Neutrophil Surface Research Articles

The role of neutrophils in tPA thrombolysis after stroke: a malicious troublemaker

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.

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.

The Internalization Pathways of Liposomes, PLGA, and Magnetic Nanoparticles in Neutrophils.

Neutrophils are emerging as promising candidates for cell-based nanodrug delivery to tumors due to their unique biological properties. This study aims to investigate the mechanisms of nanoparticle internalization by neutrophils, specifically focusing on liposomes, poly(lactic-co-glycolic acid) (PLGA), and magnetite nanoparticles. Understanding these mechanisms could enhance the efficiency of neutrophil-based nanodrug delivery for cancer treatment. Neutrophils were isolated from the peripheral blood of mice bearing 4T1 mammary adenocarcinoma. Confocal microscopy, transmission electron microscopy, and flow cytometry were employed to evaluate the uptake of liposomes, PLGA, and magnetite nanoparticles by neutrophils. The effects of cultivation conditions, such as the presence or absence of plasma in the growth medium, were also examined. Additionally, the roles of immunoglobulins (IgG/IgM) and cell surface receptors (Fc and scavenger receptors) in nanoparticle internalization were explored. All types of nanoparticles were successfully internalized by neutrophils, though the mechanisms of uptake varied. Plasma presence in the medium significantly influenced nanoparticle binding, particularly for PLGA nanoparticles. Internalization of PLGA nanoparticles was found to depend on the presence of IgG/IgM in the medium and Fc receptors on neutrophil surfaces, while scavenger receptors were not involved. Understanding the distinct endocytosis pathways for different nanoparticles can improve the efficacy of neutrophil loading with nanodrugs, potentially advancing the development of neutrophil-based cancer therapies. The findings underscore the importance of the extracellular environment in modulating nanoparticle uptake.

Neutrophil CD10 and CD16 as markers of generalized infection development in newborns

BACKGROUND: Neonatal sepsis remains a critical concern. Thus, predictors of infection development and generalization should be determined. AIM: This study aimed to determine novel neutrophil surface biomarkers for early prediction of the infections in newborns. MATERIALS AND METHODS: This observational, single-center, prospective, selective, uncontrolled, unblinded experimental study included 261 newborns, with a mean postconceptual age of 38.7 (38.4–39.0) weeks and a mean gestation age of 38.0 (37.7–38.2) weeks. Blood samples were collected into vacutainers on hospitalization day 1. Patients were enrolled between April 2022 and December 2023. The primary endpoints were length of stay in the ICU and total length of hospitalization in patients with normal and decreased values of CD16 and CD10 neutrophils and HLA-DR monocytes. The expression of CD16 on CD62Lhigh neutrophils, total neutrophil CD10, and monocyte HLA-DR were evaluated by flow cytometry. RESULTS: We assessed infants in the “control” (n = 96), “localized infection” (n = 95), and “generalized infection” (n = 70) subgroups. In all patients, a decrease in CD16 was associated with an increase in the median ICU stay from 4 to 8 days (p = 9.33 × 10–8) and total stay from 14 to 22 days (p = 1.58 × 10–7). A decrease in CD10 was associated with an increase in median ICU stay from 4 to 8 days (p = 3.01 × 10–6) and in the total stay from 14 to 19 days (p = 2.78 × 10–5). A decrease in monocytic HLA-DR was associated with a longer ICU and total hospital stay: 4 vs 8 days (p = 7.16 × 10–5) and 14 vs 21 days (p = 4.03 × 10–5), respectively. The median ICU stay in patients with a decrease in CD16 but normal CD10 was 4 days, whereas a decrease in both indicators was associated with prolonged hospital stay to 11 days (p = 2.13 × 10–5). The median hospital stay in patients with decreased CD16 but normal CD10 was 16 days, whereas the drop of both markers was related with increase of hospitalization stay to 23 days (p = 3.36 × 10–6). In the “localized infection” subgroup, low CD16 was associated with an increased median ICU stay from 4 to 6 days (p = 0.010) and the median hospital stay from 13 to 19 days (p = 4.14 × 10–4). In the “generalized infection” subgroup, decreased CD10 was related with prolongation of the median ICU stay from 7 to 11 days (p = 0.011) and the median total duration of hospitalization from 19 to 27 days (p = 0.037). CONCLUSIONS: A decrease in CD10 and CD16 on the neutrophils at the start of clinical is an unfavorable prognostic factor of infectious and septic complications in newborns.

