Download Human Intestinal Epithelial Cells in Innate Immunity

Umeå University, Medical Dissertations
New Series No. 1242-ISSN 0346-6612-ISBN 978-91-7264-721-3
Editor: The Dean of the Faculty of Medicine
From the Departments of Clinical Microbiology, Immunology
and Clinical Sciences, Pediatrics
Umeå University, Umeå, Sweden
Human Intestinal Epithelial Cells
in Innate Immunity
Interactions with normal microbiota and pathogenic bacteria
by
Gangwei Ou
Umeå 2009
ii
Copyright©Gangwei Ou
ISBN: 978-91-7264-721-3
ISSN: 0346-6612, New Series nr: 1242
Tryck/Printed by: Arkitektkopia
Umeå, Sweden 2009
Table of Contents
List of Papers…………………………………………………….….
Abstract……………………………………………………………..
Abbreviations………………………………………………………..
1. Introduction
2
3
4
5
1.1 The human intestine and mucosal immunity………………… 6
1.2 Hurdles for intestinal pathogens…………………………….. 8
1.3 Innate immunity...…………………………………………… 10
1.3.1 TLRs and their signaling……………………………… 10
1.3.2 TLR-independent pathogen recognition………………. 12
1.3.3 TLR and NOD signaling in the human intestine……… 13
1.4 Mucins………………………………………………………. 14
1.5 The carcinoembryonic antigen family……………………… 15
1.6 Antimicrobial peptides and lysozyme……………………… 17
1.7 Cytokines…………………………………………………… 20
1.7.1 Interferon-γ..….………………………………………. 21
1.7.2 Tumor necrosis factor-α.…..………………………… 22
1.7.3 Interleukin-1β………………………………………… 23
1.7.4 Interleukin-6…………………………………………. 24
1.7.5 Interleukin-8…………………………………………. 25
1.8 Intestinal microflora……………………………………….. 26
1.9 Vibrio cholerae……………………………………………. 28
1.10 Celiac disease…………………………………………….. 31
2. Aims of this Thesis………………………………………………. 36
3. Results and Discussion…………………………………………... 37
3.1 Proximal small intestinal microbiota and identification of
rod-shaped bacteria associated with childhood celiac disease
(Paper I)………………………………………………………...
3.2 Contribution of intestinal epithelial cells to innate
immunity of the human gut - studies on polarized monolayers
of colon carcinoma cells (Paper II)…………………………….
3.3 Role of V. cholerae protease, PrtV - from C. elegans to
human intestine………………………………………………...
3.3.1 PrtV protects V. cholerae from natural predator
grazing and is responsible for killing of infected C. elegans
(Paper III)…………………………………………………..
3.3.2 V. cholera cytolysin (VCC) is the secreted
proinflammatory factor and its activity is modulated by
PrtV (Paper IV)…………………………………………….
37
43
45
45
45
4. Conclusions……………………………………………………… 48
5. Acknowledgements……………………………………………... 49
6. References……………………………………………………….. 51
7. Papers I – IV...………………………………………Appendix I - IV
List of Papers
This thesis is based on the following papers referred to in the text by their
roman numbers (I-IV).
I.
Ou G, Hedberg M, Hörstedt P, Baranov V, Forsberg G, Drobni
M, Sandström O, Wai SN, Johansson I, Hammarström ML,
Hernell O, Hammarström S. Proximal Small Intestinal
Microbiota and Identification of Rod-shaped Bacteria associated
with Childhood Celiac Disease. 2009. (submitted)
II.
Ou G, Baranov V, Lundmark E, Hammarström S, Hammarström
ML. 2009. Contribution of intestinal epithelial cells to innate
immunity of the human gut - studies on polarized monolayers of
colon carcinoma cells. Scand J Immunol. 69:150-61.
III.
Vaitkevicius K, Lindmark B, Ou G, Song T, Toma C, Iwanaga
M, Zhu J, Andersson A, Hammarström ML, Tuck S, Wai SN.
2006. A Vibrio cholerae protease needed for killing of
Caenorhabditis elegans has a role in protection from natural
predator grazing. Proc Natl Acad Sci U S A. 103:9280-5.
IV.
Ou G, Rompikuntal PK, Bitar A, Lindmark B, Vaitkevicius K,
Bhakdi S, Wai SN, Hammarström ML. Vibrio cholerae cytolysin
causes an inflammatory response in human intestinal epithelial
cells that is modulated by the protease PrtV secreted by the same
bacterium. 2009. (submitted)
-2-
Abstract
Rod-shaped bacteria were previously shown to be associated with the small intestinal
epithelium of children with celiac disease (CD). Using culture-dependent and independent
methods, we characterized the microbiota of small intestine in children with CD and
controls. The normal microbiota constitutes an unique organ-specific biofilm. Dominant
bacteria are Streptococcus, Neisseria, Veillonella, Gemella, Actinomyces, Rothia and
Haemophilus. Altogether 162 Genus Level Operational Taxonomic Units (GELOTU) of six
different phyla were identified in a total of 63 children. In biopsies collected during 20042007 we did not find major differences in the microbiota between CD patients and controls.
However, in biopsies collected earlier from children born during the “Swedish CD
epidemic” and demonstrated to have rod-shaped bacteria by electron microscopy, we found
that unclassified-Clostridales and Prevotella species were associated with CD. These
anaerobic, rod-shaped bacteria showed marked affinity for the intestinal epithelium.
Changes in breast-feeding practice and/or regiments for introduction of gluten containing
food probably affect the composition of the bacterial flora in small intestine. We hypotesize
that these bacteria contribute to contraction of CD.
An in vitro model for studies of immune mechanisms of the intestinal epithelium
was established. Polarized tight monolayers of the human colon carcinoma cell lines, T84
and Caco2, were developed by culture in a two-chamber system. The two cell lines showed
the features of mature- and immature columnar epithelial cells respectively. Polarized
monolayers were challenged with bacteria and proinflammatory cytokines. Immune
responses were estimated as quantitative changes in mRNA expression levels of a secreted
mucin (MUC2), glycocalyx components (CEACAMs, MUC3), antimicrobial factors and
cytokines (IFN-γ, TNF-α, IL-6 and IL-8). Tight monolayer cells were more resistant to
bacterial attack than ordinary tissue culture cells and only B. megaterium induced the
defensin, hBD2. Tight monolayer cells responded to cytokine challenge suggesting
awareness of basolateral attack. TNF-α induced markedly increased levels of IL-8 and TNFα itself in both cell lines suggesting recruitment and activation of immune cells. Cytokine
challenge also increased levels of CEACAM1, which includes two functionally different
forms, CEACAM1-L and CEACAM1-S. In T84 cells, IFN-γ was selective for CEACAM1L while TNF-α upregulated both forms. Increased CEACAM1 expression may influence
epithelial function and communication between epithelial cells and intraepithelial
lymphocytes.
As a pathogenic enteric bacterium, Vibrio cholerae secretes cholera toxin that is the
major factor of cholera diarrhea. However, some strains of O1 serogroup lacking the cholera
toxin still cause enterocolitis and most V. cholerae vaccines candidates exhibit
reactogenicity in clinical trails. An extracellular metalloprotease PrtV was characterized. It
was associated with killing of bacteria predators such as the nematode Caenorhabditis
elegans. Its role in human intestine was addressed by using the T84 tight monolayer in vitro
model. We found that Vibrio Cholera Cytolysin (VCC), a pore-forming toxin, induces an
inflammatory response in intestinal epithelial cells that includes increased epithelial
permeability and induction of IL-8 and TNF-α and hence could be responsible for
enterocolitis. The inflammatory response was abolished by PrtV thus VCC is indeed an
autologous substrate for PrtV. In protein rich environment PrtV degradation of VCC was
inhibited, suggesting that the magnitude of the inflammatory response is modulated by the
milieu in the small intestine. Thus, VCC is likely to be part of the pathogenesis of cholera
diarrhea and the causative agent of enteropathy in V. cholerae strains lacking the cholera
toxin.
-3-
Abbreviations
AMP
CD
CEA
CEACAM
CLR
CTX
DC
FDC
HBD
HD
HLA
IEC
IEL
IFN
IL
ITIM
LP
LPS
LRR
MHC
NF-κB
NLR
NOD
PAMP
PP
PRR
PrtV
RLR
STAT
TLR
TNF
tTG
VCC
Antimicrobial peptide
Celiac disease
Carcinoembryonic antigen
Carcinoembryonic antigen cell adhesion molecule
C-type lectin receptor
Vibrio cholera toxin
Dendritic cell
Follicular dendritic cell
Human β defensin
Human α defensin
Human leukocyte antigen
Intestinal epithelial cell
Intraepithelial lymphocyte
Interferon
Interleukin
Immunoreceptor tyrosine-based inhibition motif
Lamina propria
Lipopolysaccharide
Leucin-rich repeat
Major histocompatibility complex
Nuclear factor kappa B
NOD-like receptor
Nucleotide oligomerization domain
Pathogen associated molecular pattern
Peyer´s patches
Pattern recognition receptor
Protease of V. cholerae
Retinoic acid-inducible gene (RIG)-I-like receptor
Signal transducer and activator of transcription
Toll-like receptor
Tumor necrosis factor
Tissue transglutaminase
Vibrio cholerae cytolysin
-4-
1. Introduction
The intestine serves several functions, notably ingestion, digestion and
absorption of food and defecation of waste products. It is also the host of the
largest and most complex part of the immune system indispensable for the
survival of the individual. The intestinal mucosal surface is lined by a single
layer of epithelial cells. In humans it covers an area of over 100 m2 that is
continuously exposed to different antigens in the form of food constituents,
commensal microflora, episodic pathogens, and to noxious compounds. The
epithelial cell layer constitutes the interface between the internal milieu and
the external environment. It is a highly regulated, selectively permeable
barrier that supports digestion and absorption of nutrients, electrolytes, and
water from the lumen. It also provides an essential barrier protecting against
infection and disease. This barrier is not simply physical, but is part of a
complex mucosal immune system (Figure 1). At the apical side of the
polarized monolayer, the intestinal epithelium directly confronts both dietary
and non-dietary luminal antigens, sensing their signals and subsequently
transmitting the information to the diverse populations of cells in the
intraepithelial compartment and the underlying tissue, the lamina propria
(LP). At the basolateral side the intestinal epithelial cells (IECs) receive
signals from various immune cells, nerve cells and stromal cells that
influence their barrier function, immune activity state and/or differentiation
state.
-5-
1.1 The human intestine and mucosal immunity
The intestine consists of the small intestine and the large intestine. The small
intestine is divided into three anatomic sections: duodenum, jejunum and
ileum measuring about 6 meters in total length. The large intestine is about
1.5 meters long and consists of the caecum, appendix, colon and rectum. In
cross-section four distinct layers can be distinguished. Counting from the
inside they are: mucosa, submucosa, muscularis, and serosa. The mucosa, in
turn can be divided into an epithelial compartment, the LP, and muscularis
mucosae. Muscularis consists of circular and longitudinal muscles.
The mucosa of the small intestine has villi and shallow crypts facing the gut
lumen, while the large intestine lacks villi and has deeper crypts. The
intestinal villi are finger-like or leaf-like projections. The villi consist of a
core of loose connective tissue covered by a single layer columnar
epithelium. The core of the villus is an extension of the LP, which contains
numerous fibroblasts, smooth muscle cells, lymphocytes, plasma cells,
macrophages, dendritic cells (DCs), eosinophils and a network of blood
capillaries as well as blind-ending lymphatic capillary, the lacteal. The
intestinal crypts are composed of a single layer columnar epithelium that is
continuous with the epithelium of the villi. The crypts are simple tubular
structures that extend from the muscularis mucosae through the thickness of
the LP, where they open to the luminal surface of the intestine at the base of
the villi. Additionally, LP is enervated.
The epithelial compartment contains epithelial cells and lymphocytes (Figure
2). The epithelial cells are joined firmly together by tight junctions, which
create a semipermeable diffusion barrier between individual cells (Liévin-Le
Moal and Servin, 2006). At least five types of epithelial cells can be
distinguished: enterocytes, goblet cells, Paneth cells, enteroendocrine cells
and microfold (M) cells. All epithelial cells, both in the small and large
intestine, are derived from stem cells located towards the base of the crypts.
In small intestine enterocytes and goblet cells migrate upward from the crypt
to the villus and are shed at the tip of the villus after 5 to 6 days. In colon the
cells are shed at the free luminal surface. Enterocytes are the predominant
cell type of the villus epithelium and are highly specialized for nutrient
absorption. At the apical surface, enterocytes are lined by densely packed
microvilli (~1 µm long by 0.1 µm in diameter) termed the brush border.
Large amounts of digested nutrients can be taken up because the microvilli
greatly increase the apical surface area of the plasma membrane. The
membrane of the microvilli is rich in protein, cholesterol as well as
glycolipids and contains digestive enzymes such as disaccharidases and
peptidases. Goblet cells are large mucus-secreting cells interspersed between
-6-
enterocytes, both in the crypts and at the villus/luminal surface. Interspersed
between the enterocytes are also endocrine cells that secrete neuropeptides
such as polypeptide YY and substance P. Paneth cells, only present in the
small intestine, are located at the base of the crypts. They contain granules
with antimicrobial peptides, antibacterial enzymes as well as antibacterial
glycoproteins and most likely play a role in regulating the density and
composition of the intestinal bacterial flora. A highly specialized epithelial
cell is the M cell, located in the epithelium covering lymphoid follicles. The
M cell has an antigen sampling function by transporting antigens from the
lumen to the immune cells in the underlying follicles. The enterocytes
dominate over goblet cells in small intestine and most of colon. In colon the
ratio decreases, approaching 1:1 near the rectum.
Gut associated lymphoid tissue (GALT) is made up of three components:
organized lymphoid aggregates [e.g. Peyer’s patches (PPs), the appendix,
solitary follicles], diffuse collections of lymphocytes, macrophages, and
plasma cells in the LP and intraepithelial lymphocytes (IELs) scattered
within the epithelium. PPs are aggregations of lymphoid follicles present in
small intestine (Cornes, 1965) while solitary follicles occur mainly in colon.
The lymphoid follicles are inductive sites for the specific immune response
and contain two major cell types, T and B cells and in addition macrophages,
DCs and follicular dendritic cells (FDCs) (Brandtzaeg and Bjerke, 1990;
-7-
Yeung et al., 2000; Wittig and Zeitz, 2003). The lymphoid follicles have a
germinal center structure rich in B cells with interspaced FDCs and
surrounded by a T cell area containing DCs (Yeung et al., 2000). The
lymphoid follicles are located beneath a single layer epithelium called the
follicle-associated epithelium (FAE) containing the highly specialized M
cells (Brandtzaeg and Bjerke, 1990; Kraehenbuhl and Neutra, 2000).
The LP contains most cellular components of the immune system, with large
numbers of B cells, plasma cells, macrophages, DCs, mucosal mast cell and
T cells. The vast majority of LP T cells are αβ T cells of both the CD4 and
CD8 subsets and a small subset of CD4+CD8+ cells (Melgar, et al., 2002;
Wittig and Zeitz, 2003). LP T cells secrete cytokines as IL-2, IFN-γ, and
TNF-α (Beagley and Elson, 1992; Forsberg et al., 2002; Melgar et al., 2003).
Most plasma cells, 70-90%, produce antibodies of the IgA class (Brantzaeg
et al., 1999). Recently it was shown that plasma cells also secrete βdefensins (Rahman et al., 2007).
In contrast to the LP, all immune cells within the epithelium are
lymphocytes. These IELs are in close contact with the epithelial cells and
more frequent in the small intestine compared with large intestine (Lundqvist
et al., 1995). In the human small intestine, there are 10-20 IELs per 100
villus epithelial cells. IELs are a heterogeneous mixture of T lymphocyte
subsets that are chiefly of activated/memory phenotype (CD45RO+ cells). In
the small intestine, the CD8+ αβT cells dominate while in the large intestine
there are almost equal proportions of CD8+ αβT cells, CD4+ αβT cells and
CD4/CD8 double negative αβT cells (Lundqvist et al., 1995). In both small
and large intestine, about 10% of IELs are γδT cells (Lundqvist et al., 1995).
The majority are CD4/CD8 double negate and preferably use Vδ1 and Vγ8
in their T cell receptor as opposed to peripheral blood γδT cells which use
Vδ2 and Vγ9 (Lundqvist et al., 1995; Söderström et al., 1994). The Vδ1+ γδT
cells have the ability to kill stressed cells (Groh et al., 1998) and tumor cells
of epithelial origin (Maeurer et al., 1996). IELs probably play multiple
functions including cytotoxic activities, induction and maintenance of oral
tolerance, surveillance of the IECs, and immune protection (Wershil and
Furuta, 2008).
1.2 Hurdles for intestinal pathogens
Immune defense works at three levels in the gut. Secreted factors to the gut
lumen, at the lumen/epithelial compartment interface and inside the mucosal
tissue. (Hooper and Gordon, 2001; Guarner and Malagelada, 2003; Mowat,
2003; Magalhaes et al., 2007). To colonize mucosal surfaces and invade the
-8-
host, microorganisms typically must first penetrate the secreted mucus layer
in order to reach the apical surface of epithelial cells or release toxins that
disrupt epithelial integrity. A broad range of bacterial pathogens can produce
mucus-degrading enzymes, such as glycosulfatases, sialidases and mucinases
to destabilize the mucus gel and remove mucin decoy carbohydrates for
adhesion (Linden et al., 2008). As mucins bind pathogens, increasing the
secretion of mucins is likely to benefit the host by trapping and moving
pathogens away from the epithelial surface. Underneath the secreted mucus
layer, the cells have a dense forest of highly diverse glycoproteins and
glycolipids, which form the glycocalyx. Membrane-anchored cell-surface
mucins and Carcinoembryonic antigen (CEA)-related cell adhesion
molecules (CEACAMs) are major components of glycocalyx situated at the
top of the microvilli (Hammarström and Baranov, 2001). The
mucus/glycocalyx compartment also contains a spectrum of antimicrobial
proteins including secretory IgA with specificity for microbial antigens,
defensins, lysozyme, lactoferrin etc. (Magalhaes et al., 2007). Together,
these layers provide a first line of defense barrier preventing interactions
between microorganisms and IECs (MacDonald, 2003; Newberry and
Lorenz, 2005). Another important factor preventing access for pathogens is
the commensal microbiological flora, particularly well developed in colon.
Pathogens have to fight commensals, which are well adopted for the
environment, for nutrients, space etc.
The IECs take an active part in immune defense. This includes both
production of factors of innate immunity, e.g. antimicrobial peptides and
crosstalk between IECs and immune cells. The enterocytes serve as afferent
sensors of danger within the luminal microenvironment by secreting
chemokines and cytokines that alert and direct innate and adaptive immune
responses to the infected site (Shanahan, 2005; Akira et al., 2006). M cells
overlying lymphoid follicles directly take up and transport antigens from the
gut lumen to underlying subadjacent DCs and other antigen-presenting cells
(Miller et al., 2007). In addition, DCs are believed to sample antigen directly
from the intestinal lumen by forming tight-junction-like structures with IECs
and projecting dendrites through the epithelial-cell layer and into the lumen
(Rescigno et al., 2001; Coombes and Powrie, 2008). The consequence of
these mechanisms for antigen sensing is alerting of immune effector cells
within the epithelium and LP to humoral and cell mediated defense
responses, e.g. cytotoxicity and production of IgA as well as recruitment of
phagocytic cells like neutrophils and macrophages.
-9-
1.3 Innate immunity
To protect the host from invasion by microorganisms and neutralize their
virulence factors, all multi-cellular organisms have developed defense
mechanisms that utilize germline-encoded receptors for the detection of a
wide range of ligands derived from microorganisms. These phylogenetically
ancient defense mechanisms are collectively known as the innate immune
system. The recognition strategy is based on the detection of conserved
molecular patterns that are unique to microbes. They are called pathogenassociated molecular patterns (PAMPs) and are present in bacteria, fungi,
protozoa and viruses. PAMPs include lipopolysaccharide (LPS),
peptidoglycan, lipoteichoic acid (LTA), terminal mannose (a terminal sugar
common in microbial glycolipids, glycoproteins and polysaccharides but rare
in those of humans), bacterial flagellin, N-formylmethionine, doublestranded RNA (dsRNA) and single-stranded RNA (ssRNA), unmethylated
CpG DNA and fungal glucans etc (Akira et al., 2006; Kumagai et al., 2008).
Because all microbes, not just pathogenic microbes, have PAMPs, they are
sometimes referred to as microbe-associated molecular patterns (MAMPs)
(Mackey and McFall, 2006).
The receptors on host cells recognizing PAMPs are called patternrecognition receptors (PRRs) (Medzhitov and Janeway, 1997). There are
several structurally and functionally distinct classes of PRRs: Toll-like
receptors (TLRs), nucleotide oligomerization domain (NOD)-like receptors
(NLRs), retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) and Ctype lectin receptors (CLRs). They induce partly different host defense
pathways.
In contrast to innate immunity, adaptive immunity is dependent on antigen
receptors expressed on lymphocytes that are acquired by somatic
recombination of the antigen receptor genes during B and T cell
development. This system has a huge repertoire and is not limited to PAMPs.
However, a major drawback with the adaptive immune response is that it
requires a considerable time to become fully effective. At the first encounter
of antigen it takes up to 6-7 days due to differentiation and maturation of the
effector cells. During evolution adaptive immunity occurs first in primitive
fishes. In man innate and adaptive immunity works in parallel and can
enhance each other. Since my thesis deals with innate immunity I will not
discuss adaptive immunity further.
1.3.1 TLRs and their signaling
- 10 -
The evolutionary conserved TLRs are a family of single membranespanning receptors. Ten members of the human- and 13 members of the
mouse TLR family have been identified (Roach et al., 2005; Akira et al.,
2006; Kumagai et al., 2008). TLRs are expressed as homo- or heterodimers.
All TLR family members are made up of multiple leucin-rich repeat motifs
(LRRs) at the N-terminal of the molecule, followed by a cysteine-rich
region, a transmembrane domain, and an intracellular domain at the Cterminal, the Toll/interleukin-1 (IL-1) receptor (TIR) domain. The TLRs are
either located to the cell membrane (TLR1, TLR2, TLR4, TLR5, TLR6 and
TLR10) or to intracellular membranes (TLR3, TLR7, TLR8, and TLR9).
The C-terminal TIR domain located in the cytosol is able to recruit TIRcontaining intracellular proteins that mediate their signaling (Brikos and
O'Neill, 2008; Kumagai et al., 2008). Ligand binding to the LRR, either
directly or by means of accessory molecules, is believed to induce a
conformational change that brings the intracellular TIR domains in the dimer
together (Latz et al., 2007).
Different TLRs recognize distinct PAMPs. TLR2 plays a crucial role in the
recognition of peptidoglycan, LTA, and lipoproteins (Takeuchi et al., 1999).
TLR2 forms a heterodimer with either TLR1 or TLR6, and these
heterodimers recognize lipoproteins with different lipid moieties respectively
(Takeuchi et al., 2001; 2002). TLR1-TLR2 heterodimer recognizes
triacylated lipoproteins (Takeuchi et al., 2002; Jin et al., 2007), whereas the
TLR2-TLR6 heterodimer recognizes diacylated lipoproteins and LTA
(Takeuchi et al., 2001). LPS, a cell-wall component of Gram-negative
bacteria (Poltorak et al., 1998) is recognized by TLR4 (Triantafilou and
Triantafilou, 2002), which involves the accessory molecule myeloid
differentiation protein 2 (MD-2) (Nagai et al., 2002; Kim et al., 2007),
membrane-bound CD14 (Jiang et al., 2005), and LPS-binding protein (LBP)
(Jack et al., 1997). TLR5 is a receptor for bacterial flagellin (Hayashi et al.,
2001; Uematsu et al., 2006). TLR3, TLR7, TLR8 and TLR9, localized on
cytoplasmic membranes of the endoplasmic reticulum or endosomes, are
receptors for nucleic acids and their derivatives. TLR3 is a receptor for viral
dsRNA (Alexopoulou et al., 2001). TLR7 and TLR8 recognize ssRNA from
RNA viruses (Hemmi et al., 2002; Jurk et al., 2002; Diebold et al., 2004;
Heil et al., 2004). TLR9 is a receptor for DNA with an unmethylated CpGmotif (CpG-DNA) (Barton GM, et al. 2006), which is abundant in bacterial
DNA, whereas the frequency of CpG motifs in mammalian genome DNA is
low and mostly methylated (Ishii and Akira, 2006).
