Human Peripheral Blood Neutrophils Research Articles

Oxysterols Suppress Release of DNA from Granulocytes into Extracellular Space After Stimulation with Phorbol Myristate Acetate.

Neutrophils eject their DNA strings and cellular proteins into the extracellular space upon treatment with various stimulants. In the present study, we examined the effects of four major oxidized cholesterol metabolites on DNA release from granulocytes. When oxysterols were added to HL-60-derived granulocytes stimulated with phorbol 12-myristate 13-acetate (PMA), they suppressed the release of DNA and myeloperoxidase from the cells. Among the four oxysterols tested, 7-ketocholesterol was the most effective. Addition of the same concentration of 7-ketocholesterol did not induce any cytotoxic effects, as evaluated based on the release of lactate dehydrogenase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) assays. DNA release from human peripheral blood neutrophils after PMA stimulation was also suppressed by 7-ketocholesterol. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis was used to quantify sterol content in the cells. The addition of oxysterols increased the cellular content of the corresponding compounds by more than 10-fold compared to those at baseline. Treatment of HL-60-derived granulocytes with methyl-β-cyclodextrin that removes sterol compounds from the membranes increased DNA release from the cells in a dose-dependent manner. These results suggest that oxysterols have suppressive effects on DNA release from granulocytes stimulated with PMA.

Unveiling signaling pathways inducing MHC class II expression in neutrophils.

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

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

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

Bone morphogenetic protein 4 inhibits corneal neovascularization by blocking NETs-induced disruption to corneal epithelial barrier

Corneal neovascularization (CoNV) is the second leading cause of visual impairment worldwide, and current drugs have certain limitations. Inflammatory response is the core pathological process of CoNV. Neutrophil extracellular traps (NETs) are generated after neutrophil activation, which promotes neovascularization. Prior studies demonstrated that bone morphogenetic protein 4 (BMP4) could significantly reduce inflammation and CoNV formation, its exact molecular mechanism remains unclear. Therefore, we stimulated human peripheral blood neutrophils with phorbol myristate acetate (PMA) or deoxyribonuclease I (DNase I) to induce or inhibit NETs formation. By using corneal sutures and subconjunctival injections of NETs or DNase I, rat CoNV models were established. Compared with the suture group, NETs formation and inflammatory cell infiltration in the corneal stroma were significantly increased, corneal edema was aggravated, and the length, area and diameter of CoNV were significantly enhanced in the NETs group. Furthermore, by curetting the corneal epithelial apical junctional complexes (AJCs), a crucial component in preserving the function of the corneal epithelial barrier, we discovered that the damage of AJCs had a significant role in inducing CoNV formation. NETs could induce CoNV formation by injuring corneal epithelial AJCs. Finally, by comparing the aforementioned indicators after the intervention of BMP4, BMP4 inhibitor Noggin and NADPH oxidase (NOX) inhibitor, we finally demonstrated that BMP4 could inhibit NETs-induced inflammation and corneal epithelial AJC injury, repair corneal epithelial barrier function and eventually inhibit CoNV formation by blocking NOX-2-dependent NETs formation.

FAM3D regulation of head and neck squamous cell carcinoma behaviour through the EMT pathway.

Head and neck squamous cell carcinoma present challenges in effective treatment, with 50%-60% of cases exhibiting recurrence or metastasis, often resistant to surgery alone. Immunotherapy, a promising approach, does not guarantee benefits for all patients. Thus, the imperative lies in identifying reliable biomarkers for predicting immunotherapy efficacy. FAM3D, a protein-coding gene known for its potent chemotactic activity in human peripheral blood monocytes and neutrophils, plays a crucial role in regulating tumour immune responses and holds promise as an immune biomarker. We employed comprehensive database analysis to scrutinise FAM3D, evaluating its gene expression, mutation profiles and prognostic implications in head and neck squamous cell carcinoma, along with its associations with clinical characteristics and immune cell infiltration. Complementary functional experiments were conducted to delve into the potential mechanisms governed by FAM3D. Our findings establish a significant correlation between low FAM3D expression and the invasiveness and metastatic potential of head and neck squamous cell carcinoma. FAM3D likely exerts its influence through the regulation of epithelial-mesenchymal transition. FAM3D emerges as a valuable biomarker for predicting the responsiveness of patients with head and neck squamous cell carcinoma to immunotherapy, holding substantial clinical diagnostic and therapeutic relevance.

