Search Results (74)

Background/Objectives: The human cathelicidin LL-37 (37 residues; net charge +6) couples broad-spectrum antimicrobial activity with immunomodulatory function, but rapid proteolysis and peptidylarginine deiminase (PAD)-mediated citrullination limit its therapeutic use. We aimed to enhance enzymatic stability and to test whether substitution with 2,3-diaminopropionic acid (Dap) building blocks bearing an oxa-acid spacer (DAPEG) shifts the functional profile of the LL-37 scaffold. Methods: Six LL-37 analogs were synthesized using Fmoc/tBu solid-phase peptide synthesis, replacing all five arginine, all six lysine, or three isoleucine residues with Dap bearing a one- or two-unit oxa-acid spacer. Identity, purity and secondary structure were assessed via mass spectrometry, ultra-performance liquid chromatography and circular dichroism. Proteolytic stability against human proteinase 3 (PR3), resistance to PAD2/PAD4 citrullination, DNA binding (dynamic light scattering), antibacterial activity, MTT cytotoxicity, and lipopolysaccharide-induced degranulation (PR3 and myeloperoxidase) in differentiated HL-60 cells were evaluated. Results: Proteolytic stability against PR3 increased 5.7-fold for the arginine-substituted Dap(GO1) analog, and both arginine analogs resisted PAD2- and PAD4-mediated citrullination, with Dap(GO2) being the most resistant. DNA binding was qualitatively retained, although dynamic light scattering showed weaker complex compaction for lysine analogs. All six analogs lost direct antibacterial activity and were non-cytotoxic at 1–10 µM. In differentiated HL-60 cells, the lysine-substituted Dap(O1) and Dap(O2) analogs suppressed lipopolysaccharide-induced secretion of both PR3 and myeloperoxidase from azurophilic granules, whereas isoleucine analogs were suppressed partially and arginine analogs behaved like native LL-37. Conclusions: The DAPEG framework shifts LL-37 from microbicidal toward degranulation-suppressive activity, providing a modular route to tune cathelicidin function. The intracellular mechanism (TLR4/FPR2 engagement, cytokine signaling) was not assayed. Full article

Objective: TNFSF13B, which encodes B-cell-activating factor (BAFF) and peptidylarginine deiminase 4 (PADI4), plays crucial roles in the pathogenesis of ANCA-associated vasculitis (AAV). This study investigated the associations of single-nucleotide polymorphisms (SNPs) in TNFSF13B/BAFF and PADI4 genes with AAV susceptibility, clinical phenotypes, and disease activity in a Guangxi Chinese population. Methods: A case–control study included 324 AAV patients and 324 healthy controls. After propensity score matching (201 pairs), genomic DNA was genotyped for TNFSF13B/BAFF rs3759467 (formerly rs386492354) and rs1041569, and PADI4 rs11203366 and rs874881 using multiplex PCR and high-throughput sequencing. Genetic associations were analyzed via logistic regression, subgroup, haplotype, and clinical correlation analyses. For each of the four SNPs separately, machine learning models (logistic regression, SVM, Random Forest, XGBoost) were built and evaluated via 5-fold cross-validation. No formal adjustment for multiple comparisons was applied due to the exploratory nature of this study. Results: For TNFSF13B/BAFF, the rs3759467 C allele was protective (dominant model OR = 0.60, p = 0.011; log-additive OR = 0.71, p = 0.020; CA haplotype OR = 0.71, p = 0.019), while the rs1041569 T allele was a risk factor (dominant model OR = 1.70, p = 0.016). Subgroup analysis revealed stronger protective effects of rs3759467 in females, Han ethnicity, and MPA patients, and stronger risk effects of rs1041569 in Han ethnicity and MPA patients. Haplotype CA was protective (OR = 0.71, p = 0.019), and TT was risk-associated (OR = 1.55, p = 0.017). Both TNFSF13B/BAFF SNPs were associated with rash and hemoptysis incidence (p < 0.05). rs1041569 was also associated with RBC (red blood cell) count and HB (hemoglobin) levels (p < 0.05). For PADI4, rs11203366 and rs874881 showed no association with AAV susceptibility (all p > 0.05). However, their genotypes were associated with disease activity (BVAS, Birmingham Vasculitis Activity Score), RBC count, and HB levels (p < 0.05). Although machine learning was applied to explore predictive patterns, its performance was suboptimal (AUC < 0.6), indicating limited clinical applicability. Accordingly, the primary findings rely on the genetic model analysis, and the machine learning results should not be overinterpreted as clinically actionable. SHAP analysis indicated that risk-associated genotypes contributed most to model predictions. Conclusions:TNFSF13B/BAFF gene polymorphisms rs3759467 and rs1041569 were associated with AAV susceptibility in this Guangxi cohort, influencing clinical manifestations like rash, hemoptysis, and anemia severity. PADI4 polymorphisms rs11203366 and rs874881 are not associated with susceptibility but may correlate with disease activity and hematological parameters. These findings highlight the ethnic and clinical subtype specificity of genetic influences in AAV. Due to the lack of external validation, these findings are exploratory and require replication. Full article

