Neutrophil-like Cell Line Research Articles

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.

The hybrid CL-SP-D molecule has the potential to regulate xenogeneic rejection by human neutrophils more efficiently than CD47

ObjectiveInnate immunity plays a vital role in xenotransplantation. A CD47 molecule, binding to the SIRPα expressed on monocyte/macrophage cells, can suppress cytotoxicity. Particularly, the SIRPα contains ITIM, which delivers a negative signal. Our previous study demonstrated that the binding between CL-P1 and surfactant protein-D hybrid (CL-SP-D) with SIRPα regulates macrophages' phagocytic activity. In this study, we examined the effects of human CD47 and CL-SP-D expression on the inhibition of xenograft rejection by neutrophils in swine endothelial cells (SECs). MethodsWe first examined SIRPα expression on HL-60 cells, a neutrophil-like cell line, and neutrophils isolated from peripheral blood. CD47-expressing SECs or CL-SP-D-expressing SECs were generated through plasmid transfection. Subsequently, these SECs were co-cultured with HL-60 cells or neutrophils. After co-culture, the degree of cytotoxicity was calculated using the WST-8 assay. The suppressive function of CL-SP-D on neutrophils was subsequently examined, and the results were compared with those of CD47 using naïve SECs as controls. Additionally, we assessed ROS production and neutrophil NETosis. ResultsIn initial experiments, the expression of SIRPα on HL-60 and neutrophils was confirmed. Exposure to CL-SP-D significantly suppressed the cytotoxicity in HL-60 (p = 0.0038) and neutrophils (p = 0.00003). Furthermore, engagement with CD47 showed a suppressive effect on neutrophils obtained from peripheral blood (p = 0.0236) but not on HL-60 (p = 0.4244). The results of the ROS assays also indicated a significant downregulation of SEC by CD47 (p = 0.0077) or CL-SP-D (p = 0.0018). Additionally, the suppression of NETosis was confirmed (p = 0.0125) in neutrophils co-cultured with S/CL-SP-D. ConclusionThese results indicate that CL-SP-D is highly effective on neutrophils in xenogeneic rejection. Furthermore, CL-SP-D was more effective than CD47 at inhibiting neutrophil-mediated xenograft rejection.

Fusobacterium nucleatum elicits subspecies-specific responses in human neutrophils.

Fusobacterium nucleatum as a Gram-negative anaerobe plays a key bridging role in oral biofilms. It is involved in periodontal and extraoral diseases, the most prominent being colorectal cancer. Five subspecies are recognised: animalis, fusiforme, nucleatum, polymorphum and vincentii. Subspecies interact with neutrophils constantly patrolling tissues to remove microbial intruders. Neutrophil antimicrobial activities include generation of reactive oxygen species (ROS), formation of neutrophil extracellular traps (NETs) and release of cytokines and neutrophil enzymes. Subspecies-specific differences in immunogenicity have previously been observed in a neutrophil-like cell line but were not investigated in human neutrophils. Additionally, neutrophil responses to planktonic and biofilm-grown F. nucleatum have not been studied to date. The aims of this study were to compare the immunogenicity of planktonic and biofilm-grown F. nucleatum and to investigate potential differences in human neutrophil responses when stimulated with individual F. nucleatum subspecies. Human neutrophils isolated from peripheral blood were stimulated with planktonic and biofilm-grown F. nucleatum subspecies. Generation of ROS and NET formation were quantified by luminescence and fluorescence assays, respectively. Secretion of cytokines (IL-1β, TNF-α, IL-6, IL-8), neutrophil elastase and matrix metalloproteinase-9 was quantified by enzyme-linked immunosorbent assay (ELISA). Neutrophil responses showed biofilm-grown bacteria induced a significantly higher total and intracellular ROS response, as well as shorter time to total ROS release. Biofilm-grown F. nucleatum led to significantly lower IL-1β release. We found significant differences among individual subspecies in terms of total, intracellular ROS and extracellular superoxide. Subspecies polymorphum stimulated the highest mean amount of NET release. Amounts of cytokines released differed significantly among subspecies, while no differences were found in lysosomal enzyme release. Immunogenicity of F. nucleatum in human neutrophils is highly subspecies-specific in vitro with regard to ROS release and cytokine production. Understanding subspecies-specific immunogenicity of F. nucleatum may facilitate the discovery of novel therapeutic targets in F. nucleatum-mediated diseases.

