Iguratimod inhibits protein citrullination and inflammation by downregulating NBCe2 in patients with rheumatoid arthritis.

BACKGROUND: Bicarbonate has recently been identified as a crucial factor affecting peptidylarginine deiminase (PAD) activity; however, the mechanism underlying its role in rheumatoid arthritis (RA) remains unclear. Iguratimod (IGU), a small-molecule disease-modifying anti-rheumatic drug, requires further investigation. This study aimed to explore the mechanism by which bicarbonate affects citrullination and inflammation in RA and identify new targets for IGU. METHODS: We enrolled 20 patients with RA in the study. Sodium bicarbonate cotransporter 2 (NBCe2) was detected in the peripheral blood neutrophils and peripheral blood mononuclear cells (PBMCs) of these patients. The effects of varying concentrations of IGU, methotrexate (MTX), dexamethasone (DXM), and S0859 (an NBCe2 inhibitor) on NBCe2, PAD2, PAD4, and citrullinated histone H3 (cit-H3) levels in, migration ability of, and cytokine production from neutrophils and PBMCs were examined. RESULTS: Our findings showed that in patients with RA, citrullinated protein production by peripheral blood neutrophils instead of PBMCs, which showed higher NBCe2 expression levels, increased with an increase in the bicarbonate concentration. In addition, tumor necrosis factor-alpha (TNF-α) promoted NBCe2 expression in neutrophils from patients with RA. Furthermore, we revealed that the inhibitory effects of IGU on neutrophil NBCe2 and cit-H3 levels, degrees of inhibition of neutrophil and PBMC migration, and suppression of interleukin 6, TNF-α, and metalloproteinase-9 secretion from neutrophil-like differentiated HL-60 cells did not substantially differ from those of MTX, DXM, and S0859 at specific doses. CONCLUSIONS: Bicarbonate promotes protein citrullination and inflammation in RA via NBCe2, and IGU can downregulate NBCe2.

Belimumab Decreases Autophagy and Citrullination in Peripheral Blood Mononuclear Cells from Patients with Systemic Lupus Erythematosus.

Belimumab (BLM) is a B lymphocyte stimulator (BLyS) inhibitor approved for the treatment of systemic lupus erythematosus (SLE). Autophagy is a cell survival mechanism involved in the pathogenesis of SLE. Citrullination is a post-translational modification catalyzed by peptidylarginine deiminase (PAD) enzymes. Autophagy and citrullination may generate neoepitopes, evoking an autoimmune response. No previous studies have investigated the connection of these processes, and how BLM could affect them, in SLE. Ex vivo autophagy and protein citrullination were analyzed by western blot in lysates from 26 SLE patients' PBMCs at baseline and after 2, 4, and 12 weeks of BLM administration, and from 16 healthy donors' PBMCs. Autophagic PBMCs were identified by the immunofluorescent detection of the autophagy-associated proteins LC3B (LC3 puncta) and LAMP-1. Autophagosome accumulation was evaluated in CD14- (PBLs) and CD14+ (monocytes) SLE cells. The presence of the BLyS receptors BAFF-R, BCMA, and TACI on SLE CD4+, CD8+ T cells and monocytes, as well as serum IL-18 levels, was also assessed. Following BLM administration, we observed a decrease in autophagy and citrullination, with a lowering of LC3-II, citrullinated vimentin, and PAD4 expression levels in PBMCs from SLE patients. LC3-II levels showed a correlation with the SLE Disease Activity Index 2000 (SLEDAI-2K) after 12 weeks of therapy. The LC3B/LAMP-1 analysis confirmed the reduction in autophagy. A lesser autophagosome accumulation occurred in PBLs and monocytes which, in turn, seemed to be the main cellular populations contributing to autophagy. A reduction in patients' serum IL-18 concentrations occurred. CD4+ and CD8+ cells weakly expressed BAFF receptors; monocytes expressed only BAFF-R. BLM could impact on autophagy and citrullination, offering an opportunity for a deeper understanding of these mechanisms in SLE, and a possible tool for the clinical management of SLE.

IL-22 Downregulates Peptidylarginine Deiminase-1 in Human Keratinocytes: Adding Another Piece to the IL-22 Puzzle in Epidermal Barrier Formation.

