PLB-985 Cells Research Articles

Inositol hexaphosphate-induced cellular response in myeloid leukemia cells is mediated by nicotinamide adenine dinucleotide phosphate oxidase activation.

The objective of this study was to identify the role of reactive oxygen species (ROS) generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, in inositol hexaphosphate (IP6)-induced metabolic disruption in human leukemia PLB-985 cells. PLB-985 and X chromosome linked gp91-phox gene knockout (X-CGD) cells were treated with 5, 10, or 20mM IP6 for 24 to 72h. Cell growth was assayed using a highly water-soluble tetrazolium salt. The rate of apoptotic and necrotic cell death was determined with an Annexin V-fluorescein isothiocyanate/propidium iodide kit. The expression of CD11b as a marker of monocytic property and of LC3 as an autophagy marker was tested, using flow cytometry combined with fluorescent antibodies. Treatment with 5 and 10mM IP6 for 24h was found to suppress the growth of both cell lines, though the effect was more dramatic in PLB-985 cells. After 6-h treatment with 20mM IP6, the necrosis rate of PLB-985 cells was significantly greater than that of X-CGD cells. Further, after 72-h treatment with 10mM IP6, CD11b expression was observed in PLB-985 cells but inhibited in X-CGD cells. Autophagy monitoring after 6-h treatment with 10mM IP6 revealed that LC3 expression was suppressed in PLB-985 cells, whereas it was somewhat increased in X-CGD cells. Our results suggest that NADPH oxidase activation mediates IP6-induced metabolic disruption associated with necrosis, differentiation, cell growth, and autophagy in PLB-985 cells.

The X-CGD PLB-985 Cell Model for NOX2 Structure-Function Analysis.

Structure-function analysis of specific regions of NOX2 can be carried out after stable expression of site-directed mutagenesis-modified NOX2 in the X0-CGD PLB-985 cell model. Indeed, the generation of this human cellular model by Prof. MCDinauer's team gave researchers the opportunity to gain a deeper understanding of functional regions of NOX2. With this model cell line, the functional impact of X+-CGD or of new mutations in NOX2 can be highlighted, as the biological material is not limited. PLB-985 cells transfected with various NOX2 mutations can be easily cultured and differentiated into neutrophilsor monocytes/macrophages. Several measurements in intact mutated NOX2 PLB-985 cells can be carried out such as NOX2 expression, cytochrome b 558 spectrum, enzymatic activity, and assembly of the NADPH oxidase complex. Purified membranes or purified cytochrome b 558 from mutated NOX2 PLB-985 cells can be used for the study of the impact of specific mutations on NADPH oxidase or diaphorase activity, FAD incorporation, and NADPH or NADH binding in a cell-free assay system. Here, we describe a method to generate mutated NOX2 PLB-985 cells in order to analyze NOX2 structure-function relationships.

Genetic manipulation of PLB-985 cells and quantification of chemotaxis using the underagarose assay.

Neutrophils are the most common leukocyte in human blood and are the first cells to respond to injury and infection. Improper neutrophil chemotaxis can have deleterious effects on human health, including autoimmune diseases, poor innate immune response, and cancer. Therefore, gaining a better understanding of the signaling pathways governing chemotactic responses in these cells is important. One of the main challenges of working with primary human neutrophils is their short lifespan (about 1 day), making genetic manipulations not feasible. PLB-985 cells, which are pluripotent hematopoietic cells that can easily be differentiated to neutrophil-like cells, are amenable to genetic manipulations, including the expression of fluorescently tagged proteins-of-interest (POI) and gene editing using the CRISPR/CAS9 system to delete genes-of-interest (GOI). The use of PLB-985 cells can therefore greatly facilitate our understanding of the molecular mechanisms governing neutrophil biology during chemotaxis and serve as a good system to complement results gained from pharmacological inhibition of primary neutrophils. To better study the role and localization of proteins during chemotaxis, the underagarose assay has become a widely used and quantitative assay for measuring several aspects of chemotaxis. The objective of this chapter is to provide protocols for (1) the generation of genetically altered PLB-985 cell lines, (2) the set-up of an underagarose chemotaxis assay, and (3) the analysis of cell movement in chemotactic gradients from an underagarose experiment.