At ligand binding to TLR the cytoplasmic TIR domains dimerize to form a
platform for the recruitment of adapter proteins and additional signaling
molecules (Trinchieri and Sher, 2007). Through a series of intracellular
- 11 -
molecular reactions nuclear factor-kappa B (NF-κB) and/or interferon
regulatory factor (IRFs) are activated, leading to the expression of various
genes associated with host defense, including proinflammatory cytokines,
chemokines and defensins. Depending on cell type, the TLR-mediated
immune response may differ. Moreover, the different signaling pathways
used by distinct TLRs and the presence of different PAMPs on invading
microbes contributes to a complex multifunctional innate immune response.
1.3.2 TLR-independent pathogen recognition
Microbial components are also recognized by the intracellular cytosolic
receptor NLRs, the cytosolic RLRs and the surface expressed CLRs.
The NLR family has over 30 members in humans and is characterized by the
presence of a central NOD domain and a C-terminal LRR domain and a Nterminal domain responsible for the signaling, either the caspase recruit
domain (CARD) or the putative protein-protein interaction (PYRIN) domain
(Kanneganti et al., 2007; Franchi et al., 2008).
The NLRs recognize different bacterial components notably γ-D-glutamylmesodiaminopimelic acid and muramyl dipeptide, respectively leading to
activation of NF-κB (Franchi et al., 2008; Chamaillard et al., 2003; Girardin
et al., 2003). Members of the NALP subfamily have a PYRIN domain
instead of a CARD domain. Fourteen members (NALP1-14) have been
described in humans (Kanneganti et al., 2007). NALP1-3 were shown to be
involved in Caspase-1-mediated cleavage of proIL-1β, proIL-18 and proIL33 into their mature forms in response to stimuli (Agostini et al., 2004;
Martinon et al., 2002; Keller et al., 2008). NALP3 is activated by LPS,
lipoprotein, CpG-DNA, bacterial RNA, uric acid crystals and by ATP
(Kanneganti et al., 2006; Mariathasan et al., 2006; Martinon et al., 2006).
The cytosolic flagellin is capable of activating caspase-1 in an NLRC4dependent manner (Sutterwala et al., 2007).
RLRs are DExD/H box RNA helicases located in the cytoplasm where they
mediate recognition of virus-specific RNA and then activate IRFs inducing
expression of type I interferon (IFN). Three members of RLRs have been
described in mammals: retinoic acid inducible gene I protein (RIG-I),
melanoma differentiation associated gene-5 protein (MDA5) and laboratory
of genetics and physiology protein-2 (LGP2) (Bowie and Fitzgerald, 2007;
Yoneyama and Fujita, 2007; 2008; Xiao, 2008). Interestingly, microbial
ligands for members of the RLR-, NLR- and TLR-families may be the same.
However, the molecular bases of their receptor-ligand interactions and the
outcome of the interactions may be quite different. The cross-talk between
- 12 -
these sensing-systems probably plays an important role in immunological
homeostasis.
CLRs are a family of membrane-associated receptors that are able to
recognize carbohydrate structures present on the pathogens. So far, more
than 60 CLRs have been identified in humans. The extra-cellular portion of
CLR consists of one or more C-type (Ca2+ dependent) carbohydrate
recognition domains (CRDs), involved in carbohydrate recognition
(Zelensky and Gready, 2005). Although microbes and vertebrate cells are
able to produce similar polysaccharide chains, the density of carbohydrates
on microbes is generally much higher than on vertebrate cells. At least partly
due to this difference, carbohydrates present on microorganisms can be
specifically recognized by the CLRs (Gijzen et al., 2006; van Vliet et al.,
2008). By binding to carbohydrate moieties, CLRs mediate biological
events, such as cell-cell or cell-matrix adhesion and glycoprotein turnover.
Although the majority CLRs are primarily involved in antigen uptake, some
CLRs such as Dectin-1 have been shown to trigger intracellular signaling
cascades upon ligand binding resulting in NF-κB activation and production
of cytokines (Rogers et al., 2005; Gross et al., 2006).
1.3.3 TLR and NOD signaling in the human intestine
TLRs and NODs are expressed in human small and large intestine and play
important roles of immune and non-immune functions in the mucosa
(Melmed et al., 2003; Otte et al., 2004; Abreu et al., 2005; Sanderson and
Walker, 2007). Thus, IECs and lamina propria macrophages express TLR4
and TLR2 (Hausmann et al., 2002) and jejunal smooth muscle cells express
TLR4 (Rumio et al., 2006). NOD1 is ubiquitously expressed in many tissues
and cells including IEC while NOD2 is constitutively or inducibly expressed
in monocytes, macrophages, T and B cells, DCs, as well as IECs, including
Paneth cells. (Sanderson and Walker, 2007).
In order to maintain intestinal mucosal homeostasis against tissue damage,
commensal microflora induced host modulatory effects seem to require
functional TLRs and NODs (Rakoff-Nahoum et al., 2004; Kobayashi et al.,
2005). Under normal conditions, the intestinal mucosa is constantly exposed
to TLR/NOD ligands derived from the commensal microflora and a basal
state of activation of downstream signaling pathways is achieved, thus
ensuring rapid compensation and limited inflammatory responses (RakoffNahoum et al., 2004). Deficient TLR or NOD signaling may elicit an
imbalance, facilitating injury and leading to disease. There is evidence to
show that commensal microflora directly assist the host to strengthen
- 13 -
intestinal epithelial barrier resistance (Hooper et al., 2001; Madsen et al.,
2001; Otte et al., 2004). Altered expression of TLRs and NODs in IECs has
been reported in chronic inflammation. Thus, increased expression of TLR4
has been demonstrated in inflammatory bowel disease (IBD), while the
expression of TLR2 and TLR5 remained unchanged (Cario and Podolsky,
2000). Moreover, proinflammatory cytokines such as IFN-γ and tumor
necrosis factor-α (TNF-α) increase expression of TLR4 and MD-2 resulting
in increased LPS responsiveness in human colonic epithelial cells (Abreu et
al., 2001; Suzuki et al., 2003). Polymorphisms and mutations within the
NOD2 gene have been associated with the development of Crohns’ disease
in a subgroup of patients ( Hugot et al., 2001; Ogura et al., 2001) and
persons homozygous for a variant NOD2 may have a 20-fold higher
susceptibility to Crohns’ disease (Cuthbert et al., 2002).
1.4 Mucins
The intestinal mucosal surface in humans is covered by a layer of hydrated
viscous mucus ranging in thickness from 50 to 700 µm. The mucus is a
mixture of mucins, free protein, salts, and water. The major components of
mucus are secreted gel-forming mucins produced by goblet cells (Strugala et
al., 2003). This viscous, sticky layer traps particles, bacteria, and viruses,
which are expelled by the peristaltic process of the gut.
Mucins are family of heavily glycosylated proteins. Based on their
properties, the mucins can be divided into three distinct subfamilies (Linden
et al., 2008): (a) Secreted gel-forming mucins, which are the major
component of mucus and confer its viscoelastic properties. They contain Nand C-terminal cysteine-rich domains that are involved in homooligomerization mediated by inter-molecular disulfide bonds (Gum et al.,
1994; Perez-Vilar et al., 1998). (b) Cell surface mucins, also called
membrane-bound mucins, which have a single membrane-spanning domain
and a short cytoplasmic tail in addition to the extensive extracellular domain.
(c) Secreted non-gel-forming mucins, which are relatively small
glycoproteins and incapable of forming a gel (Andrianifahanana et al.,
2006). Classically, mucins are known to play a central role in the protection,
lubrication and hydration of the external surfaces of epithelial tissue layers
lining the intricate network of ducts and lumens within the human body
(Strous and Dekker, 1992; Van Klinken et al., 1995). Mucins have direct and
indirect roles in defense against infection. Different mucins are expressed in
varying proportions throughout the normal digestive tract and the complexity
of the mucin mixture increases from esophagus to rectum (Corfield et al.,
- 14 -
2001; de Bolos et al., 2001). In the small intestine, MUC2 is predominantly
expressed by the goblet cells, while MUC1, MUC3, and MUC4 are mostly
found in enterocytes, and to a lesser extent in goblet cells (Audie et al., 1993;
Jass and Roberton, 1994). MUC17 (Gum et al., 2002) and MUC20 (Higuchi
et al., 2004) have also been detected in the small intestine. The colorectal
part of the intestine exhibits a more diverse pattern of mucin expression.
Here large amounts of MUC2, MUC4, and MUC11 are found in the goblet
cells, whereas the membrane-bound mucins, MUC1, MUC3, MUC4, and
MUC12 are detected in colonocytes (Audie et al., 1993; Jass and Roberton,
1994; Tytgat et al., 1994). MUC13 (Williams et al., 2001), MUC17 (Gum et
al., 2002) and MUC20 (Higuchi et al., 2004) have also been observed in
colon.
Gene expression of mucins in the mucosal epithelium is both constitutive
and inducible. The mucosal epithelial cells continuously produce sufficient
mucins to maintain the mucus layer, whereas the inducable pathway affords
a massive discharge as a response to environmental and/or
(patho)physiological stimuli, including cholinergic stimuli, inflammatory
cytokines, prostaglandins, LPS, bile salts and nitric oxide (Andrianifahanana
et al., 2006; Linden et al., 2008). Moreover, infections can induce alterations
in mucins, such as increased mucin production through goblet cell
hyperplasia, increased mucin secretion and altered mucin glycosylation
affecting microbial adhesion and the ability of microorganisms to degrade
mucus. These alterations work in concert with other defense processes to
prevent infection (Linden et al., 2008).
1.5 The Carcinoembryonic antigen family
Carcinoembryonic antigen (CEA) was identified as a prominent tumorassociated antigen in human colon cancer in 1965 (Gold and Freedman,
1965). CEA and related genes make up the CEA gene family belonging to
the immunoglobulin gene superfamily. The gene products are highly
glycosylated glycoproteins. In human, the CEA family consists of 29 genes,
18 of which normally are expressed, including seven CEACAMs,
(CEACAM1, CEACAM3-CEACAM8) and 11 pregnancy specific
glycoproteins (PSG1-PSG11), and 11 PSG-related pseudogenes
(Hammarström, 1999). Recently, a group of genes more distantly related to
CEA than the CEACAMs discussed above was identified in the human
genome (Zebhauser et al., 2005). CEACAMs are also found in other
mammals and CEACAM1 appears to be the phylogenetically most ancient
member of the CEACAM subfamily. According to the new nomenclature
CEA should be called CEACAM5 (Beauchemin et al., 1999). However, the
- 15 -
name CEA is kept for historical and practical reason. Thus, in the extensive
literature on the use of CEA/CEACAM5 as a clinical biomarker the name
CEA is used. Clinical aspects of the utility of CEA as a biomarker will not
be discussed here.
CEACAMs are linked to the cell surface by a glycosylphosphatidylinositol
(GPI) anchor (CEA, CEACAM6-8) or through a transmembrane region with
a cytosolic signaling domain (CEACAM1, CEACAM3 and CEACAM4),
while PSGs are secreted glycoproteins (Hammarström, 1999; Beauchemin et
al., 1999). There are two major CEACAM1 isoforms, which differ with
respect to the length of their cytoplasmic domain. CEACAM1-L has a long
cytoplasmic domain (71-73 amino acids) and CEACAM1-S has a short
cytoplasmic domain (10-12 amino acids). CEACAM1-L contains two
immunoreceptor tyrosine-based inhibition motifs (ITIM) while CEACAM1S lacks ITIM sequences (Gray-Owen and Blumberg, 2006).
It has been established that four CEACAMs subgroup genes are expressed in
human intestinal epithelial cells (Frängsmyr et al., 1999). In colon,
CEACAM1, CEA, CEACAM6 and CEACAM7 are expressed at high level
in mature and highly differentiated enterocytes at the crypt mouth and free
luminal surface (Frängsmyr et al., 1995; 1999). CEA/CEACAM5 is also
present in the mucus droplets of goblet cells. Immunoelectron microscopy
studies of colon demonstated that all four molecules are localized to the
microvilli and the glycocalyx (the fuzzy coat) at the top of the microvilli
(Hammarström and Baranov, 2001). Within this compartment the 4
molecules show a different distribution. In the small intestine CEACAM1 is
expressed by absorptive epithelial cells while CEA is only produced by
goblet cells (Fahlgren et al., 2003). CEACAM6 and CEACAM7 appear not
to be expressed in small intestine (Schölzel et al., 2000).
The CEACAM family members are multifunctional molecules involved in
cell adhesion, tumor suppression, regulation of signal transduction and
probably also innate immunity. Several CEACAM family members have
also been identified as receptors for host-specific viruses and bacteria in
mice and humans, respectively, making these proteins interesting targets of
pathogen (Hammarstrom, 1999; Gray-Owen and Blumberg, 2006; Kuespert
et al., 2006; Griffiths et al., 2007; Hemmila et al., 2004). Deletion of CEA
family members can render the host resistant to particular pathogens
(Kuespert et al., 2006). The majority of the currently characterized
meningococcal and gonococcal adhesions, the opacity-associated (Opa)
proteins display binding specificity for human surface receptors of the
CEACAMs family (OpaCEA proteins). All OpaCEA proteins bind to the nonglycosylated C′CFG-face of the immunoglobulin-domain fold of the Nterminal domain of CEACAM1, CEACAM3, CEA and CEACAM6. Binding
- 16 -
of OpaCEA proteins to CEACAM molecules is sufficient to induce the
internalization of the bacteria into several cell types in vitro (Heyderman and
Virji, 2007).
Essentially all CEA family members, including GPI-anchored CEA and
CEACAM6, function as intercellular adhesion molecules in vitro
(Benchimol et al., 1989; Oikawa et al., 1989). CEACAM1 is primarily an
activation-induced cell-surface molecule that functions as a co-inhibitory
receptor. Homophilic ligation of CEACAM1 on T cells leads to a signaling
mechanism, which results in inhibition of a broad range T-cell functions
(Nagaishi et al., 2006). CEACAM1-L and CEACAM1-S have different
functions. CEACAM1-L can regulate cell proliferation, invasion, cell
migration, angiogenesis, lymphangiogenesis, and cytotoxicity. CEACAM1L, containing two ITIMs, generally transmit inhibitory signals while
CEACAM1-S contains sequences that can bind calmodulin (Edlund et al.,
1996), tropomyosin and globular actin (Schumann et al., 2001), indicating a
regulated interaction with the cytoskeleton. In the breast, CEACAM1-S
mediates mammary lumen formation via an apoptotic and cytoskeletal
reorganization mechanism (Kirshner et al., 2004; Chen et al., 2007). In most
cell types and tissues, CEACAM1-L and CEACAM1-S are expressed
together, but at characteristically different ratios (Hauck et al., 2006). The
abundance of CEACAM1 and the relative ratio of CEACAM1-L to
CEACAM1-S isoforms are not static, and can vary substantially according to
the cell type, its phase of growth and activation state (Singer et al., 2000;
Greicius et al., 2003). Moreover, in vitro studies performed using polarized
epithelial cells clearly show that the CEACAM1 cytoplasmic tail governs
both the distribution of the receptor between intracellular compartments and
the cell membrane, and the movement of the receptor between apical and
basolateral surfaces (Sundberg et al., 2004). An optimal ratio in the
expression level of CEACAM1-L and CEACAM1-S isoforms were found to
be essential for some of its biological functions, e.g. the suppression of cell
proliferation (Obrink et al., 2002). Recent data demonstrated a high ratio of
CEACAM1-S to CAECAM1-L isoforms in normal breast tissues versus
breast cancer suggesting that altered splicing of CEACAM1 may play an
important role in tumorogenesis (Gaur et al., 2008).
1.6 Antimicrobial peptides and lysozyme
Although the alimentary tract is colonized by, and exposed to a great number
of different bacteria, intestinal infection or translocation of bacterial agents is
the exception, not the rule, and is largely limited to pathogenic bacteria or
predisposing disease states (Liévin-Le Moal and Servin, 2006; Wehkamp et
- 17 -
al., 2007). Intestinal epithelial cells in higher organisms not only form a
physical barrier but also a chemical barrier. This chemical barrier is
composed of secreted anti-infectious substances, including mucus,
antimicrobial peptides (AMPs), and other anti-microbial molecules, such as
lysozyme, which together with resident microbiota provide the front line of
defense against pathogenic microorganisms (Ganz, 2002). AMPs are small
molecular weight proteins with broad-spectrum antimicrobial activity against
bacteria, fungi, protozoa and some enveloped viruses. Based on common
structural and functional features, the AMPs present in the human intestinal
tract have been divided into two major families: defensins and cathelicidins
(Otte et al., 2003).
The human defensins are small cysteine containing cationic peptides (range
from 28 to 50 amino acid residues) containing three intra-molecular disulfide
bonds (Pazgier et al., 2007). The positions of the disulfide bonds define two
subclasses: α-defensins and β-defensins (Cunliffe, 2003; Ganz, 2003). All of
them have anti-microbial activity against bacteria, and some also against
fungi, viruses and protozoa (Lehrer et al., 1993).
Human α-defensins are 29-35 amino acid residues in length and form a
triple-stranded β-sheet. Four of the α-defensins are produced by neutrophils
and are named human neutrophil peptides 1-4 (HNP1-4) (Ganz et al., 1985).
The primary source of two additional α-defensins, human α-defensin-5 and 6
(HD-5 and HD-6), is Paneth cells in the small intestine, where these cells are
located at the crypt base (Jones and Bevins, 1992; 1993; Ganz, 1999; Porter
et al., 1997).
Genomic studies have revealed that there are over 40 different β-defensin
genes arranged in 4 different clusters in the human genome (Liu et al., 1998).
Four different β-defensins have been isolated and characterized human βdefensin 1-4 (hBD 1-4) present in the intestine. β-defensins form three βstrands arranged as an anti-parallel sheet held together by three intramolecular disulfide bonds (Dann and Eckmann, 2007; Taylor et al., 2008). In
contrast to the high and relatively stable expression level of HD-5 and HD-6
in the small intestine and low expression in other regions of the
gastrointestinal tract, β-defensins expression is much more variable with
respect to the individual peptides, anatomical location, and pathogenic
condition. hBD1 is constantly expressed throughout the gastrointestinal tract
and remains invariable even in inflammation or infections (O'Neil et al.,
1999; Fahlgren et al., 2003; 2004). In normal intestine, there is little hBD-2
expression, but it is strongly upregulated in inflammatory bowel disease
(Fahlgren et al., 2003; Ganz, 2002). It was reported that in the colonic IECs
from patients with ulcerative colitis, the levels of mRNA expression of HD- 18 -
5, HD-6, hBD-2, hBD-3, hBD-4 increased significantly compared with in
the IECs from control colon (Fahlgren et al., 2003; 2004). HD-5, HD-6 was
produced by metaplastic Paneth cells in colon of patients with ulcerative
colitis (Fahlgren et al., 2003). hBD-2, hBD-3 and hBD-4 also were
produced by mature IgA and IgG producing colonic plasma cells (Rahman et
al., 2007). mRNAs expression levels of HD-5 and HD-6 were also increased
in proximal small intestinal IECs from children with active celiac disease
(CD) but levels returned to normal in treated patients. The increased
production was a consequence of Paneth cell metaplasia, which in turn
correlated to the increased production of IFN-γ by IELs in CD (Forsberg et
al., 2004). The increased expression of β-defensins is explained by the
inducibility of these peptides by proinflammatory cytokines and bacteria
probably via TLRs. The induction of hBD2, hBD3 and hBD4 is mediated by
proinflammatory factors such as IL-1β, IFN-γ, mostly through NF-κBdependent and activator protein (AP-1) -dependent pathways (O'Neil et al.,
1999; Wehkamp et al., 2003; Fahlgren et al., 2004). The signaling pathways
also include TLRs, especially TLR2 and TLR4, that recognize and bind
PAMPs and mitogen-activated protein kinases (Froy, 2005). Complementing
their role as antibacterial agents in innate immune defense, defensins also act
as links to the adaptive immune system. hBD2 can bind to DCs that express
the chemokine receptor CCR6; thereby recruiting DCs and memory T cells
by chemotaxis to sites of infection (Yang et al., 1999). HD-5 can block the
release of IL-1β from LPS-activated monocytes (Shi et al., 2007).
Cathelicidins are α-helical cationic antimicrobial peptides that consist of a
highly conserved N-terminal structural domain, cathelin, linked to a variable
C-terminal peptide with antimicrobial activity. LL-37 is the only cathelicidin
identified in humans, found predominantly in neutrophils and various
epithelial cells lining the stomach, lower small intestine and throughout the
colon (Gudmundsson et al., 1996; Hase et al., 2002; 2003; Schauber et al.,
2003; Kai-Larsen and Agerberth, 2008). The name LL-37 derives from the
first two leucines (L) residues at the N-terminus and the number of amino
acids in the peptide (Gudmundsson et al., 1996). The expression is most
prominent in the lower small intestine and colon with high peptide
abundance in superficial epithelial cells (Schauber et al., 2003). Apart from
exhibiting a broad antimicrobial spectrum, it is now evident that LL-37
possesses several additional functions, which are chemotaxi, endotoxin
neutralization, angiogenesis and wound healing (Kai-Larsen and Agerberth,
2008).
Lysozyme is an enzyme that acts by breaking the β1, 4 linkage between Nacetylglucosamine and N-acetylmuramic acid in the peptidoglycan layer of
- 19 -
bacterial cell walls. Paneth cells display prominent cytoplasmic granules,
containing antibacterial proteins such as lysozyme, secretory phospholipase
A2 type IIA, HD-5 and HD-6, which are released into the intestinal lumen in
response to a range of stimuli (Keshav, 2006). Wehkamp and colleagues
reported that lysozyme mRNA expression was high in the duodenum, and
that the protein localized to both Brunner's glands in the lamina propria and
Paneth cells (Wehkamp et al., 2006). It also has been recognized that
lysozyme mRNA expression is increased in colonic intestinal epithelial cells
from ulcerative colitis patients (Fahlgren et al., 2003; 2004) and in the
proximal small intestinal mucosa of patients with active CD (Forsberg et al.,
2004).
1.7 Cytokines
Cytokines are a group of small soluble signaling proteins that are released
mainly by immune cells and used extensively in cellular communication
through cell-surface receptor-ligand interaction. The cytokine superfamily of
proteins is an integral part of the signaling network between cells and is
essential in generating and regulating the immune system. These signaling
molecules can act in an autocrine, paracrine and endocrine manner that have
important local and systemic effects. By binding to specific membrane
receptors and acting in networks or cascades synergistically or
antagonistically, cytokines signal the target cell and alter its functions, often
including activation, proliferation, and expression and secretion of effectors
molecules. Cytokines generally are pleiotropic and redundant molecules,
which means that a particular cytokine not only acts on a number of different
types of cells rather than on a single cell type but also that a number of
different cytokines carry out the same function. Because of their central role
in the immune system, aberrant expression levels of cytokines usually signal
ongoing patho-physiological processes (Clark and Coopersmith, 2007).
On the basis of their presumed function, cell of secretion, or target of action,
cytokines have been classified as lymphokines, chemokines, interleukins,
and interferons. Some cytokines promote inflammation and are therefore
called proinflammatory cytokines, including TNF-α, IL-1β, IFN-γ and IL18, where other cytokines suppress the activity of proinflammatory cytokines
and are therefore called anti-inflammatory cytokines. IL-10 and IL-6 are
pleiotropic cytokines with both pro- and anti-inflammatory effects.
Chemokines are cytokines with the primary function of regulating
leukocytes recruitment. IL-8 (CXCL8) is one of more than 50 chemokines
identified to date. Through binding to specific cell surface receptors,
- 20 -
cytokines act on their target cells and initiate signals that are critical to a
diverse spectrum of functions.
Of the many cell types involved in the intestinal inflammatory response,
IECs function as an integral component of the immune defence. Indeed,
IECs form a critical mucosal barrier between the host’s internal milieu and
the external environment. In the course of inflammation, intestinal epithelial
cells serve as immuno-effector cells and are capable of up-regulating surface
molecules and secreting cytokines and chemokines to facilitate antigen
presentation to immune cells. The induction of inflammatory gene
expression in the intestinal epithelial compartment leads to impaired barrier
function, cell differentiation, and proliferation (Acheson and Luccioli, 2004).
Additionally, enterocytes can act as antigen presenting cells themselves and
regulate lymphocyte responses in the intestine (Hershberg and Mayer, 2000).
Enterocytes are also in constant communication with IELs and T cells within
the lamina propria and has been shown to exhibit increased major
histocompatibility complex (MHC) class II molecules at the basolateral
membrane in states of inflammation (Kaiserlian et al., 1989). The polarized
expression of levels of MHC class II molecules on enterocytes suggests that
antigen exposure on the luminal side as opposed to the basolateral side elicits
different immunologic responses (Hershberg and Mayer, 2000).
