Proteomic analysis of the response of murine bone marrow derived macrophages to IFN-γ [IFN gamma] stimulation and infection with Staphylococcus aureus [Elektronische Ressource] / vorgelegt von Dinh Hoang Dang Khoa

Tran Bich Thu

Sujets

• Gesundheit

Informations

• Publié par: ernst-moritz-arndt-universitat_greifswald
• Publié le: 01 janvier 2010
• Nombre de lectures: 62
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Proteomic analysis of the response of murine bone

marrow derived macrophages to IFN-γ stimulation
and infection with Staphylococcus aureus


Inauguraldissertation
zur
Erlangung des akademischen Grades
Doktor rerum naturalium (Dr. rer. nat.)
die Mathematisch-Naturwissenschaftliche Fakultät
der Ernst-Moritz-Arndt-Universität Greifswald

Vorgelegt von Dinh Hoang Dang Khoa
geboren am 12.06.1981
in Binh Thuan - Vietnam



Greifswald, July 2010


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Dekan: Prof. Dr. Klaus Fesser .....................................................................................................


1. Gutachter 1: Prof. Uwe Völker ...............................................................................................
2. Gutachter 2: Prof. Bhanu Sinha
Tag der Promotion: 9.12.2010 .....................................................................................................
......................................................................................................................................................

ii
Content
Abbreviations ................................................................................................................................... i
List of Figures and Tables ........... iii
Summary ......................................................................................................................................... 1
1. Introduction ............................... 3
1.1. Macrophages ......................................................................................................................... 3
1.1.1. Macrophage origin and morphology ...................................... 3
1.1.2. Immunological function of macrophages ............................... 3
1.1.2.1. Microbial pathogen phagocytosis ................................. 4
1.1.2.2. Antigen presentation ..................................................... 5
1.1.2.3. Immune modulation ...................................................... 5
1.1.3. Other functions of macrophages ............................................ 6
1.2. IFN gamma activation of macrophages ................ 7
1.2.1. IFN gamma ............................................................................. 7
1.2.2. Effects of IFN-γ on macrophages ........................................... 7
1.3. Proteomics studies of macrophages ...................................................... 9
1.3.1. Strategies of proteomics analysis ........... 9
1.3.2. Macrophage proteomics ....................................................... 11
1.4. Interaction of Staphylococcus aureus and macrophages .................................................... 12
1.5. A reproducible experimental system - Bone marrow derived macrophages in serum-free
culture ............................................................................................................ 14
2. Materials and Methods ........................................... 15
2.1. Materials ............................................................................................................................. 15
2.1.1. Chemicals ............................................................................. 15
2.1.2. Instruments ........................................... 16
2.1.3. Software ............... 17
2.2. Methods .............................................................................................................................. 17
2.2.1. Sample preparation ............................... 17
2.2.1.1. Stem cell preparation, cultivation, and differentiation to macrophages .... 17
2.2.1.2. Interferon-γ activation of bone marrow derived macrophages .................. 18
iii
2.2.1.3. S. aureus infection ....................................................................................... 18
2.2.1.4. BMM protein extraction for proteome analysis .......... 19
2.2.1.5. Determination of protein concentration ..................... 19
2.2.2. 2D-DIGE approach .............................................................................................. 19
2.2.2.1. CyDye labeling reaction for DIGE experiment .......................................... 19
2.2.2.2. Rehydration . 20
2.2.2.3. IEF separation ............................................................................................ 20
2.2.2.4. Equilibration ............................... 21
2.2.2.5. Second dimension separation ..................................................................... 21
2.2.3. Protein spot visualization ..................................................................................... 21
2.2.3.1. CyDye DIGE scanning ................ 21
2.2.3.2. Colloidal coomassie staining ...................................................................... 22
2.2.4. Spot detection and quantification ......... 22
2.2.5. Mass spectrometry analysis .................................................................................. 23
2.2.5.1. MALDI-TOF-MS/MS 23
2.2.5.1.1. Preparative gels ................................................................................................ 23
2.2.5.1.2. Protein identification by MALDI-TOF/TOF MS .............. 23
2.2.5.2. Quantitative LC-MS/MS analysis ............................... 24
2.2.6. Functional classification of proteins .................................................................... 26
2.2.7. Transcriptomic analysis ........................................................ 26
3. Results ....................................................................... 28
3.1. 2-DE protein reference map of BMMs ............................................................................... 30
3.2. IFN-γ effect on BALB/c and C57BL/6 macrophages ........................ 36
3.2.1. IFN-γ regulated proteins identified by 2D-DIGE technique ................................ 37
3.2.2. IFN-γ regulated proteins identified by LC-MS/MS and comparison with
transcriptomic results. ............................................................................................................... 45
3.3. Comparative proteome analysis of BALB/c and C57BL/6 macrophages ..............................
............................................ 59
3.3.1. Differences in proteomic profiles of BMM-BALB/c and BMM-C57BL/6
identified with the 2D – DIGE technique ................................................................................. 60
3.3.2. Comparison of LC-MS/MS and transcriptomic data ........... 64
iv
3.4. Effects of S. aureus infection on the proteome pattern of IFN-γ stimulated BMM-
C57BL/6 ......................................................................................................................................... 70
4. Discussion . 79
4.1. 2-DE proteome reference map of bone marrow derived macrophages .............................. 79
4.2. IFN-γ effects on BMM-BALB/c and BMM-C57BL/6 identified by proteomic 2D-DIGE
and LC-MS/MS approaches ........................................................................................................... 80
4.2.1. Transcription regulation ....................... 81
4.2.2. p47 and p65 GTPases ........................................................................................... 82
4.2.3. Antigen presentation ............................ 83
4.2.4. Metabolism ........................................................................................................... 85
4.2.5. Cell survival ......... 86
4.2.6. Secretion of cathepsin L and metalloelastase ....................................................... 87
4.2.7. Well known, immunologically important proteins not influenced by IFN-γ
treatment .............................................................................................. 88
4.3. Changes in the proteome of IFN-γ stimulated BMM-C57BL/6 due to S. aureus infection ...
............................................................................................................................................ 88
4.3.1. Anti-microbial proteins ........................ 88
4.3.2. Inflammatory regulation proteins ......... 89
4.3.3. Cell-cell interaction .............................................................................................. 91
4.3.4. Metabolism ........................................... 91
4.3.4.1. Protein and glucose uptake ......................................... 91
4.3.4.2. Lipid metabolism ......................................................................................... 92
4.3.4.3. Cellular iron homeostasis ........... 92
4.3.5. Immune-responsive gene 1 protein ...................................................................... 94
4.4. Differences in proteome of BMMs derived from strain BALB/c and C57BL/6 ................ 94
Conclusion ..................................................................... 96
References ..................................................................... 97
Affidavit
Curriculum Vitae
Acknowledgments
Supplements
v
Abbreviations

2-DE : Two dim