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Ulcerative colitis (UC) is characterized by chronic non-recessive inflammation of the intestinal mucosa involving both innate and adaptive immune responses. Currently, new targeted therapies are urgently needed for UC, and neutrophil extracellular traps (NETs) are new therapeutic options. NETs are DNA-based networks released from neutrophils into the extracellular space after stimulation, in which a variety of granule proteins, proteolytic enzymes, antibacterial peptides, histones, and other network structures are embedded. With the deepening of the studies on NETs, their regulatory role in the development of autoimmune and autoinflammatory diseases has received extensive attention in recent years. Increasing evidence indicates that excess NETs exacerbate the inflammatory response in UC, disrupting the structure and function of the intestinal mucosal barrier and increasing the risk of thrombosis. Although NETs are usually assigned a deleterious role in promoting the pathological process of UC, they also appear to have a protective role in some models. Despite such progress, comprehensive reviews describing the therapeutic promise of NETs in UC remain limited. In this review, we discuss the latest evidence for the formation and degradation of NETs, focusing on their double-edged role in UC. Finally, the potential implications of NETs as therapeutic targets for UC will be discussed. This review aims to provide novel insights into the pathogenesis and therapeutic options for UC. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is immune-mediated, inflammatory disease that affects synovial joints, and characterized by inflammatory changes in synovial tissue, cartilage, bone, and less commonly in extra-articular structures. Docetaxel (DTX) is a semi-synthetic anti-neoplastic medication. Peptidyl-arginine deiminase type 4 (PAD4) is expressed in macrophages and neutrophils in RA synovial membrane. Their effectiveness is in producing anti-cyclic citrullinated peptide antibodies (ACPA)-targeted citrullinated neoepitopes. Aim: To evaluate the anti-inflammatory effects of DTX in RA and the effect of methotrexate on PAD4 to investigate its potential as an RA biomarker. Methods: Forty male Wistar rats were divided into five groups of eight rats. Healthy rats formed the control group. The Second Group to Fifth group were induced with Complete Freund's adjuvant. The third group received DTX at a dosage of 1 mg/kg on alternate days, as determined by a preliminary experiment. The fourth group was given 1 mg/kg/week of methotrexate intraperitoneally. The fifth group was treated with a half dose of DTX and methotrexate simultaneously. Results: Significant Arthritis index and knee joint circumference decrease in the DTX group. No significant difference in body weight, platelet–lymphocyte ratio, and white blood cell count between the groups. Neutrophile lymphocyte ratio showed weak correlation with ACPA, while PAD4 showed good correlation with RA markers. Level of ACPA, PAD4, TNF-α, IL-1β, and VEGF significantly decreased in the DTX group than induction group (p < 0.05). Conclusion: DTX reduces the progression and joint destruction in rats induced by Complete Freund's Adjuvant which may due to inhibition of PAD4, TNF-α, IL-1β, VEGF, and ACPA. Also, methotrexate exhibited anti PAD4 effect. [ABSTRACT FROM AUTHOR]

Abstract Background Sepsis is life-threatening organ dysfunction caused by a dysregulated host response to infection. Acute respiratory distress syndrome (ARDS) is a common sepsis-associated injury that can increase postoperative mortality but the mechanism is still unclear. Main text The role of neutrophils in the pathophysiology of sepsis was deeply challenged after the discovery of NETosis, a process resulting in neutrophil extracellular traps (NETs) release. NETs can support thrombin generation and the concept of immunothrombosis has emerged as a new innate response to infection. Immunothrombosis leads to thrombosis in microvessels and supports immune cells together with specific thrombus-related molecules. ARDS is a common sepsis-associated organ injury. Immunothrombosis participates in thrombosis in pulmonary capillaries. Intervention regarding immunothrombosis in ARDS is a key scientific problem. PAD4 is the key enzyme regulating the NET skeleton protein histone H3 to citrulline histone to form NETs in immune thrombosis. This review summarizes NETosis and immunohaemostasis, ARDS and therapeutic opportunities targeting PAD4 via PAD4 inhibitors and lncRNAs potentially, providing future therapies. Conclusions We identified and summarized the fundamental definition of ARDS and the concept of immune thrombosis and its composition. NETs activation has become particularly relevant in the formation of immune thrombosis. The taskforce highlighted the intervention targets of PAD4, including noncoding RNAs, potentially providing future therapeutic targets to confront the high postoperative mortality of ARDS.

Chronic obstructive pulmonary disease (COPD), characterised by persistent airflow limitation during breathing, is considered to be the third leading cause of death worldwide. Among the mechanisms involved in this pathology is the excessive generation of neutrophil extracellular traps (NETs), which can induce an unwanted inflammatory response. These traps have been reported to be generated by the enzyme peptidyl arginine deiminase 4 (PAD4). The aim of this work is therefore to evaluate the effect of the administration of a siRNA targeting PAD4 on lung damage in a COPD animal model. Wistar rats weighing 300–350 g were administered cadmium chloride (5 mg/kg i.p.) every 24 h. Then, following one week of the administration of cadmium chloride, the PAD4-targeted siRNA was administered, and at the second week, lung function was measured, as were lung and heart weights, as well as PAD4 expression by RT-PCR. Our results showed that cadmium administration generated a COPD model, which increased PAD4 expression and decreased lung and heart weights and respiratory function. SiRNA administration partially reversed the changes associated with the COPD model. In conclusion, our results suggest that administration of an siRNA targeting PAD4 could improve respiratory function by decreasing lung and heart damage. [ABSTRACT FROM AUTHOR]

Protein arginine deiminase 4 (PAD4) plays an important role in cancer progression by participating in gene regulation, protein modification, and neutrophil extracellular trap (NET) formation. Many reversible and irreversible PAD4 inhibitors have been reported recently. In this review, we summarize the structure–activity relationships of newly investigated PAD4 inhibitors to bring researchers up to speed by guiding and describing new scaffolds as optimization and development leads for new effective, safe, and selective cancer treatments. In addition, some recent reports have shown evidence that PAD4 inhibitors are expected to trigger antitumor immune responses, regulate immune cells and related immune factors, enhance the effects of immune checkpoint inhibitors, and enhance their antitumor efficacy. Therefore, PAD4 inhibitors may potentially change tumor immunotherapy and provide an excellent direction for the development and clinical application of immunotherapy strategies for related diseases. [ABSTRACT FROM AUTHOR]

Arabidopsis PHYTOALEXIN DEFICIENT 4 (PAD4) has an essential role in pathogen resistance as a heterodimer with ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1). Here we investigated an additional PAD4 role in which it associates with and promotes the maturation of the immune-related cysteine protease RESPONSIVE TO DEHYDRATION 19 (RD19). We found that RD19 and its paralog RD19c promoted EDS1- and PAD4-mediated effector-triggered immunity to an avirulent Pseudomonas syringae strain, DC3000, expressing the effector AvrRps4 and basal immunity against the fungal pathogen Golovinomyces cichoracearum. Overexpression of RD19, but not RD19 protease-inactive catalytic mutants, in Arabidopsis transgenic lines caused EDS1- and PAD4-dependent autoimmunity and enhanced pathogen resistance. In these lines, RD19 maturation to a pro-form required its catalytic residues, suggesting that RD19 undergoes auto-processing. In transient assays, PAD4 interacted preferentially with the RD19 pro-protease and promoted its nuclear accumulation in leaf cells. Our results lead us to propose a model for PAD4-stimulated defense potentiation. PAD4 promotes maturation and nuclear accumulation of processed RD19, and RD19 then stimulates EDS1–PAD4 dimer activity to confer pathogen resistance. This study highlights potentially important additional PAD4 functions that eventually converge on canonical EDS1–PAD4 dimer signaling in plant immunity. [ABSTRACT FROM AUTHOR]