1.7.1 Interferon-γ
The interferons (IFNs), initially described as a family of antiviral proteins
(Isaacs and Lindenmann, 1957). There are three types of IFNs, type I (α, β, ω
and τ), type II (γ) and type III (λ), which signal through different receptors to
produce distinct, but overlapping, cellular effects (Boehm et al., 1997). Type
II interferon is also called interferon-γ (IFN-γ). Human IFN-γ is a
homodimeric glycoprotein containing 146-amino acid subunits and is
secreted by activated immune cells, primarily T and NK cells (Schroder et
al., 2004). IFNγ is a pleiotropic cytokine that stimulates a wide range of
cellular responses promoting cell-mediated immune responses, increased
antigen presentation and production of proinflammatory cytokines.
(Schroder et al., 2006; van Boxel-Dezaire and Stark, 2007). Other cellular
responses include regulation of cell proliferation and stimulation of
apoptosis. The biological effects of IFN-γ are elicited through activation of
intracellular molecular signaling networks, the best characterized of which is
the Janus kinase (JAK)/Signal Transducer and Activator of Transcription
(STAT) signaling pathway providing the primary mechanism through which
gene expression is induced. There are, however, differential patterns of
activation of STAT1, STAT3, and STAT5 in different cells, and activation
- 21 -
of transcription factors other than STATs yielding cell type specific
regulation of biological function (Darnell et al., 1994; Levy and Darnell,
2002; van Boxel-Dezaire and Stark, 2007).
1.7.2 Tumor Necrosis Factor-α
Tumor necrosis factor-α (TNF-α), which was first identified in 1975 as a
cytokine with anti-tumor effects in mice (Carswell et al., 1975), plays a
crucial role in the immune system as it mediates inflammation, cell growth,
apoptosis, and enhances the cellular immune response (Fukushima et al.,
2004; Thomson and Lotze, 2003). Human TNF-α cDNA was cloned and
expressed in 1985 (Pennica et al., 1985). Monocytes and macrophages
produce TNF-α. However, other cell-types, including T cells and IECs have
the ability to produce TNF-α in response to bacterial toxins, inflammatory
products, and other stimuli (Tracey and Cerami, 1993; Bazzoni and Beutler,
1996). TNF-α is primarily synthesized as a 233-amino acid type II
transmembrane precursor arranged in stable homotrimers that is
subsequently cleaved proteolytically in the extracellular domain to release
soluble TNF-α (17 kDa) (Beutler and Cerami, 1986; Tang et al., 1996). The
biologically active soluble form of TNF-α is a non-covalently bound trimer.
The cell membrane-associated form of TNF-α also possesses biological
activity. The two forms of TNF-α may have distinct biological activities. A
matrix metalloprotease, TNF-α-converting enzyme (TACE), not only
mediates release of TNF-α from the cell membrane-bound precursor (Black
et al., 1997) but is also involved in processing several cell-membraneassociated proteins, including TNF-α receptors. The released soluble TNF-α
receptor forms can neutralize the actions of TNF-α (Wang et al., 2003).
TNF-α exerts the biological functions via interaction with its cognate
membrane receptors, comprising the TNF-α receptors (Locksley et al.,
2001). Two types of TNF-α receptors, type I (TNFR1) and type II (TNFR2)
are present on the plasma membrane of virtually all cell types except
erythrocytes and bind both soluble and membrane-associated TNF-α. Each
TNF-α trimer binds three receptor molecules to produce a hexameric (TNFα)3-(TNFR)3 structure triggering the intracellular signaling pathway (Idriss
and Naismith, 2000). TNFR1 is expressed in most tissues, and can be fully
activated by both membrane-bound and soluble TNF-α, whereas TNFR2 is
found only in cells of the immune system, and respond to the membranebound form of the TNF-α. TNFR1 and TNFR2 share structural homology in
the extracellular TNF-α binding domains and exhibit similar binding affinity
for TNF-α, but they show no sequence homology, and induce separate
- 22 -
cytoplasmic signaling pathways following receptor-ligand interaction
(Wallach et al., 1999; Ledgerwood et al., 1999).
All known responses to TNF-α are triggered by binding to one of these two
receptors (Al-Lamki et al., 2001). Binding causes a conformational change
in the receptor, leading to the dissociation of the inhibitory protein called
“silencer of death domain” (SODD) from the intracellular death domain.
This dissociation enables the adaptor protein TNFR-associated-death-domain
(TRADD) protein to bind to the death domain, serving as a platform for
subsequent protein binding. Following TRADD binding, these pathways can
be initiated, including activation of NF-κB and mitogen-activated protein
kinases (MAPK) pathway and induction of death signaling (Wajant et al.,
2003; Chen and Goeddel, 2002). Based on cell culture work and studies with
receptor knockout mice, both the proinflammatory and the programmed cell
death pathways that are activated by TNF-α, and associated with tissue
injury, are largely mediated through TNFR1. The consequences of TNFR2
signaling are less well characterized, but TNFR2 has been shown to mediate
signals that promote tissue repair and angiogenesis (Bradley, 2008).
1.7.3 Interleukin-1β
Interleukins are cytokines first found to be expressed by leukocytes. The IL1 family has the following member: IL-1α, IL-1β, IL-1 receptor antagonist
(IL-1RA), IL-18 and IL-33. IL-1β is the prototypic pro-inflammatory
cytokine involved in immune defense against infection (Arend et al., 2008).
IL-1β is produced in a variety of cells including macrophages, monocytes,
neutrophils, keratinocytes and epithelial cells. In the human, IL-1β is
synthesized as a 31-kDa precursor which is processed and released from
cytoplasm. Pro-IL-1β is biologically inactive and must be converted to the
mature 17 kDa IL-1β by caspase-1 to acquire the ability to bind to receptors
and to activate cells. Both IL-1α and IL-1β bind to the same receptor and
have similar if not identical biological properties. There are two types of
receptors for IL-1, IL-1 receptor type I (IL-1RI) and IL-1 receptor type II
(IL-1RII), and an IL-1R accessory protein (IL-1RAcP) (Eisenberg et al.,
1991; Martin and Falk, 1997; May and Ghosh, 1998). IL-1RI possesses a
long cytoplasmic domain of 213 amino acids and is capable of activating
cells, whereas IL-1RII has only a short intracellular domain consisting of 29
amino acids and may act as a suppressor of IL-1 activity by competing for
IL-1 binding (Kuno and Matsushima, 1994; Vigers et al., 2000). After IL-1
binds to IL-1RI, IL-1RAcP joins with IL-1/IL-1RI to form a complex,
leading to cell activation mediated by the cytoplasmic domains of both
receptor chains. This complex recruits a number of intracellular adapter
- 23 -
molecules, including MyD88, IL-1R-associated kinase (IRAK), and TNF
receptor-associated factor 6 (TRAF6), to activate signal transduction
pathways such as NF-κB, activator protein-1 (AP-1), JNK, and mitogenassociated protein kinase (p38 MAPK) (O'Neill and Greene, 1998;
Verstrepen et al., 2008). IECs are the major source of IL-1RI in the human
mucosa. Upon inflammation they respond with upregulation of IL-1RI
production instead of IL-1RII (Daig et al., 2000). IL-1 can induce IECs to
produce several cytokines, suggesting that IEC is important in a cytokine
network at the intestinal mucosa to amplify the effects of IL-1 during an
inflammatory response (McGee et al., 1996).
1.7.4 Interleukin-6
The IL-6 is a 185 amino acid multifunctional protein that acts as both a proinflammatory and anti-inflammatory cytokine. It is produced by many cell
types including macrophages, fibroblasts, endothelial cells, keratinocytes and
epithelial cells (Hirano et al., 1986; Xu et al., 1997). IL-6 expression is
induced during the acute-phase response by a variety of stimuli, which
include cytokines such as IL-1, TNF-α, and platelet-derived growth factor
(PDGF). Bacterial and viral infection and microbial components such as LPS
are also potent inducers of IL-6 (Taga and Kishimoto, 1997; Heinrich et al.,
1990; Kishimoto et al., 1995; Kamimura et al., 2003; Kishimoto, 2005). IL-6
signals through the IL-6 receptor (IL-6R), which is composed of two
different subunits, an alpha subunit, IL-6 receptor subunit alpha (IL-6Rα)
and glycoprotein 130 (gp130), a receptor subunit for signal transduction
(Paonessa et al., 1995; Hirano, 1998). IL-6Rα is the binding component
specific to IL-6. In contrast, gp130 transmits signals not only of IL-6 but also
of IL-6-related cytokines such as IL-11, ciliary neurotrophic factor,
cardiotrophin-1, cardiotrophin-like cytokine, leukemia inhibitory factor,
neuropoietin, oncostatin M, IL-27 and IL-31 (Taga and Kishimoto, 1997;
Derouet et al., 2004; Dillon et al., 2004; Pflanz et al., 2004). Binding of IL-6
to IL-6Rα leads to homo- and hetero-dimerization of gp130 and these
complexes bring together the intracellular regions of gp130 to initiate a
signal transduction cascade through initiating cellular events including
activation of gp130-associated JAK kinases and activation of ras-mediated
signaling. The tyrosine-phosphorylated gp130 further transmits signals by
recruiting Src homology region 2 (SH2) domain-containing signaling
molecules such as the protein tyrosine phosphatase SHP-2 and STAT1 and
STAT3. These factors and other transcription factors like AP-1 and SRF
(serum response factor) that respond to many different signaling pathways
- 24 -
come together to regulate a variety of complex promoters and enhancers
that respond to IL-6 and other signaling factors (Heinrich et al., 2003;
Murray, 2007; O'Sullivan et al., 2007; Xu and Qu, 2008).
Together with TNF-α and IL-1, IL-6 is also considered a major
proinflammatory cytokine important in the protection from pathogens during
an infection. In the intestine, the major sources of IL-6 are macrophages and
epithelial cells. In human colon, IL-6 expression is induced locally in
epithelial cells and macrophages in response to PAMPs (Stadnyk and
Waterhouse, 1997; Kusugami et al., 1995; Reinecker et al., 1993). IL-6
deficiency causes more severe infection-associated mucosal inflammation
and ulceration, indicating that this cytokine was protective against colitis
caused by C. rodentium infection in mice (Dann et al., 2008).
1.7.5 Interleukin-8
IL-8 or CXCL8 is a cytokine belonging to the CXC chemokine or αchemokine family. It has leukocyte chemotactic properties (Baggiolini et al.,
1994). IL-8 is produced by macrophages, fibroblasts, endothelial cells and
epithelial cells. Expression of IL-8 is regulated by different stimuli including
inflammatory signals, chemical and environmental stresses, and steroid
hormones (Ben-Baruch et al., 1995). IL-8 is a major mediator of immune
reactions in the innate immune system. The major sources of IL-8 in the
intestinal mucosa are macrophages, epithelial cells, and fibroblasts. A large
number of studies have shown increased levels of IL-8 in infected mucosal
(Daig et al., 1996; Rogler and Andus, 1998). In inflammation, IL-8 is
secreted at high levels to attract and activate neutrophils to the diseased site
(Eckmann and Kagnoff, 2005; Jung et al., 1995). Neutrophils are effector
cells that kill bacteria by phagocytosis and intracellular killing mechanisms
but also destroy affected tissues through the release of enzymes from
granules. IL-8 also enhances the expression of adhesion molecules that
promote of neutrophils recruitment into different tissue sites (Middleton et
al., 1997; Kobayashi, 2008). While neutrophil granulocytes are the primary
target cells for IL-8, endothelial cells, macrophages, mast cells and epithelial
cells also respond to this chemokine.
There are several receptors of the cell surface capable to binding IL-8. The
most frequently studied are two G protein-coupled receptors, CXCR1 and
CXCR2 (Holmes et al., 1991; Murphy and Tiffany, 1991). After activation
of heterotrimeric G proteins, IL-8 signaling promotes activation of the
primary effectors phosphatidyl-inositol-3-kinase or phospholipase C. In
addition, IL-8 signaling activates members of the Rho GTPase family and a
number of nonreceptor tyrosine kinases that regulate the architecture of the
- 25 -
cell cytoskeleton and its interaction with the surrounding extracellular
environment (Waugh and Wilson, 2008).
1.8 Intestinal microflora
The terms ‘intestinal microflora or microbiota’ are used to describe the entire
population of microorganisms in the intestine regardless of type and number.
The adult human intestinal microbiota is composed of an estimated 1014
commensal bacteria (Ley et al., 2006) with a total weight of approximately
1.5 kg. Somewhere between 500 and 1000 different species, of which most
have not yet been properly characterized, live in the intestine based on
molecular biology analysis of the collective genome, termed the
microbiome. (Guarner and Malagelada, 2003; Sears, 2005; Eckburg et al.,
2005; Gill et al., 2006).
In human duodenum, the density of bacteria is very low, typically less then
103 colony-forming units (CFU) per gram of contents, adhering to the
mucosal surface or in transit. Along the jejunum and distal ileum, the density
of bacteria ranges from 104 to 107 CFU per gram of intestinal content.
Further down, in the colon, the numbers increase up to around 1012 CFU per
gram of luminal content (Guarner, 2006). Within each compartment the
bacteria live either close to the epithelial cells or in the lumen (Schultsz et
al., 1999). Both DNA and RNA viruses are present in the intestine. In adult
fecal samples, the majority of the DNA viruses are phages, while the
majority of the RNA viruses are plant viruses. In the feces of infants,
Adenovirus, Astrovirus, Bocavirus, Enterovirus, Norovirus, Picobirnavirus,
Rotavirus, Sapovirus, and Torovirus were detected by using specific PCR
methods (Breitbart et al., 2003; Zhang et al., 2006; Breitbart et al., 2008).
Fungi and protozoa are also part of the intestinal flora, but little is known
about their numbers and distribution.
Population densities of intestinal bacterial species vary widely among
individuals but stay fairly constant within an individual over time (Guarner
and Malagelada, 2003; Eckburg et al., 2005; Gill et al., 2006; Li et al., 2008).
The vast majority (over 99%) of human intestinal bacteria belong to the
phyla: Firmicutes, Actinobacteria, Bacteroides, Proteobacteria and
Fusobacteria. The proportions of the different phyla differ between
compartments within the same individual (Wang et al., 2005; Eckburg et al.,
2005; Li et al., 2008; Andersson et al., 2008). The greatest diversity of
bacteria is found in colon, where the predominant bacterial genera are:
Bacteroides, Clostridium, Fusobacterium, Eubacterium, Ruminococcus,
Peptococcus, Peptostreptococcus, and Bifidobacterium. Other genera such as
- 26 -
Escherichia and Lactobacillus are present to a lesser extent (Ley et al.,
2006; Eckburg et al., 2005; Artis, 2008).
The colonization of gastrointestinal tract starts immediately at birth. A germfree infant is immediately exposed to the microflora of the mother and of the
surrounding environment irrespective of vaginal delivery or Caesarian
section. In the beginning, the composition and temporal pattern of the
microbial communities varies widely from baby to baby. By the end of the
first year of life, the idiosyncratic microbial ecosystem in each baby,
although still distinct, has converged toward a profile characteristic of the
adult gastrointestinal tract (Palmer et al., 2007). By the second year of life
the fecal microflora resembles that of adults (Mackie et al., 1999). The
composition of the commensal bacterial communities is strictly regulated by
competition for nutrients and space. This process is dependent on complex
regulatory mechanisms, which involve the microflora itself, the surrounding
environment, diet, and the innate and adaptive immunity (Sanderson, 1999;
McCracken and Lorenz, 2001).
The intestinal commensal microflora fulfills a host of useful functions,
including digestion of unutilized carbohydrates and nitrogenous substrates;
production of essential nutrients and short chain fatty acids (SCFAs);
stimulating and modulating development and function of the intestinal and
systemic immune system; repressing the growth of harmful microorganisms;
training the immune system to respond only to pathogens and preventing
allergy (Guarner and Malagelada, 2003; Macpherson and Harris, 2004;
Calder et al., 2005; Sears, 2005; Artis, 2008).
Alterations in the acquisition or the composition of the intestinal microflora
may trigger or influence not only the course of various metabolic and
inflammatory disease states but also susceptibility to or progression of,
immune-mediated diseases. Ley and colleagues have demonstrated that the
relative proportion of commensals of Bacteroidales is reduced both in obese
individuals and in inbred mouse strains that are genetically predisposed to
obesity. The intestinal commensal bacteria isolated from obese mice were
more effective at releasing calories from food, and these bacteria transmitted
this trait to germ free mice, resulting in greater deposition of adipose tissue.
(Bäckhed et al., 2004; 2007; Ley et al., 2005; 2006; 2006; Turnbaugh et al.,
2006). Currently, Kalliomäki and colleagues have indicated that alterations
in the gut microbiota may be linked not only to obesity in children but also to
the development of allergy (Kalliomäki et al., 2008). Alterations in the
intestinal microflora have also been seen both in different patient groups and
murine models of airway inflammation, arthritis, inflammatory bowel
disease and necrotizing enterocolitis (Swidsinski et al., 2002; 2005; 2005;
Guarner and Malagelada, 2003; Kalliomäki et al., 2001; 2008; Noverr and
- 27 -
Huffnagle, 2004; Tlaskalová-Hogenová et al., 2004; de la Cochetiere et al.,
2004; Ott et al., 2004; Noverr and Huffnagle, 2005; De Hertogh et al., 2006).
The intestinal microflora displays a wide range of metabolic activities, which
could produce potentially harmful products including toxins and carcinogens
that have been implicated in such conditions as multisystem organ failure,
sepsis, colon cancer, and inflammatory bowel disease. Thus, a major factor
in health is the balance of bacterial numbers, species and distribution
(Guarner and Malagelada, 2003; Guarner, 2006).
1.9 Vibrio cholerae
Vibrio cholerae is a facultative anaerobic, motile, gram negative, curved rodshaped bacterium with a polar flagellum, first described and named V.
cholerae by the Italian doctor, Filippo Pacini, in 1854 (Kaper and Morris,
1995). There are more than 200 serogroups of V. cholerae identified on the
basis of their lipopolysaccharide O side chain antigenic structures (Osawa et
al., 1997). Two serogroups of V. cholerae O1 and O139 infect humans and
cause epidemic and pandemic cholera. The serogroup V. cholerae O1 is
subdivided into two biotypes, classical and El Tor depending on biochemical
properties and phage sensitivity. Each biotype can be divided into three
serotypes depending on expression of three O-antigens (A, B, and C):
Ogawa (A and B), Inaba (A and C) and Hikojima (A, B and C) (Kaper et al.,
1995).
Cholera is a severe diarrheal disease caused by V. cholerae infection in the
small bowel, generally contracted by intake of contaminated food or water.
WHO estimates 3 to 5 million cases worldwide every year and that more
than
120,000
people
die
from
cholera
(WHO,
http://www.who.int/topics/cholera/en/). V. cholerae is widely distributed in
aquatic environments and cholera outbreaks are associated with
contaminated food and water supplies. The seasonality of cholera has been
associated with physico-chemical and biological factors (Lipp et al., 2002).
However, many factors affect the survival of V. cholerae in aquatic
environments such as attachment to plankton, entering into and resuscitation
from viable but non-culturable state, and predation (Cottingham et al., 2003).
Factors regulating the level of viable V. cholerae O1 and O139 in aquatic
environment are still being investigated (Faruque et al., 2005). The infective
dose of V. cholerae to cause cholera is approximately 108 live cells (Sack et
al., 2004). Therefore, the bacteria need a biological reservoir in order to
grow and survive in high concentrations to infect humans. Finding of aquatic
reservoirs of V. cholerae is an important factor in the epidemiology of the
disease.
- 28 -
V. cholerae can secrete virulence factors in two ways either soluble
reagents or carried by vesicles (Mashburn-Warren and Whiteley, 2006). V.
cholerae adhere to the intestinal epithelial lining by means of toxincoregulated pili (TCP). Cholera enterotoxin (CTX) that is produced by the
adherent vibrios and secreted across the bacterial outer membrane into the
extracellular environment is a major agent involved in severe diarrheal
disease. It is composed of a catalytically active heterodimeric A-subunit
linked with a homopentameric B-subunit. The B subunit binds to the CTX
receptor ganglioside GM1 on the intestinal epithelial cell leading to
conformational alterations of the toxin structure which to release the Asubunit that enters inside the epithelial cell. The intracellular glutathione
reduces the disulfide bond of the A subunit that dissociates into A1 and A2
(Lencer and Saslowsky, 2005; Chinnapen et al., 2007). The A1 subunit
hydrolyzes nicotinamide adenine dinucleotide to ADP-ribose and
nicotinamide. The ADP-ribose activates adenylate cyclase to hydrolyze ATP
to cyclic AMP resulting in a substantial increase in intracellular
concentrations of cyclic AMP, followed by a protein kinase -mediated
phosphorylation of the major chloride channel of intestinal epithelial cells.
Osmotic movement of large quantities of water accompanies the net increase
in Cl− secretion into the intestinal lumen. The subsequent loss of water and
electrolytes leads to the severe diarrhea and dehydration that are
characteristic of cholera (Vanden Broeck et al., 2007). However, the loss or
absence of the ctx gene coding for the CTX does not eliminate all disease
symptoms from EI Tor strains (Ninin et al., 2000; Saka et al., 2008).
Naturally occurring CTX-negative strains both of the EI Tor biotype and of
non-O1 types have been associated with enterocolitis, as well as septicemia
and extraintestinal infections (Anderson et al., 2004). Additionally, studies
with CTX-negative vaccine strains resulted in mild to severe diarrhea and
evidence of inflammation in human volunteers (Silva et al., 1996; Tacket et
al., 1997). All these findings strongly suggested the existence of additional
virulence factors associated with EI Tor and non-O1 V. cholerae
pathogenesis. One of the additional virulence factors is the pore-forming
Vibrio cholerae cytolysin (VCC), also referred to as hemolysin A (HlyA),
which is encoded by a hlyA gene located on the V. cholerae chromosome II
(Heidelberg et al., 2000). VCC/HlyA was shown to be synthesized and
secreted as a pro-cytolysin monomer of about 80 kDa molecular weight that
is activated by proteolytical cleavage by different proteases removing a 15
kDa polypeptide at the N-terminus (Alm et al., 1988; Nagamune et al., 1996;
1997). In the presence of cholesterol and ceramide-rich membranes active
VCC/HlyA forms heptameric oligomers, which insert into lipid bilayers
generating membrane pores with an internal diameter of 1.2 nm (Zitzer et al.,
- 29 -
1999; Harris et al., 2002; Pantano and Montecucco, 2006). It has been
reported that VCC/HlyA is capable of causing several cytotoxic effects such
as cell lysis and extensive vacuolization in vitro, depending upon the cell
line studied and the toxin concentration (Moschioni et al., 2002; Alm et al.,
1991; Figueroa-Arredondo et al., 2001; Coelho et al., 2000; Mitra et al.,
2000; Zitzer et al., 1997; Valeva et al., 2008; Saka et al., 2008). Recently,
Gutierrez et al. (Gutierrez et al., 2007) described that the vacuolization
phenomenon associated to VCC/HlyA was an autophagic response of the
cells against this toxin and was related to a protective cell response upon
VCC/HlyA intoxication.
CTX-negative and TCP-negative V. cholerae strains were isolated from
patients suffering cholera-like diarrhea. In addition to gastroenteritis, noncholera toxigenic V. cholerae infections can cause extraintestinal infections
such as cellulitis, meningitis, wound infections, septicemia and bacteremia
(Anderson et al., 2004; Hlady and Klontz, 1996; Ou et al., 2003). No definite
virulence factor(s) has yet been identified to explain their disease producing
ability. It has been proposed that VCC/HlyA is implicated in the
pathogenesis of these diseases. However, studies so far are contradictory
regarding the real role of VCC/HlyA (Alm et al., 1991; Figueroa-Arredondo
et al., 2001; Ichinose et al., 1987; Fullner et al., 2002; Levine et al., 1988;
Faruque et al., 2004). The finding that purified fractions of VCC/HlyA
produced fluid accumulation in ligated rabbit ileal loops supports the notion
of a role for VCC/HlyA in gastroenteritis (Ichinose Y, et al. 1987). Recently
Saka and colleagues (Saka et al., 2008) reported that VCC/HlyA may trigger
apoptotic cell death during infection in vivo and they suggested that the
apoptotic cell death pathway plays the major role in pathogenesis of a CTX
negative V. cholerae non-O1, non-O139 strain.
