Supernatants Of Neutrophils Research Articles

Revealing the Therapeutic Potential of Muscle-Derived Mesenchymal Stem/Stromal Cells: An In Vitro Model for Equine Laminitis Based on Activated Neutrophils, Anoxia-Reoxygenation, and Myeloperoxidase.

Laminitis in horses is a crippling condition marked by the deterioration of the dermal-epidermal interface, leading to intense lameness and discomfort, often necessitating euthanasia. This study aimed to establish an in vitro model of laminitis using a continuous keratinocyte cell line exposed to anoxia-reoxygenation and an activated neutrophil supernatant. A significant decrease in the keratinocytes' metabolism was noted during the reoxygenation period, indicative of cellular stress. Adding muscle-derived mesenchymal stem/stromal cells during the reoxygenation demonstrated a protective effect, restoring the keratinocytes' metabolic activity. Moreover, the incubation of the keratinocytes with either an activated neutrophil supernatant or myeloperoxidase alone induced increased keratinocyte myeloperoxidase activity, which was modulated by stem cells. These findings underscore the potential of muscle-derived mesenchymal stem/stromal cells in mitigating inflammation and restoring keratinocyte metabolism, offering insights for future cell therapy research in laminitis treatment.

Optimization and verification of conditions for induction of neutrophil extracellular traps in vitro

This study aims to optimize the conditions for the formation of neutrophil extracellular traps(NETs) in vitro, so as to establish a relatively stable experimental research platform. Different conditions were compared, including commonly used laboratory animals(rats and mice) and a variety of cell sources(bone marrow neutrophils and peripheral blood neutrophils separated by percoll density gradient centrifugation). Different inducers like lipopolysaccharide(LPS) and phorbol 12-myristate 13-acetate(PMA) were used for induction in vitro. Myeloperoxidase(MPO)/citrullinated histone H3(CitH3)/DAPI immunofluorescence and cell free DNA(cf-DNA) content determination were used for comprehensive evaluation to screen the optimal conditions for the formation of NETs induced in vitro. Furthermore, the stability of the selected conditions for inducing the formation of NETs in vitro was evaluated by tetramethylpyrazine(TMP), an active component in Chinese herbal medicines. The results showed that coated poly-D-lysine(PDL) induced the formation of NETs in bone marrow neutrophils of mice to a certain extent. Both LPS and PMA significantly up-regulated the protein levels of MPO and CitH3 in mouse bone marrow neutrophils and elevated the cfDNA level in the supernatant of rat peripheral blood neutrophils. The cfDNA level in the PMA-induced group increased more significantly than that in the LPS-induced group(P<0.05). The results of immunofluorescence staining showed that the expression of MPO and CitH3 in mouse bone marrow neutrophils, rat bone marrow neutrophils, and rat peripheral blood neutrophils were significantly increased after PMA induction, especially in rat peripheral blood neutrophils. TMP significantly down-regulated the protein levels of MPO, CitH3, and neutrophil elastase(NE) in rat peripheral blood neutrophils induced by PMA. In conclusion, treating the peripheral blood neutrophils of rats with PMA is the optimal condition for inducing the formation of NETs in vitro. This study provides an optimal platform for in vitro studies based on NETs and a basis for studying the effects of traditional Chinese medicines targeting NETs.

Dysfunctional neutrophil type 1 interferon responses in preschool children with recurrent wheezing and IL-4–mediated aeroallergen sensitization

