Human Neutrophil-like HL-60 Cells Research Articles

MicroRNA-193a-5p Rescues Ischemic Cerebral Injury by Restoring N2-Like Neutrophil Subsets

Circulating neutrophils are activated shortly after stroke and in turn affect the fate of ischemic brain tissue, and microRNAs (miRNA) participate in regulating neuroinflammation. We probed the role of neutrophilic miRNA in ischemic stroke. miR-193a-5p was decreased in circulating neutrophils of acute ischemic stroke (AIS) patients and healthy controls. In another set of AIS patients treated with recombinant tissue plasminogen activator, higher neutrophilic miR-193a-5p levels were associated with favorable outcomes at 3 months and non-symptomatic intracerebral hemorrhage. An experimental stroke model and human neutrophil-like HL-60 cells were further transfected with agomiR-193a-5p/antagomiR-193a-5p or ubiquitin-conjugating enzyme V2 (UBE2V2)-siRNA prior to model induction for in vivo and in vitro studies. Results of 2,3,5-triphenyl tetrazolium chloride staining and neurological function evaluations at post-experimental stroke showed that intravenous agomiR-193a-5p transfusion protected against ischemic cerebral injury in the acute stage and promoted neurological recovery in the subacute stage. This protective role was suggested to correlate with neutrophil N2 transformation based on the N2-like neutrophil proportions in the bone marrow, peripheral blood, and spleen of the experimental stroke model and the measurement of neutrophil phenotype-associated molecule levels. Mechanistically, analyses indicated that UBE2V2 might be a target of miR-193a-5p. Cerebral injury and neuroinflammation aggravated by miR-193a-5p inhibition were reversed by UBE2V2 silencing. In conclusion, miR-193a-5p protects against cerebral ischemic injury by restoring neutrophil N2 phenotype-associated neuroinflammation suppression, likely, in part, via UBE2V2 induction.

Filamin A promotes efficient migration and phagocytosis of neutrophil-like HL-60 cells

The primary defense machinery to combat inflammation involves neutrophil granulocytes which in order to execute their functions rely on the efficiency of different cellular mechanisms including adhesion, spreading, migration in different environments, and phagocytosis. These functions require an accurately regulated actin network as well as the activation and adjustment of various signaling pathways. Mammalian filamins (FLNs) comprise three highly homologous large actin-binding proteins that are obvious candidates to control these processes as FLNs have been described to play a role in migration, spreading and adhesion in a variety of different cell types. The present study analyzed the role of filamin A (FLNa) in human neutrophil-like HL-60 cells. We found a strong enrichment of FLNa at the uropod of migrating neutrophils, and show that deficiency of FLNa caused a decrease in speed of migration both in 2D and 3D that is accompanied by a reduced activation of myosin-II. In addition, we show that FLNa plays a role in neutrophil phagocytosis. We also identified a hitherto unknown interaction of FLNa with coronin 1A that is mediated by FLNa repeats 9–18. FLNa deficiency had no or only minor effects on cell adhesion and spreading. In summary, deficiency of FLNa in human neutrophil-like HL-60 cells resulted in a surprisingly subtle phenotype. Our data indicate that FLNa is not essential for the regulation of mechanical properties during migration, but contributes to motility in a modulatory manner probably through its action at the uropod.

Abstract B096: Optogenetics-based dissection of chemotactic signaling in neutrophils

Abstract Neutrophils comprise a crucial component of the innate immune system and rely on chemotaxis (directed migration) to track down, engulf, and destroy pathogens. A variety of tumor-produced chemokines also recruit neutrophils to sites of tumor growth where, through mechanisms that remain unclear, they exhibit both beneficial and detrimental effects on tumor clearance. Effective chemotaxis requires that neutrophils restrict activity of the GTPase Rac to a single location to properly form a leading edge. However, multiple GEFs function redundantly to activate Rac and it has been difficult to determine where within the signaling cascade each specific GEF is required. Here, we develop an optogenetic tool to drive PIP3 production, which itself is sufficient to activate Rac in human neutrophil-like HL-60 cells. In cells where we used CRISPR-mediated editing to knockout the Rac GEF PREX1, we find that optogenetic stimulation of PIP3 production fails to activate Rac. In contrast, when these PREX1null neutrophils are instead treated with chemoattractant, other GEFs that do not explicitly rely on PIP3 production can still activate Rac. While signaling cascades often feature multiple redundant pathways functioning in parallel, our optogenetics-based approach reveals the location within such a network where a particular factor is necessary for the transmission of information. A more nuanced understanding of chemotactic signaling in neutrophils should lead to the development of treatments that can more precisely modulate the interactions between neutrophils and the tumor microenvironment. Citation Format: Brian R. Graziano, Delquin Gong, Karen E. Anderson, Orion D. Weiner. Optogenetics-based dissection of chemotactic signaling in neutrophils [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B096.

