Trap Formation Research Articles

Neutrophils in Ocular Diseases.

Neutrophils, traditionally viewed as first responders to infection or tissue damage, exhibit dynamic and diverse roles in ocular health and disease. This review elaborates on previous findings that showed how neutrophils contribute to ocular diseases. In ocular infections, neutrophils play a pivotal role in host defense by orchestrating inflammatory responses to combat pathogens. Furthermore, in optic nerve neuropathies and retinal degenerative diseases like age-related macular degeneration (AMD) and diabetic retinopathy (DR), neutrophils are implicated in neuroinflammation and tissue damage owing to their ability to undergo neutrophil extracellular trap formation (NETosis) and secretion of inflammatory molecules. Targeting neutrophil-dependent processes holds promise as a therapeutic strategy, offering potential avenues for intervention in ocular infections, cancers, and retinal degenerative diseases. Understanding the multifaceted roles of neutrophils in ocular diseases is crucial for developing targeted therapies to improve patient outcomes.

Investigating the role of palladium electrical contacts in interactions with carbyne nanomaterial solid matter

Introduction: Traps at the interface between carbyne and palladium nanocoatings, produced at different growth conditions, are explored by current-voltage characteristics, scanning electron microscopy and thermal stimulation of charges for evaluation of their nature. It was found that the Pd films can form an Ohmic contact with the carbyne at certain deposition conditions and such deviated from the Ohmic behavior according to the RF sputtering voltage. This growth parameter was found to affect the interfacial traps formation on the carbyne surface, which is important feature for the charge trapping and releasing properties for hydrogen isotopes in the context of the energy release applications.Methods, Results and Discussion: The sputtering voltages of 0.5 kV and 0.7 kV were found unsuitable for controlled trap formation. Based on the currentvoltage and thermally stimulated current (TSC) measurements, a sputtering voltage of 0.9 kV appeared to be more favorable compared to 0.5 kV and 0.7 kV. At 0.9 kV thermal activation of charge carriers are enabled at lower thermal energies, showing a distinct change in TSC behavior correlated to trap activation.

DHXT1, a Virulence Factor of Dactylellina haptotyla, Regulates Pathogenicity by Participating in Trap Formation and Metabolite Synthesis.

The capsule-associated protein 10 gene (CAP10) is indispensable due to its involvement in pod formation and virulence maintenance in Cryptococcus neoformans. The function of the CAP10 gene in nematode-predatory fungi remains unreported. As a typical nematode-trapping fungus, Dactylellina haptotyla efficiently captures nematodes using adhesive knobs, which has potential applications in the biological control of plant-parasitic nematodes. In this study, we investigated the function of DHXT1 (a CAP10 homologous protein) in D. haptotyla-nematode interactions based on the disruption and overexpression of DHXT1, phenotypic analysis and metabolomic analysis. As a result, it was shown that the disruption of the DHXT1 gene causes a marked decrease in the number of adhesive knobs, and on the contrary, the overexpression of the DHXT1 gene causes a substantial increase in the number of adhesive knobs. Interestingly, the variety of metabolites increased with the disruption of the DHXT1 and decreased with the overexpression of the DHXT1 gene. The results suggest that DHXT1 effects pathogenicity through its involvement in adhesive knobs' formation and metabolite synthesis and serves as a key virulence factor in D. haptotyla.

Efficient and Stable Air-Processed Organic Solar Cells Enabled by an Antioxidant Additive.

Current high-efficiency organic solar cells (OSCs) are generally fabricated in an inert atmosphere that limits their real-world scalable manufacturing, while the efficiencies of air-processed OSCs lag far behind. The impacts of ambient factors on solar cell fabrication remain unclear. In this work, the effects of ambient factors on cell fabrication are systematically investigated, and it is unveiled that the oxidation and doping of organic light absorbers are the dominant reasons causing cell degradation when fabricated in air. To address this issue, a new strategy for fabricating high-performance air-processed OSCs by introducing an antioxidant additive (4-bromophenylhydrazine, BPH) into the precursor solutions, is developed. BPH can effectively inhibit oxygen infiltration from the ambient to the photoactive layer and suppress trap formation caused by oxidation. Compared with conventional air-processed OSCs, this strategy remarkably increases the cell power conversion efficiency (PCE) from 16.7% to 19.3% (independently certified as 19.2%), representing the top value of air-processed OSCs. Furthermore, BPH significantly improves the operational stability of the cells in air by two times with a T80 lifetime of over 500 h. This study highlights the potential of using antioxidant additives to fabricate high-efficiency and stable OSCs in air, significantly promoting the industrialization of OSCs.