Increased presence of IL-22+ cells in the skin is a characteristic finding in skin barrier defects, such as psoriasis and atopic dermatitis. However, mechanistic insight into effects of IL-22 on epidermal functioning is yet to be elucidated. One crucial step during epidermal differentiation is deimination or citrullination. Here, we show reduced levels of peptidylarginine deiminase 1, an enzyme that converts peptidylarginine into citrulline in lesional psoriatic skin. IL-22 signaling through the IL-22 receptor complex was found to suppress expression of peptidylarginine deiminase 1 in epidermal keratinocytes. Subsequently, total peptidylarginine deiminase activity and extent of protein deimination in keratinocytes treated with IL-22 were reduced together with a significant decrease in deimination of keratin 1 and FLG, both important for epidermal differentiation. Vitamin D and acitretin partly restored the peptidylarginine deiminase 1 defect caused by IL-22. Collectively, we show that IL-22 downregulates deimination, thus identifying a potential target for treatment of skin barrier defects.

Epitope-Based Chicken-Derived Novel Anti-PAD2 Monoclonal Antibodies Inhibit Citrullination.

The aberrant upregulation of protein arginine deiminase 2- (PAD2-) catalyzed citrullination is reported in various autoimmune diseases (rheumatoid arthritis and multiple sclerosis) and several cancers. Currently, there are no anti-PAD2 monoclonal antibodies (mAbs) that can inhibit the citrullination reaction. Here, an epitope 341YLNRGDRWIQDEIEFGY357 was examined as an antigenic site of PAD2. Chickens were immunized with this epitope, and the generated mAbs were screened for its reactivity against the full-length PAD2. Enzyme-linked immunosorbent assay revealed that six mAbs, which were screened from the phage display library, crossreacted with mouse PAD2. Kinetic analysis revealed that mAbs are bound to PAD2 in the nanomolar range, which indicated a strong binding. Results of the in vitro citrullination inhibition assay revealed that the half-maximal effective concentration values of mAbs for the inhibition of histone or benzoyl-L-arginine ethyl ester citrullination were in the range of 6-75 nM which supports strong inhibition capabilities. Alanine scanning of epitope revealed that the peptide fragment 344RGDRWIQDEIEF355 was responsible for generating strong antibody responses that inhibit the PAD2-catalyzed citrullination reaction. These antibodies can aid in understanding the extracellular PAD2 function and treating diseases associated with aberrant citrullination.

Regulation of polyamine homeostasis through an antizyme citrullination pathway.

This study reveals an uncovered mechanism for the regulation of polyamine homeostasis through protein arginyl citrullination of antizyme (AZ), a natural inhibitor of ornithine decarboxylase (ODC). ODC is critical for the cellular production of polyamines. AZ binds to ODC dimers and promotes the degradation of ODC via the 26S proteasome. This study demonstrates the protein citrullination of AZ catalyzed by peptidylarginine deiminase type 4 (PAD4) both in vitro and in cells. Upon PAD4 activation, the AZ protein was citrullinated and accumulated, leading to higher levels of ODC proteins in the cell. In the PAD4-overexpressing and activating cells, the levels of ODC enzyme activity and the product putrescine increased with the level of citrullinated AZ proteins and PAD4 activity. Suppressing cellular PAD4 activity reduces the cellular levels of ODC and downregulates cellular polyamines. Furthermore, citrullination of AZ in the C-terminus attenuates AZ function in the inhibition, binding, and degradation of ODC. This paper provides evidence to illustrate that PAD4-mediated AZ citrullination upregulates cellular ODC and polyamines by retarding ODC degradation, thus interfering with the homeostasis of cellular polyamines, which may be an important pathway regulating AZ functions that is relevant to cancer biology.

Citrullination-Resistant LL-37 Is a Potent Antimicrobial Agent in the Inflammatory Environment High in Arginine Deiminase Activity.

LL-37, the only member of the mammalian cathelicidin in humans, plays an essential role in innate immunity by killing pathogens and regulating the inflammatory response. However, at an inflammatory focus, arginine residues in LL-37 can be converted to citrulline via a reaction catalyzed by peptidyl-arginine deiminases (PAD2 and PAD4), which are expressed in neutrophils and are highly active during the formation of neutrophil extracellular traps (NETs). Citrullination impairs the bactericidal activity of LL-37 and abrogates its immunomodulatory functions. Therefore, we hypothesized that citrullination-resistant LL-37 variants would retain the functionality of the native peptide in the presence of PADs. To test this hypothesis, we synthetized LL-37 in which arginine residues were substituted by homoarginine (hArg-LL-37). Bactericidal activity of hArg-LL-37 was comparable with that of native LL-37, but neither treatment with PAD4 nor exposure to NETs affected the antibacterial and immunomodulatory activities of hArg-LL-37. Importantly, the susceptibilities of LL-37 and hArg-LL-37 to degradation by proteases did not significantly differ. Collectively, we demonstrated that citrullination-resistant hArg-LL-37 is an attractive lead compound for the generation of new agents to treat bacterial infections and other inflammatory diseases associated with enhanced PAD activity. Moreover, our results provide a proof-of-concept for synthesis of therapeutic peptides using homoarginine.

