Differential regulation of IL-12p70 and IL-23 in murine dendritic cells [Elektronische Ressource] / vorgelegt von Cornelia Richter

Conny Keyber

Sujets

• Gesundheit

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2009
• Nombre de lectures: 31
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

Aus dem Fachbereich Medizin
der Johann Wolfgang Goethe-Universität
Frankfurt am Main

Institut für Allgemeine Pharmakologie und Toxikologie
Direktor: Prof. Dr. Josef M. Pfeilschifter


“Differential regulation of IL-12p70
and IL-23 in murine dendritic cells”


Dissertation
zur Erlangung des Doktorgrades der theoretischen Medizin
des Fachbereichs Medizin der
Johann Wolfgang Goethe-Universität Frankfurt am Main


vorgelegt von
Cornelia Richter
aus Dresden



Frankfurt am Main, 2009
Index Richter




























Dekan: Prof. Dr. Josef M. Pfeilschifter
Referent: Prof. Dr. Heinfried H. Radeke
Koreferent: Prof. Dr. Wolf-Henning Boehncke


Tag der mündlichen Prüfung: 8. April 2010
Index Richter

1 SUMMARY 1
2 INTRODUCTION 1
2.1 Innate and adaptive immunity 1
2.1.1 Innate immunity – antigen presenting cells 1
2.1.2 Adaptive immunity – T lymphocytes 2
2.2 Autoimmunity 3
2.3 Dendritic cells 6
2.3.1 Conventional dendritic cells 6
2.3.2 Plasmacytoid dendritic cells 7
2.4 Toll-like receptors 7
2.4.1 Toll-like receptor 4 10
2.4.2 Toll-like receptor 9 10
2.5 Cytokines 11
2.5.1 Interleukin-12 12
2.5.2 Interleukin-23 14
2.5.3 Interferon-α 15
2.5.4 Interferon-γ 16
2.5.5 Interleukin-17 17
2.6 Selenium 17
phox2.7 Different roles of p47 18
phox2.7.1 p47 as component of the NADPH oxidase 18
phox2.7.2 p47 in autoimmune diseases 20
phox2.7.3 p47 in TLR signaling 20
2.8 Vitamin D 21 3
2.9 Objectives 22
3 MATERIAL AND METHODS 24
3.1 Mouse and rat strains 24
3.1.1 C57BL/6 strain 24
3.1.2 BALB/c strain 24
phox-/-3.1.3 p47 mice 24
phox3.1.2 Natural mutated p47 mice 25
phox-/-3.1.3 gp91 mice 25
-/-3.1.4 TRIF mice 25
I
Index Richter

-/-3.1.5 MyD88 mice 26
phox3.1.6 p47 mutated rats 26
3.2 Methods of cell biology 27
3.2.1 Cultivation and stimulation of the immature Langerhans cell line
XS-52 27
3.2.2 Isolation of primary cells from spleen and lymph nodes 27
+3.2.3 Purification of CD11c dendritic cells from the spleen 28
3.2.4 Isolation and differentiation of BM-derived cells 29
3.2.5 Cultivation and stimulation of primary and BM-derived,
differentiated cells 30
3.2.6 FACS analyses 30
3.2.7 Concentration of the supernatant 31
3.2.8 Protein isolation from whole cells 31
3.2.9 Protein assays 31
3.3.10 Detection of Reactive Oxygen Species (ROS) 32
3.3 Immunological methods 32
3.3.1 SDS PAGE 32
3.3.2 Western Blot 33
3.3.3 Enzyme-linked immunosorbent assay 35
3.3.4 ELISPOT assay 36
3.4 Molecular biological methods 36
3.4.2 cDNA synthesis 37
3.4.4 Standard PCR 37
3.4.3 Real time PCR 39
3.4.4 Polyacrylamidgel-electrophoresis 40
3.5 In vivo immunization 40
4 RESULTS 41
4.1 Regulation of IL-23 by Selenium 41
4.1.1 Regulation of IL-23 by different TLR ligands in combination
with Selenium 41
4.1.2 Regulation of IL-23 subunits p19 and p40 by Selenium 42
phox4.2 Regulation of IL-12p70 and IL-23 by p47 46
4.2.1 IL-12p70 regulation upon TLR stimulation in primary spleen cells 46
phox4.2.1.1 Regulation of IL-12p70 in p47 knockout mice 46
II
Index Richter