PAD4 expression and activity were significantly up-regulated in lung cancer tissues suggesting that PAD4 could be a possible target for lung cancer treatment. In this study we had demonstrated that PAD4 expression was higher in lung cancer patients whom with lymphnode metastasis and pleural invasion. Inhibiting PAD4 with a small molecular inhibitor could induce apoptosis and suppress growth in lung cancer cells. We used RNA-sequencing to further investigate transcriptional changes that induced by PAD4 inhibition, and results suggested its affected mostly on the cell cycle, mitotic cell cycle process, p53 signaling pathway. By using image flow cytometry analysis, we found that PAD4 inhibited by YW3-56 could accumulate cells in the G1/G0 phases and reducing the fraction of G2/M and S phase cells. Quantification of different phase of mitosis in cells treated with YW3-56 revealed an increasing trend of telophase and prophase cells. Taken together, our data indicated that PAD4 inhibitor could affect cell cycle and mitosis of lung cancer cells, and targeting PAD4 could be a promising strategy for discovery novel anti-NSCLC treatments.

Neutrophil adhesion to endothelia, entry into tissues and chemotaxis constitute essential steps in the immune response to infections that drive inflammation. Neutrophils bind to other cells and migrate via adhesion receptors, notably the αMβ2 integrin dimer (also called Mac-1, CR3 or CD11b/CD18). Here, the response of neutrophils to integrin engagement was examined by monitoring the activity of peptidylarginine deiminase 4 (PAD4). Histone H3 deimination was strongly stimulated by manganese, an integrin-activating divalent cation, even in the absence of additional inflammatory stimuli. Manganese-induced cell attachment resulted in neutrophil swarm formation that paralleled histone deimination, whereas antibodies that impair integrin binding prevented both cell adhesion and histone deimination. Manganese treatment led to putative deimination of profilin, a protein that functions as an actin-organizing hub, as detected by two-dimensional gel electrophoresis and citrulline immunoblotting. Cl-amidine, a covalent inhibitor of PAD4, and GSK484, a specific PAD4 inhibitor, blocked profilin deimination. Neutrophil migration toward leukotriene B4 and toward synovial fluid from a rheumatoid arthritis patient were inhibited by chloramidine, thus supporting the contribution of deimination to chemotaxis. The data, based on a simplified system for integrin activation, imply a mechanism whereby integrin attachment coordinates neutrophil responses to inflammation and orchestrates deimination of nuclear and cytoskeletal proteins. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'. [ABSTRACT FROM AUTHOR]

Introduction: Colorectal cancer (CRC) is the third most common malignancy and a major cause of cancer‐related deaths. Peptidyl arginine deiminase 4 (PAD4 or PADI4) is expressed in neutrophils that, when activated, can drive the formation of neutrophil extracellular traps (NETs). PAD4 has been found to be upregulated in CRC patients and to predict a poor prognosis. This study is aimed at exploring the role of PAD4 inhibitor (GSK484) in NET formation and radioresistance in CRC. Methods: Reverse transcriptase quantitative polymerase chain reaction and western blotting were used to measure PAD4 expression in CRC tissues and cells. GSK484, an inhibitor of PAD4, was investigated in the following functional assays in vitro: western blotting, clonogenic survival, colony formation, TUNEL, flow cytometry and transwell assays. Nude mouse xenograft models were applied to evaluate the effect of GSK484 on tumor growth in CRC in vivo. The formation of NETs influenced by GSK484 was also investigated. Results: We showed upregulation of PAD4 mRNA and protein in CRC tissues and cells. High expression of PAD4 was related to a poor prognosis in CRC patients. GSK484 treatment promoted the radiosensitivity of CRC cells and induced cell death by promoting DNA double‐strand breaks. Rescue experiments further verified that GSK484 inhibited the effects of PAD4 overexpression in irradiated CRC cells. Moreover, GSK484 injection strengthened the radiosensitivity of CRC and inhibited NET formation in vivo. Conclusions: PAD4 inhibitor GSK484 promotes the radiosensitivity of CRC and inhibits NET formation in vivo and in vitro. [ABSTRACT FROM AUTHOR]

Abstract Background Peptidylarginine deiminase enzymes (PADs) convert arginine residues to citrulline in a process called citrullination or deimination. Recently, two PADs, PAD2 and PAD4, have been linked to hormone signaling in vitro and the goal of this study was to test for links between PAD2/PAD4 and hormone signaling in vivo. Methods Preliminary analysis of Padi2 and Padi4 single knockout (SKO) mice did not find any overt reproductive defects and we predicted that this was likely due to genetic compensation. To test this hypothesis, we created a Padi2/Padi4 double knockout (DKO) mouse model and tested these mice along with wild-type FVB/NJ (WT) and both strains of SKO mice for a range of reproductive defects. Results Controlled breeding trials found that male DKO mice appeared to take longer to have their first litter than WT controls. This tendency was maintained when these mice were mated to either DKO or WT females. Additionally, unsexed 2-day old DKO pups and male DKO weanlings both weighed significantly less than their WT counterparts, took significantly longer than WT males to reach puberty, and had consistently lower serum testosterone levels. Furthermore, 90-day old adult DKO males had smaller testes than WT males with increased rates of germ cell apoptosis. Conclusions The Padi2/Padi4 DKO mouse model provides a new tool for investigating PAD function and outcomes from our studies provide the first in vivo evidence linking PADs with hormone signaling.

Coffee, a widely consumed important agricultural product, is mainly produced from two species, Coffea arabica (Arabica coffee) and C. canephora (Robusta coffee). Timor Hybrid (HDT) is a population resulting from a natural cross between C. arabica and C. canephora. HDT derivatives have a high spectrum of resistance to different races of Hemileia vastatrix (Hv), the causal agent of coffee leaf rust. A RNAseq database, obtained from HDT832/2 leaves inoculated with Hv (Host Resistance) and Uromyces vignae (Uv, Nonhost Resistance), showed the presence of genes implicated in the hypersensitive response and salicylic acid pathway. Hypersensitive Induced Reaction (HIR) gene family, Enhanced Disease Susceptibility1 gene (EDS1), and Phytoalexin Deficient 4 (PAD4) gene are involved in host and nonhost resistance. Relative expression calculated by RT-qPCR was used to confirm and expand the transcriptome analysis. HDTHIR4, HDTEDS1, and HDTPAD4 showed the highest upregulation in response to Hv and Uv inoculation, confirming a similar trend in host and nonhost resistance in HDT. HIR and EDS1/PAD4 gene families were characterized for the first time in the three available Coffea genomes. HIR genes were quite conserved between Coffea species. Surprisingly, EDS1 and PAD4 genes revealed major differences in gene structure. The PAD4 predicted protein from C. arabica does not include both conserved domains of the EDS1/PAD4 family, and the EDS1 putative protein from C. canephora includes a formin domain unusual in the same protein family. The variability shown by EDS1/PAD4 gene family may impact the disease resistance response of Coffea species, which can be surveyed for the gene sequences that will produce a more resistant phenotype. [ABSTRACT FROM AUTHOR]

