scholarly journals NADPH Oxidase Activation and Endothelial Nitric Oxide Synthase Uncoupling Contribute to Endothelial Dysfunction in Antiphospholipid Syndrome

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4639-4639
Author(s):  
Meifang Wu ◽  
Suman Kundu ◽  
Keith R. McCrae

Abstract Introduction: Antiphospholipid syndrome (APS) is characterized by thrombosis and/or recurrent fetal loss in the presence of persistently elevated antiphospholipid antibodies (APLA). The majority of pathologic APLA are directed against β2-glycoprotein I (β2GPI). APLA cause endothelial dysfunction, though the underlying mechanisms have not been clearly delineated. Methods: Endothelial cells (EC) were incubated with β2GPI and either control antibodies or anti-β2GPI antibodies affinity-purified from sera of patients with APS, in the absence or presence of diapocynin, an NADPH oxidase (NOX) inhibitor, or siRNA against NOX1, NOX2, or NOX4. Generation of reactive oxygen species (ROS) in EC and conditioned medium were measured using fluorescent dyes (CM-H2DCFDA and CellROX Deep Red) or chemiluminescent substrate. NOX1, NOX2, NOX4, 3-nitrotyrosine, and thioredoxin reductase 1 (TrxR1) expression were analyzed by western blot. eNOS monomer and dimer were detected using cold (4°C) SDS-PAGE and immunoblot. Immunoprecipitation of TrxR1 was performed using protein A/G agarose. EC activation was assessed by measuring expression of E-selectin. Results: Incubation of EC with β2GPI and anti-β2GPI antibodies stimulated ROS generation in EC, as well as the release of ROS into conditioned medium. Expression of NOX2, but not NOX1 or NOX4, was significantly increased in EC exposed to anti-β2GPI antibodies, but not control IgG. Preteatment of endothelial cells with diapocynin, a NOX inhibitor, or siRNA against NOX2 and NOX4, but not NOX1, inhibited endothelial cell activation by anti-β2GPI antibodies. Furthermore, anti-β2GPI antibody-treated EC generated more peroxynitrite, as determined by 3-nitrotyrosine expression. Treatment of EC with anti-β2GPI antibodies increased eNOS monomer/dimer ratio, suggesting eNOS uncoupling. Compared to control human IgG, the TrxR1 immunoprecipitate from EC treated with β2GPI and anti-β2GPI antibodies contained more 3-nitrotyrosine level, suggesting TrxR1 tyrosine residue modification by nitration and possible loss of function. Conclusions: β2GPI and anti-β2GPI antibodies stimulate ROS generation in EC, with release into the conditioned medium. The impairment of EC activation by diapocynin, NOX2 siRNA, or NOX4 siRNA suggests that NOX mediate EC activation by anti-β2GPI antibodies. Anti-β2GPI antibodies induce nitrative stress in EC, which might be explained by eNOS uncoupling induced by these antibodies. The nitration of TrxR1, an enzyme that prevents eNOS uncoupling, may result in the inactivation of TrxR1 and contribute to eNOS uncoupling. Taken together, these studies provide preliminary evidence of the contribution of NOX activation and eNOS uncoupling in mediating oxidative and nitrative stress and induction of EC dysfunction by APLA. Disclosures McCrae: Janssen: Membership on an entity's Board of Directors or advisory committees; Syntimmune: Consultancy; Momenta: Consultancy; Halozyme: Membership on an entity's Board of Directors or advisory committees.

2010 ◽  
Vol 108 (6) ◽  
pp. 1745-1756 ◽  
Author(s):  
Hsiu-Chung Ou ◽  
Tuzz-Ying Song ◽  
Yueh-Chiao Yeh ◽  
Chih-Yang Huang ◽  
Shun-Fa Yang ◽  
...  

