ray crystallography.

The Ca2+-dependent enzyme peptidyl-arginine deiminase type III (PAD3) catalyses the deimination of arginine residues to form citrulline residues in proteins such as keratin, filaggrin and trichohyalin. This is an important post-translation modification that is required for normal hair and skin formation in follicles and keratocytes. The structure of apo human PAD3 was determined by X-ray crystallography to a resolution of 2.8 Å. The structure of PAD3 revealed a similar overall architecture to other PAD isoforms: the N-terminal and middle domains of PAD3 show sequence and structural variety, whereas the sequence and structure of the C-terminal catalytic domain is highly conserved. Structural analysis indicates that PAD3 is a dimer in solution, as is also the case for the PAD2 and PAD4 isoforms but not the PAD1 isoform.

Crosstalk between B cells and neutrophils in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease without known cure that primarily affects synovial joints. RA has a prevalence of approximately 1% of the population worldwide. A vicious circle between two critical immune cell types, B cells and neutrophils, develops and promotes disease. Pathogenic anti‐citrullinated protein antibodies (ACPA) directed against a range of citrullinated epitopes are abundant in both plasma and synovial fluid of RA patients. In addition to stimulating numerous cell types, ACPA and other autoantibodies, notably rheumatoid factor, form immune complexes (ICs) that potently activate neutrophils. Attracted to the synovium by abundant chemokines, neutrophils are locally stimulated by ICs. They generate cytokines and release cytotoxic compounds including neutrophil extracellular traps (NETs), strands of decondensed chromatin decorated with citrullinated histones and granule‐derived neutrophil proteins, which are particularly abundant in the synovial fluid. In this way, neutrophils generate citrullinated epitopes and release peptidylarginine deiminase (PAD) enzymes capable of citrullinating extracellular proteins in the rheumatic joint, contributing to renewed ACPA generation. This review article focusses on the central function of citrullination, a post‐translational modification of arginine residues in RA. The discussion includes ACPA and related autoantibodies, somatic hypermutation‐mediated escape from negative selection by autoreactive B cells, promotion of the dominance of citrullinated antigens by genetic and lifestyle susceptibility factors and the vicious circle between ACPA‐producing pathogenic B cells and NET‐producing neutrophils in RA.

Inhibition of Neutrophil Extracellular Trap Release through the Disruption of PAD4 Leads to Decreased Neutrophil and Platelet Accumulation at Sites of Venular Vascular Injury in a Murine Model of Heparin-Induced Thrombocytopenia

