Neutrophil Activation Research Articles

NINJ1-mediated Macrophage Plasma Membrane Rupture and Neutrophil Extracellular Trap Formation Contribute to Oxalate Nephropathy.

Oxalate nephropathy is characterized by calcium oxalate crystals deposition, which triggers necrosis of renal tubular epithelial cells, initiates an inflammatory cascade characterized by neutrophil and macrophage activation within the renal microenvironment. Despite the close association of immune cells with acute oxalate nephropathy, the underlying mechanisms still remain unclear. Nerve injury-induced protein 1 (NINJ1) plays an essential role in the induction of plasma membrane rupture (PMR), leading to damage-associated molecular patterns (DAMPs) release and triggering inflammation. We hypothesize that NINJ1-mediated high mobility group box 1 (HMGB1) release from macrophage PMR and neutrophil extracellular traps (NETs) formation synergistically contribute to the progression of acute oxalate nephropathy. Using a murine model of acute oxalate nephropathy, myeloid cell-specific deletion of Ninj1 mice (Ninj1fl/flvavcre) and their wild-type littermate control mice (Ninj1wt/wtvavcre) were administered intraperitoneal injection of 100 mg/kg sodium oxalate followed by drinking water with 3% sodium oxalate. Evaluation was conducted on tubular injury and inflammatory cell infiltration. In vitro studies involved isolation and culture of renal proximal tubular epithelial cells (RTECs), bone marrow-derived macrophages, and neutrophils to investigate NETs formation and HMGB1 release. Targeted deletion of Ninj1 in myeloid cells significantly mitigated oxalate-induced acute kidney injury by suppressing both HMGB1 release and NETs formation in vivo. In vitro investigations demonstrated that HMGB1 release from macrophage PMR and NETs formation in neutrophils mediated by NINJ1 oligomerization, which consequently coordinated to enhance renal tubular epithelial cell death. Our findings elucidate the pivotal role of NINJ1-dependent macrophage PMR and NETs formation in the progression of acute oxalate nephropathy, providing novel insights for its prevention and therapeutic targets.

Neutrophil extracellular traps activate hepatic stellate cells and monocytes via NLRP3 sensing in alcohol-induced acceleration of MASH fibrosis

ObjectiveAlcohol use in metabolic dysfunction-associated steatohepatitis (MASH) is associated with an increased risk of fibrosis and liver-related death. Here, we aimed to identify a mechanism through which repeated alcohol binges...

7972 A Shared Phenotype, Transcriptome, and Methylome in Turner Syndrome Across Various Karyotypes with Concurrent Chronic Neutrophil Activation