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), originally identified as the major receptor for oxidized low-density lipoprotein (oxLDL) in endothelial cells, plays a major role in the pathology of vascular diseases. Green tea consumption is associated with reduced cardiovascular mortality in some epidemiological studies. In the present study, we hypothesized that the most abundant polyphenolic compound in tea, epigallocatechin-3-gallate (EGCG), can downregulate parameters of endothelial dysfunction by modulating LOX-1-regulated cell signaling. In cultured human umbilical vein endothelial cells (HUVECs), exposure to oxLDL (130 μg/ml), which led to an increase in LOX-1 expression at the RNA and protein levels, was abrogated by addition of EGCG or DPI, a well-known inhibitor of flavoproteins, suggesting the involvement of NADPH oxidase. Furthermore, oxLDL rapidly activated the membrane translocation of Rac-1 and p47phox and the subsequent induction of ROS generation, which was suppressed markedly by pretreatment with EGCG or anti-LOX-1 monoclonal antibody. OxLDL also increased p38 MAPK phosphorylation and decreased phosphorylation of the amino-terminal region of Akt, with maximal induction at about 30 min, and NF-κB phosphorylation within 1 h, resulting in redox-sensitive signaling. In addition, oxLDL diminished the expression of endothelial nitric oxide synthase (eNOS), enhanced the expression of endothelin-1 and adhesion molecules (ICAM, E-selectin, and monocyte chemoattractant protein-1), and increased the adherence of monocytic THP-1 cells to HUVECs. Pretreatment with EGCG, however, exerted significant cytoprotective effects in all events. These data suggest that EGCG inhibits the oxLDL-induced LOX-1-mediated signaling pathway, at least in part, by inhibiting NADPH oxidase and consequent ROS-enhanced LOX-1 expression, which contributes to further ROS generation and the subsequent activation of NF-κB via the p38 MAPK pathway. Results from this study may provide insight into a possible molecular mechanism by which EGCG suppresses oxLDL-mediated vascular endothelial dysfunction.


Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1447-1447
Author(s):  
Meifang Wu ◽  
Keith R. McCrae

Abstract Introduction: Antiphospholipid Syndrome (APS) is characterized by thrombosis and/or recurrent fetal loss in the presence of persistently elevated antiphospholipid antibodies (APLA). The majority of pathologic APLA are directed against β2-glycoprotein I (β2GPI), an abundant plasma phospholipid binding protein. APLA/anti-β2GPI antibodies activate endothelial cells in a β2GPI-dependent manner, though the underlying mechanisms are not well defined. Objective: To define the role of NOX1 in the generation of ROS and activation of endothelial cells by anti-β2GPI antibodies. Methods: Endothelial cells were incubated with β2GPI and either control or affinity-purified anti-β2GPI antibodies in the absence or presence of diphenyleneiodonium (DPI), an NADPH oxidase (NOX) inhibitor. Generation of reactive oxygen species (ROS) in treated cells and conditioned medium were measured by using fluorescent dyes (CM-H2DCFDA and CellROX Deep Red) or luminescent substrate. NOX mRNA and protein expression were assessed using quantitative PCR and immunoblot. Endothelial cell activation was measured by increased expression of E-selectin. Results: Incubation of endothelial cells with β2GPI and anti-β2GPI antibodies stimulated ROS generation in endothelial cells, as well as the release of ROS into conditioned medium. The expression of NOX1 mRNA and protein levels were significantly increased in endothelial cells exposed to anti-β2GPI antibodies, but not control IgG (Figure 1). The ability of β2GPI and anti-β2GPI antibodies to induce endothelial cell E-selectin mRNA expression was blocked by pretreatment of cells with DPI (Figure 2), suggesting that ROS is required for downstream events underlying endothelial cell activation. Conclusions: Endothelial cells exposed to β2GPI and anti-β2GPI antibodies generate ROS, which is subsequently released into the conditioned medium. NOX1 appears to be essential for ROS generation. The impairment of endothelial cell activation by DPI suggests that NOX is also essential for endothelial cell activation by anti-β2GPI antibodies. We hypothesize that production of ROS by NOX plays a central role in APLA-induced endothelial dysfunction. Acknowledgment: This work was supported by an ASH Bridge Grant Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.


Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
Dan Wang ◽  
Christopher S Wilcox

Introduction and hypothesis: Following bodily entry, the SARS-CoV-2 virus undergoes pulmonary replication with release of circulating viral spike protein 1 (SP1) into the bloodstream. Uptake of SP1 by endothelial cells might provoke vascular dysfunction and thrombosis. We hypothesized that spironolactone could prevent microvascular complications from circulating SP1 in COVID-19. Methods: male C57Bl/6 mice received spironolactone (100 mg · kg -1 · d -1 PO x 3d) or vehicle and intravenous injections of recombinant full-length human SP1 (10 μg per mouse) or vehicle. They were euthanized after 3 days. Mesenteric resistant arterioles (n=4 per group) were dissected and mounted on isometric myographs. Acetylcholine-induced EDRF responses and L-NAME-inhibitable NO generation (DAF-FM fluorescence) were studied in pre-constricted vessels and contraction to endothelin 1 (ET1) or thromboxane (U-46, 619) and ET1-induced ROS (PEG-SOD inhibitable ethidium: dihydroethidium fluorescence) were studied by fluorescence microscopy in other vessels. Results: SP1 reduced acetylcholine-induced EDRF (17 ± 3 vs 27 ± 5 % mean ± sem; P < 0.05) and NO generation (0.21 ± 0.03 vs 0.36 ± 0.04, F 1 /F 0 ; P < 0.05) while increasing contraction to ET1 (10 -7 mol·l -1 : 124 ± 13 vs 89 ± 4 %; P < 0.05) and U-46, 619 (10 -6 mol·l -1 :114± 5 vs 87± 6 %; P < 0.05) and ET1-induced ROS generation(0.30± 0.08 vs 0.09± 0.03; P < 0.05). Spironolactone did not modify any of these responses in vessels from normal mice but prevented all the effects of SP1. Conclusion: these preliminary studies provide a novel model to study COVID-19 vasculopathy. They indicate that spironolactone can provide protection from microvascular oxidative stress, endothelial dysfunction and enhanced contractility and might thereby moderate COVID-19 complications.


2019 ◽  
Vol 8 (6) ◽  
pp. 918-927 ◽  
Author(s):  
Li Pang ◽  
Ping Deng ◽  
Yi-dan Liang ◽  
Jing-yu Qian ◽  
Li-Chuan Wu ◽  
...  

Abstract Paraquat (PQ) is a widely used herbicide in the agricultural field. The lack of an effective antidote is the significant cause of high mortality in PQ poisoning. Here, we investigate the antagonistic effects of alpha lipoic acid (α-LA), a naturally existing antioxidant, on PQ toxicity in human microvascular endothelial cells (HMEC-1). All the doses of 250, 500 and 1000 μM α-LA significantly inhibited 1000 μM PQ-induced cytotoxicity in HMEC-1 cells. α-LA pretreatment remarkably diminished the damage to cell migration ability, recovered the declined levels of the vasodilator factor nitric oxide (NO), elevated the expression level of endothelial nitric oxide synthases (eNOS), and inhibited the upregulated expression of vasoconstrictor factor endothelin-1 (ET-1). Moreover, α-LA pretreatment inhibited reactive oxygen species (ROS) generation, suppressed the damage to the mitochondrial membrane potential (ΔΨm) and mitigated the inhibition of adenosine triphosphate (ATP) production in HMEC-1 cells. These results suggested that α-LA could alleviate PQ-induced endothelial dysfunction by suppressing oxidative stress. In summary, our present study provides novel insight into the protective effects and pharmacological potential of α-LA against PQ toxicity in microvascular endothelial cells.


2005 ◽  
Vol 289 (6) ◽  
pp. L954-L961 ◽  
Author(s):  
Qunwei Zhang ◽  
Ikuo Matsuzaki ◽  
Shampa Chatterjee ◽  
Aron B. Fisher

Previous studies have shown endothelial cell membrane depolarization and generation of reactive oxygen species (ROS) in endothelial cells with abrupt reduction in shear stress (ischemia). This study evaluated the role of ATP-sensitive potassium (KATP) channels and NADPH oxidase in the ischemic response by using Kir6.2−/− and gp91phox−/− mice. To evaluate ROS generation, we subjected isolated perfused mouse lungs labeled with 2′,7′-dichlorodihydrofluorescein (DCF), hydroethidine (HE), or diphenyl-1-pyrenylphosphine (DPPP) to control perfusion followed by global ischemia. In wild-type C57BL/6J mice, imaging of subpleural endothelial cells showed a time-dependent increase in intensity for all three fluorescence probes with ischemia, which was blocked by preperfusion with cromakalim (a KATP channel agonist) or diphenyleneiodonium (DPI, a flavoprotein inhibitor). Endothelial cell fluorescence with bis-oxonol, a membrane potential probe, increased during lung ischemia indicating cell membrane depolarization. The change in membrane potential with ischemia in lungs of gp91phox−/− mice was similar to wild type, but ROS generation did not occur. Lungs from Kir6.2−/− showed marked attenuation of the change in both membrane potential and ROS production. Thus membrane depolarization during lung ischemia requires the presence of a KATP channel and is required for activation of NADPH oxidase and endothelial ROS generation.


