Search Results (42)

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

Cancer remains a major global health challenge requiring the development of diagnostic and therapeutic strategies. Liquid biopsy is considered a promising minimally invasive tool for cancer screening, prognosis and treatment monitoring. Recent studies suggest that neutrophil extracellular traps (NETs) may also be potential liquid biopsy markers. NETs are web-like chromatin structures released by neutrophils in response to various stimuli to trap and neutralize pathogens. However, excessive or dysregulated NET formation has been implicated in tumor progression and metastasis. Elevated levels of NETs have been observed in patients with various types of cancer and correlate with disease stage and prognosis. The presence of NET markers such as citrullinated histone H3 (H3Cit), neutrophil elastase (NE) and myeloperoxidase (MPO) has been associated with higher tumor burden and poorer clinical outcomes. Several studies have shown a positive correlation between NET markers and circulating free DNA (cfDNA) levels, suggesting that NETs may increase the sensitivity of liquid biopsy in detecting and monitoring cancer progression. This review examines the role of NETs in the tumor microenvironment, their contribution to cancer progression and metastasis, and their potential use in liquid biopsy and cancer therapy. 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

Hepatitis B virus (HBV) infection is one of the most dangerous viral diseases, with innate immunity representing the first line of defense against the virus. In this branch of the immune system, neutrophils are considered key cellular mediators. To better understand the implication of neutrophils in the distinct stages of the disease, HBV-infected patients were enrolled in this study and categorized into three groups: patients with acute infection, chronic infection under treatment, and at early cirrhotic stage. To elucidate the role of inflammatory mediators and cellular mechanisms of neutrophilic origin in the course of the infection, both ex vivo and in vitro studies were performed. Increased levels of C-C motif chemokine ligand 2 (CCL2), interleukin (IL)-18, IL-33, and citrullinated histone H3 (CitH3)—an accurate marker of neutrophil extracellular traps (NETs)—were detected in the circulation of patients with acute infection or early cirrhosis. In parallel, sera from the aforementioned patient groups induced the formation of IL-1b-bearing NETs in neutrophils from healthy individuals. These inflammatory NETs affected primary fibroblasts towards acquiring a pro-fibrotic phenotype. These results suggest that NETs could be regarded as mediators in hepatitis B manifestations, while their therapeutic targeting could enhance the management of early-stage cirrhotic patients. Full article

Activation of cGAS, a cytosolic receptor recognizing double-stranded DNA, in macrophages is important in sepsis (a life-threatening condition caused by infection). The responses against sepsis induced by subcutaneous implantation of the Pseudomonas-contaminated catheters in cGAS-deficient (cGAS^−/−) mice were lower than in wild-type (WT) mice as indicated by liver enzymes, white blood cell count, cytokines, and M1-polarized macrophages in the spleens. Likewise, a lethal dose of lipopolysaccharide (LPS) induced less severe sepsis severity as determined by mortality, organ injury, cell-free DNA, and serum cytokines. Patterns of the transcriptome of lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages were clearly different between cGAS^−/− and WT cells. Gene set enrichment analysis (GSEA; a computational statistical determination of the gene set) indicated more prominent enrichment of oxidative phosphorylation (OXPHOS; the mitochondrial function) and mTORC1 pathways in LPS-activated cGAS^−/− macrophages compared with WT. Meanwhile, LPS upregulated cGAS and increased cGAMP (a cGAS inducer) only in WT macrophages along with less severe inflammation in cGAS^−/− macrophages, as indicated by supernatant cytokines, pro-inflammatory molecules (nuclear factor kappa B; NF-κB), M1 polarization (IL-1β, CD80, and CD86), and macrophage extracellular traps (METs; web-like structures composed of DNA, histones, and other proteins) through the detection of citrullinated histone 3 (CitH3) in supernatant and immunofluorescent visualization. In conclusion, less prominent pro-inflammatory responses of cGAS^−/− macrophages than WT were demonstrated in mice (catheter-induced sepsis and LPS injection model) and in vitro (transcriptomic analysis, macrophage polarization, and METs). Full article