Peptidylarginine deiminases (PADs) are a family of five isozymes (PAD1–4, PAD6) in humans, with PAD2, 3 and 4 associated with the central nervous system. PAD-mediated post-translational citrullination/deimination of target proteins contributes to pathobiological processes, including in the central nervous system (CNS), where the potential of PAD inhibitor treatment has been reported. This study aimed to identify PAD-dependent pro-regenerative responses in neuronal and astrocytic cells, respectively, using human cellular in vitro models to assess the therapeutic effects of pan-PAD, PAD2- and PAD4 isozyme-specific inhibitors in an oxygen–glucose deprivation/reperfusion model of ischaemia (OGD/R) at different time windows (30 min, 1 h and 4 h) in conjunction with scratch injury and LPS stimulation. Key findings suggest that pan-PAD inhibitor Cl-amidine promotes CNS regeneration through enhancing wound-healing of both neuronal and astrocytic cells, indicating roles for several PAD isozymes in acute CNS injury. Astrocyte cells showed the most prominent PAD4 detection, with significantly lower levels of PAD1, PAD2, PAD3 and PAD6, while differentiated SH-SY5Y neuronal cells showed the highest detection of PAD3, followed by PAD2 and PAD1, as well as strong PAD6 positivity, but negligible PAD4 detection. Histone H3 citrullination was significantly reduced in response to Cl-amidine treatment in both cell types, indicating changes in histone H3-dependent events in CNS injury. Cl-amidine treatment modulated key neuronal (beta-3 tubulin) and astrocytic (GFAP) markers and also reduced inflammatory cytokine IL-6 levels in astrocytes following 4 h OGD/R in conjunction with LPS stimulation. This study indicates roles for several PAD isozymes, with differing prominence in neurons and astrocytes, and emphasises the potential for pharmacological PAD inhibitor treatment in CNS injury. Full article

Excessive neutrophil extracellular trap (NET) formation (NETosis), frequently associated with reactive oxygen species (ROS), exacerbates cutaneous inflammation induced by acute ultraviolet B (UVB) exposure. Although hispidin has potent antioxidant activity, its protective effects against acute UVB-induced skin inflammation and its relationship with NET-associated responses remain unclear. We investigated the effects of topical hispidin on acute UVB-induced skin injury in mice and examined its effects on ROS-associated NET-related responses in differentiated HL-60 cells. In a mouse model, topical hispidin (0.1% and 0.5%) ameliorated UVB-induced skin damage in a dose-dependent manner, as evidenced by improved clinical and histological findings. Hispidin treatment was associated with reduced systemic oxidative stress and decreased cutaneous expression of CXCL2, C5a, IL-1β, NLRP3, Ly6G, PAD4, and citrullinated histone H3. In differentiated HL-60 cells, hispidin reduced ROS-associated signals and suppressed PMA-triggered extracellular DNA release, but did not suppress A23187-triggered extracellular DNA release under experimental conditions. Cell viability analysis showed that hispidin did not significantly affect differentiated HL-60 cell viability at tested concentrations under the present experimental conditions. Topical hispidin alleviates acute UVB-induced skin inflammation by suppressing neutrophil infiltration and NET-related inflammatory responses. Hispidin may therefore represent a promising candidate as a topical modulator of oxidative stress- and NET-associated skin inflammation. Full article