Protein arginine deiminase 4 (PAD4) plays an important role in cancer progression by participating in gene regulation, protein modification, and neutrophil extracellular trap (NET) formation. Many reversible and irreversible PAD4 inhibitors have been reported recently. In this review, we summarize the structure–activity relationships of newly investigated PAD4 inhibitors to bring researchers up to speed by guiding and describing new scaffolds as optimization and development leads for new effective, safe, and selective cancer treatments. In addition, some recent reports have shown evidence that PAD4 inhibitors are expected to trigger antitumor immune responses, regulate immune cells and related immune factors, enhance the effects of immune checkpoint inhibitors, and enhance their antitumor efficacy. Therefore, PAD4 inhibitors may potentially change tumor immunotherapy and provide an excellent direction for the development and clinical application of immunotherapy strategies for related diseases.

Chronic obstructive pulmonary disease (COPD), characterised by persistent airflow limitation during breathing, is considered to be the third leading cause of death worldwide. Among the mechanisms involved in this pathology is the excessive generation of neutrophil extracellular traps (NETs), which can induce an unwanted inflammatory response. These traps have been reported to be generated by the enzyme peptidyl arginine deiminase 4 (PAD4). The aim of this work is therefore to evaluate the effect of the administration of a siRNA targeting PAD4 on lung damage in a COPD animal model. Wistar rats weighing 300–350 g were administered cadmium chloride (5 mg/kg i.p.) every 24 h. Then, following one week of the administration of cadmium chloride, the PAD4-targeted siRNA was administered, and at the second week, lung function was measured, as were lung and heart weights, as well as PAD4 expression by RT-PCR. Our results showed that cadmium administration generated a COPD model, which increased PAD4 expression and decreased lung and heart weights and respiratory function. SiRNA administration partially reversed the changes associated with the COPD model. In conclusion, our results suggest that administration of an siRNA targeting PAD4 could improve respiratory function by decreasing lung and heart damage.

Abstract Plant intracellular nucleotide-binding leucine-rich repeat (NLR) receptors with an N-terminal Toll/Interleukin-1 receptor (TIR) domain detect pathogen effectors to produce TIR-catalyzed signaling molecules for activation of plant immunity. Plant immune signaling by TIR-containing NLR (TNL) proteins converges on Enhanced Disease Susceptibility 1 (EDS1) and its direct partners Phytoalexin Deficient 4 (PAD4) or Senescence-Associated Gene 101 (SAG101). TNL signaling also require helper NLRs N requirement gene 1 (NRG1) and activated disease resistance 1 (ADR1). In two recent remarkable papers published in Science, the authors show that the TIR-containing proteins catalyze and produce two types of signaling molecules, ADPr-ATP/diADPR and pRib-AMP/ADP. Importantly, they demonstrate that EDS1-SAG101 and EDS1-PAD4 modules are the receptor complexes for ADPr-ATP/diADPRp and Rib-AMP/ADP, respectively, which allosterically promote EDS1-SAG101 interaction with NRG1 and EDS1-PAD4 interaction with ADR1. Thus, two different small molecules catalyzed by TIR-containing proteins selectively activate the downstream two distinct branches of EDS1-mediated immune signalings. These breakthrough studies significantly advance our understanding of TNL downstream signaling pathway.

Infections represent a common health problem in people at any age. Usually, a local reaction develops in response to the infection, and intensive treatment is not required. However, sometimes the response to infection is inadequate and may lead to organ dysfunction; such condition is defined as septic state. The pathophysiology of sepsis is multifactorial, and the immune system is directly involved in development of pathological processes accompanying septic conditions. Dysregulatory disorders in sepsis may be observed, in particular, those affecting immune system and, immediately, innate immunity, which presents the first line of immune defense. Currently, neutrophilic granulocytes are considered important players in development and maintenance of inflammatory processes, which is due to their ability to produce various inflammatory mediators. At the same time, at different stages of pathogenesis, immune factors and mechanisms act both as a generating factors and effectors of damage reactions, as well as the main components of the defense responses. Uncontrolled release of endogenous inflammatory mediators induced by microorganisms, and lack of damagelimiting mechanisms mutually synergize when promoting the organ dysfunction. Hence, dysfunction of the immune system may develop and deepen upon any inappropriate strategy of response to distinct infectious pathogen. The aim of our study was to assess the spectrum of microorganisms, levels of neutrophilic extracellular traps (NVL), PAD4, IL-6, IL-8 and their correlations in the patients with sepsis and septic shock, depending on the outcome of the disease. The study included 44 patients with sepsis and septic shock, and 20 apparently healthy donors. Peripheral blood samples were taken from the ulnar vein serially in dynamics, from the moment of diagnosis to clinical outcome. Microbiological and immunological studies have been carried out. The obtained data were processed with “SPSS Statistic 23.0” software. Development of septic conditions was found to be accompanied by a significant changes in the levels of studied immunological parameters (NLV, IL-6, IL-8 and PAD4). Toxicity of lysosomal enzymes and oxygen metabolites present in neutrophilic extracellular traps leads to endothelial damage, appearance of focal intravascular inflammation, which, along with damaging effects of microorganisms and their toxins, leads to development of organ failure and onset. High burden of these factors is most likely to cause lethal outcomes.

Excessive neutrophil influx and activation in lungs during infections, such as manifest during the ongoing SARS CoV-2 pandemic, have brought neutrophil extracellular traps (NETs) and the concomitant release of granule contents that damage surrounding tissues into sharp focus. Neutrophil proteases, which are known to participate in NET release, also enable the binding of the viral spike protein to cellular receptors and assist in the spread of infection. Blood and tissue fluids normally also contain liver-derived protease inhibitors that balance the activity of proteases. Interestingly, neutrophils themselves also express the protease inhibitor alpha-1-antitrypsin (AAT), the product of the SERPINA-1 gene, and store it in neutrophil cytoplasmic granules. The absence of AAT or mutations in the SERPINA-1 gene promotes lung remodeling and fibrosis in diseases such as chronic obstructive pulmonary disease (COPD), and acute respiratory distress syndrome (ARDS) and increases the risk of allergic responses. Recent observations point to the fact that reduced activity of AAT presents a major susceptibility factor for severe COVID-19. Here, we focus attention on the mechanism of neutrophil elastase (NE) in NET release and its inhibition by AAT as an additional factor that may determine the severity of COVID-19. [ABSTRACT FROM AUTHOR]