Icaritin inhibits the progression of urothelial cancer by suppressing PADI2-mediated neutrophil infiltration and neutrophil extracellular trap formation

Tumor relapse and metastasis are the major causes of mortality associated with urothelial cancer. In the tumor microenvironment, negative regulatory molecules and various immune cell subtypes suppress antitumor immunity. The inflammatory microenvironment, associated with neutrophils and neutrophil extracellular traps (NETs), promotes tumor metastasis. However, no drugs are currently available to specifically inhibit neutrophils and NETs. In this study, we first demonstrated that icaritin (ICT), a Chinese herbal remedy that is a first-line treatment for advanced and incurable hepatocellular carcinoma, reduces NETs caused by suicidal NETosis and prevents neutrophil infiltration in the tumor microenvironment. Mechanistically, ICT binds to and inhibits the expression of PADI2 in neutrophils, thereby suppressing PADI2-mediated histone citrullination. Moreover, ICT inhibits ROS generation, suppresses the MAPK signaling pathway, and inhibits NET-induced tumor metastasis. Simultaneously, ICT inhibits tumoral PADI2-mediated histone citrullination, which consequently suppresses the transcription of neutrophil-recruiting genes such as GM-CSF and IL-6. The downregulation of IL-6 expression, in turn, forms a regulatory feedback loop through the JAK2/STAT3/IL-6 axis. Through a retrospective study of clinical samples, we found a correlation between neutrophils, NETs, UCa prognosis, and immune evasion. Combining ICT with immune checkpoint inhibitors may have synergistic effects. In summary, our study demonstrated that ICT could be a novel inhibitor of NETs and a novel UCa treatment.

TSG-6 Is Involved in Fibrous Structural Remodeling after the Injection of Adipose-derived Stem Cells.

Although aesthetic treatments can rejuvenate the skin, they often cause specific forms of tissue damage. Unlike wounding, which typically results in fibrotic scar tissue, damage from aesthetic treatments induces a distinct histological rejuvenation. The mechanisms that drive this rejuvenation are not yet fully understood. Here, we were interested in cellular responses following aesthetic treatments injecting adipose-derived stem cells (ASCs) subcutaneously. Through investigation with an ex vivo experimental model, a key gene was identified that orchestrates fibrous structural changes and tissue remodeling. Using fresh human subcutaneous adipose tissue co-cultured with ASCs, the changes in the fibrous architecture of the tissue were sequentially mapped. The key regulatory genes involved in remodeling were identified using gene expression and computational analyses. We identified the regulatory elements that are crucial for tissue remodeling. Among those, we found that tumor necrosis factor-stimulated gene-6 (TSG-6) is a paracrine mediator essential for the collagen activity. It not only alleviates tissue inflammation but also promotes collagen replacement ex vivo. This is primarily achieved by inhibiting the formation of neutrophil extracellular traps, which are known to promote fibrosis. TSG-6 is a key factor modulating tissue inflammation. As our results demonstrate, after ASCs treatment, this factor directs skin healing away from fibrosis by reducing neutrophil extracellular trap formation in subcutaneous adipose tissue and promotes fibrous rejuvenation.

IL-8 Induces Neutrophil Extracellular Trap Formation in Severe Thermal Injury.

