- Email: [email protected]
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Abstract / Cytokine 63 (2013) 243–314 66 Signal-specific, genome-wide regulation of nuclear factor-kappa B activation by epidermal growth factor and interleukin-1 Josephine K.T. Dermawan a,b, George R Stark b, a Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, USA, b Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Epidermal Growth Factor Receptor (EGFR) and Nuclear Factor kappa B (NF-jB) both play key roles in promoting tumorigenesis in a variety of cancers but their connection is not yet well understood. We have used Western assays, transcription analysis of known NF-jB target genes, and chromatin immunopreciptiation (ChIP) assays to show that EGF activates NF-jB-dependent signaling robustly, through I kappa B (IjB) and IjB kinase (IKK) phosphorylation. We then studied how NF-jB-dependent transcription is regulated differentially by EGFR, compared to the Interleukin-1 receptor (IL-1R), a classical NF-jB activator. Our preliminary global analysis of NF-jB promoter enrichment of EGF- and IL-1-activated genes leads us to propose that EGF induces distinct NF-jB targets that are not induced by IL-1. We are currently testing this hypothesis by global ChIP-seq and RNA-seq analyses, to identify target genes that are differentially activated by the two signals and have identified a novel set of NFjB-dependent genes that are induced by EGF but not by IL-1. We will investigate the underlying mechanisms of differential NF-jB gene regulation by analyzing post-translational modifications of NF-jB and induction of specific transcriptional co-regulators that are activated differentially by the two signals. We expect that this work will lead to a better understanding of how EGF-dependent pathways contribute to NF-jB-dependent gene expression in normal and cancer cells, and we hope that it leads to improved treatment of EGFR-driven cancers, in which NF-jB is known to play a key role in mediating resistance to therapy. http://dx.doi.org/10.1016/j.cyto.2013.06.069
67 Assessment of b2-adrenergic receptors on leukocyte subpopulations using imaging cytometry Alex P. Di Battista a, Shawn G. Rhind b, Maria Y. Shiu a, Andrew Baker c, Ruth A. Lanius d, Donald J. Richardson d,e, Rakesh Jetly f, a Graduate Program, University of Toronto, Toronto, ON, Canada, b Defence Research & Development (DRDC) Canada Toronto, Toronto, ON, Canada, c Dept of Critical Care, Trauma & Neurosurgery Program, St. Michael’s Hospital, University of Toronto, Toronto, ON, Canada, d Dept of Psychiatry, Schulich School of Medicine, Western University, London, ON, Canada, e Parkwood Operational Stress Injury Clinic, St. Joseph’s Health Care, London, ON, Canada, f Canadian Forces Health Services, Directorate of Mental Health, Dept of National Defence, Canada Introduction: b2-Adrenergic receptors (AR) are the primary subtype of adrenergic receptors found on blood leukocytes, and are recognized as key mediators of interaction between the sympathetic nervous system and immune system. This biological pathway is central in understanding neuroimmune signaling in a number of disease states, spanning mental disorders to traumatic brain injury. Traditionally, b2-ARs have been primarily assessed using radio-ligand binding assays, where results are limited in their ability to provide in depth cell-signaling information on specific leukocyte subtypes. As a result, current knowledge of b2-AR distribution, reactivity and potential implications in disease pathology or drug therapy are limited. This study evaluated b2-AR expression on circulating leukocyte subsets with various commercially available antibodies (Abs) using imaging cytometry. Methods: Whole blood was labeled with CD14 and CD16 surface markers for leukocyte subtype phenotyping. Four different b2-AR antibodies, sc-81578 – FITC (Santa Cruz), sc-81577 – PE (Santa Cruz), PA5-19649 (Pierce Biotechnology, USA) and MCA2784 (Serotec, USA) were assessed. DAPI stain was added to all samples to determine nuclear morphology. Based on cell area and aspect ratio, 100,000 single-cell events were acquired on an ImageStreamX Mark IIÒ Cytometer (Amnis, Seattle, WA). Monocytes and lymphocytes were identified using side scatter (SSC) and CD14 expression. Neutrophils and eosinophils were identified using SSC and CD16. Results: b2 AR expression levels on leukocyte subpopulations in whole blood were detectable using image cytometery. Leukocyte subsets identified according to surface markers, size, granularity, cell and nuclear morphology displayed varying amounts of b2-AR expression. Furthermore, the PA5-19649 Ab performed best in terms of binding specificity and signal strength. Conclusion: Imaging cytometry is a valuable technique for evaluating b2-ARs on leukocyte subsets. The capability of this technology to provide in-depth, high throughput cell signaling information offers an attractive alternative to current methods used in the assessment of adrenergic receptors. http://dx.doi.org/10.1016/j.cyto.2013.06.070
