Neutrophil Function Research Articles

APEX1 in intestinal epithelium triggers neutrophil infiltration and intestinal barrier damage in ulcerative colitis

Ulcerative colitis (UC) can lead to the generation of large amounts of reactive oxygen species and DNA damage. DNA repair caused by base excision repair (BER) enzymes is an important mechanism for maintaining genomic integrity. However, the specific relationship between the function of BER enzymes and UC remains unclear. To address this, we conducted a study on non-cancerous colon tissue from patients with UC, focusing on the role of apurinic/apyrimidinic endonuclease 1 (APEX1) in BER to explore its significance in the progression of UC. Our research found that the expression of APEX1 in epithelium cells was significantly correlated to the severity of inflammatory bowel disease (IBD) and the infiltration and function of neutrophils in human UC and mouse models, particularly in relation to neutrophil extracellular traps (NETs) and the degranulation processes. APEX1 deficiency resulted in decreased production of the chemokines CXCL1 by the NF-κB pathway in epithelium cells, leading to reduced accumulation and activation of neutrophils associated with colitis in colon tissue, as well as decreased levels of IL-1β. Furthermore, APEX1 deficiency reduced symptoms of colitis by decreasing epithelial cell apoptosis and altering the gut microbiome. Studies related to the redox activity of APEX1 have shown that the combination of the redox inhibitor E3330 with 5-aminosalicylic acid (5-ASA) can effectively alleviate colitis, indicating that APEX1 has promising prospects for clinical treatment of IBD. APEX1 is required for interactions between neutrophil and intestinal epithelial cells. This study provided a mechanism demonstrating that APEX1 protein triggered the risk of UC by promoting neutrophil infiltration and compromising intestinal epithelial barrier function.

Glucocorticoids Suppress NF-κB-Mediated Neutrophil Control of Aspergillus fumigatus Hyphal Growth.

Glucocorticoids are a major class of therapeutic anti-inflammatory and immunosuppressive drugs prescribed to patients with inflammatory diseases, to avoid transplant rejection, and as part of cancer chemotherapy. However, exposure to these drugs increases the risk of opportunistic infections such as with the fungus Aspergillus fumigatus, which causes mortality in >50% of infected patients. The mechanisms by which glucocorticoids increase susceptibility to A. fumigatus are poorly understood. In this article, we used a zebrafish larva Aspergillus infection model to identify innate immune mechanisms altered by glucocorticoid treatment. Infected larvae exposed to dexamethasone succumb to infection at a significantly higher rate than control larvae. However, both macrophages and neutrophils are still recruited to the site of infection, and dexamethasone treatment does not significantly affect fungal spore killing. Instead, the primary effect of dexamethasone manifests later in infection with treated larvae exhibiting increased invasive hyphal growth. In line with this, dexamethasone predominantly inhibits neutrophil function rather than macrophage function. Dexamethasone-induced mortality also depends on the glucocorticoid receptor. Dexamethasone partially suppresses NF-κB activation at the infection site by inducing the transcription of IκB via the glucocorticoid receptor. Independent CRISPR/Cas9 targeting of IKKγ to prevent NF-κB activation also increases invasive A. fumigatus growth and larval mortality. However, dexamethasone treatment of IKKγ crispant larvae further increases invasive hyphal growth and host mortality, suggesting that dexamethasone may suppress other pathways in addition to NF-κB to promote host susceptibility. Collectively, we find that dexamethasone acts through the glucocorticoid receptor to suppress NF-κB-mediated neutrophil control of A. fumigatus hyphae in zebrafish larvae.

Unveiling signaling pathways inducing MHC class II expression in neutrophils.