Proteases constitute important bacterial virulence factors. They may function
in invasion, immune evasion, nutrition, and reproduction in a host
environment (Armstrong, 2006). Several proteases produced by pathogens
are able to modulate other bacterial virulence factors, for example the V.
cholerae hemagglutinin/protease (Hap) can process and activate VCC/HlyA
(Booth et al., 1984; Nagamune et al., 1996). One newly described V.
cholerae protease, PrtV (protease of V. cholerae), was shown to confer
protection against grazing by natural bacteriovorous predators (Paper III).
PrtV belongs to an M6 family of metallopeptidases, with the best
characterized member of the family being an immune inhibitor A (InA or
InhA) from Bacillus thuringiensis, a bacterium widely used in agriculture to
control insect pests (Ogierman et al., 1997). Vaitkevicius et al. recently
reported that PrtV is able to degrade mammalian extracellular matrix
components and plasminogen (Vaitkevicius et al., 2008).
- 30 -
1.10 Celiac disease
Celiac disease (CD) is a gluten-dependent enteropathy that occurs in
genetically predisposed individuals (Troncone et al., 2008).
Clinical symptoms. The classical clinical manifestations of CD include
chronic diarrhea, weight loss and anemia, but while CD is primarily an
intestinal disorder, intestinal symptoms may also be limited or even absent.
A significant proportion of CD patients present with extra-intestinal
symptoms, such as skin lesions, bone pains and fractures, infertility, apthous
ulceration, and polyneuropathia etc (Farrell and Kelly, 2002; Fasano, 2005).
However, there are also some atypical forms with negligible symptoms
(silent CD patients), but still with a positive serologic test and small
intestinal villous atrophy (Gasbarrini et al., 2008).
Diagnosis. Traditionally, the diagnosis of CD has required a biopsy of the
mucosa taken from proximal small intestine. Histological characteristics
show villus atrophy, crypt hyperplasia, and an increased number of
lymphocytes in the lamina propria and within the epithelium. In 1970, the
European Society of Pediatric Gastroenterology, Hepatology, and Nutrition
(ESPGHAN) established a three-biopsy procedure as a standard for the
diagnosis of celiac disease. The three-biopsy procedure includes one initial
biopsy on suspicion of CD during active disease, a second biopsy on glutenfree diet (GDF) and a third biopsy after challenge with gluten-containing diet
(Meeuwisse, 1970). Although the three-biopsy procedure has been
abandoned in many centers due to the introduction of serological tests, at
least one biopsy from proximal small intestine is generally required. The
serologic tests for diagnosis and screening of CD include anti-gliadin
antibodies (AGA), anti-endomysium antibodies (EMA) and anti-tissue
transglutaminase antibodies (Carlsson et al., 2001).
Prevalence. Serologic screening studies have revealed that the prevalence of
CD approaches 1% in the Caucasian populations, but may be even higher
due to under-diagnosis. A growing portion of CD-diagnosis is being made in
asymptomatic individuals (West et al., 2003; 2007; van Heel and West,
2006). CD is one of the most common chronic childhood disorders in
Sweden. The prevalence of CD in our study series was at least 1.0%, but
may be as high as 2.0% (Carlsson et al., 2001). In the total childhood
population (0-15 years old) the annual incidence rate of clinically diagnosed
CD was 10-45 cases per 100 000 person during the period 1973 to 2003.
However, among children of different age groups the annual incidence rate
varied considerably (Olsson et al., 2008). In the group of children under 2
years of age the annual incidence rate varied from 65 during 1973 to 1984, to
around 200 during the time period 1985 to 1995 (Ivarsson et al., 2000), and
- 31 -
then to 98 from 2001 to 2003 (Olsson et al., 2008). In the group of children
over 2 years of age, the incidence rate varied from 14 to 52. The observed
differences in CD incidence rates in relation to year of presentation with CD
possibly indicate that some environmental factors, such as an infectious
component (Ivarsson et al., 2003), infant feeding style (breast feeding versus
bottle feeding), or amount and/or timing of gluten administration in infancy
(Ivarsson et al., 2000; 2002), influence disease incidence.
Genetics. CD has a tendency to occur more often in some families. The
concordance rate of CD is about 10% in first-degree relatives, about 30% in
siblings and 75% to 100% in monozygotic twins, a rate higher than in any
other condition with a multi-factorial basis (Sollid and Thorsby, 1993;
Houlston and Ford, 1996; Greco et al., 2002). The principal determinants of
genetic susceptibility are alleles within the highly variable human MHC
complex, also called human leukocyte antigen (HLA) system1. Over 97% of
CD patients are HLA-DQ2 or HLA-DQ8. The reason why these genes are
associated with CD is that the peptide-binding groove of HLA-DQ2 and
HLA-DQ8 favors the binding of gluten peptides with negatively charged
residues at key anchor positions (Sollid, 2000; Hourigan, 2006; Kagnoff,
2007). However, up to 30% of the normal population, most of them eating
food containing gluten, carry the HLA-DQ2 and/or HLA-DQ8 alleles, but do
not develop CD (Sollid, 2000; Hourigan, 2006). Thus, exposure to gliadin
and related prolamins from wheat, barley or rye and appropriate HLA-DQ
haplotypes are necessary, but not sufficient to develop the disease. Apart
from HLA genes, genes involved in intestinal permeability and immunity
have been proposed to be risk factors for CD (Wapenaar et al., 2008; Hunt et
al., 2008). There is no doubt that additional inherited and/or environmental
factors and/or epigenetic factors are involved in contraction of CD (Green
and Jabri, 2006).
Gluten. Almost 60 years ago, the Dutch pediatrician, Wim Dicke recognized
for the first time gluten as a trigger of CD (Losowsky, 2008). Gluten is found
in the seeds of cereal grains of the tribe Triticeae and is a mixture of
prolamins of a group of plant storage proteins. Prolamins are characterized
by a high glutamine and proline content that dissolve only in alcohol
solutions. They are resistant to proteases and peptidases in the intestine.
1
The functions of MHC are to act as recognition molecules and to present antigen to effector cells in
the immune system. There are two classes of MHC molecules, MHC class I, which is present on
almost every nucleated cell in the body and MHC class II molecules, which is present on the cell
surface of antigen presenting cells, such as DCs, B-cells, macrophages, and also on IECs of small
intestine. HLA-DQ is a αβ-heterodimer of the MHC class II type. The α and β chains are encoded by
HLA-DQA1 and HLA-DQB1 loci respectively and contain many alleles. There are 7 HLA DQ
variants (DQ2 and DQ4 through 9). Different DQ isoforms can bind to and present different antigens
to T-cells.
- 32 -
Some prolamins, notably wheat gliadin, and similar proteins and are only
partly degraded resulting in long peptides in barley and rye induce CD in
genetically predisposed individuals (Shewry and Halford, 2002; Green and
Cellier, 2007; van Heel and West, 2006). There is also evidence of a direct
toxic effect of gluten peptides in several biological models. However, the
failure to control the inflammatory response may be one of the factors
underlying gluten intolerance in these individuals. Many different gliadin
peptides have been shown to express epitopes for gliadin-specific T-cell
clones and also to be active when intestinal biopsies or isolated cells from
intestinal biopsies of CD patients are challenged with them in vitro (Molberg
et al., 1997; van de Wal et al., 1998; Sjöström et al., 1998; Mantzaris and
Jewell, 1991; de Ritis et al., 1988; Maiuri et al., 1996).
Transglutaminase. Because of their high glutamine content and specific
sequence patterns, prolamins are excellent substrates for deamidation by
tissue transglutaminase (tTG). Tissue transglutaminase or transglutaminase 2
(TG2), a member of the vast TG enzyme family, is a ubiquitously expressed
multifunctional enzyme and present in almost all cell types in the body. In
the intestine, tTG is found intra-cellularly in enterocytes and myofibroblasts.
Extra-cellularly, this enzyme is located in the membranes of enterocytes and
in the subepithelial connective tissue of the small intestine (Biagi, et al.,
2006). tTG catalyzes an ordered and specific deamidation of certain
glutamine residues of food gliadins and deamidated gluten peptides bind
more effectively to HLA-DQ2 and HLA-DQ8 and trigger T cell responses
(van de Wal et al., 1998; Johansen et al., 1996; Godkin et al., 1997;
Anderson et al., 2000). Recently, Lammers and colleagues reported that one
region of α-gliadin binds to the chemokine receptor CXCR3 and leads to
MyD88-dependent zonulin release and increased intestinal permeability.
Disruption of tight junctions allows entry for large peptides (Lammers et al.,
2008).
Immuno-pathology. Gluten reactive T lymphocytes in the jejunal mucosa
play a central role in the pathogenesis of CD. The inflammatory process,
mediated by T cells, leads to disruption of the normal tissue architecture
such as villous atrophy, crypt hyperplasia, and increased infiltration by
intraepithelial lymphocytes and dysfunction of the small intestinal mucosal
lining, leading to malabsorption. To delineate local T lymphocytes response
to gluten, our group investigated that the gene expression of cytokines,
including IL-2, IFN-γ, TNF-α, IL-4, IL-10, and TGF-β1, in jejunal T cell
subsets, from pediatric CD patients with active disease (i.e. untreated- and
gluten challenged CD patients) and asymptomatic CD patients and healthy
controls. The results showed that the high production levels of IFN-γ and IL10 by intraepithelial T lymphocytes in CD patients could be the reasons for
- 33 -
both recruitment of intraepithelial lymphocytes and a leaky epithelium,
leading to a vicious circle with amplified immune activity and establishment
of intestinal lesions (Forsberg et al., 2002). The excessive production of IFNγ is believed to drive the formation of intestinal lesions in active CD.
CD94−CD8+αβ T cells within the epithelium constitute the major sourse of
IFN-γ in active disease. The production of IL-10 may be a common feature
of IELs producing proinflammatory cytokines, thereby attempting to limit
inflammation in an autocrine fashion. Active CD is associated with increased
frequencies of αβTCR+ and γδTCR+ IEL and CD4+αβTCR+ cells in the
lamina propria of the small intestinal mucosa, including increased
frequencies of IEL expressing the NK-receptors CD94 and NKG2D and
lamina propria lymphocytes (LPL) expressing CD25 (Halstensen and
Brandtzaeg, 1993; Forsberg et al., 2007). Other groups have reported that the
cytokine network involved in CD may include IL-15, IL-18, IL-21, and
perhaps IL-27, which can drive the inflammatory response through STAT1
and STAT5 pathways (Benahmed et al., 2007; Garrote et al., 2008).
It could be argued that the pathogenic activity of T cells in the small
intestinal mucosa of CD patients is caused by an impaired local T cell
development and/or failure of adequate T cell receptor (TCR)-editing upon
encounter with dietary antigens, leading to an abrogated development of oral
tolerance. Our group also observed two signs of extrathymic TCR-genes
rearrangement in the jejunal mucosal, including recombination activating
gene-1 (RAG1) and preTα-chain gene. Expression levels of the four RAG1
mRNA splice forms and preTα-chain mRNA were determined in γδTCR+
IEL, αβTCR+ IEL, and TCR−CD2+CD7+ IEL of children with CD and
controls. The results show that (1) RAG1 is normally expressed in both
TCR+ and TCR− IEL while preTα-chain is only expressed in the immature
TCR− IEL and (2) “extrathymic” splice forms of RAG1 mRNA and preTαchain mRNA are down-regulated in IEL of CD patients regardless of disease
activity suggesting that CD patients have impaired TCR-gene rearrangement
in their jejunal mucosa which could be a contributing factor for CD (Bas et
al., 2003; 2009).
Intestinal microbiota. It has been suggested that intestinal bacterial infection
or microflora alterations might be involved in the pathogenesis of CD. An
intestinal infection could cause inflammatory response and damage to the
intestinal mucosa, thereby raising the activity of tTG, possibly increase the
permeability of gluten peptides across the epithelial barrier, and stress the
epithelial cells to express major inflammatory factors. By scanning electron
microscopy, our group observed that rod-shaped bacteria were associated
with the intestinal epithelium in children with CD but not in controls
- 34 -
(Forsberg et al., 2004). Furthermore, by studying short chain fatty acids in
fecal samples from CD children and healthy controls, Tjellström et al. found
an altered pattern of these fatty acids in both treated and untreated CD, as
compared to controls (Tjellström et al., 2005; 2007). They suggested that
their findings might reflect a deviant gut flora in CD patients. Moreover, by
fluorescent in situ hybridization (FISH) coupled with flow cytometry, Nadal
and co-workers reported that the higher incidence of Gram-negative and
potentially proinflammatory bacteria in the duodenal microbiota of celiac
children was linked to the symptomatic presentation of the disease and could
favor the pathological process of the disorder (Nadal et al., 2007).
Alterations of the intestinal microflora in fecal samples from CD children
were demonstrated by FISH (Collado et al., 2007) and by PCR coupled with
denaturing gradient gel electrophoresis (DGGE) analysis (Sanz et al., 2007).
To understand the relationship between intestinal bacteria and CD, it is
necessary to perform a detailed characterization of the intestinal microflora
from both patients with CD and healthy individuals.
Treatment. At present, the only effective treatment for CD is a lifelong strict
gluten-free diet (Kupper, 2005). In practice, however, this generally means a
supposedly harmless level of gluten rather than a complete absence
(Akobeng and Thomas, 2008). The strict gluten avoidance allows the
intestinal mucosal restitution and improvement of symptoms in most cases
and can also eliminate the heightened risk of osteoporosis and intestinal
cancer (Treem, 2004).
- 35 -
2. Aims of the Thesis
The purposes of this thesis were to study the composition of the microbiota
at the proximal small intestine in children with or without celiac disease and
also to study the immune responses of intestinal epithelial cells to different
stimuli especially Vibrio cholerae virulence factors. The specific aims were:
• To characterize the composition of microflora at proximal small
intestine in children with celiac disease and controls by cultureindependent and culture-dependent methods.
• To identify the rod-shaped bacterium/a previously detected by scanning
electron microscopy in proximal small intestine of children with celiac
disease.
• To establish an in vitro model for studies of innate defense mechanisms
of human intestinal epithelial cells.
• To study the roles of a Vibrio cholerae protease, PrtV and its putative
substrate Vibrio cholerae cytolysin, VCC as secreted pathogenic factors
for human intestine in Vibrio cholerae infection.
- 36 -
3. Results and Discussion
3.1 Proximal small intestinal microbiota and identification of rodshaped bacteria associated with childhood celiac disease (Paper I)
As discussed earlier, the fact that an individual expresses HLA-DQ2 or
HLA-DQ8 on her/his immune cells and is exposed to gluten in the diet is not
sufficient to develop CD. Obviously other factors, genetic, epigenetic or
environmental, are also required to precipitate disease. One such factor may
the composition of the intestinal microbiota. Indeed a previous investigation
in our laboratory, using scanning electron microscopy (SEM), revealed that
rod-shaped bacteria were associated with the epithelium of proximal small
intestine of children with CD in contrast to controls (Forsberg et al., 2004).
Moreover, even children with CD on a gluten-free diet and with no clinical
symptoms of CD retained these bacteria indicating that it may be an inherent
property to harbor rod-shaped bacteria in the small intestine.
In order to identify these bacterium/a and to find possible links between
specific bacterial populations and the development of CD, we have
determined the composition of the bacterial flora of proximal small intestine
in children with CD and controls, using culture-dependent and cultureindependent methods. In total, we analyzed biopsy samples from 63
children, including 18 biopsies from clinical controls, (3 of them from
healthy siblings of CD patients) and 33 biopsies from children with CD
collected during 2004 to 2007. The remaining 12 biopsies were “historical”
biopsies from children with CD born during the Swedish CD epidemic.
These biopsies had been stored frozen at -80°C. The fresh biopsies were
washed repeatedly with PBS containing dithiothreitol (DTT) to dissolve the
mucus layer. Among these biopsies, 19 were from untreated CD patients, 12
from CD patients treated on a gluten-free diet and 3 from CD patients
challenged with gluten containing diet. To allow different types of analysis,
biopsies were divided into 4 parts, each part treated as required by the
particular method utilized.
The proximal small intestine is a unique ecological niche. As one of the
culture independent methods we used sequencing of the bacterial 16S rRNA
gene. Totally we collected 2,247 sequences of an about 440 base-pair long
fragment of the bacterial 16S rRNA gene from 63 individuals. In Table 1 the
data are displayed as the percentage of bacteria in proximal small intestine
belonging to different phyla. Here we have pooled the results from CD and
controls since, as discussed below there was no major difference between
- 37 -
CD and controls in biopsies collected during 2004-2007. Comparing with
the microbiota from other sites, the composition of the bacterial biota in
proximal small intestine is different from those of the other sites indicating
that it is a distinct ecological niche. At the phylum level, Firmicutes,
Proteobacteria and Actinobacteria dominate the bacterial community while
Bacteriodetes and Fusobacteria constitute small populations. One hundred
and sixty-two Genus Level Operational Taxonomic Units (GELOTUs),
named jej201 to jej362, of six different phyla were identified. Thirty-five
sequences were sufficiently different from all previously known bacterial
sequences (i.e. ≤ 97% homology) to most probably represent previously
uncharacterized phylotypes (Figure 2 and supplementary Table 2 in Paper I).
The most diverse phyla were Firmicutes and Proteobacteria the least diverse
phylum was TM7.
Composition and properties of the normal biota of proximal small intestine.
These results also show that the mucosa-associated microbiota in proximal
small intestine of Swedish children with normal intestinal mucosa is
dominated by two bacterial genera namely Streptococcus and Neisseria.
Together with a limited number of other genera, i.e. Veillonella, Gemella,
Actinomyces, Rothia and Haemophilus they constitute almost the entire
bacterial community. In total 42 different GELOTUs were detected of which
30 had not been described previously in proximal small intestine, 18 of
which are unknown. Our findings are in general agreement with, but
substantially expand previous studies on adults using culture-based methods
(Sullivan et al., 2003; Zilberstein et al., 2007) also showing a predominance
of Streptococci. To ensure that bacteria identified by 16S rDNA sequencing
and electron microscopy are alive and potentially able to exert biological
effects at the epithelial lining, we attempted to cultivate bacteria from
biopsies. Ten consecutive biopsies collected during 2007 were investigated
using selective conditions for anaerobic and aerobic growth. Live bacteria
were identified in all ten biopsies. Bacteria from eight of the biopsies (5 CD
patients and 3 controls) were subjected to quantitative cultivation methods
and characterized by standard bacteriological tests. The results (Table 3 in
Paper I) demonstrated that all genera identified by 16S rDNA sequencing in
controls could be cultivated except Gemella, certifying that they indeed were
alive. Most of the bacteria in the “majority community” of controls were
cocci or coccobacilli. The only rods regularly found were bacteria of the
genus Actinomyces. The bacteria are to a significant degree associated to the
mucosal surface since they were identified in biopsies washed with buffer
containing DTT, a solution that will dissolve the outer mucin layer. We
therefore conclude that the bacteria of the “majority community” are not just
- 38 -
transient passengers in the gut lumen but constitute an organ-specific
biofilm. It is reasonable to assume that it is protective, limiting access for
potential pathogens to the mucosal surface and possibly influencing the
maturation of the local immune system. Apparently lactobacilli are transient
visitors in proximal small intestine of children since they were not found
regardless of detection method (Table 4 and Figure 2 in Paper I) while
abundantly seen when the intestinal content was included (Sullivan et al.,
2003; Zilberstein et al., 2007).
Microbiota of children with CD, results based on biopsies collected during
2004-2007. Using bacterial 16S rDNA sequencing we compared the
composition of the bacterial flora in non-CD children with that of children
with CD (supplementary Table 2 in Paper I). There was no statistically
significant difference between CD patients and controls in frequency of
biopsies containing any of the 38 different bacterial genera discovered in the
material. However, a difference between the two groups was that
Haemophilus was more common and Neisseria less common in CD than in
controls. Although it cannot be excluded that subtle differences exist that
would have been revealed if a larger number of clones had been analyzed we
conclude that the proximal small intestine microbiota is essentially the same
in the two groups. Moreover, dividing the group of CD patients into those,
which were on gluten-free diet and those, which presented with CD we did
not find major differences in the microbiota either. Similarly only small
differences were seen in the flora between young untreated- and older
untreated CD patients.
Almost all of these biopsies were also investigated by SEM for presence of
rod-shaped bacteria. Only one biopsy in each group contained such bacteria.
This finding was unexpected since the previous investigation showed that
rod-shaped bacteria were present in 20-40% of the children with CD in
contrast to controls, which essentially lacked such bacteria (Forsberg et al.,
2004). From this study we tentatively conclude that the differences seen
between CD and controls in the previous study cannot be seen in the
collection of current biopsies.
Microbiota of children with CD, results based on “historical” biopsies.
Fortunately parts of nine CD-biopsies, which showed presence of rod-shaped
bacteria by SEM (Figure 3 in Paper I) in the earlier study and which had
been stored frozen at -80°C were available for analysis. When these biopsies
were compared with CD biopsies lacking rod-shaped bacteria as defined by
SEM, three differences stand out: bacteria similar to unclassified
Clostridales, Prevotella species and Actinomyces graevenitzii were
- 39 -
significantly more common in the former biopsies than in the latter (Table 1
in Paper I). It is interesting to note that all 3 bacteria are rods. At the phylum
level the two groups differed in that particularly Bacteroidetes and
Fusobacteria were more common in the group of “historical” CD biopsies. It
could be said that the microbiota is more colon-like. It should be pointed out
that this Table displays only those genera that were different either in
frequency of positive biopsies or number of clones between the two groups.
Thus, bacteria similar to Streptococcus pseudopneumonie and Neisseria
polysaccharea both occurred at high frequencies and in all biopsies of both
groups. However, there was no statistically significant difference between
the groups. Since the frozen biopsies could not be washed it could be argued
that the difference between the two groups was due to lack of washing in one
group and buffer/DTT washing in the other. We therefore studied the relative
binding affinity of different bacterial genera to the epithelial lining. Ten
DTT-washed biopsies and their corresponding wash-fluids were analyzed.
The DTT-washed biopsy is expected to contain bacteria actually attached to,
or inside, the mucosal tissue while the wash fluid would contain bacteria
from the gut lumen or from inside the mucous layer that covers the epithelial
cells. An adhesion index i.e. bacteria in biopsy/ bacteria in washings could
be calculated (Table 2 in Paper I). Seven genera showed preferential binding
to the epithelial lining, including jej203 (similar to unclassified Clostridiales
species), jej273 (similar to Actinomyces species) and jej 308 (similar to
Prevotella species). Taken together these results suggest that any or all of the
bacteria of the genera Clostridium, Prevotella and Actinomyces could be the
rod-shaped bacteria seen by SEM in the earlier study. On the basis of SEM
morphology (Figure 3 in Paper I) it is, however, not possible to discriminate
between these possibilities.
Most interestingly, bacteria of the three genera mentioned above could be
isolated from two current CD biopsies (Table 4 in Paper I). These biopsies
were from a girl born in 1995 and a boy born in 1994, both on gluten-free
diet when the biopsies were taken in 2007. Detailed analysis revealed that
the Clostridium species and one of the Prevotella species were heretounknown species while the Actinomyces species was identified as
Actinomyces graevenitzii. Rod-shaped bacteria probably expressing both a
mucinase, allowing passage through the mucin layer, and forming an
intimate contact with microvilli of jejunum epithelial cells was detected by
transmission electron microscopy (TEM) (Figure 4 in Paper I). Four current
biopsies were analyzed by TEM. In two of them (1 CD patient and 1 sibling
to a CD patient), cocci and rods were found in close contact with the
microvilli. Presently we do not know whether the isolated bacteria have
mucinase activity and can bind to the proximal small intestine mucosa.
- 40 -
Further work with artificial tight monolayers of human small intestine as
developed for colonocytes in paper II will hopefully tell us whether any of
these bacteria will bind to and affect the cells biologically.
Relationship to Swedish CD epidemic. Investigations have shown an
epidemic pattern of childhood CD in Sweden during 1985-1996, in children
younger then two years of age (Ivansson et al., 2001; Olssen et al., 2008).
This epidemic coincided with not only changes in the recommendations of
gluten-containing food and breast-feeding to infants (Olssen et al., 2008) but
also with the frequency of CD patients with rod-shaped bacteria associated
to the small intestinal mucosa (Forsberg et al., 2004; Paper I). It should be
noted that the genetic background in the population before, during and after
the epidemic is almost the same. These results support the notion of an
infectious component in the pathogenesis of CD.