Immune-mediated inflammatory diseases, such as rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus, impose a severe and growing global health burden, where current therapies are limited by poor specificity and significant side effects. The peptidylarginine deiminase (PAD)/citrullination axis, in which protein citrullination catalyzed by PADs drives autoantigen generation and sustains inflammation, has emerged as a critical therapeutic target. This review outlines a comprehensive strategy for targeting this axis using natural products. We first detail the established role of natural compounds as direct PAD inhibitors, covering their chemical diversity, inhibitory mechanisms, and therapeutic applications in disease models. Subsequently, the discussion extends to their broader, indirect modulatory functions, highlighting how these compounds can suppress pathogenic citrullination by regulating upstream processes like NETosis and inflammatory signaling. Furthermore, the review introduces the innovative substrate-centric intervention strategy, which represents a paradigm shift toward shielding key arginine residues on autoantigens, thereby preventing the formation of immunogenic neoepitopes. The translational challenges and future directions for each of these avenues are outlined, addressing persistent obstacles including achieving isoform selectivity and biomarker validation. By integrating these multifaceted strategies, from direct inhibition and indirect modulation to substrate protection, this work provides a strategic roadmap for advancing the next generation of more precise, effective, and safe anti-citrullination therapies, ultimately moving beyond conventional enzyme inhibition toward targeted immunomodulation in immune-mediated inflammatory diseases. Full article

Bioresorbable scaffolds (BRS; also referred to as bioresorbable vascular scaffolds, BVS) represent a promising approach in interventional cardiology, offering theoretical advantages such as temporary mechanical support followed by complete resorption. However, clinical experience has revealed challenges, including late-stage scaffold thrombosis and heterogeneous scaffold discontinuity during degradation, prompting investigation into host immune responses. Neutrophil extracellular traps (NETs), which are network-like structures composed of decondensed chromatin decorated with antimicrobial proteins, have emerged as critical mediators of vascular inflammation and thrombosis. This review explores the intersection between NET biology and BRS performance, investigating how NETosis affects biocompatibility, degradation kinetics, and device-related complications. We discuss the molecular mechanisms that trigger neutrophil activation and NET formation in scaffold materials, the effect of NET components on polymeric and metallic scaffold degradation, and emerging biomarkers to monitor NET-mediated complications. We also evaluate therapeutic strategies targeting NET pathways, including DNase-based therapies, peptidylarginine deiminase 4 (PAD4) inhibitors, and anti-inflammatory coatings that can optimize next-generation BRS outcomes. Understanding the immunological environment surrounding bioresorbable vascular devices is crucial for developing scaffolds that deliver predictable degradation while minimizing adverse inflammatory responses. Full article