Exacerbation of symptomatic arthritis by cigarette smoke in experimental arthritis.

INTRODUCTION: Epidemiologically, cigarette smoking is a well-known risk factor for the pathogenesis of rheumatoid arthritis (RA). However, there has been few plausible explanations why cigarette smoking aggravated RA. We investigated the causal effect of smoking in experimental model of arthritis development. METHODS: During induction of experimental arthritis with collagen challenge, mice were exposed to a smoking environment with 3R4F cigarettes. Generated smoke was delivered to mice through a nose-only exposure chamber (ISO standard 3308). Human cartilage pellet was challenged by cigarette smoke extract to identify citrullinating potential in vitro. RESULTS: Cigarette smoke exacerbated arthritis in a collagen-induced arthritis (CIA) model. Exposure to smoke accelerated the onset of arthritis by 2 weeks compared to the conventional model without smoke. Citrullination of lung tissue as well as tarsal joints were revealed in smoke-aggravated CIA mice. Interestingly, tracheal cartilage was a core organ regarding intensity and area size of citrullination. The trachea might be an interesting organ in viewpoint of sharing cartilage with joint and direct smoke exposure. Anti-CCP antibodies were barely detected in the serum of CIA mice, they were significantly elevated in cigarette smoke group. Citrullinated antigens were increased in the serum of smoke-exposed mice. Lastly, a cigarette smoke extract enhanced human cartilage citrullination in vitro. CONCLUSIONS: Missing link of arthritic mechanism between smoke and RA could be partially explained by tracheal citrullination. To control tracheal cartilage citrullination may be beneficial for preventing arthritis development or aggravation if cigarette smoke is becoming a risk factor to pre-arthritic individual.

GnRH impairs diabetic wound healing through enhanced NETosis.

It has been reported that neutrophil extracellular traps (NETs) impair wound healing in diabetes and that inhibiting NET generation (NETosis) improves wound healing in diabetic mice. Gonadotropin-releasing hormone (GnRH) agonists are associated with a greater risk of diabetes. However, the role of GnRH in diabetic wound healing is unclear. We determined whether GnRH-promoted NETosis and induced more severe and delayed diabetic wound healing. A mouse model of diabetes was established using five injections with streptozotocin. Mice with blood glucose levels >250 mg/dL were then used in the experiments. GnRH agonist treatment induced delayed wound healing and increased NETosis at the skin wounds of diabetic mice. In contrast, GnRH antagonist treatment inhibited GnRH agonist-induced delayed wound healing. The expression of NETosis markers PAD4 and citrullinated histone H3 were increased in the GnRH-treated diabetic skin wounds in diabetic mice and patients. In vitro experiments also showed that neutrophils expressed a GnRH receptor and that GnRH agonist treatment increased NETosis markers and promoted phorbol myristate acetate (PMA)-induced NETosis in mouse and human neutrophils. Furthermore, GnRH antagonist treatment suppressed the expression of NETosis markers and PMA-induced NETosis, which were increased by GnRH treatment. These results indicated that GnRH-promoted NETosis and that increased NETosis induced delayed wound healing in diabetic skin wounds. Thus, inhibition of GnRH might be a novel treatment of diabetic foot ulcers.

Asymmetric dimethylation and citrullination in the LEW.1AR1-iddm rat, an animal model of human type 1 diabetes, and effects of anti-TCR/anti-TNF-α antibody-based therapy.

The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes (T1D). We determined by GC-MS the extent of asymmetric dimethylation (prADMA) and citrullination (prCit) of L-arginine residues in organ proteins (pr) of normoglycaemic control (ngCo, n = 6), acutely diabetic (acT1D, n = 6), chronically diabetic (chT1D, n = 4), and cured (cuT1D, n = 4) rats after anti-TCR/anti-TNF-α therapy. Pancreatic prCit and prADMA did not differ between the groups but were correlated (r = 0.728, P = 0.0003, n = 20). acT1D rats had lower prCit levels in spleen and kidney than ngCo rats. cuT1D rats had higher prADMA levels than chT1D rats only in the spleen. Combination therapy re-established normoglycaemia and increased prADMA in the spleen without altering pancreatic prADMA and prCit. Western blotting demonstrated the presence of different prADMA pattern, especially an ≈ 50-kDa prADMA in spleen and pancreas, and an ≈ 25-kDa prADMA in the pancreas only, with the kidney showing only a very faint and small prADMA. Besides the changes in the pancreas during different metabolic states, the spleen may play a stronger role for the recognition of metabolic changes in T1D than thought thus far.