A map of gene expression in neutrophil-like cell lines

BackgroundHuman neutrophils are central players in innate immunity, a major component of inflammatory responses, and a leading model for cell motility and chemotaxis. However, primary neutrophils are short-lived, limiting their experimental usefulness in the laboratory. Thus, human myeloid cell lines have been characterized for their ability to undergo neutrophil-like differentiation in vitro. The HL-60 cell line and its PLB-985 sub-line are commonly used to model human neutrophil behavior, but how closely gene expression in differentiated cells resembles that of primary neutrophils has remained unclear.ResultsIn this study, we compared the effectiveness of differentiation protocols and used RNA sequencing (RNA-seq) to compare the transcriptomes of HL-60 and PLB-985 cells with published data for human and mouse primary neutrophils. Among commonly used differentiation protocols for neutrophil-like cell lines, addition of dimethyl sulfoxide (DMSO) gave the best combination of cell viability and expression of markers for differentiation. However, combining DMSO with the serum-free-supplement Nutridoma resulted in increased chemotactic response, phagocytic activity, oxidative burst and cell surface expression of the neutrophil markers FPR1 and CD11b without a cost in viability. RNA-seq analysis of HL-60 and PLB-985 cells before and after differentiation showed that differentiation broadly increases the similarity in gene expression between the cell lines and primary neutrophils. Furthermore, the gene expression pattern of the differentiated cell lines correlated slightly better with that of human neutrophils than the mouse neutrophil pattern did. Finally, we created a publicly available gene expression database that is searchable by gene name and protein domain content, where users can compare gene expression in HL-60, PLB-985 and primary human and mouse neutrophils.ConclusionsOur study verifies that a DMSO-based differentiation protocol for HL-60 and PLB-985 cell lines gives superior differentiation and cell viability relative to other common protocols, and indicates that addition of Nutridoma may be preferable for studies of chemotaxis, phagocytosis, or oxidative burst. Our neutrophil gene expression database will be a valuable tool to identify similarities and differences in gene expression between the cell lines and primary neutrophils, to compare expression levels for genes of interest, and to improve the design of tools for genetic perturbations.

P-309 - Crosstalk between mitochondria and NOX2 in vitamin D3-induced differentiation in PLB-985 myeloid cells

To elucidate the role of reactive oxygen species (ROS) in immune enhancement, we focused on the redox signaling in vitamin D3-induced monocytic differentiation of leukemia cells. ROS generation in the cells was analyzed with ESR spin-trapping method. As a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was used. Human myeloid PLB-985 cells were stimulated with 200 nM vitamin D3 and/or 100 nM phorbol 12-myristate 13-acetate. Immediately after vitamin D3-stimulation, ESR-spin trapping indicated the spectrum of DMPO-OH adduct. Flow cytometry showed the expression of CD11b/Mac1–alpha in the cells incubated with vitamin D3 for three days. Diphenyleneiodonium (NOX2 inhibitor:100 uM) and actinomycin A (mitochondrial electron transport chain complex III inhibitor:10 uM) have suppressed vitamin D3-induced the expression of CD11b and ROS generation. Our results indicate that vitamin D3-induced expression of CD11b have occurred through a close interaction between NOX2 and mitochondria.

Autophagy Mediates Interleukin-1β Secretion in Human Neutrophils.

Interleukin-1β (IL-1β), a major pro-inflammatory cytokine, is a leaderless cytosolic protein whose secretion does not follow the classical endoplasmic reticulum-to-Golgi pathway, and for which a canonical mechanism of secretion remains to be established. Neutrophils are essential players against bacterial and fungi infections. These cells are rapidly and massively recruited from the circulation into infected tissues and, beyond of displaying an impressive arsenal of toxic weapons effective to kill pathogens, are also an important source of IL-1β in infectious conditions. Here, we analyzed if an unconventional secretory autophagy mechanism is involved in the exportation of IL-1β by these cells. Our findings indicated that inhibition of autophagy with 3-methyladenine and Wortmannin markedly reduced IL-1β secretion induced by LPS + ATP, as did the disruption of the autophagic flux with Bafilomycin A1 and E64d. These compounds did not noticeable affect neutrophil viability ruling out that the effects on IL-1β secretion were due to cell death. Furthermore, VPS34IN-1, a specific autophagy inhibitor, was still able to reduce IL-1β secretion when added after it was synthesized. Moreover, siRNA-mediated knockdown of ATG5 markedly reduced IL-1β secretion in neutrophil-differentiated PLB985 cells. Upon LPS + ATP stimulation, IL-1β was incorporated to an autophagic compartment, as was revealed by its colocalization with LC3B by confocal microscopy. Overlapping of IL-1β-LC3B in a vesicular compartment peaked before IL-1β increased in culture supernatants. On the other hand, stimulation of autophagy by cell starvation augmented the colocalization of IL-1β and LC3B and then promoted neutrophil IL-1β secretion. In addition, specific ELISAs indicated that although both IL-1β and pro-IL-1β are released to culture supernatants upon neutrophil stimulation, autophagy only promotes IL-1β secretion. Furthermore, the serine proteases inhibitor AEBSF reduced IL-1β secretion. Moreover, IL-1β could be also found colocalizing with elastase, suggesting both some vesicles containing IL-1β intersect azurophil granules content and that serine proteases also regulate IL-1β secretion. Altogether, our findings indicate that an unconventional autophagy-mediated secretory pathway mediates IL-1β secretion in human neutrophils.