Neutrophil extracellular traps (NETs) are webs of decondensed nuclear DNA decorated with histones that facilitate microbial killing but also promote thrombosis. NET release is dependent on chromatin decondensation mediated by the enzyme peptidylarginine deiminase (PAD) 4 that is expressed predominantly in granulocyte nuclei and citrullinates histone arginine residues, reducing their positive charge, and decreasing their affinity for DNA. Mice deficient in PAD4 are incapable of NET release and have been found to be protected from deep venous thrombosis formation. We have shown that PF4 physically compacts released NETs, causing them to become resistant to DNase digestion. Moreover PF4/NETs bind to heparin-induced thrombocytopenia (HIT)-specific antibodies further stabilizes PF4/NET complexes, enhancing their endonuclease resistance. We therefore speculated that these immunogenic and longer-lasting NETs may contribute to the prothrombotic nature of HIT. To investigate this hypothesis, we used CRISPR technology to disrupt Exon 2 of the Padi4 gene in embryos obtained from mice transgenic for human CXCL4 and FCGR2A, termed “HIT mice”, that have previously been found to develop thrombocytopenia and thrombosis when exposed to the monoclonal HIT-like antibody, KKO. We studied clot formation after cremaster vessel laser injury in PAD4-/- compared toPAD4+/+ HIT mice, quantifying both neutrophil and platelet accumulation. We initially noted PAD4+/+ HIT mice treated with KKO had a significant increase in neutrophil thrombus incorporation 5 minuntes and 60 minutes following venule (but not arteriole) laser injury, compared to mice injected with the isotype control antibody, TRA [8.5±1.3(n=31) vs. 3.1±1.0(n=11), p=0.026 at 5 minutes, 20.1±2.2(n=46) vs. 7.5±1.8(n=17) p=0.002 at 60 minutes). Compared to TRA, KKO infusion also led to a significant increase in venule thrombus platelet volumes 60 minutes following laser injury (p=0.0035, Figure 1). Although KKO exposed PAD4-/- mice had similar thrombus platelet volumes and neutrophil thrombus infiltration compared to PAD4+/+ animals 5 minutes after injury, by 60 minutes, the PAD4-/- animals had significantly smaller platelet volumes (p<0.004, Figure 1) and significantly fewer incorporated neutrophils (p<0.007, Figure 1). To determine if the rise in platelet volumes and increase in neutrophil-thrombus infiltration in HIT was the result of chemokine release by activated platelets, we treated PAD4+/+ mice with the CXCR2 antagonist sch527123 prior to HIT induction. We observed that these mice had a marked reduction in the number of neutrophils in their thrombi, but no decrease in platelet volumes. These findings suggest that PAD4-mediated NET release plays a unique role in the prothrombotic nature of HIT, enhancing neutrophil recruitment while also augmenting platelet accumulation through a chemokine-independent mechanism. We therefore propose that NET release is important for the venular prothrombotic nature of HIT. Furthermore, inhibition of NET-release prevents thrombus growth more effectively than the blockade of chemokine-based neutrophil-platelet interactions and merits further investigation as a treatment strategy in HIT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Citrullination and PAD Enzyme Biology in Type 1 Diabetes - Regulators of Inflammation, Autoimmunity, and Pathology.

The generation of post-translational modifications (PTMs) in human proteins is a physiological process leading to structural and immunologic variety in proteins, with potentially altered biological functions. PTMs often arise through normal responses to cellular stress, including general oxidative changes in the tissue microenvironment and intracellular stress to the endoplasmic reticulum or immune-mediated inflammatory stresses. Many studies have now illustrated the presence of ‘neoepitopes’ consisting of PTM self-proteins that induce robust autoimmune responses. These pathways of inflammatory neoepitope generation are commonly observed in many autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and type 1 diabetes (T1D), among others. This review will focus on one specific PTM to self-proteins known as citrullination. Citrullination is mediated by calcium-dependent peptidylarginine deiminase (PAD) enzymes, which catalyze deimination, the conversion of arginine into the non-classical amino acid citrulline. PADs and citrullinated peptides have been associated with different autoimmune diseases, notably with a prominent role in the diagnosis and pathology of rheumatoid arthritis. More recently, an important role for PADs and citrullinated self-proteins has emerged in T1D. In this review we will provide a comprehensive overview on the pathogenic role for PADs and citrullination in inflammation and autoimmunity, with specific focus on evidence for their role in T1D. The general role of PADs in epigenetic and transcriptional processes, as well as their crucial role in histone citrullination, neutrophil biology and neutrophil extracellular trap (NET) formation will be discussed. The latter is important in view of increasing evidence for a role of neutrophils and NETosis in the pathogenesis of T1D. Further, we will discuss the underlying processes leading to citrullination, the genetic susceptibility factors for increased recognition of citrullinated epitopes by T1D HLA-susceptibility types and provide an overview of reported autoreactive responses against citrullinated epitopes, both of T cells and autoantibodies in T1D patients. Finally, we will discuss recent observations obtained in NOD mice, pointing to prevention of diabetes development through PAD inhibition, and the potential role of PAD inhibitors as novel therapeutic strategy in autoimmunity and in T1D in particular.

Progesterone stimulates histone citrullination to increase IGFBP1 expression in uterine cells.

