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P118 IFN-A transcriptional response in endothelial cells: Key modulators and gene regulatory modules
Cytokine 59 (2012) 557–564
Contents lists available at SciVerse ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
Interferons and innate immunity P117 Regulation of actin dynamics by Protein Kinase R control of gelsolin enforces basal innate immune defense A.T. Irving, B.R. Williams, A.J. sadler, Monash Institute of Medical Research, Monash University, melbourne, Australia Introduction. The primary resistance to pathogens is heavily reliant on the innate immune response. This resistance depends upon both basal and inducible defenses. Contrasting to inducible resistance through cytokine induction, basal defense mechanisms are much less evident. Here we detail how the antiviral effector Protein Kinase R (PKR) interacts with the key actin-regulatory protein gelsolin to regulate actin dynamics and control cytoskeletal functions under homeostatic conditions. In addition, up-regulation of PKR by Interferon can enhance this interaction. Methods. The interaction was confirmed by recombinant proteins, Immunoprecipitations, FRET and bi-molecular Fluorescence (Bi-FC). Investigation of actin dynamics was performed with recombinant proteins and in cells via live cell microscopy and FRAP. Endocytosis and uptake of viral particles/bacteria was examined via live cell microscopy, 3D modelling and replicated with in vivo mouse models. Results. Through this mechanism, PKR directly inhibits Gelsolin, blocking its ability to sever actin filaments and affecting actin-dependent processes that include membrane ruffling, migration, endocytosis and accordingly, PKR counteracts pathogen entry into the cell. This also impacts the direct function of cells of the immune compartment including phagocytosis and activation. Conclusion. These findings identify a new layer of host resistance, with the regulation of actin-modifying proteins during the innate immune response bolstering firstline defense against intracellular pathogens and having a sustained effect on virus production and the overall consequence of infection. This response can also be enhanced by Interferon treatment to up-regulate PKR. Moreover, modulating the cell cytoskeleton with specific targets could potentially be used to elicit broad protection against pathogens. Disclosure of interest: None declared. http://dx.doi.org/10.1016/j.cyto.2012.06.209
P118 IFN-A transcriptional response in endothelial cells: Key modulators and gene regulatory modules A. Grassi 1,2, F. Ciccarese 1,3, B. Di Camillo 4, G. Toffolo 4, S. Indraccolo 1, 1 Istituto Oncologico Veneto – IRCSS, Italy, 2 Institute of Biomedical Engineering, Italian National Research Council, Italy, 3 Department of Surgical, Oncological and Gastroenterological Sciences, Italy, 4 Department of Information Engineering, University of Padova, Padova, Italy Introduction: . Inference of gene regulation from expression data may help to unravel regulatory mechanisms involved in complex diseases or in the action of specific drugs. Interferon-a (IFN-a) is a pleiotropic cytokine endowed with potent biological activities, achieved through the up-regulation of hundreds of interferon-stimulated genes (ISGs). Aim of this study was to extend previous work on IFN-a transcriptional response in endothelial cells, by identifying IFN-a modulators and inferring putative gene regulatory modules involving them. Methods. Primary endothelial cells (HUVEC) were pulsed in vitro with human recombinant IFN-a under different perturbation conditions: siRNA inactivation of target genes. A panel of 96 transcripts, including ISGs belonging to IFN-a top signature and genes related to the IFN-a signalling pathway, were screened by custom TaqMan Low-Density Arrays. Stealth siRNA were used for RNAi-mediated knockdown of seven candidate IFN-a modulators: STAT1, IRF1, IRF7, GBP1, OAS2, IFIH1 and USP18. The effects of each siRNA were evaluated with respect to a calibrator siRNA using the comparative threshold cycle method (DDCT method). A selection procedure, testing the null hypothesis of no difference in the effects of a siRNA targeting a candidate modulator and
http://dx.doi.org/10.1016/S1043-4666(12)00453-X
the calibrator siRNA, was used to assign a significance value (p-value) to each regulation. Significant regulations were defined fixing a cut-off of 0.05 on the Bonferroni corrected p-values. Regulatory modules, built by different feed-forward loops (FFLs), the basic three-node building blocks of biological networks, were then inferred by combining the significant regulations, induced by different couples of IFN-a modulators. Results. Our analysis showed both mainly positive (STAT1, IFIH1) and mainly negative (USP18, GBP1, IRF1) modulators of IFN-a transcriptional response, while IRF7 and OAS2 were found to act equally in both directions. STAT1 was confirmed as the primary positive modulator of IFN-a; following its silencing several genes were significantly down-regulated and only few genes up-regulated. Conversely, USP18 was found to be the strongest negative modulator and its silencing caused a massive up-regulation of genes, including some involved in cell-to-cell adhesion. An interesting regulatory module in which STAT1 activates IFIH1 and they both regulate six genes, including IFNa-R1, was reconstructed by our inference method. Some of the inferred FFLs are currently being validated. Conclusion. The investigation of IFN-a transcriptional modulators through our method has led to a clear picture, depicting the transcriptional impact of each considered modulator in terms of sign of regulation, strength and target genes. The inferred gene regulatory modules, involving key modulators, may help to unravel the mechanisms of action of IFN-a and discover future target of intervention in knock-out experiments. Disclosure of interest: None declared. http://dx.doi.org/10.1016/j.cyto.2012.06.210