Gram-negative bacillary bacteremia poses a significant threat, ranking among the most severe infectious diseases capable of triggering life-threatening sepsis. Despite the unambiguous involvement of neutrophils in this potentially fatal disease, there are limited data about the molecular signaling mechanisms, phenotype, and function of human neutrophils during the early phase of gram-negative bacillary bacteremia. By using an unbiased proteomics and flow cytometry approach, we identified an antigen-presenting cell (APC)-like phenotype in human peripheral blood neutrophils (PMN) with MHC class II molecule expression in the early phase of bacteremia. Using an in-vitro model of GM-CSF-mediated induction of APC-like phenotype in PMN, we investigated downstream signaling pathways leading to MHC class II expression. GM-CSF stimulation of neutrophils leads to the activation of three major signaling pathways, the JAK-STAT, the mitogen-activated protein kinase (MAPK), and the phosphoinositide 3-kinase (PI3K)-Akt-mTOR pathways, while MHC class II induction is mediated by a MAPK-p38-MSK1-CREB1 signaling cascade and the MHC class II transactivator CIITA in a strictly JAK1/2 kinase-dependent manner. This study provides new insights into the signaling pathways that induce MHC class II expression in neutrophils, highlighting the potential for therapeutic targeting of JAK1/2 signaling in the treatment of gram-negative bacteremia and sepsis. Understanding these mechanisms may open up novel approaches for managing inflammatory responses during sepsis.

Effects of Pressure, Hypoxia, and Hyperoxia on Neutrophil Granulocytes

Background: The application of normo- and hyperbaric O2 is a common therapy option in various disease patterns. Thereby, the applied O2 affects the whole body, including the blood and its components. This study investigates influences of pressure and oxygen fraction on human blood plasma, nutrient media, and the functions of neutrophil granulocytes (PMNs). Methods: Neutrophil migration, reactive oxygen species (ROS) production, and NETosis were examined by live cell imaging. The treatment of various matrices (Roswell Park Memorial Institute 1640 medium, Dulbecco’s Modified Eagle’s Medium, H2O, human plasma, and isolated PMNs) with hyperbaric oxygen (HBO) was performed. In addition, the expression of different neutrophil surface epitopes (CD11b, CD62L, CD66b) and the oxidative burst were investigated by flow cytometry (FACS). The application of cold atmospheric plasma (CAP) to normoxic and normobaric culture media served as a positive control. Soluble reaction products such as H2O2, reactive nitrogen species (RNS: NO2− and NO3−), and ROS-dependent dihydrorhodamine oxidation were quantified by fluoro- and colorimetric assay kits. Results: Under normobaric normoxia, PMNs migrate slower and shorter in comparison with normobaric hyper- or hypoxic conditions and hyperbaric hyperoxia. The pressure component has less effect on the migration behavior of PMNs than the O2 concentration. Individual PMN cells produce prolonged ROS at normoxic conditions. PMNs showed increased expression of CD11b in normobaric normoxia, lower expression of CD62L in normobaric normoxia, and lower expression of CD66b after HBO and CAP treatment. Treatment with CAP increased the amount of ROS and RNS in common culture media. Conclusions: Hyperbaric and normobaric O2 influences neutrophil functionality and surface epitopes in a measurable way, which may have an impact on disorders with neutrophil involvement. In the context of hyperbaric experiments, especially high amounts of H2O2 in RPMI after hyperbaric oxygen should be taken into account. Therefore, our data support a critical indication for the use of normobaric and hyperbaric oxygen and conscientious and careful handling of oxygen in everyday clinical practice.

Assessing the Effects of Surgical Irrigation Solutions on Human Neutrophil Interactions with Nascent Staphylococcus aureus Biofilms.

Staphylococcus aureus (S. aureus) is the leading cause of surgical site infections (SSIs) and is capable of biofilm growth on implanted foreign devices. The use of surgical irrigation solutions has become a common strategy to combat bacterial contamination events that occur during surgery. Despite their antimicrobial activity, SSI rates remain consistent, suggesting that low-level contamination persists. In these cases, circulating neutrophils must traffic from the blood to contamination sites to aid in bacterial clearance. The influence of irrigation solutions on neutrophils' ability to engage with bacteria has not been explored. The effects of three commonly used irrigation solutions: Xperience (sodium lauryl sulfate), Irrisept (chlorhexidine gluconate), and Betadine® (povidone-iodine) on nascent S. aureus biofilms alone and in the presence of human neutrophils were assessed at manufactured and diluted concentrations. All three solutions, at a 10% dilution, inhibited bacterial growth as demonstrated by culture assays and confocal video microscopy of bacterial aggregate formation. The effects of 10% dilutions of each of these solutions on neutrophil membrane integrity (by flow cytometry and propidium iodide staining) and motility (by confocal video microscopy of neutrophil track length) were investigated with differing outcomes for each irrigation solution. At this concentration only Irrisept preserved neutrophil membrane integrity and motility. Together, this study examines an overlooked aspect of surgical irrigation solutions by investigating their impact on innate immunity and highlights the feasibility of formulations wherein solution effectiveness is complemented by neutrophil function to reduce risks of infection.