Peptidylarginine deiminase type4 (PAD4) is a pivotal pro-inflammatory protein within the human immune system, intricately involved in both inflammatory processes and immune responses. Its role extends to the generation of diverse immune cell types, including T cells, B cells, natural killer cells, and dendritic cells. PAD4 has recently garnered attention due to its association with a spectrum of inflammatory and autoimmune disorders, notably rheumatoid arthritis (RA). Mutations in the PAD4 gene, leading to the conversion of arginine to citrulline, have emerged as significant factors in the pathogenesis of RA and related conditions. As a calcium-dependent enzyme, PAD4 is central to the citrullination process, a crucial post-translational modification implicated in disease pathophysiology. Its critical role in autoimmune disorders and inflammation makes PAD4 a prime candidate for therapeutic intervention in RA. Inhibiting PAD4 presents a promising avenue for mitigating inflammatory responses and curtailing joint degradation and impairment. To explore its therapeutic potential, a structure-based virtual screening (SBVS) approach was employed, harnessing an array of marine natural products (MNPs) sourced from databases such as CMNPD, MNPD, and Seaweed. Notably, MNPD10752, CMNPD12680, and CMNPD2751 emerged as potential hit molecules, exhibiting adherence to essential pharmacokinetic properties and favorable toxicity profiles. Quantum mechanics studies using density functional theory (DFT) calculations revealed the inhibitory potential of these identified natural products. Further structural elucidation through molecular dynamics simulations (MDS) and principal component-based free energy landscape (FEL) analysis shed light on the stability of MNP-bound PAD4 complexes. In conclusion, this computational study serves as a stepping stone for further experimental evaluation, aiming to explore the potential of MNPs in addressing PAD4-related human pathologies. Probing Marine natural products to halt the catalytic activity of PAD4. [Display omitted] • Virtual screening study highlights the potential of marine-derived natural products (MNPs) as alternative therapeutic agents for autoimmune disorders, particularly rheumatoid arthritis (RA), by targeting PAD4. • Three hit molecules—MNPD10752, CMNPD12680, and CMNPD2751—were identified, showing favorable binding poses, pharmacokinetic properties, and toxicity profiles. • Structural dynamics analysis confirmed that these hits demonstrated stronger inhibition potential compared to the control compound. • CMNPD12680 emerged as the most promising candidate for future in vitro studies aimed at developing PAD4-targeted RA therapies. [ABSTRACT FROM AUTHOR]

Background: Nasopharyngeal carcinoma (NPC) has high incidence in Southern China and is derived from the mucosal epithelium of the nasopharynx. Accumulating evidence has revealed that peptidyl arginine deiminase 4 (PAD4) exerts carcinogenic effect on certain cancers. We designed this study to probe the specific role that PAD4 plays in NPC and its molecular mechanism. Methods: PAD4 expression in NPC cells was detected by RT-qPCR analysis. MTT, colony formation, flow cytometry, TUNEL staining, and LC3-II punctuation experiments were done to probe into the biological functions of PAD4 on NPC cellular behaviors in vitro. Subsequently, the upstream regulatory mechanism of PAD4 was investigated by luciferase reporter, RNA pull-down, and RIP assays. The impact of PAD4 on NPC tumor growth in mice was assessed by in vivo xenograft tumor assay. Results: PAD4 was upregulated in NPC cells. PAD4 knockdown suppressed proliferative ability and promoted apoptosis and autophagy in NPC cells. Additionally, PAD4 expression was negatively regulated by microRNA 3164 (miR-3164). LINC00324 positively upregulated PAD4 expression by interacting with miR-3164 and recruiting HuR protein. The LINC00324/miR-3164/PAD4 axis modulated the PI3K/AKT pathway in NPC cells. Moreover, PAD4 upregulation countervailed the influences of LINC00324 deficiency on NPC cell proliferation, apoptosis, and autophagy and on NPC tumor growth in mice. Conclusion: LINC00324 promoted NPC malignancy by upregulation of PAD4 to activate the PI3K/AKT pathway. [ABSTRACT FROM AUTHOR]

Forsythiaside B (FTB) is one of the main components of Forsythia suspensa (Thunb.) Vahl and exerts anti-inflammatory and anti-oxidative effects. However, its mechanism of action as a treatment for sepsis remains unclear. In this study, we developed a rat model of sepsis using cecal ligation and puncture (CLP) to investigate the effects of FTB on sepsis-associated coagulopathies. Using rats with sepsis, we investigated the effects of FTB on neutrophil extracellular trap (NETs) formation and peptidylarginine deiminase 4 (PAD4) expression in neutrophils. NET (DNase1) and PAD4 (Cl-amidine) inhibitors were used to further investigate whether FTB mitigates sepsis-associated coagulopathies by inhibiting PAD4-dependent NETs production. Our results showed that treatment with FTB increased the survival rate, ameliorated the CLP-induced inflammatory response and multiple organ dysfunction, and reduced CLPinduced pathological changes. FTB also alleviated the associated coagulopathies. Additionally, we demonstrated that treatment with FTB inhibited NETs formation and downregulated PAD4 expression in peripheral neutrophils. The effects of FTB on coagulopathies were similar to those of monotherapy with NET or PAD4 inhibitors. In conclusion, our study confirmed that FTB can alleviate coagulopathies in rats with sepsis. The underlying mechanism of FTB’s effect consists in inhibition of PAD4-dependent NETs formation. [ABSTRACT FROM AUTHOR]

Background: Peptidylarginine deiminase enzymes (PADs) convert arginine residues to citrulline in a process called citrullination or deimination. Recently, two PADs, PAD2 and PAD4, have been linked to hormone signaling in vitro and the goal of this study was to test for links between PAD2/PAD4 and hormone signaling in vivo. Methods: Preliminary analysis of Padi2 and Padi4 single knockout (SKO) mice did not find any overt reproductive defects and we predicted that this was likely due to genetic compensation. To test this hypothesis, we created a Padi2/Padi4 double knockout (DKO) mouse model and tested these mice along with wild-type FVB/NJ (WT) and both strains of SKO mice for a range of reproductive defects. Results: Controlled breeding trials found that male DKO mice appeared to take longer to have their first litter than WT controls. This tendency was maintained when these mice were mated to either DKO or WT females. Additionally, unsexed 2-day old DKO pups and male DKO weanlings both weighed significantly less than their WT counterparts, took significantly longer than WT males to reach puberty, and had consistently lower serum testosterone levels. Furthermore, 90-day old adult DKO males had smaller testes than WT males with increased rates of germ cell apoptosis. Conclusions: The Padi2/Padi4 DKO mouse model provides a new tool for investigating PAD function and outcomes from our studies provide the first in vivo evidence linking PADs with hormone signaling. [ABSTRACT FROM AUTHOR]