P119 Type I and III interferon are attenuated in a human in vitro model of alternatively activated macrophages and is mediated by IRF4 A. El Fiky, R. Perreault, R.L. Rabin, Laboratory of Immunobiochemistry – Division of Bacterial Parasitic and Allergenic Products – Office of Vaccine Research and Review, United States Food and Drug Administration, Bethesda, MD, United States Introduction. Human monocytes may be polarized in vitro into classically or alternatively activated macrophages (CAM and AAM) after treatment with IFN-g or IL-4, respectively [1]. In vivo, AAM are closely associated with amplifying Th2 responses, and are implicated in the pathogenesis of allergic diseases and asthma. A current impediment to investigating the biology of human AAM is wide donor-to-donor variability and the quantity of primary macrophages that survive in vitro polarization. To overcome this impediment, we have successfully established a THP-1 derived -CAM and -AAM populations demonstrating typical macrophage-oriented morphological characteristics, gene and protein expression profile in vitro. The THP1-AAM model preferentially expressed interferon alpha/beta pathway genes, type I and III interferon genes, in addition to IRF4 compared to THP1-CAM. Methods. THP-1 cells were activated with PMA for 48 h and polarized for 96 h with either IFN-g (500 IU/mL) or IL-4 (100 IU/mL). After polarization, the THP1-CAM and THP1-AAM were treated with LPS (100 ng/mL) and expression of IFN and inflammatory genes were measured by qRT-PCR or ELISA. Expression of IRF4 in THP1-AAM and -CAM was determined by qRT-PCR. To determine the role of IRF4 in THP1-AAM, the gene for IRF4 was silenced with siRNA prior to polarization of the THP-1 cells, and expression of interferon and inflammatory genes were measured in the IRF-silenced vs. control cells. Results. THP1-AAM preferentially expressed mannose receptor and select chemokines including CCL18 and CCL22 while THP1-CAM expressed CXCL10 and TNF-alpha among other phenotype dependent markers. Upon LPS treatment, THP1-AAM preferentially expressed IL-10 in the supernatant while THP1-CAM expressed IFN-g, and IL1Beta. Using THP1-AAM to understand interferon subtype expression in response to TLR4 challenge, we demonstrated that THP1-AAM express significantly attenuated level of interferon alpha/beta pathway, IFN-Beta, and IFN-Lambda1 genes compared to THP1-CAM upon LPS treatment. Expression of phenotype-oriented genes and IFN
Contents lists available at SciVerse ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
Interferons and innate immunity P117 Regulation of actin dynamics by Protein Kinase R control of gelsolin enforces basal innate immune defense A.T. Irving, B.R. Williams, A.J. sadler, Monash Institute of Medical Research, Monash University, melbourne, Australia Introduction. The primary resistance to pathogens is heavily reliant on the innate immune response. This resistance depends upon both basal and inducible defenses. Contrasting to inducible resistance through cytokine induction, basal defense mechanisms are much less evident. Here we detail how the antiviral effector Protein Kinase R (PKR) interacts with the key actin-regulatory protein gelsolin to regulate actin dynamics and control cytoskeletal functions under homeostatic conditions. In addition, up-regulation of PKR by Interferon can enhance this interaction. Methods. The interaction was confirmed by recombinant proteins, Immunoprecipitations, FRET and bi-molecular Fluorescence (Bi-FC). Investigation of actin dynamics was performed with recombinant proteins and in cells via live cell microscopy and FRAP. Endocytosis and uptake of viral particles/bacteria was examined via live cell microscopy, 3D modelling and replicated with in vivo mouse models. Results. Through this mechanism, PKR directly inhibits Gelsolin, blocking its ability to sever actin filaments and affecting actin-dependent processes that include membrane ruffling, migration, endocytosis and accordingly, PKR counteracts pathogen entry into the cell. This also impacts the direct function of cells of the immune compartment including phagocytosis and activation. Conclusion. These findings identify a new layer of host resistance, with the regulation of actin-modifying proteins during the innate immune response bolstering firstline defense against intracellular pathogens and having a sustained effect on virus production and the overall consequence of infection. This response can also be enhanced by Interferon treatment to up-regulate PKR. Moreover, modulating the cell cytoskeleton with specific targets could potentially be used to elicit broad protection against pathogens. Disclosure of interest: None declared. http://dx.doi.org/10.1016/j.cyto.2012.06.209
