The present review describes the mechanisms of NETosis and its role in myocardial ischemia-reperfusion injury (MIRI), focusing on the release of neutrophil extracellular traps (NETs) by activated neutrophils. NETs, composed of depolymerized chromatin and granule proteins, are crucial for pathogen entrapment, infection control and immune regulation. However, NET formation, linked to neutrophil death (NETosis), exacerbates MIRI by promoting inflammation and tissue damage. To address therapeutic strategies for NETosis in MIRI, several potential clinically significant approaches were explored, including peptidylarginine deaminase 4 inhibition, DNase therapy, antioxidants, inflammation modulation, and antithrombotic treatments, which not only provide novel diagnostic biomarkers and therapeutic targets in MIRI, but are also expected to improve patient prognosis and advance the development of personalised medicine.

The DNA component of neutrophil extracellular traps (NET-DNA) is associated with cancer metastasis and chemotherapy resistance. However, recent studies have suggested that NET-DNA contributes to the activation of dendritic cells (DCs) and promotes the innate immune response to anticancer immunity. Therefore, exploring therapeutic approaches to inhibit NET-mediated tumor progression while maintaining antitumor immunity is essential. Our groups recently identified CCDC25 as a specific NET-DNA sensor on the cytoplasmic membrane of cancer cells that promotes cancer metastasis. In this study, we performed small-molecule compound screening and revealed that mitoxantrone (MTO) could block the interaction between NET-DNA and CCDC25. Molecular docking results indicated that MTO competed with NET-DNA by binding with the amino acid residues Tyr24 (Y24), Glu25 (E25), and Asp28 (D28) of the crystal structure of CCDC25. More importantly, we conjugated MTO with palmitoleic acids such as di-Pal-MTO to increase its residence time on the cytoplasmic membrane, which increased its inhibitory efficiency and decreased its cytotoxicity. In addition, di-Pal-MTO markedly inhibited the RAC1-CDC42 cascade to alleviate the NET-induced cytoskeleton arrangement and chemotactic migration of cancer cells. In multiple mouse models, di-Pal-MTO can suppress breast cancer metastasis and have synergistic effects with chemotherapeutics. Moreover, di-Pal-MTO promotes NET-DNA-dependent DC activation, leading to the subsequent expression of various chemokines that facilitate the infiltration of CD8+ T cells. Overall, we successfully identified a small molecule inhibitor, di-Pal-MTO, with dual effects on tumor repression and the antitumor immune response, which provides a novel therapeutic strategy against breast cancer.

Background: Sepsis is a life-threatening clinical condition often seen in intensive care units, leading to multi-organ dysfunction. Myocardial injury is a prevalent complication, significantly increasing mortality among sepsis patients. Although heparin is used in sepsis management, its specific effects on myocardial injury and the role of neutrophil extracellular traps (NETs) in this context remain insufficiently understood. Aim: This study investigates the role of unfractionated heparin (UFH) combined with DNase I in reducing myocardial injury in a septic mouse model. Methods: A cecal ligation and puncture (CLP)-induced sepsis model was established in C57BL/6 mice to study myocardial injury. The experimental groups included treatments with UFH, UFH with DNase I, and NETs introduction. Myocardial injury was assessed using hematoxylin and eosin staining, enzyme linked immunosorbent assay for injury markers (creatine kinase MB [CK-MB] and lactate dehydrogenase [LDH]), and Western blotting for inflammatory proteins (TNF-α and IL-6). Differential proteomic analysis using data independent acquisition mass spectrometry and pathway enrichment analysis (Gene Ontology and Kyoto Encyclopedia of Genes and Genomes) were conducted to identify molecular pathways and key proteins affected by the treatments. Results: Single UFH treatment increased the formation of NETs, upregulated TNF-α and IL-6, and increased CK-MB and LDH, worsening myocardial injury. The combination of UFH and DNase I significantly reduced myocardial injury, suppressing NETs formation and inflammation. Proteomic analysis identified crucial pathways related to NETs, metabolism, and complement and coagulation cascades, with proteins Ccn1 and Tagln highlighted as potential therapeutic targets. Conclusion: UFH combined with DNase I effectively alleviates myocardial injury in septic mice by modulating NETs formation and associated inflammatory processes. This study may provide new insights and options for the early use of heparin in the treatment of septic patients, particularly in cases with a higher risk of myocardial injury.

Mechanisms controlling innate immune responses and coagulation are interdependent, evolutionarily entangled and make a complex network to form immuno-thrombosis axis which is an integral part of host-defence response. During infections, immunothrombosis generates intravascular scaffold enabling recognition, trap and destruction of pathogens facilitating tissue integrity. However, the accompanying dysregulation fosters into pathologies associated with thrombosis and regulates severity, morbidity and mortality in infections. Several extrinsic and intrinsic factors such as (epi)genetic mechanisms, age, metabolism and lifestyle regulate immunothrombosis during infections. Mounting evidence demonstrates that homocysteine, a metabolic intermediate of methionine synthesis pathway activate cells participating in immuno-thrombosis such as neutrophils, platelets, monocytes and endothelial cells. Interestingly, multiple infections are significantly associated with perturbed homocysteine metabolism. In the present review, we describe mechanistic insights into how homocysteine drives immuno-thrombotic crosstalk that generate a vicious cycle of inflammation and coagulation that fuels organ failure during infections with an emphasis on sepsis, COVID-19, and other infectious diseases caused by parasites, viral, and bacterial pathogens. Subsequently, we discuss therapeutic strategies targeting homocysteine metabolism that may improve clinical outcomes in infections.