Clinical and pathogenetic significance of the phenomenon of functional heterogeneity of oxygen-dependent functions of neutrophils in ankylosing spondylitis

Neutrophils are able to exert a variety of effects on other cells of the immune system, which indicates their heterogeneity. In various rheumatic diseases, neutrophils are involved in the immunopathological process. The study of the phenomenon of functional heterogeneity of neutrophils associated with the pathogenesis of rheumatic diseases is a promising area of research in immunology and rheumatology. The purpose of the research: study of factors influencing oxygen-dependent neutrophil functions in ankylosing spondylitis (AS). A total of 82 patients with AS were examined. The functional activity of neutrophils was assessed according to the data of determining the indicators of oxygen-dependent functions by the chemiluminescence method. Functional neutrophil reserve (FNR) was assessed by activation coefficients (the ratio of chemiluminescence induced by suspension of heat-killed staphylococcus cells to spontaneous value). Statistical data processing was carried out using Statistica 10.0 program. An increase in oxygen-dependent neutrophil functions in AS was accompanied by an increase in disease activity. The greatest influence on the increase in the functional activity of cells was exerted by the age of patients, the stage of the disease and the level of circulating immune complexes. The process of stabilizing the metabolic activity of neutrophils was significantly influenced by the age of patients, ESR, ã-globulins, total cholesterol, and low-density lipoprotein cholesterol. Among the laboratory parameters, the majority of AS patients with moderate and high FNR had elevated levels of ESR, CRP, and IgM; there was a tendency to increase IgA in AS patients with high FNR. Statistical analyses showed an association between FNR and disease activity; there was no statistically significant dependence of FNR on the stage, course, and form of AS. The study of the metabolic activity of neutrophils during dynamic observation revealed a group of patients with stabilization of FNR parameters against the background of therapy with the achievement of a normal level of oxygen-dependent metabolism. Thus, in ankylosing spondylitis, the functional heterogeneity of neutrophils associated with disease activity was established according to the determination of oxygen-dependent metabolism.

The cGAS-STING Pathway Is Essential in Acute Ischemia-Induced Neutropoiesis and Neutrophil Priming in the Bone Marrow.

Acute myocardial ischemia triggers a rapid mobilization of neutrophils from the bone marrow to peripheral blood, facilitating their infiltration into the infarcted myocardium. These cells are critical for inducing inflammation and contributing to myocardial repair. While neutrophils in infarcted tissue are better characterized, our understanding of whether and how ischemia regulates neutrophil production, differentiation, and functionality in the bone marrow remains limited. This study investigates these processes and the influence of the cGAS-STING pathway in the context of myocardial infarction. The cGAS-STING pathway detects aberrant DNA within cells, activates STING, and initiates downstream signaling cascades involving NFKB and IRF3. We analyzed neutrophils from bone marrow, peripheral blood, and infarct tissues using MI models generated from wild-type, Cgas -/- , and Sting -/- mice. These models are essential for studying neutropoiesis (neutrophil production and differentiation), as it involves multiple cell types. RNA sequencing analysis revealed that ischemia not only increased neutrophil production but also promoted cytokine signaling, phagocytosis, chemotaxis, and degranulation in the bone marrow before their release into the peripheral blood. Inhibition of the cGAS-STING pathway decreased neutrophil production after MI and down-regulated the same pathways activated by ischemia. Neutrophils lacking cGAS or STING were less mature, exhibited reduced activation, and decreased degranulation. Deletion of cGAS and STING decreased the expression of a large group of IFN-stimulated genes and IFIT1+ neutrophils from peripheral blood and the infarct tissue, suggesting that cGAS-STING plays an essential role in neutrophils with the IFN-stimulated gene signature. Importantly, transcriptomic analysis of Cgas -/- and Sting -/- neutrophils from bone marrow and MI tissues showed downregulation of similar pathways, indicating that the functionality developed in the bone marrow was maintained despite infarct-induced stimulation. These findings highlight the importance of neutropoiesis in dictating neutrophil function in target tissues, underscoring the critical role of the cGAS-STING pathway in neutrophil-mediated myocardial repair post-ischemia.

