Identification of a Novel CEBPE Enhancer Essential for Granulocytic Differentiation

CEBPE is a member of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors essential for granulocytic differentiation. CEBPE is expressed in a stage-specific manner during myeloid differentiation and regulates transition from the promyelocyte to the myelocyte stage. It is essential for secondary and tertiary granule formation in granulocytes. We and others found germline mutations of the CEBPE gene in patients with neutrophil-specific granule deficiency. Their neutrophils display atypical bilobed nuclei, lack expression of granule proteins and these patients often have frequent bacterial infections. Cebpe knock-out mice resemble this clinical phenotype displaying a block in terminal differentiation and absence of secondary granule proteins. Given the tissue specific expression of CEBPE, we were interested in identifying genomic regions and factors that could regulate its lineage specific expression. Our CEBPE ChIP-seq in murine bone marrow cells showed binding of CEBPE to a region 6kb upstream of Cebpe gene. Chromosome conformation capture-on-chip (4C-seq) demonstrated an interaction between this putative regulatory element (6kb upstream region) and the core promoter of Cebpe. Analysis of available DNase-seq data sets revealed that the region bound by CEBPE displayed an open chromatin only in myeloid lineage cells. Further examination revealed binding of a myriad of hematopoietic transcription factors to the +6kb enhancer in HPC-7 (hematopoietic progenitor cells) and in 416B (myeloid progenitor cells), indicating that this region/enhancer might regulate the expression of CEBPE. Targeting of this region using dCas9-KRAB in murine 32D cells caused significant downregulation of RNA and protein levels of CEBPE compared to control cells. These targeted cells also exhibited impaired granulocytic differentiation with lower transcript levels of secondary granule proteins (Ltf and Ngp). To investigate further the role of the +6kb enhancer region in myelopoiesis, mice were generated with deletion of this region using CRISPR/Cas9 technology. Germ line deletion of the +6kb enhancer resulted in reduced levels of CEBPE and its target genes, accompanied by a severe block in granulocytic differentiation and a complete absence of CD11b+/Gr1hi population. This phenotype is nearly identical to our Cebpe KO mice. In summary, we have identified a novel enhancer crucial for regulating Cebpe, and required for normal granulocytic differentiation. Disclosures No relevant conflicts of interest to declare.

Yersinia pseudotuberculosis Blocks Neutrophil Degranulation

Neutrophils are essential components of immunity and are rapidly recruited to infected or injured tissue. Upon their activation, neutrophils release granules to the cell's exterior, through a process called degranulation. These granules contain proteins with antimicrobial properties that help combat infection. The enteropathogenic bacteriumYersinia pseudotuberculosissuccessfully persists as an extracellular bacterium during infection by virtue of its translocation of virulence effectors (Yersiniaouter proteins [Yops]) that act in the cytosol of host immune cells to subvert phagocytosis and proinflammatory responses. Here, we investigated the effect ofY. pseudotuberculosison neutrophil degranulation upon cell contact. We found that virulentY. pseudotuberculosiswas able to prevent secondary granule release. The blocking effect was general, as the release of primary and tertiary granules was also reduced. Degranulation of secondary granules was also blocked in primed neutrophils, suggesting that this mechanism could be an important element of immune evasion. Further, wild-type bacteria conferred a transient block on neutrophils that prevented their degranulation upon contact with plasmid-cured, avirulentY. pseudotuberculosisandEscherichia coli. Detailed analyses showed that the block was strictly dependent on the cooperative actions of the two antiphagocytic effectors, YopE and YopH, suggesting that the neutrophil target structures constituting signaling molecules needed to initiate both phagocytosis and general degranulation. Thus, via these virulence effectors,Yersiniacan impair several mechanisms of the neutrophil's antimicrobial arsenal, which underscores the power of its virulence effector machinery.

miRNA-130a regulates C/EBP-ε expression during granulopoiesis

Key Points miRNA-130a is expressed in myeloblasts and promyelocytes and inhibits translation of CEBPE mRNA encoding transcription factor C/EBP-ε. Regulation of CEBPE mRNA by miRNA-130a is required for timed expression of secondary granule proteins and cell cycle exit.