Milk fat globule epidermal growth factor-factor 8-derived peptide attenuates organ injury and improves survival in sepsis.

IntroductionSepsis involves overwhelming inflammatory responses with subsequent immune-suppression that can lead to multiple organ dysfunction and ultimately death. Milk fat globule epidermal growth factor-factor 8 (MFG-E8) is a secretory protein found to have multiple biological activities against autoimmune and inflammatory diseases. MFG-E8 contains an Arg-Gly-Asp (RGD) motif involved in cell-cell and cell-matrix interactions. In sepsis, excessive neutrophils migration through endothelial cells and matrix to sites of inflammation results in organ damage. We hypothesized that MFG-E8-derived short peptides (MSP) flanking its RGD motif could provide protection against organ injury in sepsis.MethodsThe differentiated human neutrophil-like HL-60 cells (dHL60) were incubated with a series of peptides flanking the RGD motif of human MFG-E8 for a cell adhesion assay to fibronectin or human pulmonary artery endothelial cells (PAECs). For the induction of sepsis, male C57BL/6 mice (20–25 g) were subjected to cecal ligation and puncture (CLP). Peptide MSP68 (1 mg/kg body weight) or normal saline (vehicle) was injected intravenously at 2 h after CLP. Blood and tissue samples were collected at 20 h after CLP for various measurements.ResultsAfter screening, peptide MSP68 (VRGDV) had the highest inhibition of dHL-60 cell adhesion to fibronectin by 55.8 % and to PAEC by 67.7 %. MSP68 treatment significantly decreased plasma levels of organ injury marker AST by 37.1 % and the proinflammatory cytokines IL-6 and TNF-α by 61.9 % and 22.1 %, respectively after CLP. MSP68 improved the integrity of microscopic architectures, decreased IL-6 levels in the lungs by 85.1 %, and reduced apoptosis. MSP68 treatment also significantly reduced the total number of neutrophil infiltration by 61.9 % and 48.3 % as well as MPO activity by 40.8 % and 47.3 % in the lungs and liver, respectively, after CLP. Moreover, the number of bacteria translocated to mesenteric lymph nodes was decreased by 57 % with MSP68 treatment. Finally, the 10-day survival rate was increased from 26 % in the vehicle group to 58 % in the MSP68-treated group.ConclusionsMSP68 effectively inhibits excessive neutrophils infiltrating to organs, leading to moderate attenuation of organ injury and significantly improved survival in septic mice. Thus, MSP68 may be a potential therapeutic agent for treating sepsis.

Store-operated Ca2+ entry mediated regulation of polarization in differentiated human neutrophil-like HL-60 cells under hypoxia

The regulation of neutrophil polarization by calcium entry is critical for maintaining an effective host response. Hypoxia has a major effect on the apoptosis of neutrophils, however the role of store-operated Ca2+ entry (SOCE) in neutrophil polarization under hypoxia remains to be elucidated. In the present study, we examined the polarization of differentiated human neutrophil-like HL-60 (dHL-60) cells exposed to hypoxia (3% O2) and the results demonstrated that the percentage of polarized cells following exposure to an N-formyl-Met-Leu-Phe (fMLP) gradient in the Zigmond chamber was increased. We examined stromal interaction molecule 1 (STIM1) and Orai1 expression in dHL-60 cells during hypoxia, and it was observed that the expression of STIM1 and Orai1 was significantly reduced at day 2. However, no apparent change was observed on the first day, indicating that this effect is dependent on stimulation time. Fluo-4/acetoxymethyl (AM) ester imaging also demonstrated that SOCE was decreased in dHL-60 cells. The plasmid overexpression assay demonstrated that the response of polarization was returned to the control level. We demonstrated the inhibitory role of SOCE on the polarization of dHL-60 cells under hypoxic conditions, which may be the mechanism for the adaptation of neutrophils to hypoxia. SOCE is also suggested to be a key modulator of immune deficiency under hypoxic conditions and is potentially a therapeutic target.

