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Molecular mechanisms for the inhibition of the nuclear factor {_k63B [kappa-B] signal transduction in intestinal epithelial cells under conditions of chronic inflammation [Elektronische Ressource] / Pedro Antonio Ruiz-Castro

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Published 01 January 2007
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Fachgebiet für Experimentelle Ernährungsmedizin



Molecular mechanisms for the inhibition of the nuclear factor
κB signal transduction in intestinal epithelial cells under
conditions of chronic inflammation



Pedro Antonio Ruiz-Castro



Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.



Vorsitzender: Univ.-Prof. Dr. Michael Schemann

Prüfer der Dissertation: 1. Univ.-Prof. Dr. Dirk Haller
2. Univ.-Prof. Dr. Hannelore Daniel
3. Univ.-Prof. Dr. Roland M. Schmid


Die Dissertation wurde am 08.02.2007 bei der Technischen Universität München
eingereicht und durch die Fakultät für Ernährung, Landnutzung und Umwelt am
30.07.2007 angenommen.









































A mis padres,
Rosa María Castro Abad y Pedro Ruiz Gil
por su constante ánimo y apoyo


The presented thesis is based on the following peer-reviewed original papers and
submitted manuscripts:

I. Ruiz, PA, Kim, SC, Sartor, RB and Haller, D
“15-deoxy-delta12,14-prostaglandin J2-mediated ERK signaling inhibits gram-
negative bacteria-induced RelA phosphorylation and interleukin-6 gene
expression in intestinal epithelial cells through modulation of protein phosphatase
2A activity” J. Biol. Chem., 2004, Aug 20; 279(34):36103-11

II. Ruiz, PA*, Shkoda, A*, Kim, SC, Sartor, RB and Haller, D
“IL-10 gene-deficient mice lack TGF- β/Smad signaling and fail to inhibit pro-
inflammatory gene expression in intestinal epithelial cells after the colonization
with colitogenic Enterococcus faecalis” J. Immunol., 2005, 174: 2990-2999
*Authors contributed equally

III. Ruiz, PA, Hoffmann, M, Szcesny, S, Blaut, M and Haller, D
“Innate mechanisms for Bifidobacterium lactis to activate transient pro-
inflammatory host responses in intestinal epithelial cells after the colonization of
germfree rats” Immunology, 2005, 115, 441-450

IV. Ruiz, PA and Haller, D
"Functional Diversity of Flavonoids in the Inhibition of the Pro-inflammatory NF-
κB, IRF, and Akt Signaling Pathways in Murine Intestinal Epithelial Cells" J.
Nutr., 2006, 136:664-671

V. Shkoda, A*, Ruiz, PA*, Daniel, H, Kim, SC, Rogler, G, Sartor, RB and Haller, D
“Interleukin 10 blocked endoplasmic reticulum stress in intestinal epithelial cells:
impact on chronic inflammation” Gastroenterology, 2007, 132(1): 190-207
*Authors contributed equally

VI. Ruiz, PA, Braune, A, Hölzlwimmer, G, Quintanilla-Fend and Haller, D
“Quercetin inhibits TNF-induced NF-κB transcription factor recruitment to pro-
inflammatory gene promoters in murine intestinal epithelial cells” Submitted to J.
Nutr.


















“What are the facts? Again and again and again—what are the facts? Shun
wishful thinking, ignore divine revelation, forget what "the stars foretell," avoid

opinion, care not what the neighbors think, never mind the unguessable
"verdict of history"—what are the facts, and to how many decimal places? You
pilot always into an unknown future; facts are your single clue. Get the facts!”

Robert A. Heinlein
(1907-1988)
Table of Contents vi

TABLE OF CONTENTS
SUMMARY ....................................................................................................................... 9
PROLOGUE ................................................................................................................... 10
INTRODUCTION ............................................................................................................ 11
1 Ecology of the gastrointestinal tract .......................................................................... 11
1.1 Host-microbial interactions.............................................................................. 11
1.2 Assembling of the intestinal mucosa............................................................... 13
1.3 Gut-associated lymphoid tissue ...................................................................... 14
2 Inflammatory bowel disease (IBD) ............................................................................ 16
2.1 Models of mucosal inflammation ..................................................................... 17
2.2 Probiotics and IBD........................................................................................... 19
2.3 Terapeutic role of flavonoids in IBD ................................................................ 22
3 IECs and the mucosal immune system 24
4 NF-κB signaling......................................................................................................... 26
4.1 Rel/NF-κB and IκBα families........................................................................... 28
4.2 NF-κB signaling pathway ................................................................................ 30
5 TLR-mediated NF-κB activation 34
5.1 TLR4................................................................................................................ 36
5.2 TLR2 36
5.3 TLR-mediated signaling pathways .................................................................. 37
6 MAPKs ...................................................................................................................... 39
7 Cytokine signaling and IBD....................................................................................... 41
7.1 TGF-β/Smad signaling .................................................................................... 41
7.2 IL-10 signaling pathway................................................................................... 43
AIMS OF THE WORK .................................................................................................... 46
RESULTS AND DISCUSSION 48
12,141 15-deoxy-Δ -prostaglandin J inhibits Gram-negative bacteria-induced 2
pro-inflammatory responses in IECs through modulation of protein
phosphatase 2A activity ............................................................................................ 48
Table of Contents vii

