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Abstract / Cytokine 70 (2014) 28–79
173 Probiotics in a gnotobiotic piglet model of preterm infants Alla Splichalova, Igor Splichal, Institute of Microbiology, ASCR, Prague 4 – Krc, Czech Republic Aims: Mucosal surfaces of newborns are colonized by microbes in dependence on their delivery and nutrition. Are probiotics safety to Caesarean section derived immunocompromised preterm infants? Methods: Hysterectomy derived preterm germ-free piglets, either treated or nontreated with serum, were colonized with Lactobacillus rhamnosus (LGG) four hours after hysterectomy. Bacterial translocation of LGG, its protection against subsequent infection of the piglets with Salmonella Typhimurium, and induction of plasma and intestinal levels of inflammatory cytokines IL-1 beta, IL-6, IL-8, IL-10, IFN-gamma, TNF-alpha (xMAP), and HMGB1 (ELISA) were estimated. Results: LGG did not translocate in one week old preterm piglets. It was found in mesenteric lymph nodes in piglets subsequently infected with S. Typhimurium only but it did not translocate to other organs (blood, liver, spleen, and lungs). Inflammatory cytokine values were not induced by LGG alone but they were dramatically increased in piglets infected with Salmonella. The piglets treated with mixed serum of adult sows showed reduced levels of inflammatory cytokines as in the intestine so in plasma. Conclusions: It is necessary to develop highly sensitive animal models to evaluate a safety of different probiotics for immunocompromised preterm infants. A high attention should be paid to a selection of suitable markers to evaluate possible risk of these probiotics. Inflammatory cytokines in the intestine/feces of preterm newborns should be a suitable candidates of such markers. Acknowledgements: This work was supported by Grants 13-14736S of the Czech Science Foundation, FR-TI4/504 of the Ministry of Industry and Trade of the Czech Republic and the Institutional Research Concept RVO: 61388971 of the Institute of Microbiology.
Rationale: Asthmatics and COPD patients are more susceptible to viral infections, which in turn are a major cause of exacerbations. However, the immunological mechanisms underpinning this association are largely unknown. Anti-interleukin (IL)-13 is currently in clinical trials for asthma, but the role of this cytokine in predispositing to viral infections is unknown. Methods: BALB/c mice were subjected to Ovalbumin (Ova)-, house dust mite (HDM)- or recombinant IL-13 (rIL-13)-induced models of allergic airway disease (AAD), or cigarette smoke-induced COPD, and infected with influenza virus (A/PR/8/34 strain). Some groups were treated with anti-IL-13 neutralizing antibody, miRNA-21-specific anatgomirs or PI3K inhibitors during influenza infection. The effects of infection on hallmark features of AAD (airway hyper-responsiveness [AHR], mucus secreting cell [MSC] numbers and eosinophil infiltration), COPD (lung function, inflammation) and on antiviral responses (viral load and interferon [IFN]-a, -b, -k, -c levels) were assessed. The cellular source of IL-13 was determined using novel IL-13 reporter mice. Results: Influenza infection increased the severity of Ova-, HDMand rIL-13-induced AAD by increasing AHR, MSC numbers and lung eosinophils. Importantly, anti-IL-13 treatment during AAD returned AHR, MSC numbers and eosinophils back to control levels. Infection also exacerbated COPD. All models resulted in increased viral load, which correlated with suppressed IFN-a, -b, -k, -c and increased miR-21 and PI3K levels in the lung. Anti-IL-13 treatment also improved anti-viral responses leading to reduced viral load. IL-13 increased both miRNA-21 and PI3K. Inhibition of these molecules protected against infection. Influenza infection increased IL13-production by NKT cells, ILC2s and Th2 cells. Conclusion: IL-13 responses during AAD and COPD lead to impaired antiviral immune responses resulting in more severe influenza infection that exacerbated the underlying disease. These studies identify anti-IL-13 and miRNA-21 and PI3K inhibitors as a potential therapies in influenza infections and influenza-induced exacerbations. http://dx.doi.org/10.1016/j.cyto.2014.07.182
176 miR-17 92 and it’s role in inflammation and innate immune signalling http://dx.doi.org/10.1016/j.cyto.2014.07.180