phox4.2.1.2 Regulation of IL-12p70 in p47 mutated mice 48
phox4.2.1.3 Regulation mutated rats 49
4.2.2 IL-23 production upon TLR stimulation in primary spleen cells 50
4.2.3 Regulation of other cytokines in primary spleen cells 52
phox4.2.3.1 Regulation of IFN-α in p47 knockout mice 52
phox4.2.3.2 Regulation of IL-10 in p47 knockout and mutated mice 52
4.2.4 ROS independent feedback regulation of IL-12p70 53
phox phox4.2.4.1 Comparison of p47 and gp91 knockout spleen cells 53
4.2.4.2 Inhibition of ROS with DPI 54
4.2.5 MyD88 dependent regulation of IL-12p70 55
+4.2.6 IL-12p70 regulation in CD11c enriched spleen cells 56
4.2.7 IL-12p70 and IL-23 regulation in BM-derived DCs 58
4.2.7.1 IL-12p70 and IL-23 regulation in BM-derived mDCs 58
4.2.7.3 IL-12p70 and IL-23 regulation in BM-derived pDCs 59
4.2.7.3 Characterization of BM-derived mDC and pDC by CD antigens 60
4.2.8 Adjuvant-guided T cell response in vivo 61
4.2.8.1 Th1 response of OVA re-stimulated LN cells and spleen cells 61
4.2.8.2 IL-12p70 response in LN cells from immunized mice in vitro 64
4.2.8.3 Th17 response of OVA re-stimulated LN cells and spleen cells 66
4.2.8.4 IL-23 response LN cells from immunized mice in vitro 69
4.3 Role of Vitamin D on IL-12p70 expression 71 3
4.3.1 IL-12p70 regulation by VD in different mouse strains 71 3
4.3.1.1 IL-12p70 regulation in C57BL/6 spleen cells 71
4.3.1.2 IL-12p70 regulation in BALB/c spleen cells 72
+4.3.2 IL-12p70 regulation by VD in CD11c cells 74 3
phox-/-4.3.3 IL-12p70 regulat in p47 and WT spleen cells 75 3
5 Discussion 76
5.1 Regulation of IL-23 by Selenium 76
phox5.2 Regulation of IL-12p70 and IL-23 by p47 77
5.3 Role of Vitamin D on IL-12p70 expression 83 3
6 OUTLOOK 85
7 LITERATURE 86
8 APPENDIX 109
III
Index Richter

8.1 Abbreviations 109
8.2 Deutsche Zusammenfassung 112
8.3 Figures and Tables 116
8.4 Journal publications and congress contributions 118
9 DANKSAGUNG 119
10 CURRICULUM VITAE 120
11 SELBSTÄNDIGKEITSERKLÄRUNGFehler! Textmarke nicht definiert.
IV
1 Summary Richter
1 SUMMARY
Dendritic cells are the sentinels between the innate and the adaptive immunity.
They are professionals that capture invading pathogens, recognize specific
microbial structures and induce naïve T lymphocytes to polarize into a specific T
cell subset. To initiate the T cell polarization DCs secrete cytokines which are
induced upon Toll-like receptor activation by microbial structures. The recognition
of these structures and the discrimination between non-self and self structures by
TLRs is fine tuned, but under defined circumstances deregulation of immune
responses appears. Consequently, this can result in immune disorders such as
autoimmunity, chronic inflammatory diseases or cancer. In this thesis the
investigations are focused on the regulation of the IL-12 family members IL-12p70
and IL-23 in DCs. The objective was to investigate three different endogenous and
exogenous factors that regulate IL-12p70 or IL-23.
In the first part Selenium, an essential trace element and important factor in
several metabolic pathways including the cellular redox status and reactive oxygen
species (ROS) dependent signaling was applied as supplement in immature
Langerhans cell culture. Because Selenium also plays a role in the immune
system the TLR-induced IL-23 production of the DCs upon Selenium treatment
was analyzed. In the immature Langerhans cell line XS-52 the strongest inducer of
IL-23 was TLR4 ligand LPS. Furthermore increased levels of TLR4-induced IL-23
in cells treated with Selenium were detected in a concentration dependent
manner. Whereas the IL-23 subunit p40 was upregulated upon Selenium
treatment the second subunit p19 was completely unaffected. This effect was
detected on mRNA and protein level. In addition, as expected, IFN-γ inhibited the
TLR4-induced IL-23 secretion of both, Selenium treated and untreated cells.
phoxIn the second part of this thesis p47 , an organizing protein of the NADPH
oxidase was analyzed regarding its potential to regulate IL-12p70 and/or IL-23
phoxsecreted by different DC subtypes. Since it was demonstrated that p47
deficiency is associated with enhanced autoimmunity and chronic inflammation we
wanted to prove whether it has a function in addition to that within the NADPH
phoxoxidase. We found some hints that p47 may be interact with proteins of the
phoxTLR signaling pathway and thus we hypothesized that p47 may have a function
for the regulation of TLR-mediated cytokine production in DCs. In several
1
1 Summary Richter
phoxexperiments with DCs from the spleen of different p47 deficient mice we
detected an increased production of TLR9-induced IL-12p70 compared to wild
type cells. In contrast TLR4 stimulation with LPS displayed no significant
phoxdifferences between p47 deficient and wild type cells. In spleen cells IL-23 was
phoxnot detected. Confirming the results of this new negative feedback by p47 on
phoxIL-12p70 rats, with a single nucleotide polymorphism in the p47 gene, were
investigated. Interestingly this polymorphism is located in the phosphorylation site
of IRAK4, an important kinase in the TLR pathway. In rats with a methionine
phoxresidue at this position in the p47 protein enhanced IL-12p70 level were found,
compared to the rats with threonine, which can be phosphorylated by IRAK4. All
analyzed mice and rats have defects in the NADPH oxidase function due to a non
phoxfunctional p47 protein which results in a defective ROS production. To
determine whether the observed negative feedback mechanism is connected to
phoxthe lack of ROS production ex