P118 IFN-A transcriptional response in endothelial cells: Key modulators and gene regulatory modules A. Grassi 1,2, F. Ciccarese 1,3, B. Di Camillo 4, G. Toffolo 4, S. Indraccolo 1, 1 Istituto Oncologico Veneto – IRCSS, Italy, 2 Institute of Biomedical Engineering, Italian National Research Council, Italy, 3 Department of Surgical, Oncological and Gastroenterological Sciences, Italy, 4 Department of Information Engineering, University of Padova, Padova, Italy Introduction: . Inference of gene regulation from expression data may help to unravel regulatory mechanisms involved in complex diseases or in the action of specific drugs. Interferon-a (IFN-a) is a pleiotropic cytokine endowed with potent biological activities, achieved through the up-regulation of hundreds of interferon-stimulated genes (ISGs). Aim of this study was to extend previous work on IFN-a transcriptional response in endothelial cells, by identifying IFN-a modulators and inferring putative gene regulatory modules involving them. Methods. Primary endothelial cells (HUVEC) were pulsed in vitro with human recombinant IFN-a under different perturbation conditions: siRNA inactivation of target genes. A panel of 96 transcripts, including ISGs belonging to IFN-a top signature and genes related to the IFN-a signalling pathway, were screened by custom TaqMan Low-Density Arrays. Stealth siRNA were used for RNAi-mediated knockdown of seven candidate IFN-a modulators: STAT1, IRF1, IRF7, GBP1, OAS2, IFIH1 and USP18. The effects of each siRNA were evaluated with respect to a calibrator siRNA using the comparative threshold cycle method (DDCT method). A selection procedure, testing the null hypothesis of no difference in the effects of a siRNA targeting a candidate modulator and
http://dx.doi.org/10.1016/S1043-4666(12)00453-X
the calibrator siRNA, was used to assign a significance value (p-value) to each regulation. Significant regulations were defined fixing a cut-off of 0.05 on the Bonferroni corrected p-values. Regulatory modules, built by different feed-forward loops (FFLs), the basic three-node building blocks of biological networks, were then inferred by combining the significant regulations, induced by different couples of IFN-a modulators. Results. Our analysis showed both mainly positive (STAT1, IFIH1) and mainly negative (USP18, GBP1, IRF1) modulators of IFN-a transcriptional response, while IRF7 and OAS2 were found to act equally in both directions. STAT1 was confirmed as the primary positive modulator of IFN-a; following its silencing several genes were significantly down-regulated and only few genes up-regulated. Conversely, USP18 was found to be the strongest negative modulator and its silencing caused a massive up-regulation of genes, including some involved in cell-to-cell adhesion. An interesting regulatory module in which STAT1 activates IFIH1 and they both regulate six genes, including IFNa-R1, was reconstructed by our inference method. Some of the inferred FFLs are currently being validated. Conclusion. The investigation of IFN-a transcriptional modulators through our method has led to a clear picture, depicting the transcriptional impact of each considered modulator in terms of sign of regulation, strength and target genes. The inferred gene regulatory modules, involving key modulators, may help to unravel the mechanisms of action of IFN-a and discover future target of intervention in knock-out experiments. Disclosure of interest: None declared. http://dx.doi.org/10.1016/j.cyto.2012.06.210
P119 Type I and III interferon are attenuated in a human in vitro model of alternatively activated macrophages and is mediated by IRF4 A. El Fiky, R. Perreault, R.L. Rabin, Laboratory of Immunobiochemistry – Division of Bacterial Parasitic and Allergenic Products – Office of Vaccine Research and Review, United States Food and Drug Administration, Bethesda, MD, United States Introduction. Human monocytes may be polarized in vitro into classically or alternatively activated macrophages (CAM and AAM) after treatment with IFN-g or IL-4, respectively [1]. In vivo, AAM are closely associated with amplifying Th2 responses, and are implicated in the pathogenesis of allergic diseases and asthma. A current impediment to investigating the biology of human AAM is wide donor-to-donor variability and the quantity of primary macrophages that survive in vitro polarization. To overcome this impediment, we have successfully established a THP-1 derived -CAM and -AAM populations demonstrating typical macrophage-oriented morphological characteristics, gene and protein expression profile in vitro. The THP1-AAM model preferentially expressed interferon alpha/beta pathway genes, type I and III interferon genes, in addition to IRF4 compared to THP1-CAM. Methods. THP-1 cells were activated with PMA for 48 h and polarized for 96 h with either IFN-g (500 IU/mL) or IL-4 (100 IU/mL). After polarization, the THP1-CAM and THP1-AAM were treated with LPS (100 ng/mL) and expression of IFN and inflammatory genes were measured by qRT-PCR or ELISA. Expression of IRF4 in THP1-AAM and -CAM was determined by qRT-PCR. To determine the role of IRF4 in THP1-AAM, the gene for IRF4 was silenced with siRNA prior to polarization of the THP-1 cells, and expression of interferon and inflammatory genes were measured in the IRF-silenced vs. control cells. Results. THP1-AAM preferentially expressed mannose receptor and select chemokines including CCL18 and CCL22 while THP1-CAM expressed CXCL10 and TNF-alpha among other phenotype dependent markers. Upon LPS treatment, THP1-AAM preferentially expressed IL-10 in the supernatant while THP1-CAM expressed IFN-g, and IL1Beta. Using THP1-AAM to understand interferon subtype expression in response to TLR4 challenge, we demonstrated that THP1-AAM express significantly attenuated level of interferon alpha/beta pathway, IFN-Beta, and IFN-Lambda1 genes compared to THP1-CAM upon LPS treatment. Expression of phenotype-oriented genes and IFN