Interferon lambda signaling in neutrophils enhances the pathogenesis of Bordetella pertussis infection

Abstract Interferon lambda plays diverse roles in bacterial infections. Previously, we showed that interferon lambda is induced in the lungs of Bordetella pertussis-infected adult mice and exacerbates inflammation. Here, we report that mice lacking the interferon lambda receptor 1 specifically on neutrophils (MRP8creIFNLR1fl/fl mice) exhibit reduced lung bacterial load and inflammation compared to wild-type mice during B. pertussis infection. In B. pertussis-infected wild-type mice, lung type I and III IFN responses were higher than in MRP8creIFNLR1fl/fl mice, correlating with increased lung inflammatory pathology. There was an increased proportion of interferon gamma-producing neutrophils in the lungs of MRP8creIFNLR1fl/fl mice compared to wild-type mice. IFNLR1−/− neutrophils incubated with B. pertussis exhibited higher killing compared to wild-type neutrophils. Treatment of wild-type neutrophils with interferon lambda further decreased their bacterial killing capacity and treatment of wild-type mice with interferon lambda increased lung bacterial loads. Contributing to the differential killing, we found that IFNLR1−/− neutrophils exhibit higher levels of reactive oxygen species, myeloperoxidase, matrix metalloproteinase-9 activity, neutrophil extracellular traps, and interferon gamma secretion than wild-type neutrophils, and inhibiting NADPH oxidase inhibited bacterial killing in IFNLR1−/− neutrophils. B. pertussis-induced interferon lambda secretion and IFNLR1 gene expression in mouse and human neutrophils and this was dependent on the bacterial virulence protein pertussis toxin. Pertussis toxin enhanced bacterial loads in wild type but not in MRP8creIFNLR1fl/fl or IFNLR1−/− mice. Thus, pertussis toxin disrupts neutrophil function by enhancing type III IFN signaling, which prevents neutrophils from effectively clearing B. pertussis during infection, leading to higher bacterial loads and exacerbation of lung inflammation.

Complex role of neutrophils in the tumor microenvironment: an avenue for novel immunotherapies.

Neutrophils, which originate from the bone marrow and are characterized by a segmented nucleus and a brief lifespan, have a crucial role in the body's defense against infections and acute inflammation. Recent research has uncovered the complex roles of neutrophils as regulators in tumorigenesis, during which neutrophils exhibit a dualistic nature that promotes or inhibits tumor progression. This adaptability is pivotal within the tumor microenvironment (TME). In this review, we provide a comprehensive characterization of neutrophil plasticity and heterogeneity, aiming to illuminate current research findings and discuss potential therapeutic avenues. By delineating the intricate interplay of neutrophils in the TME, this review further underscores the urgent need to understand the dual functions of neutrophils with particular emphasis on the anti-tumor effects to facilitate the development of effective therapeutic strategies against cancer.