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68 Metabolism of dietary flavonoids alters their effect on tumor necrosis factor-a J.L. di Gesso a,b, J.S. Kerr a,b, S.K. Yalamanchili b, A. Cassidy a, N.P. Botting c, D. O’Hagan c, Q. Zhang c, S. Raheen c, C.D. Kay a, M.A. O’Connell b, a Department of Nutrition, Norwich Medical School, University of East Anglia, Norwich, UK, b School of Pharmacy, University of East Anglia, Norwich, UK, c School of Chemistry, St. Andrews University, St. Andrews, UK Flavonoids are polyphenolic secondary metabolites formed by many plants including fruits and vegetables, and are therefore commonly consumed in the diet. Epidemiological evidence suggests that consuming a diet high in fruits and vegetables is positively correlated with a lower incidence of cardiovascular disease (CVD). It has long been hypothesised that flavonoids are central to these beneficial effects, however the exact mechanisms of action remain unclear. Upon consumption of a parent flavonoid, degradation and metabolism occur within the body. We hypothesise that the metabolites are responsible for the health benefits as opposed to their parent compounds. Here 6 parent flavonoids, 4 B-ring degradation products and 10 conjugated flavonoid metabolites were screened for ability to reduce lipopolysaccharide-induced tumour necrosis factor-a (TNF-a) secretion in a human monocytic cell line (THP-1). Physiologically relevant levels of treatments (0.1–10 lM) were pre-incubated in the culture model for 30 min prior to LPS stimulation. These concentrations were not cytotoxic as established using the WST-1 assay. Quercetin, epicatechin and cyanidin-3-glucoside form a common degradation product, protocatechuic acid (PCA), following ingestion. PCA can be metabolised by the liver to form glucuronide and sulfate conjugates. PCA-3-sulfate significantly decreased (38.7%, p < 0.05) TNF-a protein secretion compared to control. This reduction was greater than was elicited by treatment with quercetin (25.6%), epicatechin (21.3%), cyanidin-3-glucoside (13.7%) or PCA (22.1%). Furthermore, sulfation at carbon-3 gave rise to a greater reduction in TNF-a than sulfation at carbon-4 (9.6%) or by glucuronidation at either of these two sites (19.9%, 10.3% respectively). Intriguingly these protein data are not corroborated at the mRNA level, suggesting the treatments are having a post-translational effect. In conclusion, metabolism of parent flavonoids may increase their anti-inflammatory bioactivity. The possibile post-translational effects of flavonoids on TNF-a are currently the focus of our research.
http://dx.doi.org/10.1016/j.cyto.2013.06.071
69 Interferon-lambda is produced by and induces autocrine expression of interferon-stimulated genes in human bronchial epithelial cells Harold Dickensheets a, Faruk Sheikh a, Rachel Shepard b, Philippa Hillyer b, Ronald L. Rabin b, Raymond P. Donnelly a, a Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA, b Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA Interferons play a key role in the innate immune response to viral infection. In this study, we determined which IFN genes are expressed by primary human bronchial epithelial (HBE) cells following infection with respiratory syncytial virus (RSV) strain A2. Infection of HBE cells with RSV induced co-expression of the IFN-lambda genes (IFNL1, IFNL2 andIFNL3) in a dose- and time-dependent manner. It also induced expression of the IFNB1 gene and low levels of the IFNA1 gene but not other type-I IFN genes. Induction of IFNB1 andIFNL gene expression correlated with marked increases in the levels of IFN-b and IFN-k protein in the culture supernatants. The newly expressed IFN-b and IFN-k proteins in turn induced activation of STAT1 and STAT2 and subsequent expression of mulitple IFN-stimulated genes (ISGs), including MX1, OAS1, IFIT1, IFIT3, IFI44 andIRF7. Using neutralizing antibodies that selectively inhibit the activity of type-I versus type-III IFNs, we determined that ISG expression induced by RSV infection in HBE cells can be inhibited partially by either anti-type I or antitype III IFN antibodies and almost completely by co-treatment with both types of antibodies together. Recombinant human IFN-k1 induced activation of STAT1 and STAT2 in naïve HBE cells, and induced expression of the same ISGs that were induced by viral infection. Furthermore, pre-treatment with recombinant human IFN-k1 decreased the ability of RSV to infect naïve HBE cells indicating that IFN-k1 may be a useful prophylactic agent to protect against infection by respiratory viruses that preferentially infect airway epithelial cells. These findings demonstrate that bronchial epithelial cells express significant levels of both type I and type III IFN following viral infection. Furthermore, the newly expressed IFN-b and IFN-k proteins combine to mobilize a potent antiviral response in HBE cells by inducing autocrine expression of multiple ISGs.