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

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

Discovery of Novel Potential Reversible Peptidyl Arginine Deiminase Inhibitor.

Citrullination, a posttranslational modification, is catalyzed by peptidylarginine deiminases (PADs), a unique family of enzymes that converts peptidyl-arginine to peptidyl-citrulline. Overexpression and/or increased PAD activity is observed in rheumatoid arthritis (RA), Alzheimer's disease, multiple sclerosis, and cancer. Moreover, bacterial PADs, such as Porphyromonas gingivalis PAD (PPAD), may have a role in the pathogenesis of RA, indicating PADs as promising therapeutic targets. Herein, six novel compounds were examined as potential inhibitors of human PAD4 and PPAD, and compared to an irreversible PAD inhibitor, Cl-amidine. Four of the tested compounds (compounds 2, 3, 4, and 6) exhibited a micromolar-range inhibition potency against PAD4 and no effect against PPAD in the in vitro assays. Compound 4 was able to inhibit the PAD4-induced citrullination of H3 histone with higher efficiency than Cl-amidine. In conclusion, compound 4 was highly effective and presents a promising direction in the search for novel RA treatment strategies.

Lung alveolar tissue destruction and protein citrullination in diesel exhaust-exposed mouse lungs.

Humanity faces an increasing impact of air pollution worldwide, including threats to human health. Air pollutants prompt and promote chronic inflammation, tumourigenesis, autoimmune and other destructive processes in the human body. Post-translational modification of proteins, for example citrullination, results from damaging attacks of pollutants, including smoking, air pollution and others, rendering host tissues immunogenic. Citrullinated proteins and citrullinating enzymes, deiminases, are more prevalent in patients with COPD and correlate with ongoing inflammation and oxidative stress. In this study, we installed an in-house-designed diesel exhaust delivery and cannabidiol vaporization system where mice were exposed to relevant, urban traffic-related levels of diesel exhaust for 14 days and assessed integrity of alveolar tissue, gene expression shifts and changes in protein content in the lungs and other tissues of exposed mice. Systemic presence of modified proteins was also tested. The protective effect of phytocannabinoids was investigated as well. Data obtained in our study show subacute effects of diesel exhaust on mouse lung integrity and protein content. Emphysematous changes are documented in exposed mouse lungs. In parallel, increased levels of citrulline were detected in the alveolar lung tissue and peripheral blood of exposed mice. Pre-treatment with vaporized cannabidiol ameliorated some damaging effects. Results reported hereby provide new insights into subacute lung tissue changes that follow diesel exhaust exposure and suggest possible dietary and/or other therapeutic interventions for maintaining lung health and healthy ageing.

Development of Activity-Based Proteomic Probes for Protein Citrullination.

Protein arginine deiminases (PADs) catalyze the post-translational deimination of peptidyl arginine to form peptidyl citrulline. This modification is increased in multiple inflammatory diseases and in certain cancers. PADs regulate a variety of signaling pathways including apoptosis, terminal differentiation, and transcriptional regulation. Activity-based protein profiling (ABPP) probes have been developed to understand the role of the PADs in vivo and to investigate the effect of protein citrullination in various pathological conditions. Furthermore, these ABPPs have been utilized as a platform for high-throughput inhibitor discovery. This review will showcase the development of ABPPs targeting the PADs. In addition, it provides a brief overview of PAD structure and function along with recent advances in PAD inhibitor development.

Interrogating the Roles of Post-Translational Modifications of Non-Histone Proteins.

Post-translational modifications (PTMs) allot versatility to the biological functions of highly conserved proteins. Recently, modifications to non-histone proteins such as methylation, acetylation, phosphorylation, glycosylation, ubiquitination, and many more have been linked to the regulation of pivotal pathways related to cellular response and stability. Due to the roles these dynamic modifications assume, their dysregulation has been associated with cancer and many other important diseases such as inflammatory disorders and neurodegenerative diseases. For this reason, we present a review and perspective on important post-translational modifications on non-histone proteins, with emphasis on their roles in diseases and small molecule inhibitors developed to target PTM writers. Certain PTMs' contribution to epigenetics has been extensively expounded; yet more efforts will be needed to systematically dissect their roles on non-histone proteins, especially for their relationships with nononcological diseases. Finally, current research approaches for PTM study will be discussed and compared, including limitations and possible improvements.