Abstract Disclosure: J. Just: None. L. Ridder: None. E. Johannsen: None. H. Christensen: None. A. Skakkebæk: None. C.H. Gravholt: None. Turner syndrome (TS) is a clinical diagnosis with a karyotype containing one X chromosome and complete or partial absence of the second X chromosome. TS is typically associated with clinical manifestations such as short stature, hypergonadotropic hypogonadism and metabolic conditions. The genome-wide changes in females with TS affect both transcriptome and methylome. Historically, genomic studies have primarily focused on the 45,X karyotype, leaving the impact of other karyotypes unexplored. Only 35-45% of the entire TS population has the 45,X karyotype, while the remaining TS women have a range of other karyotypes - i.e. 45,X/46,XX, 46,X,i(Xq), 46,X,i(Xp), 45,X/46,XY, ring X. This study aimed to bridge this gap by comparing TS individuals with the standard 45,X karyotype (TS 45,X) (n = 32) to those with alternative karyotypic forms (TS other karyotypes) (n = 24), alongside age-matched 46,XX controls (n = 33). Specifically, we analyzed the DNA methylation and gene expression profiles from whole-blood samples and explored the genetic and clinical similarities between these groups. Overall findings revealed a shared phenotype and an identical autosomal transcriptome and methylome between TS 45,X and TS other karyotypes. Furthermore, TS 45,X and TS other karyotypes shared the same expression profile for all pseudoautosomal 1 (PAR1) genes on the X chromosome. In addition, the expression of XIST from the q-arm of the X chromosome did not affect the autosomal transcriptome or methylome. Thus, the X chromosomal p-arm, and perhaps in particular PAR1, appear to be a main driver influencing the genome-wide transcriptome and methylome Combining DNA methylation, gene expression and white blood cell counts, we observed a higher neutrophil count (p < 0.01), and increased neutrophil activation in the combined TS group. The increased neutrophil activity, based on increased gene expression of neutrophil activation markers (myeloperoxidase, neutrophil elastase, S100A8/9, p < 0.01), suggested a pathological activation of neutrophils with concomitant release of pro-inflammatory mediators. We further validated the increase of neutrophils in TS women in an independent cohort using flow cytometry (p < 0.01). Emerging evidence has highlighted the substantial involvement of neutrophils in chronic inflammatory processes associated with metabolic diseases such as obesity and diabetes, which triggers a sustained low-grade inflammation partially driven by activated neutrophils. The neutrophil increase and activation in TS women may offer an explanation for this phenotypic trait observed in TS women. Therefore, identifying TS women with increased neutrophil activation in order to initiate anti-inflammatory treatment earlier could potentially mitigate the progression towards more severe metabolic disturbances and associated co-morbidities. Presentation: 6/1/2024

Nanoengineered Neutrophil as 19F-MRI Tracer for Alert Diagnosis and Severity A of Acute Lung Injury.

Acute lung injury (ALI) is a severe complication in clinical settings. Alert diagnosis and severity assessment of ALI is pivotal to ensure curative treatment and increase survival rates. However, the development of a precise ALI diagnostic strategy remains a pending task. Here, leveraging neutrophil's inflammation-homing and physiological barrier-navigating capability, a facile strategy is proposed for achieving targeted 19F-MRI detection of ALI based on the nanoengineered neutrophil internalized with perfluorocarbon nanoemulsion (Neu@PFC). The remodeling process poses a negligible impact on the neutrophil's inherent activation and transmigration functions. The migratory behavior of Neu@PFC toward pneumonia is confirmed in vivo using an LPS-induced ALI murine model. Direct intratracheal (i.t.) administration contributes to a vast deposition of Neu@PFC within the lung, allowing for real-time 19F-MRI visualization and the potential to predict progressive pneumonia. Furthermore, intravenous (i.v.) administration of Neu@PFC enables quantitative assessment of the extent of ALI due to the chemokine-guided neutrophil migration. This study not only provides a pathway to diagnose ALI, but also sheds light on the neutrophil recruitment and activation cues in different tissues and inflammatory conditions, which is a prerequisite for developing potential therapeutic approaches.

Neutrophils in nasal polyps exhibit transcriptional adaptation and proinflammatory role depend on local polyp milieu.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is an inflammatory upper airway disease, divided into eosinophilic CRSwNP (eCRSwNP) and noneosinophilic CRSwNP (neCRSwNP) according to eosinophilic levels. Neutrophils are major effector cells in CRSwNP. but their role in different inflammatory environments remain largely unclear. We performed an integrated transcriptome analysis of polyp-infiltrating neutrophils from CRSwNP patients, using healthy donor blood as a control. Flow cytometry and in vitro studies showed that neutrophils are activated in both CRSwNP type. The scRNA-sequencing analysis demonstrated that neutrophils were classified into five functional subsets, with GBP5+ neutrophils occurring mainly in neCRSwNPs and a high proportion of CXCL8+ neutrophils in both subendotypes. GBP5+ neutrophils exhibited significant IFN-I pathway activity in neCRSwNPs. CXCL8+ neutrophils displayed increased neutrophil activation scores and mainly secrete Oncostatin M (OSM), which facilitates communication with other cells. In vitro experiments revealed that OSM could enhance IL-13- or IL-17-mediated immune responses in nasal epithelial cells and fibroblasts. Our findings revealed that neutrophils exhibited transcriptional plasticity and activation when exposed to polyp tissue and exert their proinflammatory role in the pathogenesis of CRSwNP by releasing OSM to interact with epithelial cells and fibroblasts in a manner dependent on the inflammatory milieu.