BackgroundThe innate mechanisms associated with viral exacerbations in preschool children with recurrent wheezing are not understood. ObjectiveWe assessed differential gene expression in blood neutrophils from preschool children with recurrent wheezing, stratified by aeroallergen sensitization, at baseline and after exposure to polyinosinic-polycytidylic acid (poly(I:C)). We also examined whether poly(I:C)-stimulated blood neutrophils influenced airway epithelial gene expression. MethodsBlood neutrophils were purified and cultured overnight with poly(I:C) and underwent next-generation sequencing with Reactome pathway analysis. Primary human small airway epithelial cells were treated with poly(I:C)-treated neutrophil culture supernatants and were analyzed for Type 1 interferon gene expression with a targeted array. Symptoms and exacerbations were assessed in participants over 12 months. Results436 genes were differently expressed in neutrophils from children with versus without aeroallergen sensitization at baseline, with significant downregulation of Type 1 interferons. These Type 1 interferons were significantly upregulated in sensitized children after poly(I:C) stimulation. Confirmatory experiments demonstrated similar upregulation of Type 1 interferons in IL-4 treated neutrophils stimulated with poly(I:C). Poly(I:C)-treated neutrophil supernatants from children with aeroallergen sensitization also induced a Type 1 interferon response in epithelial cells. Children with aeroallergen sensitization also had higher symptom scores during exacerbations and these symptom differences persisted for three days after prednisolone treatment. ConclusionsType 1 interferon responses are dysregulated in preschool children with aeroallergen sensitization, which is in turn associated with exacerbation severity. Given the importance of Type 1 interferon signaling in viral resolution, additional studies of neutrophil Type 1 interferon responses are needed in this population.

Quantitative evaluation of citrullinated fibrinogen for detection of neutrophil extracellular traps.

Activated neutrophils release neutrophil extracellular traps (NETs) composed of chromatin filaments containing bactericidal proteins and enzymes. This process, known as NETosis, is an innate host defense mechanism. However, NET accumulation can lead to uncontrolled inflammation and organ damage. Therefore, NET detection provides clinically important information for the assessment of inflammatory conditions. We investigated whether quantification of citrullinated fibrinogen (C-Fbg), which is catalyzed by peptidylarginine deiminase (PAD) released during NETosis, can be used to detect NETs. Human neutrophils were stimulated with fibrinogen using phorbol 12-myristate 13-acetate (PMA). The myeloperoxidase (MPO)-DNA complex and C-Fbg concentrations in the culture supernatants were quantified using an enzyme-linked immunosorbent assay. The protein levels of peptidylarginine deiminase 2 and 4 in culture supernatants and mRNA levels in PMA-stimulated neutrophils were also assessed. The levels of the MPO-DNA complex in the supernatants of PMA-stimulated neutrophils increased, indicating NETosis. C-Fbg level also increased, which was suppressed by both NETosis and PAD inhibitors. PAD2 was detected in the culture supernatant; however, PAD4, but not PAD2, mRNA levels increased in PMA-stimulated neutrophils. This study quantitatively demonstrates that fibrinogen is citrullinated by PAD derived from PMA-stimulated neutrophils upon NETosis. Although further studies are needed for clinical application, quantification of C-Fbg in blood may help detect the presence of NETs.

Lead activates neutrophil degranulation to induce early myocardial injury in mice

Lead (Pb) is a pervasive toxic metal contaminant associated with a high risk of myocardial injury. However, the precise mechanism underlying Pb-induced myocardial injury has yet to be fully elucidated. In this study, a murine model of Pb exposure (0, 1, 5, and 10 mg/kg) was employed to investigate the involvement of neutrophil degranulation in the induction of myocardial injury. Notably, serum levels of cardiac troponin I (cTnI) and creatine kinase-MB (CK-MB) increased significantly in Pb-exposed mice, whereas cTnI levels in cardiomyocytes decreased, suggesting that Pb exposure may cause early myocardial injury. Moreover, Pb exposure was found to promote neutrophil degranulation, as evidenced by elevated myeloperoxidase (MPO) and neutrophil elastase (NE) concentrations in both the serum of Pb-exposed workers and Pb-exposed mice, as well as the extracellular supernatant of neutrophils following exposure. However, we found that serum level of cTnI enhanced by Pb exposure is associated with increased NE levels in the serum, but not with MPO levels. Upon treatment with NE inhibitor (sivelestat), the serum level of cTnI markedly reduced in Pb-exposed mice, we found that early myocardial injury is associated with NE levels in the serum. At the molecular level, western blotting analysis revealed an upregulation of ERK1/2 expression in vitro following Pb exposure, suggesting that the activation of the ERK1/2 signaling pathway may underlie the participation of neutrophil degranulation in Pb-induced myocardial injury. In summary, our findings demonstrate that Pb exposure can initiate early myocardial injury by promoting the neutrophil degranulation process, thereby highlighting the potential role of this process in the pathogenesis of Pb-associated myocardial injury.