Icariside II alleviates lipopolysaccharide-induced acute lung injury by inhibiting lung epithelial inflammatory and immune responses mediated by neutrophil extracellular traps

AimsAcute lung injury (ALI) is a life-threatening lung disease characterized by inflammatory cell infiltration and lung epithelial injury. Icariside II (ICS II), one of the main active ingredients of Herba Epimedii, exhibits anti-inflammatory and immunomodulatory effects. However, the effect and mechanism of ICS II in ALI remain unclear. The purpose of the current study was to investigate the pharmacological effect and underlying mechanism of ICS II in ALI. Main methodsModels of neutrophil-like cells, human peripheral blood neutrophils, and lipopolysaccharide (LPS)-induced ALI mouse model were utilized. RT-qPCR and Western blotting determined the gene and protein expression levels. Protein distribution and quantification were analyzed by immunofluorescence. Key findingsICS II significantly reduced lung histopathological damage, edema, and inflammatory cell infiltration, and it reduced pro-inflammatory cytokines in ALI. There is an excessive activation of neutrophils leading to a significant production of NETs in ALI mice, a process mitigated by the administration of ICS II. In vivo and in vitro studies found that ICS II could decrease NET formation by targeting neutrophil C-X-C chemokine receptor type 4 (CXCR4). Further data showed that ICS II reduces the overproduction of dsDNA, a NETs-related component, thereby suppressing cGAS/STING/NF-κB signalling pathway activation and inflammatory mediators release in lung epithelial cells. SignificanceThis study suggested that ICS II may alleviate LPS-induced ALI by modulating the inflammatory response, indicating its potential as a therapeutic agent for ALI treatment.

Abstract 5329: Ovarian cancer predisposes neutrophils to form neutrophil extracellular traps(NETs)

Abstract Objective: Neutrophil extracellular trap (NETs) are neutrophil-derived extracellular DNA released in response to inflammation. In contrast to their primary host-defensive role, NETs have been recently reported to promote cancer progression. The aim of this study is to investigate the role of NETs in ovarian cancer progression. Method: The clinical data of ovarian cancer patients who underwent primary surgery at Osaka University Medical Hospital were retrospectively reviewed (n=340). The relationship between neutrophilia, peritoneal dissemination and prognosis were analyzed. Prior to initial treatment, peripheral blood was obtained from newly diagnosed ovarian cancer patients and examined for CBC and granulocyte-colony stimulating factor (G-CSF) concentration by ELISA. To evaluate NETs formation capacity, isolated neutrophils were stimulated with G-CSF and incubated in vitro to monitor NETs formation. The ascites was collected at the initial surgery and assessed for G-CSF concentration and for neutrophils population using flow cytometry. The omental tissues were pathologically examined for NETs using H&E-staining and immunostaining with anti-Citrullinated Histon3 (CitH3) antibody. To test NETs induction capacity, we treated human peripheral blood neutrophils with the conditioned media from 20 ovarian cancer cell lines and the serum from ovarian cancer patients. Then human ovarian cancer OVCAR8 cells were co-cultured with NETs to evaluate the effect of NETs on cancer cell adhesion. Results: Neutrophilia (neutrophils>7000/µl) was found in 39 patients (39/340,11.5%). Patients with neutrophilia were associated with advanced disease accompanied by peritoneal dissemination and compromised survival compared to patients without neutrophilia (5-year PFS 40.0 vs 59.9 %, p 0.0027). In these patients, serum G-CSF was upregulated. Neutrophils from ovarian cancer patients formed NETs significantly earlier than those from benign tumor patients. Ovarian cancer patients with neutrophilia showed elevated G-CSF concentration and increased neutrophils proportion in their ascites. Omental tissues from patients with neutrophilia showed NETs foci close to the disseminated lesions. G-CSF strongly induced neutrophils to form NETs in vitro, which was observed in some conditioned media from ovarian cancer cell lines and serum from ovarian cancer patients as well. The adhesion of ovarian cancer cell was increased by co-incubation with NETs. Conclusion: Neutrophilia is associated with peritoneal dissemination and poor prognosis in ovarian cancer. One of the underlying mechanism of neutrophilia is upregulated G-CSF, which stimulate neutrophils to form NETs. NETs may have a causative effect on ovarian cancer dissemination. Citation Format: Gaku Yamamoto, Mahiru Kawano, Michiko Bun, Koutaro Shimura, Aska Toda, Koji Nakamura, Yasuto Kinose, Michiko Kodama, Kae Hashimoto, Kenjiro Sawada, Tadashi Kimura. Ovarian cancer predisposes neutrophils to form neutrophil extracellular traps(NETs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5329.