High-grade serous ovarian cancer development and anti-PD-1 resistance is driven by IRE1α activity in neutrophils

ABSTRACT High-grade serious ovarian cancer (HGSOC) is an aggressive malignancy that remains refractory to current immunotherapies. While advanced stage disease has been extensively studied, the cellular and molecular mechanisms that promote early immune escape in HGSOC remain largely unexplored. Here, we report that primary HGSO tumors program neutrophils to inhibit T cell anti-tumor function by activating the endoplasmic reticulum (ER) stress sensor IRE1α. We found that intratumoral neutrophils exhibited overactivation of ER stress response markers compared with their counterparts at non-tumor sites. Selective deletion of IRE1α in neutrophils delayed primary ovarian tumor growth and extended the survival of mice with HGSOC by enabling early T cell-mediated tumor control. Notably, loss of IRE1α in neutrophils sensitized tumor-bearing mice to PD-1 blockade, inducing HGSOC regression and long-term survival in ~ 50% of the treated hosts. Hence, neutrophil-intrinsic IRE1α facilitates early adaptive immune escape in HGSOC and targeting this ER stress sensor might be used to unleash endogenous and immunotherapy-elicited immunity that controls metastatic disease.

A-307 Clinical utility of serum calprotectin in febrile infants presenting to the Emergency Department

Abstract Background Antimicrobial stewardship involves a delicate balance between the risk of undertreating individuals and the potential societal burden of overprescribing antimicrobials. However, early and accurate diagnosis of bacterial infections in critically ill patients is challenging, as the clinical manifestation is non-specific. Furthermore, the results from microbial cultures may be delayed, resulting in delayed antibiotic treatment, which is associated with increased mortality. Neutrophil activation is a major response to bacterial infection and calprotectin is the most abundant protein in the cytosolic fraction of neutrophils, shown as an early marker for neutrophil activation. The objective of this study was to evaluate the clinical performance of serum calprotectin in identifying bacterial infection in febrile infants presenting to an Emergency Department (ED) relative to other common biomarkers of infection. Methods 141 infants aged 28-90 days with suspected infectious disease presenting to the ED at The Hospital for Sick Children in Toronto, were included in this non-interventional investigation. All biomarkers except calprotectin were used in the routine clinical adjudication of the final diagnosis. Residual serum specimens collected as part of the standard care pathway were stored at -80°C prior to analysis of serum calprotectin using GCAL® assay (Gentian AS, Norway). Chart review was completed for key variables including, age (days), sex, chief complaint and temperature at ED presentation, and available laboratory test results (i.e., C-reactive protein and procalcitonin (Architect, Abbott Diagnostics), WBC count and neutrophil count (XN3000, Sysmex Europe GmbH), urinary leukocytes and nitrates, blood and/or urine bacterial culture). Final diagnosis, admission status, and antibiotic prescription at ED discharge were also extracted. Results Final discharge diagnosis in patient population included bacterial infection (n=23), viral infection (n=22), fever of unknown source (n=54) and other/unknown diagnosis (n=42). Antibiotics were prescribed in 36 cases, although bacterial infection could only be confirmed in 23 cases when final diagnosis was made. Statistically significant differences were observed in serum calprotectin (p<0.001), procalcitonin (p<0.001), and CRP (p<0.001) in patients with bacterial infections compared to patients with other discharge diagnoses. ROC curve analysis was performed for the identification of bacterial infections in the entire clinical cohort (n=141) and in only patients with laboratory confirmed bacterial or viral infection (n=41). Calprotectin and procalcitonin showed higher specificity than CRP and WBC count for differentiation between bacterial and viral infection in both cohorts. Performance of calprotectin in detection of bacterial infections was comparable to the performance of CRP, slightly better than performance of procalcitonin and superior to WBC count. Conclusions In this cohort, one third of the children that were prescribed antibiotics did not have confirmed bacterial infection. Hence, there is a need for adequate diagnostic tools to help discriminate between various kinds of infections. This study confirms serum calprotectin as a valuable biomarker for differentiation between types of infection and estimation of disease severity in febrile infants. Access to fast and accurate analysis of circulating calprotectin, combined with good clinical performance has the potential to make this biomarker a valuable complement to the current diagnostics of patients with bacterial infections and sepsis.