Background: Although endothelial mesenchymal transition (EndMT) has been characterized as a basic process in embryogenesis, EndMT is the mechanism that accelerates the development of cardiovascular diseases, including heart failure, aging, and complications of diabetes or hypertension as well. Endothelial cells lose their distinct markers and take on a mesenchymal phenotype during EndMT, expressing distinct products. Methods: In this study, type 1 Diabetes mellitus (T1DM) was induced in rats with streptozotocin (STZ) by intraperitoneal injection at a 60 mg/kg dose. Diabetic rats were randomly divided into two groups, namely, control and diabetic rats, for 4 weeks. Heart, aorta, and plasma samples were collected at the end of 4 weeks. Sequentially, biochemical parameters, cytokines, reactive oxygen species (ROS), protein expression of EndMT markers (Chemokine C-X-C motif ligand-1 (CXCL-1), vimentin, citrullinated histone H3 (H3Cit), α-smooth muscle actin (α-SMA), and transforming growth factor beta (TGF-β) and versican), components of the extracellular matrix (matrix metalloproteinase 2 (MMP-2), tissue inhibitor of metalloproteinase-1(TIMP-1), and discoidin domain tyrosine kinase receptor 2 (DDR-2)) were detected by ELISA or Western blot, respectively. Results: Cytokines and ROS were increased in diabetic hearts, which induced partial EndMT. Among EndMT markers, histone citrullination, α-SMA, and CXCL-1 were increased; vimentin was decreased in DM. The endothelial marker endothelin-1 was significantly higher in the aortas of DM rats. Interestingly, TGF-β showed a significant decrease in the diabetic heart, plasma, and aorta. Additionally, MMP-2/TIMP-1 levels also decreased in DM. Conclusions: To sum up, the identification of molecules and regulatory pathways involved in EndMT provided novel therapeutic approaches for cardiac pathophysiological conditions. Full article

Enhancing or protecting sperm motility has always been a pivotal approach to improving the ewe pregnancy rate. Sperm motility is highly susceptible to the immune status of the reproductive tract. Neutrophil extracellular traps (NETs) have been demonstrated to capture sperm and impair its motility in human, swine, and goat species. Quercetin is a flavonoid derived from Cuscuta Chinensis Lam., which can protect sperm from oxidative damage. In this study, we investigated whether inflammation decreases sperm motility and tried to clarify the potential protective mechanism of quercetin on goat sperm motility. Sperm-triggered NETs were analyzed by immunofluorescence analysis. Sperm acrosome integrity was detected by using giemsa staining. Quercetin exhibited no cytotoxicity towards sperm and PMNs within the concentration range of 20–80 μM. PMNs impaired both the survival rate and rapid linear motility of sperm, while quercetin significantly enhanced these parameters. PMNs captured sperm through NETs composed of DNA, citrullinated histone 3 (citH3), and neutrophil elastase (NE); however, quercetin effectively inhibited the release of sperm-stimulated NETs. The stimulation of PMNs with sperm resulted in a significant increase in levels of ROS and MDA, which decreased by quercetin. Moreover, PMNs caused integrity violation to both the plasma membrane and acrosome in sperm; this effect was significantly alleviated by quercetin. In conclusion, quercetin effectively ameliorated PMN-reduced sperm motility through the inhibition of NETs and oxidative stress, and preserving sperm plasma membrane and acrosome integrity, thereby providing preliminary insights into the underlying mechanisms and theoretical support for the development of potential sperm protectors. Full article