Neutrophil extracellular traps (NETs) have a dual role in the innate immune response to thermal injuries. NETs provide an early line of defence against infection. However, excessive NETosis can mediate the pathogenesis of immunothrombosis, disseminated intravascular coagulation (DIC) and multiple organ failure (MOF) in sepsis. Recent studies suggest that high interleukin-8 (IL-8) levels in intensive care unit (ICU) patients significantly contribute to excessive NET generation. This study aimed to determine whether IL-8 also mediates NET generation in patients with severe thermal injuries. IL-8 levels were measured in serum samples from thermally injured patients with ≥15% of the total body surface area (TBSA) and healthy controls (HC). Ex vivo NET generation was also investigated by treating isolated neutrophils with serum from thermal injured patients or normal serum with and without IL-8 and anti-IL-8 antibodies. IL-8 levels were significantly increased compared to HC on days 3 and 5 (p < 0.05) following thermal injury. IL-8 levels were also significantly increased at day 5 in septic versus non-septic patients (p < 0.001). IL-8 levels were also increased in patients who developed sepsis compared to HC at days 3, 5 and 7 (p < 0.001), day 10 (p < 0.05) and days 12 and 14 (p < 0.01). Serum containing either low, medium or high levels of IL-8 was shown to induce ex vivo NETosis in an IL-8-dependent manner. Furthermore, the inhibition of DNase activity in serum increased the NET-inducing activity of IL-8 in vitro by preventing NET degradation. IL-8 is a major contributor to NET formation in severe thermal injury and is increased in patients who develop sepsis. We confirmed that DNase is an important regulator of NET degradation but also a potential confounder within assays that measure serum-induced ex vivo NETosis.

Elevated Inflammation Associated with Markers of Neutrophil Function and Gastrointestinal Disruption in Pilot Study of Plasmodium fragile Co-Infection of ART-Treated SIVmac239+ Rhesus Macaques.

Human immunodeficiency virus (HIV) and malaria, caused by infection with Plasmodium spp., are endemic in similar geographical locations. As a result, there is high potential for HIV/Plasmodium co-infection, which increases the pathology of both diseases. However, the immunological mechanisms underlying the exacerbated disease pathology observed in co-infected individuals are poorly understood. Moreover, there is limited data available on the impact of Plasmodium co-infection on antiretroviral (ART)-treated HIV infection. Here, we used the rhesus macaque (RM) model to conduct a pilot study to establish a model of Plasmodium fragile co-infection during ART-treated simian immunodeficiency virus (SIV) infection, and to begin to characterize the immunopathogenic effect of co-infection in the context of ART. We observed that P. fragile co-infection resulted in parasitemia and anemia, as well as persistently detectable viral loads (VLs) and decreased absolute CD4+ T-cell counts despite daily ART treatment. Notably, P. fragile co-infection was associated with increased levels of inflammatory cytokines, including monocyte chemoattractant protein 1 (MCP-1). P. fragile co-infection was also associated with increased levels of neutrophil elastase, a plasma marker of neutrophil extracellular trap (NET) formation, but significant decreases in markers of neutrophil degranulation, potentially indicating a shift in the neutrophil functionality during co-infection. Finally, we characterized the levels of plasma markers of gastrointestinal (GI) barrier permeability and microbial translocation and observed significant correlations between indicators of GI dysfunction, clinical markers of SIV and Plasmodium infection, and neutrophil frequency and function. Taken together, these pilot data verify the utility of using the RM model to examine ART-treated SIV/P. fragile co-infection, and indicate that neutrophil-driven inflammation and GI dysfunction may underlie heightened SIV/P. fragile co-infection pathogenesis.

Oxidized Cholesterol Derivatives in Fraction B Prepared from Gulf Catfish (Arius bilineatus, Val.) Skin Regulate Calcium Response and Neutrophil Extracellular Traps (NETs) Formation.

