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Immunity Aug 2020Granulocyte-monocyte progenitors (GMPs) have been previously defined for their potential to generate various myeloid progenies such as neutrophils and monocytes....
Granulocyte-monocyte progenitors (GMPs) have been previously defined for their potential to generate various myeloid progenies such as neutrophils and monocytes. Although studies have proposed lineage heterogeneity within GMPs, it is unclear if committed progenitors already exist among these progenitors and how they may behave differently during inflammation. By combining single-cell transcriptomic and proteomic analyses, we identified the early committed progenitor within the GMPs responsible for the strict production of neutrophils, which we designate as proNeu1. Our dissection of the GMP hierarchy led us to further identify a previously unknown intermediate proNeu2 population. Similar populations could be detected in human samples. proNeu1s, but not proNeu2s, selectively expanded during the early phase of sepsis at the expense of monocytes. Collectively, our findings help shape the neutrophil maturation trajectory roadmap and challenge the current definition of GMPs.
Topics: Animals; Granulocyte Precursor Cells; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Monocytes; Myelopoiesis; Neutrophils; Single-Cell Analysis
PubMed: 32579887
DOI: 10.1016/j.immuni.2020.06.005 -
Cell Feb 2022Single-cell (sc)RNA-seq, together with RNA velocity and metabolic labeling, reveals cellular states and transitions at unprecedented resolution. Fully exploiting these...
Single-cell (sc)RNA-seq, together with RNA velocity and metabolic labeling, reveals cellular states and transitions at unprecedented resolution. Fully exploiting these data, however, requires kinetic models capable of unveiling governing regulatory functions. Here, we introduce an analytical framework dynamo (https://github.com/aristoteleo/dynamo-release), which infers absolute RNA velocity, reconstructs continuous vector fields that predict cell fates, employs differential geometry to extract underlying regulations, and ultimately predicts optimal reprogramming paths and perturbation outcomes. We highlight dynamo's power to overcome fundamental limitations of conventional splicing-based RNA velocity analyses to enable accurate velocity estimations on a metabolically labeled human hematopoiesis scRNA-seq dataset. Furthermore, differential geometry analyses reveal mechanisms driving early megakaryocyte appearance and elucidate asymmetrical regulation within the PU.1-GATA1 circuit. Leveraging the least-action-path method, dynamo accurately predicts drivers of numerous hematopoietic transitions. Finally, in silico perturbations predict cell-fate diversions induced by gene perturbations. Dynamo, thus, represents an important step in advancing quantitative and predictive theories of cell-state transitions.
Topics: Algorithms; Female; Gene Expression Regulation; HL-60 Cells; Hematopoiesis; Hematopoietic Stem Cells; Humans; Kinetics; Models, Biological; RNA, Messenger; Single-Cell Analysis; Staining and Labeling; Transcriptome
PubMed: 35108499
DOI: 10.1016/j.cell.2021.12.045 -
Proceedings of the National Academy of... Mar 2020Neutrophil extracellular traps (NETs) are web-like DNA structures decorated with histones and cytotoxic proteins that are released by activated neutrophils to trap and...
Neutrophil extracellular traps (NETs) are web-like DNA structures decorated with histones and cytotoxic proteins that are released by activated neutrophils to trap and neutralize pathogens during the innate immune response, but also form in and exacerbate sterile inflammation. Peptidylarginine deiminase 4 (PAD4) citrullinates histones and is required for NET formation (NETosis) in mouse neutrophils. While the in vivo impact of NETs is accumulating, the cellular events driving NETosis and the role of PAD4 in these events are unclear. We performed high-resolution time-lapse microscopy of mouse and human neutrophils and differentiated HL-60 neutrophil-like cells (dHL-60) labeled with fluorescent markers of organelles and stimulated with bacterial toxins or to induce NETosis. Upon stimulation, cells exhibited rapid disassembly of the actin cytoskeleton, followed by shedding of plasma membrane microvesicles, disassembly and remodeling of the microtubule and vimentin cytoskeletons, ER vesiculation, chromatin decondensation and nuclear rounding, progressive plasma membrane and nuclear envelope (NE) permeabilization, nuclear lamin meshwork and then NE rupture to release DNA into the cytoplasm, and finally plasma membrane rupture and discharge of extracellular DNA. Inhibition of actin disassembly blocked NET release. Mouse and dHL-60 cells bearing genetic alteration of PAD4 showed that chromatin decondensation, lamin meshwork and NE rupture and extracellular DNA release required the enzymatic and nuclear localization activities of PAD4. Thus, NETosis proceeds by a stepwise sequence of cellular events culminating in the PAD4-mediated expulsion of DNA.