Cyclic stretch enhances neutrophil extracellular trap formation

BackgroundNeutrophils, the most abundant leukocytes circulating in blood, contribute to host defense and play a significant role in chronic inflammatory disorders. They can release their DNA in the form of extracellular traps (NETs), which serve as scaffolds for capturing bacteria and various blood cells. However, uncontrolled formation of NETs (NETosis) can lead to excessive activation of coagulation pathways and thrombosis. Once neutrophils are migrated to infected or injured tissues, they become exposed to mechanical forces from their surrounding environment. However, the impact of transient changes in tissue mechanics due to the natural process of aging, infection, tissue injury, and cancer on neutrophils remains unknown. To address this gap, we explored the interactive effects of changes in substrate stiffness and cyclic stretch on NETosis. Primary neutrophils were cultured on a silicon-based substrate with stiffness levels of 30 and 300 kPa for at least 3 h under static conditions or cyclic stretch levels of 5% and 10%, mirroring the biomechanics of aged and young arteries.ResultsUsing this approach, we found that neutrophils are sensitive to cyclic stretch and that increases in stretch intensity and substrate stiffness enhance nuclei decondensation and histone H3 citrullination (CitH3). In addition, stretch intensity and substrate stiffness promote the response of neutrophils to the NET-inducing agents phorbol 12-myristate 13-acetate (PMA), adenosine triphosphate (ATP), and lipopolysaccharides (LPS). Stretch-induced activation of neutrophils was dependent on calpain activity, the phosphatidylinositol 3-kinase (PI3K)/focal adhesion kinase (FAK) signalling and actin polymerization.ConclusionsIn summary, these results demonstrate that the mechanical forces originating from the surrounding tissue influence NETosis, an important neutrophil function, and thus identify a potential novel therapeutic target.

Enterotoxigenic Escherichia coli heat labile enterotoxin affects neutrophil effector functions via cAMP/PKA/ERK signaling

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal illness in humans and animals, induced by enterotoxins produced by these pathogens. Despite the crucial role of neutrophils in combatting bacterial infections, our understanding of how enterotoxins impact neutrophil function is limited. To address this knowledge gap, we used heat-labile enterotoxin (LT) and heat-stable enterotoxin a (STa) to investigate their impact on the effector functions of neutrophils. Our study reveals that pSTa does not exert any discernible effect on the function of neutrophils. In contrast, LT altered the migration and phagocytosis of neutrophils and induced the production of inflammatory factors via activation of cAMP/PKA and ERK1/2 signaling. LT also attenuated the release of neutrophil extracellular traps by neutrophils via the PKA signaling pathway. Our findings provide novel insights into the impact of LT on neutrophil function, shedding light on the underlying mechanisms that govern its immunoregulatory effects. This might help ETEC in subverting the immune system and establishing infection.

Activation induces shift in nutrient utilization that differentially impacts cell functions in human neutrophils

Neutrophils utilize a variety of metabolic sources to support their crucial functions as the first responders in innate immunity. Here, through in vivo and ex vivo isotopic tracing, we examined the contributions of different nutrients to neutrophil metabolism under specific conditions. Human peripheral blood neutrophils, in contrast to a neutrophil-like cell line, rely on glycogen storage as a major metabolic source under resting state but rapidly switch to primarily using extracellular glucose upon activation with various stimuli. This shift is driven by a substantial increase in glucose uptake, enabled by rapidly increased GLUT1 on cell membrane, that dominates the simultaneous increase in gross glycogen cycling capacity. Shifts in nutrient utilization impact neutrophil functions in a function-specific manner: oxidative burst depends on glucose utilization, whereas NETosis and phagocytosis can be flexibly supported by either glucose or glycogen, and neutrophil migration and fungal control are enhanced by the shift from glycogen utilization to glucose utilization. This work provides a quantitative and dynamic understanding of fundamental features in neutrophil metabolism and elucidates how metabolic remodeling shapes neutrophil functions, which has broad health relevance.