Nutrients, Antioxidant Capacity and Safety of Hot Water Extract from Sugar Maple (Acer saccharum M.) and Red Maple (Acer rubrum L.) Bark.

Sugar maple (Acer saccharum M.) and red maple (Acer rubrum L.) barks were treated with hot water to extract nutrients in order to explore, for the first time, its potential as safe dietary antioxidants. The organic and inorganic nutrients of these extracts, as well as their safety on human PLB-985 cells differentiated into neutrophils-like cells, were determined. Proximate analysis showed that both bark extracts were low in moisture and fat. Sugar maple bark extract (SM-BX) showed crude protein and ash content higher than those found in red maple bark extract (RM-BX). In addition, SM-BX had total sugars higher than those evaluated in RM-BX, while complex sugars (oligo- and/or poly-saccharides) were similarly abundant in both bark extracts. Furthermore, SM-BX demonstrated a wide array of vital minerals (K, Ca, Mg, P, Na, Fe and Cu) in quantity larger than that evaluated in RM-BX, whereas RM-BX have Zn and Mn levels higher than those found in SM-BX. Phytochemical analyses showed that RM-BX exhibited total phenolic and flavonoid contents higher than those measured in SM-BX. Consequently, RM-BX presented an antioxidant activity higher than that of SM-BX: 2.85-fold ABTS radical cation scavenging capacity and 1.9-fold oxygen radical absorbance capacity. Finally, RM-BX and SM-BX were greatly safe since, at concentration up to 100μg/ml, they did not modify the viability of neutrophils as determined by flow-cytometry assay using Annexin V-FITC/Propidum Iodide as markers. In conclusion, our in vitro studies indicate that both red and sugar maple bark extracts have a real potential as food additives.

Mechanotransduction through High-Affinity LFA-1 is a Minimum Requirement to Induce Kindlin-3/RACK1/OraI1 to Mediate Intracellular Calcium Flux and Outside-In Signaling

Absence of Kindlin-3 impairs β2-integrin function on neutrophils, results in bleeding and resistance to arterial thrombosis and immunodeficiency. Kindlin-3 binds LFA-1 β-integrin tail independent of Talin-1 and is associated with integrin conversion to a high-affinity and the facilitation of micro-cluster formation that allow for long-lived shear resistance bonds with ICAM-1. However, the molecular and mechanical events that link LFA-1 to Kindlin-3 at spatially distinct sites to allow for subsequent engagement with scaffolding protein RACK1 and calcium release activated channel Orai1 have not been rigorously shown. We show that an allosteric shift of LFA-1 due to force generation on the β2-integrin cytodomain promotes interaction with Kindlin-3 F3 subdomain, and when sufficient force is transduced through this mechanical linkage induces calcium flux. We utilize real time two-line total internal reflection fluorescent and epifluorescence microscopy on neutrophil-like PLB-985 cells arrested to ICAM-1 within our microfluidic vascular mimetic we have been able to identify the stoichiometric binding rate of Kindlin-3 and subsequent RACK-1 recruitment to high-affinity LFA-1 sites in response to increasing shear profiles prior to calcium flux. In addition to outside-in signaling we have shown that force acting on LFA-1 amplifies calcium influx during CXCR1/2 ligation in a Kindlin-3 dependent manner. To assess the spatial proximity and association between Kindlin-3/RACK1 with focal adhesion clusters we are developing a FRET pair between the Kindlin-3 PH domain and RACK1 scaffolds 5 to 7 to further elucidate the spatiotemporal events and requirements for outside-in signaling and neutrophil progression to a migratory phenotype. This has allowed for us to identify key forces and proteins required for outside-in signaling during neutrophil transition from rolling to arrest which will help further glyocomimetic development to treat various disease states.