http://dx.doi.org/10.1016/j.cyto.2013.06.072
67 Assessment of b2-adrenergic receptors on leukocyte subpopulations using imaging cytometry Alex P. Di Battista a, Shawn G. Rhind b, Maria Y. Shiu a, Andrew Baker c, Ruth A. Lanius d, Donald J. Richardson d,e, Rakesh Jetly f, a Graduate Program, University of Toronto, Toronto, ON, Canada, b Defence Research & Development (DRDC) Canada Toronto, Toronto, ON, Canada, c Dept of Critical Care, Trauma & Neurosurgery Program, St. Michael’s Hospital, University of Toronto, Toronto, ON, Canada, d Dept of Psychiatry, Schulich School of Medicine, Western University, London, ON, Canada, e Parkwood Operational Stress Injury Clinic, St. Joseph’s Health Care, London, ON, Canada, f Canadian Forces Health Services, Directorate of Mental Health, Dept of National Defence, Canada Introduction: b2-Adrenergic receptors (AR) are the primary subtype of adrenergic receptors found on blood leukocytes, and are recognized as key mediators of interaction between the sympathetic nervous system and immune system. This biological pathway is central in understanding neuroimmune signaling in a number of disease states, spanning mental disorders to traumatic brain injury. Traditionally, b2-ARs have been primarily assessed using radio-ligand binding assays, where results are limited in their ability to provide in depth cell-signaling information on specific leukocyte subtypes. As a result, current knowledge of b2-AR distribution, reactivity and potential implications in disease pathology or drug therapy are limited. This study evaluated b2-AR expression on circulating leukocyte subsets with various commercially available antibodies (Abs) using imaging cytometry. Methods: Whole blood was labeled with CD14 and CD16 surface markers for leukocyte subtype phenotyping. Four different b2-AR antibodies, sc-81578 – FITC (Santa Cruz), sc-81577 – PE (Santa Cruz), PA5-19649 (Pierce Biotechnology, USA) and MCA2784 (Serotec, USA) were assessed. DAPI stain was added to all samples to determine nuclear morphology. Based on cell area and aspect ratio, 100,000 single-cell events were acquired on an ImageStreamX Mark IIÒ Cytometer (Amnis, Seattle, WA). Monocytes and lymphocytes were identified using side scatter (SSC) and CD14 expression. Neutrophils and eosinophils were identified using SSC and CD16. Results: b2 AR expression levels on leukocyte subpopulations in whole blood were detectable using image cytometery. Leukocyte subsets identified according to surface markers, size, granularity, cell and nuclear morphology displayed varying amounts of b2-AR expression. Furthermore, the PA5-19649 Ab performed best in terms of binding specificity and signal strength. Conclusion: Imaging cytometry is a valuable technique for evaluating b2-ARs on leukocyte subsets. The capability of this technology to provide in-depth, high throughput cell signaling information offers an attractive alternative to current methods used in the assessment of adrenergic receptors. http://dx.doi.org/10.1016/j.cyto.2013.06.070
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68 Metabolism of dietary flavonoids alters their effect on tumor necrosis factor-a J.L. di Gesso a,b, J.S. Kerr a,b, S.K. Yalamanchili b, A. Cassidy a, N.P. Botting c, D. O’Hagan c, Q. Zhang c, S. Raheen c, C.D. Kay a, M.A. O’Connell b, a Department of Nutrition, Norwich Medical School, University of East Anglia, Norwich, UK, b School of Pharmacy, University of East Anglia, Norwich, UK, c School of Chemistry, St. Andrews University, St. Andrews, UK Flavonoids are polyphenolic secondary metabolites formed by many plants including fruits and vegetables, and are therefore commonly consumed in the diet. Epidemiological evidence suggests that consuming a diet high in fruits and vegetables is positively correlated with a lower incidence of cardiovascular disease (CVD). It has long been hypothesised that flavonoids are central to these beneficial effects, however the exact mechanisms of action remain unclear. Upon consumption