The intestinal microflora is constructed during first two years after birth and
the composition of commensal bacterial communities is regulated by
competition for nutrients and space (Mackie et al., 1999; Palmer et al.,
2007). This process is dependent on complex regulatory mechanisms, which
involve the microbes themselves, diet, the surrounding environment, and
innate and adaptive immunity (Sanderson, 1999; McCracken and Lorenz,
2001). The feeding-style and recommended food are capable of altering the
composition of intestinal microflora. The intestinal microflora participates in
stimulating and modulating the development and function of the intestinal
and systemic immune system, training the immune system to respond to
pathogens and preventing allergy and thus, possibly also CD (Guarner and
Malagelada 2003; Macpherson and Harris, 2004; Sears, 2005; Artis, 2008).
Our results suggest that changes in the composition of the proximal small
intestinal microflora, especially the presence of adhering rod-shaped bacteria
is one factor contributing to contraction of CD.
- 41 -
Table 1. Distribution of bacterial phyla in proximal small intestine and other
sites of the human body
Percentage of 16S rDNA clones
Phylum
Actinobacteria
Bacteroidetes
Firmicutes
Fusobacteria
Proteobacteria
Subtotal
Proximal
Mouth Esophagus Stomach Small
Intestine
10.9
4.3
8.9
14.2
9.3
20.2
10.5
6.1
26.1
69.6
25.3
44.8
14.0
2.2
3.1
3.3
13.4
2.2
52.0
31.2
73.7
98.6
99.8
99.7
Colon
Skin
0.2
47.7
50.8
0.1
0.6
99.3
51.4
1.6
23.8
0
19.4
96.2
TM7
Deferribacteres
Spirochaetes
Unclassified bacteria
Verrucomicrobia
DeinococcusThermus
Cyanobacteria
Thermomicrobia
1.4
3.4
21.3
0.2
0
0
1.4
0
0
0
0
0
0.1
0.1
0
0
0
0.1
0.3
0
0
0
0
0
0
0
0
0
0.6
0
0
0
0
0.7
0
2.9
0
0
0
0
0
0
0
0
0
0
0.2
0.1
No of clones analyzed
No of individuals
analyzed
Reference
2,522
31
900
4
1,833
23
2,247
63
1
2
3
Paper I
11,831 1,221
3
6
4
References: (1) Paster et al., 2001; (2) Pei et al., 2004; (3) Bik et al., 2006; (4)
Eckburg et al., 2005; (5) Gao et al., 2007.
- 42 -
5
3.2 Contribution of intestinal epithelial cells to innate immunity of the
human gut - studies on polarized monolayers of colon carcinoma cells
(Paper II)
In the intestine a single layer of epithelial cells separate the bodily tisssue
from the external environment. The functional intactness of this epithelium
is essential for survival. Not only is the epithelium a physical barrier
protecting the host from microbial invasion it also provides direct and
indirect defence functions since the epithelium must be seen as a component
of both innate and adaptive immunity. The purpose of this work was to
establish an in vitro model for functional studies of human intestinal
epithelium in innate immunity.
We attempted to cultivate several colon carcinoma cell lines in a twochamber system in which the chambers are connected by a semi-permeable
membrane. Cells were grown on the semi-permeable membrane of the upper
chamber and medium added both to the lower and upper chambers. As the
cells grow some cell lines form a tight monolayer of polarized epithelial
cells. To monitor development of a tight cell layer transepithelial resistance
(TER) is measured. Two colonic carcinoma cell lines, T84 and Caco2 cells
could be successfully transformed to tight monlayers. When TER reached a
plateau both T84 and Caco2 cells formed a single layer of polarized
epithelial cells, developed microvilli at the apical side and tight junctions
between the cells (Figure 1 and Figure 2 in Paper II).
For T84 cells, the monolayers exhibited several features typical for the
mature colonic epithelium. Those were: 1) a distinct apical glycocalyx that
expressed large amount of CEA, which is also seen on colonocytes at the
free luminal surface of normal colon (Frängsmyr et al., 1999; Hammarström,
1999; Hammarström and Baranov, 2001); 2) cells with mucous granules that
are characteristic for intestinal goblet cells (Figure 2 in Paper II), 3)
expression levels of mRNA for the apical surface components/glycocalyx
components CEACAM1, CEA, CEACAM6 and CEACAM7 similar to those
expressed in normal colon IECs; 4) a normal ratio between CEACAM1-L
and CEACAM1-S. As CEACAM1-L has tumor suppressor functions
(Hammarström, 1999; Kunath et al., 1995; Öbrink, 1997) it is likely that
malignant features of the tumor cells are repressed in the polarized T84 cells
and that they indeed can be expected to give a good reflection of normal IEC
functions (Table 1 in Paper II). In one respect, however, the tight monolayer
was different from normal mature colon epithelium namely in the expression
levels of MUC2 mRNA and number of goblet cells, which were significantly
lower.
- 43 -
The Caco2 tight monolayer culture was different. It showed barely any
CEA expressing microvilli/glycocalyx and the levels of mRNA for CEA,
CEACAM6 and CEACAM7 were low while the CEACAM1 mRNA levels
and CEACAM1-L/CEACAM1-S ratio were normal. The MUC2 mRNA
level was low compared to normal IEC indicating a low frequency of fully
differentiated goblet cells. Thus, Caco2 tight monolayer culture most likely
is a model for immature columnar epithelial cells in the lower part of the
colonic crypts.
The polarized monolayers were challenged with laboratory strains of Gram+
(B. megaterium and M. luteus), Gram− (E. coli and S. typhimurium) bacteria
and E. coli derived LPS from the apical side in order to mimic microbial
attack from the gut lumen. They were also challenged by proinflammatory
IFN-γ, TNF-α and IL-1β from the basolateral side to mimic signaling from
immune cells in gut immune reactions. Immune responses were estimated as
changes in mRNA expression levels for mucous component MUC2,
glycocalyx components the four CEA family members and MUC3,
antimicrobial factors hBD1, hBD2, hBD3, and lysozyme, chemokine IL-8,
and cytokines IL-6 and TNF-α. mRNA levels for these protein components
were determined by specific real-time quantitative reverse transcriptasepolymerase chain reactions with copy standards.
We found that both tight monolayer cells generally were unresponsive to
challenge with these bacteria except for Bacillus megaterium, which
significantly increased hBD2 mRNA levels in both cell lines. Several
bacterial strains and LPS also upregulated TNF-α levels in T84 tight
monolayer cells. Tight monolayer cells responded to cytokine challenge
suggesting awareness of basolateral attack. Thus, TNF-α induced markedly
increased levels of IL-8 and of TNF-α itself in both cell lines suggesting that
the epithelium participates in recruitment and activation of immune cells in
the underlying mucosa in vivo.
IFN-γ and TNF-α challenge most interestingly increased the level of
CEACAM1-L mRNA in T84 monolayer cells while other CEACAMs or
MUC3 were not affected. Although not statistically significant TNF-α also
upregulated the CEACAM1-S form. Thus if the ratio CEACAM1L/CEACAM1-S is calculated it is evident that the two cytokines have
different effects, IFN-γ selectively upregulating the form with downregulatory properties. Increased CEACAM1 expression may influence
epithelial function and communication between epithelial cells and
intraepithelial lymphocytes.
In future studies we will use this system for biological analysis of bacteria
associated to CD.
- 44 -
3.3 Role of V. cholerae protease, PrtV - from C. elegans to human
intestine
3.3.1 PrtV protects V. cholerae from natural predator grazing and is
responsible for killing of infected C. elegans (Paper III)
By using C. elegans as a potential model system for studies of cholera
diarrhea we found that C. elegans with an intestinal infection were killed by
the bacterium. The killing efficiency was dependent on quorum sensing
regulators. Thus, the main virulence factor of V. cholerae, cholera toxin
(CTX) did not affect the C. elegans killing, while hapR mutated strains were
attenuated (Figures 1 and 3 in Paper III). After testing in this C. elegans
killing assay it was evident that only the depletion of a new protease, PrtV
clearly attenuated the ability of V. cholerae to cause a lethal infection
(Figure 3 in Paper III). Further studies on PrtV showed that it belongs to
family of metallopeptidases, M6 (Vaitkevicius et al., 2008), with the best
characterized member of the family being the immune inhibitor A (InA)
from Bacillus thuringiensis, a bacterium widely used in agriculture to control
insect pests (Ogierman et al., 1997). Moreover, PrtV conferred protection
against grazing by natural bacteriovorous predators. It was recently reported
that PrtV is a Zn2+ dependent protease able to degrade mammalian
extracellular matrix components and plasminogen (Vaitkevicius et al., 2008).
In order to study whether PrtV was also toxic to human intestine an in vitro
model system consisting of polarized tight monolayers of the human colonic
adenocarcinoma cell line T84 was utilized (described in Paper II). The
chemokine IL-8 was used as a marker of inflammatory response.
Unexpectedly, supernatants of the ΔprtV derivates did not induce increased
permeability or IL-8 expression (Figure 6 and supplementary Figure 11 in
Paper III). These results suggest a role for this protease in modulating the
human intestinal epithelial cell response. Considering the proteolytic activity
of the PrtV protein, the observed effect could be indirect and might result
from processing of other secreted V. cholerae factor(s).
3.3.2 V. cholera cytolysin (VCC) is the secreted proinflammatory factor
and its activity is modulated by PrtV (Paper IV)
To throw light on the contrasting effects of PrtV in human intestinal
epithelial cells compared to in invertebrate intestine, we aimed at identifying
secreted component(s) of V. cholerae O1 strain C6706 that induces an
epithelial inflammatory response in human intestinal epithelial cells and
- 45 -
define whether the active component(s) are substrates for PrtV. Culture
supernatants from V. cholerae C6706 and genetically modified strains were
analyzed for capacity to induce changes in cytokine production,
permeability and cell viability in the polarized tight monolayer in vitro
model for human intestinal epithelium. The culture supernatants were also
immunochemically characterized and monitored for capacity to lyse red
blood cells.
Culture supernatants from the V. cholerae ∆prtV strain when grown in
Luria-Bertani broth caused permeability increase and increased expression
of IL-8 and TNF-α while supernatants from wild-type bacteria did not
(Paper III and IV). Experiments with supernatants from CTX deletion
mutants showed that CTX was not the stimulatory component. Thus, PrtV
influences the stability of one or more secreted factors other than CTX. This
influence by PrtV was abolished when supernatants from bacteria cultured
in brain heart infusion broth were used (Figure 2 in Paper IV), suggesting
that this broth in some way inhibits PrtV degradation of the factor(s) causing
the inflammatory response. VCC was found to be the sole active component
causing the inflammatory response as revealed by VCC deletion mutant
experiments (Figure 2 in Paper IV). The hemolytic activity of supernatants
from wild-type V. cholera and its mutants showed exactly the same pattern
(Figure 3 in Paper IV). Furthermore pure VCC protein was able to induce an
inflammatory response of the same magnitude as the culture supernatant
(Figure 5 in Paper IV). High doses of VCC were cytotoxic for epithelial
cells. The inflammatory effect of VCC could be abolished by treatment with
PrtV (Figure 6 in Paper IV). That PrtV is the major component causing
degradation of VCC is indicated by the fact that supernatants from a
derivative carrying an inframe deletion mutation in the gene for a regulator
of V. cholerae proteases expression (∆hapR) caused the same changes in the
inflammatory response as the ∆prtV mutant.
VCC most likely is a pathogenetic factor in cholera. At high concentrations
it caused hemolysis and epithelial cell death, which is in line with previous
studies in a mouse model (Olivier et al., 2007). At low doses VCC induced
an inflammatory response in intestinal epithelial cells that included
increased epithelial permeability and induction of IL-8 and TNF-α
suggesting that intestinal epithelial cells initially execute a front-line defense
reaction to V. cholerae infection involving recruitment of immune cells to
the infected site by IL-8 and activation of epithelial cells, intraepithelial
lymphocytes, and lamina propria macrophages by TNF-α. Paracellular
leakage due to decreased tight junction stability and/or formation of
- 46 -
transmembrane pores by VCC could be the mechanism behind the
increased permeability in tight monolayers seen here.
The reason why the bacterium produces VCC and PrtV simultaneously is
unclear but the difference in VCC activity in different types of broth
suggests that this might be an adaptation for different environments. In the
human intestine PrtV is probably inactivated either by chelators or by high
protein/peptide content allowing VCC to exert its toxic actions and yield
access for the bacterium to the bodily tissues.
- 47 -
4. Conclusions
1. A unique bacterial flora inhabits the normal proximal jejunum of children.
This flora is not just transient passengers in the jejunum but constitutes an
organ-specific biofilm. The predominant bacterial genera are Streptococcus,
Neisseria, Veillonella, Gemella, Actinomyces, Rothia and Haemophilus.
2. The rod-shaped bacteria previously found in children with CD during the
CD epidemic is likely to be one or more of a previously unidentified
unclassified-Clostridales species, a previously unidentified Prevotella
species or a pathogenic variant of Actinomyces graevenitzii.
3. The polarized tight monolayer of both colon carcinoma cell lines T84 and
Caco2 appear to be suitable in vitro models for studies of innate defense
mechanisms of human intestinal epithelium. The two cell lines seem to
represent intestinal epithelial cells at two different sites in the colonic
mucosa, namely the luminal surface in the case of T84 and the lower crypts
in the case of Caco2 cells.
4. Polarized intestinal monolayer cells seem “more resistant” to bacterial
attack than ordinary tissue culture cells.
5. The inhibitory and tumor suppressive variant of CEACAM1, namely
CEACAM1-L, is strongly upregulated in polarized T84 cells by stimulation
with IFN-γ. This indicates that intraepithelial lymphocytes can influence
epithelial cell proliferation during an immune response.
6. Vibrio cholera cytolysin (VCC) was found to be solely responsible for
induction of an inflammatory response in intestinal epithelial cells that
includes increased epithelial permeability and production of IL-8 and TNFα.
7. PrtV degrades VCC thereby modulating the inflammatory and cytotoxic
response.
- 48 -
5. Acknowledgements
If life is a long march, this thesis is the milestone of my journey. I have not
traveled lonely during my trip through the years. There are so many people
who have made this trip easier for me. I wish to express my sincere gratitude
and deep appreciation to all of you who helped me along the way.
First of all, I would like to thank my supervisors: Professor Sten
Hammarström and Professor Marie-Louise Hammarström for providing
me this opportunity to share your knowledge about mucosal immunology
and favorite molecules CEACAMs. Thank you for your endless support,
enthusiasm, encouragement and understanding both in academic field and
daily life during these years. Professor Olle Hernell, thank you for support,
enthusiasm, encouragement, discussion and the cooperation in the celiac
disease project.
Many thanks to Dr. Vladimir Baranov for sharing your knowledge and for
all the nice electron micrographs, and for giving constructive suggestions.
Professor Torgny Stigbrand for your hospitality and all the terrific foods
that we had at your place.
Kristina Lejon for interesting discussion.
Lucia Mincheva Nilsson, thank you for interesting discussion and
suggestions.
Many thanks to Lena Hallström-Nylén for making everyday life much
easier for me.
Anne Israelsson, Marianne Sjöstedt, and Elisabeth Granström for your
invaluable help on my bench work and for well-being concerns.
Birgitta Sjöberg for making everyday life at the lab so much easier for all of
us.
I thank David, Lina, Veronika, Mia, Lotta, Aziz, Rifat, Therese, Erica
and Christoffer for your friendship. I will always remember the PhD parties
and Journal Club! I wish you good luck with your studies and a wonderful
time.
Many thanks also to the former group members and colleagues: Annas, Ali
Sheikholvaezin, Arman, Silvia, Niklas, Homa, Guang-Qian, Bi-Feng and
Lili for friendship and happiness.
Thanks to all members at the Department of Immunology for friendship and
for a series of nice dinners with excellent food and discussions.
- 49 -
Göte Forsberg, Maria Hedberg, Per Hörstedt, Mirva Drobni, Olof
Sandström, Ingegerd Johansson and Elena Lundmark for cooperation
and coauthorship.
Sun Nyunt Wai and your group members: Karolis Vaitkevicius, Barbro
Lindmark, Tianyan Song, Pramod Kumar Rompikuntal, for
collaboration and coauthorship.
Sucharit Bhakdi for coauthorship.
Special thanks to all Chinese friends in Umeå, Teacher Gu, families of
Zhang Jiexie’s, Xiong’s, Mei Yafang’s, Chen Sa’s, Gong’s, and Lao Lai’s
are acknowledged for help and friendship.
I am especially grateful to Prof. Jin Taiyi for introducing me to Umeå and
for never-failing encouragement and support.
I would like to thank Lu Jian and Yuan Ming and Xiao Hu, Bo, Jufang,
Zijiu, Qingyin and Haiyang, Daping and Yaoyi, Changchun and
ShengMing, Lao Huang, Junfa, Shenyue, Yihuai, Xuhao-for all your
friendship and help.
I wish to thank my friends and relatives who always concern me for patience
and encouragement.
I also wish to thank my former colleagues in Zunyi Medical College for
patience, enthusiasm, encouragement and friendship.
My mom and my sisters and young brother, I love you!
This thesis is dedicated to the loving memory of my father.
Last but not least, Jun and Ran, without your love, it would have been
impossible to show this thesis here.
- 50 -
6. References
Abreu MT, Fukata M, Arditi M. 2005. TLR signaling in the gut in health and disease. J
Immunol. 174:4453-60.
Abreu MT, Vora P, Faure E, Thomas LS, Arnold ET, Arditi M. 2001. Decreased expression
of Toll-like receptor-4 and MD-2 correlates with intestinal epithelial cell protection
against dysregulated proinflammatory gene expression in response to bacterial
lipopolysaccharide. J Immunol. 167:1609-16.
Acheson DW, Luccioli S. 2004. Microbial-gut interactions in health and disease. Mucosal
immune responses. Best Pract Res Clin Gastroenterol. 18:387-404.
Agostini L, Martinon F, Burns K, McDermott MF, Hawkins PN, Tschopp J. 2004. NALP3
forms an IL-1beta-processing inflammasome with increased activity in Muckle-Wells
autoinfl ammatory disorder. Immunity. 20:319-25.
Akira S, Uematsu S, Takeuchi O. 2006. Pathogen recognition and innate immunity. Cell.
124:783-801.
Akobeng AK, Thomas AG. 2008. Systematic review: tolerable amount of gluten for people
with coeliac disease. Aliment Pharmacol Ther. 27:1044-52.
Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. 2001. Recognition of double-stranded
RNA and activation of NF-kappaB by Toll-like receptor 3. Nature. 413:732-8.
Al-Lamki RS, Wang J, Skepper JN, Thiru S, Pober JS, Bradley JR. 2001. Expression of
tumor necrosis factor receptors in normal kidney and rejecting renal transplants. Lab
Invest. 81: 1503-15.
Alm RA, Mayrhofer G, Kotlarski I, Manning PA. 1991. Amino-terminal domain of the El
Tor haemolysin of Vibrio cholerae O1 is expressed in classical strains and is cytotoxic.
Vaccine. 9:588-94.
Alm RA, Stroeher UH, Manning PA. 1988. Extracellular proteins of Vibrio cholerae:
nucleotide sequence of the structural gene (hlyA) for the haemolysin of the haemolytic
El Tor strain 017 and characterization of the hlyA mutation in the non-haemolytic
classical strain 569B. Mol Microbiol. 2:481-8.
Andersson AF, Lindberg M, Jakobsson H, Bäckhed F, Nyrén P, Engstrand L. 2008.
Comparative analysis of human gut microbiota by barcoded pyrosequencing. PLoS
ONE. 3:e2836.
Anderson AM, Varkey JB, Petti CA, Liddle RA, Frothingham R, Woods CW. 2004. NonO1 Vibrio cholerae septicemia: case report, discussion of literature, and relevance to
bioterrorism. Diagn Microbiol Infect Dis. 49:295-7.
Anderson RP, Degano P, Godkin AJ, Jewell DP, Hill AV. 2000. In vivo antigen challenge in
celiac disease identifies a single transglutaminase-modified peptide as the dominant Agliadin T-cell epitope. Nat Med. 6:337-42.
Andrianifahanana M, Moniaux N, Batra SK. 2006. Regulation of mucin expression:
mechanistic aspects and implications for cancer and inflammatory diseases. Biochim
Biophys Acta. 1765:189-222.
Arend WP, Palmer G, Gabay C. 2008. IL-1, IL-18, and IL-33 families of cytokines.
Immunol Rev. 223:20-38.
- 51 -
Armstrong PB. 2006. Proteases and protease inhibitors: a balance of activities in hostpathogen interaction. Immunobiology. 211:263-81.
Artis D. 2008. Epithelial-cell recognition of commensal bacteria and maintenance of
immune homeostasis in the gut. Nat Rev Immunol. 8:411-20.
Audie JP, Janin A, Porchet N, Copin MC, Gosselin B, Aubert JP. 1993. Expression of
human mucin genes in respiratory, digestive, and reproductive tracts ascertained by in
situ hybridization. J Histochem Cytochem. 41:1479-85.
Baggiolini M, Dewald B, Moser B. 1994. Interleukin-8 and related chemotactic cytokines CXC and CC chemokines. Adv Immunol. 55:97-179.
Bas A, Forsberg G, Sjöberg V, Hammarström S, Hernell O, Hammarström ML. 2009.
Aberrant extrathymic T cell receptor gene rearrangement in the small intestinal mucosa:
a risk factor for celiac disease? Gut. 58:189-95.
Bas A, Hammarström SG, Hammarström ML. 2003. Extrathymic TCR gene rearrangement
in human small intestine: identification of new splice forms of recombination activating
gene-1 mRNA with selective tissue expression. J Immunol. 171:3359-71.
Bazzoni F, Beutler B. 1996. The tumor necrosis factor ligand and receptor families. N Engl
J Med. 334:1717-25.
Beagley KW, Elson CO. 1992. Cells and cytokines in mucosal immunity and inflammation.
Gastroenterol Clin North Am. 21:347-66.
Beauchemin N, Draber P, Dveksler G, Gold P, Gray-Owen S, Grunert F, Hammarström S,
Holmes KV, Karlsson A, Kuroki M, Lin SH, Lucka L, Najjar SM, Neumaier M, Obrink
B, Shively JE, Skubitz KM, Stanners CP, Thomas P, Thompson JA, Virji M, von Kleist
S, Wagener C, Watt S, Zimmermann W. 1999. Redefined nomenclature for members of
the carcinoembryonic antigen family. Exp Cell Res. 252:243-9.
Beaugerie L, Petit JC. 2004. Microbial-gut interactions in health and disease. Antibioticassociated diarrhoea. Best Pract Res Clin Gastroenterol. 18:337-52.
Benahmed M, Meresse B, Arnulf B, Barbe U, Mention JJ, Verkarre V, Allez M, Cellier C,
Hermine O, Cerf-Bensussan N. 2007. Inhibition of TGF-beta signaling by IL-15: a new
role for IL-15 in the loss of immune homeostasis in celiac disease. Gastroenterology.
132:994-1008.
Ben-Baruch A, Michiel DF, Oppenheim JJ. 1995. Signals and receptors involved in
recruitment of inflammatory cells. J Biol Chem. 270:11703-6.
Benchimol S, Fuks A, Jothy S, Beauchemin N, Shirota K, Stanners CP. 1989.
Carcinoembryonic antigen, a human tumor marker, functions as an intercellular
adhesion molecule. Cell. 57:327-34.
Beutler B, Cerami A. 1986. Cachectin and tumour necrosis factor as two sides of the same
biological coin. Nature. 320:584-8.
Biagi F, Campanella J, Laforenza U, Gastaldi G, Tritto S, Grazioli M, Villanacci V, Corazza
GR. 2006. Transglutaminase 2 in the enterocytes is coeliac specific and gluten
dependent. Dig Liver Dis. 38:652-8.
Bik EM, Eckburg PB, Gill SR, Nelson KE, Purdom EA, Francois F, Perez-Perez G, Blaser
MJ, Relman DA. 2006. Molecular analysis of the bacterial microbiota in the human
stomach. Proc Natl Acad Sci U S A. 103:732-7.
Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson MF, Castner BJ,
Stocking KL, Reddy P, Srinivasan S, Nelson N, Boiani N, Schooley KA, Gerhart M,
Davis R, Fitzner JN, Johnson RS, Paxton RJ, March CJ, Cerretti DP. 1997. A
metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells.
Nature. 385:729-33.
- 52 -
Boehm U, Klamp T, Groot M, Howard JC. 1997. Cellular responses to interferon-gamma.
Annu Rev Immunol. 15:749-95.
Booth BA, Boesman-Finkelstein M, Finkelstein RA. 1984. Vibrio cholerae
hemagglutinin/protease nicks cholera enterotoxin. Infect Immun. 45:558-60.
