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«Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany ...»

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submitted to the

Combined Faculties for the Natural Sciences and for Mathematics

of the Ruperto-Carola University of Heidelberg, Germany

for the degree of

Doctor of Natural Sciences

presented by

Diplom-Biochemist Maria A. Ermolaeva

Born in Puschino, Moscow Region, Russia

Oral examination: ___________________

Analysis of the physiological function of TNF Receptor I

Associated Death Domain Protein (TRADD) and

Familial Cylindromatosis Protein (CYLD) by using

conditional gene targeting in mice.

Referees: Dr. Walter Witke Prof. Dr. Günter Hämmerling Acknowledgments.

This dissertation was started at the EMBL Mouse Biology Unit in Monterotondo, Italy and then continued at the Department of Mouse Genetics and Inflammation, Institute for Genetics, University of Cologne, Germany. During the course of my dissertation I was a member of the EMBL International PhD Programme and MUGEN PhD Research School. I would like to thank these institutions for providing me with excellent technical support and an extremely stimulating international scientific environment. I am grateful to the Louis-Jeantet Foundation for supporting my work scientifically and financially through a pre-doctoral fellowship.

Foremost, I would like to express my special gratitude to my supervisor Prof. Manolis Pasparakis who directed my pre-doctoral training and gave me the opportunity to work in a very challenging research context. I would like to thank Prof. Pasparakis for introducing me into the theoretical and methodological basis of my research, for his support and constructive criticism.

My next acknowledgments go to the present and former members of the Pasparakis group. My entire project would certainly not be possible without your help and support at both a scientific and personal level. Thank you very much.

I would like to thank the members of my Thesis Advisory Committee - Dr. Walter Witke, Dr. Anne Ephrussi and Prof. Denis Duboule, for the critical evaluation of my work, helpful suggestions and encouragement.

I would like to thank our collaborators:

Prof. Jurg Tschopp and Dr. Marie-Cécile Michallet (Department of Biochemistry, University of Lausanne) for the analysis of TNFR1- and TLR-complex formation.

Prof. George Kollias and Dr. Ksanthi Kranidioti (Institute of Immunology, Biomedical Sciences Research Center "Alexander Fleming", Athens) for the structural and functional analysis of secondary lymphoid organs of TRADD deficient mice.

Dr. Olaf Utermöhlen (Institute for Medical Microbiology, Immunology and Hygiene, Medical Center of the University of Cologne) and Dr. Nikoletta Papadopoulou (Department of Mouse Genetics and Inflammation, Institute for Genetics, University of Cologne) for the in vivo experiments with Listeria monocytogenes.

Dr. Gilles Courtois and Dr. Helene Sebban (INSERM U697, Paris) for the biochemical analysis of CYLD mutant cells.

I would like to thank my family and my friends for their patience and constant support of my professional efforts.

Furthermore I would like to express my gratitude to all my present and former colleagues and supervisors, to my fellow graduate students at EMBL and in Germany, to all the student assistants, and to the general support staff.

Analysis of the physiological function of TNF Receptor I Associated Death Domain Protein (TRADD) and Familial Cylindromatosis Protein (CYLD) by using conditional gene targeting in mice.

Maria A. Ermolaeva. EMBL-Mouse Biology Unit, Monterotondo, Italy; Department of Mouse Genetics and Inflammation, Institute for Genetics, University of Cologne, Cologne, Germany.

The aim of the project was to apply the conditional gene targeting approach based on the usage of Cre/LoxP system of site-specific DNA recombination to investigate physiological function of two putative mediators of inflammation – TNF Receptor 1 Associated Death Domain Protein (TRADD) and Familial Cylindromatosis Protein (CYLD), in a mouse model.

TRADD is an adaptor molecule postulated to be essential for signal transduction through TNF Receptor 1 (TNFR1). The in vivo physiological role of TRADD has not been determined so far. CYLD is a tumor suppressor that has been described as a negative regulator of TNFR1-mediated signaling and signaling by Toll like Receptors (TLRs). TLRs are essential components of mammalian innate immunity belonging to a group of sensors that directly recognize bacterial and viral products as well as markers of tissue stress. Upon activation TLRs induce intracellular signaling events leading to production of cytokines, chemokines and other mediators that promote protective responses. Tumor Necrosis Factor (TNF) is a pleiotropic cytokine produced by a variety of cells upon TLR stimulation. It plays a key role in the amplification of the initial immune response. The majority of effects that are induced by TNF are dependant on TNFR1.

TLR signaling and TNF signaling through TNFR1 share common mechanism of negative regulation that is based on the the removal of K63-linked polyubiquitin chains from specific key components of receptor-associated complexes. Two de-ubiquitinating enzymes – A20 and CYLD are currently known to be responsible for this process. The physiological function of A20 is well characterized by gene knockout studies while the precise role of CYLD remains enigmatic.