High Efficiency Gene Correction in Hematopoietic Cells by Donor-Template-Free CRISPR/Cas9 Genome Editing.

The CRISPR/Cas9 prokaryotic adaptive immune system and its swift repurposing for genome editing enables modification of any prespecified genomic sequence with unprecedented accuracy and efficiency, including targeted gene repair. We used the CRISPR/Cas9 system for targeted repair of patient-specific point mutations in the Cytochrome b-245 heavy chain gene (CYBB), whose inactivation causes chronic granulomatous disease (XCGD)—a life-threatening immunodeficiency disorder characterized by the inability of neutrophils and macrophages to produce microbicidal reactive oxygen species (ROS). We show that frameshift mutations can be effectively repaired in hematopoietic cells by non-integrating lentiviral vectors carrying RNA-guided Cas9 endonucleases (RGNs). Because about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that up to a quarter of all patients suffering from monogenic blood disorders could benefit from gene therapy employing personalized, donor template-free RGNs.

IFN-γ alters the expression of diverse immunity related genes in a cell culture model designed to represent maturing neutrophils.

The cytokine interferon-γ (IFN-γ) is approved as a drug to treat chronic granulomatous disease (CGD) and osteopetrosis and is also used in hyperimmunoglobulin E syndromes. Patients with CGD have defects in proteins of the NOX2 NADPH oxidase system. This leads to reduced production of microbicidal ROS by PMNs and recurrent life threatening infections. The goal of this study was to better understand how IFN-γ might support phagocyte function in these diseases, and to obtain information that might expand potential uses for IFN-γ. Neutrophils mature in the bone marrow and then enter the blood where they quickly undergo apoptotic cell death with a half-life of only 5–10 hours. Therefore we reasoned that IFN-γ might exert its effects on neutrophils via prolonged exposure to cells undergoing maturation in the marrow rather than by its brief exposure to short-lived circulating cells. To explore this possibility we made use of PLB-985 cells, a myeloblast-like myeloid cell line that can be differentiated into a mature, neutrophil-like state by treatment with various agents including DMSO. In initial studies we investigated transcription and protein expression in PLB-985 cells undergoing maturation in the presence or absence of IFN-γ. We observed IFN-γ induced differences in expression of genes known to be involved in classical aspects of neutrophil function (transmigration, chemotaxis, phagocytosis, killing and pattern recognition) as well as genes involved in apoptosis and other mechanisms that regulating neutrophil number. We also observed differences for genes involved in the major histocompatibility complex I (MHCI) and MHCII systems whose involvement in neutrophil function is controversial and not well defined. Finally, we observed significant changes in expression of genes encoding guanylate binding proteins (Gbps) that are known to have roles in immunity but which have not as yet been linked to neutrophil function. We propose that changes in the expression within these classes of genes could help explain the immune supportive effects of IFN-γ. Next we explored if the effect of IFN-γ on expression of these genes is dependent on whether the cells are undergoing maturation; to do this we compared the effects of IFN-γ on cells cultured with and without DMSO. For a subset of genes the expression level changes caused by IFN-γ were much greater in maturing cells than non-maturing cells. These findings indicate that developmental changes associated with cell maturation can modulate the effects of IFN-γ but that this is gene specific. Since the effects of IFN-γ depend on whether cells are maturing, the gene expression changes observed in this study must be due to more than just prolonged application of IFN-γ and are instead the result of interplay between cell maturation and changes caused by the chemokine. This supports our hypothesis that the effects of IFN-γ on developing neutrophils in the bone marrow may be very different from its effects on mature cells in the blood. Collectively the findings in this study enhance our understanding of the effects of IFN-γ on maturing myeloid cells and indicate possible mechanisms by which this cytokine could support immune function.