PSII-3 Sheep Neutrophil Activation in Response to Garlic in Vitro

Abstract St. Croix hair sheep are known to resist parasitic infection. Parasitic resistance is in part due to the increased number and ability of neutrophil leukocytes to fight pathogens. Dietary supplements such as garlic (Allium sativum L.) are antimicrobial immune modulators that can enhance production. This study sought to evaluate the impact of fresh filtered garlic on sheep neutrophils through In vitro treatment. The study collected blood from the jugular vein of five clinically healthy female St. Croix sheep at NC A&T State University Farm and isolated neutrophils from 10 mL whole blood samples from each animal. The neutrophils were treated with fresh filtered garlic after the total cell count was measured, and 1 million cells were taken for treatment. The total protein concentration in the neutrophil supernatant was measured before and after treatment. Garlic (10 ug/mL PBS) was added to neutrophils, incubated (30 minutes, 37 °C) and centrifuged. Total RNA was extracted from the pellet using the TRIZOL RNA Isolation Protocol (Sigma-Aldrich). The RNA concentration and purity were assessed using a Nanodrop Spectrophotometer (Thermo Scientific Inc.). Pooled RNA from each group was used for cDNA synthesis, followed by qPCR analysis of the expression of 84 test genes related to the innate and adaptive immune response (RT2 profiler array) of the host. Total protein concentration in the supernatant was determined using the BCA Assay (Pierce BCA Protein Assay Kit). In vitro treatment with garlic extract resulted in increased total secreted protein concentrations from treated neutrophils, as well as upregulation of 10 genes involved in immune response and downregulation of 4 genes associated with inflammation and immune regulation. The study findings indicate that garlic stimulates gene transcription and protein secretion in neutrophils from St. Croix sheep in vitro. Studies are needed on the possible impacts on neutrophil function and animal response to infection for sustainable use of garlic in animal production.

122 Response of Ruminant Neutrophils on Fresh Filtered Garlic Preparation

Abstract The purpose of this study was to evaluate the effect of fresh filtered garlic preparation on ruminant neutrophils in-vitro. Blood was collected from the jugular vein of clinically healthy female St Croix sheep (n = 3) and Boer x Spanish goats (n = 3) at NC A&T State University Farm. Neutrophils were isolated from 10 mL whole blood sample of each animal. Fresh filtered garlic were prepared to stimulate the neutrophils. Before treating the neutrophils, the total cell count was measured, and 1 million cells were taken for treatment. Total protein concentration at different dilution levels of fresh filtered garlic were analyzed using BCA assay for the volumetric determination of garlic to be used at 10 ug/mL concentration. Before and after treatment the total protein concentration in the neutrophil supernatant were also measured. RNA was extracted from treated and untreated neutrophils pellet using TRIZOL RNA Isolation Protocol. The pooled RNA of goat and sheep samples were used for cDNA synthesis followed by qPCR analysis of galectin (Galectin-1, -3, -9) gene modulation in relation to indicators of animal health and production. Due to treatment, the total protein concentrations were increased. The magnitude of galectin gene expression was also observed in both sheep and goat neutrophils treated with fresh filtered garlic preparation. Galectin-3 and -9 expression increased manifolds in both sheep and goat neutrophils as compared with the control (untreated neutrophils) whereas galectin-1 increased in goat but decreased in sheep neutrophils. These results indicate that garlic had no adverse effect and differentially modulated galectin secretion in vitro. Galectins regulate biologic functions, immunity and disease pathogenesis and can serve as biomarkers. Further studies are warranted to determine the health and production benefits of the observed galectin modulation using garlic in small & large ruminants.

Neutrophils modulate natural killer-mediated osteoclastogenesis during Aggregatibacter actinomycetemcomitans (JP2 clone) infection

The study investigates the interplay of neutrophils and natural-killer cells (NK) in mediating osseoresorption during infection of molar-incisor-pattern-periodontitis (MIPP). Human neutrophils from periodontally healthy and MIPP patients were inoculated with the periopathogen Aggregatibacter-actinomycetemcomitans (JP2) and their supernatants were exposed to NK to study their function and osteoclastogenesis promotion. A mouse MIPP model was used to compare disease progression following NK versus neutrophils depletion. The exposure of primary NK to supernatants of neutrophils inoculated with JP2 led to NK cell arrest and activation with enhanced osteoprotegerin expression. Incubation of monocytes with NK led to osteoclastogenesis, whereas NK that were pre-exposed to healthy neutrophil supernatant showed reduced osteoclastogenesis. In mice, NK depletion led to the similar bone phenotype as the neutrophil's depletion highlighting their role on osseoprotection. The present study portrays a key crosstalk between neutrophils and NK cells during JP2 infection as a central mechanism that regulates bone loss.