Psoriasis is an inflammatory skin disease with various symptoms of differing severities and with the reported prominent involvement of neutrophil extracellular traps (NETs). The excitation of neutrophils, e.g., by interleukin 8 (IL-8) or lipopolysaccharide (LPS), leads to the citrullination of histones and the release of protein–DNA complexes into the extracellular space, where they are digested by DNases. Our aim was to explore data on the levels of protein-complexed DNAs neutrophil elastase–DNA (NE-DNA) and myeloperoxidase–DNA (MPO-DNA), citrullinated histones (citH2, citH3, citH4), and NET-degrading enzyme DNase I in the serum of psoriatic patients with varying severities of clinical symptoms assessed with the Psoriasis Area Severity Index (PASI), Body Surface Area (BSA), and Dermatology Life Quality Index (DLQI) scores. The levels of factors were detected in 52 patients with psoriasis and 22 healthy volunteers by the enzyme-linked immunosorbent assay (ELISA). The results showed the elevated levels of NE-DNA, MPO-DNA, citH3, and DNase I in the patients with psoriasis compared to healthy volunteers (p < 0.05). Additionally, changes were noticed in the levels of NE-DNA, citH3, and DNase I, depending on the severity of symptoms (p < 0.05). In mild psoriasis (PASI < 10, BSA < 10, DLQI < 10), the suppressing activity of the enzyme caused the impaired ability to remove the physiological level of NETs, whereas in moderate to severe psoriasis (PASI ≥ 10, BSA ≥ 10, DLQI ≥ 10), the enhanced activity of DNase I failed to remove NETs due to the observed overexpression. It may, thus, be concluded that the mechanism of action of NETs, which play an undeniable role in psoriatic diseases, seem to follow two different paths depending on the severity of disease, which may be crucial in selecting potential anti-NET treatment methods. Full article

There is a complex interplay between viral infection and host innate immune response regarding disease severity and outcomes. Neutrophil hyperactivation, including excessive release of neutrophil extracellular traps (NETs), is linked to exacerbated disease in acute COVID-19, notably in hospitalized patients. Delineating protective versus detrimental neutrophil responses is essential to developing targeted COVID-19 therapies and relies on high-quality translational animal models. In this study, we utilize a previously established feline model for COVID-19 to investigate neutrophil dysfunction in which experimentally infected cats develop clinical disease that mimics acute COVID-19. Specific pathogen-free cats were inoculated with SARS-CoV-2 (B.1.617.2; Delta variant) (n = 24) or vehicle (n = 6). Plasma, bronchoalveolar lavage fluid, and lung tissues were collected at various time points over 12 days post-inoculation. Systematic and temporal evaluation of the kinetics of neutrophil activation was conducted by measuring markers of activation including myeloperoxidase (MPO), neutrophil elastase (NE), and citrullinated histone H3 (citH3) in SARS-CoV-2-infected cats at 4 and 12 days post-inoculation (dpi) and compared to vehicle-inoculated controls. Cytokine profiling supported elevated innate inflammatory responses with specific upregulation of neutrophil activation and NET formation-related markers, namely IL-8, IL-18, CXCL1, and SDF-1, in infected cats. An increase in MPO-DNA complexes and cell-free dsDNA in infected cats compared to vehicle-inoculated was noted and supported by histopathologic severity in respiratory tissues. Immunofluorescence analyses further supported correlation of NET markers with tissue damage, especially 4 dpi. Differential gene expression analyses indicated an upregulation of genes associated with innate immune and neutrophil activation pathways. Transcripts involved in activation and NETosis pathways were upregulated by 4 dpi and downregulated by 12 dpi, suggesting peak activation of neutrophils and NET-associated markers in the early acute stages of infection. Correlation analyses conducted between NET-specific markers and clinical scores as well as histopathologic scores support association between neutrophil activation and disease severity during SARS-CoV-2 infection in this model. Overall, this study emphasizes the effect of neutrophil activation and NET release in SARS-CoV-2 infection in a feline model, prompting further investigation into therapeutic strategies aimed at mitigating excessive innate inflammatory responses in COVID-19. Full article

