Induce Neutrophil Activation Research Articles

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.

The Role of Platelet Activation in the Development and Metastasis of Solid Tumors

The blood coagulation system is actively involved in the development of cancer. It is known that many solid tumors express tissue factor, a “trigger” of the cascade of plasma coagulation reactions, which leads to an increased risk of cancer-associated thrombosis and venous thrombosis in cancer patients. It has also long been known that platelets - small cellular fragments that are the basis of blood clots - play a critical role in metastasis by binding to the tumor cell after it enters the blood vessel, “shielding” it from the immune system and promoting the adhesion and extravasation of the tumor cell into tissues and the formation metastasis. In addition, platelets, being mobile “storehouses” of growth factors, are actively attracted and, in some cases, consumed by the tumor, which contributes to its development and vascularization. Platelet attraction occurs both through activation of the blood coagulation system in the tumor area and through exposure of the adhesive surface by the tumor. Activated in the tumor vicinity, platelets attract and induce neutrophil activation and the formation of neutrophil extracellular traps (NETs), thereby modulating the tumor microenvironment. When activated, platelets are known to secrete a variety of growth factors that promote both tumor development and vascularization. In addition to direct interaction, platelets and tumor cells exchange mRNA, micro-RNA and other regulatory molecules through microvesicles, while platelets are containers for the spread of tumor genetic material (circulating nucleic acids) throughout the body. In this review, we consider the molecular mechanisms of platelet participation in the development and metastasis of solid tumors, and also discuss possible options for pharmacological interruption of this interaction.

FTY720 ameliorates experimental MPO-ANCA-associated vasculitis by regulating fatty acid oxidation via the neutrophil PPARα-CPT1a pathway.

Increasing studies demonstrated the importance of C5a and anti-neutrophil cytoplasmic antibody (ANCA)-induced neutrophil activation in the pathogenesis of ANCA-associated vasculitis (AAV). Sphingosine-1-phosphate (S1P) acts as a downstream effector molecule of C5a and enhances neutrophil activation induced by C5a and ANCA. The current study investigated the role of a S1P receptor modulator, FTY720, in experimental autoimmune vasculitis (EAV) and explored the immunometabolism-related mechanisms of FTY720 in modulating ANCA-induced neutrophil activation. The effects of FTY720 in EAV were evaluated by quantifying haematuria, proteinuria, crescent formation, tubulointerstitial injury and pulmonary haemorrhage. RNA sequencing of renal cortex and gene enrichment analysis were performed. The proteins of key identified pathways were analysed in neutrophils isolated from peripheral blood of patients with active AAV and normal controls. We assessed the effects of FTY720 on ANCA-induced neutrophil respiratory burst and neutrophil extracellular traps formation (NETosis). FTY720 treatment significantly attenuated renal injury and pulmonary haemorrhage in EAV. RNA sequencing analyses of renal cortex demonstrated enhanced fatty acid oxidation (FAO) and peroxisome proliferator-activated receptor (PPAR) signalling in FTY720-treated rats. Compared with normal controls, patients with active AAV showed decreased FAO in neutrophils. FTY720-treated differentiated HL-60 cells showed increased expression of carnitine palmitoyltransferase 1a (CPT1a) and PPARα. Blocking or knockdown of CPT1a or PPARα in isolated human neutrophils and HL-60 cells reversed the inhibitory effects of FTY720 on ANCA-induced neutrophil respiratory burst and NETosis. FTY720 attenuated renal injury in EAV through upregulating FAO via the PPARα-CPT1a pathway in neutrophils, offering potential immunometabolic targets in AAV treatment.

HBeAg induces neutrophils activation impairing NK cells function in patients with chronic hepatitis B.

The role of neutrophils in hepatitis B virus (HBV) infection has been a subject of debate due to their involvement in antiviral responses and immune regulation. This study aimed to elucidate the neutrophil characteristics in patients with chronic hepatitis B (CHB). Through flow cytometry and ribonucleic acid-sequencing analysis, the phenotypes and counts of neutrophils were analyzed in patients with CHB. Moreover, the effects of HBeAg on neutrophils and the corresponding pattern recognition receptors were identified. Simultaneously, the cross-talk between neutrophils and natural killer (NK) cells was investigated. Neutrophils were activated in patients with CHB, characterized by higher expression levels of programmed death-ligand 1 (PD-L1), cluster of differentiation 86, and interleukin-8, and lower levels of CXC motif chemokine receptor (CXCR) 1 and CXCR2. Hepatitis B e antigen (HBeAg) partially induces neutrophil activation through the Toll-like receptor 2 (TLR2). A consistent upregulation of the TLR2 and HBeAg expression was observed in patients with CHB. Notably, the genes encoding molecules pivotal for NK-cell function upon NK receptor engagement enriched in neutrophils after HBeAg activation. The HBeAg-activated neutrophils demonstrated the ability to decrease the production of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) in NK cells, while the PD-1 and PD-L1 pathways partially mediated the immunosuppression. The immunosuppression of neutrophils induced by HBeAg suggests a novel pathogenic mechanism contributing to immune tolerance in patients with CHB.