In this study, we present in vitro actions of pure commercial preparations of oxidized and/or dehydrated metabolites of cholesterol (OS) identified in the lipid fraction of Fraction B (FB) prepared from a catfish skin preparation on calcium transients and on the formation of human neutrophil extracellular traps (NETs). These investigations are part of an ongoing effort to understand the important roles these compounds play as components of FB when FB is applied to accelerate the healing of wounds and the healing of highly infected non-healing diabetic foot ulcers, without the use of antibiotics. Our aim was to determine potential therapeutic interventions for various disease states. Our results reveal interesting findings, demonstrating specific actions of the individual compounds. Compounds 7α-hydroxy-cholesterol (S3), Cholestane-3,5,6-triol (S5), 5-cholesten-3β-ol-7-one (S8) and Cholesta-3,5 dien-7-one (S10) are inhibitory, while Cholesterol 5β,6β-epoxide (S4) and 5α-cholestane-3,6-dione (S11) activate the response for calcium influx in human neutrophils. A somewhat similar response is observed in dHL60 cell lines, where S3, S5, S7, S8, and cholesta-2,4-diene (S14) inhibit the calcium influx, although S4, S10, and S11 activate the response in this cell line. Furthermore, we observed a relationship between actions against NETosis and calcium transients. Interestingly, relative to the vehicle control, S3, Cholesta-3,5 diene (S9), and S14 appeared to significantly stimulate DNA release (NETosis), while S2, 7α-hydroxy-cholesterol (S6) and cholesta-3,5 dien-7-one (S10) caused lesser stimulation. We provide the IC50 activities for each compound tested in each assay. Calcium influx and NETs formation (NETosis) correlate with diseases exacerbation. These findings offer valuable insights into the potential therapeutic applications of individual OS for various diseases, highlighting their importance in future interventions.

Urea regulates soil nematode population by enhancing the nematode-trapping ability of nematode-trapping fungi

As the most abundant animal in the soil, nematodes are directly or indirectly involved in almost all soil ecological processes. Studying soil nematode population regulation is essential to understanding soil ecological processes. This study found urea combines nematode-trapping fungi to regulate the population of soil nematodes. In soil, compared with no urea, adding 0.2 mg/mL urea after applying Arthrobotrys oligospora and Dactylellina ellipsospora reduced the number of nematodes by 34.7% and 31.7%. Further, the mechanism of urea couple nematode-trapping fungi to regulate the nematode population was explored in the medium environment. The results showed that the addition of 0.2 mg/ml urea accelerated the trap formation of A. oligospora and D. ellipsosporas by 50% and 46.5%, and increased the yield of traps of A. oligospora and D. ellipsosporas by 39.5% and 40.6%, thus, the predatory efficiency of A. oligospora and D. ellipsospora on nematodes was increased by 34.2% and 32.7%. In conclusion, urea regulates the predation ability of A. oligospora and D. ellipsosporas to regulate the soil nematode population. This study deepens the understanding of the regulatory pathways of the soil nematodes but also provides a potential new strategy for harmful nematode bio-control.

Blocking the TRAIL-DR5 Pathway Reduces Cardiac Ischemia-Reperfusion Injury by Decreasing Neutrophil Infiltration and Neutrophil Extracellular Traps Formation.

Acute myocardial infarction (AMI) is a leading cause of mortality. Neutrophils penetrate injured heart tissue during AMI or ischemia-reperfusion (I/R) injury and produce inflammatory factors, chemokines, and extracellular traps that exacerbate heart injury. Inhibition of the TRAIL-DR5 pathway has been demonstrated to alleviate cardiac ischemia-reperfusion injury in a leukocyte-dependent manner. However, it remains unknown whether TRAIL-DR5 signaling is involved in regulating neutrophil extracellular traps (NETs) release. This study used various models to examine the effects of activating the TRAIL-DR5 pathway with soluble mouse TRAIL protein and inhibiting the TRAIL-DR5 signaling pathway using DR5 knockout mice or mDR5-Fc fusion protein on NETs formation and cardiac injury. The models used included a co-culture model involving bone marrow-derived neutrophils and primary cardiomyocytes and a model of myocardial I/R in mice. NETs formation is suppressed by TRAIL-DR5 signaling pathway inhibition, which can lessen cardiac I/R injury. This intervention reduces the release of adhesion molecules and chemokines, resulting in decreased neutrophil infiltration and inhibiting NETs production by downregulating PAD4 in neutrophils. This work clarifies how the TRAIL-DR5 signaling pathway regulates the neutrophil response during myocardial I/R damage, thereby providing a scientific basis for therapeutic intervention targeting the TRAIL-DR5 signaling pathway in myocardial infarction.

Novel Mouse Model of Myocardial Infarction, Plaque Rupture, and Stroke Shows Improved Survival With Myeloperoxidase Inhibition.