Peptidylarginine deiminases (PAD) enzymes were initially characterized in uteri, but since then little research has examined their function in this tissue. PADs post-translationally convert arginine residues in target proteins to citrulline and are highly expressed in ovine caruncle epithelia and ovine uterine luminal epithelial (OLE)-derived cell line. Progesterone (P4) not only maintains the uterine epithelia but also regulates the expression of endometrial genes that code for proteins that comprise the histotroph and are critical during early pregnancy. Given this, we tested whether P4 stimulates PAD-catalyzed histone citrullination to epigenetically regulate expression of the histotroph gene insulin-like growth factor binding protein 1 (IGFBP1) in OLE cells. 100 nM P4 significantly increases IGFBP1 mRNA expression; however, this increase is attenuated by pre-treating OLE cells with 100 nM progesterone receptor antagonist RU486 or 2 µM of a pan-PAD inhibitor. P4 treatment of OLE cells also stimulates citrullination of histone H3 arginine residues 2, 8, and 17 leading to enrichment of the ovine IGFBP1 gene promoter. Since PAD2 nuclear translocation and catalytic activity require calcium, we next investigated whether P4 triggers calcium influx in OLE cells. OLE cells were pre-treated with 10 nM nicardipine, an L-type calcium channel blocker, followed by stimulation with P4. Using fura2-AM imaging, we found that P4 initiates a rapid calcium influx through L-type calcium channels in OLE cells. Furthermore, this influx is necessary for PAD2 nuclear translocation and resulting citrullination of histone H3 arginine residues 2, 8, and 17. Our work suggests that P4 stimulates rapid calcium influx through L-type calcium channels initiating PAD-catalyzed histone citrullination and an increase in IGFBP1 expression.

Cl-Amidine Improves Survival and Attenuates Kidney Injury in a Rabbit Model of Endotoxic Shock.

Objective: Sepsis causes millions of deaths on a global scale annually. Activation of peptidylarginine deiminase (PAD) enzymes in sepsis causes citrullination of histones, which results in neutrophil extracellular trap formation and sepsis progression. This study evaluates pan-PAD inhibitor, Cl-amidine, in a model of lipopolysaccharide (LPS)-induced endotoxic shock in rabbits. We hypothesized that Cl-amidine would improve survival and attenuate kidney injury. Methods: In the survival model, rabbits were injected injected intravenously with 1 mg/kg of LPS, and then randomly assigned either to receive dimethyl sulfoxide (DMSO; 1 mcL/g) or Cl-amidine (10 mg/kg diluted in 1 mcL/g DMSO). They were then monitored for 14 days to evaluate survival. In the non-survival experiment, the same insult and treatment were administered, however; the animals were euthanized 12 hours after LPS injection for kidney harvest. Acute kidney injury (AKI) scoring was performed by a histopathologist who was blinded to the group assignment. Serial blood samples were also collected and compared. Results: Rabbits that received Cl-amidine had a higher survival (72%) compared with the rabbits that received DMSO (14%; p < 0.05). Cl-amidine-treated rabbits had lower (p < 0.05) histopathologic AKI scores, as well as plasma creatinine and blood urea nitrogen (BUN) levels 12 hours after insult. Conclusions: Pan-PAD inhibitor Cl-amidine improves survival and attenuates kidney injury in LPS-induced endotoxic shock in rabbits.

The Central Role of Acute Phase Proteins in Rheumatoid Arthritis: Involvement in Disease Autoimmunity, Inflammatory Responses, and the Heightened Risk of Cardiovascular Disease.

Rheumatoid arthritis (RA) is an autoimmune disease of complex etiopathogenic origin and traditionally characterized by chronic synovitis and articular erosions. Furthermore, there is strong evidence that infectious agents, including those that become dormant within the host, play a major role in much of the etiology of RA and its hallmark of inflammation. A combination of genetic predisposition, environmental exposure, and presence of infectious agents may therefore lead to a loss of immune tolerance to citrullinated proteins, which present as self-antigens to the human immune system. This results in generation of highly RA-specific autoantibodies, known as anti-citrullinated protein antibodies (ACPAs). Protein citrullination occurs via posttranslational deamination of arginine residues by peptidylarginine deiminase enzymes, which have confirmed sources of both endogenous and infectious origins. A recognized plasma protein target of citrullination and RA autoantibody generation is fibrin and its soluble precursor fibrinogen, both key components of hemostasis and acute phase reaction. Increased titers of ACPAs that accompany rapid progression to clinical RA disease have been shown to drive a variety of proinflammatory processes, and therefore results in aberrant fibrin clot formation and increased cardiovascular risk. However, the full extent to which hemostasis is affected in RA remains controversial, owing to the differential impact that citrullinated fibrin(ogen) and concurrent systemic inflammation may have on resulting hemostatic outcome. This review highlights key events in initiation of autoimmune-driven inflammatory events, including the role of bacterial infectious agents, which subsequently result in clinical RA disease and associated secondary cardiovascular disease risk, with specific focus on plasma proteins that are heavily involved throughout the immunopathological progression process.