A Pilot Study to Investigate Peripheral Low-Level Chronic LPS Injection as a Model of Neutrophil Activation in the Periphery and Brain in Mice.

Lipopolysaccharide-induced (LPS) inflammation is used as model to understand the role of inflammation in brain diseases. However, no studies have assessed the ability of peripheral low-level chronic LPS to induce neutrophil activation in the periphery and brain. Subclinical levels of LPS were injected intraperitoneally into mice to investigate its impacts on neutrophil frequency and activation. Neutrophil activation, as measured by CD11b expression, was higher in LPS-injected mice compared to saline-injected mice after 4 weeks but not 8 weeks of injections. Neutrophil frequency and activation increased in the periphery 4-12 h and 4-8 h after the fourth and final injection, respectively. Increased levels of G-CSF, TNFa, IL-6, and CXCL2 were observed in the plasma along with increased neutrophil elastase, a marker of neutrophil extracellular traps, peaking 4 h following the final injection. Neutrophil activation was increased in the brain of LPS-injected mice when compared to saline-injected mice 4-8 h after the final injection. These results indicate that subclinical levels of peripheral LPS induces neutrophil activation in the periphery and brain. This model of chronic low-level systemic inflammation could be used to understand how neutrophils may act as mediators of the periphery-brain axis of inflammation with age and/or in mouse models of neurodegenerative or neuroinflammatory disease.

An IL-1β-driven neutrophil-stromal cell axis fosters a BAFF-rich protumor microenvironment in individuals with multiple myeloma.

Human bone marrow permanently harbors high numbers of neutrophils, and a tumor-supportive bias of these cells could significantly impact bone marrow-confined malignancies. In individuals with multiple myeloma, the bone marrow is characterized by inflammatory stromal cells with the potential to influence neutrophils. We investigated myeloma-associated alterations in human marrow neutrophils and the impact of stromal inflammation on neutrophil function. Mature neutrophils in myeloma marrow are activated and tumor supportive and transcribe increased levels of IL1B and myeloma cell survival factor TNFSF13B (BAFF). Interactions with inflammatory stromal cells induce neutrophil activation, including BAFF secretion, in a STAT3-dependent manner, and once activated, neutrophils gain the ability to reciprocally induce stromal activation. After first-line myeloid-depleting antimyeloma treatment, human bone marrow retains residual stromal inflammation, and newly formed neutrophils are reactivated. Combined, we identify a neutrophil-stromal cell feed-forward loop driving tumor-supportive inflammation that persists after treatment and warrants novel strategies to target both stromal and immune microenvironments in multiple myeloma.

Neutrophil extracellular traps promote acetaminophen-induced acute liver injury in mice via AIM2.

Excessive acetaminophen (APAP) can induce neutrophil activation and hepatocyte death. Along with hepatocyte dysfunction and death, NETosis (a form of neutrophil-associated inflammation) plays a vital role in the progression of acute liver injury (ALI) induced by APAP overdose. It has been shown that activated neutrophils tend to migrate towards the site of injury and participate in inflammatory processes via formation of neutrophil extracellular traps (NETs). In this study we investigated whether NETs were involved in hepatocyte injury and contributed to APAP-induced ALI progression. ALI mouse model was established by injecting overdose (350 mg/kg) of APAP. After 24 h, blood and livers were harvested for analyses. We showed that excessive APAP induced multiple programmed cell deaths of hepatocytes including pyroptosis, apoptosis and necroptosis, accompanied by significantly increased NETs markers (MPO, citH3) in the liver tissue and serum. Preinjection of DNase1 (10 U, i.p.) for two consecutive days significantly inhibited NETs formation, reduced PANoptosis and consequently alleviated excessive APAP-induced ALI. In order to clarify the communication between hepatocytes and neutrophils, we induced NETs formation in isolated neutrophils, and treated HepaRG cells with NETs. We found that NETs treatment markedly increased the activation of GSDMD, caspase-3 and MLKL, while pre-treatment with DNase1 down-regulated the expression of these proteins. Knockdown of AIM2 (a cytosolic innate immune receptor) abolished NETs-induced PANoptosis in HepaRG cells. Furthermore, excessive APAP-associated ALI was significantly attenuated in AIM2KO mice, and PANoptosis occurred less frequently. Upon restoring AIM2 expression in AIM2KO mice using AAV9 virus, both hepatic injury and PANoptosis was aggravated. In addition, we demonstrated that excessive APAP stimulated mtROS production and mitochondrial DNA (mtDNA) leakage, and mtDNA activated the TLR9 pathway to promote NETs formation. Our results uncover a novel mechanism of NETs and PANoptosis in APAP-associated ALI, which might serve as a therapeutic target.