Effect of Neutrophil–Platelet Interactions on Cytokine-Modulated Expression of Neutrophil CD11b/CD18 (Mac-1) Integrin Complex and CCR5 Chemokine Receptor in Stable Coronary Artery Disease: A Sub-Study of SMARTool H2020 European Project

Atherosclerosis is an inflammatory disease wherein neutrophils play a key role in plaque evolution. We observed that neutrophil CD11b was associated with a higher necrotic core volume in coronary plaques. Since platelets modulate neutrophil function, we explored the influence of neutrophil–platelet conjugates on the cytokine-modulated neutrophil complex CD11b/CD18 and CCR5 receptor expression. In 55 patients [68.53 ± 7.95 years old (mean ± SD); 71% male], neutrophil positivity for CD11b, CD18 and CCR5 was expressed as Relative Fluorescence Intensity (RFI) and taken as a dependent variable. Cytokines and chemokines were assessed by ELISA. Following log-10-based logarithmic transformation, they were used as independent variables in Model 1 of multiple regression together with Body Mass Index and albumin. Model 1 was expanded with the RFI of neutrophil CD41a+ (model 2). The RFI of neutrophil CD41a+ correlated positively and significantly with CD11b, CD18, and CCR5. In Model 2, CCR5 correlated positively only with the RFI of neutrophil CD41a+. Albumin maintained its positive effect on CD11b in both models. These observations indicate the complexity of neutrophil phenotypic modulation in stable CAD. Despite limitations, these findings suggest there is a role played by neutrophil–platelet interaction on the neutrophil cytokine-modulated expression of adhesive and chemotactic receptors.

Prostaglandins suppress neutrophil function after sexual intercourse and may promote urinary tract infections

Sexually active women have an increased risk of urinary tract infections (UTIs), which may be influenced by components in seminal plasma. Prostaglandins in seminal plasma are key modulators of the immune system and maternal-fetal immunological tolerance, influencing cells like platelets and neutrophils. Specifically, prostaglandin I2 (PGI2) inhibits platelet aggregation, while prostaglandin E2 (PGE2) alters neutrophil activity, including the production of reactive oxygen species (ROS) and the formation of neutrophil extracellular traps (NETs). Given that the NET response is associated with UTIs, and PGI2 is known to modulate NETs, these interactions may contribute to the higher incidence of UTIs in sexual activity in women. This study aims to investigate whether UTIs following sexual activity are facilitated by an imbalance in NET formation mediated by prostaglandins. We employed four search strategies: Sex and UTI, NETs and UTIs, Prostaglandins and NETs, and Inflammasome and NETs, alongside a review of original research publications. By understanding this mechanism, we hope to reveal how prostaglandin-mediated immune modulation may increase susceptibility to UTIs after sexual activity.

Neutrophil phenotype, effector functions and microbicidal activity in SARS-CoV-2-associated ARDS patients.

Critically ill patients admitted to the intensive care unit (ICU) for SARS-CoV-2-induced acute respiratory distress syndrome (ARDS) are at increased risk of bacterial and fungal secondary pulmonary infections due to acquired immune dysfunction. Given that the activity of neutrophils has not been described in these patients, we aimed to investigate the function of neutrophils at ICU admission and on Day 7 (D7) post admission. Neutrophil maturation and several functional indicators were investigated. We detected a significant decrease in reactive oxygen species production at D7, but we did not observe any other significant alterations in neutrophil function. Furthermore, bronchoalveolar lavage obtained from patients displayed no inhibitory effect on the function of neutrophils from healthy donors. These findings indicate that patients admitted to the ICU for SARS-CoV-2-induced ARDS do not acquire neutrophil dysfunction within the first week of their stay, which suggests that nosocomial infections among these patients are not due to acquired neutrophil dysfunctions.

Seminal plasma inhibits Chlamydia trachomatis infection in vitro, and may have consequences on mucosal immunity