Influence of SARS-COV-2 Infection on Cytokine Production by Mitogen-Stimulated Peripheral Blood Mononuclear Cells and Neutrophils in COVID-19 Intensive Care Unit Patients.

We sought to investigate the influence of SARS-CoV-2 infection on the cytokine profiles of peripheral blood mononuclear cells (PBMCs) and neutrophils from coronavirus disease 2019 (COVID-19) intensive care unit (ICU) patients. Neutrophils and PBMCs were separated and stimulated with the mitogen phytohemagglutinin. Culture supernatants of mitogen-stimulated PBMCs and neutrophils from 88 COVID-19 ICU patients and 88 healthy controls were evaluated for levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-α, IFN-γ, interleukin (IL)-2, -4, -5, -6, -9, -10, -12, -17A, and tumor necrosis factor (TNF)-α using anti-cytokine antibody MACSPlex capture beads. Cytokine profiles of PBMCs showed significantly lower levels of GM-CSF, IFN-γ, IL-6, IL-9, IL-10, IL-17A, and TNF-α (p < 0.0001) in COVID-19 ICU patients. In contrast, COVID-19 ICU patients showed higher median levels of IL-2 (p < 0.001) and IL-5 (p < 0.01) by PBMCs. As for neutrophils, COVID-19 ICU patients showed significantly lower levels of GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-17A, IL-12, TNF-α (p < 0.0001), and IFN-α (p < 0.01). T-helper (Th)1:Th2 cytokine ratios revealed lower inflammatory cytokine for PBMCs and neutrophils in COVID-19 ICU patients. Cytokine production profiles and Th1:Th2 cytokine ratios suggest that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has an immunomodulatory effect on PBMCs and neutrophils. This study also suggests that the increased levels of several cytokines in the serum are not sourced from PBMCs and neutrophils.

Comparative characteristics of cytokine content in neutrophil supernatants after incubation with daily cultures of <i>S. aureus</i> with or without <i>spA</i> gene

The study was aimed for assessment of cytokine profile in the whole blood neutrophil supernatants stimulated by Staphylococcus aureus, with and without the spА gene.
 20 clinical isolates of S. aureus were tested in vitro. Ten of them carried spА gene and other 10 were without the spА gene. We selected the strains with very high ability to secrete cytokine-like substances (CLS), both in terms of the level and spectrum of the cytokines. The S. aureus strains were divided into two groups depending on the spА status according to the data from the spА genotyping databank (http://spa.ridom.de/). The neutrophils of donors were isolated in a double gradient of Ficoll-Verografin, according to the standard method, at a concentration of 5 106. The bacteria were taken at a ratio of 1:20 to neutrophils (at a concentration of 108 bacteria/mL). The effect of staphylococci on cytokine secretion by neutrophils was determined with a Magpix-100 device (USA), using immunofluorescence multiplex kits from BioRad (USA) for the detection of 17 cytokines (G-CSF, GM-CSF, IFN, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17A, TNF, MCP-1, MIP-1) after 1 hour incubation with the neutrophils. Results: It has been shown that staphylococci with a high degree of CLS activity, regardless of presence or absence of the spА gene, affected the ability of neutrophils to secrete cytokines. At the same time, S. aureus carrying the spА gene caused increased secretion of IL-1, TNF, IL-6 (pro-inflammatory cytokines), but, at the same time, it reduced secretion of IL-8, MIP-1 (chemokines). S. aureus strains devoid of spА genes have a more diverse spectrum and more pronounced effect on cytokine secretion of proinflammatory cytokines by neutrophils, e.g., IL-10, IL-12p70, IL-17A, IL-1, IL-2, TNF and the G-CSF growth factor. Both variants of staphylococci caused reduction of IL-6 secretion.