Ficolin-A/2 Aggravates Severe Lung Injury through Neutrophil Extracellular Traps Mediated by Gasdermin D–Induced Pyroptosis

Neutrophil extracellular traps (NETs) and pyroptosis are critical events in lung injury. We aimed to investigate whether Ficolin-A influences NET formation through pyroptosis to exacerbate lipopolysaccharides (LPS)-induced lung injury. The expression of Ficolin-A/2, NETs, and pyroptosis-related molecules was investigated in animal and cell models. Knockout and knockdown (recombinant protein) methods were employed to elucidate regulatory mechanisms. Pearson's correlation coefficient was utilized to analyze the correlation between Ficolins and pyroptosis- and NET-related markers in clinical samples. In this study, Ficolin-2 (similar to Ficolin-A) showed significant overexpression in patients with acute respiratory distress syndrome (ARDS). In vivo, knockout of Ficolin-A, but not Ficolin-B, attenuated lung inflammation and inhibited NET formation in LPS-induced mouse model. DNaseI further alleviated lung inflammation and NET formation in Ficolin-A knockout mice. In vitro, neutrophils derived from Fcna-/- mice exhibited less pyroptosis and necroptosis than those from the control group after LPS stimulation. Additionally, gasdermin D (GSDMD) knockdown or Nod-like receptor protein 3 (NLRP3) inhibitor reduced NET formation. Addition of recombinant Ficolin-2 protein to human peripheral blood neutrophils promoted NET formation and pyroptosis after LPS stimulation, whereas Ficolin-2 knockdown had opposite effects. ARDS patients exhibited increased levels of pyroptosis- and NET-related markers, which were positively correlated with Ficolin-2 levels. In conclusion, these results suggested that Ficolin-A/2 exacerbated NET formation and LPS-induced lung injury via the GSDMD-mediated pyroptosis.

Antioxidant and Anti-Inflammatory Activity of Fractionated Artemia Urmiana Extract in Human Peripheral Blood Neutrophils System