Down-regulation of NOX2 activity in phagocytes mediated by ATM-kinase dependent phosphorylation

NADPH oxidases (NOX) have many biological roles, but their regulation to control production of potentially toxic ROS molecules remains unclear. A previously identified insertion sequence of 21 residues (called NIS) influences NOX activity, and its predicted flexibility makes it a good candidate for providing a dynamic switch controlling the NOX active site. We constructed NOX2 chimeras in which NIS had been deleted or exchanged with those from other NOXs (NIS1, 3 and 4). All contained functional heme and were expressed normally at the plasma membrane of differentiated PLB-985 cells. However, NOX2-ΔNIS and NOX2-NIS1 had neither NADPH-oxidase nor reductase activity and exhibited abnormal translocation of p47phox and p67phox to the phagosomal membrane. This suggested a functional role of NIS. Interestingly after activation, NOX2-NIS3 cells exhibited superoxide overproduction compared with wild-type cells. Paradoxically, the Vmax of purified unstimulated NOX2-NIS3 was only one-third of that of WT-NOX2. We therefore hypothesized that post-translational events regulate NOX2 activity and differ between NOX2-NIS3 and WT-NOX2. We demonstrated that Ser486, a phosphorylation target of ataxia telangiectasia mutated kinase (ATM kinase) located in the NIS of NOX2 (NOX2-NIS), was phosphorylated in purified cytochrome b558 after stimulation with phorbol 12-myristate-13-acetate (PMA). Moreover, ATM kinase inhibition and a NOX2 Ser486Ala mutation enhanced NOX activity whereas a Ser486Glu mutation inhibited it. Thus, the absence of Ser486 in NIS3 could explain the superoxide overproduction in the NOX2-NIS3 mutant. These results suggest that PMA-stimulated NOX2-NIS phosphorylation by ATM kinase causes a dynamic switch that deactivates NOX2 activity. We hypothesize that this downregulation is defective in NOX2-NIS3 mutant because of the absence of Ser486.

CRISPR/Cas9-generated p47phox-deficient cell line for Chronic Granulomatous Disease gene therapy vector development

Development of gene therapy vectors requires cellular models reflecting the genetic background of a disease thus allowing for robust preclinical vector testing. For human p47phox-deficient chronic granulomatous disease (CGD) vector testing we generated a cellular model using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to introduce a GT-dinucleotide deletion (ΔGT) mutation in p47phox encoding NCF1 gene in the human acute myeloid leukemia PLB-985 cell line. CGD is a group of hereditary immunodeficiencies characterized by impaired respiratory burst activity in phagocytes due to a defective phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In Western countries autosomal-recessive p47phox-subunit deficiency represents the second largest CGD patient cohort with unique genetics, as the vast majority of p47phox CGD patients carries ΔGT deletion in exon two of the NCF1 gene. The established PLB-985 NCF1 ΔGT cell line reflects the most frequent form of p47phox-deficient CGD genetically and functionally. It can be differentiated to granulocytes efficiently, what creates an attractive alternative to currently used iPSC models for rapid testing of novel gene therapy approaches.

Phosphoinositol 3-phosphate acts as a timer for reactive oxygen species production in the phagosome.

Production of reactive oxygen species (ROS) in the phagosome by the NADPH oxidase is critical for mammalian immune defense against microbial infections and phosphoinositides are important regulators in this process. Phosphoinositol 3-phosphate (PI(3)P) regulates ROS production at the phagosome via p40phox by an unknown mechanism. This study tested the hypothesis that PI(3)P controls ROS production by regulating the presence of p40phox and p67phox at the phagosomal membrane. Pharmacologic inhibition of PI(3)P synthesis at the phagosome decreased the ROS production both in differentiated PLB-985 cells and human neutrophils. It also releases p67phox, the key cytosolic subunit of the oxidase, and p40phox from the phagosome. The knockdown of the PI(3)P phosphatase MTM1 or Rubicon or both increases the level of PI(3)P at the phagosome. That increase enhances ROS production inside the phagosome and triggers an extended accumulation of p67phox at the phagosome. Furthermore, the overexpression of MTM1 at the phagosomal membrane induces the disappearance of PI(3)P from the phagosome and prevents sustained ROS production. In conclusion, PI(3)P, indeed, regulates ROS production by maintaining p40phox and p67phox at the phagosomal membrane.

Virtual Screening of DrugBank Reveals Two Drugs as New BCRP Inhibitors

The breast cancer resistance protein (BCRP) is an ABC transporter playing a crucial role in the pharmacokinetics of drugs. The early identification of substrates and inhibitors of this efflux transporter can help to prevent or foresee drug-drug interactions. In this work, we built a ligand-based in silico classification model to predict the inhibitory potential of drugs toward BCRP. The model was applied as a virtual screening technique to identify potential inhibitors among the small-molecules subset of DrugBank. Ten compounds were selected and tested for their capacity to inhibit mitoxantrone efflux in BCRP-expressing PLB985 cells. Results identified cisapride (IC50 = 0.4 µM) and roflumilast (IC50 = 0.9 µM) as two new BCRP inhibitors. The in silico strategy proved useful to prefilter potential drug-drug interaction perpetrators among a database of small molecules and can reduce the amount of compounds to test.