Dual Affinity Nanoparticles for the Transport of Therapeutics from Carrier Cells to Target Cells under Physiological Flow Conditions.

In this study, a novel two-stage nanoparticle delivery platform was developed based on the dual functionalization of a liposome with moieties that have fundamentally different strengths of adhesion and binding kinetics. The essential concept of this system is that the nanoparticles are designed to loosely bind to the carrier cell until they come into contact with the target cell, to which they bind with greater strength. This allows the nanoparticle to be transferred from one cell to another, circulating for longer periods of time in the blood and delivering the therapeutic agent to the target circulating tumor cell. Liposomes were prepared using the lipid cake and extrusion technique, then functionalized with E-selectin (ES), anti-cell surface vimentin antibody fragments, and TRAIL via click chemistry. The binding of dual affinity (DA) liposomes was confirmed with the neutrophil-like cell line PLB985, the colorectal cancer cell line HCT116, and healthy granulocytes isolated from peripheral whole blood under physiologically relevant fluid shear stress (FSS) in a cone-and-plate viscometer. Transfer of the DA liposomes from PLB985 to HCT116 cells under FSS was greater compared to all of the control liposome formulations. Additionally, DA liposomes demonstrated enhanced apoptotic effects on HCT116 cells in whole blood under FSS, surpassing the efficacy of the ES/TRAIL liposomes previously developed by the King Lab.

Whole-genome screens reveal regulators of differentiation state and context-dependent migration in human neutrophils

Neutrophils are the most abundant leukocyte in humans and provide a critical early line of defense as part of our innate immune system. We perform a comprehensive, genome-wide assessment of the molecular factors critical to proliferation, differentiation, and cell migration in a neutrophil-like cell line. Through the development of multiple migration screen strategies, we specifically probe directed (chemotaxis), undirected (chemokinesis), and 3D amoeboid cell migration in these fast-moving cells. We identify a role for mTORC1 signaling in cell differentiation, which influences neutrophil abundance, survival, and migratory behavior. Across our individual migration screens, we identify genes involved in adhesion-dependent and adhesion-independent cell migration, protein trafficking, and regulation of the actomyosin cytoskeleton. This genome-wide screening strategy, therefore, provides an invaluable approach to the study of neutrophils and provides a resource that will inform future studies of cell migration in these and other rapidly migrating cells.

TGF-β1 activates neutrophil signaling and gene expression but not migration.

Tumor-associated neutrophils are found in many types of cancer and are often reported to contribute to negative outcomes. The presence of transforming growth factor-beta (TGF-β) in the tumor microenvironment reportedly contributes to the skewing of neutrophils to a more pro-tumor phenotype. The effects of TGF-β on neutrophil signaling and migration are, however, unclear. We sought to characterize TGF-β signaling in both primary human neutrophils and the neutrophil-like cell line HL-60 and determine whether it directly induces neutrophil migration. We found that TGF-β1 does not induce neutrophil chemotaxis in transwell or underagarose migration assays. TGF-β1 does activate canonical signaling through SMAD3 and noncanonical signaling through ERK1/2 in neutrophils in a time- and dose-dependent manner. Additionally, TGF-β1 present in the tumor-conditioned media (TCM) of invasive breast cancer cells results in SMAD3 activation. We discovered that TCM induces neutrophils to secrete leukotriene B4 (LTB4), which is a lipid mediator important for amplifying the range of neutrophil recruitment. However, TGF-β1 alone does not induce secretion of LTB4. RNA-sequencing revealed that TGF-β1 and TCM alter gene expression in HL-60 cells, including the mRNA levels of the pro-tumor oncostatin M (OSM) and vascular endothelial growth factor A (VEGFA). These new insights into the role and impact of TGF-β1 on neutrophil signaling, migration, and gene expression have significant implications in the understanding of the changes in neutrophils that occur in the tumor microenvironment.

Closing the gap between murine neutrophils and neutrophil-like cell lines.

Advantages of cloned Hoxb8 neutrophil-like cells are discussed and contrasted with weaknesses of human HL-60 and PLB-985 neutrophil-like cell lines, and shared and distinct features of primary murine and human neutrophils are summarized.

Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils.

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and oxidative burst, and create neutrophil extracellular traps (NETs) for microbial clearance. New insights into the metabolic activities of neutrophils challenge the early concept that they primarily rely on glycolysis. Precise measurement of metabolic activities can unfold different metabolic requirements of neutrophils, including the tricarboxylic acid (TCA) cycle (also known as the Krebs cycle), oxidative phosphorylation (OXPHOS), pentose phosphate pathway (PPP), and fatty acid oxidation (FAO) under physiological conditions and in disease states. This paper describes a step-by-step protocol and prerequirements to measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration on mouse bone marrow-derived neutrophils, human blood-derived neutrophils, and the neutrophil-like HL60 cell line, using metabolic flux analysis on a metabolic extracellular flux analyzer. This method can be used for quantifying the mitochondrial functions of neutrophils under normal and disease conditions.

Role of the phospholipid binding sites, PX of p47phox and PB region of Rac1, in the formation of the phagocyte NADPH oxidase complex NOX2

In phagocytes, superoxide anion (O2−), the precursor of reactive oxygen species, is produced by the NADPH oxidase complex to kill pathogens. Phagocyte NADPH oxidase consists of the transmembrane cytochrome b558 (cyt b558) and four cytosolic components: p40phox, p47phox, p67phox, and Rac1/2. The phagocyte activation by stimuli leads to activation of signal transduction pathways. This is followed by the translocation of cytosolic components to the membrane and their association with cyt b558 to form the active enzyme.To investigate the roles of membrane-interacting domains of the cytosolic proteins in the NADPH oxidase complex assembly and activity, we used giant unilamellar phospholipid vesicles (GUV). We also used the neutrophil-like cell line PLB-985 to investigate these roles under physiological conditions. We confirmed that the isolated proteins must be activated to bind to the membrane. We showed that their membrane binding was strengthened by the presence of the other cytosolic partners, with a key role for p47phox. We also used a fused chimera consisting of p47phox(aa 1–286), p67phox(aa 1–212) and Rac1Q61L, as well as mutated versions in the p47phox PX domain and the Rac polybasic region (PB). We showed that these two domains have a crucial role in the trimera membrane-binding and in the trimera assembly to cyt b558. They also have an impact on O2.- production in vitro and in cellulo: the PX domain strongly binding to GUV made of a mix of polar lipids; and the PB region strongly binding to the plasma membrane of neutrophils and resting PLB-985 cells.

Staphylococcus aureus Infection Induces the Production of the Neutrophil Chemoattractants CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7 by Murine Osteoblasts.

Staphylococcus aureus is the principal causative agent of osteomyelitis, a serious bacterial infection of bone that is associated with progressive inflammatory damage. Bone-forming osteoblasts have increasingly been recognized to play an important role in the initiation and progression of detrimental inflammation at sites of infection and have been demonstrated to release an array of inflammatory mediators and factors that promote osteoclastogenesis and leukocyte recruitment following bacterial challenge. In the present study, we describe elevated bone tissue levels of the potent neutrophil-attracting chemokines CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7 in a murine model of posttraumatic staphylococcal osteomyelitis. RNA sequencing (RNA-Seq) gene ontology analysis of isolated primary murine osteoblasts showed enrichment in differentially expressed genes involved in cell migration and chemokine receptor binding and chemokine activity following S. aureus infection, and a rapid increase in the expression of mRNA encoding CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7, in these cells. Importantly, we have confirmed that such upregulated gene expression results in protein production with the demonstration that S. aureus challenge elicits the rapid and robust release of these chemokines by osteoblasts and does so in a bacterial dose-dependent manner. Furthermore, we have confirmed the ability of soluble osteoblast-derived chemokines to elicit the migration of a neutrophil-like cell line. As such, these studies demonstrate the robust production of CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7 by osteoblasts in response to S. aureus infection, and the release of such neutrophil-attracting chemokines provides an additional mechanism by which osteoblasts could drive the inflammatory bone loss associated with staphylococcal osteomyelitis.

