The role of cytosolic RNA and DNA recognition in systemic autoimmunity and immune complex glomerulonephritis [Elektronische Ressource] / vorgelegt von Ramanjaneyulu Allam

Allam, Ramanjaneyulu

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 24
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Aus der Medizinischen Poliklinik – Innenstadt
der Ludwig-Maximilians-Universität München
Komm. Direktor: Prof. Dr. med. Martin Reincke









The role of cytosolic RNA and DNA recognition
in systemic autoimmunity and
immune complex glomerulonephritis





Dissertation
zum Erwerb des Doktorgrades der Humanbiologie
an der Medizinischen Fakultät der
Ludwig-Maximilians-Universität zu München




vorgelegt von
Ramanjaneyulu Allam
Vempalli, India
2010








Mit Genehmigung der Medizinischen Fakultät
der Universität München







1.Berichterstatter: Prof. Dr. med. Hans- Joachim. Anders

2.Berichterstatter: Prof. Dr. Ludger Klein

1.Mitberichterstatter: Priv. Doz. Dr. Lutz T. Weber

2.Mitberichterstatter: Prof. Dr. W Lange

Dekan: Prof. Dr. med. Dr. h.c. M. Reiser, FACR, FRCR

Tag der mündlichen Prüfung: 20.05.2010




























Ramanjaneyulu Allam M. Sc.
Med. Poliklinik, Klinische Biochemie,
Ludwig-Maximillians University (LMU),
Schiller straße-42, Munich- 80336,
Germany
[e-mail]

DECLARATION

I here by declare that the present work embodied in this thesis was carried out by me under
the supervision of Prof. Dr. Hans Joachim Anders, Internist-Nephrologe-Rheumatologie,
Medizinische Poliklinik-Innenstadt Klinikum der Universität München. This work has not
been submitted in part or full to any other university or institute for any degree or diploma.
This work has been published in two journals.
1) Allam R et. al. Viral RNA and DNA Trigger Common Antiviral Responses in Mesangial
Cells. J Am Soc Nephrol. 2009; 20(9):1986-96.

2) Allam R et. al. Viral 5'-triphosphate RNA and non-CpG DNA aggravate autoimmunity and
lupus nephritis via distinct TLR-independent immune responses. Eur J Immunol. 2008;
38(12):3487-98.







Ramanjaneyulu Allam

Date: 20-05-2010






ACKNOWLEDGEMENTS


I would like to sincerely thank my supervisor Prof. Dr. Hans-Joachim Anders for his support
and guidance during my work in the laboratory. I am indebted to him for giving me scientific
freedom and formulating my ideas in right direction.
It is my pleasure to thank Prof. Dr. Stefan Endres, Leader of GRAKO1202, LMU, for
allowing me to become a member of graduate students network during my Ph.D. tenure, and
Deutsche Forschungsgemeinschaft (DFG) for the grant that supported me during the course of
my research term (2006-2009).
I wish to thank all the staff members, and colleagues of the Department of Klinische
Biochemie, Nephrologisches Zentrum, Medical Policlinic, for their help, co-operation and for
providing a friendly environment.
I wish to thank all researchers and their encouraging discussions during meetings and
seminars of GRAKO1202.
I wish to express my profound gratitude to, Dan Draganovici, Ewa Radomska and Jana
Mandelbaum for providing skillful technical assistance to carry out the research work
successfully.
I would like to thank my past and current colleagues of this institute: Rahul, Onkar, Julia,
Sufyan, Anela, Nuru, Mi, Maciej, Olga, Dilip, Veronika, Stephanie, Robert, Anil, Murthy,
Peter, Kathi, Lilli, Henny, Holger, Ali, Anne, Christian, christoph, Pati, Sara, Liliana, Ilka,
Farha and Khader.
I would like to take this opportunity to mention here few of the best pals during my stay in
Munich: Nagendran, Rajesh, Pandu, Ravi, Pallavi, Praveen, Vishal, Arun, Tarun, Shiva,
Johny.
I would like thank my childhood friends Amarnath, Nageswara Rao, Dakka sreenu,
Ramchand, Hari, Sunil and Sagar
Many thanks to my brother Siva sankar, my uncle Venu Gopal, Annapurna, Manisha and
other family members.

