We successfully generated mice carrying “floxed” (modified by the insertion of LoxP sites and extra DNA fragments at specific locations) alleles of TRADD and CYLD by using homologous recombination in embryonic stem cells. In case of TRADD the deletion of the LoxP-flanked sequence would generate a null TRADD allele; in case of CYLD the last exon of the gene would be replaced by a mutated copy resulting in the expression of C-terminally truncated form of CYLD that is lacking catalytic activity. We then generated TRADD knockout and CYLD complete mutant mice by crossing the homozygous “floxed” animals to a ubiquitous Cre-Deleter strain.

By analyzing TRADD knockout mice we could observe that TNFR1-mediated apoptosis was completely blocked in these mice while TNF-induced pro-inflammatory and anti-bacterial responses were dramatically reduced but still present.

We obtained similar results by evaluating the response of TRADD deficient primary cells to TNF. To our surprise we discovered that TRADD knockout mice had impaired immediate responses to stimulation of Toll like receptors 3 and 4.

Consistent with this observation TRADD deficient primary cells demonstrated reduced cytokine production as well as impaired activation of NF-κB and MAP kinases upon stimulation with poly(I:C) and LPS. On the basis of co-expression experiments performed in HEK293T cells we propose that TRADD is recruited to TLR adaptor TRIF via ReceptorInteracting Protein 1 (RIP1) and acts as a mediator of TRIF-dependant TLR signaling.

To our surprise CYLD homozygous mutant mice did not survive until the age of weaning. By carefully following the pups we observed that the mutants died within minutes after birth showing signs of cyanosis and respiratory distress.

Mutant pups were smaller then control littermates and demonstrated altered morphology of the tail. We then produced mutant mouse embryonic fibroblasts (MEFs) and analyzed the response of these cells to cytokines. Consistent with the role of CYLD as a negative regulator of pro-inflammatory signaling, mutant cells showed elevated activation of NF-κB and JNK cascades upon stimulation with TNF and IL-1β.

Analyse der physiologischen Funktion des TNF Rezeptor I Associated Death Domain Proteins (TRADD) und des Familial Cylindromatosis Proteins (CYLD) durch konditionelles „Gene Targeting“ in Mäusen.

Maria A. Ermolaeva. EMBL-Mouse Biology Unit, Monterotondo, Italy; Department of Mouse Genetics and Inflammation, Institut für Genetik, Universität zu Köln, Köln, Deutschland.

Das Ziel dieser Studie war die physiologische Funktion von zwei mutmaßlichen Inflammationsmediatoren – TNF Receptor 1 Associated Death Domain Protein (TRADD) und Familial Cylindromatosis Protein (CYLD) – in Mausmodelsystemen zu erforschen. Hierfür wurde die Methode des konditionellen „Gene Targetings“ angewandt, welches auf der zielgerichteten Rekombination der DNA durch das Cre/LoxP Systemberuht.

Von dem Adapter Molekül TRADD wird angenommen, dass es für die Signaltransduktion durch TNF Rezeptor 1 (TNFR1) essentiell ist. Die physiologische Rolle von TRADD wurde in vivo bisher noch nicht bestimmt. CYLD ist ein Tumorsuppressor, der TNFR1- und Toll like Receptor (TLR) vermittelte Signale inhibiert. TLRs sind essentielle Sensoren des unspezifischen Immunsystems, die sowohl bakterielle und virale Produkte als auch Stressmarker erkennen.

Werden TLRs aktiviert, so führt dies zu einer intrazellulären Signaltransduktionskaskade, die die Produktion von Zytokinen, Chemokinen und anderen Mediatoren als Schutzantwort bewirkt. Tumor Nekrose Faktor (TNF) ist ein vielseitiges Zytokin, das von verschiedenen Zelltypen zur Antwort auf TLR-Stimulation hergestellt wird. TNF spielt eine Schlüsselrolle in der Verstärkung der initialen Immunantwort. Der Großteil der TNF vermittelten Effekte ist von TNFR1 abhängig. Die von TLR- und TNFR1- vermittelten Signalkaskaden haben einen negativen Regulationsmechanismus gemeinsam. Dieser beruht auf der Abspaltung K63 verbundener Polyubiquitinketten von rezeptorassoziierten Schlüsselkomponenten. Bisher sind zwei deubiquitinierende Enzyme – A20 und CYLD – bekannt, die für diesen Prozess verantwortlich sind. Während die physiologische Funktion von A20 durch Inaktivierung des Gens gut charakterisiert wurde, ist die genaue Rolle von CYLD noch rätselhaft.