Identification of NET formation and the renoprotective effect of degraded NETs in lupus nephritis.

To explore molecular biomarkers associated with the pathophysiology and therapy of lupus nephritis (LN), we conducted a joint analysis of transcriptomic data from 40 peripheral blood mononuclear cells (PBMCs) (GSE81622) and 21 kidney samples (GSE112943) from the Gene Expression Omnibus database using bioinformatics. A total of 976 and 2,427 differentially expressed genes (DEGs) were identified in PBMCs and renal tissues. Seven and two functional modules closely related to LN were identified. Further enrichment analysis revealed that the neutrophil activation pathway was highly active in both PBMCs and the kidney. Subsequently, 16 core genes closely associated with LN were verified by protein-protein interaction screening and quantitative PCR. In vitro cell models and MRL/lpr mouse models confirmed that the abnormal expression of these core genes was closely linked to neutrophil extracellular traps (NETs) generated by neutrophil activation, while degradation of NETs led to downregulation of core gene expression, thereby improving pathological symptoms of LN. Therefore, identification of patients with systemic lupus erythematosus exhibiting abnormal expression patterns for these core genes may serve as a useful indicator for kidney involvement. In addition, targeting neutrophils to modulate their activation levels and inhibit aberrant expression of these genes represents a potential therapeutic strategy for treating LN. NEW & NOTEWORTHY The mechanisms by which immune cells cause kidney injury in lupus nephritis are poorly understood. We integrated and analyzed the transcriptomic features of PBMCs and renal tissues from the GEO database to identify key molecular markers associated with neutrophil activation. We confirmed that neutrophil extracellular traps (NETs) formed by neutrophil activation promoted the upregulation of key genes in cell and animal models. Targeted degradation of NETs significantly ameliorated kidney injury in MRL/lpr mice.

A Description of the THRIVE (“The Study of Host-Bacterial Relationships in Different Vaginal Environments”) Bacterial Vaginosis Observational Study

ObjectivesBacterial vaginosis (BV) contributes to poor reproductive health and is characterized by a displacement of Lactobacillus in the vaginal microbiome. However, treatment for BV is limited to antibiotics and half of the women treated experience recurrence within a year. THRIVE is a prospective study in XXXXX, which is designed to capture the daily variation of the microbiome and host mucosal immunity during treatment. The objective of this study is to identify host and bacterial factors that associate with vaginal microbiome stability to better inform therapeutic interventions. MethodsWomen treated for BV, and controls, are followed for 6 months collecting daily vaginal swabs and monthly questionnaires. Comprehensive mucosal sampling, including swabs, cytobrushes, biopsies, and blood are collected at baseline, months 1 and 6 post-enrolment. ResultsWe performed analysis on the first 52 participants, (19 BV+, 33 BV–). Molecular profiling by 16s RNA sequencing showed 20 women with non-Lactobacillus-dominant microbiomes and 32 with Lactobacillus-dominant microbiomes, with increased microbial diversity in non-Lactobacillus-dominant microbiomes (P = 3.1E-05). A pilot analysis in 2 participants demonstrates that multi-omics profiling of self-collected daily swabs provides high-quality data identifying 73 bacterial species, 1773 mucosal proteins and 117 metabolites. Initial flow cytometry analysis showed an increased cluster of differentiation (CD)4+ T cells and neutrophil activation (CD11b+CD62Lneg/dim) in the positive participant at baseline, while after treatment these shifted and resembled the control participant. ConclusionsThis study provides a framework to comprehensively investigate the kinetics of vaginal mucosal microbiome alterations, providing further insight into host and molecular features predicting BV recurrence.