Neutrophil extrusion of neutrophil extracellular traps (NETs) in a process called NETosis provides immune defense against extracellular bacteria. It has been observed that bacteria are capable of activating neutrophils to release NETs that subsequently kill them or at least prevent their local spread within host tissue. However, existing studies have mainly focused on the isolated function of NETs, with less attention given to their anti-bacterial mechanisms through interactions with other immune cell populations. The net effect of these complex intercellular interactions, which may act additively, synergistically, or antagonistically, is a critical determinant in the outcomes of host-pathogen interactions. This review summarizes the mechanisms underlying classic NET formation and their crosstalk with the immune system, offering novel insights aimed at balancing the anti-microbial function with their potential inflammatory risks.

Neutrophil extracellular traps (NETs), web-like complex structures secreted by neutrophils, have emerged as key players in the modulation of immune responses and the immunopathogenesis of immune disorders. Initially described for their antimicrobial function, NETs now play a part in the fundamental processes of cancer biology, including cancer initiation, metastatic dissemination, and immune evasion strategies. NETs hijack anti-tumor immunity by entrapping circulating cancer cells, fostering the growth of tumors, and reorganizing the tumor microenvironment such that it is pro-malignancy. Emerging evidence emphasizes the role of NETosis coupled with non-coding RNAs-long non-coding RNAs (lncRNAs) and microRNAs (miRNAs)-as key regulators of gene expression and controllers of processes vital for cancer growth, such as immune response and programmed cell death processes like apoptosis, necroptosis, pyroptosis, and ferroptosis. Aberrantly expressed non-coding RNAs have been attributed to immune dysregulation and excessive NET production, promoting tumor growth. NETs are also associated with a myriad of pathological conditions, such as autoimmune disorders, cystic fibrosis, sepsis, and thrombotic disorders. New therapeutic approaches-such as DNase therapy and PAD4 inhibitors-target NET production and their degradation to modify immune function and the efficiency of immunotherapies. Further clarification of the intricate interactions of NETosis, lncRNAs, and miRNAs has the potential to establish new strategies for the suppression of the growth of tumors and preventing immune evasion. This review seeks to elucidate the interactions between NETosis and the regulatory networks involving non-coding RNAs that significantly contribute to the immunopathogenesis of cancer.

Neutrophils are important components of the immune system and play a key role in defending against pathogenic infections and responding to inflammatory cues, including cancer. Their dysregulation indicates potential disease risk factors. However, their functional importance in disease progression has often been underestimated due to their short half-life, especially as there is limited information on the role of intratumoral neutrophils. Recent studies on their prominent role in cancer have led to a paradigm shift in our understanding of the functional diversity of neutrophils. These studies highlight that neutrophils have emerged as key components of the tumor microenvironment, where they can play a dual role in promoting and suppressing cancer. Moreover, several approaches to therapeutically target neutrophils have emerged, and clinical trials are investigating their efficacy. In this review, we discussed the involvement of neutrophils in cancer initiation and progression. We summarized recent advances in therapeutic strategies targeting neutrophils and, most importantly, suggested future research directions that could facilitate the manipulation of neutrophils for therapeutic purposes in cancer patients.

Beta-lactams (BLs) are the predominant cause of immediate allergic reactions to drugs. Immediate hypersensitivity reactions (IHR) with positive provocation tests and negative skin and in vitro tests have undetermined mechanisms. We evaluated whether biomarkers of IgG-dependent neutrophil activation could help to assess this subgroup of BL IHRs.

We evaluated biomarkers of neutrophil activation and neutrophil extracellular traps (NETs) in the serum of 26 BL IHR patients presenting with a positive provocation test, negative skin and serum specific IgE, and positive specific sIgG, and 8 perioperative BL IHR cases with positive skin tests and negative sIgE compared to 19 non-allergic matched controls.

We observed increased levels of DNase activity, neutrophil elastase (NE), myeloperoxidase (MPO)-DNA, IL8 and decreased IL4 and IL13 in patients, compared to matched controls, in the first 15 min of IHRs. DNAse activity, NE and MPO-DNA were maintained at high levels 2 h later (T0 + 2), while cell-free DNA and CXCR2 decreased significantly. IgG-related activation of neutrophils was suggested by significant correlations between NE, IL8 and CXCR2 axis and a single cluster associating BL sIgG antibodies and NE at T0 + 2, in principal factor analysis of all biomarkers.

Biomarkers of neutrophil activation and NETs were increased in BL IHRs with negative skin tests, positive sIgG and negative serum sIgE, and positive provocation test. We propose DNAse activity and NE as biomarkers for the biological assessment of BL IHRs and provocation tests and to consider IgG-related neutrophil activation as one of the mechanisms involved in BL IHRs with undetermined cause.

Neutrophil extracellular traps (NETs) are web-like structures released by neutrophils to trap and kill microbes. While NETs are crucial in host defense, excessive formation can lead to autoimmune diseases. Currently, no specific drugs target NETs directly; however, some medications can indirectly modulate their formation. The secretory leukocyte protease inhibitor (SLPI) is a small protein that inhibits the activity of neutrophil elastase (NE), an enzyme essential for NETs formation. By inhibiting NE activity, SLPI prevents the chromatin from decondensing, which is necessary for NETs to form; this suggests that SLPI may protect by preventing excessive NETs development. However, evidence indicates that NE can inactivate SLPI by cleaving its N-terminus. This action can create a protease/antiprotease imbalance, potentially leading to detrimental consequences for the host. In this study, we produced a recombinant variant of SLPI (SLPI-S15G-A16G: rSLPIv) using recombinant DNA technology, expressed in Escherichia coli SHuffle T7. The protein was tagged with the small metal-binding protein (SmbP) to facilitate its expression and purification through immobilized metal-affinity chromatography. Our results demonstrated that rSLPIv exhibited immunomodulatory activity at a concentration of 10 nM in neutrophils that had been prestimulated with PMA. It reduced NETs formation by 30 % and maintained this effect for up to 6 h. Confocal microscopy confirmed these findings, revealing a rSLPIv-dependent reduction in neutrophil nuclear expansion. Thus, rSLPIv shows significant suppressive activity on NETs formation at low concentrations, making it a potential candidate as an immunotherapeutic agent.