Seminal plasma (SP) is the main vector of C. trachomatis (CT) during heterosexual transmission from male to female. It has immunomodulatory properties and impacts the susceptibility to HIV-1 infection, but its role has not been explored during CT infection. In the female reproductive tract (FRT), CT infection induces cytokine production and neutrophil recruitment. The role of neutrophils during CT infection is partially described, they could be at the origin of the pathology observed during CT infection. During this study, we developed an experimental in vitro model to characterize the impact of CT infection and SP on endocervical epithelial cell immune response in the FRT. We also studied the impact of the epithelial cell response on neutrophil phenotype and functions. We showed that the production by epithelial cells of pro-inflammatory cytokines increased during CT infection. Moreover, the pool of SP as well as individuals SP inhibited CT infection in a dose-dependent manner. The pool of SP inhibited cytokine production in a dose-dependent manner. The pool of SP altered gene expression profiles of infected cells. The culture supernatants of cells infected or not with CT, in presence or not of the pool of SP, had an impact on neutrophil phenotype and functions: they affected markers of neutrophil maturation, activation and adhesion capacity, as well as the survival, ROS production and phagocytosis ability. This study proposes a novel approach to study the impact of the environment on the phenotype and functions of neutrophils in the FRT. It highlights the impact of the factors of the FRT environment, in particular SP and CT infection, on the mucosal inflammation and the need to take into account the SP component while studying sexually transmitted infections during heterosexual transmission from male to female.

Impact of Hypoxia on Neutrophil Degranulation and Inflammatory Response in Alpha-1 Antitrypsin Deficiency Patients.

Alpha-1 antitrypsin deficiency (AATD) is an inflammatory disorder where neutrophils play a key role. Excessive neutrophil activation leads to local hypoxia and tissue damage. Most research on neutrophil function has been conducted under atmospheric conditions (21% O2), which may not represent physiological or pathological conditions. This study aimed to determine the effects of hypoxia on neutrophil degranulation and cytokine production in AATD patients. Neutrophils isolated from 54 AATD patients (31 MZ; 8 SZ; 15 ZZ) and 7 controls (MM) were exposed to hypoxia (1% O2) for 4 h. Neutrophil degranulation was assessed by measuring elastase (NE), myeloperoxidase (MPO), lactoferrin, and matrix metalloproteinase-9 (MMP-9) levels using immunoassay-based methods. Pro-inflammatory (IL-8, IL-1 beta, IL-6, and TNF-alpha) and anti-inflammatory (IL-4 and IL-10) cytokine levels were assessed by a Luminex-based method. Our results indicate a significantly increased release of NE (p = 0.015), MPO (p = 0.042), lactoferrin (p = 0.015), and MMP-9 (p = 0.001) compared to controls. Pro-inflammatory cytokines show a significant rise in IL-8 (p = 0.019), a trend towards increased IL-1 beta (p = 0.3196), no change in IL-6 (p = 0.7329), and reduced TNF-alpha (p = 0.006). Anti-inflammatory cytokines show increased IL-4 (p = 0.057) and decreased IL-10 (p = 0.05703). Increased neutrophil degranulation and inflammatory phenotype are observed in AATD neutrophils under physiological hypoxia.

Differential roles for the oxygen sensing enzymes PHD1 and PHD3 in the regulation of neutrophil metabolism and function.

Neutrophils are essential in the early innate immune response to pathogens. Harnessing their antimicrobial powers, without driving excessive and damaging inflammatory responses, represents an attractive therapeutic possibility. The neutrophil population is increasingly recognised to be more diverse and malleable than was previously appreciated. Hypoxic signalling pathways are known to regulate important neutrophil behaviours and, as such, are potential therapeutic targets for regulating neutrophil antimicrobial and inflammatory responses. We used a combination of in vivo and ex vivo models, utilising neutrophil and myeloid specific PHD1 or PHD3 deficient mouse lines to investigate the roles of oxygen sensing prolyl hydroxylase enzymes in the regulation of neutrophilic inflammation and immunity. Mass spectrometry and Seahorse metabolic flux assays were used to analyse the role of metabolic shifts in driving the downstream phenotypes. We found that PHD1 deficiency drives alterations in neutrophil metabolism and recruitment, in an oxygen dependent fashion. Despite this, PHD1 deficiency did not significantly alter ex vivo neutrophil phenotypes or in vivo outcomes in mouse models of inflammation. Conversely, PHD3 deficiency was found to enhance neutrophil antibacterial properties without excessive inflammatory responses. This was not linked to changes in the abundance of core metabolites but was associated with increased oxygen consumption and increased mitochondrial reactive oxygen species (mROS) production. PHD3 deficiency drives a favourable neutrophil phenotype in infection and, as such, is an important potential therapeutic target.

