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Die Rolle Der Angeborenen Immunität Für Die Yersinia-Infektion Im

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Die Rolle Der Angeborenen Immunität Für Die Yersinia-Infektion Im Aus dem Max von Pettenkofer-Institut der Ludwig-Maximilians-Universität München Die Rolle der angeborenen Immunität für die Yersinia- Infektion im Mausmodell Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Fakultät für Biologie der Ludwig-Maximilians-Universität München . vorgelegt von Ekaterina Lenk geb. Vlasova 2011 Die vorliegende Arbeit wurde in der Abteilung Bakteriologie am Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie der Ludwig-Maximilians-Universität München unter der Leitung von Prof. Dr. Dr. J. Heesemann angefertigt. Die Dissertation wurde bei der Ludwig-Maximilians-Universität München eingereicht und durch die Fakultät für Biologie angenommen am: 18. August 2011 Tag der mündlichen Prüfung: 6. Dezember 2011 Vorsitzende : Prof. Dr. Elisabeth Weiß Prüfer/-in der Dissertation: 1. Gutachterin: Prof. Dr. Elisabeth Weiß 2. Gutachter: Prof. Dr. Heinrich Jung Ehrenwörtliche Versicherung Ich versichere hiermit ehrenwörtlich, dass die vorgelegte Dissertation von mir selbständig und ohne unerlaubte Hilfe angefertigt ist. München, den ............................................ ............................................................. (Unterschrift) Erklärung Hiermit erkläre ich, (x) dass die Dissertation nicht ganz oder in wesentlichen Teilen einer anderen Prüfungskommission vorgelegt worden ist. (x) dass ich mich anderweitig einer Doktorprüfung ohne Erfolg nicht unterzogen habe. ( ) dass ich mich mit Erfolg der Doktorprüfung im Hauptfach ……………………………….. und in den Nebenfächern……………………………………………………………………... bei der Fakultät für ..................................... der ……………………………………………. (Hochschule/Universität) unterzogen habe. ( ) dass ich ohne Erfolg versucht habe, eine Dissertation einzureichen oder mich der Doktorprüfung zu unterziehen. München, den............................... ........................................................................................ (Unterschrift) Meiner Familie Abkürzungsverzeichnis .................................................................................................................. V Zusammenfassung ........................................................................................................................ 1 1 Einleitung ................................................................................................................................... 3 1.1 Die Gattung Yersinia ............................................................................................................... 3 1.1.1 Yersinia enterocolitica ............................................................................................... 4 1.1.2 Infektion und Klinik der Yersiniose ............................................................................ 4 1.1.3 Pathogenese und Virulenz von Y. enterocolitica ....................................................... 5 1.1.4 Yersinia -Injektisom/Ysc-T3SS ................................................................................... 5 1.1.5 Yersinia -Effektorproteine (Yops) ............................................................................... 7 1.1.6 Das Virulenz-Antigen LcrV ...................................................................................... 10 1.2 Mechanismen der Infektionsabwehr ...................................................................................... 11 1.2.1 Toll-like Rezeptoren ................................................................................................ 12 1.2.2 NOD-like Rezeptoren .............................................................................................. 15 1.2.3 Neutrophile Polymorphkernige Leukozyten (PMN) .................................................. 17 1.2.4 Zytokine und Chemokine als Effektoren der angeborenen Immunantwort............... 20 1.2.5 IL-10 als anti-inflammatorisches Zytokin der angeborenen und adaptiven…………. Immunität................................................................................................................ 22 Zielsetzung .................................................................................................................................. 25 2 Material und Methoden ............................................................................................................. 26 2.1 Reagenzien ........................................................................................................................... 26 2.2 Geräte ................................................................................................................................... 27 2.3 Bakterien ............................................................................................................................... 27 I 2.3.1 Yersinienstämme .................................................................................................... 27 2.3.2 Nährmedien und Zusätze ........................................................................................ 28 2.3.3 Kultivierung von Yersinien ...................................................................................... 28 2.3.4 Mauspassage von Y. enterocolitica ......................................................................... 28 2.3.5 Bestimmung der Bakterienzahl für Mausinfektionsversuche ................................... 29 2.3.6 Bestimmung der Bakterienzahl für Zellkulturinfektionsversuche .............................. 29 2.4 Mausgenotypisierung ............................................................................................................ 29 2.4.1 Mauslinien .............................................................................................................. 29 2.4.2 DNA-Isolierung ....................................................................................................... 30 2.4.3 Polymerase-Kettenreaktion (PCR) .......................................................................... 31 2.4.4 Primer für Mausgenotypisierung ............................................................................. 33 2.4.5 Agarosegelelektrophorese ...................................................................................... 35 2.5 Tierexperimentelle Arbeiten ................................................................................................... 36 2.5.1 Medien und Lösungen ............................................................................................ 36 2.5.2 Gewinnung von neutrophilen Granulozyten aus der Peritonealhöhle ...................... 36 2.5.3 Gewinnung von Knochenmarkzellen ....................................................................... 37 2.5.4 Zellzahlbestimmung ................................................................................................ 37 2.5.5 Mausinfektionsmodell ............................................................................................. 37 2.5.6 Anfertigen von Gewebepräparaten ......................................................................... 39 2.5.7 Immunfluoreszenzfärbung von Gewebeschnitten .................................................... 39 2.6 Allgemeine immunologische Techniken ................................................................................. 41 2.6.1 FACS-Analyse ........................................................................................................ 41 2.6.2 Cytometric Bead Array (CBA) ................................................................................. 42 2.6.3 Stimulation von Knochenmarkzellen mit Y. enterocolitica ....................................... 44 2.6.4 Zytokinbestimmung mittels ELISA........................................................................... 45 2.6.5 ROS-Test ................................................................................................................ 46 2.6.7 Statistische Auswertungen ...................................................................................... 47 3. Ergebnisse .............................................................................................................................. 48 3.1 Untersuchungen zur Yersinia -Infektionsempfänglichkeit von Mäusen mit definierten Defekten der angeborenen Immunität ................................................................................... 48 II 3.1.1 Differentielle Anfälligkeit der ko -Mausstämme für Y. enterocolitica ......................... 50 3.1.2 Veränderung des Milzgewichtes während einer Yersinia -Infektion .......................... 52 3.1.3 Immunhistopathologische Untersuchung der Milzstruktur ....................................... 54 3.1.4 Zytologische Charakterisierung der Milzzellzusammensetzung vor und nach der Yersinia -Infektion .................................................................................................... 62 3.1.5 Charakterisierung der hämatopoetischen Zellen in der Bauchhöhle uninfizierter Mäuse ................................................................................................................... 66 3.1.6 Analyse des Infektionsverlaufs bei C57BL/6, IL-10 -/- und MyD88 -/- Mäusen ............ 69 3.1.7 Zytokinkinetik von Yersinia -infizierten C57BL/6, IL-10 -/- und MyD88 -/- Mäusen ........ 73 3.1.8 Analyse des Infektionsverlaufs bei C57BL/6, MyD88 -/- und TLR2 -/-/TLR4 -/- Mäusen…………………………………………………………………………………….. 77 3.1.9 Analyse des Infektionsverlaufs bei C57BL/6, TLR2 -/-, CD14 -/- und TLR2 -/-/CD14 -/- Mäusen .................................................................................................................
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