174 Transgenic mice expressing interferon-regulated human influenza virus restriction factor MxA Christoph Deeg, Pascal Mutz, Lara Rheinemann, Carsten Kallfass, Cindy Nuernberger, Mirjam Schilling, Sébastien Soubies, Peter Staeheli, Institute for Virology, University Medical Center Freiburg, Freiburg, Germany In mice, influenza virus resistance is strongly dependent on the interferon (IFN)regulated Mx1 gene which encodes a potent restriction factor that inhibits a poorly defined early step of the viral life cycle. Humans possess two IFN-regulated Mx genes with antiviral activity. The human Mx1 gene (encoding MxA protein) confers resistance to a broad range of RNA viruses in cell culture, including influenza A viruses, whereas the human Mx2 gene (encoding MxB protein) has been shown to inhibit HIV-1. To determine whether human MxA plays a decisive role in defending the intact organism against influenza A viruses, we developed a mouse strain that lacks functional endogenous Mx genes but carries the complete human Mx locus as a transgene. MxA-transgenic mice exhibited solid resistance to infection with highly pathogenic H5N1 and H7N7 avian influenza viruses, as well as influenza-like Thogoto virus. However, transgenic mice differed only slightly from non-transgenic littermates with regard to resistance to H1N1 and H3N2 influenza viruses of human origin, suggesting that seasonal human influenza viruses have acquired adaptive mutations which permit MxA evasion. To identify adaptive mutations which confer Mx resistance, we passaged mouseadapted H7N7 avian influenza virus SC35M in MxA-transgenic mice. Here we will describe an escape variant of SC35M resulting from this screen that induces fatal disease in MxA-transgenic mice and exhibits enhanced virulence for mice with intact endogenous Mx1 genes.
James H. Stunden 1, Michael P. Gantier 2, Joachim L. Schultze 3, Eicke Latz 1, 1 Institute for Innate Immunity, Bonn, Germany, 2 Centre for Cancer Research, MIMR-PHI Institute of Medical Research, Clayton, Vic, Australia, 3 Life and Medical Sciences Institute, University of Bonn, Bonn, Germany The importance of microRNA regulation of genes is already well accepted, yet we are still to understand the role that miRNA play in the regulation of inflammation. We have previously have shown that miR-19 has pro-inflammatory effects by targeting several negative regulators of NF-kB, and thus resulting in prolonged, chronic inflammatory signalling [1]. However, miR-19 is also part of the miR-17 92 cluster, a group of miRNA that are transcriptionally activated together, and they may each be able to target many other genes simultaneously. Our experiments show that miR-19 is induced following LPS treatment in macrophages. Furthermore, removal of the cluster also results in a reduced response to multiple TLR agonists, indicating that the miRNA target more NF-kB signal regulators than previously thought. We wish to further evaluate the role of these miRNA on the regulation of inflammation, by examining the effect of deleting the whole miRNA cluster in vivo using the miR-17 92flox mouse. We will also examine the effect of deleting the cluster on the transcriptome, by performing a microarray technology to assess which genes are expressed in modified titres from basal levels, which will provide a wealth of information to understand how this miRNA cluster affects the cell.
Reference [1] Gantier MP et al.. A miR-19 regulon that controls NF-jB signaling. Nucl Acids Res 2012;40:8048–58. http://dx.doi.org/10.1016/j.cyto.2014.07.183
http://dx.doi.org/10.1016/j.cyto.2014.07.181
177 The NLRP3 inflammasome is regulated by phosphorylation and ubiquitinylation
175 Interleukin-13 predisposes mice to more severe influenza infection by suppressing interferon responses and activating microRNA-21/PI3K
Andrea Stutz 1, Gabor L. Horvath 1, Rainer Stahl 1, Bernardo S. Franklin 1, Carl-Christian L. Kolbe 1, Matthias Geyer 2, Felix Meissner 3, Eicke Latz 1,4,5, 1 Institute of Innate Immunity, University Hospital Bonn, Bonn, Germany, 2 Group Physical Biochemistry, Center of Advanced European Studies and Research (caesar), Bonn, Germany, 3 Dept. Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried/ Munich, Germany, 4 Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA, USA, 5 Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Bonn, Germany
Malcolm Starkey, Irwan Hanish, Kamal Dua, Prema Nair, Tattjhong Haw, Alan Hsu, Paul Foster, Darryl Knight, Jay Horvat, Peter Wark, Phil Hansbro, University of Newcastle Australia, New Lambton Heights, NSW, Australia