Following myocardial infarction (MI), patients with type 2 diabetes mellitus (T2DM) have poorer prognosis which may be linked to increased susceptibility of coronary microvessels to injury. Interleukin-36 (IL-36) may mediate this injury but its role in the microcirculation of the chronically hyperglycaemic injured heart is unknown. Intravital and laser speckle imaging of the anaesthetised mouse beating heart evaluated the impact of a 16-week high fat diet (HFD)-induced hyperglycaemia ± myocardial ischaemia-reperfusion injury (IR) injury on coronary microvessels. Neutrophil/platelet recruitment, neutrophil extracellular trap formation, cellular necrosis, vascular leakage, vascular tonal changes, functional capillary density, overall ventricular perfusion and levels of circulating inflammatory cytokines were assessed alongside the vasculoprotective ability of an IL-36 receptor antagonist (IL-36Ra). Whether heightened microvessel damage in injured HFD mice was permanent or reversible was investigated after normalising hyperglycaemia through diet reversal (DR). Microcirculatory events assessed were perturbed basally in HFD mice and further after injury. IL-36Ra mitigated these effects and improved infarct size. DR was also beneficial, decreasing neutrophil recruitment to levels below those seen in untreated mice. Mechanistically, benefits of both IL-36Ra and DR could be explained by decreased endothelial oxidative stress and VCAM-1 expression and possibly by raised levels of IL-4/IL-13. Basal changes in chronically hyperglycaemic coronary microvessels that are heightened in the aftermath of reperfusion may explain the poorer outcomes in MI patients with T2DM. These findings are the first to highlight the specific benefits of IL-36 inhibition and reversing hyperglycaemia through dietary modification on the coronary microcirculation in a preclinical model of T2DM.

Urinary tract infection (UTI), a common clinical infectious disease, is marked by high incidence and frequent recurrence. Recurrent UTIs can cause severe complications, negatively affecting health. The emergence and spread of drug-resistant bacteria present significant challenges to UTI treatment. This article systematically reviews the key immune mechanisms in the body's defense against UTI pathogens. It discusses various immune response components, such as the urinary tract mucosal epithelium, neutrophils, macrophages, dendritic cells, mast cells, innate lymphocytes, T cells, and B cells, with the aim of providing insights for future UTI research.

Neutrophil extracellular traps (NETs) are web-like, bactericidal structures produced by neutrophils and are composed principally of extracellular DNA, histones, elastase, and myeloperoxidase, among other components. NET formation is an innate immune response that is beneficial for pathogen killing and clearance. However, excessive NET formation and clearance defects can lead to inflammation and induce damage to host organs. NETs are also implicated in the development of noninfectious inflammatory disorders, such as liver injury in chronic liver diseases. The liver parenchyma contains hepatocytes, liver sinusoidal endothelial cells, Kupffer cells, and hepatic stellate cells. Each of these cells possesses unique structures and functions, and their interactions with NETs result in pathophysiological changes contributing to liver injury. This review updates the findings related to the modes of action and molecular mechanisms by which NETs modulate the pathophysiology of various hepatic cells and potentiate liver injury. The article also reviews the roles of NETs in hepatic ischemia reperfusion injury, hepatocellular carcinoma pathogenesis, and cancer metastasis. Last, we examine data to determine whether NETs induce crosstalk among various hepatic cells during liver injury and to identify future research directions.

In the mammalian cardiovascular system, endothelial glycocalyx is a gel-like layer that covers the luminal surface of endothelial cells (ECs) and plays crucial roles in vascular homeostasis, permeability and leukocyte adhesion. Degradation of this structure occurs early in sepsis and becomes accordingly dysfunctional. In severe cases, it is not self-regulated by the organism. However, the relationship between the glycocalyx and the occurrence and development of sepsis remains poorly understood. One possibility is that thinned glycocalyx promotes leukocyte recognition and adhesion, thereby facilitating the elimination of pathogens from infected areas. This may represent a protective mechanism developed by the organism during through evolutionary processes. However, if the damage persists and disrupts the dynamic balance of the microcirculation, interstitial edema or organ failure can occur. Thus, we asked the questions, what is the precise composition and structure of the glycocalyx? How is it degraded? What animal models are available to study the relationship between the glycocalyx and sepsis? What glycocalyx biomarkers are found in the blood of patients with sepsis? To determine whether sepsis can be treated by interfering with the glycocalyx, this study provides a systematic summary and discussion of the latest progress in addressing these questions.