Thromboembolic events, including myocardial infarction (MI) or stroke, caused by the rupture or erosion of unstable atherosclerotic plaques are the leading cause of death worldwide. Although most mouse models of atherosclerosis develop lesions in the aorta and carotid arteries, they do not develop advanced coronary artery lesions. Moreover, they do not undergo spontaneous plaque rupture with MI and stroke or do so at such a low frequency that they are not viable experimental models to study late-stage thrombotic events or to identify novel therapeutic approaches for treating atherosclerotic disease. This has stymied the development of more effective therapeutic approaches for reducing these events beyond what has been achieved with aggressive lipid lowering. Here, we describe a diet-inducible mouse model that develops widespread advanced atherosclerosis in coronary, brachiocephalic, and carotid arteries with plaque rupture, MI, and stroke. We characterized a novel mouse model with a C-terminal mutation in the scavenger receptor class B, type 1 (SR-BI), combined with Ldlr knockout (designated SR-BI∆CT/∆CT/Ldlr-/-). Mice were fed Western diet (WD) for 26 weeks and analyzed for MI and stroke. Coronary, brachiocephalic, and carotid arteries were analyzed for atherosclerotic lesions and indices of plaque stability. To validate the utility of this model, SR-BI∆CT/∆CT/Ldlr-/- mice were treated with the drug candidate AZM198, which inhibits myeloperoxidase, an enzyme produced by activated neutrophils that predicts rupture of human atherosclerotic lesions. SR-BI∆CT/∆CT/Ldlr-/- mice show high (>80%) mortality rates after 26 weeks of WD feeding because of major adverse cardiovascular events, including spontaneous plaque rupture with MI and stroke. Moreover, WD-fed SR-BI∆CT/∆CT/Ldlr-/- mice displayed elevated circulating high-sensitivity cardiac troponin I and increased neutrophil extracellular trap formation within lesions compared with control mice. Treatment of WD-fed SR-BI∆CT/∆CT/Ldlr-/- mice with AZM198 showed remarkable benefits, including >90% improvement in survival and >60% decrease in the incidence of plaque rupture, MI, and stroke, in conjunction with decreased circulating high-sensitivity cardiac troponin I and reduced neutrophil extracellular trap formation within lesions. WD-fed SR-BI∆CT/∆CT/Ldlr-/- mice more closely replicate late-stage clinical events of advanced human atherosclerotic disease than previous models and can be used to identify and test potential new therapeutic agents to prevent major adverse cardiac events.

Bacterial endometritis-induced changes in the endometrial proteome in mares: Potential uterine biomarker for bacterial endometritis

Equine endometritis is one of the main causes of subfertility in the mare. Unraveling the molecular mechanisms involved in this condition and pinpointing proteins with biomarker potential could be crucial in both diagnosing and treating this condition. This study aimed to identify the endometritis-induced changes in the endometrial proteome in mares and to elucidate potential biological processes in which these proteins may be involved. Secondly, biomarkers related to bacterial endometritis (BE) in mares were identified. Uterine lavage fluid samples were collected from 28 mares (14 healthy: negative cytology and culture, and no clinical signs and 14 mares with endometritis: positive cytology and culture, in addition to clinical signs). Proteomic analysis was performed with a UHPLC-MS/MS system and bioinformatic analysis was carried out using Qlucore Omics Explorer. Gene Ontology enrichment and pathway analysis (PANTHER and KEGG) of the uterine proteome were performed to identify active biological pathways in enriched proteins from each group. Quantitative analysis revealed 38 proteins differentially abundant in endometritis mares when compared to healthy mares (fold changes >4.25, and q-value = 0.002). The proteins upregulated in the secretome of mares with BE were involved in biological processes related to the generation of energy and REDOX regulation and to the defense response to bacterium. A total of 24 biomarkers for BE were identified using the biomarker workbench algorithm. Some of the proteins identified were related to the innate immune system such as isoforms of histones H2A and H2B involvement in neutrophil extracellular trap (NET) formation, complement C3a, or gelsolin and profilin, two actin-binding proteins which are essential for dynamic remodeling of the actin cytoskeleton during cell migration. The other group of biomarkers were three known antimicrobial peptides (lysosome, equine cathelicidin 2 and myeloperoxidase (MPO)) and two uncharacterized proteins with a high homology with cathelicidin families.Findings in this study provide the first evidence that innate immune cells in the equine endometrium undergo reprogramming of metabolic pathways similar to the Warburg effect during activation. In addition, biomarkers of BE in uterine fluid of mares including the new proteins identified, as well as other antimicrobial peptides already known, offer future lines of research for alternative treatments to antibiotics.