Evaluation of the cytokine profile of the supernatants of donor neutrophils irradiated with a low-intensity laser with a wavelength of 450 nm (in vitro study)

The influence of a low-intensity laser on the dynamics of the content of cytokines in the supernatants of intact neutrophils and activated by a low-intensity laser was studied. Under experimental conditions, the stimulating effect of the laser on the process of cytokine production was shown under experimental conditions.

The promoting effect of neutrophil-derived BAFF molecule on the proliferation and life span of CAL-27 oral squamous carcinoma cells

Tumor-associated neutrophils (TANs) are the major cellular component of the tumor microenvironment and have been shown to release of different bioactive molecules such as B-cell activating factor (BAFF). The data on the interactions between OSCC cells and neutrophils are limited and do not explain the actual role of the BAFF in the development of the OSCC. In the present study we examined the direct effect of neutrophils-derived BAFF on the OSCC cell line CAL-27 proliferation and apoptosis.PMNs of OSCC patients and healthy control were isolated from whole blood and separated by magnetic selection with monoclonal anti-human CD16 antibodies. CD-16 – positive neutrophils were incubated in the presence of TGF-β and/or LPS as well as flavonoids (luteolin and quercetin). CAL-27 cells were co-incubated with supernatants of neutrophils. BAFF expression in neutrophils, BAFF-R expression on CAL-27 cells and apoptosis of CAL-27 cells were assessed by flow cytometry. To determine the CAL-27 cells proliferation, the MTT test was used. Expression of select mitochondrial proteins in CAL-27 cells were measured by Western blot.Neutrophils from OSCC patients showed significantly higher expression of BAFF than those from the healthy controls. The results obtained revealed upregulation of the proliferation and downregulation of the apoptosis of the CAL-27 cells in the presence of the supernatants of TGF-β-treated neutrophils. Flavonoids reduced BAFF expression in neutrophils of patients with OSCC and control group. Lower intensity of apoptosis in CAL-27 cells was associated with the increased expression of anti-apoptotic Bcl-2, Mcl-1 and activated form of PI3K kinase (pPI3K) and simultaneously reduced expression of pro-apoptotic Bax protein in the presence of rhBAFF, as well as of supernatants of neutrophils derived from OSCC patients. In conclusion, the data presented confirm the previously suggested role of neutrophil-derived BAFF in OSCC development. The favorable effects of examined flavonoids on tumor-promoting BAFF expression in neutrophils suggest that they might be promising candidates as chemo-preventive agents in the therapy of patients with OSCC.

Hypoxia Increases the Potential for Neutrophil-mediated Endothelial Damage in Chronic Obstructive Pulmonary Disease.

Rationale: Patients with chronic obstructive pulmonary disease (COPD) experience excess cardiovascular morbidity and mortality, and exacerbations further increase the risk of such events. COPD is associated with persistent blood and airway neutrophilia and systemic and tissue hypoxia. Hypoxia augments neutrophil elastase release, enhancing capacity for tissue injury. Objective: To determine whether hypoxia-driven neutrophil protein secretion contributes to endothelial damage in COPD. Methods: The healthy human neutrophil secretome generated under normoxic or hypoxic conditions was characterized by quantitative mass spectrometry, and the capacity for neutrophil-mediated endothelial damage was assessed. Histotoxic protein concentrations were measured in normoxic versus hypoxic neutrophil supernatants and plasma from patients experiencing COPD exacerbation and healthy control subjects. Measurements and Main Results: Hypoxia promoted PI3Kγ-dependent neutrophil elastase secretion, with greater release seen in neutrophils from patients with COPD. Supernatants from neutrophils incubated under hypoxia caused pulmonary endothelial cell damage, and identical supernatants from COPD neutrophils increased neutrophil adherence to endothelial cells. Proteomics revealed differential neutrophil protein secretion under hypoxia and normoxia, and hypoxia augmented secretion of a subset of histotoxic granule and cytosolic proteins, with significantly greater release seen in COPD neutrophils. The plasma of patients with COPD had higher content of hypoxia-upregulated neutrophil-derived proteins and protease activity, and vascular injury markers. Conclusions: Hypoxia drives a destructive "hypersecretory" neutrophil phenotype conferring enhanced capacity for endothelial injury, with a corresponding signature of neutrophil degranulation and vascular injury identified in plasma of patients with COPD. Thus, hypoxic enhancement of neutrophil degranulation may contribute to increased cardiovascular risk in COPD. These insights may identify new therapeutic opportunities for endothelial damage in COPD.

