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Project 4
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Project 4

Olaf Utermöhlen, Martin Krönke

Antimicrobial effector and signaling activities of NADPH oxidases

Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne

Publications

Brief description in German:
Aufbauend auf den Befunden der ersten Förderperiode wollen wir in diesem Projekt die Mechanismen der differenziellen Aktivierung der gewebespezifisch exprimierten Nox1- und Nox2 sowie Duox1 und -2 Isoenzyme der NADPH Oxidase (Nox) durch pathogenassoziierte und wirtszelleigene Stimuli aufklären. Die antimikrobiellen Effektormechanismen und Signaltransduktionsfunktionen der durch die NADPH Oxidasen gebildeten reaktiven Sauerstoffspezies (ROS) sollen in vitro und in vivo im Hinblick auf zell-autonome und gewebsspezifische Abwehrreaktionen charakterisiert werden.

Summary
Based on our observations of the previous funding period we will now characterize the mechanisms of differential activation of the tissue-specifically expressed Nox1 and -2 as well as Duox1 and -2 isoenzymes of NADPH oxidase (Nox) by pathogen-associated and host cell-derived stimuli. Further, the direct oxidative effector mechanisms and signaling functions of NADPH oxidase-derived reactive oxygen species (ROS) will be characterized in vitro and in vivo with respect to cell-autonomous and tissue-specific defense.

During the first funding period we have unravelled the mechanism of TNF-triggered production of ROS by identifying the riboflavin kinase (RFK) as a novel TNF receptor 1 (TNFR1)-binding protein. RFK physically links TNFR1 with NADPH oxidase and thereby provides to NADPH oxidase the prosthetic group FAD which is required for maximal production of the oxidative antimicrobial effector molecules ROS.

In addition we demonstrated that the TNF-activated acid sphingomyelinase (ASMase) essentially contributes to non-oxidative antimicrobial effector mechanisms by allowing proper fusion of phagosomes with lysosomes resulting in the rapid delivery of acid lysosomal hydrolases to phagosomes containing microbial pathogens. In unpublished data we could show that ROS produced by NADPH oxidase functions as a transmembrane signaling complex required for activation of ASMase within phagosomes and lysosomes. Thus, ROS contribute to cell-autonomous defense against phagocytosed microbes directly by exerting oxidative antimicrobial effector activity and indirectly by activating non-oxidative effector mechanisms.

Based on these fundamental findings, this project aims at elucidating the contributions of four isoenzymes of NADPH oxidase, Nox1 and -2 and Duox1 and -2, that have previously been implicated in tissue-specific host defense against microbial infections. Of special note for this project is that the isoenzymes Nox1, Nox2, Duox1, and Duox2 have distinct activation requirements and that during infection in vivo host cells are exposed simultaneously or consecutively to a variety of stimuli that can differentially influence the activity of NADPH oxidases. These stimuli include on the one hand pathogen-associated molecular patterns (PAMP) activating Toll-like receptors (TLR) or NOD-like receptors (NLR) and on the other hand host-derived danger-associated molecular patterns (DAMP), e.g. ATP as well as cytokines.

In view of these complex premises, we aim at dissecting the direct and indirect antimicrobial effector activities of Nox1, Nox2 as well as of Duox1 and -2 NADPH oxidase isoenzymes. Specifically, we will stimulate phagocytic and non-phagocytic host cells with pathophysiologically relevant combinations of agonists for TLRs, NLRs together with DAMP and cytokines and, thereafter, characterize qualitatively and quantitatively the direct, oxidative antimicrobial activities and the signaling effects of these NADPH oxidase isoenzymes on cell-autonomous effector mechanisms, including activation of ASMase, (auto-) phagosomal maturation, and inflammasome activation.

The expected results of our study will elucidate the complex regulatory networks that allow the temporally and spatially adequate production of appropriate amounts of highly reactive ROS during cell-autonomous defense reactions against infectious pathogens.

 

List of publications resulting from the project
Peer-reviewed publications:

Daniel Erny D, Anna Lena Hrabě de Angelis, Diego Jaitin, Peter Wieghofer, Ori Staszewski , Eyal David, Hadas Keren-Shaul, Tanel Mahlakoiv, Kristin Jakobshagen, Thorsten Buch, Vera Schwierzeck, Olaf Utermöhlen, Eunyoung Chun, Wendy S. Garrett, Kathy D. McCoy, Andreas Diefenbach, Peter Staeheli, Bärbel Stecher, Odo Amit, Marco Prinz. Host microbiota constantly control maturation and function of microglia in the CNS. Nat Neurosci. 2015 Jun 1. doi:10.1038/nn.4030

Lars Ketscher, Ronny Hannß, David Morales, Anja Basters, Susana Guerra, Tobias Goldmann, Annika Hausmann, Marco Prinz, Ronald Naumann, Andrew Pekosz, Olaf Utermöhlen, Deborah J. Lenschow, Klaus-Peter Knobeloch. Selective inactivation of USP18 isopeptidase activity in vivo enhances ISG15 conjugation and viral resistance. Proc Natl Acad Sci U S A. 112:1577-82. 2015