IFN-γ Alters the Expression of Diverse Immunity Related Genes in a Cell Culture Model of Maturing Neutrophils

The cytokine IFN-γ is used as a drug to treat chronic granulomatous disease (CGD), hyperimmunoglobulin E syndromes and osteopetrosis. The goal of this study was to better understand how IFN-γmight support PMN function in these diseases, and to obtain information that might expand potential uses for IFN-γ.Neutrophils mature in the bone marrow and then enter the blood where they quickly undergo apoptotic cell death; a range of human studies have estimated the neutrophil half-life at only 5-10 hours. Therefore we reasoned that IFN-γ might exert its effects on neutrophils via prolonged exposure to cells undergoing maturation toward a terminally differentiated state in the marrow rather than by its brief exposure to short-lived circulating cells. To explore this possibility we made use of PLB-985 cells which belong to a myeloblast-like myeloid cell line that can be differentiated into a mature, neutrophil-like state by treatment with various agents including DMSO. In initial studies we used microarrays and western blotting to compare transcription and protein expression in PLB-985 cells matured, using DMSO, in the presence or absence of IFN-γ. We observed changes in a significant number of mRNAs and proteins, that fall into the following categories, which could help explain the immune supportive effects of IFN-γ.1 Genes known to be involved in classical aspects of neutrophil function, i.e. transmigration, chemotaxis, phagocytosis and pathogen killing2 Genes involved in neutrophil clearance and homeostasis, including some involved in apoptosis.3 Genes encoding innate immune receptors4 Genes encoding guanylate binding proteins, a family of GTPases implicated in antimicrobial activity in different cell types but not, to our knowledge, neutrophils. These genes were among the most upregulated observed in this study, for example Gbp5 was increased 337 fold.5 Genes involved in antigen presentation by MHCI and MHCII systems. Although neutrophils are generally considered to be involved in innate immunity a number of studies have suggested that they might act as antigen presenting cells and our finding of IFN-g induced upregulation of MHCII components is consistent with this. Some MHCI related genes were amongst the most upregulated genes in this study, for example HLA-DRA was increased 75 fold.Next we explored if the effect of IFN-γ on expression of these genes depends on whether the cells are undergoing maturation. This was done by comparing the effects of IFN-γ on maturing cells (i.e. those cultured in the presence of DMSO) versus non-maturing cells. To do this, the fold changes in mRNAs for DMSO plus IFN-γ treated cells versus DMSO treated cells were compared to the fold changes for IFN-γ treated cells versus untreated cells. In general, for genes showing the largest changes in mRNA expression in response IFN-γ, it was found that even larger changes occurred in maturing cells. This suggests that the effects of IFN-γon maturing cells are due to more than just prolonged application of the chemokine and are enhanced by ongoing developmental differentiation of the cells.Collectively the findings in this study enhance our understanding of the effects of IFN-γ on myeloid cells and indicate possible mechanisms by which this cytokine could support immune function. DisclosuresNo relevant conflicts of interest to declare.

High Efficiency Gene Correction in Hematopoietic Cells By Template-Free Crispr/Cas9 Genome Editing