Сomparative analysis of cytokine determination in neutrophil supernatants stimulated by S. aureus supernatants with and without the SPA gene

This study presents the results of the determination of cytokines by multiplex analysis in the blood neutrophil supernatants of donors stimulated by Staphylococcus aureus supernatants, depending on the presence or absence of the staphylococcal protein A (spa) gene in bacteria. Cytokine indication was carried out by multiplex analysis using BioRad kits (USA) for the determination of 17 human cytokines (G-CSF, GM-CSF, INF-γ, TNF-alfa, MCP-1, MIP-1beta, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-7,IL-8, IL-10, IL-12p70, IL-13, IL-17A) after 1 hour incubation of staphylococcal supernatants with neutrophils. It was shown that S. aureus, which had the staphylococcal protein A (spa) gene, significantly stimulated the secretion of neutrophils G-CSF (growth factor), IL-12p70, IL-17A, IFN-gamma, IL-13, IL-1b, IL-4, IL-6, IL-7, TNF-alfa (proinflammatory cytokines), MCP-1 (chemokine). But at the same time, they significantly reduced the secretion of IL-10 (anti-inflammatory cytokine), IL-8, MIP-1beta (chemokines). S. aureus supernatants, which did not have spa, also acted almost identically. IFN-gamma increased less significantly under their action, and IL-1b, on the contrary, more significantly compared to S. aureus supernatants having the staphylococcal protein A gene. It was found that the supernatants of all studied clinical isolates of S. aureus with a pronounced ability to produce cytokine-like substances, regardless of the presence/absence of the spa gene, affect the ability of neutrophils to secrete cytokines. Thus, the presence of cytokine-like activity in S. aureus affects the response of neutrophils to secrete a pool of cytokines upon contact of phagocytic cells with staphylococcal supernatants. In particular, pro-inflammatory interleukins, some chemokines and growth factors. This indicates that the interaction of neutrophils with extracellular cytokine-like products of S. aureus can significantly modulate the inflammatory process.

Study on anti-inflammatory activity and mechanism of indolealkylamines in toad skin on LPS-activated neutrophils

Indolealkylamines(IAAs) are the main hydrophilic substances in toad skin, mainly including free N-methyl-5-hydroxytryptamine, bufotenine, bufotenidine, dehydrobufotenine, and binding bufothionine. In this study, the LPS-activated neutrophils were used to investigate the structure-activity relationship and anti-inflammatory mechanism of the above-mentioned five monomers from the toad skin in vitro. The neutrophils were divided into the control group, model group(1 μg·mL~(-1) LPS), positive drug group(100 μg·mL~(-1) indometacin), as well as the low-(50 μg·mL~(-1)), medium-(100 μg·mL~(-1)) and high-dose(200 μg·mL~(-1)) free N-methyl-5-hydroxytryptamine, bufotenine, bufotenidine, dehydrobufotenine, and binding bufothionine groups. The levels of IL-6, TNF-α and IL-1β in the neutrophil supernatant of each group was measured by enzyme-linked immunosorbent assay(ELISA) after LPS stimulation, followed by the detection of apoptosis in each group after Annexin V/PI staining. The protein expression levels of caspase-3, Bax, Bcl-2, beclin1, LC3-I, and LC3-Ⅱ were assayed by Western blot. The results showed that IAAs reduced the excessive secretion of inflammatory cytokines caused by LPS compared with the model group. Besides, the activity of each free IAAs(N-methyl-5-hydroxytryptamine, bufotenine, bufotenidine and dehydrobufotenine), especially bufotenine, was stronger than that of the binding bufothionine. As revealed by Annexin V/PI staining, LPS delayed the early apoptosis of neutrophils compared with the control group, while bufotenine promoted the apoptosis of neutrophils in a dose-dependent manner, which might be related to the elevated expression of apoptosis-related protein Bax/Bcl-2. In addition, LPS activated the autophagy pathways in neutrophils. This study confirmed the efficacy of IAAs in reducing the secretion of inflammatory cytokines in neutrophils induced by LPS for the first time. For instance, bufotenine exerts the anti-inflammatory effect possibly by inducing the apoptosis of neutrophils.