The rheology of interactions between leukocytes, platelets and the vessel wall in thrombo-inflammation.

Leukocytes and platelets must adhere to the wall of blood vessels to carry out their protective functions in inflammation and haemostasis. Recruitment is critically dependent on rheological variables (wall shear rate and stress, red cell aggregation and haematocrit) which affect delivery to the vessel wall as well as velocities and forces experienced there. Leukocyte recruitment is efficient only up to wall shear rates of about 300s-1 and usually restricted to low-shear post-capillary venules in inflammation. Being smaller, platelets experience lower velocities and shear forces adjacent to the wall and can adhere at much higher shear rates for haemostasis in arteries. In addition, we found quite different effects of variations in haematocrit or red cell aggregation on attachment of neutrophils or platelets, which also assist their separate recruitment in venules or arteries. However, it has become increasingly evident that inflammatory and thrombotic responses may occur together, with platelets promoting the adhesion and activation of neutrophils and monocytes. Indeed, it is 30 years since we demonstrated that platelets could cause neutrophils to aggregate in suspension and, when attached to a surface, could support selectin-mediated rolling of all leukocytes. Thrombin-activated platelets could further induce neutrophil activation and immobilisation. In some conditions, platelets could bind to intact endothelial monolayers and capture neutrophils or monocytes. Subsequently, we found that extracellular vesicles released by activated platelets (PEV) fulfilled similar functions when deposited on surfaces or bound to endothelial cells. In murine models, platelets or PEV could act as bridges for monocytes in inflamed vessels. Thus, leukocytes and platelets are rheologically adapted for their separate functions, while novel thrombo-inflammatory pathways using platelets or PEV may underlie pathogenic leukocyte recruitment.

Hemogram Derived Clinical Traits Evinced Differential Contribution to Explained Variance of Mortality Risk Model at Index Hospitalization for Sars-Cov-2 Infection across Wuhan/D614G, Delta, Omicron and Omicron BA.x Variants

Introduction SARS-CoV-2 infection promulgates an immune dysregulation that drives an inflammatory cascade therefore inducingmorbidity and mortality across the COVID-19 presentation spectrum. Hemogram (CBC) derived traits associated with COVID-19 severity have been reported (Meizlish ML et al., 2021, Dhinata KS et al., 2021), but their relative impact across SARS-CoV-2 variants is unclear. We employed sequential machine learning approachesto identify significantly contributing traits with attendant portion of explained variance (EV%) in hospital mortality risk models across predominant SARS-CoV-2 variants. Methods Our retrospective design, analyses and interpretations followed constructs detailed in the Strengthening the Reporting of Observational Studies in Epidemiology reporting guideline. Adult patients with laboratory confirmed COVID-19 who underwent index hospitalization during Wuhan/D614G (March 14, 2020- June 18, 2021), Delta (June 19, 2021- December 18, 2021), Omicron (December 19-March 30, 2022) or Omicron BA.x (March 31, 2022 - April 14, 2023) variant waves at an 820-bed academic public health trust hospital in Florida were studied. Demographics, laboratory results (within 24h of presentation), ICD-10-CM-based comorbidity, COVID-19 directed treatment and administrative data were extracted from electronic medical records under IRB exemption. Generalized regression with adaptive LASSO identified hemogram-derived traits significantly associated with mortality in at least one of the variants while controlling for presentation vital signs, age, sex, extant comorbidities and ultimate COVID-19 directed treatment. Boosted Tree modeling computed within-variant proportion contributed by each trait to model's accuracy (R 2) representing EV% of mortality risk. Traits contributing to one or more variants for at least 1% portion of EV were retained in model. A Bonferroni corrected two-tailed p < .0125 was considered significant. Results We included 6490 consecutively discharge patients distributed across Wuhan/D614G (n=2249), Delta (n=1196), Omicron (n=953) and Omicron BA.x (n=2092) variants with respective mortality of 9.3%, 13.0%, 4.4%, and 2.4% (p< .0001). Patient characteristics and on-presentation laboratory medicine clinical traits are in Table 1. Table 2 provides ranked contribution for each of the 7 traits retained in the model with attendant presentation mortality risk accuracy (R 2) across variant waves. Potent synergy can be generated with biomarkers ratios, as seen with Segmented neutrophil/monocyte ratio (SMR) and lymphocyte/monocyte ratio (LMR), compared to studying these variables individually. Conclusion Sequential machine-learning approaches identified differential expression intensity across SARS-CoV-2 variants in presentation hemogram-derived traits associated with hospital mortality risk modeling at index infection. These findings suggest that COVID-19 presentation risk and level of care assessment tools employing hemogram-derived traits may not generalize across pandemic variants. Platelets counts were associated with mortality in Delta, Wuhan/D614G and Omicron variants. Although it was also associated in Omicron Bx.5, the R 2 is lower, this can be explained because in this study the variant of Omicron Bx.5 had the lowest mortality rate. Studies have shown that platelet activation generates a cascade with more thromboxane A2 and platelet thrombofactor A which increases prothrombotic states (Dhinata KS et al., 2021). The absence of eosinophils was seen in Wuhan/D614G and Delta, the cause of which cannot be ascertained. A theory is that the inflammation caused by SARS-CoV-2 infection releases certain cytokines that induce eosinophil apoptosis (Dhinata KS et al., 2021). Immature neutrophils evidenced across variants can be explained by the inflammatory cascade inducing G-CSF and IL-8 production, which induces neutrophil activation (Meizlish ML et al., 2021).Lymphopenia seen throughout variants contributes to an increase in LMR. One presumption is that the SARS-CoV-2 affects T-Lymphocytes through ACE-2 receptor and CD117 spike proteins (Meizlish ML et al., 2021). Overall, our study brings to attention how hemogram-derived traits express differently across SARS-CoV-2 variants, which could guide the stratification of COVID-19 severity and assist in the care of patients before decompensation is noticed.