PAD enzymes in rheumatoid arthritis: pathogenic effectors and autoimmune targets

Peptidylarginine deiminases (PADs) have an important role in the pathogenesis of rheumatoid arthritis (RA) owing to their ability to generate citrullinated proteins - the hallmark autoantigens of RA. Of the five PAD enzyme isoforms, PAD2 and PAD4 are the most strongly implicated in RA at both genetic and cellular levels, and PAD inhibitors have shown therapeutic efficacy in mouse models of inflammatory arthritis. PAD2 and PAD4 are additionally targeted by autoantibodies in distinct clinical subsets of patients with RA, suggesting anti-PAD antibodies as possible biomarkers for RA diagnosis and prognosis. This Review weighs the evidence that supports a pathogenic role for PAD enzymes in RA as both promoters and targets of the autoimmune response, as well as discussing the mechanistic and therapeutic implications of these findings in the wider context of RA pathogenesis. Understanding the origin and consequences of dysregulated PAD enzyme activity and immune responses against PAD enzymes will be important to fully comprehend the pathogenic mechanisms involved in this disease and for the development of novel strategies to treat and prevent RA.

Identification and Characterization of the Lactating Mouse Mammary Gland Citrullinome.

Citrullination is a post-translational modification (PTM) in which positively charged peptidyl-arginine is converted into neutral peptidyl-citrulline by peptidylarginine deiminase (PAD or PADI) enzymes. The full protein citrullinome in many tissues is unknown. Herein, we used mass spectrometry and identified 107 citrullinated proteins in the lactation day 9 (L9) mouse mammary gland including histone H2A, α-tubulin, and β-casein. Given the importance of prolactin to lactation, we next tested if it stimulates PAD-catalyzed citrullination using mouse mammary epithelial CID-9 cells. Stimulation of CID-9 cells with 5 µg/mL prolactin for 10 min induced a 2-fold increase in histone H2A citrullination and a 4.5-fold increase in α-tubulin citrullination. We next investigated if prolactin-induced citrullination regulates the expression of lactation genes β-casein (Csn2) and butyrophilin (Btn1a1). Prolactin treatment for 12 h increased β-casein and butyrophilin mRNA expression; however, this increase was significantly inhibited by the pan-PAD inhibitor, BB-Cl-amidine (BB-ClA). We also examined the effect of tubulin citrullination on the overall polymerization rate of microtubules. Our results show that citrullinated tubulin had a higher maximum overall polymerization rate. Our work suggests that protein citrullination is an important PTM that regulates gene expression and microtubule dynamics in mammary epithelial cells.

P067 Proteins citrullination and Crohn’s disease: PAD4 but not PAD2 is a strong marker of ileal inflammation