Enterohepatic circulation of nanoplastics induced hyperplasia, epithelial-mesenchymal transition, and neutrophil extracellular traps in gallbladder

Increasing concerns surround nanoplastic’s health risks owing to global exposure emphasize clear understanding of their dynamic distribution and organ-specific molecular effects. We assessed the health risks associated with nanoplastics (100 nm) following oral ingestion. Using a fluorescent tracking system, we demonstrated their recyclability through gastrointestinal tract-liver-gallbladder axis, with specific accumulation in the gallbladder. Pathological alterations in the gallbladder and single-cell RNA sequencing data indicated that short-term (three weeks) exposure induces gallbladder epithelial hyperplasia, whereas long-term exposure (six weeks) induces progressive hyperplasia, epithelial-mesenchymal transition (EMT), and fibrosis. Nanoplastic exposure facilitates neutrophil extracellular trap (NET) formation. Various nanoplastics were identified in human gallbladder bile samples using scanning electron microscopy (SEM) and Raman spectroscopy. Consistently, epithelial hyperplasia and neutrophil infiltration were observed in the gallbladder tissues, alongside nanoplastic detection in the bile. Our findings offer insights into the understanding of the enterohepatic-biliary recycling route of nanoplastics and their potential toxic consequences.

Neutrophil Depletion Changes the N-Glycosylation Pattern of IgG in Experimental Murine Sepsis.

Sepsis is a life-threatening condition with a rising disease burden worldwide. It is a multifactorial disease and is defined as a dysregulated host response to infection. Neutrophils have been shown to be involved in the pathogenesis of sepsis by exacerbating inflammation. However, the exact effector mechanism of action still remains a mystery. Changes in the glycosylation pattern of the immunoglobulin G (IgG) Fc region are described for several diseases including meningococcal sepsis. In this study, we investigated the possible contribution of neutrophils and neutrophil implication, potentially related to degranulation or neutrophil extracellular trap (NET) formation in changing the IgG Fc N-glycosylation pattern in a murine sepsis model. We have measured the serum level of cytokines/chemokines and immunoglobulins, the serum activity of neutrophil elastase (NE), and analyzed the IgG Fc glycosylation pattern by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) and Lectin enzyme-linked immunosorbent assay (ELISA). We observed an increased activity of NE- and neutrophil-associated cytokines such as keratinocyte chemoattractant (KC) with the development of sepsis. Regarding the IgG Fc N-glycosylation, we observed an increase in fucosylation and α1,3-galactosylation and a decrease for sialyation. Interestingly, these changes were not uniform for all IgG subclasses. After depletion of neutrophils, we saw a change in the exposure of fucose and α2,6-linked sialic acid during the time course of our experimental sepsis model. In conclusion, neutrophils can influence changes in the IgG glycosylation pattern in experimental sepsis.

Polymerized Salicylic Acid Microparticles Reduce the Progression and Formation of Human Neutrophil Extracellular Traps (NET)s.

Neutrophils can contribute to inflammatory disease propagation via innate mechanisms intended for inflammation resolution. For example, neutrophil extracellular traps (NETs) are necessary for trapping pathogens but can contribute to clot formation and blood flow restriction, that is, ischemia. Currently, no therapeutics in the clinic directly target NETs despite the known involvement of NETs contributing to mortality and increased disease severity. Vascular-deployed particle-based therapeutics are a novel and robust alternative to traditional small-molecule drugs by enhancing drug delivery to cells of interest. This work designs a high-throughput assay to investigate the immunomodulatory behavior and functionality of salicylic acid-based polymer-based particle therapeutics against NETosis in human neutrophils. Briefly, this work finds that polymeric composition plays a role, and particle size can also influence rates of NETosis. Salicylate-based polymeric (Poly-SA) particles are found to functionally inhibit NETosis depending on the particle size and concentration exposed to neutrophils. This work demonstrates the high throughput method can help fast-track particle-based therapeutic optimization and design, more efficiently preparing this innovative therapeutics for the clinic.