Number of blood neutrophil granulocytes is associated with diffuse coronary involvement in patients with increased lipoprotein (a)

Aim. To assess the level of lipoprotein (a) (Lp(a)) and other lipid profile parameters, the composition of blood leukocytes in patients with focal and diffuse coronary atherosclerosis who underwent coronary artery bypass surgery (CABG), as well as to study the effect of Lp(a) on the activation of leukocytes in vitro.Material and methods. The study included 120 male patients (mean age, 67±5 years) with multivessel coronary artery disease after elective CABG surgery. According to the type of coronary artery involvement, patients were divided into groups of diffuse (n=77) and focal (n=43) atherosclerosis. In peripheral blood samples, the content of the main fractions of lipoproteins, including Lp(a), was determined. Activation of neutrophil granulocytes upon addition of Lp(a) was assessed in whole blood and in primary cell culture.Results. In patients with diffuse coronary stenosis, there was a tendency to higher Lp(a) levels; no changes in other lipid profile parameters were detected. In this group of patients, an increase in the absolute leukocyte count was noted due to neutrophilic granulocytes. An absolute neutrophil count >2,9 million/mL was associated with diffuse disease (AUC, 0,69 (95% confidence interval (CI) 0,600,77), p=0,0002, sensitivity 85%, specificity 42%). In patients with Lp(a) ³30 mg/dL, the diagnostic value of a neutrophil count >2,9 million/mL for diffuse stenosis was higher (AUC, 0,74 (95% CI 0,60-0,86), p= 0,005, sensitivity 90%, specificity 48%). According to logistic regression analysis with the introduction of neutrophils and conventional risk factors (age, body mass index, hypertension and smoking) into the model, only the number of neutrophils above the threshold level remained an independent factor in diffuse coronary stenosis. Lp(a) can induce neutrophil activation, detected by increased CD66b overexpression in whole blood, as well as the formation of neutrophil extracellular traps in primary cell culture.Conclusion. Neutrophil granulocytes can participate in the formation of diffuse atherosclerotic coronary lesions. One of the neutrophil activators may be Lp(a). The combination of high neutrophil count and Lp(a) level in the blood increases the risk of diffuse coronary atherosclerosis.

CLEC5A expression can be triggered by spike glycoprotein and may be a potential target for COVID-19 therapy.