Background: Diabetic wound healing is hampered by persistent inflammation and excessive neutrophil extracellular traps (NET) formation. Peptidylarginine deiminase 4 (PAD4) is a key enzyme driving this pathology. This study developed a thermosensitive chitosan/β-glycerophosphate hydrogel for the local delivery of a novel PAD4 inhibitor, YJ-2, to promote diabetic wound repair. Methods: A YJ-2-loaded hydrogel (CGY) was synthesized and characterized. In vitro studies used HaCaT cells and macrophages to assess proliferation, migration, NETs (via H3cit), and polarization. Efficacy was evaluated in diabetic C57 mouse wound models. Results: CGY exhibited temperature-sensitive gelation and sustained YJ-2 release. In vitro, YJ-2 inhibited NETs formation, reduced pro-inflammatory markers, promoted HaCaT migration, and induced M2 macrophage polarization. In vivo, CGY treatment significantly accelerated wound closure. Conclusions: Local hydrogel delivery of the PAD4 inhibitor YJ-2 effectively mitigates inflammation and NETs, promoting healing in diabetic wounds. This strategy represents a promising targeted therapy for diabetic wounds. Full article

The peptidylarginine deiminase (PAD) family includes five isozymes (PAD1–4 and PAD6) with unique tissue distributions and substrate specificities. These enzymes facilitate citrullination, a post-translational modification where positively charged arginine residues are converted into neutral citrulline residues in the presence of calcium ions. This process significantly changes protein properties, affecting molecular interactions, structural stability, and biological functions. Over the past six years (2019–2025), there has been significant progress in understanding PAD activity mechanisms and their therapeutic potential. Recent discoveries include the regulated nuclear translocation of PAD2, PAD4’s specific role in forming cancer extracellular chromatin networks (CECNs), and the development of next-generation inhibitors with greatly improved pharmacological profiles. PAD4 is crucial in forming neutrophil extracellular traps (NETs). Citrullination of histones H3 and H4 by PAD4 destabilizes chromatin, helping release DNA-protein networks as an antibacterial defense. However, excessive NET formation can contribute to autoimmune diseases and thrombosis. Similarly, the bacterial peptidylarginine deiminase from Porphyromonas gingivalis (PPAD)—the only known prokaryotic citrullinating enzyme—plays a key role. Working with R-gingipains, PPAD triggers pathological citrullination of host proteins, leading to immune tolerance breakdown and linking periodontal disease with systemic autoimmune disorders such as rheumatoid arthritis, atherosclerosis, and Alzheimer’s disease. Once thought to be a rare post-translational modification, citrullination is now understood as a vital regulatory mechanism in both normal physiology and disease, involving both internal processes of homeostasis and external mechanisms of bacterial pathogenesis. Full article

Although female (XX) and male (XY) placentas generally function the same, it is evident that there are sex-specific postnatal health outcomes following placental dysfunction and pregnancy complications. Although the underlying causes for these sex differences are unclear, it is postulated that differences in XX and XY placental function are involved due to sex chromosomes and/or sex steroids. Studies in breast and prostate cancer cells demonstrated a role for the citrullination enzyme peptidylarginine deiminase 2 (PAD2) in post-translational regulation of estrogen (ESR) and androgen receptor (AR) signaling. The goal of this study is to determine if PAD2 is present in mouse placentas and if XX versus XY differences exist in the relative level of PAD2. Fetuses and placentas were collected from three pregnant mice (C57BL6) at 14 days of gestation. Total RNA and protein were isolated from XX and XY placentas, and relative mRNA and protein were analyzed by real-time PCR and Western blot. AR and PAD2 levels were significantly higher in XY than in XX placentas. This study is the first to demonstrate XX and XY differences in PAD2 and AR in the placenta. It suggests a role for PAD2 regulation of androgen receptor signaling in the XY placenta. Full article