Abstract Background Citrullination is a post-translational modification of proteins, mediated by enzymes called PAD (peptidylarginine deiminases). The immune system can attack citrullinated proteins, leading to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and ulcerative colitis, and the activity of PAD2 and PAD4 in innate immune cells has been demonstrated for these disorders. Recently, high levels of PAD2 have been described in activated fibroblasts in the context of liver fibrosis. We therefore investigated the role of PAD2 and PAD4, both in inflammatory and fibrotic contexts of ileal Crohn’s disease (CD). Methods We obtained ileal transmural samples from patients operated for stricturing ileal CD. Three different macroscopic areas within each resection specimen (i.e. proximal normal ileum, inflamed ileum and fibrotic ileum) were selected and histologically confirmed by an expert pathologist. Patients undergoing ileocolic resection for other conditions (e.g. right colon cancer) and with healthy terminal ileum were used as controls. For each region (normal CD, inflamed CD, fibrotic CD and control), immunohistochemistry (IHC), RNA and protein evaluations for PAD2 and PAD4 were performed. Multiplex immunofluorescence (IF) for PAD2, PAD4, myeloperoxidase, neutrophil elastase, CD68, vimentin and α-smooth muscle actin were carried out to investigate the enzymes-expressing cells. Additional IF was performed to study citrullinated histone 3 (H3cit) expression, the product of PAD4 activity in neutrophils and component of neutrophil extracellular traps (NETs). Statistical analysis was carried out with Kruskal–Wallis test and post hoc Mann–Whitney test. Results Resection specimens from 13 CD and 11 controls were included. IHC and IF showed an increased expression of both PAD2 and PAD4 in the neutrophils of inflamed areas, in cytoplasm and nucleus, respectively (Figure 1). Activated fibroblasts (vimentin+ and α-smooth muscle actin+) were negative for both enzymes. PAD4 mRNA expression was increased in inflamed tissue (p = 0.001, p = 0.008 and p = 0.028 vs. normal CD, fibrotic CD and controls, respectively), and confirmed using Western Blot (Figure 2). H3cit was increased in the ileal inflammatory infiltrates too (Figure 3), confirming high PAD4 expression. For PAD2, no significant changes were observed at RNA and protein level, mainly due to its reduced expression in epithelial cells from normal to diseased tissue (Figure 4). Conclusion Both PAD2 and PAD4 are strongly expressed in neutrophils of CD ileal resection specimens, but only PAD4 shows a significantly higher expression in the inflammatory context which translates in the formation of NETs. No direct correlation was observed between PAD enzymes and intestinal fibroblasts.

INCREASED SERUM LEVEL OF PEPTIDYL ARGININE DEIMINASE TYPE 4 IN VARIOUS MOLECULAR BIOLOGICAL SUBTYPES OF BREAST CANCER

Level of enzyme peptidyl arginine deiminase type 4 (PAD-4) was determined in serum samples collected from 98 females with primary breast cancer within January, 2017 – April, 2018 divided into 5 groups according to immunohistochemistry data: group 1 – luminal A cancer, 2 – luminal B Her-2 negative, 3 – luminal B Her-2 positive, 4 – non-luminal Her-2 positive, 5 – triple negative cancer. Samples were collected prior to the onset of any anti-cancer treatment, and in 41 cases – 15-24 days after performing radical surgery. Control samples were obtained from 20 healthy females. Serum PAD-4 level was measured with Human PAD-4 ELISA Kit (Wuhan Fine Biotech Co., Ltd, China) on automatic analyzer ADALTIS Personal LAB (Adaltis S.r.l., Italy). Statistical analysis was performed by using software IBM SPSS Statistics 19. Mean serum PAD-4 level before treatment was 9.0 ng/ml that did not change after surgery. However, mean PAD-4 level for healthy females was 1.5 ng/ml (0.0-2.0) ng/ml that significantly differed from cancer group (Mann–Whitney U test, U = 38.500, p0.001). in contrast PAD-4 level in various cancer groups was: group 1 – 11.05 ng/ml, group 2 – 11.9 ng/ml, group 3 – 10.8 ng/ml, group 4 – 7.99 ng/ml, group 5 – 9.9 ng/ml. Thus, level of serum PAD-4 was higher in groups with more favorable luminal cancer, but further research is needed to make definitive conclusions.

Modulating neutrophil extracellular traps for wound healing.