Neutrophil extracellular traps (NETs) formation, namely NETosis, is implicated in antiphospholipid syndrome (APS)-related thrombosis in various autoimmune disorders such as systemic lupus erythematosus (SLE) and APS. Human parvovirus B19 (B19V) infection is closely associated with SLE and APS and causes various clinical manifestations such as blood disorders, joint pain, fever, pregnancy complications, and thrombosis. Additionally, B19V may trigger the production of autoantibodies, including those against nuclear and phospholipid components. Thus, exploring the connection between B19V, NETosis, and thrombosis is highly relevant. An in vitro NETosis model using differentiated HL-60 neutrophil-like cells (dHL-60) was employed to investigate the effect of B19V-VP1u IgG on NETs formation. A venous stenosis mouse model was used to test how B19V-VP1u IgG-mediated NETs affect thrombosis in vivo. The NETosis was observed in the dHL-60 cells treated with rabbit anti-B19V-VP1u IgG and was inhibited in the presence of either 8-Br-cAMP or CGS216800 but not GSK484. Significantly elevated reactive oxygen species (ROS), myeloperoxidase (MPO), and citrullinated histone (Cit-H3) levels were detected in the dHL60 treated with phorbol myristate acetate (PMA), human aPLs IgG and rabbit anti-B19V-VP1u IgG, respectively. Accordingly, a significantly larger thrombus was observed in a venous stenosis-induced thrombosis mouse model treated with PMA, human aPLs IgG, rabbit anti-B19V-VP1u IgG, and human anti-B19V-VP1u IgG, respectively, along with significantly increased amounts of Cit-H3-, MPO- and CRAMP-positive infiltrated neutrophils in the thrombin sections. This research highlights that anti-B19V-VP1u antibodies may enhance the formation of NETosis and thrombosis and implies that managing and treating B19V infection could lower the risk of thrombosis. Full article

The complement system and neutrophils play crucial roles in innate immunity. Neutrophils release neutrophil extracellular traps (NETs), which are composed of decondensed DNA entangled with granular contents, as part of their innate immune function. Mechanisms governing complement-mediated NET formation remain unclear. In this study, we tested a two-step NETosis mechanism, as follows: classical complement-mediated neutrophil activation in serum and subsequent NET formation in serum-free conditions, using neutrophils from healthy donors, endothelial cells, and various assays (Fluo-4AM, DHR123, and SYTOX), along with flow cytometry and confocal microscopy. Our findings reveal that classical complement activation on neutrophils upregulated the membrane-anchored complement regulators CD46, CD55, and CD59. Additionally, complement activation increased CD11b on neutrophils, signifying activation and promoting their attachment to endothelial cells. Complement activation induced calcium influx and citrullination of histone 3 (CitH3) in neutrophils. However, CitH3 formation alone was insufficient for NET generation. Importantly, NET formation occurred only when neutrophils were in serum-free conditions. In such environments, neutrophils induced NADPH oxidase-dependent reactive oxygen species (ROS) production, leading to NET formation. Hence, we propose that complement-mediated NET formation involves a two-step process, as follows: complement deposition, neutrophil priming, calcium influx, CitH3 formation, and attachment to endothelial cells in serum. This is followed by NADPH-dependent ROS production and NET completion in serum-free conditions. Understanding this process may unveil treatment targets for pathologies involving complement activation and NET formation. Full article

Background and Objective: This randomized controlled trial investigated the influence of perioperative lidocaine administration on the postoperative inflammatory response in patients undergoing robot-assisted radical prostatectomy, with the results having potential implications for postoperative recovery and cancer recurrence via neutrophil extracellular trapping (NETosis). Materials and Methods: In total, 58 patients with localized prostate cancer were randomly assigned to receive an intravenous infusion of 2% lidocaine or a saline placebo intraoperatively. Serum levels of interleukin (IL)-6, IL-10, and IL-17, tumor necrosis factor(TNF)-α, interferon(IFN)-γ, neutrophil elastase (NE), citrullinated histone3 (CitH3), and myeloperoxidase (MPO) were determined preoperatively and at 24 h postoperatively. Biochemical recurrence (BCR) was assessed over a follow-up period of 2 years. Results: The lidocaine group showed a significant change in MPO, a greater reduction in IL-10 level, and a smaller increase in the NE level compared to the placebo group, suggesting a modulatory effect of lidocaine on certain anti-inflammatory and neuroendocrine pathways. No significant difference in the BCR rate was observed between the two groups. Conclusions: Perioperative lidocaine administration selectively modulates certain inflammatory and neuroendocrine responses after robot-assisted radical prostatectomy surgery, potentially influencing recovery outcomes. These findings highlight the need for further investigations of the role of lidocaine in Enhanced Recovery After Surgery protocols, particularly in oncologic surgeries. Full article