In recent decades, chemotherapy has significantly improved cancer survival, yet its adverse effects on non-cancerous tissues raise increasing concerns. In this context, growing attention has been focused on natural compounds that may be useful in mitigating the undesirable effects of chemotherapeutic agents. Here, we aimed to demonstrate that Cytosporone B (CsnB) is a potent agent for counteracting the cardiovascular effects induced by Imatinib. To this end, 12-week-old male Wistar rats were studied; they were divided into three groups as follows: (1) control, (2) Imatinib-treated (Imatinib: 60 mg/kg/day, per os), (3) Imatinib + CsnB-treated (CsnB: 5 mg/kg/day, i.p.). After the two-week-long experimental period, rats were euthanized. Their hearts were used for the following biochemical measurements: NADPH oxidase (NOX4), high mobility group box 1 (HMGB1), peptidylarginine deiminase 4 (PAD4), inducible nitric oxide synthase (iNOS) expression, tumor necrosis factor-alpha (TNF-α) level, and myeloperoxidase (MPO) activity. Imatinib caused a marked upregulation of key inflammatory and oxidative markers, including HMGB1, TNF-α, MPO, iNOS, PAD4, and NOX4 in cardiac tissue; however, CsnB treatment mitigated these elevations, implying its role in opposing Imatinib-induced inflammatory and oxidative processes in the heart. Our findings suggest that CsnB holds promise as a cardioprotective agent capable of modulating Imatinib-induced adverse cardiac effects. Full article

Tenrecs are heterothermic burrowing mammals, which are capable of withstanding extreme environmental stressors, including during hibernation. Their phylogenetic position as reminiscent of an ancestral placental mammal makes tenrecs a unique model for evolutionarily conserved traits, with potential translatability to human physiology and pathobiology, including adaptations to extreme environments. In this study, we compared tenrec plasma for post-translational protein citrullination profiles (citrullinomes) and extracellular vesicle (EV) characteristics, including selected microRNA cargoes (miR-21, miR-155, miR-206, miR-210), between baseline active and hibernating states at low (12 °C) and high (28 °C) ambient temperatures. Our findings show considerable changes in citrullinome plasma profiles and associated Gene Ontology and KEGG pathway analysis linked to physiological and inflammatory processes, comparing hibernating and active states, also differing between the two ambient temperature groups. We furthermore identified modified EV profiles with respect to stress-related (miR-21, miR-155), hypoxia (miR-210) and metabolic/muscle related (miR-206) microRNA cargoes, which showed significant differences between active and hibernating animals, also comparing the two ambient temperature groups. Our findings show novel roles for post-translational protein citrullination in regulating immune and metabolic associated pathways in the tenrec, and highlight EV profiles, based on microRNA cargoes, as indicators for stress and metabolic responses in active versus hibernating states, including at different temperatures. Collectively our data highlights the tenrec as an evolutionary model for regulating pathobiological responses in extreme environments and may have translatable potential for human physiology and pathologies. Full article

Peptidylarginine deiminase 4 (PAD4) catalyzes protein citrullination, a post-translational modification implicated in type 1 diabetes mellitus (T1DM). This study examined PAD4 expression and activity in the pancreas of streptozotocin (STZ)-induced diabetic Wistar rats. Animals were divided into three groups: (A) STZ-induced diabetic rats (60 mg/kg, i.p.), (B) non-diabetic controls, and (C) diabetic rats treated with Cl-amidine (5 mg/kg), a pan-PAD inhibitor, from week six post-induction. Analyses included PAD4 mRNA and protein expression, citrullinated histone H3 (CitH3), calcium concentration, and neutrophil elastase activity. Diabetic rats exhibited increased PAD4 expression, CitH3 levels, and NETosis markers, alongside reduced pancreatic calcium, suggesting calcium consumption during PAD4 activation. Cl-amidine treatment attenuated NETosis. These results implicate PAD4 in T1DM pathogenesis via NETosis and support the utility of STZ-induced diabetic rats as a model for PAD4-targeted studies. Cl-amidine may represent a promising therapeutic approach to reduce pancreatic inflammation in T1DM. Full article