Neutrophil extracellular traps (NETs) are associated with diseases linked to aberrant coagulation. The blood clotting cascade involves a series of proteases, some of which induce NET formation via a yet unknown mechanism. We hypothesized that this formation involves signaling via a factor Xa (FXa) activation of the protease-activated receptor 2 (PAR2). Our findings revealed that NETs can be triggered in vitro by enzymatically active proteases and PAR2 agonists. Intravital microscopy of the liver vasculature revealed that both FXa infusion and activation of endogenous FX promoted NET formation, effects that were prevented by the FXa inhibitor, apixaban. Unlike classical NETs, these protease-induced NETs lacked bactericidal activity and their proteomic signature indicates their role in inflammatory disorders, including autoimmune diseases and carcinogenesis. Our findings suggest a novel mechanism of NET formation under aseptic conditions, potentially contributing to a self-amplifying clotting and NET formation cycle. This mechanism may underlie the pathogenesis of disseminated intravascular coagulation and other aseptic conditions.

Staphylococcus aureus (S. aureus) is a Gram-positive bacterium of significant clinical importance, known for its versatility and ability to cause a wide array of infections, such as osteoarticular, pulmonary, cardiovascular, device-related, and hospital-acquired infections. This review describes the most recent evidence of the pathogenic potential of S. aureus, which is commonly part of the human microbiota but can lead to severe infections. The prevalence of pathogenic S. aureus in hospital and community settings contributes to substantial morbidity and mortality, particularly in individuals with compromised immune systems. The immunopathogenesis of S. aureus infections involves intricate interactions with the host immune and non-immune cells, characterized by various virulence factors that facilitate adherence, invasion, and evasion of the host's defenses. This review highlights the complexity of S. aureus infections, ranging from mild to life-threatening conditions, and underscores the growing public health concern posed by multidrug-resistant strains, including methicillin-resistant S. aureus (MRSA). This article aims to provide an updated perspective on S. aureus-related infections, highlighting the main diseases linked to this pathogen, how the different cell types, virulence factors, and signaling molecules are involved in the immunopathogenesis, and the future perspectives to overcome the current challenges to treat the affected individuals.

Neutrophils are very important cells of the immune system, and every year, new nuances of their functional activity in the body and participation in various pathological processes are discovered [...].

Skin infiltration by neutrophils is a hallmark of the chronic inflammatory skin disease psoriasis, yet the mechanisms underlying neutrophil recruitment and positioning in chronically inflamed skin remain poorly understood. In this study, we demonstrate the significant impact of a total genetic deficiency of secretory leukocyte protease inhibitor (SLPI) on neutrophil migration in mouse skin. Without SLPI, neutrophils displayed an unconventional migratory pattern, characterized by altered interactions with vessel walls and reduced efficiency in extravasating from blood vessels into skin tissue during the early stages of experimental psoriasis. This was associated with changes in tissue motility, positioning neutrophils farther from the skin entry vessels and closer to the skin surface. Neutrophil diapedesis was partially dependent on SLPI within the neutrophils themselves. The impact of SLPI on neutrophil movement was further supported by the increased migration of human neutrophils in the presence of neutrophil-penetrant recombinant SLPI. Additionally, our data suggest that neutrophils with varying capacities for vessel wall interaction are released from the bone marrow into circulation in an SLPI-dependent manner. These findings establish a role for SLPI in regulating the spatiotemporal infiltration of neutrophils into the skin in psoriasis, highlighting its relevance to psoriasis pathophysiology.

Neutrophil extracellular traps (NETs) participate in both host defense and the pathogenesis of various diseases, such as infections, thrombosis, and tumors. While they help capture and eliminate pathogens, NETs' excessive or dysregulated formation can lead to tissue damage and disease progression. Therapeutic strategies targeting NET modulation have shown potential, but challenges remain, particularly in achieving precise drug delivery and maintaining drug stability. Nanoparticle (NP)-based drug delivery systems offer innovative solutions for overcoming the limitations of conventional therapies. This review explores the biological mechanisms of NET formation, their interactions with NPs, and the therapeutic applications of NP-based drug delivery systems for modulating NETs. We discuss how NPs can be designed to either promote or inhibit NET formation and provide a comprehensive analysis of their potential in treating NET-related diseases. Additionally, we address the current challenges and future prospects for NP-based therapies in NET research, aiming to bridge the gap between nanotechnology and NET modulation for the development of novel therapeutic approaches.

The process of NETosis is observed in a range of inflammatory conditions. Progesterone (P4) has been shown to alleviate inflammation caused by viral infections such as influenza and SARS-CoV-2. However, the precise molecular mechanisms responsible for this effect are not yet fully understood. Therefore, the present investigation aims to explore whether P4 can exert its anti-inflammatory properties by inhibiting NETosis and the related molecular pathways.

Airway inflammation caused by rhinovirus serotype-1b (RV-1b) was induced in male BALB/c mice. The inflammation was assessed through histological examination and calculation of inflammatory cells present in the bronchoalveolar lavage fluid. Flow cytometry was used to analyze the inflammatory cells and NETotic neutrophils. Western blotting analysis was conducted to detect proteins associated with NETosis, inflammasome activation, and signaling. Furthermore, confocal microscopy was utilized to observe neutrophil extracellular trap (NET) structures in vivo tissues and in vitro neutrophils, neutrophil infiltration, and inflammasome formation.

The administration of P4 proved to be an effective treatment for reducing airway inflammation and the production of NETs caused by RV-1b infection. The infection triggered the activation of NLRP3 inflammasomes in neutrophils, which led to the maturation of IL-1β and subsequent activation of both the NF-κB and p38 signaling pathways. The activation of NF-κB signaling resulted in the secretion of downstream chemokines CCL3 and IL-6, which led to an increase in neutrophil infiltration into the lung airways. Moreover, the activation of p38 signaling led to the generation of reactive oxygen species, resulting in NETosis. However, the administration of P4 inhibited the activation of the NLRP3 inflammasome, which subsequently led to the deactivation of both the IL-1β-NF-κB and IL-1β-p38 axes. As a result, there was a reduction in neutrophil infiltration and NETosis. Furthermore, TGF-β-activated kinase 1 (TAK1) was identified as an intermediary enzyme. P4 inhibits both the NF-κB and IL-1β-p38 pathways by suppressing the activity of TAK1.