Neutrophil extracellular traps (NETs) released from neutrophils are related to cancer progression. However, the relationship between the therapeutic effects of immune checkpoint inhibitors (ICIs) such as anti-PD-1 and anti-PD-L1 antibodies and plasma NET concentration in patients with non-small cell lung cancer (NSCLC) is poorly understood. In this study, concentrations of citrullinated histone H3 (CitH3), a surrogate marker of NETs, in plasma before/after treatment were examined in patients with advanced or recurrent NSCLC undergoing ICI treatment (n = 185). The clinical significances of NET levels before/after treatment and posttreatment changes were statistically evaluated. As a result, multivariate Cox analysis showed that high NET levels before treatment were statistically significant predictors of unfavorable overall survival (OS; p < 0.001, HR 1.702, 95% CI 1.356–2.137) and progression-free survival (PFS; p < 0.001, HR 1.566, 95% CI 1.323–1.855). The Kaplan-Meier curves showed significant separation between the high- and low-NET groups in OS (p = 0.002) and PFS (p < 0.001). Additionally, high NET levels after treatment were also significantly associated with worse OS (p < 0.001) and PFS (p < 0.001) by multivariate Cox analysis. Notably, the pretreatment NET levels were significantly correlated with the plasma levels of NET-related inflammatory cytokines, such as IL-6 and IL-8, and with NET-related gene expression and immune-suppressive profile in peripheral blood mononuclear cells. Our findings suggest that NETs released from activated neutrophils might reduce the clinical efficacy of ICIs in patients with NSCLC. Full article

The objective of this study was to develop an in vitro model that mimics inflammatory reactions and neutrophil extracellular traps (NETs) formation by polymorphonuclear leukocytes (PMNs) in dairy cows. This model was used to examine the effect of carprofen (CA) on lipopolysaccharide (LPS)-induced NETs formation and expression of inflammatory factors. Peripheral blood samples were collected from 24 Holstein cows (3–11 days postpartum) and PMNs were isolated. In three replicates, PMNs were exposed to various treatments to establish an appropriate in vitro model, including 80 μg/mL of LPS for 2 h, followed by co-incubation for 1 h with 60 μmol/L CA and 80 μg/mL LPS. The effects of these treatments were evaluated by assessing NETs formation by extracellular DNA release, gene expression of pro-inflammatory cytokines, reactive oxygen species (ROS) production, and the expression of NETs-related proteins, including histone3 (H3), citrullinated histone (Cit-H3), cathepsin G (CG), and peptidyl arginine deiminase 4 (PAD4). The assessment of these parameters would elucidate the specific mechanism by which CA inhibits the formation of NETs through the PAD4 pathway instead of modulating the Nox2 pathway. This highlights CA’s effect on chromatin decondensation during NETs formation. Statistical analyses were performed utilizing one-way ANOVA with Bonferroni correction. The results demonstrated that LPS led to an elevated formation of NETs, while CA mitigated most of these effects, concurrent the PAD4 protein level increased with LPS stimulating and decreased after CA administration. Nevertheless, the intracellular levels of ROS did not change under the presence of LPS. LPS supplementation resulted in an upregulation of H3 and Cit-H3 protein expression levels. Conversely, the CA administration inhibited their expression. Additionally, there was no change in the expression of CG with either LPS or LPS + CA co-stimulation. The gene expression of pro-inflammatory cytokines (tumor necrosis factor -α, interleukin (IL)-18, IL-1β, and IL-6) upregulated with LPS stimulation, while the treatment with CA inhibited this phenomenon. In conclusion, CA demonstrated a pronounced inhibitory effect on both LPS-induced NETs formation as well as the associated inflammatory response. Full article