Author(s):  
Keyvan Karimi Galougahi ◽  
Chia‐Chi Liu ◽  
Carmine Gentile ◽  
Cindy Kok ◽  
Andrea Nunez ◽  
...  

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 783-783
Author(s):  
Bradley Wayne Blaser ◽  
Jessica Moore ◽  
Brian LI ◽  
Owen J. Tamplin ◽  
Vera Binder ◽  
...  

Abstract The microenvironment is an important regulator of hematopoietic stem and progenitor cell (HSC/HSPC) engraftment during development and in recipients of hematopoietic stem cell transplantation (HSCT). Factors secreted by the hematopoietic microenvironment that promote HSC/HSPC engraftment in the developing zebrafish may therefore be therapeutic targets for enhancing HSC engraftment in patients undergoing HSCT. We previously described a novel behavior we called endothelial cuddling in which sinuosoidal endothelial cells of the niche make intimate interactions with stem cells. To find candidate extracellular factors regulating this behavior, gene expression profiling was performed on sorted zebrafish endothelial cells. Gene set enrichment analysis showed that expression of chemokines and TNF family members was significantly enriched in all endothelial cells. The leading edge gene sets included 16 chemokines and chemokine receptors. Thirteen of these genes were used as candidates in a gain-of-function screen to test whether overexpression was sufficient to stimulate the hematopoietic niche in favor of HSC engraftment. High level, global gene expression was induced at 36 and 48 hours post fertilization (hpf) using a heat shock-inducible system. One gene, CXCR1, enhanced HSC/HSPC engraftment when globally overexpressed (p=0.03, N=63). CXCR1 is a specific receptor for the chemokine IL-8/CXCL8 in higher vertebrates. Zebrafish IL-8 was used in similar gain of function experiments and was also sufficient to enhance HSC/HSPC engraftment (p=0.003, N=41). CXCR2 is a promiscuous chemokine receptor for IL-8, Gro-α and Gro-β and did not enhance HSC/HSPC engraftment in this system. To further characterize the effects of CXCR1 on HSC engraftment, it was overexpressed in transgenic zebrafish carrying a stem-cell specific reporter gene, Runx1:mCherry. HSC engraftment in the CHT was enhanced when CXCR1 expression was induced beginning at 36 hpf (3.0 +/- 2.0 vs 7.4 +/- 2.6 HSC per CHT) or 48 hpf (4.3 +/- 1.1 vs 9.4 +/- 3.6 HSC per CHT). Inhibition of CXCR1 signaling from 48 to 72 hpf using the selective CXCR1/2 antagonist, SB225002, decreased HSC engraftment in Runx1:mCherry animals (1.2 +/- 0.39 vs 0.4 +/- 0.2 HSC per CHT, p=0.03). We next hypothesized that overexpression of CXCR1 might also have effects on the endothelial cell niche itself. Using FLK1(VEGFR2):mCherry reporter zebrafish and 3-dimensional reconstruction of the CHT, we found that global overexpression of CXCR1 increased the volume of the endothelial cell niche (2.0 +/- 0.09 x 106 vs 2.4 +/- 0.1 x 106 μm3, p=0.005) while treatment with SB225002 reduced its volume (6.3 +/- 0.3 x 105 vs 4.9 +/- 0.5 x 105 µm3, p=0.04). Finally, we asked if CHT remodeling would still be enhanced if CXCR1 were constitutively expressed only within the endothelial cell niche. FLK1:CXCR1; FLK1:mCherry double transgenic animals had significantly increased CHT volume when compared with FLK1:mCherry single transgenic animals (1.1 +/- 0.05 x 106 vs 1.3 +/- 0.06 x 106 um3, p=0.02). These findings suggest a model whereby HSC/HSPCs actively participate in the remodeling of the endothelial niche via CXCR1/IL-8 in order to promote their own engraftment. Further, they suggest that CXCR1/IL-8 is a potential therapeutic target for enhancing HSC/HSPC engraftment in patients undergoing HSCT. Disclosures Zon: FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.