The capacity of P4 to mitigate rhinovirus-induced airway inflammation is attributed to its ability to impede the infiltration of neutrophils and NETosis. As inflammation mediated by NETosis is widespread in diverse disorders, our findings propose that P4 could potentially function as a universal therapeutic agent in the management of such ailments.

Although surgical resection of tumor mass remains the mainstay of curative therapeutic management for solid tumors, accumulating studies suggest that these procedures promote tumor recurrence and metastasis. Regarded as the first immune cells to fight against infectious or inflammatory insults from surgery, neutrophils along with their ability of neutrophil extracellular traps (NETs) production has attracted much attention. A growing body of evidence suggests that NETs promote cancer metastasis by stimulating various stages, including local invasion, colonization, and growth. Therefore, we discussed the mechanism of NETosis induced by surgical stress and tumor cells, and the contribution of NETs on tumor metastasis: aid in the tumor cell migration and proliferation, evasion of immune surveillance, circulating tumor cell adhesion and establishment of a metastatic niche. Lastly, we summarized existing NET-targeting interventions, offering recent insights into potential targets for clinical intervention.

During intestinal and liver invasion by the protozoan parasite Entamoeba histolytica, extensive tissue destruction linked to large neutrophil infiltrates is observed. It has been proposed that microbicidal components of neutrophils are responsible for the damage, however, the mechanism by which they are released and act in the extracellular space remains unknown. In previous studies, we have shown that E. histolytica trophozoites induce NET formation, leading to the release of neutrophil granule content into extruded DNA. In this work, we evaluate the possible participation of NETs in the development of amoeba-associated pathology and analyze the contribution of anti-microbial components of the associated granules. E. histolytica-induced NETs were isolated and their effect on the viability and integrity of HCT 116 colonic and Hep G2 liver cultures were evaluated. The results showed that simple incubation of cell monolayers with purified NETs for 24 h resulted in cell detachment and death in a dose-dependent manner. The effect was thermolabile and correlated with the amount of DNA and protein present in NETs. Pretreatment of NETs with specific inhibitors of some microbicidal components suggested that serine proteases, are mostly responsible for the damage caused by NETs on HCT 116 cells, while the MPO activity was the most related to Hep G2 cells damage. Our study also points to a very important role of DNA as a scaffold for the activity of these proteins. We show evidence of the development of NETs in amoebic liver abscesses in hamsters as a preamble to evaluate their participation in tissue damage. In conclusion, these studies demonstrate that amoebic-induced NETs have potent cytotoxic effects on target cells and, therefore, may be responsible for the intense damage associated with tissue invasion by this parasite.

Ischemic heart disease (IHD) is a prevalent cardiovascular condition that remains the primary cause of death due to its adverse ventricular remodelling and pathological changes in end-stage heart failure. As a complex pathologic condition, it involves intricate regulatory processes at the cellular and molecular levels. The immune system and cardiovascular system are closely interconnected, with immune cells playing a crucial role in maintaining cardiac health and influencing disease progression. Consequently, alterations in the cardiac microenvironment are influenced and controlled by various immune cells, such as macrophages, neutrophils, dendritic cells, eosinophils, and T-lymphocytes, along with the cytokines they produce. Furthermore, studies have revealed that Gata6+ pericardial cavity macrophages play a key role in regulating immune cell migration and subsequent myocardial tissue repair post IHD onset. This review outlines the role of immune cells in orchestrating inflammatory responses and facilitating myocardial repair following IHD, considering both macro and micro views. It also discusses innovative immune cell-based therapeutic strategies, offering new insights for further research on the pathophysiology of ischemic heart disease and immune cell-targeted therapy for IHD.

Traditional methods for treating diabetic wounds are limited in effectiveness because of their long healing times, the risk of immune rejection, and susceptibility to infection. Suppressing neutrophil extracellular traps (NETs) is an effective strategy for reducing persistent inflammation in diabetic wounds. Although disulfiram (DSF) can inhibit the significant increase of NETs in diabetic wounds, oral DSF suffers from rapid and harmful metabolism in the liver. To address these challenges, we developed a nanomedicine formulation in which DSF was incorporated into the hydrogel.

In this study, we developed a DSF-laden sodium alginate hydrogel wound dressing, DEP@SA, and characterized its composition, properties, and performance. We examined the effects of DEP@SA on inflammatory phase-related markers such as NETs and their pathway proteins, inflammatory factors, and macrophage phenotypes in a high-glucose environment in vivo and in vitro. In addition, the effects of DEP@SA on tissue regenerative capacity such as epidermal proliferative migration and angiogenesis, were also assessed.

The results showed that by utilizing extracellular vesicles as a drug delivery system, we effectively mitigated the degradation of DSF via direct contact with aqueous solutions and ensured the stability of DSF@SA, which could then be applied to diabetic wounds. The inflammatory phase-related indicators revealed that DSF@SA effectively reduced inflammation levels, decreased NETs formation, suppressed the Caspase-1/GSDMD pathway in neutrophils, and promoted the polarization of M2 macrophages. Moreover, the hydrogel accelerated wound healing by promoting angiogenesis and re-epithelialization, thereby shortening the diabetic wound healing time.

This study confirmed that the DSF@SA composite dressing has the potential to enhance diabetic wound repair and offers a novel approach for drug reutilization.