Uns ist es durch homologe Rekombination in embryonalen Stammzellen gelungen, Mäuse zu generieren, die „gefloxte“ (modifiziert durch die Insertion von LoxP Sequenzen und zusätzlicher DNA Fragmente an spezifischen Positionen) Allele von TRADD und CYLD tragen. Im Falle von TRADD ergibt die Deletion der LoxP flankierten Sequenz ein TRADD Nullallel; im Fall von CYLD wird das letzte Exon durch eine Variante ersetzt, die zur Expression einer Cterminal verkürzten Form von CYLD führt, die keine katalytische Aktivität mehr besitzt. Die Kreuzung homozygot „gefloxter“ Tiere mit einem generellen CRE-Deleter Stamm ergab dann Nachkommen, denen entweder TRADD vollständig fehlte, oder in denen das Wildtyp CYLD durch das mutierte CYLD ersetzt war.

Die Analyse der TRADD knockout Mäuse ergab, dass die TNFR1-vermittelte Apoptose in diesen Tieren vollständig blockiert war, wohingegen die TNF-induzierte proinflammatorische und antibakterielle Antwort zwar dramatisch reduziert, aber prinzipiell vorhanden war. TNF-Behandlung von TRADD defizienten Zellkulturen zeigte ähnliche Ergebnisse. Überraschenderweise war die frühe Immunantwort nach Stimulation von TLR3 und TLR4 in TRADD knockout Mäusen eingeschränkt. Übereinstimmend mit dieser Beobachtung war die verminderte Zytokinproduktion und eingeschränkte Aktivität von NF- B und MAP Kinase nach poly(I:C) Stimulation in TRADD defizienten Zellen.

Basierend auf den Ergebnissen von Koexpressionsexperimenten in Hek293T Zellen nehmen wir an, dass TRADD via RIP zum TLR Adapter TRIF rekrutiert wird und als Vermittler in der TRIF-abhängigen TLR Signalübertragung agiert.

Mäuse, die homozygote Träger des mutierten CYLD Allels waren, starben überraschenderweise innerhalb von Minuten nach der Geburt mit Zyanose- und Atemnotsymptomen, wobei die Tiere mit mutiertem CYLD Gen kleiner als Wildtypmäuse waren und eine veränderte Schwanzmorphologie aufwiesen. Deshalb analysierten wir die Reaktion auf Zytokinstimulation in mutierten embryonalen Mausfibroblasten. In den Zellen mit mutiertem CYLD war erhöhte NFkB und JNK Aktivität nach TNF und IL-1b Stimulation feststellbar, was mit einer Funktion von CYLD als negativer Regulator der proinflammatorischen Signalvermittlung im Einklang steht.

–  –  –

Table of contents.

1. Abbreviations

2. Introduction.

2.1 Tumor Necrosis Factor (TNF). TNF receptors – TNF receptor 1 (TNFR1, p55) and TNF receptor 2 (TNFR2, p75).

2.2 The role of TNF and TNF receptors in physiological and pathological processes.

2.2.1 The role of TNF in mammalian development.

2.2.2 TNF in inflammation and immunity.

2.2.3 TNF in autoimmune diseases and chronic inflammation.

2.2.4 Non-immune functions of TNF.

2.3 Tumor Necrosis Factor Receptor 1 (TNFR1).

2.4 TRADD – TNFR1 Associated Death Domain Protein.

2.5 Toll like receptors (TLRs). Signal transduction through TLR3 and TLR4.

2.6 Negative regulation of TNF Receptor I and Toll like receptor signaling

2.7 CYLD – a product of the gene mutated in Brook-Spiegler syndrome.

2.8 The familial cylindromatosis protein (CYLD) acts as a de-ubiquitinating enzyme specific for various targets.

3. Conditional targeting of TNFR1 associated Death Domain protein (TRADD) and Familial Cylindromatosis protein (CYLD). Aim of the study.

3.1 Part A: Conditional targeting of TRADD.

3.2 Part B: Conditional targeting of CYLD.

4. Materials and Methods

4.1 Generation of mutant mice.

4.1.1 Generation of TRADD deficient mice. Construction of the targeting vector. Targeting of the locus of interest in ES cells. Analysis of ES clones that were obtained as a result of targeting for the insertion of the modified allele into the appropriate genomic location.

Table of contents Preparation of ES cells for blastocyst injections. Solutions and media for ES work.

4.1.2 Generation of CYLD 932 mice Construction of the targeting vector. Targeting of the cyld gene in ES cells.

4.1.3 Genotyping of TADD deficient and CYLD ∆932 mice. TRADD CYLD∆932

4.1.4 Other mutant mice.

4.2 In vivo experiments.

4.2.1 In vivo infection of mice with Listeria monocytogenes.

4.2.2 SRBC immunization.

4.2.3 Mouse models of acute liver failure.

4.2.4 In vivo treatment of mice with TLR ligands.

4.2.5 Preparation of serum from blood.

4.3 In vitro cell culture experiments

4.3.1 Preparation of mouse embryonic fibroblasts (MEFs).

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