S100A9 in adult asthmatic patients: a biomarker for neutrophilic asthma

The biomarkers and therapeutic targets of neutrophilic asthma (NA) are poorly understood. Although S100 calcium-binding protein A9 (S100A9) has been shown to correlate with neutrophil activation, its role in asthma pathogenesis has not been clarified. This study investigated the mechanism by which S100A9 is involved in neutrophil activation, neutrophil extracellular trap (NET)-induced airway inflammation, and macrophage polarization in NA. The S100A9 levels (by ELISA) in sera/culture supernatant of peripheral blood neutrophils (PBNs) and M0 macrophages from asthmatic patients were measured and compared to those of healthy controls (HCs). The function of S100A9 was evaluated using airway epithelial cells (AECs) and PBNs/M0 macrophages from asthmatic patients, as well as a mouse asthma model. The serum levels of S100A9 were higher in NA patients than in non-NA patients, and there was a positive correlation between serum S100A9 levels and sputum neutrophil counts (r = 0.340, P = 0.005). Asthmatic patients with higher S100A9 levels had lower PC20 methacholine values and a higher prevalence of severe asthma (SA) (P < .050). PBNs/M0 macrophages from SA released more S100A9 than those from non-SA patients. PBNs from asthmatic patients induced S100A9 production by AECs, which further activated AECs via the extracellular signal-regulated kinase (ERK) pathway, stimulated NET formation, and induced M1 macrophage polarization. Higher S100A9 levels in sera, bronchoalveolar lavage fluid, and lung tissues were observed in the mouse model of NA but not in the other mouse models. These results suggest that S100A9 is a potential serum biomarker and therapeutic target for NA.

Viral mimetic poly(I:C) induces neutrophil extracellular traps via PAD4 to promote inflammation and thrombosis

Neutrophil extracellular traps (NETs) are extracellular webs of DNA, histones and granular contents that are released by neutrophils to control infections. However, NETs that is not properly regulated can propagate inflammation and thrombosis. It was recognized that viruses can induce NETs. As a synthetic analog of viral double-stranded (ds) RNA, polyinosinic-polycytidylic acid [poly(I:C)] is known to induce inflammation and thrombosis. However, whether and how poly(I:C) modulates NETs remains unclear. Here, we have demonstrated that poly(I:C) induced extracellular DNA traps in human neutrophils in a dose-dependent manner. Further, poly(I:C) or dsRNA virus elevated the levels of myeloperoxidase-DNA complexes and citrullinated histone H3, which are specific markers of NETs, in both neutrophil supernatants and mouse plasma. Interestingly, a potent peptidylarginine deiminase 4 (PAD4) inhibitor, BB-CL-Amidine (BB-CLA) or PAD4 knockdown effectively prevented poly(I:C)-induced NETs formation and release. In addition, BB-CLA abrogated poly(I:C)-triggered neutrophil activation and infiltration, and vascular permeability in lungs. BB-CLA also attenuated poly(I:C)-induced thrombocytopenia in circulation, fibrin deposition and thrombus formation in tissues. Taken together, these results suggest that viral mimetic poly(I:C) may induce NETs-dependent inflammation and thrombosis through PAD4, and that inhibiting PAD4 may become a good strategy to protect against viral infection-caused inflammation/thrombosis-related pathological conditions of diseases.

High Ambient Temperature Aggravates Experimental Autoimmune Uveitis Symptoms.

