ALBERT

Description: CRISPR/Cas9-based genome editing has yet to be reported in species of the Platyhelminthes. We tested this approach by targeting omega-1 (ω1) of Schistosoma mansoni as proof of principle. This secreted ribonuclease is crucial for Th2 polarization and granuloma formation. Schistosome eggs were exposed to Cas9 complexed with guide RNA complementary to ω1 by electroporation or by transduction with lentiviral particles. Some eggs were also transfected with a single stranded donor template. Sequences of amplicons from gene-edited parasites exhibited Cas9-catalyzed mutations including homology directed repaired alleles, and other analyses revealed depletion of ω1 transcripts and the ribonuclease. Gene-edited eggs failed to polarize Th2 cytokine responses in macrophage/T-cell co-cultures, while the volume of pulmonary granulomas surrounding ω1-mutated eggs following tail-vein injection into mice was vastly reduced. Knock-out of ω1 and the diminished levels of these cytokines following exposure showcase the novel application of programmed gene editing for functional genomics in schistosomes.

Description: Abstract 4228 Human erythropoiesis is a dynamic complex multistep process that involves differentiation of early erythroid progenitors to enucleated red blood cells. Importantly, erythroid differentiation involves lineage-specific activation and restriction of gene expression. However, the mechanisms that play a role in erythropoiesis still remain incompletely understood. We previously demonstrated that erythropoietin-stimulated clone-1 (EP1), which is selectively expressed in normal human erythroid lineage cells shares 99.5% identity with the malignant fibrous histiocytoma amplified sequence 1(MFHAS1 or MASL1). MASL1 is an important oncogene that is highly expressed in malignant fibrous histiocytomas (MFH). MASL1 protein has several domains; ras, three leucine zipper, ATP/GTP-binding site and leucine-rich tandem repeat motif which are important structural or functional elements for interactions among proteins related to the cell cycle. However, the function of the MASL1 gene in erythropoiesis has not been studied. We hypothesized that MASL1 gene plays a role in the erythroid differentiation, and used a human liquid erythroid culture system to explore this concept. MASL1 mRNA and protein expression levels were significantly increased during the erythroid differentiation of CD34+ cells following EPO treatment. Conversely, small interfering RNA-mediated knock down of MASL1 in CD34+ cells resulted in a reduction of a double positive of transferrin receptor and glycophorin A (GPA) (14.1 ± 4.7%) when compared with mock (77.9 ± 4.4%) or control vector-transfected CD34+ cells (76.7 ± 8.8%) at day 14. May-Grunwald-Giemsa staining confirmed a phenotype of MASL1 knockdown CD34+ cells that most of cells were pro- or basophilic erythroblasts. Western blotting also showed a significant decrease in hemoglobin protein levels in MASL1 knockdown CD34+ cells. In addition, MASL1-knockdown in CD34+ cells demonstrated an interruption of Raf/MEK/ERK signaling pathway. Inhibition assay of SH3 domain of Son of Sevenless (SOS) which is an upstream adapter protein in EPO-induced erythroid differentiation, confirmed that a suppression of MASL1 reduced the phosphorylation of Raf/MEK/ERK kinases and delays erythroid differentiation of Epo-induced CD34+ cells. Taken together, our study provides novel insights into MASL1-regulated erythropoiesis through the Raf/MEK/ERK signaling pathway. Disclosures: No relevant conflicts of interest to declare.

Description: Current gene therapy approaches for treatment of hemoglobinopathies involve viral transduction of hematopoietic stem cells with "antisickling" globin genes. Hemoglobin A2 (HbA2, α2δ2), expressed at a low level due to the lack of Eklf binding motif in its promoter region, is fully functional and could be a valid substitute for hemoglobin A in β-thalassemia, as well as an "anti-sickling" agent in sickle cell disease. We have previously demonstrated that two Eklf-GATA1 fusion proteins, which were recruited to the GATA1 binding motif at the δ-globin promoter, can significantly activate δ-globin expression in K562 cells and hematopoietic stem CD34+ cells. Here, we report that enforced expression of Eklf-GATA1 fusion protein in sickle trait CD34+ cells significantly increased δ-globin expression, as determined by quantitative PCR and HPLC. Upon deoxygenation, the percentage of sickling cells was lower in Eklf-GATA1-transduced red blood cells as compared with mock-transduced cells. By a series of flow cytometry analyses, which includes BRDU incorporation assay, CD71/GPA, and thiazole orange staining, we found that erythroid cells proliferation, differentiation and enucleation were not affected by Eklf-GATA1 expression. To assess the potential off-target effects of our fusion protein constructs, we analyzed differentially expressed genes (and proteins) in vector-only and Eklf-GATA-1 transduced-CD34+ cells by microarray and proteomic changes by liquid chromatography-mass spectrometry (LC-MS). We found that over-expression of Eklf-GATA1 resulted in a less than 2-fold change in the gene expression profile related to bone marrow hematopoiesis, and there were no significant changes that were detected in proteomic profiling. These results indicate that these fusion constructs could be a valuable genetic therapeutic tool for hemoglobinopathies, and warrant further preclinical study and evaluation. Disclosures No relevant conflicts of interest to declare.