Marine organisms are great resources for discovering bioactive compounds with new antioxidant properties. The aim of this work was to purify and isolate the protein fractions of Artemia urmiana and to evaluate antioxidant activities of them on human peripheral blood neutrophils response. For these purposes, crude extracts protein was extracted from napulii of A. urmiana and sequentially fractionated by ion exchange. The antioxidant activities of the samples were determined by DPPH free radical scavenging, reducing power ability assays in comparison with ascorbic acid. In the second phase, the human peripheral blood neutrophils (HPBN) were separated and cultured. The cells were treated with increasing concentrations of crude and partially purified fractions for 24 hrs. After that, the pellet and supernatant of treated cells were separately collected and assayed for Nitro Blue Tetrazolium (NBT) reduction, the activity of Super Oxide Dismutase (SOD) and Nitric oxide content. The results showed that the IC50 values of ascorbic acid, crude extracts, partially purified protein fractions(F1-F8) were 150, 126, 141, 295, 299, 114, 164 and 132 µg/ml respectively in the DPPH radical scavenging activity. The reducing power activity at the highest concentration (200 µg/ml) based on the optical density of the sample listed, were 2.8, 1.4, 0.13, 0.14, 0.17, 0.14, 0.13, 0.66, 0.18 and 0.35 respectively. In NBT reduction and production of Nitric oxide, treated cells did not show significant difference between different concentrations of protein extract and untreated control cells. The activity of SOD was increased in presence of 50-100 µg/ml of crud extract and fractions 1, 2, 3, 4 and 5 in comparison to untreated control cells (p<0.05).

Formylated Peptide Receptor-1-Mediated Gut Inflammation as a Therapeutic Target in Inflammatory Bowel Disease.

Formylated peptide receptor (FPR)-1 is a G-coupled receptor that senses foreign bacterial and host-derived mitochondrial formylated peptides (FPs), leading to innate immune system activation. We sought to investigate the role of FPR1-mediated inflammation and its potential as a therapeutic target in inflammatory bowel disease (IBD). We characterized FPR1 gene and protein expression in 8 human IBD (~1000 patients) datasets with analysis on disease subtype, mucosal inflammation, and drug response. We performed in vivo dextran-sulfate sodium (DSS) colitis in C57/BL6 FPR1 knockout mice. In ex vivo studies, we studied the role of mitochondrial FPs and pharmacological blockade of FPR1 using cyclosporin H in human peripheral blood neutrophils. Finally, we assess mitochondrial FPs as a potential mechanistic biomarker in the blood and stools of patients with IBD. Detailed in silico analysis in human intestinal biopsies showed that FPR1 is highly expressed in IBD (n = 207 IBD vs 67 non-IBD controls, P < .001), and highly correlated with gut inflammation in ulcerative colitis (UC) and Crohn's disease (CD) (both P < .001). FPR1 receptor is predominantly expressed in leukocytes, and we showed significantly higher FPR1+ve neutrophils in inflamed gut tissue section in IBD (17 CD and 24 UC; both P < .001). Further analysis in 6 independent IBD (data available under Gene Expression Omnibus accession numbers GSE59071, GSE206285, GSE73661, GSE16879, GSE92415, and GSE235970) showed an association with active gut inflammation and treatment resistance to infliximab, ustekinumab, and vedolizumab. FPR1 gene deletion is protective in murine DSS colitis with lower gut neutrophil inflammation. In the human ex vivo neutrophil system, mitochondrial FP, nicotinamide adenine dinucleotide dehydrogenase subunit-6 (ND6) is a potent activator of neutrophils resulting in higher CD62L shedding, CD63 expression, reactive oxygen species production, and chemotactic capacity; these effects are inhibited by cyclosporin H. We screened for mitochondrial ND6 in IBD (n = 54) using ELISA and detected ND6 in stools with median values of 2.2 gg/mL (interquartile range [IQR] 0.0-4.99; range 0-53.3) but not in blood. Stool ND6 levels, however, were not significantly correlated with paired stool calprotectin, C-reactive protein, and clinical IBD activity. Our data suggest that FPR1-mediated neutrophilic inflammation is a tractable target in IBD; however, further work is required to clarify the clinical utility of mitochondrial FPs as a potential mechanistic marker for future stratification.