Neutrophils are essential components of innate immunity, executing a range of effector functions including phagocytosis, degranulation, and the release of neutrophil extracellular traps (NETs). A key hallmark of NET formation is the presence of citrullinated histone H3 (CitH3), produced by peptidylarginine deiminases (PAD2 and PAD4) to facilitate chromatin decondensation. While NETs play critical antimicrobial roles, excessive or dysregulated NET formation, termed NETosis, can drive tissue injury, chronic inflammation, and organ dysfunction across a wide spectrum of diseases. Beyond its structural role within NETs, CitH3 acts as a damage-associated molecular pattern (DAMP), amplifying immune activation and pathological inflammation. Elevated CitH3 levels have been identified as biomarkers in sepsis, viral infections, ischemia–reperfusion injury, organ transplantation, diabetic wounds, autoimmune diseases, and cancer. Despite increasing recognition of CitH3’s pathogenic contributions, its therapeutic potential remains largely untapped. This review summarizes recent advances in understanding the role of CitH3 in NETosis and immune dysfunction, highlights emerging strategies targeting CitH3 therapeutically, and identifies critical knowledge gaps. Collectively, these insights position CitH3 as a promising druggable biomarker for the diagnosis, prognosis, and treatment of acute and chronic inflammatory diseases. Full article

Neutrophil extracellular traps (NETs) are associated with the extracellular release of nuclear chromatin decorated with cytoplasmic proteins. Excessive release of NETs has been reported in chronic lung diseases, including chronic obstructive pulmonary disease (COPD). However, the role of NETs in the pathogenesis of COPD remains unclear. Peptidylarginine deaminase 4 (PAD4) contributes to NET formation. Therefore, in an elastase (ELS)-induced emphysema mouse model, we examined the role of PAD4 using Padi4 gene knockout (KO) mice. First, we confirmed that ELS induced NET formation in the parenchyma of the lungs. PAD4 deficiency suppressed ELS-induced NET expression and tended to ameliorate the lung tissue injury. The cellular profile of bronchoalveolar lavage fluid (BALF) did not differ between the two groups. Additionally, PAD4 deficiency ameliorated emphysema and apoptosis in lung cells. Finally, we examined the effects of PAD4 on comprehensive gene expression signatures using RNA sequencing. Enrichment analysis of the transcriptomic data revealed that the expression of several genes associated with COPD pathogenesis was altered in the KO mice. Overall, the results suggest that PAD4 deficiency improves NET formation and emphysema in the lungs; this pathway can be a potential therapeutic target for the treatment of COPD. Full article

Inflammation contributes to myocardial injury in ST-elevation myocardial infarction (STEMI). Interleukin-6 receptor (IL-6R) inhibition has been shown to mitigate myocardial injury and reduce levels of the prothrombotic and inflammatory mediator, neutrophil extracellular traps (NETs). The enzyme peptidylarginine deiminase 4 (PAD4) is central in NET formation. We hypothesized that PAD4 links IL-6R activation and NET formation. Methods: We conducted thrombus aspiration and peripheral blood sampling in 33 STEMI patients. In thrombi and leukocytes, we quantified the mRNA of IL-6, IL-6R, and PAD4. In peripheral blood, the protein levels of IL-6, IL-6R, PAD4, dsDNA, H3Cit, MPO-DNA, and troponin T were quantified. Results: In thrombi and circulating leukocytes, PAD4 mRNA was associated with IL-6R mRNA (thrombi: β = 0.34, 95% CI [0.16–0.53], p = 0.001, circulating leukocytes: β = 0.92, 95% CI [0.07–1.77], p = 0.036). There were no correlations between PAD4 and IL-6 in thrombi and leukocytes. The protein levels of IL-6R were associated with the NET marker H3Cit (r_s = 0.40, p = 0.02). In thrombi, PAD4 mRNA was associated with high levels of troponin T (β = 1.15 95% CI [0.27–2.04], p = 0.013). Conclusion: We demonstrate an association between PAD4, IL-6R, and troponin release in STEMI patients. Our findings indicate a PAD4-mediated connection between IL-6R and NET formation and highlight PAD4 as a potential treatment target for mitigating inflammation and myocardial injury in STEMI. Full article