The immune response is crucial for coronavirus disease 19 (COVID-19) progression, with the participation of proinflammatory cells and cytokines, inducing lung injury and loss of respiratory function. CLEC5A expression on monocytes can be triggered by viral and bacterial infections, leading to poor outcomes. Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) is able to induce neutrophil activation by CLEC5A and Toll-like receptor 2, leading to an aggressive inflammatory cascade, but little is known about the molecular interactions between CLEC5A and SARS-CoV-2 proteins. Here, we aimed to explore how CLEC5A expression could be affected by SARS-CoV-2 infection using immunological tools with in vitro, in vivo, and in silico assays. The findings revealed that high levels of CLEC5A expression were found in monocytes from severe COVID-19 patients in comparison with mild COVID-19 and unexposed subjects, but not in vaccinated subjects who developed mild COVID-19. In hamsters, we detected CLEC5A gene expression during 3-15 days of Omicron strain viral challenge. Our results also showed that CLEC5A can interact with SARS-CoV-2, promoting inflammatory cytokine production, probably through an interaction with the receptor-binding domain in the N-acetylglucosamine binding site (NAG-601). The high expression of CLEC5A and high levels of proinflammatory cytokine production were reduced in vitro by a human CLEC5A monoclonal antibody. Finally, CLEC5A was triggered by spike glycoprotein, suggesting its involvement in COVID-19 progression; therapy with a monoclonal antibody could be a good strategy for COVID-19 treatment, but vaccines are still the best option to avoid hospitalization/deaths.

CCR7/dendritic cell axis mediates early bacterial dissemination in Orientia tsutsugamushi-infected mice.

Scrub typhus is a life-threatening zoonosis caused by the obligate intracellular bacterium Orientia tsutsugamushi (Ot) that is transmitted by the infected larvae of trombiculid mites. However, the mechanism by which Ot disseminates from the bite site to visceral organs remains unclear; host innate immunity against bacterial dissemination and replication during early infection is poorly understood. In this study, by using an intradermal infection mouse model and fluorescent probe-labeled Ot, we assessed the dynamic pattern of innate immune cell responses at the inoculation site. We found that neutrophils were the first responders to Ot infection and migrated into the skin for bacterial uptake. Ot infection greatly induced neutrophil activation, and Ot-neutrophil interaction remarkably promoted cell death both in vitro and in vivo. Depletion of neutrophils did not alter bacterial dissemination in mice, as evidenced by similar bacterial burdens in the skin and draining lymph nodes (dLN) at day 3, as well as in the lungs and brains at day 14, as compared to the control mice. Instead, dendritic cells (DCs) and macrophages played a role as a Trojan horse and transmitted Ot from the skin into dLN. Importantly, the absence of homing receptor CCR7 or neutralization of its ligand, CCL21, significantly impaired DC migration, resulting in reduced bacterial burdens in dLN. Taken together, our study sheds light on a CCR7/dendritic cell-mediated mechanism of early Ot dissemination and provides new insights into therapeutic and vaccine development strategies for scrub typhus.

355: RHINOVIRUS-ASSOCIATED ANCA VASCULITIS: A RARE ASSOCIATION

Introduction: The anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitides (AAV) are immune mediated diseases, associated with antigen exposure and host immune response. Approximately 52–80% of patients with AAV have pulmonary abnormalities on chest CT, mainly interstitial lung disease (ILD), granuloma, or alveolar hemorrhage. Myeloperoxidase (MPO) antineutrophil cytoplasmic antibody (ANCA) has been particularly associated with interstitial lung disease and the majority of patients present with pulmonary symptoms prior to the diagnosis of ANCA vasculitis. ILD is a relatively uncommon manifestation of MPO-ANCA, but signifies a poor prognosis. We present a patient with rhinovirus infection associated ANCA vasculitis. Description: A 42-year-old female with a past medical history of COPD, bipolar disorder, and intravenous drug use presented to the emergency department in acute respiratory distress. Computed tomography of the chest revealed bilateral diffuse, patchy ground glass opacities. Respiratory PCR testing was positive for rhinovirus. The patient was admitted to the ICU for acute hypoxic respiratory failure. Her respiratory status continued to decline during the ICU stay. Serum studies including DsDNA, SSA, SSB, anticentromere, myositis, aldolase and ANCA were obtained. The studies were positive for MPO-ANCA. The patient was treated with high dose methylprednisolone. Subsequently, the oxygen demand improved and the patient made a full recovery, with plan for outpatient biopsies. Discussion: MPO-ANCA is a marker which may be induced by various drugs, dust inhalation, smoking, heavy metal exposure, or silica exposure. The hypothesized mechanism involves cytokines produced by antigens to induce MPO expression within the neutrophil cell membrane. The ANCA component binds to MPO to induce neutrophil activation which then activates the inflammatory cascade. The overproduction of cytokines causes vascular endothelial cell damage and ultimately vasculitis. One proposed mechanism of MPO-ANCA involves the alteration in degradation of neutrophil extracellular traps (NETs) likely due to infections. This mechanism leads to an increase in MPO-ANCA binding. NETs have a proliferative effect on lung fibroblasts, and thus are seen alongside fibroblasts in patients with MPO-ANCA interstitial pneumonia.