Acute ischemic stroke represents a critical, life-threatening condition affecting the central nervous system. Intravenous thrombolysis with tissue plasminogen activator (tPA) remains a cornerstone for achieving vascular recanalization in such patients; however, its therapeutic utility is limited, with only approximately 10% of patients benefiting due to the narrow therapeutic window and significant risk of hemorrhagic transformation. Enhancing the efficacy of tPA thrombolysis is therefore imperative. Neutrophils have been identified as key modulators of thrombolytic outcomes, interacting with tPA post-stroke to influence treatment effectiveness. The binding of tPA to low-density lipoprotein receptor-related protein 1 (LRP-1) on neutrophil surfaces induces degranulation and formation of neutrophil extracellular traps (NETs). Conversely, neutrophils impede the thrombolytic action of tPA by obstructing its interaction with fibrin and activating platelets. These findings suggest that targeting neutrophils may hold promise for improving thrombolysis outcomes. This review explores the role of neutrophils in tPA-mediated thrombolysis following acute ischemic stroke, examines neutrophil-associated biomarkers, and outlines potential strategies for enhancing tPA efficacy.

The acute inflammatory response is an inherent protective mechanism, its unsuccessful resolution can contribute to disease pathogenesis and potentially lead to death. Innate immune cells are the first line of host defenders and play a substantial role in inflammation initiation, amplification, resolution, or subsequent disease progression. As the resolution of inflammation is an active and highly regulated process, modulating innate immune cells, including neutrophils, monocytes and macrophages, and endothelial cells, and their interactions offer opportunities to control excessive inflammation. Nanobiomaterials have shown superior therapeutic potential in inflammation-related diseases by manipulating inflammatory responses because nanobiomaterials can target and interact with innate immune cells. Versatile nanobiomaterials can be designed for targeted modulation of specific innate immune responses. Nanopro-resolving medicines have been prepared both with pro-resolving lipid mediators and peptides each demonstrated to active resolution of inflammation in animal disease models. Here, we review innovative nanobiomaterials for modulating innate immunity and alleviating inflammation. We summarise the strategies converging the design of nanobiomaterials and the nano-bio interaction in modulating innate immune profiles and propelling the advancement of nanobiomaterials for inflammatory disease treatments. We also propose the future perspectives and translational challenges of nanobiomaterials that need to be overcome in this swiftly rising field.

Objective : Vascular endothelial cells (ECs) sense and respond to both trauma factors (histone proteins) and sepsis signals (bacterial lipopolysaccharide, LPS) with elevations in calcium (Ca 2+ ), but it is not clear if the patterns of activation are similar or different. We hypothesized that within seconds of exposure, histones but not LPS would produce a large EC Ca 2+ response. We also hypothesized that histones would produce different spatio-temporal patterns of Ca 2+ events in veins than in arteries. Methods : We studied cultured ECs (EA.hy926) and native endothelial cells from surgically opened murine blood vessels. High-speed live cell imaging of Ca 2+ events were acquired for 5 min before and after stimulation of cultured ECs with histones or LPS alone or in combination. Histone-induced EC Ca 2+ events were also compared in native endothelial cells from resistance-sized arteries and veins. Ca 2+ activity was quantified as "Ca 2+ prevalence" using custom spatiotemporal analysis. Additionally, cultured ECs were collected after 6 h of exposure to histones or LPS for RNA sequencing. Results : ECs-both in culture and in blood vessels-rapidly increased Ca 2+ activity within seconds of histone exposure. In contrast, LPS exposure produced only a slight increase in Ca 2+ activity in cultured ECs and no effect on blood vessels over 5-min recording periods. Histones evoked large aberrant Ca 2+ events (>30 s in duration) in both veins and arteries, but with different spatio-temporal patterns. Ca 2+ activity in arterial ECs often appeared as "rosettes", with Ca 2+ events that propagated from one cell to all adjacent surrounding cells. In veins, ECs responded individually without spreading. Surprisingly, exposure of cultured ECs to LPS for 5 min before histones potentiated EC Ca 2+ activity by an order of magnitude. Exposure of ECs to histones or LPS both increased gene expression, but different mRNAs were induced. Conclusions : LPS and histones activate ECs through mechanisms that are distinct and additive; only histones produce large aberrant Ca 2+ events. ECs in arteries and veins display different patterns of Ca 2+ responses to histones.

Fluorescence intravital microscopy captures large data sets of dynamic multicellular interactions within various organs such as the lungs, liver, and brain of living subjects. In medical imaging, edge detection is used to accurately identify and delineate important structures and boundaries inside the images. To improve edge sharpness, edge detection frequently requires the inclusion of low-level features. Herein, a machine learning approach is needed to automate the edge detection of multicellular aggregates of distinctly labeled blood cells within the microcirculation. In this work, the Structured Adaptive Boosting Trees algorithm (AdaBoost.S) is proposed as a contribution to overcome some of the edge detection challenges related to medical images. Algorithm design is based on the observation that edges over an image mask often exhibit special structures and are interdependent. Such structures can be predicted using the features extracted from a bigger image patch that covers the image edge mask. The proposed AdaBoost.S is applied to detect multicellular aggregates within blood vessels from the fluorescence lung intravital images of mice exposed to e-cigarette vapor. The predictive capabilities of this approach for detecting platelet-neutrophil aggregates within the lung blood vessels are evaluated against three conventional machine learning algorithms: Random Forest, XGBoost and Decision Tree. AdaBoost.S exhibits a mean recall, F-score, and precision of 0.81, 0.79, and 0.78, respectively. Compared to all three existing algorithms, AdaBoost.S has statistically better performance for recall and F-score. Although AdaBoost.S does not outperform Random Forest in precision, it remains superior to the XGBoost and Decision Tree algorithms. The proposed AdaBoost.S is widely applicable to analysis of other fluorescence intravital microscopy applications including cancer, infection, and cardiovascular disease.