Abstract / Cytokine 70 (2014) 28–79
173 Probiotics in a gnotobiotic piglet model of preterm infants Alla Splichalova, Igor Splichal, Institute of Microbiology, ASCR, Prague 4 – Krc, Czech Republic Aims: Mucosal surfaces of newborns are colonized by microbes in dependence on their delivery and nutrition. Are probiotics safety to Caesarean section derived immunocompromised preterm infants? Methods: Hysterectomy derived preterm germ-free piglets, either treated or nontreated with serum, were colonized with Lactobacillus rhamnosus (LGG) four hours after hysterectomy. Bacterial translocation of LGG, its protection against subsequent infection of the piglets with Salmonella Typhimurium, and induction of plasma and intestinal levels of inflammatory cytokines IL-1 beta, IL-6, IL-8, IL-10, IFN-gamma, TNF-alpha (xMAP), and HMGB1 (ELISA) were estimated. Results: LGG did not translocate in one week old preterm piglets. It was found in mesenteric lymph nodes in piglets subsequently infected with S. Typhimurium only but it did not translocate to other organs (blood, liver, spleen, and lungs). Inflammatory cytokine values were not induced by LGG alone but they were dramatically increased in piglets infected with Salmonella. The piglets treated with mixed serum of adult sows showed reduced levels of inflammatory cytokines as in the intestine so in plasma. Conclusions: It is necessary to develop highly sensitive animal models to evaluate a safety of different probiotics for immunocompromised preterm infants. A high attention should be paid to a selection of suitable markers to evaluate possible risk of these probiotics. Inflammatory cytokines in the intestine/feces of preterm newborns should be a suitable candidates of such markers. Acknowledgements: This work was supported by Grants 13-14736S of the Czech Science Foundation, FR-TI4/504 of the Ministry of Industry and Trade of the Czech Republic and the Institutional Research Concept RVO: 61388971 of the Institute of Microbiology.
Rationale: Asthmatics and COPD patients are more susceptible to viral infections, which in turn are a major cause of exacerbations. However, the immunological mechanisms underpinning this association are largely unknown. Anti-interleukin (IL)-13 is currently in clinical trials for asthma, but the role of this cytokine in predispositing to viral infections is unknown. Methods: BALB/c mice were subjected to Ovalbumin (Ova)-, house dust mite (HDM)- or recombinant IL-13 (rIL-13)-induced models of allergic airway disease (AAD), or cigarette smoke-induced COPD, and infected with influenza virus (A/PR/8/34 strain). Some groups were treated with anti-IL-13 neutralizing antibody, miRNA-21-specific anatgomirs or PI3K inhibitors during influenza infection. The effects of infection on hallmark features of AAD (airway hyper-responsiveness [AHR], mucus secreting cell [MSC] numbers and eosinophil infiltration), COPD (lung function, inflammation) and on antiviral responses (viral load and interferon [IFN]-a, -b, -k, -c levels) were assessed. The cellular source of IL-13 was determined using novel IL-13 reporter mice. Results: Influenza infection increased the severity of Ova-, HDMand rIL-13-induced AAD by increasing AHR, MSC numbers and lung eosinophils. Importantly, anti-IL-13 treatment during AAD returned AHR, MSC numbers and eosinophils back to control levels. Infection also exacerbated COPD. All models resulted in increased viral load, which correlated with suppressed IFN-a, -b, -k, -c and increased miR-21 and PI3K levels in the lung. Anti-IL-13 treatment also improved anti-viral responses leading to reduced viral load. IL-13 increased both miRNA-21 and PI3K. Inhibition of these molecules protected against infection. Influenza infection increased IL13-production by NKT cells, ILC2s and Th2 cells. Conclusion: IL-13 responses during AAD and COPD lead to impaired antiviral immune responses resulting in more severe influenza infection that exacerbated the underlying disease. These studies identify anti-IL-13 and miRNA-21 and PI3K inhibitors as a potential therapies in influenza infections and influenza-induced exacerbations. http://dx.doi.org/10.1016/j.cyto.2014.07.182
176 miR-17 92 and it’s role in inflammation and innate immune signalling http://dx.doi.org/10.1016/j.cyto.2014.07.180