2 Mechanisms for Bifidobacterium lactis BB12-induced transient pro-
inflammatory processes in the intestinal epithelium.................................................. 54
3 TGF-β1 inhibits TLR2-mediated Enterococcus faecalis induction of NF-κB
signaling pathway in IECs......................................................................................... 58
4 IL-10 abrogates ER stress response by blocking ATF-6 recruitment to the
Grp78 promoter......................................................................................................... 61
5 Functional diversity of flavonoids and its bacterial metabolites in the
inhibition of pro-inflammatory signaling pathways in IECs ........................................ 66
ZUSAMMENFASSUNG.................................................................................................. 74
REFERENCES ............................................................................................................... 75
LIST OF ABBREVIATIONS.......................................................................................... 103
LIST OF FIGURES ....................................................................................................... 107
LIST OF TABLES......................................................................................................... 108
APPENDIX 1................................................................................................................. 110
APPENDIX 2 120
APPENDIX 3 131
APPENDIX 4 142
APPENDIX 5 151
APPENDIX 6 170
ACKNOWLEDGEMENTS............................................................................................. 180
CURRICULUM VITAE .................................................................................................. 182






























Summary 9

SUMMARY
Intestinal bowel disease (IBD) is an inflammatory disorder which results from an
exacerbated mucosal immune response induced by the indigenous microbiota. The
nuclear factor (NF)-κB is responsible for the expression of many genes involved in
immunity and inflammation. In the present work, animal models as well as cell lines were
used to characterize the molecular mechanisms for the downregulation of the NF-κB
signalling pathway in intestinal epithelial cells (IECs), which play a crucial role in mucosal
immune homeostasis. Non-pathogenic Gram-negative Bacteroides vulgatus was found
to induce NF-κB activation in germ-free rats as well as in cultured IECs. Studies in vitro
12,14evidenced a mechanism by which the anti-inflammatory mediator 15-deoxy-Δ -
prostaglandin J (15d-PGJ ) reversed Gram-negative bacteria-induced NF-κB 2 2
phosphorylation through activation of protein phosphatase (PP)2A. Paradoxically, the
probiotic bacterium Bifidobacterium lactis strain BB12 triggered a higher transient pro-
inflammatory gene expression than B. vulgatus in the intestinal epithelium of germ-free
rats. This response was concomitant with suppressive mechanisms, pointing to
tolerance-inducing processes in the intestinal epithelium. Reconstitution of the IEC line
Mode K with Toll-like receptor (TLR)4 and stable transfection of non-functional TLR2
helped us to identify the latter as the target for B. lactis BB12 activation of NF-κB. The
same was found to be true for the colitogenic Enterococcus faecalis. Transforming
growth factor (TGF)-β had the ability to abrogate E. faecalis-induced interleukin (IL)-6
expression by blocking NF-κB recruitment to its promoter as shown by chromatin
immunoprecipitation (ChIP) analysis in vitro. Together with TGF-β, IL-10 plays a
predominant role in the control of inflammation. Experiments with IL-10 receptor (IL10R)-
reconstituted Mode K cells demonstrated the ability of IL-10 to inhibit tumor necrosis
factor (TNF)-induced recruitment of activating transcription factor (ATF)-6 to the
promoter of the glucose regulated protein (Grp)78, a crucial mediator of endoplasmatic
reticulum (ER) stress response. An additional major focus in the present work was the
characterization of the molecular anti-inflammatory effects of flavonoids. These plant-
derived polyphenols presented a striking functional diversity in the inhibition of TNF-
induced NF-κB signalling cascade. In addition, the flavonol quercetin inhibited pro-
ΔAREinflammatory gene expression in TNF mice, an animal model for chronic ileitis,
pointing to its possible therapeutic use in chronic inflammation.
Prologue 10

PROLOGUE
There is a war going on. An unheeded war taking place on the surfaces of the intestinal
mucosa from the very moment of our birth. Lined up on the one side we have the enteric
bacteria, outnumbering by a factor of ten the cells of our whole body. They are faded on
the other side by the mucosal immune system, always on the edge and in a constant
pre-inflammatory status, ready to launch a devastating attack on the intruders. In the
middle of this battlefield are the epithelial cells, sentinels taking active part in the decision
of whether or not to wage war. Normally there are repressive mechanisms which curb
the aggression, peacemakers abrogating the total war. This is fortunate, because not
only is this a war we cannot hope to win, but also a war we do not want to win. However,
some people either harbour bacteria which are too vicious, have an immune system
which is too aggressive, or perhaps have peacemakers performing their duties
insufficiently. This results in the break out of hostilities and the inevitable rise of war in
periodic waves with dramatic consequences for the host.

Approximately 4 million people worldwide suffer from IBD, an intestinal inflammatory
condition of unknown origin. Therapies which rely on anti-inflammatories and
immunosuppressants are effective in inducing and maintaining remission, but side
effects and lack of response in some patients limit their utility. The inflammatory
response consists of the sequential release of mediators and the recruitment of
circulating leukocytes, which become activated at the inflammatory site and in turn
release more pro-inflammatory mediators. In most cases, the inflammatory response is
resolved by the action of anti-inflammatory cytokines as well as the increased production
of intracellular suppressive factors. Nevertheless, the persistent accumulation and
activation of leukocytes are a hallmark of chronic inflammation, pointing to a dysfunction
of negative regulatory mechanisms. An emerging line of research stresses the crucial
contribution of IECs to intestinal homeostasis and inflammation due to their strategic
location in the complex network of the mucosal immune system and their ability to
respond to a wide range of stimuli. Rapid cellular response, which is critical for host
defence, is coordinated by a regulatory network of signal transduction pathways leading
to selective activation of transcription factors. These control the expression of a set of
target genes, depending on the cell type and the nature of the stimuli. Molecular biology
approaches leading to the understanding of cellular mechanisms involved in
inflammation could potentially give rise to new targets for therapeutic intervention.