Legacy and emerging per- and polyfluoroalkyl substances suppress the neutrophil respiratory burst

Per- and polyfluoroalkyl substances (PFASs) are used in a multitude of processes and products, including nonstick coatings, food wrappers, and fire-fighting foams. These chemicals are environmentally-persistent, ubiquitous, and can be detected in the serum of 98% of Americans. Despite evidence that PFASs alter adaptive immunity, few studies have investigated their effects on innate immunity. The report here presents results of studies that investigated the impact of nine environmentally-relevant PFASs [e.g. perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid potassium salt (PFOS-K), perfluorononanoic acid (PFNA), perfluorohexanoic acid (PFHxA), perfluorohexane sulfonic acid (PFHxS), perfluorobutane sulfonic acid (PFBS), ammonium perfluoro(2-methyl-3-oxahexanoate) (GenX), 7H-perfluoro-4-methyl-3,6-dioxa-octane sulfonic acid (Nafion byproduct 2), and perfluoromethoxyacetic acid sodium salt (PFMOAA-Na)] on one component of the innate immune response, the neutrophil respiratory burst. The respiratory burst is a key innate immune process by which microbicidal reactive oxygen species (ROS) are rapidly induced by neutrophils in response to pathogens; defects in the respiratory burst can increase susceptibility to infection. The study here utilized larval zebrafish, a human neutrophil-like cell line, and primary human neutrophils to ascertain whether PFAS exposure inhibits ROS production in the respiratory burst. It was observed that exposure to PFHxA and GenX suppresses the respiratory burst in zebrafish larvae and a human neutrophil-like cell line. GenX also suppressed the respiratory burst in primary human neutrophils. This report is the first to demonstrate that these PFASs suppress neutrophil function and support the utility of employing zebrafish larvae and a human cell line as screening tools to identify chemicals that may suppress human immune function.

PLB-985 Neutrophil-Like Cells as a Model To Study Aspergillus fumigatus Pathogenesis.

ABSTRACTFungal infections remain a major global concern. Emerging fungal pathogens and increasing rates of resistance mean that additional research efforts and resources must be allocated to advancing our understanding of fungal pathogenesis and developing new therapeutic interventions. Neutrophilic granulocytes are a major cell type involved in protection against the important fungal pathogen Aspergillus fumigatus, where they employ numerous defense mechanisms, including production of antimicrobial extracellular vesicles. A major drawback to work with neutrophils is the lack of a suitable cell line system for the study of fungal pathogenesis. To address this problem, we assessed the feasibility of using differentiated PLB-985 neutrophil-like cells as an in vitro model to study A. fumigatus infection. We find that dimethylformamide-differentiated PLB-985 cells provide a useful recapitulation of many aspects of A. fumigatus interactions with primary human polymorphonuclear leukocytes. We show that differentiated PLB-985 cells phagocytose fungal conidia and acidify conidia-containing phagolysosomes similar to primary neutrophils, release neutrophil extracellular traps, and also produce antifungal extracellular vesicles in response to infection. In addition, we provide an improved method for the isolation of extracellular vesicles produced during infection by employing a size exclusion chromatography-based approach. Advanced liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics revealed an enrichment of extracellular vesicle marker proteins and a decrease of cytoplasmic proteins in extracellular vesicles isolated using this improved method. Ultimately, we find that differentiated PLB-985 cells can serve as a genetically tractable model to study many aspects of A. fumigatus pathogenesis.IMPORTANCE Polymorphonuclear leukocytes are an important defense against human fungal pathogens, yet our model systems to study this group of cells remain very limited in scope. In this study, we established that differentiated PLB-985 cells can serve as a model to recapitulate several important aspects of human polymorphonuclear leukocyte interactions with the important human fungal pathogen Aspergillus fumigatus. The proposed addition of a cultured neutrophil-like cell line to the experimental toolbox to study fungal pathogenesis will allow for a more mechanistic description of neutrophil antifungal biology. In addition, the easier handling of the cell line compared to primary human neutrophils allowed us to use PLB-985 cells to provide an improved method for isolation of neutrophil-derived extracellular vesicles using size exclusion chromatography. Together, these results provide significant tools and a baseline knowledge for the future study of neutrophil-derived extracellular vesicles in the laboratory.

Electrotaxis-on-Chip to Quantify Neutrophil Migration Towards Electrochemical Gradients.