Recognition of differently expressed genes and DNA methylation markers in patients with Lupus nephritis

Abstract Background and Objectives Systemic lupus erythematosus (SLE) is distinguished by dysregulated immune system activity, resulting in a spectrum of clinical manifestations, with lupus nephritis being particularly prominent. This study endeavors to discern novel targets as potential therapeutic markers for this condition. Methods Weighted correlation network analysis (WGCNA) was used to construct the network and select the key hub genes in the co-expression module based on the gene expression dataset GSE81622. Subsequently, functional enrichment and pathway analysis were performed for SLE and lupus nephritis. In addition, also identify genes and differences in SLE with lupus nephritis and methylation site. Finally, qRT-PCR and western blot were used to verify the up-regulated expression levels of the selected key genes. Results Within the co-expression modules constructed by WGCNA, the MElightcyan module exhibited the strongest positive correlation with lupus nephritis (0.4, P = 0.003), while showing a weaker correlation with the control group SLE (0.058) and a negative correlation with the control group (-0.41, P = 0.002). Additionally, the MEgreenyellow module displayed the highest positive correlation with SLE (0.25), but its P value was 0.06, which did not reach statistical significance(P > 0.05). Furthermore, it had a negative correlation with the control group was (-0.38, P = 0.004). The module associated with lupus nephritis was characterized by processes such as neutrophil activation (neutrophil_activation), neutrophil degranulation (neutrophil_degranulation), neutrophil activation involved in immune response (neutrophil_activation_involved_in_immune_response), neutrophils mediated immune (neutrophil_mediated_immunity) and white blood cells degranulation (leukocyte_degranulation) and so on the adjustment of the process. Secondly, in the analysis of SLE samples, the identification of differentially expressed genes revealed 125 genes, with 49 being up-regulated and 76 down-regulated. In the case of lupus nephritis samples, 156 differentially expressed genes were discerned, include in 70 up-regulated and 86 down-regulated genes. When examining differential methylation sites, we observed 12432 such sites in the SLE sample analysis, encompassing 2260 hypermethylation sites and 10172 hypomethylation sites. In the lupus nephritis samples analysis, 9613 differential methylation sites were identified, comprising 4542 hypermethylation sites and 5071 hypomethylation sites. Substantiating our findings, experimental validation of the up-regulated genes in lupus nephritis confirmed increased levels of gene expression and protein expression for CEACAM1 and SLC2A5. Conclusions We have identified several genes, notably CEACAM1 and SLC2A5, as potential markers for lupus nephritis. Their elevated expression levels and reduced DNA methylation in lupus nephritis contribute to a more comprehensive understanding of the aberrant epigenetic regulation of expression in this condition. These findings hold significant implications for the diagnosis and therapeutic strategies of lupus nephritis.

Neutrophil-specific Shp1 loss results in lethal pulmonary hemorrhage in mouse models of acute lung injury.

The acute respiratory distress syndrome (ARDS) is associated with significant morbidity and mortality and neutrophils are critical to its pathogenesis. Neutrophil activation is closely regulated by inhibitory tyrosine phosphatases including Src homology region 2 domain containing phosphatase-1 (Shp1). Here, we report that loss of neutrophil Shp1 in mice produced hyperinflammation and lethal pulmonary hemorrhage in sterile inflammation and pathogen-induced models of acute lung injury (ALI) through a Syk kinase-dependent mechanism. We observed large intravascular neutrophil clusters, perivascular inflammation, and excessive neutrophil extracellular traps in neutrophil-specific Shp1 knockout mice suggesting an underlying mechanism for the observed pulmonary hemorrhage. Targeted immunomodulation through the administration of a Shp1 activator (SC43) reduced agonist-induced reactive oxygen species in vitro and ameliorated ALI-induced alveolar neutrophilia and NETs in vivo. We propose that the pharmacologic activation of Shp1 has the potential to fine-tune neutrophil hyperinflammation that is central to the pathogenesis of ARDS.