Esculetin facilitates post-stroke rehabilitation by inhibiting CKLF1-mediated neutrophil infiltration.

Esculetin (ESC) is a coumarin-derived phytochemical prevalent in traditional Chinese medicine that exhibits anti-acute ischemic stroke activities. Our previous studies demonstrate that CKLF1 is a potential anti-stroke target for coumarin-derived compound. In this study we investigated whether CKLF1 was involved in the neuroprotective effects of ESC against photothrombotic stroke in mice. The mice were treated with ESC (20, 40 or 80 mg·kg-1·d-1, i.g.) for two weeks. The therapeutic effect of ESC was assessed using MRI, neurological function evaluation, and a range of behavioral tests on D1, 3, 7 and 14 of ESC administration. We showed that oral administration of ESC dose-dependently reduced the cerebral infarction volume within one week after stroke, improved behavioral performance, and alleviated neuropathological damage within two weeks. Functional MRI revealed that ESC significantly enhanced the abnormal low-frequency fluctuation (ALFF) value of the motor cortex and promoted functional connectivity between the supplementary motor area (SMA) and multiple brain regions. We demonstrated that ESC significantly reduced the protein levels of CKLF1 and CCR5, as well as the CKLF1/CCR5 protein complex in the peri-infarcted area. We showed that ESC (0.1-10 μM) dose-dependently blocked CKLF1-induced chemotactic movement of neutrophils in the Transwell assay, reducing the interaction of CKLF1/CCR5 on the surface of neutrophils, thereby reducing neutrophil infiltration, and decreasing the expression of ICAM-1, VCAM-1 and MMP-9 in the peri-infarct tissue. Knockout of CKLF1 reduced brain infarction volume and motor dysfunction after stroke but also negated the anti-stroke efficacy and neutrophil infiltration of ESC. These results suggest that the efficacy of ESC in promoting post-stroke neural repair depends on its inhibition on CKLF1-mediated neutrophil infiltration, which offering novel perspectives for elucidating the therapeutic properties of coumarins.

INHIBITION OF INTEGRIN VLA-3 AND TETRASPANIN CD151 PROTECTS AGAINST NEUTROPHIL-MEDIATED ENDOTHELIAL DAMAGE.

Background: The recruitment of neutrophils to sites of localized injury or infection is initiated by changes on the surface of endothelial cells located in proximity to tissue damage. Inflammatory mediators, such as TNF-α, increase surface expression of adhesive ligands and receptors on the endothelial surface to which neutrophils tether and adhere. Neutrophils then transit through the activated endothelium to reach sites of tissue injury with little lasting vascular injury. However, in cases of sepsis, the interaction of endothelial cells with highly activated neutrophils can cause damage vascular damage. The identification of molecules that are essential for neutrophil diapedesis may reveal targets of therapeutic opportunity for preservation of endothelial function in the presence of critical illness. We tested the hypothesis that inhibition of neutrophil β1 integrin very late antigen-3 (VLA-3; α3β1) and/or inhibition of the tetraspanin (TM4) family member CD151 would protect against neutrophil-mediated loss of endothelial function. Methods: Blood was obtained from septic patients or healthy donors. Neutrophils were purified, and aliquots were treated with/without proinflammatory molecules. Confluent human umbilical vascular endothelial cells were activated with TNF-α. Electric cell impedance sensing was used to determine monolayer resistance over time after the addition of neutrophils that were treated with blocking antibodies against VLA-3 and/or CD151 or isotype controls. Groups (depending on relevancy) were analyzed by Mann-Whitney U test, Wilcoxon test, or repeated-measures one-way ANOVA. Results: Neutrophils from septic patients and neutrophils activated ex vivo reduced endothelial monolayer resistance to a greater extent than neutrophils from healthy donors. Antibody blockade of VLA-3 and/or CD151 significantly reduced activation-associated endothelial damage. Similar findings were demonstrated on fibronectin, collagen I, collagen IV, and laminin, suggesting that neutrophil surface VLA-3 and CD151 are responsible for endothelial damage regardless of substrata and are likely to be operative in all bodily tissues. Conclusion: This report identifies VLA-3 and CD151 on the activated human neutrophil, which are responsible for damage to endothelial function. Targeting these molecules in vivo may demonstrate preservation of organ function during critical illness.

