Peptidoglycan recognition protein 3 [Elektronische Ressource] : a novel microbial pattern recognition receptor mediating immune regulation depending on microbial and dietary factors / vorgelegt von Marwa Zenhom Mahmoud

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• Publié par: christian-albrechts-universitat_zu_kiel
• Publié le: 01 janvier 2011
• Nombre de lectures: 31
• Poids de l'ouvrage: 5 Mo

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Peptidoglycan Recognition Protein 3:
A novel Microbial Pattern Recognition Receptor
Mediating Immune Regulation Depending on
Microbial and Dietary Factors





DISSERTATION

zur Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Christian-Albrechts-Universität
zu Kiel











vorgelegt von
M.Sc. Marwa Zenhom Mahmoud


Kiel, 2011















Dekan: Prof. Dr. Lutz Kipp


1. Berichterstatter: Prof. Dr. Thomas Roeder

2. Berichterstatter: Prof. Dr. Jürgen Schrezenmeir

Tag der mündlichen Prüfung: 02/03/2011
Zum Druck genehmigt: 14/04/2011









My lovely kids TABLE OF CONTENTS
List of Figures..…………………………………………………………………………… III
Summary ……………………………………………………………..……………........... IV–V
Zusammenfassung ................................................................................................... VI–VII
Abbreviations ........................................................................................................... VIII
Chapter I: General introduction..…………………………………………………….............................. 1–14
Hypothesis and aim of the work…………………………………………………………................................... 1
Review of Literature……………………………………………………………………………………………….… 3
The intestinal environment……………………………………………………...................................... 3
Intestinal bacteria produce fatty acids…………………………………………………………………. 4
Dietary fatty acids are known to modulate immune responses in the intestine....................... 5
The interaction between intestinal epithelial cells and microbes………… 6
Peptidoglycan Recognition proteins…………………………………………………………………… 7

References……………………………………………………………………………………………..... 11
Chapter II: PPARγ-dependent Peptidoglycan recognition protein 3 (PGlyRP3)
15–53 expression regulates proinflammatory cytokines by microbial and dietary fatty acids……….
Abstract…………………………………………………………………………………………………………… 17
Introduction……………………………………………………………………………………………………… 18
Material and Methods…… 21
Cell culture………………………………………………………………………………………………….. 21
In silico analysis…………………………………………………………………………………………… 21
Electron mobility shift assay (EMSA)…………………………………………………………………. 22
Preparation of fatty acids and incubations………………………………………………………….. 23
RNA isolation, cDNA synthesis, and real time PCR………………………………………………... 23
Quantitative real time PCR analysis…………..……………….. 24
Transfection of Caco2 cells with PGlyRP3 plasmid………….…………………………………….. 24
Small interfering (si) RNA experiments………………………………………………………...…….. 25
Protein extraction and Western blot analysis……………………………………………………..… 26
Interleukin 12 protein assay……………………………………………………………..……………… 26
TransAm assay for NF-κB nuclear localization……………………………………... 26
27 Results…………………………………………………………………………………………………..…………
Detection of PGlyRP3 gene and protein in Caco2 cells…………………………………………… 27
PPARγ and PPARγ-RXRα complex bind to PGlyRP3 promoter…………………………………. 29
PGlyRP3 gene expression is regulated by a PPARγ agonist and different FFA……………... 31
FFA regulate PGlyRP3 gene expression through PPARγ……………………………..………...... 31
FFA modulate the gene expression of the cytokines IL-8, IL-12p35 and TNF-α…………….... 32
PGlyRP3 down-regulates inflammatory cytokine expression in Caco2 cells…………………. 34
FFA that upregulate PGlyRP3 inhibit cytokine gene expression……………………….………... 36
PGlyRP3 anti-inflammatory role includes the negative regulation of NF-κB pathwa………... 37