Patients coinfected with respiratory syncytial virus (RSV) and bacteria have longer hospital stays, higher risk of intensive care unit admission, and worse outcomes. We describe a model of RSV line 19F/methicillin-resistant Staphylococcus aureus (MRSA) USA300 coinfection that does not impair viral clearance, but prior RSV infection enhances USA300 MRSA bacterial growth in the lung. The increased bacterial burden post-RSV correlates with reduced accumulation of neutrophils and impaired bacterial killing by alveolar macrophages. Surprisingly, reduced neutrophil accumulation is likely not explained by reductions in phagocyte-recruiting chemokines or alterations in proinflammatory cytokine production compared with mice infected with S. aureus alone. Neutrophils from RSV-infected mice retain their ability to migrate toward chemokine signals, and neutrophils from the RSV-infected lung are better able to phagocytize and kill S. aureus ex vivo on a per cell basis. In contrast, while alveolar macrophages could ingest USA300 post-RSV, intracellular bacterial killing was impaired. The RSV/S. aureus coinfected lung promotes a state of overactivation in neutrophils, demonstrated by increased production of reactive oxygen species (ROS) that can drive formation of neutrophil extracellular traps (NETs), resulting in cell death. Mice with RSV/S. aureus coinfection had increased extracellular DNA and protein in bronchoalveolar lavage fluid and histological evidence confirmed NETosis in vivo. Taken together, these data highlight that prior RSV infection can prime the overactivation of neutrophils leading to cell death that impairs neutrophil accumulation in the lung. Additionally, alveolar macrophage killing of bacteria is impaired post-RSV. Together, these defects enhance USA300 MRSA bacterial growth in the lung post-RSV.

Neutrophils rapidly respond to inflammation resulting from infection, injury, and cancer. Intravital microscopy (IVM) has significantly advanced our understanding of neutrophil behavior, enabling real-time visualization of their migration, interactions with pathogens, and coordination of immune responses. This review delves into the insights provided by IVM studies on neutrophil dynamics in various inflammatory contexts. We also examine the dual role of neutrophils in tumor microenvironments, where they can either facilitate or hinder cancer progression. Finally, we highlight how computational modeling techniques, especially agent-based modeling, complement experimental data by elucidating neutrophil kinetics at the level of individual cells as well as their collective behavior. Understanding the role of neutrophils in health and disease is essential for developing new strategies for combating infection, inflammation and cancer.

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.

Lung metastases are the leading cause of death among cancer patients. The challenges of inefficient drug delivery, compounded by a robust immunosuppressive microenvironment, make effective treatment difficult. Here, an innovative dual-engineered macrophage-microbe encapsulation (Du-EMME) therapy is developed that integrates modified macrophages and engineered antitumor bacteria. These engineered macrophages, termed R-GEM cells, are designed to express RGD peptides on extracellular membranes, enhancing their tumor cell binding and intratumor enrichment. R-GEM cells are cocultured with attenuated Salmonella typhimurium VNP20009, producing macrophage-microbe encapsulation (R-GEM/VNP cells). The intracellular bacteria maintain bioactivity for more than 24 h, and the bacteria released from R-GEM/VNP cells within the tumor continue to exert bacteria-mediated antitumor effects. This is further supported by macrophage-based chemotaxis and camouflage, which enhance the intratumoral enrichment and biocompatibility of the bacteria. Additionally, R-GEM cells loaded with IFNγ-secreting strains (VNP-IFNγ) form R-GEM/VNP-IFNγ cells. Treatment with these cells effectively halts lung metastatic tumor progression in three mouse models (breast cancer, melanoma, and colorectal cancer). R-GEM/VNP-IFNγ cells vigorously activate the tumor microenvironment, suppressing tumor-promoting M2-type macrophages, MDSCs, and Tregs, and enhancing tumor-antagonizing M1-type macrophages, mature DCs, and Teffs. Du-EMME therapy offers a promising strategy for targeted and enhanced antitumor immunity in treating cancer metastases.

Heart failure with reduced (HFrEF) or preserved ejection fraction (HFpEF) is characterized by low-grade chronic inflammation. Circulating neutrophils regroup 2 subtypes termed high- and low-density neutrophils (HDNs and LDNs). LDNs represent less than 2% of total neutrophil under physiological conditions, but their counts increase in multiple pathologies, releasing more inflammatory cytokines and neutrophil extracellular traps (NETs). The aims of this study were to assess the differential count and role of HDNs, LDNs, and NETs-related activities in patients with heart failure (HF).

HDNs and LDNs were isolated from human blood by density gradient and purified by fluorescence-activated cell sorting (FACS) and their counts obtained by flow cytometry. Formation of NETs (NETosis) was quantified by confocal microscopy. Circulating inflammatory and NETosis biomarkers were measured by enzyme-linked immunosorbent assay (ELISA). Neutrophil adhesion onto human extracellular matrix (hECM) was assessed by optical microscopy.