PPARα suppresses growth of hepatocellular carcinoma in a high-fat diet context by reducing neutrophil extracellular traps release

Background & AimsThe role of infiltrating neutrophils in hepatocellular carcinoma (HCC) is modulated by cellular metabolism, specifically lipid homeostasis. Throughout the progression of HCC, alterations in lipid metabolism are intricately linked with various signaling that regulate neutrophil function and the release of neutrophil extracellular traps (NETs). However, the dependence of high-fat diet (HFD) on NETs for regulating the development of HCC and the potential interplay between NETs and other leukocytes remains uncertain. MethodsIn this study, the molecular mechanism of NETs release and the potential improvement of the HCC microenvironment by PPARα agonists were explored through proteomics, metabolomics, tissue microarray, immunofluorescence, flow cytometry, Western blot, and dual-luciferase reporter gene assay (n = 6 per group). ResultsOur study demonstrated a notable inhibition of PPARα signaling in HCC. Furthermore, the disruption of PPARα-mediated lipid metabolism was responsible for the release of NETs. The presence of HFD was observed to induce mitochondrial impairment in neutrophils, leading to the activation of cGAS-STING by oxidized mitochondrial DNA (Ox-mtDNA). Consequently, this activation triggered the release of NETs containing Ox-mtDNA through the enhancement of NLRP3-GSDMD-N in a NF-κB-dependent manner. Moreover, the release of NETs within HCC tissues effectively isolated cytotoxic leukocytes in the outer regions of HCC cells. ConclusionsOur study not only enriched the relationship between lipid metabolism disorders and NETs’ tumor-promoting function, but also provided an important strategic reference for multi-target or combined immunotherapy of HCC. Impact and implicationsWe have identified PPARα and its agonists as therapeutic targets for controlling the neutrophil extracellular traps associated with high lipid metabolism. Results from preclinical models suggest that PPARα can limit mitochondrial oxidative stress, inhibit cGAS-STING-NF-κB signaling, and limit the release of neutrophil extracellular traps, thereby increasing the contact of anti-tumor leukocytes and hepatocellular cancer cells, thereby limiting tumor growth.

Malondialdehyde-specific natural IgM inhibit NETosis triggered by culprit site-derived extracellular vesicles from myocardial infarction patients.

Abstract Background and Aims Neutrophil extracellular traps (NETs) trigger atherothrombosis during acute myocardial infarction (AMI), but mechanisms of induction remain unclear. Levels of extracellular vesicles (EV) carrying oxidation-specific epitopes (OSE), which are targeted by specific natural immunoglobulin M (IgM), are increased at the culprit site in AMI. This study investigated EV as inducers of NETosis and assessed the inhibitory effect of natural anti-OSE–IgM in this process. Methods Blood from the culprit and peripheral site of ST-segment elevation myocardial infarction (STEMI) patients (n = 28) was collected, and myocardial function assessed by cardiac magnetic resonance imaging (cMRI) 4 ± 2 days and 195 ± 15 days post-AMI. Extracellular vesicles were isolated from patient plasma and cell culture supernatants for neutrophil stimulation in vitro and in vivo, in the presence of a malondialdehyde (MDA)-specific IgM or an isotype control. NETosis and neutrophil functions were assessed via enzyme-linked immunosorbent assay and fluorescence microscopy. Pharmacological inhibitors were used to map signalling pathways. Neutrophil extracellular trap markers and anti-OSE–IgM were measured by ELISA. Results CD45+ MDA+ EV and NET markers were elevated at the culprit site. Extracellular vesicles induced neutrophil activation and NET formation via TLR4 and PAD4, and mice injected with EV showed increased NETosis. Malondialdehyde-specific IgM levels were inversely associated with citH3 in STEMI patient blood. An MDA-specific IgM inhibited EV-induced NET release in vitro and in vivo. CD45+ MDA+ EV concentrations inversely correlated with left ventricular ejection fraction post-AMI. Conclusions Culprit site–derived EV induce NETosis, while MDA-specific natural IgM inhibit this effect, potentially impacting outcome after AMI.