A local drug delivery system prolongs graft survival by dampening T cell infiltration and neutrophil extracellular trap formation in vascularized composite allografts.

The standard treatment for preventing rejection in vascularized composite allotransplantation (VCA) currently relies on systemic immunosuppression, which exposes the host to well-known side effects. Locally administered immunosuppression strategies have shown promising results to bypass this hurdle. Nevertheless, their progress has been slow, partially attributed to a limited understanding of the essential mechanisms underlying graft rejection. Recent discoveries highlight the crucial involvement of innate immune components, such as neutrophil extracellular traps (NETs), in organ transplantation. Here we aimed to prolong graft survival through a tacrolimus-based drug delivery system and to understand the role of NETs in VCA graft rejection. To prevent off-target toxicity and promote graft survival, we tested a locally administered tacrolimus-loaded on-demand drug delivery system (TGMS-TAC) in a multiple MHC-mismatched porcine VCA model. Off-target toxicity was assessed in tissue and blood. Graft rejection was evaluated macroscopically while the complement system, T cells, neutrophils and NETs were analyzed in graft tissues by immunofluorescence and/or western blot. Plasmatic levels of inflammatory cytokines were measured using a Luminex magnetic-bead porcine panel, and NETs were measured in plasma and tissue using DNA-MPO ELISA. Lastly, to evaluate the effect of tacrolimus on NET formation, NETs were induced in-vitro in porcine and human peripheral neutrophils following incubation with tacrolimus. Repeated intra-graft administrations of TGMS-TAC minimized systemic toxicity and prolonged graft survival. Nevertheless, signs of rejection were observed at endpoint. Systemically, there were no increases in cytokine levels, complement anaphylatoxins, T-cell subpopulations, or neutrophils during rejection. Yet, tissue analysis showed local infiltration of T cells and neutrophils, together with neutrophil extracellular traps (NETs) in rejected grafts. Interestingly, intra-graft administration of tacrolimus contributed to a reduction in both T-cellular infiltration and NETs. In fact, in-vitro NETosis assessment showed a 62-84% reduction in NETs after stimulated neutrophils were treated with tacrolimus. Our data indicate that the proposed local delivery of immunosuppression avoids off-target toxicity while prolonging graft survival in a multiple MHC-mismatch VCA model. Furthermore, NETs are found to play a role in graft rejection and could therefore be a potential innovative therapeutic target.

Rapid high-throughput method for investigating physiological regulation of neutrophil extracellular trap formation

BackgroundNeutrophils, the most abundant white blood cells in humans, play pivotal roles in innate immunity, rapidly migrating to sites of infection and inflammation to phagocytose, neutralize, and eliminate invading pathogens. Neutrophil extracellular trap (NET) formation is increasingly recognized as an essential rapid innate immune response, but when dysregulated, it contributes to pathogenesis of sepsis and immunothrombotic disease. ObjectivesCurrent NETosis models are limited, routinely employing nonphysiological triggers that can bypass natural NET regulatory pathways. Models utilizing isolated neutrophils and immortalized cell lines do not reflect the complex biology underlying neutrophil activation and NETosis that occurs in whole blood. To our knowledge, we report the first human ex vivo model utilizing naturally occurring molecules to induce NETosis in whole blood. This approach could be used for drug screening and, importantly, inadvertent activators of NETosis. MethodsHere we describe a novel, high-throughput ex vivo whole blood–induced NETosis model using combinatorial pooling of native NETosis-inducing factors in a more biologically relevant Synthetic-Sepsis model. ResultsWe found different combinations of factors evoked distinct neutrophil responses in the rate of NET generation and/or magnitude of NETosis. Despite interdonor variability, similar sets of proinflammatory molecules induced consistent responses across donors. We found that at least 3 biological triggers were necessary to induce NETosis in our system including either tumor necrosis factor-α or lymphotoxin-α. ConclusionThese findings emphasize the importance of investigating neutrophil physiology in a biologically relevant context to enable a better understanding of disease pathology, risk factors, and therapeutic targets, potentially providing novel strategies for disease intervention and treatment.