A total of 140 individuals were enrolled, including 33 healthy volunteers (HVs), 41 HFrEF (19 stable patients and 22 presenting acute decompensated HF [ADHF]), and 66 patients with HFpEF (36 stable patients and 30 presenting HF decompensation). HDNs and LDNs counts were significantly increased up to 39% and 2740%, respectively, in patients with HF compared with HVs. In patients with HF, the correlations among LDNs counts and circulating inflammatory (CRP, IL-6 and -8), troponin T, N-terminal prohormone of brain natriuretic peptide (NT-proBNP), and NETosis components were significant. In vitro, LDNs expressed more citrullinated histone H3 (H3Cit) and NETs and were more proadhesive, with ADHFpEF patients presenting the highest proinflammatory profile.

Patients with HFpEF present higher levels of circulating LDNs- and NETs-related activities, which are the highest in the context of acute HF decompensation.

Engineered bacteria-mediated antitumor approaches have been proposed as promising immunotherapies for cancer. However, the off-target bacterial toxicity narrows the therapeutic window. Living microbes will benefit from their controllable immunogenicity within tumors for safer antitumor applications. In this study, a genetically encoded microbial activation strategy is reported that uses tunable and dynamic expression of surface extracellular polysaccharides to improve bacterial biocompatibility while retaining therapeutic efficacy. Based on screening of genes associated with Salmonella survival in macrophages, a novel attenuated Salmonella chassis strain AIS (htrA gene-deficient) highly enriched in tumors after administration and rapidly cleared from normal organs are reported. Subsequently, an engineered bacterial strain, AISI-H, is constructed based on the AIS strain and an optimized quorum-sensing regulatory system. The AISI-H strain can achieve recovery of dynamic tumor-specific bacterial virulence through a novel HTRA-RCSA axis-based and quorum-sensing synthetic gene circuit-mediated increase in extracellular polysaccharide content. These strains act "off" in normal organs to avoid unwanted immune activation and "on" in tumors for precise tumor suppression in mice. The AISI-H strain shows significant tumor inhibition and potent activation of anticancer immunity in a melanoma mouse model. The AISI-H strain exhibits excellent biocompatibility. This bacterial regulation strategy expands the applications of microbe-based antitumor therapeutics.

In mice, the first liver-resident macrophages, known as Kupffer cells (KCs), are thought to derive from yolk sac (YS) hematopoietic progenitors that are specified prior to the emergence of the hematopoietic stem cell (HSC). To investigate human KC development, we recapitulated YS-like hematopoiesis from human pluripotent stem cells (hPSCs) and transplanted derivative macrophage progenitors into NSG mice previously humanized with hPSC-liver sinusoidal endothelial cells (LSECs). We demonstrate that hPSC-LSECs facilitate stable hPSC-YS-macrophage engraftment for at least 7 weeks. Single-cell RNA sequencing (scRNA-seq) of engrafted YS-macrophages revealed a homogeneous MARCO-expressing KC gene signature and low expression of monocyte-like macrophage genes. In contrast, human cord blood (CB)-derived macrophage progenitors generated grafts that contain multiple hematopoietic lineages in addition to KCs. Functional analyses showed that the engrafted KCs actively perform phagocytosis and erythrophagocytosis in vivo. Taken together, these findings demonstrate that it is possible to generate human KCs from hPSC-derived, YS-like progenitors.

Portal vein thrombosis (PVT) is a challenging and controversial complication of cirrhosis. Experimental models that reproduce cirrhotic PVT and effective pharmacological therapies are limited. We aimed to investigate the nature course and mechanisms of PVT in cirrhosis. A novel PVT model was developed via two-step total portal vein ligation in healthy and thioacetamide (TAA)-cirrhotic rats. Circulating and liver-infiltrating neutrophils were isolated from individuals with cirrhosis to examine neutrophil extracellular traps (NETs) and explore their unique characteristics and implications in PVT-associated fibrosis in cirrhosis. We further validated macrophage-myofibroblast transition (MMT) via multiplex immunofluorescence and single-cell sequencing. In the experimental model, cirrhosis promoted PVT development and portal vein intimal thickening. Interestingly, cirrhosis promoted spontaneous resolution of PVT due to instability of thrombus structure, along with pulmonary and intrahepatic clots. NETs-MMT mediate cirrhotic PVT and PVT-associated fibrosis, including fibrotic thrombus remodeling and increased hepatic collagen deposition. Mechanistically, caspase-4-dependent activation of neutrophils and GSDMD mediated the formation of NETs. The extracellular DNA of NETs promoted TGF-β1/Smad3-driven MMT. Inhibiting GSDMD with disulfiram suppressed cirrhotic PVT and prevented associated fibrosis. The cirrhotic PVT model reflected the following three main characteristics of cirrhotic PVT: spontaneous resolution, immunothrombosis, and intimal fibrosis. Targeting NETs with GSDMD inhibitors may serve as a new therapeutic concept to treat cirrhotic PVT.

Liver disease ranks as the eleventh leading cause of mortality, leading to approximately 2 million deaths annually worldwide. Neutrophils are a type of immune cell that are abundant in peripheral blood and play a vital role in innate immunity by quickly reaching the site of liver injury. They exert their influence on liver diseases through autocrine, paracrine, and immunomodulatory mechanisms. Extracellular vesicles, phospholipid bilayer vesicles, transport a variety of substances, such as proteins, nucleic acids, lipids, and pathogenic factors, for intercellular communication. They regulate cell communication and perform their functions by delivering biological information. Current research has revealed the involvement of the interaction between neutrophils and extracellular vesicles in the pathogenesis of liver disease. Moreover, more research has focused on targeting neutrophils as a therapeutic strategy to attenuate disease progression. Therefore, this article summarizes the roles of neutrophils, extracellular vesicles, and their interactions in noncancerous liver diseases.