AKT-mediated regulation of polarization in differentiated human neutrophil-like HL-60 cells

Neutrophil polarization is critical for the inflammatory response. AKT is a serine/threonine protein kinase and has been implicated in cell migration. However, it is not completely clear whether AKT affects neutrophil polarization. In this study, we tested the hypothesis that AKT regulates the polarization of neutrophil-like differentiated HL-60 cells (dHL-60) in response to fMLP. HL-60 cells were differentiated into dHL-60 by incubation in medium containing 1.3 % DMSO for up to 6 days. Polarization of dHL-60 cells and primary human neutrophils were measured by Zigmond chamber. Phospho-Akt was analyzed by immunofluorescence and Western blot analysis. F-actin polymerization was detected by Rhodamine-Phalloidine staining. Rac2 activation was evaluated using GST Pull-down assay. We found that changes in the rate of cell polarization were consistent with the changes in AKT phosphorylation levels during HL-60 cell differentiation in response to fMLP. Moreover, cell polarization and AKT phosphorylation were reduced in fMLP-stimulated dHL-60 cells pretreated with the PI3 kinase inhibitors or the AKT inhibitors, which was confirmed in the primary human neutrophils. The AKT inhibitors altered fMLP-induced F-actin polymerization. Rac2 GTPases was also decreased by the AKT inhibitors in fMLP-stimulated dHL-60 cells. This study demonstrates that AKT activation plays a crucial role in dHL-60 cell polarization.

Protein Kinase B (AKT) Mediates Phospholipase D Activation via ERK1/2 and Promotes Respiratory Burst Parameters in Formylpeptide-stimulated Neutrophil-like HL-60 Cells

Phospholipase D (PLD), a major source of lipid second messengers (phosphatidic acid, diglycerides) in many cell types, is tightly regulated by protein kinases, but only a few of them have been identified. We show here that protein kinase B (AKT) is a novel major signaling effector of PLD activity induced by the formylpeptide f-Met-Leu-Phe (fMLP) in human neutrophil-like HL-60 cells (dHL-60 cells). AKT inhibition with the selective antagonist AKTib1/2 almost completely prevented fMLP-mediated activity of PLD, its upstream effector ERK1/2, but not p38 MAPK. Immunoprecipitation studies show that phosphorylated AKT, ERK, and PLD2 form a complex induced by fMLP, which can be prevented by AKTib1/2. In cell-free systems, AKT1 stimulated PLD activity via activation of ERK. AKT1 actually phosphorylated ERK2 as a substrate (K(m) 1 μm). Blocking AKT activation with AKTib1/2 also prevented fMLP- but not phorbol 12-myristate 13-acetate-mediated NADPH oxidase activation (respiratory burst, RB) of dHL-60 cells. Impaired RB was associated with defective membrane translocation of NADPH oxidase components p67(phox) and p47(phox), ERK, AKT1, AKT2, but not AKT3. Depletion of AKT1 or AKT2 with antisense oligonucleotides further indicates a partial contribution of both isoforms in fMLP-induced activation of ERK, PLD, and RB, with a predominant role of AKT1. Thus, formylpeptides induce sequential activation of AKT, ERK1/2, and PLD, which represents a novel signaling pathway. A major primarily role of this AKT signaling pathway also emerges in membrane recruitment of NOX2 components p47(phox), p67(phox), and ERK, which may contribute to assembly and activation of the RB motor system, NADPH oxidase.