Bruton’s tyrosine kinase is a possible therapeutic target in microscopic polyangiitis

BackgroundBruton’s tyrosine kinase (Btk) is an enzyme expressed in leukocytes other than T lymphocytes and plasma cells and involved in B-cell receptor- and Fcγ receptor (FcγR)-mediated signal transduction. Btk inhibitors potentially suppress autoantibody production due to the expected inhibitory ability of B lymphocyte differentiation into antibody-producing plasma cells and reduce FcγR-mediated neutrophil activation, including the release of neutrophil extracellular traps (NETs). Microscopic polyangiitis (MPA) is a systemic small-vessel vasculitis characterized by the pathogenic autoantibody, antineutrophil cytoplasmic antibody (ANCA) that reacts with myeloperoxidase (MPO). MPO and MPO-ANCA immune complex (IC)-induced FcγR-mediated NETs are critically involved in MPA pathogenesis. This study aimed to demonstrate the therapeutic efficacy of the Btk inhibitor tirabrutinib on MPA.MethodsVarious doses of tirabrutinib or vehicle were orally administered to Sprague–Dawley rats daily. Four weeks later, the number of peripheral B lymphocytes was counted, and Btk phosphorylation in B lymphocytes was evaluated by flow cytometry. Human peripheral blood neutrophils were stimulated by MPO and anti-MPO antibody ICs (MPO and anti-MPO-ICs), and Btk and its downstream Vav phosphorylation were assessed by western blotting. The effects of tirabrutinib on MPO and anti-MPO-IC-induced NET formation were examined in vitro. Wistar Kyoto rats were immunized with human MPO to induce experimental MPA and given drug-free or tirabrutinib-containing feed (0.0037% or 0.012%) from day 0 or 28. All rats were euthanized on day 42 for serological and histological evaluation.ResultsTirabrutinib inhibited Btk phosphorylation without decreasing B lymphocytes in vivo. Neutrophil Btk and Vav were phosphorylated when stimulated with MPO and anti-MPO-ICs. Tirabrutinib suppressed MPO and anti-MPO-IC-induced NET formation in vitro and ameliorated experimental MPA in a dose-dependent manner in vivo. Although MPO-ANCA production was not affected, NET-forming neutrophils in the blood were significantly reduced by tirabrutinib.ConclusionsThe Btk inhibitor tirabrutinib suppressed MPO and anti-MPO-IC-induced NET formation in vitro and ameliorated experimental MPA by reducing NET-forming neutrophils but not decreasing MPO-ANCA titer in vivo. This study suggests that Btk is a possible therapeutic target in MPA.

Vaping-Induced Proteolysis Causes Airway Surface Dehydration.

Proteases such as neutrophil elastase cleave and activate the epithelial sodium channel (ENaC), causing airway dehydration. Our current study explores the impact of increased protease levels in vapers' airways on ENaC activity and airway dehydration. Human bronchial epithelial cultures (HBECs) were exposed to bronchoalveolar lavage fluid (BALF) from non-smokers, smokers and vapers. Airway surface liquid (ASL) height was measured by confocal microscopy as a marker of hydration. ENaC cleavage was measured by Western blotting. Human peripheral blood neutrophils were treated with a menthol-flavored e-liquid (Juul), and the resulting secretions were added to HBECs. BALF from smokers and vapers significantly and equally increased ENaC activity and decreased ASL height. The ASL height decrease was attenuated by protease inhibitors. Non-smokers' BALF had no effect on ENaC or ASL height. BALF from smokers and vapers, but not non-smokers, induced ENaC cleavage. E-liquid-treated neutrophil secretions cleaved ENaC and decreased ASL height. Our study demonstrated that elevated protease levels in vapers' airways have functional significance since they can activate ENaC, resulting in airway dehydration. Lung dehydration contributes to diseases like cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) and asthma. Thus, our data predict that vaping, like smoking, will cause airway surface dehydration that likely leads to lung disease.