A diabetic microenvironment primes neutrophils for NETosis, a process of formation of neutrophil extracellular traps (NETs) that further degrades the neutrophils and makes them unavailable for the early-stage inflammatory processes. Mechanistically, simple modification of arginine residues of histones to citrulline by peptidylarginine deiminase (PAD4) enzyme is considered to be a prerequisite for NETosis. In fact, under diabetic conditions, an increase in PAD4-mediated NET formation is considered as one of the reasons for impaired wound healing. Therefore, in the present work, an alginate-GelMa (generally recognized as safe category by FDA, USA) based hydrogel scaffold containing a tripeptide (Thr-Asp-F-amidine) that inhibits PAD4 is developed, based on the hypothesis that inhibiting PAD4 enzyme might offer a way to enhance wound healing under diabetic conditions. The scaffolds are thoroughly characterized for their physicochemical and biological properties. Furthermore, neutrophil-scaffold interactions in terms of NETosis ability and release of other related biomarkers are studied. The wound healing ability is evaluated by a cell migration assay. In vivo wound healing efficacy of the developed scaffolds is demonstrated using a diabetic rat model. The results suggest a reduction in NETosis in the presence of a PAD4 inhibitor. Thus, the study demonstrates a novel scaffold system to deliver the PAD4 inhibitor that can be used to modulate NETosis and improve wound healing.

An Overview of the Intrinsic Role of Citrullination in Autoimmune Disorders.

A protein undergoes many types of posttranslation modification. Citrullination is one of these modifications, where an arginine amino acid is converted to a citrulline amino acid. This process depends on catalytic enzymes such as peptidylarginine deiminase enzymes (PADs). This modification leads to a charge shift, which affects the protein structure, protein-protein interactions, and hydrogen bond formation, and it may cause protein denaturation. The irreversible citrullination reaction is not limited to a specific protein, cell, or tissue. It can target a wide range of proteins in the cell membrane, cytoplasm, nucleus, and mitochondria. Citrullination is a normal reaction during cell death. Apoptosis is normally accompanied with a clearance process via scavenger cells. A defect in the clearance system either in terms of efficiency or capacity may occur due to massive cell death, which may result in the accumulation and leakage of PAD enzymes and the citrullinated peptide from the necrotized cell which could be recognized by the immune system, where the immunological tolerance will be avoided and the autoimmune disorders will be subsequently triggered. The induction of autoimmune responses, autoantibody production, and cytokines involved in the major autoimmune diseases will be discussed.

An interplay of structure and intrinsic disorder in the functionality of peptidylarginine deiminases, a family of key autoimmunity-related enzymes.

Citrullination is a post-translation modification of proteins, where the proteinaceous arginine residues are converted to non-coded citrulline residues. The immune tolerance to such citrullinated protein can be lost, leading to inflammatory and autoimmune diseases. Citrullination is a chemical reaction mediated by peptidylarginine deiminase enzymes (PADs), which are a family of calcium-dependent cysteine hydrolase enzymes that includes five isotypes: PAD1, PAD2, PAD3, PAD4, and PAD6. Each PAD has specific substrates and tissue distribution, where it modifies the arginine to produce a citrullinated protein with altered structure and function. All mammalian PADs have a sequence similarity of about 70-95%, whereas in humans, they are 50-55% homologous in their structure and amino acid sequences. Being calcium-dependent hydrolases, PADs are inactive under the physiological level of calcium, but could be activated due to distortions in calcium homeostasis, or when the cellular calcium levels are increased. In this article, we analyze some of the currently available data on the structural properties of human PADs, the mechanisms of their calcium-induced activation, and show that these proteins contain functionally important regions of intrinsic disorder. Citrullination represents an important trigger of multiple physiological and pathological processes, and as a result, PADs are recognized to play a number of important roles in autoimmune diseases, cancer, and neurodegeneration. Therefore, we also review the current state of the art in the development of PAD inhibitors with good potency and selectivity.

DNA released from neutrophil extracellular traps (NETs) activates pancreatic stellate cells and enhances pancreatic tumor growth