2011 ◽  
Vol 110 (2) ◽  
pp. 520-527 ◽  
Author(s):  
X. Lu ◽  
X. Guo ◽  
C. D. Wassall ◽  
M. D. Kemple ◽  
J. L. Unthank ◽  
...  

Although elevation of shear stress increases production of vascular reactive oxygen species (ROS), the role of ROS in chronic flow overload (CFO) has not been well investigated. We hypothesize that CFO increases ROS production mediated in part by NADPH oxidase, which leads to endothelial dysfunction. In six swine, CFO in carotid arteries was induced by contralateral ligation for 1 wk. In an additional group, six swine received apocynin (NADPH oxidase blocker and anti-oxidant) treatment in conjunction with CFO for 1 wk. The blood flow in carotid arteries increased from 189.2 ± 25.3 ml/min (control) to 369.6 ± 61.9 ml/min (CFO), and the arterial diameter increased by 8.6%. The expressions of endothelial nitric oxide synthase (eNOS), p22/p47phox, and NOX2/NOX4 were upregulated. ROS production increased threefold in response to CFO. The endothelium-dependent vasorelaxation was compromised in the CFO group. Treatment with apocynin significantly reduced ROS production in the vessel wall, preserved endothelial function, and inhibited expressions of p22/p47phox and NOX2/NOX4. Although the process of CFO remodeling to restore the wall shear stress has been thought of as a physiological response, the present data implicate NADPH oxidase-produced ROS and eNOS uncoupling in endothelial dysfunction at 1 wk of CFO.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 955-955
Author(s):  
Cristiane Maria de Souza ◽  
Carolina Lanaro ◽  
Irene Pereira dos Santos ◽  
Oladele Olatunya ◽  
Sara T Olalla Saad ◽  
...  

Abstract Extracellular vesicles (EVs) are submicron structures released in blood circulation by different cell types which have been found to be increased in sickle cell disease (SCD) and are associated with clinical complications. The most abundant EVs in SCD patients derive from platelets, endothelial cells, and red blood cells (RBCs) and EVs have been explored as biomarkers of clinical severity. Crizanlizumab is a monoclonal antibody against P-selectin, an adhesion molecule expressed in activated platelets and endothelial cells. P-selectin facilitates the formation of heterocellular aggregates and is implicated in the pathophysiology of vaso-occlusive episodes (VOEs) in SCD. This study aimed to investigate the circulating levels of EVs in patients with SCD on standard of care or treated with crizanlizumab. We collected peripheral blood samples from 20 adults with SCD (Non treated group: 7 patients on hydroxyurea treatment and 7 without it. Treated group: 6 patients undergoing treatment with crizanlizumab in combination with hydroxyurea). Patients received the last dose of crizanlizumab at least a month prior to the study. EVs were identified by lactadherin+calcein stain and quantified by flow cytometry to determine the immunophenotype of their parent cell (platelet, endothelial cell, and RBC, with CD41+; CD146+/CD45-; CD235+, respectively). EV quantification was calculated in number per ml of blood as previously described by our group (Olatunya et al., 2019). We found that patients on crizanlizumab had lower total circulating EV counts than patients not receiving the drug (62.670.000,00 ± 15.600.000,00 vs 13.100.000,00 ± 3.513.000,00/mL, respectively, p=0,0076). The difference was statistically significant in platelet-derived EVs levels (5.397.000,00 ± 953.875,00 vs 2.413.000,00 ± 745.165,00/mL, p=0,0169), but not in endothelium-derived or RBC-derived EVs (345714 ± 101817 vs 220000 ± 64291, and 2.189.000,00 ± 1.648.000,00 vs 1.013.000,00 ± 572775, respectively). Crizanlizumab therapy has been shown to reduce the incidence of VOEs in SCD. EVs have been recognized as bio-effectors involved in VOEs, contributing to a hypercoagulable state, chronic inflammation, and endothelial damage. Our findings show an association between the use of crizanlizumab and lower EV levels, particularly of the platelet-derived type. While the anti-P-selectin activity of crizanlizumab could be expected to help remove platelets from circulation, clinical studies have not reported a reduction in platelet counts in patients treated with crizanlizumab. Therefore, we speculate that crizanlizumab may decrease the release of EV by activated platelets, reduce platelet activation, or contribute to EV removal from circulation. Our findings suggest that crizanlizumab therapy may modulate EV levels in the plasma of SCD patients and provide, for the first time, data to support exploring the use of extracellular vesicles as biomarkers to monitor the clinical response to this drug in patients. Further studies on EV expression of P-selectin and how crizanlizumab interacts with EVs and platelets may help clarify this particular effect of this drug. Disclosures Benites: Novartis: Honoraria. Fertrin: Sanofi Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Costa: Novartis: Consultancy.


Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 18-18
Author(s):  
David Muench ◽  
Kyle Ferchen ◽  
Giang Pham ◽  
Somchai Chutipongtanate ◽  
Pankaj Dwivedi ◽  
...  

Abstract Efforts to understand the genomic impact of human-disease-relevant genetic lesions and how they disrupt the normal sequence of cell-state transitions is hampered by a lack of defined hierarchical cellular states and corresponding networks of regulatory genes (transcription factors). Severe congenital neutropenia (SCN) patients display inherited and de novo mutations in Growth factor independent-1 (GFI1), which encodes a zinc-finger transcription factor. We identified known (e.g. N382S in zinc finger 5) and novel GFI1 sequence changes in SCN patients, then used lentiviral mediated expression to functionally evaluate them. GFI1-N382S, GFI1-K403R and GFI1-R412X mutations (in zinc finger 6) significantly elevated the expression of the Gfi1 target gene, Irf8. We generated mice with these patient-derived SCN-associated mutations in the murine Gfi1 locus. Neonatal and adult Gfi1N382S/- and Gfi1R412X/-mice are neutropenic, but Gfi1K403R/- mice have normal steady-state neutrophil levels. The resulting steady-state dysgranulopoiesis in adult mice was further pronounced in neonates. We noted that Gfi1R412X/-mice accumulate less Gfi1 protein than Gfi1+/+, while Gfi1R412X/R412Xhomozygous alleles genetically rescued both the hypomorphic protein defect and substantially restored neutrophil numbers (though not to normal). In contrast, functional challenge with neutrophil-dependent pathogens in vivo revealed a broad susceptibility for all Gfi1-mutant mice. To determine the underlying mechanism of neutropenia and immune defects, we first used novel flow cytometry analyses and Fluidigm C1 single cell RNA-Seq to establish the successive genomic states encompassing normal granulocyte specification and commitment. Independent CITE-Seq/10x sequencing analysis provided direct correlation between flow cytometry populations and genomic information, while also establishing the trajectory through genomic states traversed during terminal granulopoiesis. Next, using a novel bioinformatics algorithm (cellHarmony) we assigned Gfi1-mutant cells to their respective wild-type cell states and then determined differential gene expression. We find few genes deregulated across granulopoiesis, and that the bulk of transcriptional impact on Gfi1 target genes is specific to successive granulopoietic cell states. These insights facilitated Gfi1R412X/- Irf8+/-genetic rescue of granulocytic specification, but not post-commitment defects. We noted that a portion of Gfi1R412X/-gene deregulation unrepaired by genetic rescue was enriched for chromosome organization, proteolysis, and innate immune effectors. Electron microscopy revealed uncondensed chromatin in mature Gfi1R412X/-neutrophils while SWATH proteomics identified a loss of neutrophil granule proteins and members of the NADPH oxidase complex (potentially linking SCN with chronic granulomatous disease genes). To this end, we functionally validated impaired NADPH oxidase complex function in neutrophils from Gfi1-mutant mice. We noted Gfi1 mutant mice have consistently elevated levels of granulocyte colony stimulating factor (but not other cytokines), and so we extended our analysis of oxidative burst to GCSF-rescued human SCN patients to find profound defects; underscoring the inability of genetic or cytokine rescued specification to resolve post-commitment defects. We illustrate a work flow that can be broadly applied to molecularly dissect translationally relevant mouse models of disease, and underscores the necessity of evaluating mutations within the context of relevant cell states. Disclosures Dwivedi: Abbvie: Employment. Myers:Bellicum Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Nazor:BioLegend: Employment.


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