Topics: Animals; Chromatin; Cytoskeleton; DNA; Extracellular Traps; HL-60 Cells; Histones; Humans; Immunity, Innate; Inflammation; Mice; Microtubules; Neutrophil Activation; Neutrophils; Nuclear Envelope; Protein-Arginine Deiminase Type 4
PubMed: 32170015
DOI: 10.1073/pnas.1909546117 -
Cell Reports Jan 2022AMP-activated protein kinase (AMPK) regulates the balance between cellular anabolism and catabolism dependent on energy resources to maintain proliferation and survival....
AMP-activated protein kinase (AMPK) regulates the balance between cellular anabolism and catabolism dependent on energy resources to maintain proliferation and survival. Small-compound AMPK activators show anti-cancer activity in preclinical models. Using the direct AMPK activator GSK621, we show that the unfolded protein response (UPR) is activated by AMPK in acute myeloid leukemia (AML) cells. Mechanistically, the UPR effector protein kinase RNA-like ER kinase (PERK) represses oxidative phosphorylation, tricarboxylic acid (TCA) cycle, and pyrimidine biosynthesis and primes the mitochondrial membrane to apoptotic signals in an AMPK-dependent manner. Accordingly, in vitro and in vivo studies reveal synergy between the direct AMPK activator GSK621 and the Bcl-2 inhibitor venetoclax. Thus, selective AMPK-activating compounds kill AML cells by rewiring mitochondrial metabolism that primes mitochondria to apoptosis by BH3 mimetics, holding therapeutic promise in AML.
Topics: AMP-Activated Protein Kinases; Adolescent; Adult; Aged; Aged, 80 and over; Animals; Antineoplastic Agents; Apoptosis; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Citric Acid Cycle; Drug Evaluation, Preclinical; Female; HEK293 Cells; HL-60 Cells; Humans; Imidazoles; Leukemia, Myeloid, Acute; Male; Mice; Middle Aged; Mitochondria; Oxidative Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Pyrimidinones; Sulfonamides; THP-1 Cells; U937 Cells; Unfolded Protein Response; Young Adult; eIF-2 Kinase
PubMed: 34986346
DOI: 10.1016/j.celrep.2021.110197 -
Drug Design, Development and Therapy 2019This study aims to observe the effects of notopterol on the apoptosis and differentiation of HL-60 cells and to explore the underlying molecular mechanisms. Cell...
This study aims to observe the effects of notopterol on the apoptosis and differentiation of HL-60 cells and to explore the underlying molecular mechanisms. Cell viability was assessed using sulforhodamine B assay. Cell proliferation was determined by the trypan blue dye exclusion test. Colony-forming units were assayed in methylcellulose. Apoptosis assays were carried out by annexin V-fluorescein isothiocyanate(FITC)/propidium iodide (PI) double staining, Hoechst 33342 staining, mitochondrial membrane potential, and Western blot. Wright-Giemsa staining, nitroblue tetrazolium (NBT) reduction assay, CD11b and CD14 and Western blot were detected for induction of differentiation. In addition, cell-cycle phase distribution was analyzed by flow cytometry and Western blot. The combination therapy of notopterol and all-trans retinoic acid (ATRA) on HL-60 cells was examined. Notopterol obviously inhibited the growth of HL-60 cells with an IC value of 40.32 μM and remarkably reduced the number of colonies by 10, 20, and 40 µM. In addtion, notopterol induced the percentage of apoptotic HL-60 cells, reduced the mitochondrial membrane potential, decreased the protein expresstion of Bcl-2 and Mcl-1, and increased the expression of Bax, cleavage of caspase 9, caspase 3, and PARP. As for cell differentiation, notopterol clearly induced chromatin condensation; increased the nucleocytoplasmic ratio, nitroblue tetrazolium-positive cells, expression of CD14 and CD11b, and protein expression of c-Jun and Jun B, and decreased c-myc. Furthermore, notopterol induced the G0/G1 cell-cycle arrest as determined using flow cytometry, which may be related to the regulation of cell-cycle-related proteins p53, CDK2, CDK4, Cyclin D1, Cyclin E, and survivin. The combined use of notopterol and ATRA did not enhance the apoptotic effect as evidenced by cell viability test and Hoechst 33342. However, the combination of notopterol and ATRA enhanced the effect of inducing differentiation when compared with using either notopterol or ATRA alone, which can be evidenced by the increased nucleocytoplasmic ratio, NBT positive cells, and expression of CD14. This is the first time it has been demonstrated that notopterol could induce apoptosis, differentiation, and G0/G1 arrest in human AML HL-60 cells, suggesting that notopterol has potential therapeutic effects on AML. The combination application of notopterol (20 and 40 μM) and ATRA (2 μM) could augment differentiation of HL-60 cells.
Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Differentiation; Cell Proliferation; Cell Survival; Coumarins; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Structure-Activity Relationship
PubMed: 31239643
DOI: 10.2147/DDDT.S189969 -
Pathobiology : Journal of... 2024Disease progression in myelodysplastic syndromes (MDS), myelodysplastic-myeloproliferative neoplasms (MDS/MPN), and myeloproliferative neoplasms (MPN), altogether... (Review)
Review
Disease progression in myelodysplastic syndromes (MDS), myelodysplastic-myeloproliferative neoplasms (MDS/MPN), and myeloproliferative neoplasms (MPN), altogether referred to as myeloid neoplasms (MN), is a major source of mortality. Apart from transformation to acute myeloid leukemia, the clinical progression of MN is mostly due to the overgrowth of pre-existing hematopoiesis by the MN without an additional transforming event. Still, MN may evolve along other recurrent yet less well-known scenarios: (1) acquisition of MPN features in MDS or (2) MDS features in MPN, (3) progressive myelofibrosis (MF), (4) acquisition of chronic myelomonocytic leukemia (CMML)-like characteristics in MPN or MDS, (5) development of myeloid sarcoma (MS), (6) lymphoblastic (LB) transformation, (7) histiocytic/dendritic outgrowths. These MN-transformation types exhibit a propensity for extramedullary sites (e.g., skin, lymph nodes, liver), highlighting the importance of lesional biopsies in diagnosis. Gain of distinct mutations/mutational patterns seems to be causative or at least accompanying several of the above-mentioned scenarios. MDS developing MPN features often acquire MPN driver mutations (usually JAK2), and MF. Conversely, MPN gaining MDS features develop, e.g., ASXL1, IDH1/2, SF3B1, and/or SRSF2 mutations. Mutations of RAS-genes are often detected in CMML-like MPN progression. MS ex MN is characterized by complex karyotypes, FLT3 and/or NPM1 mutations, and often monoblastic phenotype. MN with LB transformation is associated with secondary genetic events linked to lineage reprogramming leading to the deregulation of ETV6, IKZF1, PAX5, PU.1, and RUNX1. Finally, the acquisition of MAPK-pathway gene mutations may shape MN toward histiocytic differentiation. Awareness of all these less well-known MN-progression types is important to guide optimal individual patient management.
Topics: Humans; Granulocyte Precursor Cells; Myeloproliferative Disorders; Myelodysplastic Syndromes; Mutation; Myelodysplastic-Myeloproliferative Diseases; Leukemia, Myeloid, Acute
PubMed: 37232015
DOI: 10.1159/000530940 -
International Journal of Molecular... Jul 2020In vertebrates, biomineralization is a feature considered unique to mature osteoblasts and odontoblasts by which they synthesize hydroxyapatite (HAP), which is deposited...