Assessment of mammary stress in early lactating crossbred (Alpine × Beetal) does during heat stress in conjunction with milk somatic cells and non-invasive indicators

Milk somatic cell counts (SCC) are crucial for monitoring dairy animals' mammary health and milk quality. This study was initiated to assess the mammary health of early lactating crossbred (Alpine × Beetal) does during periods of heat stress. The udder skin surface temperature (USST), total and differential milk SCC, milk composition, milk cortisol levels and phagocytic activity (PA) of milk neutrophils and macrophages were estimated in these does. Results indicated a significant (p<0.01) increase in the milk neutrophils to macrophages (N: M) ratio and milk cortisol levels with milk somatic cell score (SCS). The PA of milk neutrophils decreased significantly (p<0.01), while that of macrophages increased significantly (p<0.01) with higher SCC. On Pearson correlation, milk SCS revealed strong positive correlation (p<0.01) with N: M ratio (r=0.90), USST (r=0.98), milk cortisol (r=0.97), milk pH (r=0.83) and conductivity (r=0.92). Conversely, milk lactose exhibited a moderate negative correlation of (r= −0.48) with SCS. These findings suggest that USST infrared thermography and milk cortisol levels are promising non-invasive methods for rapidly assessing mammary stress in early lactating does during heat stress.

Features of the immune system and levels of blood transport components in residents of the arctic of the Russian Federation.

The peripheral venous blood of 191 residents of the Arctic of the Russian Federation (Murmansk Oblast) aged 21-55 analyzed comprehensively. Blood was sampled from the ulnar vein on an empty stomach in the morning. The hemogram, phagocytic activity of neutrophils, lymphocyte content with CD3, CD4, CD8, CD10, CD19, CD16, CD71, CD25, HLA-DR and CD95 phenotypes, concentrations of cytokines: TNF-α, IFN-γ, IL-6, IL-10, extracellular receptor pool: sCD71, sCD62L, sApo-1/Fas, sFasL, circulating immune complexes (CIC) and blood transport components: haptoglobin (Hp), transferrin, IgM, IgG, IgA, IgE (immunoglobulins M, G, A and E) low-density lipoproteins (LDL), and high density lipoprotein (HDL) were evaluated. The results were analyzed using descriptive statistics and comparative, factorial, and regression analyses. Residents of the Murmansk Oblast exhibit a high prevalence of erythrocytosis (62.3%), thrombocytosis (25.0%), leukocytosis (20.1%), and increased hemoglobin (Hb) concentrations (42.5%), coupled with a significantly decreased level of active phagocytes (47.6%). A significantly decreased level of lymphocytes with phenotypes CD3 (92.4%), CD4 (40.4%), CD71 (62.3%) was revealed, coupled with the activation of lymphocytes with phenotypes CD16 (50.1%), CD8 (37.5%), CD19 (15.9%) and increased concentrations of pro-inflammatory IFN-γ (47.9%), IL-6 (33.3%), and TNF-α (20.1%). Elevated levels of sCD71 (56.6%), sCD62L (32.1%), sApo-1/Fas (22.1%), sFasL (10.2%), and autoantibodies to double-stranded DNA (57.1%), RNA (10.4%), and oLDL (oxidized-modified LDL) (16.3%) were recorded in residents of the Murmansk Oblast. Arctic residents showed elevated concentrations of Hp (63.3%), IgM (63.9%), IgA (42.4%), IgE (18.8%), LDL (24.9%), and a decreased level of HDL (21.1%) owing to an ApoA-I ligand deficiency (58.1%). Elevated levels of Hp in the blood are associated with increased erythrocyte aggregation frequency, T-lymphocyte activation, and increased concentration of free receptors of the extracellular pool. Increased IgM and IgA levels in the blood are attributed to the need to bind components of the extracellular receptor pool sCD71, sCD62L, sApo-1/Fas, sFasL and are associated with decreased levels lymphocyte with CD8 and CD16 phenotypes. The humoral immune response in Arctic residents is triggered when the cellular component of immunity is under strain which compounded by ineffective clearance of metabolic byproducts owing to imbalanced LDL and HDL lipid transport complexes. The immune system of Arctic residents is characterized by excessive cytotoxic activity of lymphocytes and increased concentrations of pro-inflammatory cytokines, free forms of receptors of the extracellular pool, and autoantibodies. The change in the content of transport components of the blood system is directed at maintaining homeostasis by exhibiting antioxidant, anti-inflammatory, and immunoregulatory properties.