Dedicated to

My loving parents


















CONTENTS PAGE


1. Introduction

1.1 Systemic lupus erythematosus 1
1.2 Pathogenesis of lupus 3
1.2.1 Cell death 3
1.2.2 Nucleosomes 4
1.2.3 T and B lymphocytes 5
1.2.4 Autoantibodies 7
1.2.5 Type 1 interferons and other proinflammatory cytokines 9
1.3 Pattern recognition receptors 12
1.3.1 Toll like receptors 12
1.3.2 Cytosolic nucleic acid pattern recognition receptors 17
1.3.3 The inflammasome 22
1.4 Potential role of pattern recognition receptors in autoimmunity 23
1.5 MRLlpr/lpr mice- experimental mouse model of lupus 27

2. Hypothesis/objectives 29

3. Materials and methods 30

3.1 Materials 30
3.2 Methods 34
3.2.1 Cell culture and stimulation experiments
3.2.2 RNA isolation, cDNA synthesis and real-time –PCR 35
3.2.3 Microarray studies 40
3.2.4 RNA silencing 41
3.2.5 Western blotting 41
3.2.6 Animals and experimental protocol 43
3.2.7 Morphological and histological analysis 44
3.2.8 Evaluation of serum autoantibodies 46
3.2.9 Flow cytometry 48
3.2.10 Other methods 49
3.2.11 Statistical analysis 50

4. Results 51

4.1 Results part-I 51
4.1.1 TLR-independent IL-6 induction by 3P-RNA and non-CpG-DNA
in vio 51
4.1.2 3P-RNA and non-CpG-DNA dose dependent studies in
MRLlpr/lpr mice 51
4.1.3 Non-CpG-DNA induces serum cytokines in MRLlpr/lpr mice 53
4.1.4 Non-CpG-DNA induces lymphoproliferation and splenomegaly 53
4.1.5 Non-CpG-DNA increased negative T cells and plasma cells 55
4.1.6 Dendritic cell activation 56
4.1.7 Expression of inflammatory mediators and transcription factors
in spleen 57
4.1.8 Hypergammaglobulinemia and DNA autoantibodies 59 4.1.9 Renal inflammatory mediator mRNA expression 61
4.1.10 Glomerular IgG and complement deposits 61
4.1.11 Kidney histopathology 63
4.1.12 Localization of 3P-RNA and non-CpG-DNA in mice kidneys 65
4.1.13 3P-RNA and non-CpG-DNA induce interferon-related mediators
in kidney glomeruli of C57BL/6 mice 66
4.1.14 Backbone chemistry of DNA affects their affinity to lupus
Autoantibodies 68

4.2 Results part-II 69
4.2.1 Characterization of mesangial cells 69
4.2.2 Mesangial cells express nucleic acid-specific pattern
recognition molecules 69
4.2.3 Cationic lipids enhance the uptake of non-CpG-DNA and 3P-RNA
in mesangial cells 70
4.2.4 3P-RNA and non-CpG-DNA activate pMC to produce IL-6 71
4.2.5 d non-CpG-DNA activate pMC through a TLR-
independent pathway 73
4.2.6 Rig-1 mediates 3P-RNA but not non-CpG-DNA induced
activation of mesangial cells 74
4.2.7 Dai contributes to3P-RNA but not non-CpG-DNA induced of me
4.2.8 3P-RNA and non-CpG-DNA both activates interferon-
regulated factor-3 in mesangial cells 77
4.2.9 Unique but overlapping gene expression program triggered
by 3P-RNA and non-CpG-DNA in MC 77
4.2.10 3P-RNA and non-CpG-DNA trigger proinflammatory
cytokines in mesangial cells 80
4.2.11 d non-CpG-DNA trigger type 1 interferon and
interferon-related mediators in mesangial cells 81
4.2.12 3P-RNA and non-CpG-DNA both trigger apoptosis in MC 83

5. Discusion 85

6. Sumary 93
7. Zusamenfasung 94

8. References 95

9. Abreviatons 104

Apendix 106

Curriculum Vitae 108
1. Introduction

1.1 Systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic immune disorder, classically depicted as
a systemic autoimmune disease caused by the production of pathogenic autoantibodies to a
spectrum of nuclear antigens. The clinical manifestations include immune complex-
mediated glomerulonephritis, arthritis, vasculitis, cerebritis, pericarditis, cytopenias and
serositis [1]. SLE affects females more frequently than males, at a rate of almost 9 to 1; this
would argue that female hormones might play a role in disease incidence. The prevalence
of SLE ranges from approximately 40 cases per 100,000 persons among Northern
Europeans to more than 200 per 100,000 persons among blacks [2]. However there is a
wide variation in the prevalence of SLE worldwide, the highest prevalence was reported in
Italy, Spain, Martinique, and the UK Afro-Caribbean population and shown in Figure 1.
The recent advancements in understandi