Electric fields are generated in vivo in a variety of physiologic and pathologic settings, including wound healing and immune response to injuries to epithelial barriers (e.g. lung pneumocytes). Immune cells are known to migrate towards both chemical (chemotaxis), physical (mechanotaxis) and electric stimuli (electrotaxis). Electrotaxis is the guided migration of cells along electric fields, and has previously been reported in T-cells and cancer cells. However, there remains a need for engineering tools with high spatial and temporal resolution to quantify EF guided migration. Here we report the development of an electrotaxis-on-chip (ETOC) platform that enables the quantification of dHL-60 cell, a model neutrophil-like cell line, migration toward both electrical and chemoattractant gradients. Neutrophils are the most abundant white blood cells and set the stage for the magnitude of the immune response. Therefore, developing engineering tools to direct neutrophil migration patterns has applications in both infectious disease and inflammatory disorders. The ETOC developed in this study has embedded electrodes and four migration zones connected to a central cell-loading chamber with migration channels [10 µm X 10 µm]. This device enables both parallel and competing chemoattractant and electric fields. We use our novel ETOC platform to investigate dHL-60 cell migration in three biologically relevant conditions: 1) in a DC electric field; 2) parallel chemical gradient and electric fields; and 3) perpendicular chemical gradient and electric field. In this study we used differentiated leukemia cancer cells (dHL60 cells), an accepted model for human peripheral blood neutrophils. We first quantified effects of electric field intensities (0.4V/cm-1V/cm) on dHL-60 cell electrotaxis. Our results show optimal migration at 0.6 V/cm. In the second scenario, we tested whether it was possible to increase dHL-60 cell migration to a bacterial signal [N-formylated peptides (fMLP)] by adding a parallel electric field. Our results show that there was significant increase (6-fold increase) in dHL60 migration toward fMLP and cathode of DC electric field (0.6V/cm, n=4, p-value<0.005) vs. fMLP alone. Finally, we evaluated whether we could decrease or re-direct dHL-60 cell migration away from an inflammatory signal [leukotriene B4 (LTB4)]. The perpendicular electric field significantly decreased migration (2.9-fold decrease) of dHL60s toward LTB4 vs. LTB4 alone. Our microfluidic device enabled us to quantify single-cell electrotaxis velocity (7.9 µm/min ± 3.6). The magnitude and direction of the electric field can be more precisely and quickly changed than most other guidance cues such as chemical cues in clinical investigation. A better understanding of EF guided cell migration will enable the development of new EF-based treatments to precisely direct immune cell migration for wound care, infection, and other inflammatory disorders.

Centriole and Golgi microtubule nucleation are dispensable for the migration of human neutrophil-like cells.

Neutrophils migrate in response to chemoattractants to mediate host defense. Chemoattractants drive rapid intracellular cytoskeletal rearrangements including the radiation of microtubules from the microtubule-organizing center (MTOC) toward the rear of polarized neutrophils. Microtubules regulate neutrophil polarity and motility, but little is known about the specific role of MTOCs. To characterize the role of MTOCs on neutrophil motility, we depleted centrioles in a well-established neutrophil-like cell line. Surprisingly, both chemical and genetic centriole depletion increased neutrophil speed and chemotactic motility, suggesting an inhibitory role for centrioles during directed migration. We also found that depletion of both centrioles and GM130-mediated Golgi microtubule nucleation did not impair neutrophil directed migration. Taken together, our findings demonstrate an inhibitory role for centrioles and a resilient MTOC system in motile human neutrophil-like cells.

Neutrophil activation by nanomaterials in vitro: comparing strengths and limitations of primary human cells with those of an immortalized (HL-60) cell line