Thiocyanate Blocks Peroxidase-Dependent Extracellular Trap (ET) Formation By PMN and Eosinophils: Heme Is a Potent New Agonist For The ET Pathway

PMN and eosinophils (EO) activated by physiologic agonist such as LPS, C5a, GM-CSF and IL-5 form extracellular traps (ET) comprised of extracellular DNA, histones, and active secondary granule constituents that retain microbicidal capacity and thus contribute to host defense but also participate in the pathogenesis of sepsis, microangiopathy, vasculitis, asthma and thrombosis. Previous studies have implicated superoxide, H2O2 and myeloperoxidase (MPO) in PMN ET formation, which reflects a novel death pathway dubbed “ETosis.” Thiocyanate (SCN-) is the principle physiologic substrate for eosinophil peroxidase (EPO) and a major substrate for MPO. Because previous studies of ET were all performed in the absence of SCN- and HOSCN, the product of SCN- peroxidation, is a weak, sulfhydryl-specific oxidant markedly less toxic than HOCl or HOBr, we hypothesized SCN- blocks PMN and EO ET formation. We also hypothesized that heme, elevated levels of which occur in intravascular hemolytic states such as sickle cell disease, induces PMN ETosis because both LPS and heme signal by engaging TLR4. We assessed ET formation by human PMN stimulated with either GM-CSF + C5a or 10 µM heme in RPMI one hour using glass slide-attached leukocytes with both cell impermeant (Sytox Orange) and permeant (Syto13) DNA stains and IF localization of secondary granule proteins using confocal microscopy. ET formation in response to to both stimuli was 5-20% of PMN in stimulated cells (vs. 0% in control). The NADPH oxidase inhibitor DPI, MPO inhibitor 4-ABAH and 1 mM SCN- all diminished ET formation by >80% in response to both stimuli. Heme-dependent ET formation was inhibited 80% by the TLR4 antagonist TAK242 (see figure below in which intracellular DNA stains green and extracellular and membrane-compromised PMN DNA stains orange). PMA-induced PMN ET formation requires singlet O2 resulting from the secondary reaction of HOCl with excess H2O2. In PMN stimulated with GM-CSF + C5a, the singlet O2 scavenger edaravone (10 µM) inhibited ETs by 80%. ETs also formed in 4-8% of EOs stimulated by IL-5 + C5a, vs. 0% in control EO and 22.5% in unstimulated EO supplemented with the alternative EPO substrate 1 mM Br-. The EPO inhibitor resorcinol decreased by >90% EO ET formation stimulated with by IL-5 + C5a or by adding Br-, as did addition of SCN- or edaravone. These studies show that both PMN and EO ETosis depend upon MPO- and EPO- generated oxidants, respectively, and the presence of an alternative physiologic substrate, SCN-, markedly antagonizes ETosis. We propose that SCN- inhibits ET formation by generating HOSCN that reacts with H2O2 to yield cyanate, not singlet O2. In addition, we also demonstrate that heme is a potent inducer of PMN ETosis through a mechanism dependent upon TLR4, NADPH oxidase and MPO, raising the possibility that this pathway may directly promote the inflammation and thrombosis that contribute of the pathogenesis of sickle cell disease. Based on these findings, we speculate that dietary augmentation of SCN- to normal or supranormal levels might have clinically beneficial therapeutic effects in a variety of inflammatory states, including sickle cell disease. Disclosures: No relevant conflicts of interest to declare.

Expression of the secondary granule proteins major basic protein 1 (MBP-1) and eosinophil peroxidase (EPX) is required for eosinophilopoiesis in mice

Key Points The loss of the two most abundant eosinophil granule proteins disrupts the production of blood eosinophils from marrow progenitors. Knockout animals deficient for both MBP-1 and EPX represent a novel strain of mice with a specific and congenital loss of eosinophils.

Impaired Neutrophil Function in 24p3 Null Mice Contributes to Enhanced Susceptibility to Bacterial Infections