174 Transgenic mice expressing interferon-regulated human influenza virus restriction factor MxA Christoph Deeg, Pascal Mutz, Lara Rheinemann, Carsten Kallfass, Cindy Nuernberger, Mirjam Schilling, Sébastien Soubies, Peter Staeheli, Institute for Virology, University Medical Center Freiburg, Freiburg, Germany In mice, influenza virus resistance is strongly dependent on the interferon (IFN)regulated Mx1 gene which encodes a potent restriction factor that inhibits a poorly defined early step of the viral life cycle. Humans possess two IFN-regulated Mx genes with antiviral activity. The human Mx1 gene (encoding MxA protein) confers resistance to a broad range of RNA viruses in cell culture, including influenza A viruses, whereas the human Mx2 gene (encoding MxB protein) has been shown to inhibit HIV-1. To determine whether human MxA plays a decisive role in defending the intact organism against influenza A viruses, we developed a mouse strain that lacks functional endogenous Mx genes but carries the complete human Mx locus as a transgene. MxA-transgenic mice exhibited solid resistance to infection with highly pathogenic H5N1 and H7N7 avian influenza viruses, as well as influenza-like Thogoto virus. However, transgenic mice differed only slightly from non-transgenic littermates with regard to resistance to H1N1 and H3N2 influenza viruses of human origin, suggesting that seasonal human influenza viruses have acquired adaptive mutations which permit MxA evasion. To identify adaptive mutations which confer Mx resistance, we passaged mouseadapted H7N7 avian influenza virus SC35M in MxA-transgenic mice. Here we will describe an escape variant of SC35M resulting from this screen that induces fatal disease in MxA-transgenic mice and exhibits enhanced virulence for mice with intact endogenous Mx1 genes.
James H. Stunden 1, Michael P. Gantier 2, Joachim L. Schultze 3, Eicke Latz 1, 1 Institute for Innate Immunity, Bonn, Germany, 2 Centre for Cancer Research, MIMR-PHI Institute of Medical Research, Clayton, Vic, Australia, 3 Life and Medical Sciences Institute, University of Bonn, Bonn, Germany The importance of microRNA regulation of genes is already well accepted, yet we are still to understand the role that miRNA play in the regulation of inflammation. We have previously have shown that miR-19 has pro-inflammatory effects by targeting several negative regulators of NF-kB, and thus resulting in prolonged, chronic inflammatory signalling [1]. However, miR-19 is also part of the miR-17 92 cluster, a group of miRNA that are transcriptionally activated together, and they may each be able to target many other genes simultaneously. Our experiments show that miR-19 is induced following LPS treatment in macrophages. Furthermore, removal of the cluster also results in a reduced response to multiple TLR agonists, indicating that the miRNA target more NF-kB signal regulators than previously thought. We wish to further evaluate the role of these miRNA on the regulation of inflammation, by examining the effect of deleting the whole miRNA cluster in vivo using the miR-17 92flox mouse. We will also examine the effect of deleting the cluster on the transcriptome, by performing a microarray technology to assess which genes are expressed in modified titres from basal levels, which will provide a wealth of information to understand how this miRNA cluster affects the cell.
Reference [1] Gantier MP et al.. A miR-19 regulon that controls NF-jB signaling. Nucl Acids Res 2012;40:8048–58. http://dx.doi.org/10.1016/j.cyto.2014.07.183
http://dx.doi.org/10.1016/j.cyto.2014.07.181
177 The NLRP3 inflammasome is regulated by phosphorylation and ubiquitinylation
175 Interleukin-13 predisposes mice to more severe influenza infection by suppressing interferon responses and activating microRNA-21/PI3K
Andrea Stutz 1, Gabor L. Horvath 1, Rainer Stahl 1, Bernardo S. Franklin 1, Carl-Christian L. Kolbe 1, Matthias Geyer 2, Felix Meissner 3, Eicke Latz 1,4,5, 1 Institute of Innate Immunity, University Hospital Bonn, Bonn, Germany, 2 Group Physical Biochemistry, Center of Advanced European Studies and Research (caesar), Bonn, Germany, 3 Dept. Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried/ Munich, Germany, 4 Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA, USA, 5 Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Bonn, Germany
Malcolm Starkey, Irwan Hanish, Kamal Dua, Prema Nair, Tattjhong Haw, Alan Hsu, Paul Foster, Darryl Knight, Jay Horvat, Peter Wark, Phil Hansbro, University of Newcastle Australia, New Lambton Heights, NSW, Australia