Inflammation and Acinar Cell Dual-Targeting Nanomedicines for Synergistic Treatment of Acute Pancreatitis via Ca2+ Homeostasis Regulation and Pancreas Autodigestion Inhibition.

Severe acute pancreatitis (AP) is a life-threatening pancreatic inflammatory disease with a high mortality rate (∼40%). Existing pharmaceutical therapies in development or in clinical trials showed insufficient treatment efficacy due to their single molecular therapeutic target, poor water solubility, short half-life, limited pancreas-targeting specificity, etc. Herein, acid-responsive hollow mesoporous Prussian blue nanoparticles wrapped with neutrophil membranes and surface modified with the N,N-dimethyl-1,3-propanediamine moiety were developed for codelivering membrane-permeable calcium chelator BAPTA-AM (BA) and trypsin activity inhibitor gabexate mesylate (Ga). In the AP mouse model, the formulation exhibited efficient recruitment at the inflammatory endothelium, trans-endothelial migration, and precise acinar cell targeting, resulting in rapid pancreatic localization and higher accumulation. A single low dose of the formulation (BA: 200 μg kg-1, Ga: 0.75 mg kg-1) significantly reduced pancreas function indicators to close to normal levels at 24 h, effectively restored the cell redox status, reduced apoptotic cell proportion, and blocked the systemic inflammatory amplified cascade, resulting in a dramatic increase in the survival rate from 58.3 to even 100%. Mechanistically, the formulation inhibited endoplasmic reticulum stress (IRE1/XBP1 and ATF4/CHOP axis) and restored impaired autophagy (Beclin-1/p62/LC3 axis), thereby preserving dying acinar cells and restoring the cellular "health status". This formulation provides an upstream therapeutic strategy with clinical translation prospects for AP management through synergistic ion homeostasis regulation and pancreatic autodigestion inhibition.

Neisseria gonorrhoeae scavenges host sialic acid for Siglec-mediated, complement-independent suppression of neutrophil activation.

Neisseria gonorrhoeae, the bacterium that causes gonorrhea, is an urgent global health concern due to increasing infection rates, widespread antibiotic resistance, and its ability to thwart protective immune responses. The mechanisms by which Gc subverts protective immune responses remain poorly characterized. One way N. gonorrhoeae evades human immunity is by adding sialic acid that is scavenged from the host onto its lipooligosaccharide, using the sialyltransferase Lst. Here, we found that sialylation enhances N. gonorrhoeae survival from neutrophil assault and inhibits neutrophil activation, independently of the complement system. Our results implicate bacterial binding of sialic acid-binding lectins (Siglecs) on the neutrophil surface, which dampens neutrophil antimicrobial responses. This work identifies a new role for sialylation in protecting N. gonorrhoeae from cellular innate immunity, which can be targeted to enhance the human immune response in gonorrhea.

Systemic neutrophil degranulation and emergency granulopoiesis in patients with Clostridioides difficile infection

ObjectivesClostridioides difficile infection (CDI) is characterized by neutrophilia in blood, with a high leukocyte count accompanying severe infection. In this study, we characterized peripheral blood neutrophil activation and maturity in CDI by (i) developing a method to phenotype stored neutrophils for disease-related developmental alterations and (ii) assessing neutrophil-associated biomarkers. MethodsWe stored fixed leukocytes from blood collected within 24 h of diagnosis from a cohort of hospitalized patients with acute CDI. Additional study cohorts included recurrent CDI patients at time of and two months after FMT therapy and a control healthy cohort. We assessed levels of neutrophil surface markers CD66b, CD11b, CD16 and CD10 by flow cytometry. Plasma neutrophil elastase and lipocalin-2 were measured using ELISA, while G-CSF, GM-CSF and cytokines were measured using O-link Proteomic technology. ResultsCD66b+ neutrophil abundance assessed by flow cytometry correlated well with complete blood counts, establishing that neutrophils in stored blood are sufficiently well-preserved for phenotyping by flow cytometry. Neutrophil abundance was significantly increased in CDI patients compared to healthy controls. Emergency granulopoiesis in acute CDI patients was evidenced by lower neutrophil surface expression of CD10, CD11b and CD16. CD10+ staining of neutrophils started to recover within 3–7 days of CDI treatment. Neutrophil activation and degranulation were higher in acute CDI as assessed by plasma neutrophil elastase and lipocalin-2. Biomarker levels in immunocompetent subjects were associated with recurrence and fatal outcomes. ConclusionsNeutrophil activation and emergency granulopoiesis characterize the early immune response in acute CDI, with plasma degranulation biomarkers predictive of disease severity.