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.

Antithrombin regulates neutrophil activities through the stimulation of C-type lectin family 1A

It has been suggested that a serine proteinase inhibitor, antithrombin (AT), exerts anti-inflammatory effects on different types of cells, independent of thrombin inhibition. In the present study, we aimed to identify a specific receptor for antithrombin by a screening method using a transmembrane tethered-AT ligand expressed on HEK293T cells together with the co-expression of candidate receptors, followed by the immunoprecipitation of a complex of AT ligand with a receptor. We identified C-type lectin family 1A (CLEC1A) as a receptor for AT. We confirmed the binding of AT to the extracellular domain of CLEC1A using surface plasmon resonance. Recombinant as well as native AT concentration-dependently induced the rounding of purified human neutrophils in shape, associated with the suppression of spontaneous ROS production in vitro but argatroban did not, indicating the independence of AT effects on thrombin inhibition. Native AT maintained the passage of neutrophils through the artificial microcapillaries. Both AT also enhanced the phagocytosis of pHrodo-labeled E.coli and prolonged the viability of the neutrophils. The cellular effects of AT were similar to those of HRG which has the same CLEC1A as a receptor, and were partially inhibited by the addition of anti-CLEC1A antibody to the incubation media. These results suggested that CLEC1A is a novel receptor for AT and the stimulation of CLEC1A by AT at least in part mediates the important functional changes of human neutrophils.

The bacterial serine protease inhibitor ecotin inhibits neutrophil elastase enzymatic activity in cystic fibrosis sputa

Cystic Fibrosis (CF) airway disease is characterized by impaired mucociliary clearance, chronic, polymicrobial infections and robust, neutrophil-dominated inflammation. Pulmonary disease is the leading cause of morbidity and mortality in people with CF and is due to progressive airflow obstruction and ultimately respiratory failure. One of the earliest abnormalities in CF airway disease is the recruitment of neutrophils to the lungs. Neutrophil activation leads to the release of their intracellular content, including neutrophil elastase (NE), that damages lung tissues in CF. Our goal is to characterize a known bacterial NE inhibitor, ecotin, in the CF airway environment. Our results indicate that ecotins cloned from four Gram-negative bacterial species (Campylobacter rectus, Campylobacter showae, Escherichia coli and Pseudomonas aeruginosa) inhibit NE activity in CF sputum samples in a dose-dependent manner. Although we observed differences in the NE-inhibitory activity of the tested ecotins with the Campylobacter homologs being the most effective in NE inhibition in CF sputa, none of the ecotins impaired the ability of human neutrophils to kill major CF respiratory pathogens, P. aeruginosa or S. aureus, in vitro. Overall, we demonstrate that bacterial ecotins inhibit NE activity in CF sputa without compromising bacterial killing by neutrophils.