In vertebrates, biomineralization is a feature considered unique to mature osteoblasts and odontoblasts by which they synthesize hydroxyapatite (HAP), which is deposited in the collagen matrix to construct endoskeleton. For many decades, the mechanisms that modulate differentiation and maturation of these specialized cells have been sought as a key to understanding bone-remodeling defects. Here, we report that biomineralization is an innate ability of all mammalian cells, irrespective of cell type or maturation stage. This innate biomineralization is triggered by the concomitant exposure of living cells to three indispensable elements: calcium ion, phosphoester salt, and alkaline phosphatase. Any given somatic cell, including undifferentiated mononuclear cells, can undergo a biomineralization process to produce calcium-phosphate agglomerates. The biologically generated minerals under such conditions are composed of genuine HAP crystallites of Ca(PO)(OH) and 5-10 nanometer (nm) in size. This discovery will profoundly improve our understanding of bone metabolism and ectopic calcifications.
Topics: Alkaline Phosphatase; Animals; Biomineralization; Bone and Bones; Calcium Phosphates; Cell Differentiation; Cell Line; Cell Line, Tumor; Collagen; Durapatite; HEK293 Cells; HL-60 Cells; HeLa Cells; Humans; K562 Cells; MCF-7 Cells; Mammals; Mice; NIH 3T3 Cells; Odontoblasts; Osteoblasts; PC-3 Cells; THP-1 Cells; U937 Cells
PubMed: 32650435
DOI: 10.3390/ijms21144820 -
ELife Dec 2020Sciatic nerve crush injury triggers sterile inflammation within the distal nerve and axotomized dorsal root ganglia (DRGs). Granulocytes and pro-inflammatory Ly6C...
Sciatic nerve crush injury triggers sterile inflammation within the distal nerve and axotomized dorsal root ganglia (DRGs). Granulocytes and pro-inflammatory Ly6C monocytes infiltrate the nerve first and rapidly give way to Ly6C inflammation-resolving macrophages. In axotomized DRGs, few hematogenous leukocytes are detected and resident macrophages acquire a ramified morphology. Single-cell RNA-sequencing of injured sciatic nerve identifies five macrophage subpopulations, repair Schwann cells, and mesenchymal precursor cells. Macrophages at the nerve crush site are molecularly distinct from macrophages associated with Wallerian degeneration. In the injured nerve, macrophages 'eat' apoptotic leukocytes, a process called efferocytosis, and thereby promote an anti-inflammatory milieu. Myeloid cells in the injured nerve, but not axotomized DRGs, strongly express receptors for the cytokine GM-CSF. In GM-CSF-deficient () mice, inflammation resolution is delayed and conditioning-lesion-induced regeneration of DRG neuron central axons is abolished. Thus, carefully orchestrated inflammation resolution in the nerve is required for conditioning-lesion-induced neurorepair.
Topics: Animals; Apoptosis; Cells, Cultured; Cytokine Receptor Common beta Subunit; Disease Models, Animal; Female; Ganglia, Spinal; Gene Expression Regulation; Gene Regulatory Networks; Granulocyte-Macrophage Colony-Stimulating Factor; Inflammation Mediators; Leukocytes; Macrophages; Male; Mice, Inbred C57BL; Mice, Knockout; Monocytes; Nerve Regeneration; Neuronal Outgrowth; Peripheral Nerve Injuries; Phagocytosis; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; Sciatic Nerve; Signal Transduction
PubMed: 33263277
DOI: 10.7554/eLife.60223 -
Science Advances Jul 2019Ferroptosis is a form of nonapoptotic regulated cell death driven by iron-dependent lipid peroxidation. Autophagy involves a lysosomal degradation pathway that can...
Ferroptosis is a form of nonapoptotic regulated cell death driven by iron-dependent lipid peroxidation. Autophagy involves a lysosomal degradation pathway that can either promote or impede cell death. A high level of autophagy has been associated with ferroptosis, but the mechanisms underpinning this relationship are largely elusive. We characterize the contribution of autophagy to ferroptosis in human cancer cell lines and mouse tumor models. We show that "clockophagy," the selective degradation of the core circadian clock protein ARNTL by autophagy, is critical for ferroptosis. We identify SQSTM1 as a cargo receptor responsible for autophagic ARNTL degradation. ARNTL inhibits ferroptosis by repressing the transcription of , thus activating the prosurvival transcription factor HIF1A. Genetic or pharmacological interventions blocking ARNTL degradation or inhibiting EGLN2 activation diminished, whereas destabilizing HIF1A facilitated, ferroptotic tumor cell death. Thus, our findings reveal a new pathway, initiated by the autophagic removal of ARNTL, that facilitates ferroptosis induction.