IntroductionA significant fraction of inherited monogenic disorders is caused by patient-specific mutations dispersed over the entire locus of the affected gene. Correcting these mutations by introducing healthy gene copies into the genome of the diseased cells proved successful in several clinical gene therapy trials. Most of these trials employed retroviral vectors, which by inserting randomly throughout the genome deprived the transduced genes of their endogenous control and caused insertional mutations leading to secondary disease. The development of genome editing tools capable of modifying any prespecified genomic sequence with unprecedented accuracy opened up a wide range of new possibilities in gene manipulation including targeted gene repair. In particular, CRISPR/Cas9, a prokaryotic adaptive immune system, and its swift repurposing for genome editing was widely adopted as the hitherto simplest genome editing tool. CRISPR/Cas9 is an RNA guided endonuclease that uses RNA-DNA base pairing to target genomic DNA. Bound to its target via the guide (g)RNA, Cas9 induces DNA double strand breaks (DBS) at prespecified genomic sites that promptly activate the endogenous DNA repair machinery. DSB repair is accomplished by either non-homologous end joining (NHEJ) or homology directed repair (HDR). Thus far, correction of human mutations in hematopoietic cells relied entirely on HDR requiring gene specific donor templates in addition to the RNA guided endonucleases (RGNs). However, although the HDR offers precision, its efficiency is low and requires positive selection to enrich for gene corrected cell. Because in mammalian cells DSB repair by NHEJ significantly exceeds HDR and even more importantly, is the dominant DSB repair pathway in hematopoietic stem- and progenitor cells (HSPC), we thought to exploit NHEJ for gene therapy because in theory, approximately one third of the indels associated with NHEJ should restore the open reading frame (ORF) disrupted by a disease mutation. This would lead to a significant number of ORF reconstitutions of which some, depending on the position and type of the original mutation, should either completely or partially recover protein function.ResultsTo test gene repair efficiency by NHEJ in human hematopoietic cells, we generated PLB-985 (PLB) reporter cells expressing mutationally inactivated EGFP (mEGFP). Transduction of mEGFP expressing PLBs (mEGFP-PLB) with integrase-deficient lentiviral (IDLV) particles encoding for RGNs targeting the EGFP mutation reconstituted EGFP expression in up to 27% of the mEGFP PLB cells. Indel analysis revealed that 13 out of 28 (46%) restored the EGFP-open reading frame of which 7 (25%) reconstituted EGFP activity. To test whether the donor-template free IDLV strategy would also effectively correct bone fide disease mutations, we performed similar experiments with X-CGD PLB cells expressing transgenes encoding patient-specific frameshift, missense and nonsense CYBB mutations causing X-linked chronic granulomatous disease (X-CGD) which is an inherited, life threatening immunodeficiency disorder. Transduction of the cells with IDLVs carrying RGNs directed against each of these mutations restored CYBB function in up to 10% of cells harboring frameshift mutations which is sufficient to protect X-CGD patients from microbial infections. However, RGNs directed against the nonsense or missense mutations restored CYBB function in only 1-2% of the cells, suggesting that these mutations are less amenable to CRISPR/Cas9 mediated repair. As Cas9 frequently tolerates single nucleotide mismatches, selection against solitary nucleotide substitutions may explain this low efficiency of gene repair.ConclusionsFrameshift mutations can be effectively repaired by NHEJ in hematopoietic cells by CRISPR/Cas9 transducing IDLVs. As about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that about a quarter of patients suffering from monogenic blood disorders could benefit from personalized, template free CRISPR/Cas9 gene therapy. DisclosuresNo relevant conflicts of interest to declare.

MicroRNA-941 Expression in Polymorphonuclear Granulocytes Is Not Related to Granulomatosis with Polyangiitis.

Jumonji Domain-Containing Protein 3 (JMJD3)/lysine demethylase 6B (KDM6B) is an epigenetic modulator that removes repressive histone marks on genes. Expression of KDM6B mRNA is elevated in leukocytes from patients with ANCA-associated vasculitis (AAV) and has been suggested to be the reason for higher proteinase 3 (PR3) mRNA expression in these cells due to derepression of PRTN3 gene transcription. MicroRNA-941 (miR-941) has been shown to target KDM6B mRNA and inhibit JMJD3 production. We therefore investigated whether polymorphonuclear granulocytes (PMNs) from patients suffering from granulomatosis with polyangiitis (GPA) have lower expression of miR-941 than healthy control donors as a biological cause for higher JMJD3 levels. We found no significant difference in the degree of maturation of PMNs from GPA patients (n = 8) and healthy controls (n = 11) as determined from cell surface expression of the neutrophil maturation marker CD16 and gene expression profile of FCGR3B. The expression of PRTN3 and KDM6B mRNAs and miR-941 was not significantly different in GPA patients and healthy controls. Transfection of pre-miR-941 into the neutrophil promyelocyte cell line PLB-985 cells did not result in reduction of the KDM6B mRNA level as shown previously in a hepatocellular carcinoma cell line. The amount of PR3 in PMNs from GPA patients and healthy controls was comparable. In conclusion, we found that PRTN3 mRNA, KDM6B mRNA, and miR-941 expression levels in PMNs do not differ between GPA patients and healthy controls, and that miR-941 does not uniformly regulate KDM6B mRNA levels by inducing degradation of the transcript. Thus, decreased miR-941 expression in PMNs cannot be part of the pathogenesis of GPA.

Complement MASP-1 enhances adhesion between endothelial cells and neutrophils by up-regulating E-selectin expression.