A Role of Immunothrombosis in Acquired Antibody-Mediated Thrombophilia's: Classical Complement and Neutrophil Activation in APS and HIT

Introduction: Heparin induced thrombocytopenia (HIT) and APS (antiphospholipid syndrome) are acquired thrombophilia's characterized by both arterial and venous thromboembolic events (TE). As a common feature APS and HIT share the presence of pathological antibodies, which play a crucial role in disease pathogenesis. In HIT, antibodies are directed to platelet factor (PF) 4 and heparin, respectively, and in APS, antibodies target anti-phospholipids (anti-cardiolipin antibody, anti-Beta2- glycoprotein I and lupus anticoagulant). There is growing evidence that complement- and neutrophil activation in the form of neutrophil extracellular traps (NETs) play an important role in the pathogenesis of thrombosis, the so-called "immunothrombosis". Insufficient control with subsequent unrestrained complement activation results in thrombosis as seen in patients with paroxysmal nocturnal hemoglobinuria (PNH). Therapeutic inhibition of complement activation abrogates thrombosis in these patients, emphasizing the important role of complement activation in the pathogenesis of thrombosis. It has been demonstrated, that complement activation products efficiently induce neutrophil activation, and therapeutic complement inhibition in sepsis and PNH prevents neutrophil activation. Here, we investigate the role of complement activation and neutrophil activation in patients with APS and HIT, respectively. Methods To assess the association of complement activation and NET Formation, we measured complement activation products (C4bc, C3bc) and marks for neutrophil activation, such as nucleosomes and human neutrophil elastase-α1- antitrypsin complexes (HNE) by ELISA in plasma of 34 primary APS- and 26 HIT- patients and 31 healthy controls. Groups have been compared using the Mann- Whitney-Rank Test. Values are indicated as median and range, p<0.05 is considered statistically significant. Results Patients with APS and HIT showed sign. higher levels of complement activation products as compared to healthy controls. Complement activation mainly of the classical (C4b/c) and alternate pathway (C3b/c) in APS (11 nM and 263 nM) and in HIT (33 nM and 324 nM) were sign. higher (p<0.0001) as compared to the healthy controls (7 nM and 19 nM). C4bc levels were sign. higher in HIT as compared to APS patients (p<0001), but there was no difference in C3b/c levels between these two groups (see figure 1). Patients with HIT had a sig. (p<0.0001) higher nucleosomes (440 U/ml) and HNE (121 ng/ml) as compared to healthy controls (12 U/ml and 26 ng/ml). HNE levels were sign. (p<0.001) higher in APS patients (37 ng/ml) as compared to healthy controls (26 ng/ml), however no difference was observed in nucleosome levels (12 U/ml vs 14 U/ml) (see figure 2). Based on the number of positive antibodies, eleven APS patients have been stratified as high-risk and 24 APS patients as low- to intermediate risk. C4bc levels were sign. increased in the high-risk patients as compared to the low- to intermediate risk group, whereas no difference in C3b/c levels could be observed between the 2 groups. Similarly, HNE complexes sign. increased in the high-risk patients as compared to the low- to intermediate risk group, whereas no difference in nucleosome levels could be observed between the 2 groups. Six HIT patients (22%) had a diagnosed TE event whereas 17 patients (63%) had no diagnosed TE. In four patients (15%) no clinical data were available, and they have been excluded from subgroup analysis. Interestingly, the measured markers for complement - and neutrophil activation in HIT patient with and without thrombosis were not different. Conclusions We demonstrate complement activation in patients with HIT and APS. Based on the simultaneous raise in nucleosomes and HNE, there is evidence of NET formation in HIT, whereas only weak neutrophil activation can be found in APS. In APS classical complement activation and neutrophil activation correlates with APS risk stratification. Our results demonstrate classical complement pathway- and neutrophil activation in HIT and APS patients. These findings may form the base for new therapeutic strategies targeting complement and neutrophil activation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Altered Innate Immunity and Damaged Epithelial Integrity in Vaginal Microbial Dysbiosis.