Cell Surface Parameters for Accessing Neutrophil Activation Level with Atomic Force Microscopy.

In this study, we examine the topography and adhesion images of the cell surface of neutrophils during the activation process. Our analysis of cell surface parameters indicates that the most significant changes in neutrophils occur within the first 30 min of activation, suggesting that reactive oxygen species may require approximately this amount of time to activate the cells. Interestingly, we observed surface granular structure as early as 10 min after neutrophil activation when examining atomic force microscopy images. This finding aligns with the reorganization observed within the cells under confocal laser scanning microscopy. By analyzing the cell surface images of adhesion, we identified three spatial surface parameters that correlate with the activation time. This finding enables us to estimate the degree of activation by using atomic force microscopy maps of the cell surface.

The Role of Neutrophil Extracellular Traps in the Outcome of Malignant Epitheliomas: Significance of CA215 Involvement

Neutrophil extracellular traps (NETs) were originally discovered as a part of the innate immune response of the host to bacteria. They form a web-like structure that can immobilize microorganisms or exhibit direct antimicrobial properties, such as releasing reactive oxygen species (ROS). NETs are established when neutrophils undergo a sort of cellular death following exposure to ROS, chemokines, cytokines, or other soluble factors. This process results in the release of the neutrophil’s DNA in a web-like form, which is decorated with citrullinated histones (H3/H4-cit), neutrophil elastase (NE), and myeloperoxidase (MPO). Emerging studies have put into perspective that NETs play an important role in oncology as they were shown to influence tumor growth, malignant initiation, and proliferation, mediate the transition from endothelial to mesenchymal tissue, stimulate angiogenesis or metastasis, and can even help cancer cells evade the immune response. The role of NETs in cancer therapy resides in their ability to form and act as a mechanical barrier that will provide the primary tumor with a reduced response to irradiation or pharmaceutical penetration. Subsequently, cancer cells are shown to internalize NETs and use them as a strong antioxidant when pharmaceutical treatment is administered. In this review, we explored the role of NETs as part of the tumor microenvironment (TME), in the context of malignant epitheliomas, which are capable of an autonomous production of CA215, a subvariant of IgG, and part of the carcinoembryonic antigen (CEA) superfamily. Studies have shown that CA215 has a functional Fc subdivision able to activate the Fc-gamma-RS receptor on the surface of neutrophils. This activation may afterward stimulate the production of NETs, thus indicating CA215 as a potential factor in cancer therapy surveillance.

Neutrophil surface RNAs promote entry to inflamed sites.

Neutrophil surface RNAs promote entry to inflamed sites.

Endoplasmic Reticulum Protein 72 Regulates Integrin Mac-1 Activity to Influence Neutrophil Recruitment.

Integrins mediate the adhesion, crawling, and migration of neutrophils during vascular inflammation. Thiol exchange is important in the regulation of integrin functions. ERp72 (endoplasmic reticulum-resident protein 72) is a member of the thiol isomerase family responsible for the catalysis of disulfide rearrangement. However, the role of ERp72 in the regulation of Mac-1 (integrin αMβ2) on neutrophils remains elusive. Intravital microscopy of the cremaster microcirculation was performed to determine in vivo neutrophil movement. Static adhesion, flow chamber, and flow cytometry were used to evaluate in vitro integrin functions. Confocal fluorescent microscopy and coimmunoprecipitation were utilized to characterize the interactions between ERp72 and Mac-1 on neutrophil surface. Cell-impermeable probes and mass spectrometry were used to label reactive thiols and identify target disulfide bonds during redox exchange. Biomembrane force probe was performed to quantitatively measure the binding affinity of Mac-1. A murine model of acute lung injury induced by lipopolysaccharide was utilized to evaluate neutrophil-associated vasculopathy. ERp72-deficient neutrophils exhibited increased rolling but decreased adhesion/crawling on inflamed venules in vivo and defective static adhesion in vitro. The defect was due to defective activation of integrin Mac-1 but not LFA-1 (lymphocyte function-associated antigen-1) using blocking or epitope-specific antibodies. ERp72 interacted with Mac-1 in lipid rafts on neutrophil surface leading to the reduction of the C654-C711 disulfide bond in the αM subunit that is critical for Mac-1 activation. Recombinant ERp72, via its catalytic motifs, increased the binding affinity of Mac-1 with ICAM-1 (intercellular adhesion molecule-1) and rescued the defective adhesion of ERp72-deficient neutrophils both in vitro and in vivo. Deletion of ERp72 in the bone marrow inhibited neutrophil infiltration, ameliorated tissue damage, and increased survival during murine acute lung injury. Extracellular ERp72 regulates integrin Mac-1 activity by catalyzing disulfide rearrangement on the αM subunit and may be a novel target for the treatment of neutrophil-associated vasculopathy.