Bowie AG, Fitzgerald KA. 2007. RIG-I: tri-ing to discriminate between self and non-self
RNA. Trends Immunol. 28:147-50.
Bradley JR. 2008. TNF-mediated inflammatory disease. J Pathol. 214:149-60.
Brandtzaeg P, Bjerke K. 1990. Immunomorphological characteristics of human Peyer's
patches. Digestion. 46:S262-73.
Brandtzaeg P, Farstad IN, Johansen FE, Morton HC, Norderhaug IN, Yamanaka T. 1999.
The B-cell system of human mucosae and exocrine glands. Immunol Rev. 171:45-52.
Breitbart M, Haynes M, Kelley S, Angly F, Edwards RA, Felts B, Mahaffy JM, Mueller J,
Nulton J, Rayhawk S, Rodriguez-Brito B, Salamon P, Rohwer F. 2008. Viral diversity
and dynamics in an infant gut. Res Microbiol. 159:367-73.
Breitbart M, Hewson I, Felts B, Mahaffy JM, Nulton J, Salamon P, Rohwer F. 2003.
Metagenomic analyses of an uncultured viral community from human feces. J
Bacteriol. 185:6220-3.
Brikos C, O'Neill LA. 2008. Signalling of toll-like receptors. Handb Exp Pharmacol.
183:21-50.
Bäckhed F, Ding H, Wang T, Hooper LV, Koh GY, Nagy A, Semenkovich CF, Gordon JI.
2004. The gut microbiota as an environmental factor that regulates fat storage. Proc
Natl Acad Sci U S A. 101:15718-23.
Bäckhed F, Manchester JK, Semenkovich CF, Gordon JI. 2007. Mechanisms underlying the
resistance to diet-induced obesity in germ-free mice. Proc Natl Acad Sci U S A.
104:979-84.
Calder PC, Krauss-Etschmann S, de Jong EC, Dupont C, Frick JS, Frokiaer H, Heinrich J,
Garn H, Koletzko S, Lack G, Mattelio G, Renz H, Sangild PT, Schrezenmeir J, Stulnig
TM, Thymann T, Wold AE, Koletzko B. 2006. Early nutrition and immunity - progress
and perspectives. Br J Nutr. 96:774-90.
Cario E, Podolsky DK. 2006. Toll-like receptor signaling and its relevance to intestinal
inflammation. Ann N Y Acad Sci. 1072:332-8.
Carlsson AK, Axelsson IE, Borulf SK, Bredberg AC, Ivarsson SA. 2001. Serological
screening for celiac disease in healthy 2.5-year-old children in Sweden. Pediatrics.
107:42-5.
Carswell EA, Old LJ, Kassel RL, Green S, Fiore N, Williamson B. 1975. An endotoxininduced serum factor that causes necrosis of tumors. Proc Natl Acad Sci USA. 72:
3666-70.
Chamaillard M, Hashimoto M, Horie Y, Masumoto J, Qiu S, Saab L, Ogura Y, Kawasaki A,
Fukase K, Kusumoto S, Valvano MA, Foster SJ, Mak TW, Nuñez G, Inohara N. 2003.
An essential role for NOD1 in host recognition of bacterial peptidoglycan containing
diaminopimelic acid. Nat Immunol. 4:702-7.
Chen CJ, Kirshner J, Sherman MA, Hu W, Nguyen T, Shively JE. 2007. Mutation analysis
of the short cytoplasmic domain of the cell-cell adhesion molecule CEACAM1
identifies residues that orchestrate actin binding and lumen formation. J Biol Chem.
282:5749-60.
Chen G, Goeddel DV. 2002. TNF-R1 signaling: a beautiful pathway. Science. 296: 1634-5.
Chinnapen DJ, Chinnapen H, Saslowsky D, Lencer WI. 2007. Rafting with cholera toxin:
endocytosis and trafficking from plasma membrane to ER. FEMS Microbiol Lett.
266:129-37.
- 53 -
Clark JA, Coopersmith CM. 2007. Intestinal crosstalk: a new paradigm for understanding
the gut as the "motor" of critical illness. Shock. 28:384-93.
Coelho A, Andrade JR, Vicente AC, Dirita VJ. 2000. Cytotoxic cell vacuolating activity
from Vibrio cholerae hemolysin. Infect Immun. 68:1700-5.
Collado MC, Calabuig M, Sanz Y. 2007. Differences between the fecal microbiota of
coeliac infants and healthy controls. Curr Issues Intest Microbiol. 8:9-14.
Coombes JL, Powrie F. 2008. Dendritic cells in intestinal immune regulation. Nat Rev
Immunol. 8:435-46.
Corfield AP, Carroll D, Myerscough N, Probert CS. 2001. Mucins in the gastrointestinal
tract in health and disease. Front Biosci. 6:D1321-57.
Cornes JS. 1965. Peyer's patches in the human gut. Proc R Soc Med. 58:716.
Cottingham KL, Chiavelli DA, Taylor RK. 2003. Environmental microbe and human
pathogen: the ecology and microbiology of Vibrio cholerae. Front Ecol Environ. 1:806.
Cunliffe RN. 2003. Alpha-defensins in the gastrointestinal tract. Mol Immunol. 40:463-7.
Cuthbert AP, Fisher SA, Mirza MM, King K, Hampe J, Croucher PJ, Mascheretti S,
Sanderson J, Forbes A, Mansfield J, Schreiber S, Lewis CM, Mathew CG. 2002. The
contribution of NOD2 gene mutations to the risk and site of disease in inflammatory
bowel disease. Gastroenterology. 122:867-74.
Daig R, Andus T, Aschenbrenner E, Falk W, Schölmerich J, Gross V. 1996. Increased
interleukin-8 expression in the colon mucosa of patients with inflammatory bowel
disease. Gut. 38:216-22.
Daig R, Rogler G, Aschenbrenner E, Vogl D, Falk W, Gross V, Schölmerich J, Andus T.
2000. Human intestinal epithelial cells secrete interleukin-1 receptor antagonist and
interleukin-8 but not interleukin-1 or interleukin-6. Gut. 46:350-8.
Dann SM, Eckmann L. 2007. Innate immune defenses in the intestinal tract. Curr Opin
Gastroenterol. 23:115-20.
Dann SM, Spehlmann ME, Hammond DC, Iimura M, Hase K, Choi LJ, Hanson E, Eckmann
L. 2008. IL-6-dependent mucosal protection prevents establishment of a microbial
niche for attaching/effacing lesion-forming enteric bacterial pathogens. J Immunol.
180:6816-26.
Darnell JE Jr, Kerr IM, Stark GR. 1994. Jak-STAT pathways and transcriptional activation
in response to IFNs and other extracellular signaling proteins. Science. 264:1415-21.
de Bolos C, Real FX, Lopez-Ferrer A. 2001. Regulation of mucin and glycoconjugate
expression: from normal epithelium to gastric tumors. Front Biosci. 6:D1256-63.
De Hertogh G, Aerssens J, de Hoogt R, Peeters P, Verhasselt P, Van Eyken P, Ectors N,
Vermeire S, Rutgeerts P, Coulie B, Geboes K. 2006. Validation of 16S rDNA
sequencing in microdissected bowel biopsies from Crohn's disease patients to assess
bacterial flora diversity. J Pathol. 209:532-9.
de la Cochetiere MF, Piloquet H, des Robert C, Darmaun D, Galmiche JP, Roze JC. 2004.
Early intestinal bacterial colonization and necrotizing enterocolitis in premature infants:
the putative role of Clostridium. Pediatr Res. 56:366-70.
de Ritis G, Auricchio S, Jones HW, Lew EJ, Bernardin JE, Kasarda DD. 1988. In vitro
(organ culture) studies of the toxicity of specific A-gliadin peptides in celiac disease.
Gastroenterology. 94:41-9.
Derouet D, Rousseau F, Alfonsi F, Froger J, Hermann J, Barbier F, Perret D, Diveu C,
Guillet C, Preisser L, Dumont A, Barbado M, Morel A, deLapeyriere O, Gascan H,
Chevalier S. 2004. Neuropoietin, a new IL-6-related cytokine signaling through the
ciliary neurotrophic factor receptor Proc. Natl Acad Sci USA. 101:4827-32
- 54 -
Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C. 2004. Innate antiviral responses
by means of TLR7-mediated recognition of single-stranded RNA. Science. 303:152931.
Dillon SR, Sprecher C, Hammond A, Bilsborough J, Rosenfeld-Franklin M, Presnell SR,
Haugen HS, Maurer M, Harder B, Johnston J, Bort S, Mudri S, Kuijper JL, Bukowski
T, Shea P, Dong DL, Dasovich M, Grant FJ, Lockwood L, Levin SD, LeCiel C,
Waggie K, Day H, Topouzis S, Kramer J, Kuestner R, Chen Z, Foster D, Parrish-Novak
J, Gross JA. 2004. Interleukin 31, a cytokine produced by activated T cells, induces
dermatitis in mice. Nat Immunol. 5:752-60.
Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M, Gill SR, Nelson
KE, Relman DA. 2005. Diversity of the human intestinal microbial flora. Science.
308:1635-8.
Eckmann L, Kagnoff MF. 2005. Intestinal mucosal responses to microbial infection.
Springer Semin Immunopathol. 27:181-96.
Edlund M, Blikstad I, Obrink B. 1996. Calmodulin binds to specific sequences in the
cytoplasmic domain of C-CAM and down-regulates C-CAM self-association. J Biol
Chem. 271:1393-9.
Eisenberg SP, Brewer MT, Verderber E, Heimdal P, Brandhuber BJ, Thompson RC. 1991.
Interleukin 1 receptor antagonist is a member of the interleukin 1 gene family:
evolution of a cytokine control mechanism. Proc Natl Acad Sci U S A. 88:5232-6.
Fahlgren A, Baranov V, Frängsmyr L, Zoubir F, Hammarström ML, Hammarström S. 2003.
Interferon-gamma tempers the expression of carcinoembryonic antigen family
molecules in human colon cells: a possible role in innate mucosal defence. Scand J
Immunol. 58:628-41.
Fahlgren A, Hammarström S, Danielsson A, Hammarström ML. 2003. Increased expression
of antimicrobial peptides and lysozyme in colonic epithelial cells of patients with
ulcerative colitis. Clin Exp Immunol. 131:90-101.
Fahlgren A, Hammarstrom S, Danielsson A, Hammarstrom ML. 2004. beta-Defensin-3 and
-4 in intestinal epithelial cells display increased mRNA expression in ulcerative colitis.
Clin Exp Immunol.137:379-85.
Farrell RJ, Kelly CP. 2002. Celiac sprue. N Engl J Med. 346:180-8
Faruque SM, Chowdhury N, Kamruzzaman M, Dziejman M, Rahman MH, Sack DA, Nair
GB, Mekalanos JJ. 2004. Genetic diversity and virulence potential of environmental
Vibrio cholerae population in a cholera-endemic area. Proc Natl Acad Sci U S A.
101:2123-8.
Faruque SM, Naser IB, Islam MJ, Faruque AS, Ghosh AN, Nair GB, Sack DA, Mekalanos
JJ. 2005. Seasonal epidemics of cholera inversely correlate with the prevalence of
environmental cholera phages. Proc Natl Acad Sci U S A. 102:1702-7.
Fasano A. 2005. Clinical presentation of celiac disease in the pediatric population.
Gastroenterology. 128:S68-73.
Figueroa-Arredondo P, Heuser JE, Akopyants NS, Morisaki JH, Giono-Cerezo S, EnríquezRincón F, Berg DE. 2001. Cell vacuolation caused by Vibrio cholerae hemolysin.
Infect Immun. 69:1613-24.
Forsberg G, Fahlgren A, Hörstedt P, Hammarström S, Hernell O, Hammarström ML. 2004.
Presence of bacteria and innate immunity of intestinal epithelium in childhood celiac
disease. Am J Gastroenterol. 99:894-904.
Forsberg G, Hernell O, Hammarström S, Hammarström ML. 2007. Concomitant increase of
IL-10 and pro-inflammatory cytokines in intraepithelial lymphocyte subsets in celiac
disease. Int Immunol. 19:993-1001.
- 55 -
Forsberg G, Hernell O, Melgar S, Israelsson A, Hammarström S, Hammarström ML. 2002.
Paradoxical coexpression of proinflammatory and down-regulatory cytokines in
intestinal T cells in childhood celiac disease. Gastroenterology. 123:667-78.
Franchi L, Park JH, Shaw MH, Marina-Garcia N, Chen G, Kim YG, Núñez G. 2008.
Intracellular NOD-like receptors in innate immunity, infection and disease. Cell
Microbiol. 10:1-8.
Froy O. 2005. Regulation of mammalian defensin expression by Toll-like receptordependent and independent signalling pathways. Cell Microbiol. 7:1387-97.
Frängsmyr L, Baranov V, Hammarström S. 1999. Four carcinoembryonic antigen subfamily
members, CEA, NCA, BGP and CGM2, selectively expressed in the normal human
colonic epithelium, are integral components of the fuzzy coat. Tumour Biol. 20:277-92.
Frängsmyr L, Baranov V, Prall F, Yeung MM, Wagener C, Hammarström S. 1995. Celland region-specific expression of biliary glycoprotein and its messenger RNA in
normal human colonic mucosa. Cancer Res. 55:2963-7.
Fukushima K, Ishiyama C, Yamashita K. 2004. Recognition by TNF-alpha of the GPIanchor glycan induces apoptosis of U937 cells. Arch Biochem Biophys. 426:298-305.
Fullner KJ, Boucher JC, Hanes MA, Haines GK 3rd, Meehan BM, Walchle C, Sansonetti
PJ, Mekalanos JJ. 2002. The contribution of accessory toxins of Vibrio cholerae O1 El
Tor to the proinflammatory response in a murine pulmonary cholera model. J Exp Med.
195:1455-62.
Ganz T. 1999. Defensins and host defense. Science. 286:420-1.
Ganz T. 2002. Epithelia: not just physical barriers. Proc Natl Acad Sci U S A. 99:3357-8.
Ganz T. 2003. Defensins: antimicrobial peptides of innate immunity. Nat. Rev. Immunol.
3:710-20.
Ganz T, Selsted ME, Szklarek D, Harwig SS, Daher K, Bainton DF, Lehrer RI. 1985.
Defensins. Natural peptide antibiotics of human neutrophils. J Clin Invest. 76:1427-35.
Gao Z, Tseng CH, Pei Z, Blaser MJ. 2007. Molecular analysis of human forearm superficial
skin bacterial biota. Proc Natl Acad Sci U S A. 104:2927-32.
Garrote JA, Gómez-González E, Bernardo D, Arranz E, Chirdo F. 2008. Celiac disease
pathogenesis: the proinflammatory cytokine network. J Pediatr Gastroenterol Nutr.
47:S27-32.
Gasbarrini G, Malandrino N, Giorgio V, Fundarò C, Cammarota G, Merra G, Roccarina D,
Gasbarrini A, Capristo E. 2008. Celiac disease: what's new about it? Dig Dis. 26:121-7.
Gaur S, Shively JE, Yen Y, Gaur RK. 2008. Altered splicing of CEACAM1 in breast
cancer: identification of regulatory sequences that control splicing of CEACAM1 into
long or short cytoplasmic domain isoforms. Mol Cancer. 7:46.
Gijzen K, Cambi A, Torensma R, Figdor CG. 2006. C-type lectins on dendritic cells and
their interaction with pathogen-derived and endogenous glycoconjugates. Curr Protein
Pept Sci. 7:283-94.
Gill SR, Pop M, Deboy RT, Eckburg PB, Turnbaugh PJ, Samuel BS, Gordon JI, Relman
DA, Fraser-Liggett CM, Nelson KE. 2006. Metagenomic analysis of the human distal
gut microbiome. Science. 312:1355-9.
Girardin SE, Boneca IG, Carneiro LA, Antignac A, Jéhanno M, Viala J, Tedin K, Taha MK,
Labigne A, Zähringer U, Coyle AJ, DiStefano PS, Bertin J, Sansonetti PJ, Philpott DJ.
2003. Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan.
Science. 300:1584-7.
Godkin A, Friede T, Davenport M, Stevanovic S, Willis A, Jewell D, Hill A, Rammensee
HG. 1997. Use of eluted peptide sequence data to identify the binding characteristics of
- 56 -
peptides to the insulin-dependent diabetes susceptibility allele HLA-DQ8 (DQ 3.2).
Int Immunol. 9:905-11.
Gold P, Freedman SO. 1965. Specific carcinoembryonic antigens of the human digestive
system. J Exp Med. 122:467-81.
Gray-Owen SD, Blumberg RS. 2006. CEACAM1: contact-dependent control of immunity.
Nat Rev Immunol. 6:433-46.
Greco L, Romino R, Coto I, Di Cosmo N, Percopo S, Maglio M, Paparo F, Gasperi V,
Limongelli MG, Cotichini R, D'Agate C, Tinto N, Sacchetti L, Tosi R, Stazi MA. 2002.
The first large population based twin study of coeliac disease. Gut. 50:624-8.
Green PH, Cellier C. 2007. Celiac disease. N Engl J Med. 357:1731-43.
Green PH, Jabri B. 2006. Celiac disease. Annu Rev Med. 57:207-21.
Greicius G, Severinson E, Beauchemin N, Obrink B, Singer BB. 2003. EACAM1 is a potent
regulator of B cell receptor complex-induced activation. J Leukoc Biol. 74:126-34.
Griffiths NJ, Bradley CJ, Heyderman RS, Virji M. 2007. IFN-gamma amplifies NFkappaBdependent Neisseria meningitidis invasion of epithelial cells via specific upregulation
of CEA-related cell adhesion molecule 1. Cell Microbiol. 9:2968-83.
Groh V, Steinle A, Bauer S, Spies T. 1998. Recognition of stress-induced MHC molecules
by intestinal epithelial gammadelta T cells. Science. 279:1737-40.
Gross O, Gewies A, Finger K, Schäfer M, Sparwasser T, Peschel C, Förster I, Ruland J.
2006. Card9 controls a non-TLR signalling pathway for innate anti-fungal immunity.
Nature. 442:651-6.
Guarner F. 2006. Enteric flora in health and disease. Digestion. 73 S1:5-12.
Guarner F, Malagelada JR. 2003. Gut flora in health and disease. Lancet. 361:512-9.
Gudmundsson GH, Agerberth B, Odeberg J, Bergman T, Olsson B, Salcedo R. 1996. The
human gene FALL39 and processing of the cathelin precursor to the antibacterial
peptide LL-37 in granulocytes. Eur J Biochem. 238:325-32.
Gum JR Jr, Crawley SC, Hicks JW, Szymkowski DE, Kim YS. 2002. MUC17, a novel
membrane-tethered mucin. Biochem Biophys Res Commun. 291:466-75.
Gum JR Jr, Hicks JW, Toribara NW, Siddiki B, Kim YS. 1994. Molecular cloning of human
intestinal mucin (MUC2) cDNA. Identification of the amino terminus and overall
sequence similarity to prepro-von Willebrand factor. J Biol Chem. 269:2440-6.
Gutierrez MG, Saka HA, Chinen I, Zoppino FC, Yoshimori T, Bocco JL, Colombo MI.
2007. Protective role of autophagy against Vibrio cholerae cytolysin, a pore-forming
toxin from V. cholerae. Proc Natl Acad Sci U S A. 104:1829-34.
Halstensen TS, Brandtzaeg P. 1993. Activated T lymphocytes in the celiac lesion: nonproliferative activation (CD25) of CD4+ alpha/beta cells in the lamina propria but
proliferation (Ki-67) of alpha/beta and gamma/delta cells in the epithelium. Eur J
Immunol. 23:505-10.
Hammarström S. 1999. The carcinoembryonic antigen (CEA) family: structures, suggested
functions and expression in normal and malignant tissues. Semin Cancer Biol. 9: 67-81.
Hammarström S, Baranov V. 2001. Is there a role for CEA in innate immunity in the colon?
Trends Microbiol. 9:119-25.
Harris JR, Bhakdi S, Meissner U, Scheffler D, Bittman R, Li G, Zitzer A, Palmer M. 2002.
Interaction of the Vibrio cholerae cytolysin (VCC) with cholesterol, some cholesterol
esters, and cholesterol derivatives: a TEM study. J Struct Biol. 139:122-35.
Hase K, Eckmann L, Leopard JD, Varki N, Kagnoff MF. 2002. Cell differentiation is a key
determinant of cathelicidin LL-37/human cationic antimicrobial protein 18 expression
by human colon epithelium. Infect Immun. 70:953-63.
- 57 -
Hase K, Murakami M, Iimura M, Cole SP, Horibe Y, Ohtake T, Obonyo M, Gallo RL,
Eckmann L, Kagnoff MF. 2003. Expression of LL-37 by human gastric epithelial cells
as a potential host defense mechanism against Helicobacter pylori. Gastroenterology.
125:1613-25.
Hauck CR, Agerer F, Muenzner P, Schmitter T. 2006. Cellular adhesion molecules as
targets for bacterial infection. Eur J Cell Biol. 85:235-42.
Hausmann M, Kiessling S, Mestermann S, Webb G, Spöttl T, Andus T, Schölmerich J,
Herfarth H, Ray K, Falk W, Rogler G. 2002. Toll-like receptors 2 and 4 are upregulated during intestinal inflammation. Gastroenterology. 122:1987-2000.
Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, Eng JK, Akira S,
Underhill DM, Aderem A. 2001. The innate immune response to bacterial flagellin is
mediated by Toll-like receptor 5. Nature. 410:1099-103.
Heidelberg JF, Eisen JA, Nelson WC, Clayton RA, Gwinn ML, Dodson RJ, Haft DH,
Hickey EK, Peterson JD, Umayam L, Gill SR, Nelson KE, Read TD, Tettelin H,
Richardson D, Ermolaeva MD, Vamathevan J, Bass S, Qin H, Dragoi I, Sellers P,
McDonald L, Utterback T, Fleishmann RD, Nierman WC, White O, Salzberg SL,
Smith HO, Colwell RR, Mekalanos JJ, Venter JC, Fraser CM. 2000. DNA sequence of
both chromosomes of the cholera pathogen Vibrio cholerae. Nature. 406:477-83.
Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, Lipford G, Wagner
H, Bauer S. 2004. Species-specific recognition of single-stranded RNA via toll-like
receptor 7 and 8. Science. 303:1526-9.
Heinrich PC, Behrmann I, Haan S, Hermanns HM, Müller-Newen G, Schaper F. 2003.
Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J.
374:1-20.
Heinrich PC, Castell JV, Andus T. 1990. Interleukin-6 and the acute phase response.
Biochem J. 265:621-36.
Hemmi H, Kaisho T, Takeuchi O, Sato S, Sanjo H, Hoshino K, Horiuchi T, Tomizawa H,
Takeda K, Akira S. 2002. Small anti-viral compounds activate immune cells via the
TLR7 MyD88-dependent signaling pathway. Nat Immunol. 3:196-200.
Hemmila E, Turbide C, Olson M, Jothy S, Holmes KV, Beauchemin N. 2004. Ceacam1a-/mice are completely resistant to infection by murine coronavirus mouse hepatitis virus
A59. J Virol. 78:10156-65.
Hershberg RM, Mayer LF. 2000. Antigen processing and presentation by intestinal
epithelial cells - polarity and complexity. Immunol Today. 21:123-8.
Heyderman RS, Virji M. 2007. IFN-gamma amplifies NFkappaB-dependent Neisseria
meningitidis invasion of epithelial cells via specific upregulation of CEA-related cell
adhesion molecule 1. Cell Microbiol. 9:2968-83.
Higuchi T, Orita T, Katsuya K, Yamasaki Y, Akiyama K, Li H, Yamamoto T, Saito Y,
Nakamura M. 2004. MUC20 suppresses the hepatocyte growth factor-induced Grb2Ras pathway by binding to a multifunctional docking site of met. Mol Cell Biol.
24:7456-68.
Hirano T. 1998. Interleukin 6 and its receptor: ten years later. Int Rev Immunol. 16:249-84.
Hirano T, Yasukawa K, Harada H, Taga T, Watanabe Y, Matsuda T, Kashiwamura S,
Nakajima K, Koyama K, Iwamatsu A, Tsunasawa S, Sakiyama F, Matsui H, Takahara
Y, Taniguchi T, Kishimoto T. 1986. Complementary DNA for a novel human
interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin. Nature.
324:73-6.
Hlady WG, Klontz KC. 1996. The epidemiology of Vibrio infections in Florida, 1981-1993.
J Infect Dis. 173:1176-83.