Description: Salmonella is an intracellular bacterial pathogen that replicates within membrane-bound compartment and alters host iron metabolism for its own survival. Persistent survival and replication within phagocytes is central to the pathogenesis of Salmonella infections. Macrophages play a critical role in regulating iron metabolism for securing body iron sufficiency and controlling the availability of iron for intracellular proliferation of pathogens. However, the relationship of Salmonella-induced changes of macrophage iron metabolism to the survival and replication mechanism of this pathogen within macrophages remains poorly understood. Thus, it is critical to identify the host factors involved in the intracellular survival and replication of Salmonella in order to design more-efficient antimicrobial therapeutics. Glia maturation factor gamma (GMFG), a novel regulator of the actin-related protein-2/3 (Arp2/3) complex, is predominantly expressed in inflammatory cells. We have previously demonstrated that GMFG negatively regulate TLR4-induced proinflammatory signaling, but its function in macrophage response to intracellular bacteria infection remains unclear. In this study, we investigated the role of GMFG in Salmonella-infected murine macrophages by using small-interfering RNA (RNAi) techniques to knockdown GMFG. We found that knockdown of GMFG significantly enhanced the numbers of intracellular Salmonella growth (〉3-fold, p〈 0.008) at 24 hr postinfection compared with control siRNA transfected Raw264.7 macrophages. However, there was no significant difference in growth numbers of bacteria observed at 4 hr postinfection, indicating that GMFG does not influence bacterial phagocytosis. Immunofluorescence microscopy also revealed an accumulation of Salmonella, in GMFG knockdown macrophages at 24 hr postinfection. Knockdown of GMFG results in marked decreased the iron exporter ferroportin protein levels and increased iron storage ferritin-L protein levels in Raw264.7 macrophages. Further, the intracellular iron content was elevated in GMFG-knockdown macrophages compared with control macrophages (1.9-fold, p〈 0.05). These observations indicate that regulatory impact of GMFG in Salmonella intracellular growth may be through modulation of macrophage iron metabolism. Moreover, consistent with previous studies, we found that Raw264.7 macrophage infections with Salmonella increase the expression of the iron transporter ferroportin and ferritin, indicating this is the host defense strategy against infection with intracellular microbes by limiting their access to iron. Although there was no marked altered in GMFG protein level after 24hr infection with Salmonella, GMFG knockdown macrophage infected with Salmonella displayed increased the intracellular iron content and iron storage protein ferritin compared with control macrophages. Further analysis of cytokines expression in Salmonella-infected GMFG-knockdown macrophage revealed enhanced the proinflammatory TNF-alpha mRNA (1.86-fold, p

Description: Recruitment of monocytes is essential for effective control and clearance of invading pathogens by migrating to the sites of infection, but recruited monocytes also contribute to the pathogenesis of chronic inflammatory and degenerative diseases, such as rheumatoid arthritis, multiple sclerosis, atherosclerosis and cancer. Thus, understanding the mechanisms controlling monocytes migration within different environments is of paramount importance. Although it is clear that adhesion signaling via integrin receptors and the surrounding ECM play a significant role in regulating migration of monocytes to site of inflammation, the underlying cellular and molecular mechanisms responsible for these process is still not fully characterized. Defining the molecular circuits through which integrins regulate monocytes motility is therefore important for gaining a better understanding of monocytes function. Glia maturation factor gamma (GMFG), a novel ADF/cofilin superfamily protein that is predominantly expressed in inflammatory cells, has been implicated in regulating actin reorganization. We have previously demonstrated that GMFG plays a role in regulating neutrophil chemotaxis and migration. We now examine whether GMFG has similar effects on monocytes and the cellular mechanism for these effects by using small-interfering RNA to knockdown GMFG in human primary monocytes. Knockdown of endogenous GMFG results in significantly reduced (220.6 ± 9.4 to 89.0 ± 3.2, p

Description: Key Points Hydroxyurea activates nuclear factor–κB to transcriptionally upregulate SAR1. SAR1, in turn, activates γ-globin expression through the Giα/JNK/Jun pathway.