Vitamin D3 regulates PM-driven primary human neutrophil inflammatory responses

Recent evidence has demonstrated that both acute and chronic exposure to particulate air pollution are risk factors for respiratory tract infections and increased mortality from sepsis. There is therefore an urgent need to establish the impact of ambient particulate matter (PM) on innate immune cells and to establish potential strategies to mitigate against adverse effects. PM has previously been reported to have potential adverse effects on neutrophil function. In the present study, we investigated the impact of standard urban PM (SRM1648a, NIST) and PM2.5 collected from Chiang Mai, Thailand, on human peripheral blood neutrophil functions, including LPS-induced migration, IL-8 production, and bacterial killing. Both NIST and the PM2.5, being collected in Chiang Mai, Thailand, increased IL-8 production, but reduced CXCR2 expression and migration of human primary neutrophils stimulated with Escherichia coli LPS. Moreover, PM-pretreated neutrophils from vitamin D-insufficient participants showed reduced E. coli-killing activity. Furthermore, in vitro vitamin D3 supplementation attenuated IL-8 production and improved bacterial killing by cells from vitamin D-insufficient participants. Our findings suggest that provision of vitamin D to individuals with insufficiency may attenuate adverse acute neutrophilic responses to ambient PM.

Antigen-presenting aged neutrophils induce CD4+ T cells to exacerbate inflammation in sepsis.

Extracellular cold-inducible RNA-binding protein (eCIRP) is a key mediator of severity and mortality in sepsis. We found that stimulation of mouse bone marrow-derived neutrophils (BMDNs) with eCIRP generated a distinct neutrophil subpopulation, characterized by cell surface markers of both antigen-presenting cells and aged neutrophils as well as expression of IL-12, which we named antigen-presenting aged neutrophils (APANs). The frequency of APANs was significantly increased in the blood, spleen, and lungs of WT mice subjected to cecal ligation and puncture-induced sepsis but not in CIRP-/- mice. Patients with sepsis had a significant increase in circulating APAN counts compared with healthy individuals. Compared with non-APAN-transfered mice, APAN-transferred septic mice had increased serum levels of injury and inflammatory markers, exacerbated acute lung injury (ALI), and worsened survival. APANs and CD4+ T cells colocalized in the spleen, suggesting an immune interaction between these cells. APANs cocultured with CD4+ T cells significantly induced the release of IFN-γ via IL-12. BMDNs stimulated with eCIRP and IFN-γ underwent hyper-NETosis. Stimulating human peripheral blood neutrophils with eCIRP also induced APANs, and stimulating human neutrophils with eCIRP and IFN-γ caused hyper-NETosis. Thus, eCIRP released during sepsis induced APANs to aggravate ALI and worsen the survival of septic animals via CD4+ T cell activation, Th1 polarization, and IFN-γ-mediated hyper-NETosis.

Neutrophils in STAT1 Gain-Of-Function Have a Pro-inflammatory Signature Which Is Not Rescued by JAK Inhibition

STAT1 gain-of-function (GOF) mutations cause an inborn error of immunity with diverse phenotype ranging from chronic mucocutaneous candidiasis (CMC) to various non-infectious manifestations, the most precarious of which are autoimmunity and vascular complications. The pathogenesis centers around Th17 failure but is far from being understood. We hypothesized that neutrophils, whose functions have not been explored in the context of STAT1 GOF CMC yet, might be involved in the associated immunodysregulatory and vascular pathology. In a cohort of ten patients, we demonstrate that STAT1 GOF human ex-vivo peripheral blood neutrophils are immature and highly activated; have strong propensity for degranulation, NETosis, and platelet-neutrophil aggregation; and display marked inflammatory bias. STAT1 GOF neutrophils exhibit increased basal STAT1 phosphorylation and expression of IFN stimulated genes, but contrary to other immune cells, STAT1 GOF neutrophils do not display hyperphosphorylation of STAT1 molecule upon stimulation with IFNs. The patient treatment with JAKinib ruxolitinib does not ameliorate the observed neutrophil aberrations. To our knowledge, this is the first work describing features of peripheral neutrophils in STAT1 GOF CMC. The presented data suggest that neutrophils may contribute to the immune pathophysiology of the STAT1 GOF CMC.