Andreas Neerincx, Kristin Jakobshagen, Olaf Utermöhlen, Hildegard Büning, Viktor   Steimle, Thomas A. Kufer. The N-terminal domain of NLRC5 confers transcriptional activity for HC class I and II gene expression. J. Immunol. 193:3090-100. 2014

Maria Andree, Jens M Seeger, Stephan Schüll, Oliver Coutelle, Diana Wagner-Stippich, Katja Wiegmann, Claudia M Wunderlich, Kerstin Brinkmann, Pia Broxtermann, Axel Witt, Melanie Fritsch, Paola Martinelli, Harald Bielig, Tobias Lamkemeyer, Elena I Rugarli, Thomas Kaufmann, Anja Sterner-Kock, F Thomas Wunderlich, Andreas Villunger, L Miguel Martins, Martin Krönke, Thomas A Kufer, Olaf Utermöhlen, Hamid Kashkar. BID-dependent release of mitochondrial SMAC dampens XIAP-mediated immunity against Shigella. EMBO J, 33: 2171-2187. 2014

Michael Schramm, Katja Wiegmann, Sandra Schramm, Olaf Utermöhlen, Martin Krönke.  Riboflavin (vitamin B2) deficiency impairs phagocytic ROS production and defense against Listeria monocytogenes. Eur. J. Immunol., 44:728-41. 2014

Maria A. Ermolaeva, Alexandra Segred, Alexander Dakhovnik, Hui-Ling Ou, Jennifer I. Schneider, Olaf Utermöhlen, Thorsten Hoppe, Björn Schumacher. DNA damage in germ cells induces innate immune response triggering systemic stress resistance through enhanced proteostasis. Nature, 501: 416-20. 2013

Tobias Sydor, Kristine von Bargen, Fong-Fu Hsu, Gitta Huth, Otto Holst, Jens Wohlmann, Ulrike Becken, Tobias Dykstra, Kristina Söhl, Buko Lindner, John F. Prescott, Ulrich E. Schaible, Olaf Utermöhlen, Albert Haas. Diversion of phagosome trafficking by pathogenic Rhodococcus equi depends on mycolic acid chain length. Cellular Microbiology, 15:458-73. 2013

Oliver Liesenfeld, Iana Parvanova, Jens Zerrahn, Seong-Ji Han, Frederick Heinrich, Melba Munoz, Frank Kaiser, Toni Aebischer, Torsten Buch, Ari Waisman, Gabriela Reichmann, Olaf Utermöhlen, Esther von Stebut, Friederike D. von Loewenich, Christian Bogdan, Sabine Specht, Michael Saeftel, Achim Hoerauf, Maria M. Mota, Stephanie Könen Waisman, Stefan H.E. Kaufmann, Jonathan Howard. The resistance GTPase, Irga6, protects mice against Toxoplasma gondii, but not against Plasmodium berghei or several other intracellular pathogens. PloS ONE, 6(6):e20568. 2011. Epub 2011 Jun 17

Kristine von Bargen, Jens Wohlmann, Gregory Alan Taylor, Olaf Utermöhlen, Albert Haas. Nitric oxide-mediated intracellular growth restriction of pathogenic Rhodococcus equi can be prevented by iron. Infection and Immunity 79: 2098-2111. 2011.

Tobias Dykstra, Olaf Utermöhlen, Albert Haas. Defined particle ligands trigger specific defense mechanisms of macrophages. Innate Immunity 17:388-402. 2010.

Yazdanpanah, B., K. Wiegmann, V. Tchikov, O. Krut, C. Pongratz, M. Schramm, A. Kleinridders, T. Wunderlich, H. Kashkar, O. Utermöhlen, J.C. Brüning, S. Schütze, M. Krönke. 2009. Riboflavin Kinase couples TNF receptor 1 to NADPH oxidase. Nature, 460:1159-1163.

Herz, J., J. Pardo, J. Kashkar, M. Schramm, E. Bos, K. Wiegmann, R. Wallich, P.J. Peters, E. Schmelzer, M. Krönke, M.M. Simon, O. Utermöhlen. 2009. Acid sphingomyelinase is a critical regulator of cytotoxic granule secretion by primary T lymphocytes. Nature Immunology, 10:761-768.

Schramm, M., J. Herz, A. Haas, M. Krönke, and O. Utermöhlen. 2008. Acid sphingomyelinase is required for efficient phago-lysosomal fusion. Cell Microbiol 10:1839-1853.

Ermolaeva, M.A., M.-C. Michallet, N. Papadopoulou, O. Utermöhlen, K. Kranidioti, G. Kollias, J. Tschopp, M. Pasparakis. 2008. Role of TRADD in TNFR1-signaling and in TRIF-dependent inflammatory responses. Nature Immunology 9.1037-1046.

 

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