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Severe congenital neutropenia (SCN) is frequently associated with dominant point mutations in ELANE, the gene encoding neutrophil elastase (NE). Chronic administration of granulocyte colony-stimulating factor (G-CSF) is a first-line treatment of ELANE-mutant (ELANEmut) SCN. However, some ELANEmut patients, including patients with ELANE start codon mutations, do not respond to G-CSF. Here, through directed granulopoiesis of gene-edited isogenic normal and patient-derived iPSCs, we demonstrate that ELANE start codon mutations suffice to induce G-CSF-resistant granulocytic precursor cell death and refractory SCN. ELANE start codon-mutated neutrophil precursors express predominantly nuclear N-terminally truncated alternate NE. Unlike G-CSF-sensitive ELANE mutations that induce endoplasmic reticulum and unfolded protein response stress, we found that the mutation of the ELANE translation initiation codon resulted in NE aggregates and activated proapoptotic aggrephagy, as determined by downregulated BAG1 expression, decreased BAG1/BAG3 ratio, NE colocalization with BAG3, and localized expression of autophagic LC3B. We found that SERF1, an RNA-chaperone protein, known to localize in misfolded protein aggregates in neurodegenerative diseases, was highly upregulated and interacted with cytoplasmic NE of mutant neutrophil precursors. Silencing of SERF1 enhanced survival and differentiation of iPSC-derived neutrophil precursors, restoring their responsiveness to G-CSF. These observations provide a mechanistic insight into G-CSF-resistant ELANEmut SCN, revealing targets for therapeutic intervention.

Severe congenital neutropenia (SCN) is often associated with inherited heterozygous point mutations in ELANE, which encodes neutrophil elastase (NE). However, a lack of appropriate models to recapitulate SCN has substantially hampered the understanding of the genetic etiology and pathobiology of this disease. To this end, we generated both normal and SCN patient-derived induced pluripotent stem cells (iPSCs), and performed genome editing and differentiation protocols that recapitulate the major features of granulopoiesis. Pathogenesis of ELANE point mutations was the result of promyelocyte death and differentiation arrest, and was associated with NE mislocalization and activation of the unfolded protein response/ER stress (UPR/ER stress). Similarly, high-dose G-CSF (or downstream signaling through AKT/BCL2) rescues the dysgranulopoietic defect in SCN patient-derived iPSCs through C/EBPβ-dependent emergency granulopoiesis. In contrast, sivelestat, an NE-specific small-molecule inhibitor, corrected dysgranulopoiesis by restoring normal intracellular NE localization in primary granules; ameliorating UPR/ER stress; increasing expression of CEBPA, but not CEBPB; and promoting promyelocyte survival and differentiation. Together, these data suggest that SCN disease pathogenesis includes NE mislocalization, which in turn triggers dysfunctional survival signaling and UPR/ER stress. This paradigm has the potential to be clinically exploited to achieve therapeutic responses using lower doses of G-CSF combined with targeting to correct NE mislocalization.

Bacterial and fungal infections are a major cause of morbidity and mortality in neutropenic patients. Donor‐derived neutrophil transfusions have been used for prophylaxis or treatment for infection in neutropenic patients. However, the short half‐life and the limited availability of large numbers of donor‐derived neutrophils for transfusion remain a significant hurdle in the implementation of neutrophil transfusion therapy. Here, we investigate the in vitro and in vivo activity of neutrophils generated from human induced pluripotent stem cells (iPSC), a potentially unlimited resource to produce neutrophils for transfusion. Phenotypic analysis of iPSC‐derived neutrophils reveal reactive oxygen species production at similar or slightly higher than normal peripheral blood neutrophils, but have an ∼50%–70% reduced Escherichia coli phagocytosis and phorbol 12‐myristate 13‐acetate induced formation of neutrophil extracellular traps (NET). Signaling of granulocytic precursors identified impaired AKT activation, but not ERK or STAT3, in agonist‐stimulated iPSC‐derived neutrophils. Expression of a constitutively activated AKT in iPSC‐derived neutrophils restores most phagocytic activity and NET formation. In a model of bacterial induced peritonitis in immunodeficient mice, iPSC‐derived neutrophils, with or without corrected AKT activation, migrate similarly to the peritoneal fluid as peripheral blood neutrophils, whereas the expression of activated AKT significantly improves their phagocytic activity in vivo. Stem Cells Translational Medicine 2019;8:557–567 Human induced pluripotent stem cells can be engineered to generate human neutrophils expressing constitutively active AKT with significant activity to phagocytose bacteria in vitro and in vivo. Abbreviation: Diff., Differentiation.