Two-dimensional molybdenum disulfide nanosheets evoke nitric oxide-dependent antibacterial effects.

Nanomaterials are currently being explored as novel antimicrobial agents. In this study, we first investigated the ability of two-dimensional (2D) molybdenum disulfide (MoS2) nanosheets to trigger neutrophil extracellular traps (NETs) using neutrophil-differentiated HL-60 cells as well as primary human peripheral blood neutrophils. We then addressed whether the MoS2 nanosheets themselves function as antibacterial agents. We found that MoS2 and Na2MoO4 both triggered NETs, as evidenced by the quantification of neutrophil elastase (NE) activity and immunofluorescence staining of extracellular NE, as well as scanning electron microscopy. The release of NETs was found to be nitric oxide (NO)-dependent. We also found that the MoS2 nanosheets but not the soluble salt prompted acellular NO production in the presence of NaNO2. The acellular generation of NO, suggestive of nanozyme properties of the MoS2 nanosheets, was demonstrated by electron paramagnetic resonance analysis. Electrochemical analysis using cyclic voltammetry confirmed the redox transition of the MoS2 nanosheets. Finally, MoS2 nanosheets inhibited the growth of Escherichia coli in the presence of sodium nitrate. Taken together, MoS2 nanosheets triggered cellular effects as well as acellular antibacterial effects, and we provided evidence for nitrite reductase-like properties of MoS2.

Human Amniotic Membrane Mesenchymal Stem Cell-Synthesized PGE2 Exerts an Immunomodulatory Effect on Neutrophil Extracellular Trap in a PAD-4-Dependent Pathway through EP2 and EP4.

Human amniotic membrane mesenchymal stem cells (hAM-MSC) secrete a myriad of components with immunosuppressive activities. In the present research, we aimed to describe the effect of prostaglandin E2 (PGE2) secreted by hAM-MSCs on neutrophil extracellular trap (NET) release and to characterize the role of its receptors (EP2/EP4) in PAD-4 and NFκB activity in neutrophils. Human peripheral blood neutrophils were ionomycin-stimulated in the presence of hAM-MSC conditioned medium (CM) treated or not with the selective PGE2 inhibitor MF-63, PGE2, EP2/EP4 agonists, and the selective PAD-4 inhibitor GSK-484. NET release, PAD-4, and NFκB activation were analyzed. Ionomycin induced NET release, which was inhibited in the presence of hAM-MSC-CM, while CM from hAM-MSCs treated with MF-63 prevented NET release inhibition. PGE2 and EP2/EP4 agonists, and GSK-484 inhibited NET release. EP2/EP4 agonists and GSK-484 inhibited H3-citrullination but did not affect PAD-4 protein expression. Finally, PGE2 and EP2/EP4 agonists and GSK-484 increased NFκB phosphorylation. Taken together, these results suggest that hAM-MSC exert their immunomodulatory activities through PGE2, inhibiting NET release in a PAD-4-dependent pathway. This research proposes a new mechanism by which hAM-MSC exert their activities when modulating the innate immune response and inhibiting NET release.

Single-Cell Analysis Reveals the Range of Transcriptional States of Circulating Human Neutrophils.