of a parent flavonoid, degradation and metabolism occur within the body. We hypothesise that the metabolites are responsible for the health benefits as opposed to their parent compounds. Here 6 parent flavonoids, 4 B-ring degradation products and 10 conjugated flavonoid metabolites were screened for ability to reduce lipopolysaccharide-induced tumour necrosis factor-a (TNF-a) secretion in a human monocytic cell line (THP-1). Physiologically relevant levels of treatments (0.1–10 lM) were pre-incubated in the culture model for 30 min prior to LPS stimulation. These concentrations were not cytotoxic as established using the WST-1 assay. Quercetin, epicatechin and cyanidin-3-glucoside form a common degradation product, protocatechuic acid (PCA), following ingestion. PCA can be metabolised by the liver to form glucuronide and sulfate conjugates. PCA-3-sulfate significantly decreased (38.7%, p < 0.05) TNF-a protein secretion compared to control. This reduction was greater than was elicited by treatment with quercetin (25.6%), epicatechin (21.3%), cyanidin-3-glucoside (13.7%) or PCA (22.1%). Furthermore, sulfation at carbon-3 gave rise to a greater reduction in TNF-a than sulfation at carbon-4 (9.6%) or by glucuronidation at either of these two sites (19.9%, 10.3% respectively). Intriguingly these protein data are not corroborated at the mRNA level, suggesting the treatments are having a post-translational effect. In conclusion, metabolism of parent flavonoids may increase their anti-inflammatory bioactivity. The possibile post-translational effects of flavonoids on TNF-a are currently the focus of our research.
http://dx.doi.org/10.1016/j.cyto.2013.06.071
69 Interferon-lambda is produced by and induces autocrine expression of interferon-stimulated genes in human bronchial epithelial cells Harold Dickensheets a, Faruk Sheikh a, Rachel Shepard b, Philippa Hillyer b, Ronald L. Rabin b, Raymond P. Donnelly a, a Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA, b Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA Interferons play a key role in the innate immune response to viral infection. In this study, we determined which IFN genes are expressed by primary human bronchial epithelial (HBE) cells following infection with respiratory syncytial virus (RSV) strain A2. Infection of HBE cells with RSV induced co-expression of the IFN-lambda genes (IFNL1, IFNL2 andIFNL3) in a dose- and time-dependent manner. It also induced expression of the IFNB1 gene and low levels of the IFNA1 gene but not other type-I IFN genes. Induction of IFNB1 andIFNL gene expression correlated with marked increases in the levels of IFN-b and IFN-k protein in the culture supernatants. The newly expressed IFN-b and IFN-k proteins in turn induced activation of STAT1 and STAT2 and subsequent expression of mulitple IFN-stimulated genes (ISGs), including MX1, OAS1, IFIT1, IFIT3, IFI44 andIRF7. Using neutralizing antibodies that selectively inhibit the activity of type-I versus type-III IFNs, we determined that ISG expression induced by RSV infection in HBE cells can be inhibited partially by either anti-type I or antitype III IFN antibodies and almost completely by co-treatment with both types of antibodies together. Recombinant human IFN-k1 induced activation of STAT1 and STAT2 in naïve HBE cells, and induced expression of the same ISGs that were induced by viral infection. Furthermore, pre-treatment with recombinant human IFN-k1 decreased the ability of RSV to infect naïve HBE cells indicating that IFN-k1 may be a useful prophylactic agent to protect against infection by respiratory viruses that preferentially infect airway epithelial cells. These findings demonstrate that bronchial epithelial cells express significant levels of both type I and type III IFN following viral infection. Furthermore, the newly expressed IFN-b and IFN-k proteins combine to mobilize a potent antiviral response in HBE cells by inducing autocrine expression of multiple ISGs.
http://dx.doi.org/10.1016/j.cyto.2013.06.072