- 58 -
Holmes WE, Lee J, Kuang WJ, Rice GC, Wood WI. 1991. Structure and functional
expression of a human interleukin-8 receptor. Science. 253:1278-80.
Hooper LV, Gordon JI. 2001. Commensal host-bacterial relationships in the gut. Science.
292:1115-8.
Hooper LV, Wong MH, Thelin A, Hansson L, Falk PG, Gordon JI. 2001. Molecular
analysis of commensal host-microbial relationships in the intestine. Science. 291:881-4.
Houlston RS, Ford D. 1996. Genetics of coeliac disease. QJM. 89:737-43.
Hourigan CS. 2006. The molecular basis of coeliac disease. Clin Exp Med. 6:53-9.
Hugot JP, Chamaillard M, Zouali H, Lesage S, Cezard JP, Belaiche J, Almer S, Tysk C,
O'Morain CA, Gassull M, Binder V, Finkel Y, Cortot A, Modigliani R, Laurent-Puig P,
Gower-Rousseau C, Macry J, Colombel JF, Sahbatou M, Thomas G. 2001. Association
of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease. Nature.
411:599-603.
Hunt KA, Zhernakova A, Turner G, Heap GA, Franke L, Bruinenberg M, Romanos J,
Dinesen LC, Ryan AW, Panesar D, Gwilliam R, Takeuchi F, McLaren WM, Holmes
GK, Howdle PD, Walters JR, Sanders DS, Playford RJ, Trynka G, Mulder CJ, Mearin
ML, Verbeek WH, Trimble V, Stevens FM, O'Morain C, Kennedy NP, Kelleher D,
Pennington DJ, Strachan DP, McArdle WL, Mein CA, Wapenaar MC, Deloukas P,
McGinnis R, McManus R, Wijmenga C, van Heel DA. 2008. Newly identified genetic
risk variants for celiac disease related to the immune response. Nat Genet. 40:395-402.
Ichinose Y, Yamamoto K, Nakasone N, Tanabe MJ, Takeda T, Miwatani T, Iwanaga M.
1987. Enterotoxicity of El Tor-like hemolysin of non-O1 Vibrio cholerae. Infect
Immun. 55:1090-3.
Idriss HT, Naismith JH. 2000. TNF alpha and the TNF receptor superfamily: structurefunction relationship(s). Microsc Res Tech. 50:184-95.
Isaacs A, Lindenmann J. 1957. Virus interference I. The interferon. Proc R Soc Lond B:
Biol Sci. 147:258-67.
Ishii KJ, Akira S. 2006. Innate immune recognition of, and regulation by, DNA. Trends
Immunol. 27:525-32.
Ivarsson A, Hernell O, Nyström L, Persson LA. 2003. Children born in the summer have
increased risk for coeliac disease. J Epidemiol Community Health. 57:36-9.
Ivarsson A, Hernell O, Stenlund H, Persson LA. 2002. Breast-feeding protects against celiac
disease. Am J Clin Nutr. 75:914-21.
Ivarsson A, Persson LA, Nyström L, Ascher H, Cavell B, Danielsson L, Dannaeus A,
Lindberg T, Lindquist B, Stenhammar L, Hernell O. 2000. Epidemic of coeliac disease
in Swedish children. Acta Paediatr. 89:165-71.
Jack RS, Fan X, Bernheiden M, Rune G, Ehlers M, Weber A, Kirsch G, Mentel R, Fürll B,
Freudenberg M, Schmitz G, Stelter F, Schütt C. 1997. Lipopolysaccharide-binding
protein is required to combat a murine gram-negative bacterial infection. Nature.
389:742-5.
Jass JR, Roberton AM. 1994. Colorectal mucin histochemistry in health and disease: a
critical review. Pathol Int. 44:487-504.
Jiang Z, Georgel P, Du X, Shamel L, Sovath S, Mudd S, Huber M, Kalis C, Keck S,
Galanos C, Freudenberg M, Beutler B. 2005. CD14 is required for MyD88-independent
LPS signaling. Nat Immunol. 6:565-70.
Jin MS, Kim SE, Heo JY, Lee ME, Kim HM, Paik SG, Lee H, Lee JO. 2007. Crystal
structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated
lipopeptide. Cell. 130:1071-82.
- 59 -
Johansen BH, Gjertsen HA, Vartdal F, Buus S, Thorsby E, Lundin KE, Sollid LM. 1996.
Binding of peptides from the N-terminal region of alpha-gliadin to the celiac diseaseassociated HLA-DQ2 molecule assessed in biochemical and T cell assays. Clin
Immunol Immunopathol. 79:288-93.
Jones DE, Bevins CL. 1992. Paneth cells of the human small intestine express an
antimicrobial peptide gene. J Biol Chem. 267:23216-25.
Jones DE, Bevins CL. 1993. Defensin-6 mRNA in human Paneth cells: implications for
antimicrobial peptides in host defense of the human bowel. FEBS Lett. 315:187-92.
Jung HC, Eckmann L, Yang SK, Panja A, Fierer J, Morzycka-Wroblewska E, Kagnoff MF.
1995. A distinct array of proinflammatory cytokines is expressed in human colon
epithelial cells in response to bacterial invasion. J Clin Invest. 95:55-65.
Jurk M, Heil F, Vollmer J, Schetter C, Krieg AM, Wagner H, Lipford G, Bauer S. 2002.
Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound
R-848. Nat Immunol. 3:499.
Kagnoff MF. 2007. Celiac disease: pathogenesis of a model immunogenetic disease. J Clin
Invest. 117:41-9.
Kai-Larsen Y, Agerberth B. 2008. The role of the multifunctional peptide LL-37 in host
defense. Front Biosci. 13:3760-7.
Kaiserlian D, Vidal K, Revillard JP. 1989. Murine enterocytes can present soluble antigen to
specific class II-restricted CD4+ T cells. Eur J Immunol. 19:1513-6.
Kalliomäki M, Collado MC, Salminen S, Isolauri E. 2008. Early differences in fecal
microbiota composition in children may predict overweight. Am J Clin Nutr. 87:534-8.
Kalliomäki M, Kirjavainen P, Eerola E, Kero P, Salminen S, Isolauri E. 2001. Distinct
patterns of neonatal gut microflora in infants in whom atopy was and was not
developing. J Allergy Clin Immunol. 107:129-34.
Kamimura D, Ishihara K, Hirano T. 2003. IL-6 signal transduction and its physiological
roles: the signal orchestration model. Rev Physiol Biochem Pharmacol. 149:1-38.
Kanneganti TD, Lamkanfi M, Núñez G. 2007. Intracellular NOD-like receptors in host
defense and disease. Immunity. 27:549-59.
Kanneganti TD, Ozören N, Body-Malapel M, Amer A, Park JH, Franchi L, Whitfield J,
Barchet W, Colonna M, Vandenabeele P, Bertin J, Coyle A, Grant EP, Akira S, Núñez
G. 2006. Bacterial RNA and small antiviral compounds activate caspase-1 through
cryopyrin/Nalp3. Nature. 440:233-6.
Kaper JB, Morris JG Jr, Levine MM. 1995. Cholera. Clin Microbiol Rev. 8:48-86.
Keller M, Rüegg A, Werner S, Beer HD. 2008. Active caspase-1 is a regulator of
unconventional protein secretion. Cell. 132:818-31.
Keshav S. 2006. Paneth cells: leukocyte-like mediators of innate immunity in the intestine. J
Leukoc Biol. 80:500-8.
Kim HM, Park BS, Kim JI, Kim SE, Lee J, Oh SC, Enkhbayar P, Matsushima N, Lee H,
Yoo OJ, Lee JO. 2007. Crystal structure of the TLR4-MD-2 complex with bound
endotoxin antagonist Eritoran. Cell. 130:906-17.
Kirshner J, Hardy J, Wilczynski S, Shively JE. 2004. Cell-cell adhesion molecule
CEACAM1 is expressed in normal breast and milk and associates with beta1 integrin in
a 3D model of morphogenesis. J Mol Histol. 35:287-99.
Kishimoto T. 2005. Interleukin-6: from basic science to medicine: 40 years in immunology.
Annu Rev Immunol. 23:1-21.
Kishimoto T, Akira S, Narazaki M, Taga T. 1995. Interleukin-6 family of cytokines and
gp130. Blood. 86:1243-54.
- 60 -
Kobayashi KS, Chamaillard M, Ogura Y, Henegariu O, Inohara N, Nuñez G, Flavell RA.
2005. Nod2-dependent regulation of innate and adaptive immunity in the intestinal
tract. Science. 307:731-4.
Kobayashi Y. 2008. The role of chemokines in neutrophil biology. Front Biosci. 13:2400-7.
Kraehenbuhl JP, Neutra MR. 2000. Epithelial M cells: differentiation and function. Annu
Rev Cell Dev Biol. 16:301-32.
Kuespert K, Pils S, Hauck CR. 2006. CEACAMs: their role in physiology and
pathophysiology. Curr Opin Cell Biol. 18: 565-71.
Kumagai Y, Takeuchi O, Akira S. 2008. Pathogen recognition by innate receptors. J Infect
Chemother. 14:86-92.
Kunath T, Ordonez-Garcia C, Turbide C, Beauchemin N. 1995. Inhibition of colonic tumor
cell growth by biliary glycoprotein. Oncogene. 11:2375-82.
Kuno K, Matsushima K. 1994. The IL-1 receptor signaling pathway. J Leukoc Biol. 56:5427.
Kupper C. 2005. Dietary guidelines and implementation for celiac disease.
Gastroenterology. 128:S121-7.
Kusugami K, Fukatsu A, Tanimoto M, Shinoda M, Haruta J, Kuroiwa A, Ina K, Kanayama
K, Ando T, Matsuura T, Yamaguchi T, Morise K, Jeda M, Iokawa H, Ishihara A, Sarai
S. 1995. Elevation of interleukin-6 in inflammatory bowel disease is macrophage- and
epithelial cell-dependent. Dig Dis Sci. 40:949-59.
Lammers KM, Lu R, Brownley J, Lu B, Gerard C, Thomas K, Rallabhandi P, SheaDonohue T, Tamiz A, Alkan S, Netzel-Arnett S, Antalis T, Vogel SN, Fasano A. 2008.
Gliadin induces an increase in intestinal permeability and zonulin release by binding to
the chemokine receptor CXCR3. Gastroenterology. 135:194-204.
Latz E, Verma A, Visintin A, Gong M, Sirois CM, Klein DC, Monks BG, McKnight CJ,
Lamphier MS, Duprex WP, Espevik T, Golenbock DT. 2007. Ligand induced
conformational changes allosterically activate Toll-like receptor 9. Nat Immunol.
8:772-9.
Ledgerwood EC, Pober JS, Bradley JR. 1999. Recent advances in the molecular basis of
TNF signal transduction. Lab Invest. 79:1041-50.
Lehrer RI, Lichtenstein AK, Ganz T. 1993. Defensins: antimicrobial and cytotoxic peptides
of mammalian cells. Annu Rev Immunol. 11:105-28.
Lencer WI, Saslowsky D. 2005. Raft trafficking of AB5 subunit bacterial toxins. Biochim
Biophys Acta. 1746:314-21.
Levine MM, Kaper JB, Herrington D, Losonsky G, Morris JG, Clements ML, Black RE,
Tall B, Hall R. 1988. Volunteer studies of deletion mutants of Vibrio cholerae O1
prepared by recombinant techniques. Infect Immun. 56:161-7.
Levy DE, Darnell JE Jr. 2002. Stats: transcriptional control and biological impact. Nat Rev
Mol Cell Biol. 3:651-62.
Ley RE, Bäckhed F, Turnbaugh P, Lozupone CA, Knight RD, Gordon JI. 2005. Obesity
alters gut microbial ecology. Proc Natl Acad Sci U S A. 102:11070-5.
Ley RE, Peterson DA, Gordon JI. 2006. Ecological and evolutionary forces shaping
microbial diversity in the human intestine. Cell. 124:837-48.
Ley RE, Turnbaugh PJ, Klein S, Gordon JI. 2006. Microbial ecology: human gut microbes
associated with obesity. Nature. 444:1022-3.
Li M, Wang B, Zhang M, Rantalainen M, Wang S, Zhou H, Zhang Y, Shen J, Pang X,
Zhang M, Wei H, Chen Y, Lu H, Zuo J, Su M, Qiu Y, Jia W, Xiao C, Smith LM, Yang
S, Holmes E, Tang H, Zhao G, Nicholson JK, Li L, Zhao L. 2008. Symbiotic gut
- 61 -
microbes modulate human metabolic phenotypes. Proc Natl Acad Sci U S A.
105:2117-22.
Linden SK, Sutton P, Karlsson NG, Korolik V, McGuckin MA. 2008. Mucins in the
mucosal barrier to infection. Mucosal Immunol. 1:183-97.
Lipp EK, Huq A, Colwell RR. 2002. Effects of global climate on infectious disease: the
cholera model. Clin Microbiol Rev. 15:757-70.
Liu L, Wang L, Jia HP, Zhao C, Heng HH, Schutte BC, McCray PB Jr, Ganz T. 1998.
Structure and mapping of the human beta-defensin HBD-2 gene and its expression at
sites of inflammation. Gene. 222:237-44.
Liévin-Le Moal V, Servin AL. 2006. The front line of enteric host defense against
unwelcome intrusion of harmful microorganisms: mucins, antimicrobial peptides, and
microbiota. Clin Microbiol Rev. 19:315-37.
Locksley RM, Killeen N, Lenardo MJ. 2001. The TNF and TNF receptor superfamilies:
integrating mammalian biology. Cell. 104:487-501.
Losowsky MS. 2008. A history of coeliac disease. Dig Dis. 26:112-20.
Lundqvist C, Baranov V, Hammarström S, Athlin L, Hammarström ML. 1995. Intraepithelial lymphocytes. Evidence for regional specialization and extrathymic T cell
maturation in the human gut epithelium. Int Immunol. 7:1473-87.
MacDonald TT. 2003. The mucosal immune system. Parasite Immunol. 25:235-46.
Mackey D, McFall AJ. 2006. MAMPs and MIMPs: proposed classifications for inducers of
innate immunity. Mol Microbiol. 61:1365-71.
Mackie RI, Sghir A, Gaskins HR. 1999. Developmental microbial ecology of the neonatal
gastrointestinal tract. Am J Clin Nutr. 69:1035S-45S.
Macpherson AJ, Harris NL. 2004. Interactions between commensal intestinal bacteria and
the immune system. Nat Rev Immunol. 4:478-85.
Madsen K, Cornish A, Soper P, McKaigney C, Jijon H, Yachimec C, Doyle J, Jewell L, De
Simone C. 2001. Probiotic bacteria enhance murine and human intestinal epithelial
barrier function. Gastroenterology. 121:580-91.
Maeurer MJ, Martin D, Walter W, Liu K, Zitvogel L, Halusczcak K, Rabinowich H,
Duquesnoy R, Storkus W, Lotze MT. 1996. Human intestinal Vdelta1+ lymphocytes
recognize tumor cells of epithelial origin. J Exp Med. 183:1681-96.
Magalhaes JG, Tattoli I, Girardin SE. 2007. The intestinal epithelial barrier: how to
distinguish between the microbial flora and pathogens. Semin Immunol. 19:106-15.
Maiuri L, Troncone R, Mayer M, Coletta S, Picarelli A, De Vincenzi M, Pavone V,
Auricchio S. 1996. In vitro activities of A-gliadin-related synthetic peptides: damaging
effect on the atrophic coeliac mucosa and activation of mucosal immune response in the
treated coeliac mucosa. Scand J Gastroenterol. 31:247-53.
Mantzaris G, Jewell DP. 1991. In vivo toxicity of a synthetic dodecapeptide from A gliadin
in patients with coeliac disease. Scand J Gastroenterol. 26:392-8.
Mariathasan S, Weiss DS, Newton K, McBride J, O'Rourke K, Roose-Girma M, Lee WP,
Weinrauch Y, Monack DM, Dixit VM. 2006. Cryopyrin activates the inflammasome in
response to toxins and ATP. Nature. 440:228-32.
Martin MU, Falk W. 1997. The interleukin-1 receptor complex and interleukin-1 signal
transduction. Eur Cytokine Netw. 8:5-17.
Martinon F, Burns K, Tschopp J. 2002. The inflammasome: a molecular platform triggering
activation of infl ammatory caspases and processing of proIL-beta. Mol Cell. 10:41726.
Martinon F, Pétrilli V, Mayor A, Tardivel A, Tschopp J. 2006. Gout-associated uric acid
crystals activate the NALP3 inflammasome. Nature. 440:237-41.
- 62 -
Mashburn-Warren LM, Whiteley M. 2006. Special delivery: vesicle trafficking in
prokaryotes. Mol Microbiol. 61:839-46.
May MJ, Ghosh S. 1998. Signal transduction through NF-kappa B. Immunol Today. 19:808.
McCracken VJ, Lorenz RG. 2001. The gastrointestinal ecosystem: a precarious alliance
among epithelium, immunity and microbiota. Cell Microbiol. 3:1-11.
McGee DW, Vitkus SJ, Lee P. 1996. The effect of cytokine stimulation on IL-1 receptor
mRNA expression by intestinal epithelial cells. Cell Immunol. 168:276-80.
Medzhitov R, Janeway CA Jr. 1997. Innate immunity: the virtues of a nonclonal system of
recognition. Cell. 91:295-8.
Meeuwisse GW. 1970. Diagnostic criteria in coeliac disease. Acta Paediatr Scand, 59:461-3.
Melgar S, Bas A, Hammarström S, Hammarström ML. 2002. Human small intestinal
mucosa harbours a small population of cytolytically active CD8+ alphabeta T
lymphocytes. Immunology. 106:476-85.
Melgar S, Yeung MM, Bas A, Forsberg G, Suhr O, Oberg A, Hammarstrom S, Danielsson
A, Hammarstrom ML. 2003. Over-expression of interleukin 10 in mucosal T cells of
patients with active ulcerative colitis. Clin Exp Immunol. 134:127-37.
Melmed G, Thomas LS, Lee N, Tesfay SY, Lukasek K, Michelsen KS, Zhou Y, Hu B,
Arditi M, Abreu MT. 2003. Human intestinal epithelial cells are broadly unresponsive
to Toll-like receptor 2-dependent bacterial ligands: implications for host-microbial
interactions in the gut. J Immunol. 170:1406-15.
Middleton J, Neil S, Wintle J, Clark-Lewis I, Moore H, Lam C, Auer M, Hub E, Rot A.
1997. Transcytosis and surface presentation of IL-8 by venular endothelial cells. Cell.
91:385-95.
Miller H, Zhang J, Kuolee R, Patel GB, Chen W. 2007. Intestinal M cells: the fallible
sentinels? World J Gastroenterol. 13:1477-86.
Mitra R, Figueroa P, Mukhopadhyay AK, Shimada T, Takeda Y, Berg DE, Nair GB. 2000.
Cell vacuolation, a manifestation of the El tor hemolysin of Vibrio cholerae. Infect
Immun. 68:1928-33.
Molberg O, Kett K, Scott H, Thorsby E, Sollid LM, Lundin KE.1997. Gliadin specific, HLA
DQ2-restricted T cells are commonly found in small intestinal biopsies from coeliac
disease patients, but not from controls. Scand J Immunol. 46:103-9.
Moschioni M, Tombola F, de Bernard M, Coelho A, Zitzer A, Zoratti M, Montecucco C.
2002. The Vibrio cholerae haemolysin anion channel is required for cell vacuolation
and death. Cell Microbiol. 4:397-409.
Mowat AM. 2003. Anatomical basis of tolerance and immunity to intestinal antigens. Nat
Rev Immunol. 3:331-41.
Murphy PM, Tiffany HL. 1991. Cloning of a complimentary DNA encoding a functional
human interleukin-8 receptor. Science. 253:1280-3.
Murray PJ. 2007. The JAK-STAT signaling pathway: input and output integration. J
Immunol. 178:2623-9.
Nadal I, Donat E, Ribes-Koninckx C, Calabuig M, Sanz Y. 2007. Imbalance in the
composition of the duodenal microbiota of children with coeliac disease. J Med
Microbiol. 56:1669-74.
Nagai Y, Akashi S, Nagafuku M, Ogata M, Iwakura Y, Akira S, Kitamura T, Kosugi A,
Kimoto M, Miyake K. 2002. Essential role of MD-2 in LPS responsiveness and TLR4
distribution. Nat Immunol. 3:667-72.
- 63 -
Nagaishi T, Iijima H, Nakajima A, Chen D, Blumberg RS. 2006. Role of CEACAM1 as a
regulator of T cells. Ann N Y Acad Sci. 1072:155-75.
Nagamune K, Yamamoto K, Honda T. 1997. Intramolecular chaperone activity of the proregion of Vibrio cholerae El Tor cytolysin. J Biol Chem. 272:1338-43.
Nagamune K, Yamamoto K, Naka A, Matsuyama J, Miwatani T, Honda T. 1996. In vitro
proteolytic processing and activation of the recombinant precursor of El Tor
cytolysin/hemolysin (pro-HlyA) of Vibrio cholerae by soluble hemagglutinin/protease
of V. cholerae, trypsin, and other proteases. Infect Immun. 64:4655-8.
Newberry RD, Lorenz RG. 2005. Organizing a mucosal defense. Immunol Rev. 206:6-21.
Ninin E, Caroff N, El Kouri D, Espaze E, Richet H, Quilici ML, Fournier JM. 2000.
Nontoxigenic vibrio Cholerae O1 bacteremia: case report and review. Eur J Clin
Microbiol Infect Dis. 19:489-91.
Noverr MC, Huffnagle GB. 2004. Does the microbiota regulate immune responses outside
the gut? Trends Microbiol. 12:562-8.
Noverr MC, Huffnagle GB. 2005. The 'microflora hypothesis' of allergic diseases. Clin Exp
Allergy. 35:1511-20.
Obrink B, Sawa H, Scheffrahn I, Singer BB, Sigmundsson K, Sundberg U, Heymann R,
Beauchemin N, Weng G, Ram P, Iyengar R. 2002. Computational analysis of isoformspecific signal regulation by CEACAM1-A cell adhesion molecule expressed in PC12
cells. Ann N Y Acad Sci. 971:597-607.
Ogierman MA, Fallarino A, Riess T, Williams SG, Attridge SR, Manning PA. 1997.
Characterization of the Vibrio cholerae El Tor lipase operon lipAB and a protease gene
downstream of the hly region. J Bacteriol. 179:7072-80.
Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen FF, Ramos R, Britton H, Moran T,
Karaliuskas R, Duerr RH, Achkar JP, Brant SR, Bayless TM, Kirschner BS, Hanauer
SB, Nunez G, Cho JH. 2001. A frameshift mutation in NOD2 associated with
susceptibility to Crohn's disease. Nature. 411:603-6.
Oikawa S, Inuzuka C, Kuroki M, Matsuoka Y, Kosaki G, Nakazato H. 1989. Cell adhesion
activity of non-specific cross-reacting antigen (NCA) and carcinoembryonic antigen
(CEA) expressed on CHO cell surface: homophilic and heterophilic adhesion. Biochem
Biophys Res Commun. 164:39-45.
Olivier V, Haines III GK, Tan Y, Fullner Satchell KJ. 2007. Hemolysin and the
multifunctional autoprocessing RTX toxin are virulence factors during intestinal
infection of mice with Vibrio cholerae El Tor O1 strains. Infect Immun. 75:5035-42.
Olsson C, Hernell O, Hörnell A, Lönnberg G, Ivarsson A. 2008. Difference in celiac disease
risk between Swedish birth cohorts suggests an opportunity for primary prevention.
Pediatrics. 122:528-34
O'Neil DA, Porter EM, Elewaut D, Anderson GM, Eckmann L, Ganz T, Kagnoff MF. 1999.
Expression and regulation of the human beta-defensins hBD-1 and hBD-2 in intestinal
epithelium. J Immunol. 163:6718-24.
O'Neill LA, Greene C. 1998. Signal transduction pathways activated by the IL-1 receptor
family: ancient signaling machinery in mammals, insects and plants. J Leukoc Biol.
63:650-7.
Osawa R, Okitsu T, Sata S, and Yamai S. 1997. Rapid screening method for identification
of cholera toxin-producing Vibrio cholerae O1 and O139. J Clin Microbiol. 35:951-3.
O'Sullivan LA, Liongue C, Lewis RS, Stephenson SE, Ward AC. 2007. Cytokine receptor
signaling through the Jak-Stat-Socs pathway in disease. Mol Immunol. 44:2497-506.