Neutrophils are the most abundant leukocytes in human blood and are essential components of innate immunity. Until recently, neutrophils were considered homogeneous and transcriptionally inactive cells, but both concepts are being challenged. Single-cell RNA sequencing (scRNA-seq) offers an unbiased view of cells along a continuum of transcriptional states. However, the use of scRNA-seq to characterize neutrophils has proven technically difficult, explaining in part the paucity of published single-cell data on neutrophils. We have found that modifications to the data analysis pipeline, rather than to the existing scRNA-seq chemistries, can significantly increase the detection of human neutrophils in scRNA-seq. We have then applied a modified pipeline to the study of human peripheral blood neutrophils. Our findings indicate that circulating human neutrophils are transcriptionally heterogeneous cells, which can be classified into one of four transcriptional clusters that are reproducible among healthy human subjects. We demonstrate that peripheral blood neutrophils shift from relatively immature (Nh0) cells, through a transitional phenotype (Nh1), into one of two end points defined by either relative transcriptional inactivity (Nh2) or high expression of type I IFN-inducible genes (Nh3). Transitions among states are characterized by the expression of specific transcription factors. By simultaneously measuring surface proteins and intracellular transcripts at the single-cell level, we show that these transcriptional subsets are independent of the canonical surface proteins that are commonly used to define and characterize human neutrophils. These findings provide a new view of human neutrophil heterogeneity, with potential implications for the characterization of neutrophils in health and disease.

Endoplasmic Reticulum Stress of Oral Squamous Cell Carcinoma Induces Immunosuppression of Neutrophils

The endoplasmic reticulum (ER) stress of cancer cells not only determined cancer cell fate but also indirectly triggered proinflammatory or immunosuppressive responses of macrophages. In addition, ER stressed neutrophils were known to acquire immunosuppressive activity with surface expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). Since the importance of tumor ER stress and immunosuppressive neutrophils has been emphasized in head and neck cancers, we hypothesized that the ER stress of oral squamous cell carcinoma (OSCC) could transform neutrophils into LOX-1 expressing immunosuppressive phenotype. Two human OSCC cell lines, SCC25 and OML1, were treated with either vehicle or thapsigargin (THG), an ER stress inducer. These tumor conditioned media (TCM) were collected accordingly. Then human peripheral blood neutrophils from healthy donors were cultured in these TCM. The results showed that neutrophils cultured in THG-treated TCM had higher expression of LOX-1 compared with those cultured in vehicle-treated TCM. Moreover, by interleukin-2/anti-CD3/anti-CD28 activated autologous T cell proliferation assay, neutrophils conditioned by THG-treated TCM were shown to inhibit T cell proliferation more significantly than those conditioned by vehicle-treated TCM. These novel findings indicated that the ER stress of OSCC could be transmitted to neutrophils which in turn expressed LOX-1 and obtained immunosuppressive ability. Our findings further supported the existence of “transmissible” ER stress between tumor cells and neutrophils.

Albumin Nanoparticle Endocytosing Subset of Neutrophils for Precision Therapeutic Targeting of Inflammatory Tissue Injury.

The complex involvement of neutrophils in inflammatory diseases makes them intriguing but challenging targets for therapeutic intervention. Here, we tested the hypothesis that varying endocytosis capacities would delineate functionally distinct neutrophil subpopulations that could be specifically targeted for therapeutic purposes. By using uniformly sized (∼120 nm in diameter) albumin nanoparticles (ANP) to characterize mouse neutrophils in vivo, we found two subsets of neutrophils, one that readily endocytosed ANP (ANPhigh neutrophils) and another that failed to endocytose ANP (ANPlow population). These ANPhigh and ANPlow subsets existed side by side simultaneously in bone marrow, peripheral blood, spleen, and lungs, both under basal conditions and after inflammatory challenge. Human peripheral blood neutrophils showed a similar duality. ANPhigh and ANPlow neutrophils had distinct cell surface marker expression and transcriptomic profiles, both in naive mice and in mice after endotoxemic challenge. ANPhigh and ANPlow neutrophils were functionally distinct in their capacities to kill bacteria and to produce inflammatory mediators. ANPhigh neutrophils produced inordinate amounts of reactive oxygen species and inflammatory chemokines and cytokines. Targeting this subset with ANP loaded with the drug piceatannol, a spleen tyrosine kinase (Syk) inhibitor, mitigated the effects of polymicrobial sepsis by reducing tissue inflammation while fully preserving neutrophilic host-defense function.