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Kawasaki disease (KD) is the leading cause of acquired heart disease in children. While circulating neutrophils are increased and activated during acute KD, it is unclear whether neutrophils and neutrophil extracellular traps (NETs) contribute to the pathogenesis of KD. Peptidylarginine deiminase 4 (PAD4), an enzyme involved in protein citrullination and essential for NETs formation, is implicated in the pathogenesis of various diseases. Here, we used the Lactobacillus casei cell wall extract (LCWE)-induced mouse model of KD vasculitis to determine the contribution of PAD4 in KD vasculitis. We found that the pan-PADs inhibitor, Cl-amidine, significantly reduced LCWE-induced cardiovascular lesions, but neutrophil-specific Padi4 KO mice did not impact the development of KD vasculitis. While in vitro treatment of macrophages, which highly express Padi4, with Cl-amidine inhibited IL-1β secretion, macrophage-specific Padi4 KO mice did not reduce the lesions. Padi4-/- mice also developed KD vasculitis, AFM30a, a PAD2 inhibitor, significantly reduced KD vasculitis in Padi4-/- mice, indicating a compensatory role of PAD2 in PAD4 deficiency. We also identified several citrullinated proteins in macrophages with constitutively active NLRP3 inflammasome that were inhibited by Cl-amidine treatment, suggesting that protein citrullination participates in NLRP3 inflammasome activation. These data indicate a dispensable role for PAD4-dependent NETs formation, and a redundant role of PAD2 and PAD4 in this murine KD vasculitis. The cardioprotective effects of Cl-amidine to reduce the severity of murine KD vasculitis are not limited to PAD4 inhibition and may include decreased citrullination in the inflammasome pathway., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The pursuit of novel therapeutics is a complex and resource-intensive endeavor marked by significant challenges, including high costs and low success rates. In response, drug repositioning strategies leverage existing FDA-approved compounds to predict their efficacy across diverse diseases. Peptidyl arginine deiminase 4 (PAD4) plays a pivotal role in protein citrullination, a process implicated in the autoimmune pathogenesis of rheumatoid arthritis (RA). Targeting PAD4 has thus emerged as a promising therapeutic approach. This study employs computational and enzyme inhibition strategies to identify potential PAD4-targeting compounds from a library of FDA-approved drugs. In silico docking analyses validated the binding interactions and orientations of screened compounds within PAD4's active site, with key residues such as ASP350, HIS471, ASP473, and CYS645 participating in crucial hydrogen bonding and van der Waals interactions. Molecular dynamics simulations further assessed the stability of top compounds exhibiting high binding affinities. Among these compounds, Saquinavir (SQV) emerged as a potent PAD4 inhibitor, demonstrating competitive inhibition with a low IC50 value of 1.21 ± 0.04 µM. In vitro assays, including enzyme kinetics and biophysical analyses, highlighted significant changes in PAD4 conformation upon SQV binding, as confirmed by circular dichroism spectroscopy. SQV induced localized alterations in PAD4 structure, effectively occupying the catalytic pocket and inhibiting enzymatic activity. These findings underscore SQV's potential as a therapeutic candidate for RA through PAD4 inhibition. Further validation through in vitro and in vivo studies is essential to confirm SQV's therapeutic benefits in autoimmune diseases associated with dysregulated citrullination., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Plants have evolved a network of complex signaling pathways that allow them to cope with the fluctuations of internal and external environmental cues. GIGANTEA (GI), a well-known, highly conserved plant nuclear protein, has been shown to regulate multiple biological functions in plants such as circadian rhythm, light signaling, cold tolerance, hormone signaling, and photoperiodic flowering. Recently, the role of GI in disease tolerance against different pathogens has come to light; however, a detailed mechanism to understand the role of GI in pathogen defense remains largely unexplained. Here, we report that GIGANTEA is upregulated upon infection with a virulent oomycete pathogen, Hyaloperonospora arabidopsidis (Hpa), in Arabidopsis thaliana accession Col-0. To investigate the role of GI in Arabidopsis defense, we examined the pathogen infection phenotype of gi mutant plants and found that gi-100 mutant was highly susceptible to Hpa Noco2 infection. Notably, the quantitative real-time PCR showed that PHYTOALEXIN DEFICIENT4 (PAD4) and several PAD4-regulated downstream genes were downregulated upon Noco2 infection in gi-100 mutant as compared to Col-0 plants. Furthermore, the chromatin immunoprecipitation results show that GI can directly bind to the intronic region of the PAD4 gene, which might explain the mechanism of GI function in regulating disease resistance in plants. Taken together, our results suggest that GI expression is induced upon Hpa pathogen infection and GI can regulate the expression of PAD4 to promote resistance against the oomycete pathogen Hyaloperonospora arabidopsidis in Arabidopsis thaliana.

Peptidylarginine deiminase 4 (PAD4), a Ca2+-dependent enzyme, catalyzes the conversion of arginine to citrulline and has been strongly associated with many malignant tumors. However, the molecular mechanisms of PAD4 in the development and progression of colorectal cancer (CRC) remain unclearly defined. In our study, PAD4 expression was increased in CRC tissues and cells, and was closely related to tumor size, lymph node metastasis. Moreover, the transcription factor KLF9 directly bound to PADI4 gene promoter, leading to overexpression of PAD4 in CRC cells, which augmented cell growth and migration. We revealed that PAD4 interacted with and citrullinated glycogen synthase kinase-3β (GSK3β) in CRC cells, and GSK3β Arg-344 was the dominating PAD4-citrullination site. Furthermore, IgL2 and catalytic domains of PAD4 directly bound to the kinase domain of GSK3β in CRC cells. Mechanistically, PAD4 promoted the transport of GSK3β from the cytoplasm to the nucleus, thereby increasing the ubiquitin-dependent proteasome degradation of nuclear cyclin-dependent kinase inhibitor 1 (CDKN1A). Our study is the first to reveal the details of a critical PAD4/GSK3β/CDKN1A signaling axis for CRC progression, and provides evidence that PAD4 is a potential diagnosis biomarker and therapeutic target in CRC.

This review will briefly outline the major signaling pathways in PMA-activated neutrophils. PMA is widely used to understand neutrophil pathways and formation of NETs. PMA activates PKC; however, we highlight some isoforms that contribute to specific functions. PKC α, β and δ contribute to ROS production while PKC βII and PKC ζ are involved in cytoskeleton remodeling. Actin polymerization is important for the chemotaxis of neutrophils and its remodeling is connected to ROS balance. We suggest that, although ROS and production of NETs are usually observed together in PMA-activated neutrophils, there might be a regulatory mechanism balancing both. Interestingly, we suggest that serine proteases might determine the PAD4 action. PAD4 could be responsible for the activation of the NF-κB pathway that leads to IL-1β release, triggering the cleavage of gasdermin D by serine proteases such as elastase, leading to pore formation contributing to release of NETs. On the other hand, when serine proteases are inhibited, NETs are formed by citrullination through the PAD4 pathway. This review puts together results from the last 31 years of research on the effects of PMA on the neutrophil and proposes new insights on their interpretation. [ABSTRACT FROM AUTHOR]