Assessment of nanomaterial (NM) induced inflammatory responses has largely relied on rodent testing via measurement of leukocyte accumulation in target organs. Despite observations that NMs activate neutrophil driven inflammatory responses in vivo, a limited number of studies have investigated neutrophil responses to NMs in vitro. We compared responses between the human neutrophil-like HL-60 cell line and human primary neutrophils following exposure to silver (Ag), zinc oxide (ZnO), copper oxide (CuO) and titanium dioxide (TiO2) NMs. NM cytotoxicity and neutrophil activation were assessed by measuring cellular metabolic activity, cytokine production, respiratory burst, and release of neutrophil extracellular traps. We observed a similar pattern of response between HL-60 cells and primary neutrophils, however we report that some neutrophil functions are compromised in the cell line. Ag NMs were consistently observed to stimulate neutrophil activation, with CuO NMs inducing similar though weaker responses. TiO2 NMs did not induce a neutrophil response in either cell type. Interestingly, ZnO NMs readily induced activation of HL-60 cells but did not appear to activate primary cells. Our findings are relevant to the development of a tiered testing strategy for NM hazard assessment which promotes the use of non-rodent models. Whilst we acknowledge that HL-60 cells may not be a perfect substitute for primary cells and require further investigation regarding their ability to predict neutrophil activation, we recommend their use for initial screening of NM-induced inflammation. Primary human neutrophils can then be used for more focused assessments of neutrophil activation before progressing to in vivo models where necessary.

Membrane Dynamics and Organization of the Phagocyte NADPH Oxidase in PLB-985 Cells

Neutrophils are the first cells recruited at the site of infections, where they phagocytose the pathogens. Inside the phagosome, pathogens are killed by proteolytic enzymes that are delivered to the phagosome following granule fusion, and by reactive oxygen species (ROS) produced by the NADPH oxidase. The NADPH oxidase complex comprises membrane proteins (NOX2 and p22phox), cytoplasmic subunits (p67phox, p47phox, and p40phox) and the small GTPase Rac. These subunits assemble at the phagosomal membrane upon phagocytosis. In resting neutrophils the catalytic subunit NOX2 is mainly present at the plasma membrane and in the specific granules. We show here that NOX2 is also present in early and recycling endosomes in human neutrophils and in the neutrophil-like cell line PLB-985 expressing GFP-NOX2. In the latter cells, an increase in NOX2 at the phagosomal membrane was detected by live-imaging after phagosome closure, probably due to fusion of endosomes with the phagosome. Using super-resolution microscopy in PLB-985 WT cells, we observed that NOX2 forms discrete clusters in the plasma membrane. The number of clusters increased during frustrated phagocytosis. In PLB-985NCF1ΔGT cells that lack p47phox and do not assemble a functional NADPH oxidase, the number of clusters remained stable during phagocytosis. Our data suggest a role for p47phox and possibly ROS production in NOX2 recruitment at the phagosome.

An Evaluation of the Activity of Histidine-Rich Glycoprotein on Differentiated Neutrophil-Like Cells from Human Cell Lines.

Histidine-rich glycoprotein (HRG) treatment ameliorated the survival rate of septic mice by suppressing excess immunothrombus formation. Although such findings suggested that HRG may be one of the most useful drugs for sepsis, obtaining a stable experimental system to standardize the HRG drug product is difficult to achieve using neutrophils isolated from volunteers. This is due to the short survival time and individual differences of human neutrophils. In the present study, we determined whether the differentiated neutrophil-like cell lines exhibited similar responses to HRG compared with human purified neutrophils. All-trans retinoic acid (ATRA) was employed to induce the differentiation of the human myeloid leukemia cell lines HL-60 and NB-4. Thereafter, the cells were treated with Hank's balanced salt solution, human serum albumin, or HRG. The effects of HRG on these cells were evaluated according to cell shape, microcapillary passage, reactive oxygen species (ROS) production, neutrophil extracellular traps (NETs) formation, the expression of activated CD11b, and cell viability. HRG maintained the round shape of differentiated neutrophil-like cells, decreased the time required by cells to pass through the microcapillaries, and inhibited ROS production, NETs formation, and the expression of activated CD11b on the cell surface. Moreover, the cells could survive longer in the presence of HRG than the control. The ATRA-induced differentiated cell lines could be used as alternatives to neutrophils to investigate the effects of HRG on neutrophils. This method can thus be used as an essential standardization test in pharmaceutical development. SIGNIFICANCE STATEMENT: Human neutrophils exhibit varying responses to histidine-rich glycoprotein (HRG); however, all-trans retinoic acid-induced differentiated neutrophil-like cell lines can be used as reliably proxies to investigate the effects of HRG on neutrophils. Additionally, these cell lines can be employed in the development of therapies for the treatment of sepsis.