- 64 -
Ott SJ, Musfeldt M, Wenderoth DF, Hampe J, Brant O, Fölsch UR, Timmis KN, Schreiber
S. 2004. Reduction in diversity of the colonic mucosa associated bacterial microflora in
patients with active inflammatory bowel disease. Gut. 53:685-93.
Otte JM, Cario E, Podolsky DK. 2004. Mechanisms of cross hyporesponsiveness to Tolllike receptor bacterial ligands in intestinal epithelial cells. Gastroenterology. 126:105470.
Otte JM, Kiehne K, Herzig KH. 2003. Antimicrobial peptides in innate immunity of the
human intestine. J Gastroenterol. 38:717-26.
Otte JM, Podolsky DK. 2004. Functional modulation of enterocytes by gram-positive and
gram-negative microorganisms. Am J Physiol Gastrointest Liver Physiol. 286:G613-26.
Ou TY, Liu JW, Leu HS. 2003. Independent prognostic factors for fatality in patients with
invasive vibrio cholerae non-O1 infections. J Microbiol Immunol Infect. 36:117-22.
Palmer C, Bik EM, Digiulio DB, Relman DA, Brown PO. 2007. Development of the Human
Infant Intestinal Microbiota. PLoS Biol. 5:e177.
Pantano S, Montecucco C. 2006. A molecular model of the Vibrio cholerae cytolysin
transmembrane pore. Toxicon. 47:35-40.
Paonessa G, Graziani R, De Serio A, Savino R, Ciapponi L, Lahm A, Salvati AL, Toniatti
C, Ciliberto G. 1995. Two distinct and independent sites on IL-6 trigger gp 130 dimer
formation and signalling. EMBO J. 14:1942-51.
Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Levanos VA, Sahasrabudhe A,
Dewhirst FE. 2001. Bacterial diversity in human subgingival plaque. J Bacteriol.
183:3770-83.
Pazgier M, Li X, Lu W, Lubkowski J. 2007. Human defensins: synthesis and structural
properties. Curr Pharm Des. 13:3096-118.
Pei Z, Bini EJ, Yang L, Zhou M, Francois F, Blaser MJ. 2004. Bacterial biota in the human
distal esophagus. Proc Natl Acad Sci U S A. 101:4250-5.
Pennica D, Hayflick JS, Bringman TS, Palladino MA, Goeddel DV. 1985. Cloning and
expression in Escherichia coli of the cDNA for murine tumor necrosis factor. Proc Natl
Acad Sci USA. 82: 6060-4.
Perez-Vilar J, Eckhardt AE, DeLuca A, Hill RL. 1998. Porcine submaxillary mucin forms
disulfide-linked multimers through its amino-terminal D-domains. J Biol Chem.
273:14442-9.
Pflanz S, Hibbert L, Mattson J, Rosales R, Vaisberg E, Bazan JF, Phillips JH, McClanahan
TK, de Waal Malefyt R, Kastelein RA. 2004. WSX-1 and glycoprotein 130 constitute a
signal-transducing receptor for IL-27. J Immunol. 172:2225-31.
Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D, Alejos E, Silva
M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, Beutler B. 1998.
Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene.
Science. 282:2085-8.
Porter EM, Liu L, Oren A, Anton PA, Ganz T. 1997. Localization of human intestinal
defensin 5 in Paneth cell granules. Infect Immun. 65:2389-95.
Rahman A, Fahlgren A, Sitohy B, Baranov V, Zirakzadeh A, Hammarström S, Danielsson
A, Hammarström ML. 2007. Beta-defensin production by human colonic plasma cells:
a new look at plasma cells in ulcerative colitis. Inflamm Bowel Dis. 13:847-55.
Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R. 2004.
Recognition of commensal microflora by toll-like receptors is required for intestinal
homeostasis. Cell. 118:229-41.
- 65 -
Reinecker HC, Steffen M, Witthoeft T, Pflueger I, Schreiber S, MacDermott RP, Raedler
A. 1993. Enhanced secretion of tumour necrosis factor-alpha, IL-6, and IL-1 beta by
isolated lamina propria mononuclear cells from patients with ulcerative colitis and
Crohn's disease. Clin Exp Immunol. 94:174-81.
Rescigno M, Urbano M, Valzasina B, Francolini M, Rotta G, Bonasio R, Granucci F,
Kraehenbuhl JP, Ricciardi-Castagnoli P. 2001. Dendritic cells express tight junction
proteins and penetrate gut epithelial monolayers to sample bacteria. Nat Immunol.
2:361-7.
Roach JC, Glusman G, Rowen L, Kaur A, Purcell MK, Smith KD, Hood LE, Aderem A.
2005. The evolution of vertebrate Toll-like receptors. Proc Natl Acad Sci U S A.
102:9577-82.
Rogers NC, Slack EC, Edwards AD, Nolte MA, Schulz O, Schweighoffer E, Williams DL,
Gordon S, Tybulewicz VL, Brown GD, Reis e Sousa C. 2005. Syk-dependent cytokine
induction by Dectin-1 reveals a novel pattern recognition pathway for C type lectins.
Immunity. 22:507-17.
Rogler G, Andus T. 1998. Cytokines in inflammatory bowel disease. World J Surg. 22:3829.
Rumio C, Besusso D, Arnaboldi F, Palazzo M, Selleri S, Gariboldi S, Akira S, Uematsu S,
Bignami P, Ceriani V, Ménard S, Balsari A. 2006. Activation of smooth muscle and
myenteric plexus cells of jejunum via Toll-like receptor 4. J Cell Physiol. 208:47-54.
Sack DA, Sack RB, Nair GB, Siddique AK. 2004. Cholera. Lancet. 363:223-33.
Saka HA, Bidinost C, Sola C, Carranza P, Collino C, Ortiz S, Echenique JR, Bocco JL.
2008. Vibrio cholerae cytolysin is essential for high enterotoxicity and apoptosis
induction produced by a cholera toxin gene-negative V. cholerae non-O1, non-O139
strain. Microb Pathog. 44:118-28.
Sanderson IR. 1999. The physicochemical environment of the neonatal intestine. Am J Clin
Nutr. 69:1028S-34S.
Sanderson IR, Walker WA. 2007. TLRs in the Gut I. The role of TLRs/Nods in intestinal
development and homeostasis. Am J Physiol Gastrointest Liver Physiol. 292:G6-10.
Sanz Y, Sánchez E, Marzotto M, Calabuig M, Torriani S, Dellaglio F. 2007. Differences in
faecal bacterial communities in coeliac and healthy children as detected by PCR and
denaturing gradient gel electrophoresis. FEMS Immunol Med Microbiol. 51:562-8.
Schauber J, Svanholm C, Termén S, Iffland K, Menzel T, Scheppach W, Melcher R,
Agerberth B, Lührs H, Gudmundsson GH. 2003. Expression of the cathelicidin LL-37
is modulated by short chain fatty acids in colonocytes: relevance of signalling
pathways. Gut. 52:735-41.
Schroder K, Hertzog PJ, Ravasi T, Hume DA. 2004. Interferon-gamma: an overview of
signals, mechanisms and functions. J Leukoc Biol. 75:163-89.
Schroder K, Sweet MJ, Hume DA. 2006. Signal integration between IFNgamma and TLR
signalling pathways in macrophages. Immunobiology. 211:511-24.
Schultsz C, Van Den Berg FM, Ten Kate FW, Tytgat GN, Dankert J. 1999. The intestinal
mucus layer from patients with inflammatory bowel disease harbors high numbers of
bacteria compared with controls. Gastroenterology. 117:1089-97.
Schumann D, Chen CJ, Kaplan B, Shively JE. 2001. Carcinoembryonic antigen cell
adhesion molecule 1 directly associates with cytoskeleton proteins actin and
tropomyosin.J Biol Chem. 276:47421-33.
Schölzel S, Zimmermann W, Schwarzkopf G, Grunert F, Rogaczewski B, Thompson J.
2000. Carcinoembryonic antigen family members CEACAM6 and CEACAM7 are
- 66 -
differentially expressed in normal tissues and oppositely deregulated in hyperplastic
colorectal polyps and early adenomas. Am J Pathol. 156:595-605.
Sears CL. 2005. A dynamic partnership: celebrating our gut flora. Anaerobe. 11:247-51.
Shanahan F. 2005. Physiological basis for novel drug therapies used to treat the
inflammatory bowel diseases. I. Pathophysiological basis and prospects for probiotic
therapy in inflammatory bowel disease. Am J Physiol Gastrointest Liver Physiol. 288:
G417-21.
Shewry PR, Halford NG. 2002. Cereal seed storage proteins: structures, properties and role
in grain utilization. J Exp Bot. 53:947-58.
Shi J, Aono S, Lu W, Ouellette AJ, Hu X, Ji Y, Wang L, Lenz S, van Ginkel FW, Liles M,
Dykstra C, Morrison EE, Elson CO. 2007. A novel role for defensins in intestinal
homeostasis: regulation of IL-1beta secretion. J Immunol. 179:1245-53.
Silva TM, Schleupner MA, Tacket CO, Steiner TS, Kaper JB, Edelman R, Guerrant R.
1996. New evidence for an inflammatory component in diarrhea caused by selected
new, live attenuated cholera vaccines and by El Tor and Q139 Vibrio cholerae. Infect
Immun. 64:2362-4.
Singer BB, Scheffrahn I, Obrink B. 2000. The tumor growth-inhibiting cell adhesion
molecule CEACAM1 (C-CAM) is differently expressed in proliferating and quiescent
epithelial cells and regulates cell proliferation. Cancer Res. 60:1236-44.
Sjöström H, Lundin KE, Molberg O, Körner R, McAdam SN, Anthonsen D, Quarsten H,
Norén O, Roepstorff P, Thorsby E, Sollid LM. 1998. Identification of a gliadin T-cell
epitope in coeliac disease: general importance of gliadin deamidation for intestinal Tcell recognition. Scand J Immunol. 48:111-5.
Sollid LM. 2000. Molecular basis of celiac disease. Annu Rev Immunol. 18:53-81.
Sollid LM, Thorsby E. 1993. HLA susceptibility genes in celiac disease: genetic mapping
and role in pathogenesis. Gastroenterology. 105:910-22.
Stadnyk AW, Waterhouse CC. 1997. Epithelial cytokines in intestinal inflammation and
mucosal immunity. Curr Opin Gastroenterol. 13:510-7.
Strous GJ, Dekker J. 1992. Mucin-type glycoproteins. Crit Rev Biochem Mol Biol. 27:5792.
Strugala V, Allen A, Dettmar PW, Pearson JP. 2003. Colonic mucin: methods of measuring
mucus thickness. Proc Nutr Soc. 62:237-43.
Sundberg U, Beauchemin N, Obrink B. 2004. The cytoplasmic domain of CEACAM1-L
controls its lateral localization and the organization of desmosomes in polarized
epithelial cells. J Cell Sci. 117:1091-104.
Sutterwala FS, Mijares LA, Li L, Ogura Y, Kazmierczak BI, Flavell RA. 2007. Immune
recognition of Pseudomonas aeruginosa mediated by the IPAF/NLRC4 inflammasome.
J Exp Med. 204:3235-45.
Suzuki M, Hisamatsu T, Podolsky DK. 2003. Gamma interferon augments the intracellular
pathway for lipopolysaccharide (LPS) recognition in human intestinal epithelial cells
through coordinated up-regulation of LPS uptake and expression of the intracellular
Toll-like receptor 4-MD-2 complex. Infect Immun. 71:3503-11.
Swidsinski A, Ladhoff A, Pernthaler A, Swidsinski S, Loening-Baucke V, Ortner M, Weber
J, Hoffmann U, Schreiber S, Dietel M, Lochs H. 2002. Mucosal flora in inflammatory
bowel disease. Gastroenterology. 122:44-54.
Swidsinski A, Loening-Baucke V, Lochs H, Hale LP. 2005. Spatial organization of bacterial
flora in normal and inflamed intestine: a fluorescence in situ hybridization study in
mice. World J Gastroenterol. 11:1131-40.
- 67 -
Swidsinski A, Weber J, Loening-Baucke V, Hale LP, Lochs H. 2005. Spatial organization
and composition of the mucosal flora in patients with inflammatory bowel disease. J
Clin Microbiol. 43:3380-9.
Söderström K, Bucht A, Halapi E, Lundqvist C, Gronberg A, Nilsson E, Orsini DL, van de
Wal Y, Koning F, Hammarström ML, Kiessling R. 1994. High expression of V gamma
8 is a shared feature of human gamma delta T cells in the epithelium of the gut and in
the inflamed synovial tissue. J Immunol. 152:6017-27.
Tacket CO, Kotloff KL, Losonsky G, Nataro JP, Michalski J, Kaper JB, Edelman R, Levine
MM. 1997. Volunteer studies investigating the safety and efficacy of live oral El Tor
Vibrio cholerae O1 vaccine strain CVD 111. Am J Trop Med Hyg. 56:533-7.
Taga T, Kishimoto T. 1997. Gp130 and the interleukin-6 family of cytokines. Annu Rev
Immunol. 15:797-819.
Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T, Takeda K, Akira S. 1999.
Differential roles of TLR2 and TLR4 in recognition of gram-negative and grampositive bacterial cell wall components. Immunity. 11:443-51.
Takeuchi O, Kawai T, Mühlradt PF, Morr M, Radolf JD, Zychlinsky A, Takeda K, Akira S.
2001. Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int Immunol.
13:933-40.
Takeuchi O, Sato S, Horiuchi T, Hoshino K, Takeda K, Dong Z, Modlin RL, Akira S. 2002.
Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial
lipoproteins. J Immunol. 169:10-4.
Tang P, Hung MC, Klostergaard J. 1996. Human pro-tumor necrosis factor is a homotrimer.
Biochemistry. 35:8216-25.
Taylor K, Barran PE, Dorin JR. 2008. Structure-activity relationships in beta-defensin
peptides. Biopolymers. 90:1-7.
Thomson AW and Lotze MT. Editors, The Cytokine Handbook, Academic Press (2003), pp.
837-60.
Tjellström B, Stenhammar L, Högberg L, Fälth-Magnusson K, Magnusson KE, Midtvedt T,
Sundqvist T, Houlston R, Popat S, Norin E. 2007. Gut microflora associated
characteristics in first-degree relatives of children with celiac disease. Scand J
Gastroenterol. 42:1204-8.
Tjellström B, Stenhammar L, Högberg L, Fälth-Magnusson K, Magnusson KE, Midtvedt T,
Sundqvist T, Norin E. 2005. Gut microflora associated characteristics in children with
celiac disease. Am J Gastroenterol. 100:2784-8.
Tlaskalová-Hogenová H, Stepánková R, Hudcovic T, Tucková L, Cukrowska B, LodinováZádníková R, Kozáková H, Rossmann P, Bártová J, Sokol D, Funda DP, Borovská D,
Reháková Z, Sinkora J, Hofman J, Drastich P, Kokesová A. 2004. Commensal bacteria
(normal microflora), mucosal immunity and chronic inflammatory and autoimmune
diseases. Immunol Lett. 93:97-108.
Tracey KJ, Cerami A. Tumor necrosis factor, other cytokines and disease. 1993. Annu Rev
Cell Biol. 9:317-43.
Treem WR. 2004. Emerging concepts in celiac disease. Curr Opin Pediatr. 16:552-9.
Triantafilou M, Triantafilou K. 2002. Lipopolysaccharide recognition: CD14, TLRs and the
LPS-activation cluster. Trends Immunol. 23:301-4.
Trinchieri G, Sher A. 2007. Cooperation of Toll-like receptor signals in innate immune
defence. Nat Rev Immunol. 7:179-90.
Troncone R, Ivarsson A, Szajewska H, Mearin ML; Members of European Multistakeholder
Platform on CD (CDEUSSA). 2008. Review article: future research on coeliac disease -
- 68 -
a position report from the European multistakeholder platform on coeliac disease
(CDEUSSA). Aliment Pharmacol Ther. 27:1030-43.
Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. 2006. An
obesity-associated gut microbiome with increased capacity for energy harvest. Nature.
444:1027-31.
Tytgat KM, Büller HA, Opdam FJ, Kim YS, Einerhand AW, Dekker J. 1994. Biosynthesis
of human colonic mucin: Muc2 is the prominent secretory mucin. Gastroenterology.
107:1352-63.
Uematsu S, Jang MH, Chevrier N, Guo Z, Kumagai Y, Yamamoto M, Kato H, Sougawa N,
Matsui H, Kuwata H, Hemmi H, Coban C, Kawai T, Ishii KJ, Takeuchi O, Miyasaka
M, Takeda K, Akira S. 2006. Detection of pathogenic intestinal bacteria by Toll-like
receptor 5 on intestinal CD11c+ lamina propria cells. Nat Immunol. 7:868-74.
Vaitkevicius K, Rompikuntal PK, Lindmark B, Vaitkevicius R, Song T, Wai SN. 2008. The
metalloprotease PrtV from Vibrio cholerae. FEBS J. 275:3167-77.
Valeva A, Walev I, Weis S, Boukhallouk F, Wassenaar TM, Bhakdi S. 2008. Proinflammatory feedback activation cycle evoked by attack of Vibrio cholerae cytolysin
on human neutrophil granulocytes. Med Microbiol Immunol. 197:285-93.
van Boxel-Dezaire AH, Stark GR. 2007. Cell type-specific signaling in response to
interferon-gamma. Curr Top Microbiol Immunol. 316:119-54.
van de Wal Y, Kooy Y, van Veelen P, Peña S, Mearin L, Papadopoulos G, Koning F. 1998.
Selective deamidation by tissue transglutaminase strongly enhances gliadin-specific T
cell reactivity. J Immunol. 161:1585-8.
van Heel DA, West J. 2006. Recent advances in coeliac disease. Gut. 55:1037-46.
Van Klinken BJ, Dekker J, Büller HA, Einerhand AW. 1995. Mucin gene structure and
expression: protection vs. adhesion. Am J Physiol. 269:G613-27.
van Vliet SJ, García-Vallejo JJ, van Kooyk Y. 2008. Dendritic cells and C-type lectin
receptors: coupling innate to adaptive immune responses. Immunol Cell Biol. 86:580-7.
Vanden Broeck D, Horvath C, De Wolf MJ. 2007. Vibrio cholerae: cholera toxin. Int J
Biochem Cell Biol. 39: 1771-5.
Verstrepen L, Bekaert T, Chau TL, Tavernier J, Chariot A, Beyaert R. 2008. TLR-4, IL-1R
and TNF-R signaling to NF-kappaB: variations on a common theme. Cell Mol Life Sci.
65:2964-78.
Vigers GP, Dripps DJ, Edwards CK 3rd, Brandhuber BJ. 2000. X-ray crystal structure of a
small antagonist peptide bound to interleukin-1 receptor type 1. J Biol Chem.
275:36927-33.
Wajant H, Pfizenmaier K, Scheurich P. 2003. Tumor necrosis factor signaling. Cell Death
Differ. 10:45-65.
Wallach D, Varfolomeev EE, Malinin NL, Goltsev YV, Kovalenko AV, Boldin MP. 1999.
Tumor necrosis factor receptor and Fas signaling mechanisms. Annu Rev Immunol.
17:331-67.
Wang J, Al-Lamki RS, Zhang H, Kirkiles-Smith N, Gaeta ML, Thiru S, Pober JS, Bradley
JR. 2003. Histamine antagonizes tumor necrosis factor (TNF) signaling by stimulating
TNF receptor shedding from the cell surface and Golgi storage pool. J Biol Chem.
278:21751-60.
Wang M, Ahrné S, Jeppsson B, Molin G. 2005. Comparison of bacterial diversity along the
human intestinal tract by direct cloning and sequencing of 16S rRNA genes. FEMS
Microbiol Ecol. 54:219-31.
Wapenaar MC, Monsuur AJ, van Bodegraven AA, Weersma RK, Bevova MR, Linskens
RK, Howdle P, Holmes G, Mulder CJ, Dijkstra G, van Heel DA, Wijmenga C. 2008.
- 69 -
Associations with tight junction genes PARD3 and MAGI2 in Dutch patients point to
a common barrier defect for coeliac disease and ulcerative colitis. Gut. 57:463-7.
Waugh DJ, Wilson C. 2008. The interleukin-8 pathway in cancer. Clin Cancer Res.
14:6735-41.
Wehkamp J, Chu H, Shen B, Feathers RW, Kays RJ, Lee SK, Bevins CL. 2006. Paneth cell
antimicrobial peptides: topographical distribution and quantification in human
gastrointestinal tissues. FEBS Lett. 580:5344-50.
Wehkamp J, Harder J, Weichenthal M, Mueller O, Herrlinger KR, Fellermann K, Schroeder
JM, Stange EF. 2003. Inducible and constitutive beta-defensins are differentially
expressed in Crohn's disease and ulcerative colitis. Inflamm Bowel Dis. 9:215-23.
Wehkamp J, Schauber J, Stange EF. 2007. Defensins and cathelicidins in gastrointestinal
infections. Curr Opin Gastroenterol. 23:32-8.
Wershil BK, Furuta GT. 2008. 4. Gastrointestinal mucosal immunity. J Allergy Clin
Immunol. 121:S380-3.
West J, Logan RF, Hill PG, Khaw KT. 2007. The iceberg of celiac disease: what is below
the waterline? Clin Gastroenterol Hepatol. 5:59-62.
West J, Logan RF, Hill PG, Lloyd A, Lewis S, Hubbard R, Reader R, Holmes GK, Khaw
KT. 2003. Seroprevalence, correlates, and characteristics of undetected coeliac disease
in England. Gut. 52:960-5.
Williams SJ, Wreschner DH, Tran M, Eyre HJ, Sutherland GR, McGuckin MA. 2001.
Muc13, a novel human cell surface mucin expressed by epithelial and hemopoietic
cells. J Biol Chem. 276:18327-36.
Wittig BM, Zeitz M. 2003. The gut as an organ of immunology. Int J Colorectal Dis. 18:
181-7.
World Health Organization, Cholera Fact Sheet No. 107, Revised September 2007, Geneva,
Switzerland: World Health Organization
Xiao T. 2008. Innate immune recognition of nucleic acids. Immunol Res. Sep 23. [Epub
ahead of print]
Xu D, Qu CK. 2008. Protein tyrosine phosphatases in the JAK/STAT pathway. Front
Biosci. 13, 4925-32.
Xu GY, Yu HA, Hong J, Stahl M, McDonagh T, Kay LE, Cumming DA. 1997. Solution
structure of recombinant human interleukin-6. J Mol Biol. 268:468-81.
Yang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, Shogan J, Anderson M, Schröder
JM, Wang JM, Howard OM, Oppenheim JJ. 1999. Beta-defensins: linking innate and
adaptive immunity through dendritic and T cell CCR6. Science. 286:525-8.
Yeung MM, Melgar S, Baranov V, Öberg Å, Danielsson Å, Hammarström S, Hammarström
ML. 2000. Characterisation of mucosal lymphoid aggregates in ulcerative colitis:
immune cell phenotype and TcR-gammadelta expression. Gut. 47:215-27.
Yoneyama M, Fujita T. 2007. Function of RIG-I-like receptors in antiviral innate immunity.
2007. J Biol Chem. 282:15315-8.
Yoneyama M, Fujita T. 2008. Structural mechanism of RNA recognition by the RIG-I-like
receptors. Immunity. 29:178-81.
Zebhauser R, Kammerer R, Eisenried A, McLellan A, Moore T, Zimmermann W. 2005.
Identification of a novel group of evolutionarily conserved members within the rapidly
diverging murine CEA family.Genomics. 86:566-80.
Zelensky AN, Gready JE. 2005. The C-type lectin-like domain superfamily. FEBS J. 272:
6179-217
- 70 -
Zhang T, Breitbart M, Lee WH, Run JQ, Wei CL, Soh SW, Hibberd ML, Liu ET, Rohwer
F, Ruan Y. 2006. RNA viral community in human feces: prevalence of plant
pathogenic viruses. PLoS Biol. 4:e3.
Zitzer A, Wassenaar TM, Walev I, Bhakdi S. 1997. Potent membrane-permeabilizing and
cytocidal action of Vibrio cholerae cytolysin on human intestinal cells. Infect Immun.
65:1293-8.
Zitzer A, Zitzer O, Bhakdi S, Palmer M. 1999. Oligomerization of Vibrio cholerae cytolysin
yields a pentameric pore and has a dual specificity for cholesterol and sphingolipids in
the target membrane. J Biol Chem. 274:1375-80.
Öbrink B. 1997. CEA adhesion molecules: multifunctional proteins with signal-regulatory
properties. Curr Opin Cell Biol. 9:616-26.
- 71 -