The complement system and neutrophil granulocytes are indispensable in the immune response against extracellular pathogens such as bacteria and fungi. Endothelial cells also participate in antimicrobial immunity largely by regulating the homing of leukocytes through their cytokine production and their pattern of cell surface adhesion molecules. We have previously shown that mannan-binding lectin-associated serine protease-1 (MASP-1), a complement lectin pathway enzyme, is able to activate endothelial cells by cleaving protease activated receptors, which leads to cytokine production and enables neutrophil chemotaxis. Therefore, we aimed to investigate how recombinant MASP-1 (rMASP-1) can modify the pattern of P-selectin, E-selectin, ICAM-1, ICAM-2, and VCAM-1 adhesion molecules in human umbilical vein endothelial cells (HUVEC), and whether these changes can enhance the adherence between endothelial cells and neutrophil granulocyte model cells (differentiated PLB-985). We found that HUVECs activated by rMASP-1 decreased the expression of ICAM-2 and increased that of E-selectin, whereas ICAM-1, VCAM-1 and P-selectin expression remained unchanged. Furthermore, these changes resulted in increased adherence between differentiated PLB-985 cells and endothelial cells. Our finding suggests that complement MASP-1 can increase adhesion between neutrophils and endothelial cells in a direct fashion. This is in agreement with our previous finding that MASP-1 increases the production of pro-inflammatory cytokines (such as IL-6 and IL-8) and chemotaxis, and may thereby boost neutrophil functions. This newly described cooperation between complement lectin pathway and neutrophils via endothelial cells may be an effective tool to enhance the antimicrobial immune response.

Neutrophil-Expressed p21/waf1 Favors Inflammation Resolution in Pseudomonas aeruginosa Infection.

Neutrophil-associated inflammation during Pseudomonas aeruginosa lung infection is a determinant of morbidity in cystic fibrosis (CF). Neutrophil apoptosis is a key factor in inflammation resolution and is controlled by cytosolic proliferating cell nuclear antigen (PCNA). p21/Waf1, a cyclin-dependent kinase inhibitor, is a partner of PCNA, and its mRNA is up-regulated in human neutrophils during LPS challenge. We show here that, after 7 days of persistent infection with P. aeruginosa, neutrophilic inflammation was more prominent in p21(-/-) compared with wild-type (WT) mice. Notably, no intrinsic defect in the phagocytosis of apoptotic cells by macrophages was found in p21(-/-) compared with WT mice. Inflammatory cell analysis in peritoneal lavages after zymosan-induced peritonitis showed a significantly increased number of neutrophils at 48 hours in p21(-/-) compared with WT mice. In vitro analysis was consistent with delayed neutrophil apoptosis in p21(-/-) compared with WT mice. Ectopic expression of p21/waf1 in neutrophil-differentiated PLB985 cells potentiated apoptosis and reversed the prosurvival effect of PCNA. In human neutrophils, p21 messenger RNA was induced by TNF-α, granulocyte colony-stimulating factor, and LPS. Neutrophils isolated from patients with CF showed enhanced survival, which was reduced after treatment with a carboxy-peptide derived from the sequence of p21/waf1. Notably, p21/waf1 was detected by immunohistochemistry in neutrophils within lungs from patients with CF. Our data reveal a novel role for p21/waf1 in the resolution of inflammation via its ability to control neutrophil apoptosis. This mechanism may be relevant in the neutrophil-dominated inflammation observed in CF and other chronic inflammatory lung conditions.

Locally excitable Cdc42 signals steer cells during chemotaxis.

Neutrophils and other amoeboid cells chemotax by steering their front towards chemoattractant. While Ras, Rac, Cdc42, and RhoA small GTPases all regulate chemotaxis, it has been unclear how they spatiotemporally control polarization and steering. Using fluorescence biosensors in neutrophil-like PLB-985 cells and photorelease of chemoattractant, we show that local Cdc42 signals, but not those of Rac, RhoA or Ras, precede cell turning during chemotaxis. Furthermore, preexisting local Cdc42 signals in morphologically unpolarized cells predict the future direction of movement upon uniform stimulation. Moreover, inhibition of actin polymerization uncovers recurring local Cdc42 activity pulses, suggesting that Cdc42 has the excitable characteristic of the compass activity proposed in models of chemotaxis. Globally, Cdc42 antagonizes RhoA, and maintains a steep spatial activity gradient during migration, while Ras and Rac form shallow gradients. Thus, chemotactic steering and de novo polarization are both directed by locally excitable Cdc42 signals.