Human CD200 Suppresses the HL-60 Mediated Xenocytotoxicity

BackgroundNeutrophils play an important role in xenogeneic rejection and represent a major obstacle in clinical application of xenografts. CD200 and its receptor CD200R are both type-1 membrane glycoproteins, which are members of the immunoglobulin superfamily (IgSF) and the ligation of CD200 with CD200R induces inhibitory NPXY signaling. The expression of CD200R appears in myeloid cells such as macrophages and granulocytes. Thus, we hypothesized that human CD200 expression on porcine cells might suppress the xenogeneic neutrophil-mediated cytotoxicity against porcine cells. MethodsTo prove our hypothesis, the suppressive effect of human CD200 in neutrophil-like human cell line 60 (HL-60)–mediated xenogeneic cytotoxicity against swine endothelial cells (SECs) was examined. Cytotoxicity was assessed with water-soluble tetrazolium salt 8 (WST-8) assay. ResultsHL-60 cells differentiated into CD66b+ CD200R+ neutrophil-like cells in the presence of dimethyl sulfoxide (DMSO). HL-60-mediated cytotoxicity against SECs was significantly suppressed by human CD200 on SECs. ConclusionsThe findings in this study indicate that human CD200 may suppress neutrophil-mediated xenogeneic rejection.

Neutrophil-like HL-60 cells expressing only GFP-tagged β-actin exhibit nearly normal motility.

Observations of actin dynamics in living cells using fluorescence microscopy have been foundational in the exploration of the mechanisms underlying cell migration. We used CRISPR/Cas9 gene editing to generate neutrophil‐like HL‐60 cell lines expressing GFP‐β‐actin from the endogenous locus (ACTB). In light of many previous reports outlining functional deficiencies of labeled actin, we anticipated that HL‐60 cells would only tolerate a monoallelic edit, as biallelic edited cells would produce no normal β‐actin. Surprisingly, we recovered viable monoallelic GFP‐β‐actin cells as well as biallelic edited GFP‐β‐actin cells, in which one copy of the ACTB gene is silenced and the other contains the GFP tag. Furthermore, the edited cells migrate with similar speeds and persistence as unmodified cells in a variety of motility assays, and have nearly normal cell shapes. These results might partially be explained by our observation that GFP‐β‐actin incorporates into the F‐actin network in biallelic edited cells at similar efficiencies as normal β‐actin in unedited cells. Additionally, the edited cells significantly upregulate γ‐actin, perhaps helping to compensate for the loss of normal β‐actin. Interestingly, biallelic edited cells have only modest changes in global gene expression relative to the monoallelic line, as measured by RNA sequencing. While monoallelic edited cells downregulate expression of the tagged allele and are thus only weakly fluorescent, biallelic edited cells are quite bright and well‐suited for live cell microscopy. The nondisruptive phenotype and direct interpretability of this fluorescent tagging approach make it a promising tool for studying actin dynamics in these rapidly migrating and highly phagocytic cells.

Neutrophil Maturation and Survival Is Controlled by IFN-Dependent Regulation of NAMPT Signaling.

Granulocyte-colony stimulating factor (G-CSF)/nicotinamide phosphoribosyltransferase (NAMPT) signaling has been shown to be crucial for the modulation of neutrophil development and functionality. As this signaling pathway is significantly suppressed by type I interferons (IFNs), we aimed to study how the regulation of neutrophil differentiation and phenotype is altered in IFN-deficient mice during granulopoiesis. The composition of bone marrow granulocyte progenitors and their Nampt expression were assessed in bone marrow of type I IFN receptor knockout (Ifnar1-/-) mice and compared to wild-type animals. The impact of NAMPT inhibition on the proliferation, survival, and differentiation of murine bone marrow progenitors, as well as of murine 32D and human HL-60 neutrophil-like cell lines, was estimated. The progressive increase of Nampt expression during neutrophil progenitor maturation could be observed, and it was more prominent in IFN-deficient animals. Altered composition of bone marrow progenitors in these mice correlated with the dysregulation of apoptosis and altered differentiation of these cells. We observed that NAMPT is vitally important for survival of early progenitors, while at later stages it delays the differentiation of neutrophils, with moderate effect on their survival. This study shows that IFN-deficiency leads to the elevated NAMPT expression in the bone marrow, which in turn modulates neutrophil development and differentiation, even in the absence of tumor-derived stimuli.