The Effect of Cytokine Adsorption on Leukocyte and Platelet Activation after Extracorporeal Cardiopulmonary Resuscitation.

Post-cardiac arrest syndrome (PCAS) is a frequent complication following successful cardiopulmonary resuscitation and correlates with poor outcome. PCAS is characterized by an excessive inflammatory response to whole-body ischemia and reperfusion. Cytokine adsorption was suggested as an adjunctive treatment option for the removal of cytokines from the patients' blood to restore the physiological equilibrium of pro- and anti-inflammatory activity and thus mitigate hemodynamic instability and end-organ complications. To better understand the cellular effects of cytokine adsorption in patients receiving extracorporeal cardiopulmonary resuscitation (ECPR) after in- and out-of-hospital cardiac arrest, we compared the activation status of neutrophils, monocytes, and platelets as well as the formation of platelet-leukocyte complexes in intravenous whole blood samples from an exploratory subgroup (n = 24) from the randomized CYTER study. At 48 hours after initiation of ECPR, flow cytometry analyses did neither reveal significant differences in neutrophil (CD11b, CD66b, L-selectin, and PSGL-1) and monocyte (CD11b, L-selectin, and PSGL-1) surface molecule expression nor in circulating platelet-monocyte complexes between patients receiving cytokine adsorption and those without. Data did not show a relevant effect of cytokine adsorption on neutrophil and monocyte activation during the first 48 hours after initiation of ECPR.

ECRG4 mediates host response to cutaneous infection by regulating neutrophil recruitment and adhesion receptor expression.

Rapid neutrophil recruitment is critical for controlling infection, with dysfunctional neutrophil responses in diseases like diabetes associated with greater morbidity and mortality. We have shown that the leukocyte protein ECRG4 enhances early neutrophil recruitment to cutaneous wounds and hypothesized that ECRG4 regulates the early host response to infection. Using a cutaneous infection model, we found that ECRG4 KO mice had decreased early neutrophil recruitment with persistent larger lesions, increased bacterial proliferation and systemic dissemination. Although previous work identified ECRG4 as a negative regulator of CD44 on neutrophils, the mechanism regulating neutrophil recruitment remained unknown. We demonstrated that pro-inflammatory responses were intact in ECRG4 KO mice, but found decreased neutrophil mobilization from bone marrow and decreased migration to chemokines. ECRG4 KO mouse neutrophils demonstrated an increase in adhesion molecules that regulate recruitment, including enhanced induction of integrin CD11b and increased L-selectin and CD44 on bone marrow neutrophils. Analysis of gene expression in leukocytes from diabetic patients found decreased ECRG4 expression with similar increased L-selectin and CD44. We propose a previously unrecognized mechanism governing neutrophil recruitment, whereby ECRG4 mediates neutrophil surface adhesion molecules that determine both recruitment and outside-in signaling that modulates neutrophil response to pro-inflammatory stimuli.

Effect of pre-infusion of hypertonic saline on postoperative delirium in geriatric patients undergoing shoulder arthroscopy: a randomized controlled trial

BackgroundNeuroinflammation may be a potential mechanism of postoperative delirium (POD) in geriatric patients, and hypertonic saline (HS) has immunomodulatory properties. The purpose of this study was to investigate whether HS could reduce the incidence of POD in elderly patients and its effect on neutrophil activation and inflammatory cytokine expression.MethodsWe studied the effect of pre-infusion of 4 mL/kg 3% hypertonic saline vs. 4 mL/kg 0.9% normal saline on POD in patients undergoing shoulder arthroscopy in a prospective, randomized, double-blind, controlled trial. Neutrophil surface molecules (CD11b, CD66b and CD64) were analyzed by flow cytometry. Circulating concentrations of inflammatory factors IL-1β, IL-6, TNF-α and neurological damage factor S100β were assessed by enzyme immunoassay. The Confusion Assessment Method-Chinese Revision (CAM-CR) was applied for the assessment of POD 1–3 days after surgery.ResultsThe incidence of POD in group H was significantly lower than that in group N (7.14% vs 26.83%, P = 0.036). The expression levels of inflammatory cytokines ( IL-6 and TNF-α) and neutrophil surface markers (CD11b and CD66b) were significantly lower in group H than in group N at 24 h after surgery (P = 0.018, P < 0.001, P < 0.001, P = 0.024). There were no significant differences in postoperative pain, nausea and vomiting, infection, phlebitis, and patients satisfaction between the two groups.ConclusionPre-infusion of HS can reduce the incidence of POD and the immune-inflammatory response.Trial registrationChinese Clinical Trial Registry (14/4/2022, registration number: ChiCTR2200058681.