Description: Hemolysis as a suggested cause of hyperuricemia is based upon the fact that red blood cells contain uric acid. The development of hyperuricemia also generates from an increased synthesis of nucleic acids occurring as part of the erythropoietic response to hemolysis in hemoglobin disorders such as sickle cell anemia, α-thalassemia, and β-thalassemia. In addition, multiple genome-wide association studies (GWAS) have reported significant association between uric acid levels and specific genomic loci. However, the mechanism of hyperuricemia still remains controversial and it is also unknown whether African Americans have higher prevalence of hyperuricemia due to genetic vs environmental risk factors. Here, we used joint admixture mapping and association testing to identify genetic variants associated with serum uric acid levels in African American. Interestingly, we detected 6 SNPs (rs2855125, rs2855126, rs11036415, rs11036496, rs4320977, and rs4348933) in an intergenic region of the β–globin cluster on chromosome 11 are associated with high levels of serum uric acid in populations of African ancestry. Next, we explored the potential regulatory role of intergenic SNPs associated with hyperuricemia using luciferase reporter gene activity assays and electrophoretic mobility shift assays (EMSA). Each SNP-containing DNA fragment was amplified by PCR using human genomic DNA and inserted into a firefly luciferase reporter vector, pGL3-basic vector. 293T or K562 cells were co-transfected with these constructs and a Renilla luciferase vector to control for transfection efficiency. Expression of firefly luciferase driven by each SNP-containing DNA fragment was measured by a dual luciferase reporter assay and normalized by Renilla luciferase expression. SNPs rs2855126, rs11036496 and rs4348933 on chromosome 11 had significantly greater expression levels of firefly luciferase than pGL3-basic-transfected cells in both 293T and K562 cells. Of these, the SNP rs2855126 ancestral C allele (associated with higher serum uric acid levels) showed significantly higher luciferase activity than the derived G allele. Functionally, the luciferase activities from these constructs were determined to be very similar in both cell lines used. Alleles altering expression were further assessed for binding of nuclear extracted proteins by EMSA. We found specific gel shift bands for SNPs rs2855126, rs11036496, and rs4348933, suggesting these SNPs are situated in the binding site of potential transcription factors. These data provide new insights into the potential contribution of imbalanced β-globin gene expression to hyperuricemia. Disclosures No relevant conflicts of interest to declare.