Topics: ARNTL Transcription Factors; Animals; Apoptosis; Autophagy; Biological Clocks; Ferroptosis; Gene Expression Regulation, Neoplastic; HL-60 Cells; Heterografts; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Iron; Lipid Peroxidation; Lysosomes; Mice; Neoplasms; Reactive Oxygen Species; Sequestosome-1 Protein; Signal Transduction
PubMed: 31355331
DOI: 10.1126/sciadv.aaw2238 -
Molecular Neurodegeneration Mar 2021Recent studies suggest that microglia contribute to tau pathology progression in Alzheimer's disease. Amyloid plaque accumulation transforms microglia, the primary...
BACKGROUND
Recent studies suggest that microglia contribute to tau pathology progression in Alzheimer's disease. Amyloid plaque accumulation transforms microglia, the primary innate immune cells in the brain, into neurodegenerative microglia (MGnD), which exhibit enhanced phagocytosis of plaques, apoptotic neurons and dystrophic neurites containing aggregated and phosphorylated tau (p-tau). It remains unclear how microglia promote disease progression while actively phagocytosing pathological proteins, therefore ameliorating pathology.
METHODS
Adeno-associated virus expressing P301L tau mutant (AAV-P301L-tau) was stereotaxically injected into the medial entorhinal cortex (MEC) in C57BL/6 (WT) and humanized APP mutant knock-in homozygote (App) mice at 5 months of age. Mice were fed either chow containing a colony stimulating factor-1 receptor inhibitor (PLX5622) or control chow from 4 to 6 months of age to test the effect of microglia depletion. Animals were tested at 6 months of age for immunofluorescence, biochemistry, and FACS of microglia. In order to monitor microglial extracellular vesicle secretion in vivo, a novel lentiviral EV reporter system was engineered to express mEmerald-CD9 (mE-CD9) specifically in microglia, which was injected into the same region of MEC.
RESULTS
Expressing P301L tau mutant in the MEC induced tau propagation to the granule cell layer of the hippocampal dentate gyrus, which was significantly exacerbated in App mice compared to WT control mice. Administration of PLX5622 depleted nearly all microglia in mouse brains and dramatically reduced propagation of p-tau in WT and to a greater extent in App mice, although it increased plaque burden and plaque-associated p-tau dystrophic neurites. Plaque-associated MGnD microglia strongly expressed an EV marker, tumor susceptibility gene 101, indicative of heightened synthesis of EVs. Intracortical injection of mE-CD9 lentivirus successfully induced microglia-specific expression of mE-CD9 EV particles, which were significantly enhanced in Mac2 MGnD microglia compared to Mac2 homeostatic microglia. Finally, consecutive intracortical injection of mE-CD9 lentivirus and AAV-P301L-tau into App mice revealed encapsulation of p-tau in microglia-specific mE-CD9 EVs as determined by super-resolution microscopy and immuno-electron microscopy.
DISCUSSION
Our findings suggest that MGnD microglia hyper-secrete p-tau EVs while compacting Aβ plaques and clearing NP tau, which we propose as a novel mechanistic link between amyloid plaque deposition and exacerbation of tau propagation in App mice.
Topics: Amyloid beta-Protein Precursor; Animals; Dentate Gyrus; Entorhinal Cortex; Extracellular Vesicles; Female; Gene Knock-In Techniques; Genetic Vectors; Humans; Injections; Male; Mice; Mice, Inbred C57BL; Microglia; Mutation, Missense; Neurites; Organic Chemicals; Plaque, Amyloid; Point Mutation; Protein Aggregation, Pathological; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; Recombinant Proteins; tau Proteins
PubMed: 33752701
DOI: 10.1186/s13024-021-00440-9