Human amniotic membrane mesenchymal stem cells (hAM-MSC) secrete a myriad of components with immunosuppressive activities. In the present research, we aimed to describe the effect of prostaglandin E2 (PGE2) secreted by hAM-MSCs on neutrophil extracellular trap (NET) release and to characterize the role of its receptors (EP2/EP4) in PAD-4 and NFκB activity in neutrophils. Human peripheral blood neutrophils were ionomycin-stimulated in the presence of hAM-MSC conditioned medium (CM) treated or not with the selective PGE2 inhibitor MF-63, PGE2, EP2/EP4 agonists, and the selective PAD-4 inhibitor GSK-484. NET release, PAD-4, and NFκB activation were analyzed. Ionomycin induced NET release, which was inhibited in the presence of hAM-MSC-CM, while CM from hAM-MSCs treated with MF-63 prevented NET release inhibition. PGE2 and EP2/EP4 agonists, and GSK-484 inhibited NET release. EP2/EP4 agonists and GSK-484 inhibited H3-citrullination but did not affect PAD-4 protein expression. Finally, PGE2 and EP2/EP4 agonists and GSK-484 increased NFκB phosphorylation. Taken together, these results suggest that hAM-MSC exert their immunomodulatory activities through PGE2, inhibiting NET release in a PAD-4-dependent pathway. This research proposes a new mechanism by which hAM-MSC exert their activities when modulating the innate immune response and inhibiting NET release. [ABSTRACT FROM AUTHOR]

Arabidopsis cyclic nucleotide-gated channels (CNGC) 2 and 4 are shown to negatively regulate disease resistance and heat tolerance and to positively regulate plant growth. Whether or not their functions in these processes are interdependent is largely unknown. Here, using the mutation of phytoalexin deficient 4 (PAD4) to inhibit the enhanced defense response and programmed cell death (PCD), we assessed the contribution of the altered defense response to the heat tolerance and plant growth in the cngc2 and cngc4 single and double mutants. The pad4 mutation reverted the enhanced disease resistance of the cngc2 and cngc4 mutants at the normal temperature (22 °C) but not at the elevated temperature (28 °C). The pad4 mutation slightly alleviated the dwarfism of the cngc2 and cngc4 mutants at 22 °C but not at 28 °C, indicating a small contribution from the defense response to plant growth regulation. The pad4 mutation also reduced the enhanced heat tolerance in the cngc mutants, suggesting an involvement of PCD in heat tolerance. In addition, a higher heat tolerance was correlated with more opened stomata under heat treatment among the wild type and mutants of the PAD4, CNGC2 and CNGC4 genes. In sum, this study suggests that the regulation of heat tolerance and plant growth by CNGC2 and CNGC4 is almost independent of their regulation of disease resistance. It also reveals a PAD4-dependent role of CNGC2 and CNGC4 in stomatal aperture regulation and heat tolerance. [ABSTRACT FROM AUTHOR]

Abstract Neutrophils are the most abundant type of white blood cells circulating throughout the bloodstream and are often considered the frontline defenders in innate immunity. However, neutrophils are increasingly being recognized as having an important role in tumorigenesis and carcinogenesis due to their aberrant activation by molecules released into the tumor microenvironment. One defensive response of neutrophils that is aberrantly triggered during the neoplastic process is called NETosis, where activated neutrophils expel their DNA and intracellular contents in a web-like structure known as a neutrophil extracellular trap (NET). In cancer, NETosis has been linked to increased disease progression, metastasis, and complications such as venous thromboembolism. NET structures released by neutrophils can also serve as a scaffold for clot formation, shining new light on the role of neutrophils and NETosis in coagulation-mediated diseases. Here, we review current available knowledge regarding NET and the related NETosis process in cancer patients, with an emphasis on pre-clinical and clinical data fostering the identification and validation of biomarkers of NET with a predictive/prognostic role in cancer patients treated with immunotherapy agents. NETosis biomarkers, e.g., citH3, may integrate correlates of immunogenicity currently available (e.g., PD-L1 expression, TMB, TILs) and help select the subsets of patients who may most benefit from the use of the therapeutic weapons under discussion.

The risk to develop ACPA positive rheumatoid arthritis (RA), the most destructive type of autoimmune arthritis, is carried by HLA-DRB1 alleles containing a 5 amino acid motif: the shared epitope (SE). RA is preceded by the emergence of disease specific anti citrullinated protein antibodies (ACPA). SE positive HLA-DRB1 alleles are associated with ACPA and ACPA positive RA, not with ACPA negative RA, suggesting that ACPA contribute to the pathogenesis of RA. Understanding how HLA-DRB1 genotypes influence ACPA could lead to a curative or preventive treatment of RA. The "Shared epitope binds citrullinated peptides " hypothesis suggests that RA associated HLA-DR alleles present citrullinated peptides to T cells that help ACPA producing B cells. The "Hapten carrier model" suggests that PAD4 is the target of the T cells which help ACPA specific B cells through a hapten carrier mechanism in which PAD4 is the carrier and citrullinated peptides are the haptens. Direct binding assay of citrullinated peptides to purified HLA-DR molecules does not support the "shared epitope binds citrullinated peptides" hypothesis. The Odds Ratios to develop ACPA positive RA associated with each of 12 common HLA-DRB1 genotypes match the probability that the two HLA-DR molecules they encode can bind at least one peptide from PAD4, not from citrullinated fibrinogen. Thus, PAD4 tolerization might stop the carrier effect and switch off production of ACPA. [ABSTRACT FROM AUTHOR]

SUMMARY: Arabidopsis pathogen effector‐triggered immunity (ETI) is controlled by a family of three lipase‐like proteins (EDS1, PAD4, and SAG101) and two subfamilies of HET‐S/LOB‐B (HeLo)‐domain "helper" nucleotide‐binding/leucine‐rich repeats (ADR1s and NRG1s). EDS1‐PAD4 dimers cooperate with ADR1s, and EDS1‐SAG101 dimers with NRG1s, in two separate defense‐promoting modules. EDS1‐PAD4‐ADR1 and EDS1‐SAG101‐NRG1 complexes were detected in immune‐activated leaf extracts but the molecular determinants for specific complex formation and function remain unknown. EDS1 signaling is mediated by a C‐terminal EP domain (EPD) surface surrounding a cavity formed by the heterodimer. Here we investigated whether the EPDs of PAD4 and SAG101 contribute to EDS1 dimer functions. Using a structure‐guided approach, we undertook a comprehensive mutational analysis of Arabidopsis PAD4. We identify two conserved residues (Arg314 and Lys380) lining the PAD4 EPD cavity that are essential for EDS1‐PAD4–mediated pathogen resistance, but are dispensable for the PAD4‐mediated restriction of green peach aphid infestation. Positionally equivalent Met304 and Arg373 at the SAG101 EPD cavity are required for EDS1‐SAG101 promotion of ETI‐related cell death. In a PAD4 and SAG101 interactome analysis of ETI‐activated tissues, PAD4R314A and SAG101M304R EPD variants maintain interaction with EDS1 but lose association, respectively, with helper nucleotide‐binding/leucine‐rich repeats ADR1‐L1 and NRG1.1, and other immune‐related proteins. Our data reveal a fundamental contribution of similar but non‐identical PAD4 and SAG101 EPD surfaces to specific EDS1 dimer protein interactions and pathogen immunity. Significance Statement: The molecular determinants for EDS1‐PAD4‐ADR1 and EDS1‐SAG101‐NRG1 complex formation and function remain unknown. In a PAD4 and SAG101 interactome analysis of ETI‐activated tissues, PAD4R314A and SAG101M304R EPD variants maintain interaction with EDS1 but lose association, respectively, with helper NLRs ADR1‐L1 and NRG1.1, and other immune‐related proteins, this reveals a fundamental contribution of similar but non‐identical PAD4 and SAG101 EPD surfaces to specific EDS1 dimer protein interactions and pathogen immunity. [ABSTRACT FROM AUTHOR]