MicroRNA-375 modulates neutrophil chemotaxis via targeting Cathepsin B in zebrafish

Neutrophils are crucial for defense against numerous infections, and their migration and activations are tightly regulated to prevent collateral tissue damage. We previously performed a neutrophil-specific miRNA overexpression screening and identified several microRNAs, including miR-375, as potent modulators for neutrophil activity. Overexpression of miR-375 decreases neutrophil motility and migration in zebrafish and human neutrophil-like cells. We screened the genes downregulated by miR-375 in zebrafish neutrophils and identified that Cathepsin B (Ctsba) is required for neutrophil motility and chemotaxis upon tail wounding and bacterial infection. Pharmacological inhibition or neutrophil-specific knockout of ctsba significantly decreased the neutrophil chemotaxis in zebrafish and survival upon systemic bacterial infection. Notably, Ctsba knockdown in human neutrophil-like cells also resulted in reduced chemotaxis. Inhibiting integrin receptor function using RGDS rescued the neutrophil migration defects and susceptibility to systemic infection in zebrafish with either miR-375 overexpression or ctsba knockout. Our results demonstrate that miR-375 and its target Ctsba modulate neutrophil activity during tissue injury and bacterial infection in vivo, providing novel insights into neutrophil biology and the overall inflammation process.

Neutrophil count as a risk factor for cardiovascular diseases: how can we manage it?

Neutrophils activation plays a pivotal role in the pathogenesis of atherosclerotic plaque formation, progression and rupture. An association between the leukocyte count and the risk of developing myocardial infarction has been well known for many years; however, only recently did Mendelian randomization studies show that a high neutrophil count is a causal risk factor for atherosclerotic cardiovascular disease. On the other hand, experimental studies show that depletion of circulating neutrophils impairs plaque development. Clopidogrel, an antiplatelet agent, is widely used in combination with aspirin to reduce the incidence of ischemic events in patients treated with coronary stenting. Chronic treatment with this drug reduces inflammatory markers and neutrophil numbers, rarely causing severe leukopenia. The purpose of this review is to present recent evidence showing the link between neutrophil number and the development of cardiovascular diseases and to discuss how the clopidogrel-induced reduction in the neutrophil count may be a beneficial off-target effect of this drug.

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.

Immunogram dynamics of students under the influence of dosed physical activity

Analysis of the dynamics of the state of blood leukocytes in muscular activity is relevant due to the development of methods of differentiation and assessment of the state of these cells. The purpose of the study is to study the dynamics of the phagocytic and NBT activity of neutrophils, as well as the content of CD lymphocytes of peripheral blood in students when moving on a treadmill. For students 18-20 years old involved in athletics (3 boys and 4 girls), blood was taken from the ulnar vein before and after a 15-minute jog, as well as after a 15-minute run of moderate intensity. The average level of physical performance of the examined PWC170 was 16.25±2.21 kGm/min/kg. Assessment of phagocytosis and NBT activity of neutrophils and determination of blood CD lymphocytes by immunophenotyping were performed using flow cytometry. It was found that in girls at rest, the relative content of lymphocytes and NK cells was significantly lower, and the level of induced NBT activity of neutrophils and the relative content of CD4-CD8- and CD4+CD8+ lymphocytes was significantly higher than in boys. At the same time, the dynamics of the leukogram parameters examined during the dosed physical activity fully corresponded to the development of the lymphocytic phase of myogenic leukocytosis. With an increase in the power of repeated exercise against the background of a general increase in the number of phagocytic neutrophils, the balance of spontaneous NBT-positive and reserve potentially active neutrophils significantly increased relative to the content of NBT-negative cells in the induced test. At the same time, the level of neutrocytes in girls was significantly higher, causing a higher level of phagocyte count and absolute content of NBT-positive neutrophils in a spontaneous and induced test. At the same time, against the background of an increase in the absolute content of CD4-CD8- and TNK lymphocytes, a statistically significant increase in the relative and absolute content of NK cells was observed. In contrast to the boys after the 2nd physical exercise, the relative and absolute content of NK cells, as well as the increase in the percentage of HBT-positive cells in the induced test, were significantly higher.