Saccharomyces cerevisiaeandCandida albicansStimulate Cytokine Secretion from Human Neutrophil-Like HL-60 Cells Differentiated with Retinoic Acid or Dimethylsulfoxide

We investigated whether non-pathogenic Saccharomyces cerevisiae and human commensal opportunistic pathogenic Candida albicans stimulate cytokine responses of human neutrophil-like HL-60 cells pre-treated with either 1 microM retinoic acid or 1.25% dimethyl sulfoxide (DMSO). Intact and heat-killed S. cerevisiae enhanced secretion of interleukin (IL)-1beta, IL-6, IL-8, IL-12, IL-18, MCP-1/CCL2 and TNF-alpha from retinoic acid-treated HL-60 cells, accompanied by alterations in mRNA expression of the cytokines. Heat-killed C. albicans promoted secretion of IL-6, IL-8, IL-12, MCP-1 and TNF-alpha, while intact C. albicans slightly enhanced secretion of IL-1beta, IL-8 and IL-18. In response to yeast stimuli, retinoic acid-treated HL-60 cells generally secreted cytokines more strongly than DMSO-treated HL-60 cells. Gene expression levels of Toll-like receptor (TLR)1, TLR2, TLR4, TLR6 and dectin-1 in HL-60 cells were additionally affected by retinoic acid or DMSO and by co-culturing with S. cerevisiae or C. albicans. Our results suggest that both intact and heat-killed S. cerevisiae and C. albicans induce cytokine responses of neutrophils in the intestine, and stimulate host immune function.

Induction of Heme Oxygenase-1 In Vivo Suppresses NADPH Oxidase–Derived Oxidative Stress

Our previous studies suggest that heme oxygenase (HO)-1 induction and/or subsequent bilirubin generation in endothelial cells may suppress superoxide generation of from reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. In this study, we examined the consequence of HO-1 induction in vivo on NADPH oxidase activity. Three doses of hemin (25 mg x kg(-1), IP, every 48 hours), with or without cotreatment with the HO inhibitor tin protoporphyrin-IX (15 mg x kg(-1), IP), were given to apolipoprotein E-deficient mice, which display vascular oxidative stress. Hemin treatment increased HO-1 expression and activity in aorta (undetectable at baseline) and kidney (by 3-fold) and significantly reduced both NADPH oxidase activity (by approximately 25% to 50%) and superoxide generation in situ. The increase in HO-1 activity and inhibition of NADPH oxidase activity by hemin were reversed by tin protoporphyrin-IX and were not associated with changes in Nox2 or Nox4 protein levels. Hemin also reduced plasma F(2)-isoprostane levels by 23%. The inhibition of NADPH oxidase activity by hemin in the aorta was mimicked by bilirubin in vitro (0.01 to 1 micromol/L). Bilirubin also concentration-dependently reduced NADPH oxidase-dependent superoxide production stimulated by angiotensin II in rat vascular smooth muscle cells and by phorbol 12-myristate 13-acetate in human neutrophil-like HL-60 cells. HO-1 overexpression by plasmid-mediated gene transfer in rat vascular smooth muscle cells decreased NADPH-stimulated superoxide production. Thus, systemic expression of HO-1 suppresses NADPH oxidase activity by mechanisms at least partly mediated by the bile pigment bilirubin, thereby reducing oxidative stress.

Modulation by cADPr of Ca 2+ mobilization and oxidative response in dimethylsulfoxide- or retinoic acid-differentiated HL-60 cells

In human phagocytic cells, reactive oxygen species (ROS) generation in response to N-formyl- l-Methionyl- l-Leucyl- l-Phenylalanine (fMLF) is largely dependent on cytosolic free calcium concentration ([Ca 2+] i). Cyclic ADP-ribose (cADPr) is able to regulate Ca 2+ release from intracellular stores through the ryanodine receptor but its potential role in biological responses has so far not been determined. In this study, we examined whether extracellular and intracellular cADPr is required in fMLF-induced [Ca 2+] i rise and consequently in the oxidative response in human neutrophil-like HL-60 cells differentiated with dimethylsulfoxide or all-trans-retinoic acid (ATRA). We establish that extracellular cADPr cannot elicit [Ca 2+] i elevation. Furthermore, we demonstrate that 8-Br-cADPr, a functional antagonist of cADPr, inhibits Ca 2+ entry into HL-60 cells differentiated with ATRA and stimulated with fMLF (95 ± 4 and 148 ± 5 nM respectively, n = 3). Finally, we show that this partial inhibition of Ca 2+ mobilization is unrelated to ROS production (10.0 ± 0.3 vs. 9.6 ± 0.5 A.U., n = 3). In conclusion, we showed that cADPr can control fMLF-induced Ca 2+ influx but is unable to regulate a Ca 2+-dependent biological response, i.e. H 2O 2 production.