Whether ambient temperature influences immune responses leading to uveitis is unknown. We thus tested whether ambient temperature affects the symptoms of experimental autoimmune uveitis (EAU) in mice and investigated possible mechanisms. C57BL/6 mice were kept at a normal (22°C) or high temperature (30°C) housing conditions for 2 weeks and were then immunized with human interphotoreceptor retinoid-binding protein (IRBP651–670) peptide to induce EAU. Histological changes were monitored to evaluate the severity of uveitis. Frequency of Th1 cells and Th17 cells was measured by flow cytometry (FCM). The expression of IFN-γ and IL-17A mRNA was measured by real-time qPCR. The generation of neutrophil extracellular traps (NETs) was quantified by enzyme-linked immunosorbent assay (ELISA). Differential metabolites in the plasma of the mice kept in the aforementioned two ambient temperatures were measured via ultra-high-performance liquid chromatography triple quadrupole mass spectrometry quadrupole time of flight mass spectrometry (UHPLC-QQQ/MS). The differential metabolites identified were used to evaluate their effects on differentiation of Th1 and Th17 cells and generation of NETs in vitro. The results showed that EAU mice kept at high temperature experienced a more severe histopathological manifestation of uveitis than mice kept at a normal temperature. A significantly increased frequency of Th1 and Th17 cells in association with an upregulated expression of IFN-γ and IL-17A mRNA was observed in the splenic lymphocytes and retinas of EAU mice in high temperature. The expression of NETs as evidenced by myeloperoxidase (MPO) and neutrophil elastase (NE), was significantly elevated in serum and supernatants of neutrophils from EAU mice kept at high temperature compared to the normal temperature group. The metabolites in the plasma from EAU mice, fumaric acid and succinic acid, were markedly increased in the high temperature group and could induce the generation of NETs via the NADPH oxidase-dependent pathway, but did not influence the frequency of Th1 and Th17 cells. Our findings suggest that an increased ambient temperature is a risk factor for the development of uveitis. This is associated with the induction of Th1 and Th17 cells as well as the generation of NETs which could be mediated by the NADPH oxidase-dependent pathway.

Assessment of function of neutrophil-derived properdin in a novel functional assay

Abstract The alternative pathway (AP) of complement is an essential immune effector mechanism. Properdin (P), the only positive regulator of the AP, is required for stabilization of AP enzymatic convertases. P also binds to certain surfaces, in vitro, activating complement by recruiting a de novo C3bBb convertase. P is present in serum as dimers (P2), trimers (P3) and tetramers (P4) in a 1:2:1 ratio, with tetramers the most active, followed by trimers and dimers. Activated neutrophils release properdin from secondary granules into the cellular microenvironment. However, the distribution of neutrophil-secreted P oligomers (which would correlate with P function), and whether this distribution modulates local inflammatory processes, are unknown. We developed a novel ELISA-based assay that detects functional differences between P oligomers, based on the immobilization of P and assessment of its ability to generate C3 convertases leading to C3b deposition. Pure P2, P3, and P4 were functionally distinct in this assay and serum-derived P behaved similar to pooled P2-P4, as expected. Surprisingly, P from PMA-activated neutrophil supernatants had significantly lower activity than serum P (p&amp;lt;0.0001), and varied between healthy donors. To understand whether serum is required for enhancing P function (i.e. by allowing P polymerization), neutrophil supernatants were pre-incubated with P-depleted serum, followed by measurement of P function by ELISA. No increase in neutrophil-derived P function was observed. Additional studies are underway to understand the mechanisms involved in the lower function of neutrophil-derived P, with the aim of contributing to understanding molecular mechanisms of AP regulation in inflammatory microenvironments.

Neutrophil-derived exosome from systemic sclerosis inhibits the proliferation and migration of endothelial cells

ObjectivesSystemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy, inflammation, and extensive fibrosis in multiple organs. Exosomes (EXOs) are cell-derived vesicles contained various DNAs, RNAs and proteins, and play important roles in various diseases. Here, we aimed to investigate the roles of SSc EXOs in angiogenesis related mechanisms. MethodsEXOs were isolated from plasma, cultured peripheral blood mononuclear cells (PBMCs)/neutrophil supernatants, and identified by transmission electron microscopy. The expression of S100A8/A9 was measured by real-time PCR and ELISA. Proliferation, migration and scratch assays in human dermal microvascular endothelial cells (HDMECs) were used to study the EXOs influence. ResultsPlasma and neutrophil EXOs from SSc patients can suppress the proliferation and migration of HDMECs. High levels of S100A8/A9 were found in SSc EXOs which derived from plasma, PBMCs and neutrophils. The expression of S100A8/A9 in neutrophil EXOs was higher than that in PBMC EXOs in SSc patients. The proliferation and migration of HDMECs were possibly inhibited by S100A8/A9 of neutrophil EXOs. ConclusionsNeutrophil EXOs from SSc patients inhibits the proliferation and migration of HDMECs, S100A8/A9 might play an important role in this process.