Description: Macrophages play a key role at the crossroad of iron metabolism and immune function. They store and recycle iron derived from the phagocytosis of senescent erythrocytes. Macrophages iron homeostasis is coupled to their remarkable heterogeneity and functional plasticity. It is well known that the macrophage polarization process dictates expression profiles of genes involved in iron metabolism. M1 macrophages are characterized by increased iron retention, whereas, M2 macrophages showed increased iron recycling. However, the molecular mechanisms underlying iron metabolism link to regulation of macrophage-polarized phenotype are not fully understood. Glia maturation factor gamma (GMFG), a novel regulator of the actin-related protein-2/3 (Arp2/3) complex, is predominantly expressed in inflammatory cells. We have previously found that GMFG mediated macrophage resistance to Salmonella infection, but its function in iron metabolism and macrophage phenotype remains unclear. In this study, we explored the important role of GMFG in the regulation of iron metabolism and macrophages phenotypes interlinked. We found that GMFG expression was downregulated in a dose-dependent manner in murine bone marrow-derived macrophages (BMDM) and RAW-264.7 cells by treated with iron or heme. Immunoblotting analysis demonstrated that knockdown of GMFG in BMDM and RAW-264.7 cells lead to remarkable increased the protein levels of ferroportin (Fpn), transferrin receptor 1 (TfR1), as well as heme oxygenase 1 (HO-1), whereas decreased the ferritin light chain 1 (FtL1) compared with control siRNA transfected BMDM or Raw264.7 cells. Knockdown of GMFG display higher iron export capacity and elevated intracellular labile iron pool (LIP) compared with control macrophages. These results suggest that GMFG is the crucial regulator in macrophages iron metabolism because its downregulation caused an alteration in iron-handling proteins similar to IL-4 induced M2 polarization phenotype. Quantitative PCR analysis showed that M2 alternative activation markers Arg1, Mrc1, and Ym1 were noted to be induced in GMFG knockdown macrophages in the absence of M2-induceer cytokine treatment, confirming the skewing of these macrophages toward M2 alternative activation. Moreover, treatment of GMFG-knockdown BMDM or RAW-264.7 cells with Th2 cytokines IL-4 or IL-13 markedly enhanced the induction of several genes characteristic of M2 alternative activation, including Arg1, Mrc1, and Ym1 compared with control macrophages. Furthermore, M2 skewing was confirmed by the enhancement of the IL-4-induced Arg1 protein levels in GMFG knockdown macrophages relative to control macrophages by immunoblotting analysis. Interestingly, GMFG knockdown macrophages further markedly enhanced the IL-4-induced protein levels of HO-1 and TfR1 in M2 phenotypes, but there was no marked altered in protein level of Fpn or FtL1 compared with control macrophages. These results indicated that knockdown of GMFG might enhance the M2 phenotypes through modulation of HO-1. Finally, we observed that GMFG knockdown macrophages showed more accumulation of transcriptional factor Nrf2 in nuclear without alternation of its transcriptional expression levels compared with control macrophages, suggesting that downregulation of GMFG skewing macrophages toward a M2 phenotype might be through regulation of Nrf2-mediated HO-1 expression. Our results indicate that GMFG plays an important role in the regulation of M2 alternative activation through modulation of iron metabolism and act as a negative feedback loop in macrophages. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3598 Poster Board III-535 Neutrophils are the most abundant leukocytes in the circulation and provide a primary innate immune defense function against bacterial pathogens before the development of a specific immune response. Activation of TLR4 signaling pathways by lipopolysaccharide (LPS) leads to the production of a broad array of cytokines and mediators that coordinate the immune response. The TLR4-mediated inflammatory response is tightly regulated, since excessive activation of TLR4 may induce a variety of inflammatory diseases, so the negative regulation of TLR-triggered inflammatory response attracts much attention. Glia maturation factor gamma (GMFG), a novel factor in actin cytoskeleton reorganization, is preferentially expressed in inflammatory cells, but its function in leucocytes is undefined. In this study, we investigated whether GMFG participates in the molecular events underlying the inflammatory reaction and how GMFG mediated neutrophils inflammatory response to LPS. We show here that small interfering RNA-mediated knockdown of GMFG expression in leukocytes exhibited greater than 80% inhibition of the endogenous expression levels of proinflammatory cytokines and chemokines, including TNF-alpha, IL-1beta, IL-6, and IL-8 using quantitative RT-PCR. These decreased cytokine productions could be partially restored by stimulation with TLR4 agonist LPS, but exhibit significantly lower level in silencing of GMFG cells compared to non-targeting silencing control cells (inhibition by 71%+/−3.9% of TNF-alpha, 68%+/−6.9% of IL-1beta, 50%+/−4.3% of IL-6 and 40%+/−3.1% of IL-8). Knockdown of GMFG expression results in decreased phospho-p38 MAPK levels as well as suppressed NF-kappaB by Western blot analysis. These data suggest that endogenous expression of GMFG is required for basal cytokine and chemokine response and their induced response to LPS stimulation. Moreover, immunofluorescence analysis revealed that gene silencing of GMFG inhibited LPS-induced nuclear translocation of NF-kappaB in myeloid HL-60 and THP-1 cells. In contrast, transient overexpression of GMFG significantly enhanced endogenous level of proinflammatory cytokines by 1.8∼2.2 fold, phosphor-p38 MAPK and nuclear translocation of NF-kappaB in the absence of LPS stimulation. These results suggest that GMFG-mediated regulation of inflammatory cytokines occurs through activation of p38 MAPK and NF-kappaB signaling pathway. Taken together, our findings identify GMFG as a novel factor that participates in the regulation of TLR4-mediated control of cytokine and chemokine expression through the p38 MAPK and NF-kappaB pathway. These data, if confirmed in in vivo experiments, implicates GMFG as a potential target for therapeutic intervention in neutrophil-mediated autoimmune disease. Disclosures: No relevant conflicts of interest to declare.