Peptidylarginine deiminase 4 (PAD4) is a key regulator of inflammation but its function in infections remains incompletely understood. We investigate PAD4 in the context of malaria and demonstrate a role in regulation of immune cell trafficking and chemokine production. PAD4 regulates liver immunopathology by promoting neutrophil trafficking in a Plasmodium chabaudi mouse malaria model. In human macrophages, PAD4 regulates expression of CXCL chemokines in response to stimulation with TLR ligands and P. falciparum. Using patient samples, we show that CXCL1 may be a biomarker for severe malaria. PAD4 inhibition promotes disease tolerance and may represent a therapeutic avenue in malaria. [ABSTRACT FROM AUTHOR]

Peptidyl arginine deiminases (PAD) are proteins that modify arginine residues to citrulline. In humans there are five isoforms PAD1-4 and PAD6. PAD4 is involved in several human diseases such as Alzheimer, breast cancer, rheumatoid arthritis and therefore has been recognized as a potential pharmacological target. In this work, 3175 molecules retrieved from ZINC12 Food and Drug Administration (FDA) catalog database were assayed through a structure-based virtual screening to identify potential PAD4 binders. Three were confirmed through enzyme inhibition studies. Flow cytometry analysis were used to check for drug cytotoxic effects on blood cells from three healthy donors. Molecular docking in a wild-type PAD4 structure showed the top 20 FDA molecules with the best binding energies. Those molecules were distributed in clusters of mixed binding energies but with similar chemical structures. These patterns along with the FDA information were used to aid in the rational selection of methotrexate, testosterone and leucovorin compounds for in vitro evaluations. Time course fluorescent enzyme kinetics confirmed that methotrexate (0.01–1 mmol) and testosterone enanthate (49.9–12.5 mmol) inhibit PAD4 at similar levels to the known inhibitor BB-Cl-amidine (8.8 mmol), except leucovorin. The chosen concentrations lack cytotoxic effects on blood cells. These findings encourage repositioning campaigns in the quest of PAD4 inhibitors. [ABSTRACT FROM AUTHOR]

Abstract Background Nasopharyngeal carcinoma (NPC) is a tumor deriving from nasopharyngeal epithelium. Peptidyl-arginine deiminase 4 (PAD4) is a vital mediator of histone citrullination and plays an essential role in regulating disease process. Radiotherapy is an essential method to treat NPC. In this research, we explored the effect of PAD4 on NPC radiosensitivity. Methods We enrolled 50 NPC patients, established mice xenograft model, and purchased cell lines for this study. Statistical analysis and a series of experiments including RT-qPCR, clonogenic survival, EdU, Transwell, and wound healing assays were done. Results Our data manifested that PAD4 (mRNA and protein) presented a high expression in NPC tissues and cells. GSK484, an inhibitor of PAD4, could inhibit activity of PAD4 in NPC cell lines. PAD4 overexpression promoted the radioresistance, survival, migration, and invasion of NPC cells, whereas treatment of GSK484 exerted inhibitory effects on radioresistance and aggressive phenotype of NPC cells. Additionally, GSK484 could attenuate the effect of PAD4 of NPC cell progression. More importantly, we found that GSK484 significantly inhibited tumor size, tumor weight and tumor volume in mice following irradiation. Conclusions PAD4 inhibitor GSK484 attenuated the radioresistance and cellular progression in NPC.

The risk to develop ACPA positive rheumatoid arthritis (RA), the most destructive type of autoimmune arthritis, is carried by HLA-DRB1 alleles containing a 5 amino acid motif: the shared epitope (SE). RA is preceded by the emergence of disease specific anti citrullinated protein antibodies (ACPA). SE positive HLA-DRB1 alleles are associated with ACPA and ACPA positive RA, not with ACPA negative RA, suggesting that ACPA contribute to the pathogenesis of RA. Understanding how HLA-DRB1 genotypes influence ACPA could lead to a curative or preventive treatment of RA. The “Shared epitope binds citrullinated peptides “ hypothesis suggests that RA associated HLA-DR alleles present citrullinated peptides to T cells that help ACPA producing B cells. The “Hapten carrier model” suggests that PAD4 is the target of the T cells which help ACPA specific B cells through a hapten carrier mechanism in which PAD4 is the carrier and citrullinated peptides are the haptens. Direct binding assay of citrullinated peptides to purified HLA-DR molecules does not support the “shared epitope binds citrullinated peptides” hypothesis. The Odds Ratios to develop ACPA positive RA associated with each of 12 common HLA-DRB1 genotypes match the probability that the two HLA-DR molecules they encode can bind at least one peptide from PAD4, not from citrullinated fibrinogen. Thus, PAD4 tolerization might stop the carrier effect and switch off production of ACPA.

Most acute strokes are ischemic, and subsequent neuroinflammation promotes further damage leading to cell death but also plays a beneficial role by promoting cellular repair. Neutrophils are forerunners to brain lesions after ischemic stroke and exert elaborate functions. While neutrophil extracellular traps (NETs) possess a fundamental antimicrobial function within the innate immune system under physiological circumstances, increasing evidence indicates that NETosis, the release process of NETs, occurs in the pathogenic process of stroke. In this review, we focus on the processes of NET formation and clearance, the temporal and spatial alterations of neutrophils and NETs after ischemic damage, and how NETs are involved in several stroke-related phenomena. Generally, NET formation and release processes depend on the generation of reactive oxygen species (ROS) and the activation of nuclear peptidylarginine deiminase-4 (PAD4). The acid–base environment, oxygen concentration, and iron ions around the infarct may also impact NET formation. DNase 1 has been identified as the primary degrader of NETs in serum, while reactive microglia are expected to inhibit the formation of NETs around ischemic lesions by phagocytosis of neutrophils. The neutrophils and NETs are present in the perivascular space ipsilateral to the infarct arising after ischemic damage, peaking between 1 and 3 days postischemia, but their location in the brain parenchyma remains controversial. After the ischemic injury, NETs are involved in the destruction of neurological function primarily by disrupting the blood–brain barrier and promoting thrombosis. The potential effects of NETs on various ischemic nerve cells need to be further investigated, especially in the chronic ischemic phase. [ABSTRACT FROM AUTHOR]

Abstract Silicon (Si) has been widely employed in agriculture to enhance resistance against pathogens in many crop plants. However, the underlying molecular mechanisms of Si-mediated resistance remain elusive. In this study, the Arabidopsis-powdery mildew pathosystem was employed to investigate possible defense mechanisms of Si-mediated resistance. Because Arabidopsis lacks efficient Si transporters and thus is a low Si-accumulator, two heterologous Si influx transporters (from barley and muskmelon) were individually expressed in wild-type Arabidopsis Col-0 and a panel of mutants defective in different immune signaling pathways. Results from infection tests showed that while very low leaf Si content slightly induced salicylic acid (SA)-dependent resistance, high Si promoted PAD4-dependent but largely EDS1- and SA-independent resistance against the adapted powdery mildew isolate Golovinomyces cichoracearum UCSC1. Intriguingly, our results also showed that high Si could largely reboot non-host resistance in an immune-compromised eds1/pad4/sid2 triple mutant background against a non-adapted powdery mildew isolate G. cichoracearum UMSG1. Taken together, our results suggest that assimilated Si modulates distinct, multi-layered defense mechanisms to enhance plant resistance against adapted and no-adapted powdery mildew pathogens, possibly via synergistic interaction with defense-induced callose.