Cell Surface RNAs Control Neutrophil Function

Recently, RNAs localizing to the outer cell surface have been reported in mammalian cells, with such RNAs containing glycan modifications (referred to as GlycoRNAs). However, the function of cell surface RNAs are poorly known. In this study, we investigated whether cell surface RNAs exist in neutrophils, what their functions are, and the mechanism by which they function. We first determined that neutrophils express cell surface GlycoRNAs. We utilized two strategies to assess cell surface GlycoRNAs. First, we utilized a sialic acid homologue to metabolically label glycans inside cells. Labeled glycans can be readily detected in purified total RNAs from primary murine neutrophils. The GlycoRNAs signals were depleted upon RNase digestion but not digestion by proteinase or DNase, supporting the existence of GlycoRNAs in neutrophils. Importantly, treating live neutrophils with RNase extracellularly removed over 90% of GlycoRNA signals, supporting that the majority of GlycoRNAs were located on the surface of neutrophils. Second, we directly visualized cell surface RNAs by labeling cellular RNAs with the nucleoside homologue 5'-bromouridine (BrU) and detecting live cells with an anti-BrU antibody applied extracellularly. These data support the existence of cell surface GlycoRNAs on neutrophils. We next revealed that cell surface GlycoRNAs play important functions in neutrophils to mediate transendothelial migration both in vivo and in vitro. We utilized an acute peritonitis model in which primarily neutrophils with cell surface GlycoRNAs removed by extracellular RNase were injected into circulation in mice that were treated with thioglycolate, and the migration of these neutrophils into the peritoneal cavity was quantified and compared to mock treated cells in vivo. We observed a 9-fold decrease of migration by neutrophils treated with extracellular RNase. To determine the in vitro function of cell surface RNAs, we tested the ability of neutrophils to migrate toward a chemoattractant in a trans-well assay. While neutrophils treated with extracellular RNase were viable and migrated similarly as control neutrophils, we observed a substantial defect in migration by extracellular-RNase-treated neutrophils when an endothelial layer was present on the tanswell membrane. A similar defect was observed when assaying neutrophil attachment to endothelial cells in vitro. This defect can be replicated using control neutrophils but by pre-blocking endothelial cells with purified neutrophil GlycoRNAs or the glycan fraction of GlycoRNAs. The defect in neutrophil endothelial interaction in vivo was observed by intravital confocal microscopy. These data support the function of neutrophil cell surface GlycoRNAs in helping transendothelial migration. Lastly, we found that neutrophil GlycoRNAs are bona fide ligands for P-selectin on endothelial surface. Removal of neutrophil surface RNAs with extracellular RNase treatment did not significantly change cell surface integrin levels or reactivity, but reduced recombinant P-selectin binding. Furthermore, recombinant P-selectin, but not recombinant E-selectin, can detect glycoRNAs in purified neutrophil total RNAs. Additionally, blocking endothelial cells with an antibody against P-selectin led to a significant reduction in GlycoRNA binding. These data support that GlycoRNA-P-selectin interaction, at least in part, mediate neutrophil transendothelial migration. Our data demonstrate a critical role of cell surface RNAs in neutrophils, and reveal a new dimension that regulate the function of hematopoietic cells.