The role of neutrophils relative to vaginal dysbiosis is unclear. We hypothesize that bacterial vaginosis (BV)-associated bacteria may induce the activation and accumulation of mucosal neutrophils within the female reproductive tract (FRT), resulting in epithelial barrier damage. We collected endocervical cytobrushes from women with and without BV and assessed bacteria community type and frequency/functional phenotypes of neutrophils. We performed in vitro whole blood co-cultures with BV-associated bacteria and healthy vaginal commensals and assessed their impact on epithelial integrity using transepithelial electrical resistance. We demonstrated increased neutrophil frequency (p < 0.0001), activation (p < 0.0001), and prolonged lifespan (p < 0.0001) in the cytobrushes from women with non-Lactobacillus dominant (nLD) communities. Our in vitro co-cultures confirmed these results and identified significant barrier damage in the presence of neutrophils and G. vaginalis. Here, we demonstrate that BV-associated bacteria induce neutrophil activation and increase lifespan, potentially causing accumulation in the FRT and epithelial barrier damage.

ADVANCED GLYCATION END PRODUCTS(AGES) AND DIETARY METABOLITES RESULTED IN NEUTROPHIL EXTRACELLULAR TRAPS (NETS) AND VASCULAR CHRONIC INFLAMMATION AND STIFFNESS

Objective: Inflammaging is a risk factor for cardiovascular diseases. Advanced glycation end products (AGEs) are proteins or lipids that become glycated as a result of exposure to sugars, they are thought to be a detrimental metabolite involving in inflammation and some age-related chronic diseases. To explore the effects of vascular aging via the immune pathway. Design and method: To investigate the role of AGEs in inducing neutrophil activation and cause neutrophil extracellular traps (NET), Neutrophil cell line treated with AGEs analyzed the reactive oxygen species (ROS) production and proinflammatory cytokines expression. Whether AGEs cause the morphological and functional change in endothelial cells by using a human cell line HMEC-1. Next, we investigate if older people bear spontaneous NET in the circulation that may bring detrimental impact in the vasculature system. We enrolled volunteers &gt; 65 years-old, the serum was measured with MPO-DNA complex by ELISA. In vivo, we established an animal model by feeding mice with a high-fat diet (HFD) for long period to induce chronic intestinal inflammation. The aortic stiffness was assessed in vivo by the pulse wave velocity (PWV) after HFD feeding and normal diet (ND) feeding for 20 weeks. Results: AGE not only caused neutrophil cell line cellular ROS generation in time-dependent manners but also significantly increased expression of TNF and il-1, and AGE increased expression of inflammation markers including ICAM-1, VCAM-1, DLC-1 and reduced tube formation by HMEC-1. AGEs induced the NETs formation in primary neutrophils in a time-dependent manner. Volunteer serum MPO-DNA complex is higher than younger donors, HFD-feeding mice had increased and PWV and decreased colon of length, compared with ND-feeding mice, suggesting PWV tends to increase with higher arterial stiffness. Conclusions: In conclusion, AGEs, a metabolite from HFD is able to induce neutrophil activation and NET formation. Volunteers &gt; 65 years old have high MPO-DNA complex, suggesting the “Netting” neutrophil in the circulation would be one of the potential factors causing the inflammation in the vessel and leading to vessel stiffness. The HFD-feeding mice with intestinal inflammation display vascular stiffness.

Proof of concept: hypoxanthine from stored red blood cells induces neutrophil activation.

Red blood cell (RBC) units may contain a variety of molecules that can activate the neutrophil cascade turning neutrophils into targets for immunomodulatory molecules. Our metabolomics profiling of RBC units revealed a significant increase of hypoxanthine concentration during storage. Hypoxanthine catabolism in vivo ends with the production of uric acid through a reaction catalysed by xanthine oxidase during which reactive oxygen species are generated. Some authors have described in vitro neutrophil activation after treatment with stored RBC medium. However, the response of neutrophils to the action of xanthine oxidase upon hypoxanthine accumulation in the supernatant of RBC units has never been investigated. Neutrophils were isolated from peripheral whole blood and cultured at 37 °C in a humidified incubator with 5% CO2. Hypoxanthine and RBC supernatants were tested to verify neutrophil stimulation. To prove the involvement of hypoxanthine in neutrophil activation, xanthine oxidase was pre-incubated with or without allopurinol before addition to the neutrophil cultures. Intracellular expression of tumour necrosis factor-α (TNF-α) and interleukin-8 (IL-8) was assessed by a cytofluorimetric assay and early-stage release of IL-8 was detected by a Luminex® assay. In the presence of xanthine oxidase, hypoxanthine, alone and in combination with RBC supernatants, caused increases of TNF-α- and IL-8-positive cells after 5 hours of treatment. Moreover, IL-8 was quickly released, 30 min after stimulation. Here we show, for the first time, that neutrophil activation by stored RBC depends, in part, on the presence of hypoxanthine contained in the RBC units. Our results add hypoxanthine to the already known mediators of inflammation present in RBC units, supporting the evidence that medium from stored RBC may concur to boost inflammatory processes in transfusion recipients, potentially leading to negative post-transfusion outcomes.