Coffee, a widely consumed important agricultural product, is mainly produced from two species, Coffea arabica (Arabica coffee) and C. canephora (Robusta coffee). Timor Hybrid (HDT) is a population resulting from a natural cross between C. arabica and C. canephora. HDT derivatives have a high spectrum of resistance to different races of Hemileia vastatrix (Hv), the causal agent of coffee leaf rust. A RNAseq database, obtained from HDT832/2 leaves inoculated with Hv (Host Resistance) and Uromyces vignae (Uv, Nonhost Resistance), showed the presence of genes implicated in the hypersensitive response and salicylic acid pathway. Hypersensitive Induced Reaction (HIR) gene family, Enhanced Disease Susceptibility1 gene (EDS1), and Phytoalexin Deficient 4 (PAD4) gene are involved in host and nonhost resistance. Relative expression calculated by RT-qPCR was used to confirm and expand the transcriptome analysis. HDTHIR4, HDTEDS1, and HDTPAD4 showed the highest upregulation in response to Hv and Uv inoculation, confirming a similar trend in host and nonhost resistance in HDT. HIR and EDS1/PAD4 gene families were characterized for the first time in the three available Coffea genomes. HIR genes were quite conserved between Coffea species. Surprisingly, EDS1 and PAD4 genes revealed major differences in gene structure. The PAD4 predicted protein from C. arabica does not include both conserved domains of the EDS1/PAD4 family, and the EDS1 putative protein from C. canephora includes a formin domain unusual in the same protein family. The variability shown by EDS1/PAD4 gene family may impact the disease resistance response of Coffea species, which can be surveyed for the gene sequences that will produce a more resistant phenotype.

Scope: Neutrophils play a decisive role during the immediate defense against infections. However, as observed during rheumatoid arthritis, activated neutrophils can also cause tissue damage. Previous studies indicate that zinc supplementation may alter certain neutrophil functions. However, precise underlying mechanisms and possible effects of zinc deficiency remain incompletely understood. The objective of this study is to investigate the effects of changes in zinc status on formation of neutrophil extracellular traps (NETs) and other fundamental neutrophil functions., Methods and Results: Interleukin (IL)-17 and tumor necrosis factor (TNF)-α are used to simulate the inflammatory environment observed in autoimmune diseases. The study analyzes the impact of the zinc status on NETs release, using a fluorescence plate reader, and on the expression of peptidylarginine deiminase 4 (PAD4), S100A8/A9, and certain cytokines by PCR and western blot. These results show that zinc supplementation significantly reduces NETs formation and downregulates PAD4 protein expression. Zinc supplementation results in increased protein expression of interleukin-1 receptor antagonist (IL-1RA) and IL-8 in stimulated cells., Conclusion: The results suggest that changes in extracellular zinc availability may influence the functions of neutrophils. Therefore, maintaining an appropriate zinc level is advisable for preserving innate immunity and to prevent hyper-activation of neutrophils., (© 2024 The Author(s). Molecular Nutrition & Food Research published by Wiley‐VCH GmbH.)

Ulcerative colitis (UC) is characterized by chronic non-recessive inflammation of the intestinal mucosa involving both innate and adaptive immune responses. Currently, new targeted therapies are urgently needed for UC, and neutrophil extracellular traps (NETs) are new therapeutic options. NETs are DNA-based networks released from neutrophils into the extracellular space after stimulation, in which a variety of granule proteins, proteolytic enzymes, antibacterial peptides, histones, and other network structures are embedded. With the deepening of the studies on NETs, their regulatory role in the development of autoimmune and autoinflammatory diseases has received extensive attention in recent years. Increasing evidence indicates that excess NETs exacerbate the inflammatory response in UC, disrupting the structure and function of the intestinal mucosal barrier and increasing the risk of thrombosis. Although NETs are usually assigned a deleterious role in promoting the pathological process of UC, they also appear to have a protective role in some models. Despite such progress, comprehensive reviews describing the therapeutic promise of NETs in UC remain limited. In this review, we discuss the latest evidence for the formation and degradation of NETs, focusing on their double-edged role in UC. Finally, the potential implications of NETs as therapeutic targets for UC will be discussed. This review aims to provide novel insights into the pathogenesis and therapeutic options for UC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Long, Mao, Xu and Zhu.)

Neutrophils express protein arginine deiminase 2 and PAD4, both of which mediate the citrullination of target proteins to induce production of neutrophil extracellular traps. Although PAD-dependent NETs trigger inflammatory bowel disease, the mechanisms governing the expression of PAD2 and PAD4 are poorly understood. In this study, we tried to clarify expression mechanisms of PAD2 and PAD4 in the colonic mucosa of patients with ulcerative colitis and Crohn's disease. Administration of Cl-amidine, a pan PAD-inhibitor, attenuated the development of dextran sodium sulfate-induced colitis, the effects of which were accompanied by reduced IL-6 and TNF-α production by colonic lamina propria mononuclear cells upon exposure to Toll-like receptor ligands. The mRNA expression of colonic PAD2 and PAD4 was negatively and positively correlated with disease activity and pro-inflammatory cytokine responses in patients with UC, respectively. Reciprocal regulation of PAD2 and PAD4 mRNA expression was observed in the colonic mucosa of UC patients, but not in those of CD patients. PAD4 mRNA expression was correlated with disease activity and pro-inflammatory cytokine responses in patients with CD. Collectively, these data suggest that reciprocal regulation of PAD2 and PAD4 expression is associated with disease activity in UC patients., Competing Interests: No potential conflicts of interest were disclosed., (Copyright © 2024 JCBN.)

Chromatin remodeling proteins modulate nucleosome dynamic to affect global gene expression and other cellular processes. Their roles in the regulation of plant growth and development have been widely reported, but their roles in plant stress resistance, especially disease resistance, have not been extensively investigated. Here, we show that the Arabidopsis Immunity Switch (ISWI) chromatin-remodeling factors CHR11 and CHR17, are negative regulators of plant disease resistance. The loss of both CHR11 and CHR17 function led to upregulation of a large set of defense response genes in the absence of pathogen infection. The chr11 single mutant showed enhanced resistance against a virulent pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). Further analysis revealed that mutation of Phytoalexin Deficient4 (PAD4) reduced the upregulation of defense gene expression as well as resistance against Pst DC3000 in the chr11 chr17 double mutant. However, mutation of PAD4 does not rescue the growth defects of chr11 chr17. Together, our study revealed a function of ISWI in repressing defense response under non-pathogenic conditions and indicates distinct target genes of ISWI in regulating plant growth and plant immunity. • ISWI represses defense response gene expression under normal growth condition. • ISWI negatively regulate resistance to bacterial pathogen in Arabidopsis. • The ISWI-regulated plant immunity is partially dependent on PAD4. [ABSTRACT FROM AUTHOR]