39 Discussion………………………………………………………………………………………………………….
44 References………………………………………...…………………………………………….
49 Supplementary Data………………………………………………..……………………………………..........
I Chapter III: Prebiotic oligosaccharides exert an anti-inflammatory role depending on PPARγ
54–76 and peptidoglycan recognition protein 3 (PGlyRP3)………………………………….........................
56 Abstract……………………………………………………………………………………………………………....
57 Introduction…………………………………………………………………………………………………………
59 Material and Methods……………………………………………………………………………………………
Cell culture…………………………………………………………………………………………………….. 59
Preparation of oligosaccharides and incubation regimes…………………………………………… 60
RNA isolation, cDNA synthesis, and quantitative real time PCR analysis………………………... 60
Transfection of Caco2 cells with PGlyRP3 overexpression plasmid……………………………… 61
Small interfering (si) RNA experiments………………………………………………………………….. 62
TransAm assay for NF-κB nuclear localization………………………………………………………... 63
Protein extraction and Western blot analysis…………….. 63
Interleukin 12 protein assay……………………………………………………………………………….. 63
Statistical analyses………………………………………………………………………………………….. 63
64 Results……………...……….
Inhibition of pro-inflammatory cytokines by oligosaccharides……………………………………. 64
Dose and time-dependence of induction of PGlyRP3 by oligosaccharides……………... 66
Dependence of the anti-inflammatory effect of oligosaccharides on PGlyRP3 gene
expression……………………………………………………………………………………………………. 66
PPARγ regulates PGlyRP3 expression and triggering
the oligosaccharide-induced anti-inflammatory effects…………………………………………….. 69
71 Discussion…………………………………………………………………………………………………………
74 Reference…………………………………………………………………………………………………………..
Chapter IV: peptidoglycan recognition protein 3 (PglyRP3) has anti-inflammatory role in
78–102 intestinal epithelial cells…………………………………………………………………………………….
79 Abstract……………………………………………………………………………………………………………...
80 Introduction…………………………………………………………………………………………………………
82 Material and Methods………
Cell culture…………………………………………………………………………………………………….. 82
Preparation of PGN and incubations…………………………………………………………………….. 82
Incubation with Myeloid differentiation 88 (MyD88) inhibitor……………………………………….. 83
Transfection of Caco2 cells with PGlyRP3 plasmid…………………………………………………… 83
Small interfering RNA experiments….. 84
RNA Isolation, cDNA Synthesis, and Quantitative real time PCR analysis……………………….. 85
Protein Extraction and Western blot analysis………………………………………………………….. 86
Interleukin 12 protein assay………………………………………………………………………………... 87
Statistical analyses…... 87
87 Results……………...……………………………………………………………………………………………..…
Different PGN induced PGlyRP3 expression……………… 87
PGN induce inflammatory cytokine…………………………………………………………………….... 88
PGlyRP3 suppresses the PGN-induced inflammatory cytokines………………………………….. 89
TLR but not PGlyRP3 pathway is involved in the inflammatory response to PGNs………….... 92
95 Discussion……………...………………………………………………………………………………………….
98 References.………………………………………………………………………………………….
Chapter V: General discussion ………………………………………………………………………….. 103–123
Reference………………………………………………………………………………………………… 117
II List of Figures
Detection of PGlyRP3 gene expression in Caco2 cells 28
PPARγ and RXRα bind to PGlyRP3 promoter 30
PPARγ and FFA regulate PGlyRP3 gene expression in Caco2 cells 33
Differential effects of FFA on cytokine gene expressions 35
PGlyRP3 down regulates inflammatory cytokines’ expression in Caco2 cells 36
FFA regulate cytokine gene expression through PGlyRP3 39
A negative regulation of NF-κB pathway is involved in the anti-inflammatory PGlyRP3 role 40
Evidence of PGlyRP3 silencing / over-expression in Caco2 cells 49
FFA regulate cytokine gene expression through PGlyRP3 50
A negative regulation of NF-κB pathway is involved in the anti-inflammatory PGlyRP3 role 51
PPARγ inhibit cytokines expression 52
Inhibition of pro-inflammatory cytokine expression and NF-kB in intestinal Caco2 cells by 65
oligosaccharides
Dose and time–dependence of induction of PGlyRP3 in the intestinal Caco2 cells by oligosaccharides 67
Dependence of the anti-inflammatory effect of oligosaccharides on PGlyRP3 68
Oligosaccharides regulate PGlyRP3 expression and trigger anti-inflammatory effects through PPARγ 70
Time dependence of induction of PGlyRP3 by PGN 88
PGN induce inflammatory cytokine expression 89
PGlyRP3 overexpression reduces the PGN–induced inflammatory cytokine 91
Neutralization of the TLR-pathway by an MyD88 inhibitor suppressed the PGN–induced inflammatory 93
cytokine expression in Caco2 cells
PGlyRP3reduces the inflammatory cytokine expression despite the PGNs 94

III ABBREVIATIONS
B. subtilis Bacillus subtilis
BCFA Branched-chain fatty acid
C15 13-methylmyristic acid
C17 15-methylpalmitic acid
Human colon adenocarcinoma cell line Caco2
COX-2 Cyclooxygenase-2
Dendritic cell DC
DHA Decosahexaenoic acid
EMSA Electrophoretic mobility shift assay
EPA Eicosapentaenoic
FAE Follicle-associated epithelium
FFA Free fatty acid
FOS Fructo-oligosaccharides
Gut-associated lymphoid tissue GALT
GI Gastrointestinal
Interferon-gamma IFN- γ
IKK IkB kinase
Interleukin IL-
IRAK IL-1 receptor-associated kinase
LCFA Long-chain fatty acid
LGG Lactobacillus rhamnosus GG
LPS lipopolysaccha