Identification of potential biomarkers for sepsis based on neutrophil extracellular trap-related genes

Sepsis is a highly lethal disease that poses a serious threat to human health. Increasing evidence indicates that neutrophil extracellular traps (NETs) are key factors in the pathological progression of sepsis. This study aims to screen potential biomarkers for sepsis and delve into their regulatory function in the pathogenesis. We downloaded 6 microarray datasets from the Gene Expression Omnibus (GEO) database, with 4 as the training sets and 2 as the validation sets. NETs-related genes (NRGs) were obtained from relevant literature. Differential expression analysis was performed on four training sets separately. We intersected differentially expressed genes (DEGs) from the four training sets and NRGs, finally resulting in 19 NETs-related sepsis genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) unearthed that NETs-related sepsis genes were majorly abundant in functions and pathways such as defense response to bacterium and Neutrophil extracellular trap formation. Using the PPI network, the MCC algorithm, and the MCODE algorithm in the CytoHubba plugin, 7 sepsis hub genes (ELANE, TLR4, MPO, PADI4, CTSG, MMP9, S100A12) were identified. ROC curve for each Hub gene in the training and validation sets were plotted, which revealed that the Area Under Curve (AUC) values are all greater than 0.6, indicating good classification ability. A total of 349 miRNAs targeting Hub genes were predicted in the mirDIP database, and 620 lncRNAs targeting miRNAs were predicted in the ENCORI database. The ceRNA regulatory network was constructed using Cytoscape software. Finally, we employed the cMAP database to predict small molecular complexes as potentially effective drugs for the treatment of sepsis, such as chloroquine, harpagoside, and PD-123319. In conclusion, this project successfully identified 7 core genes, which may serve as promising candidates for novel sepsis biomarkers. Meanwhile, we constructed a related ceRNA network and predicted potential targeted drugs, providing potential therapeutic targets and treatment strategies for sepsis patients.

A novel lactylation-related gene signature for effectively distinguishing and predicting the prognosis of ovarian cancer.

Lactylation has been found to regulate several types of biological processes in cancer. However, there is limited research on lactylation-related genes in predicting the prognosis of ovarian cancer (OC). This study aimed to explore the functional roles of lactylation-related genes in OC. Based on TCGA database, we obtained RNA sequencing data and clinical characteristics of patients with OC. Fourteen lactylation-related genes were screened for bioinformatic analysis in OC. Tumor classification of OC was constructed via a consistency cluster analysis. We examined the prognosis, immune-cell infiltration, and immunotherapy in relation to a lactylation-related model for OC. A total of 707 prognostic genes and 14 key lactylation-related genes (SNRPA1, MPHOSPH6, POLDIP3, RB1, AHNAK, MAGOHB, CALM1, EP300, HDAC1, HDAC2, HDAC3, SIRT1, SIRT2, and SIRT3) were identified in TCGA-OC patients. Based on 14 genes involved in lactylation, TCGA-OC patients were split into low-risk (G1) and high-risk (G2) groups. Downregulated differentially expressed genes (DEGs) in the low-risk G1 group were associated with thermogenesis, oxidative phosphorylation, neutrophil extracellular trap formation, and interleukin 17 (IL-17) signaling pathway, whereas upregulated DEGs were associated with proteoglycans in cancer, focal adhesion, Wnt signaling pathway, extracellular matrix (ECM)-receptor interaction, and the adherens junction. The immune activity of the low-risk G1 group was lower than that of the high-risk G2 group. Gemcitabine, bleomycin, and doxorubicin had lower half-maximal inhibitory concentration (IC50) values in the high-risk G2 patients with OC, while cisplatin and paclitaxel had higher IC50 values compared to the low-risk G1 patients. The prognosis of patients with OC was also predicted with the help of an eight-lactylation-related gene prognostic model, comprising SNRPA1, MPHOSPH6, POLDIP3, RB1, HDAC1, CALM1, HDAC2, and SIRT2. The lactylation-related genes are closely related to tumor classification and immunity in patients with OC. There was good prognostic predictive performance for OC based on a lactylation-related signature. Our findings may offer new insights into the diagnosis and treatment of OC.