Abstract 833 Lipocalin 24p3 (24p3) is a neutrophil secondary granule protein. 24p3 is also a siderocalin, which binds several bacterial siderophores. It was therefore proposed that synthesis and secretion of 24p3 by stimulated macrophages or release of 24p3 upon neutrophil degranulation sequesters iron-laden siderophores to attenuate bacterial growth. Accordingly, 24p3-deficient mice are susceptible to bacterial pathogens whose siderophores are chelated by 24p3. Specific granule deficiency (SGD) is a rare congenital disorder characterized by complete absence of proteins of secondary granules. Neutrophils from SGD patients lack normal functions and are prone to bacterial infections, but the importance of 24p3 for neutrophil dysfunction in SGD is not known. Here we show that neutrophils of 24p3−/− mice are defective for many neutrophil functions. Specifically, neutrophils in 24p3−/− mice do not extravasate to sites of infection and are defective for chemotaxis. A transcriptome analysis revealed that genes that control cytoskeletal reorganization are selectively suppressed in 24p3−/− neutrophils. Additionally, small regulatory RNAs (miRNAs) that control upstream regulators of cytoskeletal proteins are also increased in 24p3−/− neutrophils. Further, 24p3−/− neutrophils failed to phagocytose bacteria, which may account for the enhanced sensitivity of 24p3−/− mice to both intracellular (Listeria monocytogenes) and extracellular (Candida albicans, Staphylococcus aureus) pathogens. Interestingly, Listeria does not secrete siderophores and additionally, the siderophore secreted by Candida is not sequestered by 24p3. Therefore, the heightened sensitivity of 24p3−/− mice to these pathogens is not due to sequestration of siderophores to limit the iron availability but as a consequence of impaired neutrophil functions. Disclosures: No relevant conflicts of interest to declare.

Endotoxin Priming of Neutrophils Requires Endocytosis and NADPH Oxidase-dependent Endosomal Reactive Oxygen Species

NADPH oxidase 2 (Nox2)-generated reactive oxygen species (ROS) are critical for neutrophil (polymorphonuclear leukocyte (PMN)) microbicidal function. Nox2 also plays a role in intracellular signaling, but the site of oxidase assembly is unknown. It has been proposed to occur on secondary granules. We previously demonstrated that intracellular NADPH oxidase-derived ROS production is required for endotoxin priming. We hypothesized that endotoxin drives Nox2 assembly on endosomes. Endotoxin induced ROS generation within an endosomal compartment as quantified by flow cytometry (dihydrorhodamine 123 and Oxyburst Green). Inhibition of endocytosis by the dynamin-II inhibitor Dynasore blocked endocytosis of dextran, intracellular generation of ROS, and priming of PMN by endotoxin. Confocal microscopy demonstrated a ROS-containing endosomal compartment that co-labeled with gp91phox, p40phox, p67phox, and Rab5, but not with the secondary granule marker CD66b. To further characterize this compartment, PMNs were fractionated by nitrogen cavitation and differential centrifugation, followed by free flow electrophoresis. Specific subfractions made superoxide in the presence of NADPH by cell-free assay (cytochrome c). Subfraction content of membrane and cytosolic subunits of Nox2 correlated with ROS production. Following priming, there was a shift in the light membrane subfractions where ROS production was highest. CD66b was not mobilized from the secondary granule compartment. These data demonstrate a novel, nonphagosomal intracellular site for Nox2 assembly. This compartment is endocytic in origin and is required for PMN priming by endotoxin.

Mouse Eosinophils Possess Potent Antibacterial Properties In Vivo

ABSTRACT Eosinophils are best known as the predominant cellular infiltrate associated with asthma and parasitic infections. Recently, numerous studies have documented the presence of Toll-like receptors (TLRs) on the surfaces of eosinophils, suggesting that these leukocytes may participate in the recognition and killing of viruses and bacteria. However, the significance of this role in the innate immune response to bacterial infection is largely unknown. Here we report a novel role for eosinophils as antibacterial defenders in the host response. Isolated mouse eosinophils possessed antipseudomonal properties in vitro. In vivo, interleukin-5 transgenic mice, which have profound eosinophilia, demonstrated improved clearance of Pseudomonas aeruginosa introduced into the peritoneal cavity. The findings of improved bacterial clearance following adoptive transfer of eosinophils, and impaired bacterial clearance in mice with a congenital eosinophil deficiency, established that this effect was eosinophil specific. The data presented also demonstrate that eosinophils mediate this antibacterial effect in part through the release of cationic secondary granule proteins. Specifically, isolated eosinophil granules had antibacterial properties in vitro, and administration of eosinophil granule extracts significantly improved bacterial clearance in vivo. These data suggest a potent yet underappreciated antibacterial role for eosinophils in vivo, specifically for eosinophil granules. Moreover, the data suggest that the administration of eosinophil-derived products may represent a viable adjuvant therapy for septic or bacteremic patients in the intensive care unit.