Severe congenital neutropenia (SCN) is often associated with inherited heterozygous point mutations in ELANE, which encodes neutrophil elastase (NE). However, a lack of appropriate models to recapitulate SCN has substantially hampered the understanding of the genetic etiology and pathobiology of this disease. To this end, we generated both normal and SCN patient-derived induced pluripotent stem cells (iPSCs), and performed genome editing and differentiation protocols that recapitulate the major features of granulopoiesis. Pathogenesis of ELANE point mutations was the result of promyelocyte death and differentiation arrest, and was associated with NE mislocalization and activation of the unfolded protein response/ER stress (UPR/ER stress). Similarly, high-dose G-CSF (or downstream signaling through AKT/BCL2) rescues the dysgranulopoietic defect in SCN patient-derived iPSCs through C/EBPβ-dependent emergency granulopoiesis. In contrast, sivelestat, an NE-specific small-molecule inhibitor, corrected dysgranulopoiesis by restoring normal intracellular NE localization in primary granules; ameliorating UPR/ER stress; increasing expression of CEBPA, but not CEBPB; and promoting promyelocyte survival and differentiation. Together, these data suggest that SCN disease pathogenesis includes NE mislocalization, which in turn triggers dysfunctional survival signaling and UPR/ER stress. This paradigm has the potential to be clinically exploited to achieve therapeutic responses using lower doses of G-CSF combined with targeting to correct NE mislocalization.

Advances in genetics and sequencing have identified a plethora of disease-associated and disease-causing genetic alterations. To determine causality between genetics and disease, accurate models for molecular dissection are required; however, the rapid expansion of transcriptional populations identified through single-cell analyses presents a major challenge for accurate comparisons between mutant and wild-type cells. Here we generate mouse models of human severe congenital neutropenia (SCN) using patient-derived mutations in the GFI1 transcription factor. To determine the effects of SCN mutations, we generated single-cell references for granulopoietic genomic states with linked epitopes 1 , aligned mutant cells to their wild-type equivalents and identified differentially expressed genes and epigenetic loci. We find that GFI1-target genes are altered sequentially, as cells go through successive states of differentiation. These insights facilitated the genetic rescue of granulocytic specification but not post-commitment defects in innate immune effector function, and underscore the importance of evaluating the effects of mutations and therapy within each relevant cell state. Mouse models of severe congenital neutropenia using patient-derived mutations in the GFI1 locus are used to determine the mechanisms by which the disease progresses.

We present JWST NIRSpec spectroscopy for 11 galaxy candidates with photometric redshifts of \\(z\\simeq9-13\\) and \\(M_{\\rm\\,UV} \\in[-21,-18]\\) newly identified in NIRCam images in the Cosmic Evolution Early Release Science (CEERS) Survey. We confirm emission line redshifts for 7 galaxies at \\(z=7.762-8.998\\) using spectra at \\(\\sim1-5\\mu\\)m either with the NIRSpec prism or its three medium resolution gratings. For \\(z\\simeq9\\) photometric candidates, we achieve a high confirmation rate of \\(\\simeq\\)90\\%, which validates the classical dropout selection from NIRCam photometry. No robust emission lines are identified in three galaxy candidates at \\(z>10\\), where the strong [OIII] and H\\(\\beta\\) lines would be redshifted beyond the wavelength range observed by NIRSpec, and the Lyman-\\(\\alpha\\) continuum break is not detected with the current sensitivity. Compared with HST-selected bright galaxies (\\(M_{\\rm\\,UV}\\simeq-22\\)) that are similarly spectroscopically confirmed at \\(z\\gtrsim8\\), these NIRCam-selected galaxies are characterized by lower star formation rates (SFR\\(\\simeq4\\,M_{\\odot}\\)~yr\\(^{-1}\\)) and lower stellar masses (\\(\\simeq10^{8}\\,M_{\\odot}\\)), but with higher [OIII]+H\\(\\beta\\) equivalent widths (\\(\\simeq\\)1100\\(Å\\)), and elevated production efficiency of ionizing photons (\\(\\log(\\xi_{\\rm\\,ion}/{\\rm\\,Hz\\,erg}^{-1})\\simeq25.8\\)) induced by young stellar populations (\\(<10\\)~Myrs) accounting for \\(\\simeq20\\%\\) of the galaxy mass, highlighting the key contribution of faint galaxies to cosmic reionization. Taking advantage of the homogeneous selection and sensitivity, we also investigate metallicity and ISM conditions with empirical calibrations using the [OIII]/H\\(\\beta\\) ratio. We find that galaxies at \\(z\\sim8-9\\) have higher SFRs and lower metallicities than galaxies at similar stellar masses at \\(z\\sim2-6\\), which is generally consistent with the current galaxy formation and evolution models.

The new capabilities that JWST offers in the near- and mid-infrared (IR) are used to investigate in unprecedented detail the nature of optical/near-IR faint, mid-IR bright sources, HST-dark galaxies among them. We gather JWST data from the CEERS survey in the EGS, jointly with HST data, and analyze spatially resolved optical-to-mid-IR spectral energy distributions (SEDs) to estimate both photometric redshifts in 2 dimensions and stellar populations properties in a pixel-by-pixel basis. We select 138 galaxies with F150W-F356W>1.5 mag, F356W<27.5 mag. The nature of these sources is threefold: (1) 71% are dusty star-forming galaxies at 2100 Gyr^-1); (2) 18% are quiescent/dormant (i.e., subject to reignition and rejuvenation) galaxies at 3

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