ABSTRACTNeutrophil extracellular trap (NET) formation results in the expulsion of granulocyte proteins and DNA into the extracellular space. This process is mediated by the enzyme peptidyl arginine deiminase 4 (PADI4) and translocation of elastase to the nucleus. NET formation, marked by increased levels of extracellular DNA, promotes pancreatic cancer proliferation and metastasis. Mice deficient in Padi4 demonstrate decreased pancreatic tumor growth, associated with a reduction in circulating extracellular DNA levels, diminished pancreatic stromal activation and improved survival in murine orthotopic pancreatic adenocarcinoma. Transplantation of Padi4−/− bone marrow into genetically engineered mice with Kras driven pancreatic adenocarcinoma (Pdx1-Cre:KrasG12D/+, KC mice) limits the frequency of invasive cancers when compared with syngeneic controls. DNA from neutrophils activates pancreatic stellate cells that form dense, fibrous stroma which can promote and enable tumor proliferation. DNase treatment diminishes murine tumor growth and stromal activation to reverse the effect of NETs within the tumor microenvironment. Furthermore, deletion of the receptor for advanced glycation end products (RAGE) in pancreatic stellate cells abrogates the effects of DNA in promoting stellate cell proliferation and decreases tumor growth. Circulating neutrophil-derived DNA correlates with the stage in patients with pancreatic ductal adenocarcinoma, confirming the role of NETs in human pancreatic cancer. These findings support further investigation into targeting of NETs, PADI4 and extracellular DNA as a potential treatment strategy in patients with pancreatic cancer.Trial Registration: This study reports correlative data from a clinical trial registered with clinicaltrials.gov, NCT01978184 (November 7, 2013).

Citrullination Controls Dendritic Cell Transdifferentiation into Osteoclasts.

An increased repertoire of potential osteoclast (OC) precursors could accelerate the development of bone-erosive OCs and the consequent bone damage in rheumatoid arthritis (RA). Immature dendritic cells (DCs) can develop into OCs, however, the mechanisms underlying this differentiation switch are poorly understood. We investigated whether protein citrullination and RA-specific anti-citrullinated protein Abs (ACPAs) could regulate human blood-derived DC-OC transdifferentiation. We show that plasticity toward the OC lineage correlated with peptidyl arginine deiminase (PAD) activity and protein citrullination in DCs. Citrullinated actin and vimentin were present in DCs and DC-derived OCs, and both proteins were deposited on the cell surface, colocalizing with ACPAs binding to the cells. ACPAs enhanced OC differentiation from monocyte-derived or circulating CD1c+ DCs by increasing the release of IL-8. Blocking IL-8 binding or the PAD enzymes completely abolished the stimulatory effect of ACPAs, whereas PAD inhibition reduced steady-state OC development, as well, suggesting an essential role for protein citrullination in DC-OC transdifferentiation. Protein citrullination and ACPA binding to immature DCs might thus promote differentiation plasticity toward the OC lineage, which can facilitate bone erosion in ACPA-positive RA.

The Influence of Porphyromonas Gingivalis Bacterium Causing Periodontal Disease on the Pathogenesis of Rheumatoid Arthritis: Systematic Review of Literature.

Periodontal pathologies are highly widespread throughout the world. Epidemiological studies have shown that as much as 1% of the population is suffering from periodontal disease. In recent years, there has been a growing number of studies linking these diseases with autoimmune diseases, especially with rheumatoid arthritis. This literature review evaluates changes in the relationship between periodontal pathologies caused by the bacterium Porphyromonas gingivalis and rheumatoid arthritis. The systematic review of the literature was performed according to the PRISMA analysis protocol. The review was performed with articles from the PubMed database. Searched articles were not older than 5 years. Only full texts and research performed with people were selected. A total of 56 results were received. A review and analysis of their full texts have been carried out and 10 articles were selected according to the established criteria. They were analyzed and results were presented. The results obtained from the literature were based on the influence of Porphyromonas gingivalis on the pathogenesis of rheumatoid arthritis. In the literature, the activity of this bacterium is explained by the analysis of its enzyme peptidylarginine deiminase and its principle of action. Studies have also been found to prove the presence of Porphyromonas gingivalis not only in the oral cavity but its DNA is also found in synovial fluid and plasma. In the researched articles, direct links between Porphyromonas gingivalis and rheumatoid arthritis have led doctors to draw attention to patients' oral hygiene and the condition of parodentium, as this may be the cause of autoimmune lesions. Treatment of periodontal disease will not only help maintain a healthy oral cavity but prevent the spread of bacteria to the surrounding tissues.