Abstract 16734: Patterns of Inflammatory Activation in Cardiovascular Disease: Insights From the Randomized Colchicine PCI Trial

INTRODUCTION: Recent studies demonstrate that neutrophils accumulate in atherosclerotic lesions and that neutrophil counts correlate with disease severity. The randomized double-blind Colchicine-PCI study demonstrated that colchicine attenuated systemic inflammation (IL-6, CRP) after percutaneous coronary intervention compared with placebo. GOALS/AIMS: We characterized neutrophil activity in patients across a spectrum of cardiovascular phenotypes, including severity of coronary artery disease (CAD) and presentation of acute myocardial infarct (AMI). METHODS: The Colchicine-PCI biomarker sub-study enrolled 478 participants with pretreatment assessment of inflammatory biomarkers. White blood cell count and differential were performed using a hematology analyzer. Soluble markers of neutrophil activity, including neutrophil extracellular traps (NETs), and systemic inflammation were assessed using multiplex assays. Neutrophil surface markers of activity and neutrophil platelet aggregates were assessed using an Accuri C6 flow cytometer. RESULTS: We observed neutrophil concentration, adhesion, transmigration, and activation, as well as increased NETs complexes; but no difference in extent of neutrophil platelet aggregation or systemic inflammation in patients with versus without obstructive CAD. Conversely, in patients with versus without AMI, neutrophil markers of adhesion and transmigration did not differ, but systemic markers of inflammation and neutrophil activation were elevated. CONCLUSION: This single-site, prospective study is the largest neutrophil biorepository in patients with cardiovascular risk factors. Inflammatory activation, and particularly neutrophil profiles, differ in patients with obstructive CAD and in the setting of AMI.

Surface Geometry of Cargo-less Gold Nanoparticles Is a Driving Force for Selective Targeting of Activated Neutrophils to Reduce Thrombosis in Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by recurrent arterial, venous, and microvascular thrombosis where activated neutrophils play a determinant role. However, neutrophils are challenging to target given their short lifespan in circulation and spontaneous activation. Screening of a small library of gold nanoparticles (AuNPs) led to the discovery of a formulation capable of targeting activated neutrophil attachment and has demonstrated that star-shaped, anti-PSGL-1-antibody-coated AuNPs (aPSGL-1-AuNPs) were more efficacious compared with other shapes of AuNPs. Our findings further revealed an exciting and safe targeting mode toward activated neutrophils in the APS mouse model induced by human-patient-derived antiphospholipid IgGs. Our studies demonstrate that targeting is dependent on the specific topographical features of the highly segregated PSGL-1 on the activated neutrophil surface for which a high affinity shape-driven nanomedicine can be designed and implemented. As such, star-shaped aPSGL-1-AuNPs serve as a promising nanoimmunotherapy for immunothrombosis associated with neutrophil adhesion in APS.

Platelet factor 4 improves survival in a murine model of antibiotic-susceptible and methicillin-resistant Staphylococcus aureus peritonitis.

The complement receptor CR3, also known as integrin Mac-1 (CD11b/CD18), is one of the major phagocytic receptors on the surface of neutrophils and macrophages. We previously demonstrated that in its protein ligands, Mac-1 binds sequences enriched in basic and hydrophobic residues and strongly disfavors negatively charged sequences. The avoidance by Mac-1 of negatively charged surfaces suggests that the bacterial wall and bacterial capsule possessing net negative electrostatic charge may repel Mac-1 and that the cationic Mac-1 ligands can overcome this evasion by acting as opsonins. Indeed, we previously showed that opsonization of Gram-negative Escherichia coli with several cationic peptides, including PF4 (Platelet Factor 4), strongly augmented phagocytosis by macrophages. Here, we investigated the effect of recombinant PF4 (rPF4) on phagocytosis of Gram-positive Staphylococcus aureus in vitro and examined its impact in a mouse model of S. aureus peritonitis. Characterization of the interaction of rPF4 with nonencapsulated and encapsulated S. aureus showed that rPF4 localizes on the bacterial surface, thus making it available for Mac-1. Furthermore, rPF4 did not have direct bactericidal and bacteriostatic activity and was not toxic to host cells. rPF4 enhanced phagocytosis of S. aureus bioparticles by various primary and cultured Mac-1-expressing leukocytes by several folds. It also increased phagocytosis of live nonencapsulated and encapsulated bacteria. Notably, the augmentation of phagocytosis by rPF4 did not compromise the intracellular killing of S. aureus by macrophages. Using a murine S. aureus peritonitis model, we showed that treatment of infected mice with rPF4 caused a significant increase in the clearance of antibiotic-susceptible S. aureus and its methicillin-resistant (MRSA) variant and markedly improved survival. These findings indicate that rPF4 binding to the bacterial surface circumvents its antiphagocytic properties, improving host defense against antibiotic-susceptible and antibiotic-resistant bacteria.