S100A8/A9 Is a Marker for the Release of Neutrophil Extracellular Traps and Induces Neutrophil Activation.

Neutrophils are the most abundant innate immune cells in the circulation and they are the first cells recruited to sites of infection or inflammation. Almost half of the intracellular protein content in neutrophils consists of S100A8 and S100A9, though there has been controversy about their actual localization. Once released extracellularly, these proteins are thought to act as damage-associated molecular patterns (DAMPs), though their mechanism of action is not well understood. These S100 proteins mainly form heterodimers (S100A8/A9, also known as calprotectin) and this heterocomplex is recognized as a useful biomarker for several inflammatory diseases. We observed that S100A8/A9 is highly present in the cytoplasmic fraction of neutrophils and is not part of the granule content. Furthermore, we found that S100A8/A9 was not released in parallel with granular content but upon the formation of neutrophil extracellular traps (NETs). Accordingly, neutrophils of patients with chronic granulomatous disease, who are deficient in phorbol 12-myristate 13-acetate (PMA)-induced NETosis, did not release S100A8/A9 upon PMA stimulation. Moreover, we purified S100A8/A9 from the cytoplasmic fraction of neutrophils and found that S100A8/A9 could induce neutrophil activation, including adhesion and CD11b upregulation, indicating that this DAMP might amplify neutrophil activation.

Visualizing hypochlorous acid production by human neutrophils with fluorescent graphene quantum dots

In living organisms, redox reactions play a crucial role in the progression of disorders accompanied by the overproduction of reactive oxygen and reactive chlorine species, such as hydrogen peroxide and hypochlorous acid, respectively. We demonstrate that green fluorescence graphene quantum dots (GQDs) can be employed for revealing the presence of the hypochlorous acid in aqueous solutions and cellular systems. Hypochlorous acid modifies the oxygen-containing groups of the GQD, predominantly opens epoxide ring C–O–C, forms excessive C=O bonds and damages the carbonic core of GQDs. These changes, which depend on the concentration of the hypochlorous acid and exposure time, manifest themselves in the absorbance and fluorescence spectra of the GQD, and in the fluorescence lifetime. We also show that the GQD fluorescence is not affected by hydrogen peroxide. This finding makes GQDs a promising sensing agent for selective detecting reactive chlorine species produced by neutrophils. Neutrophils actively accumulate GQDs allowing to visualize cells and to examine the redox processes via GQDs fluorescence. At high concentrations GQDs induce neutrophil activation and myeloperoxidase release, leading to the disruption of GQD structure by the produced hypochlorous acid. This makes the GQDs a biodegradable material suitable for various biomedical applications.

Galectin-9 mediates neutrophil capture and adhesion in a CD44 and β2 integrin-dependent manner.

Neutrophil trafficking is a key component of the inflammatory response. Here, we have investigated the role of the immunomodulatory lectin Galectin-9 (Gal-9) on neutrophil recruitment. Our data indicate that Gal-9 is upregulated in the inflamed vasculature of RA synovial biopsies and report the release of Gal-9 into the extracellular environment following endothelial cell activation. siRNA knockdown of endothelial Gal-9 resulted in reduced neutrophil adhesion and neutrophil recruitment was significantly reduced in Gal-9knockout mice in a model of zymosan-induced peritonitis. We also provide evidence for Gal-9 binding sites on human neutrophils; Gal-9 binding induced neutrophil activation (increased expression of β2 integrins and reduced expression of CD62L). Intra-vital microscopy confirmed a pro-recruitment role for Gal-9, with increased numbers of transmigrated neutrophils following Gal-9 administration. We studied the role of both soluble and immobilized Gal-9 on human neutrophil recruitment. Soluble Gal-9significantly strengthened the interaction between neutrophils and the endothelium and inhibited neutrophil crawling on ICAM-1. When immobilized, Gal-9 functioned as an adhesion molecule and captured neutrophils from the flow. Neutrophils adherent to Gal-9 exhibited a spread/activated phenotype that was inhibited by CD18 and CD44 neutralizing antibodies, suggesting a role for these molecules in the pro-adhesive effects of Gal-9. Our data indicate that Gal-9 is expressed and released by the activated endothelium and functions both in soluble form and when immobilized as a neutrophil adhesion molecule. This study paves the way for further investigation of the role of Gal-9 in leukocyte recruitment in different inflammatory settings.