Linköping University Medical Dissertation No. 1006 Barrier Function of the Follicle‐Associated Epithelium in Stress and Crohn’s disease Åsa Keita Division of Surgery, Department of Biomedicine and Surgery, Faculty of Health Sciences, SE‐581 85, Linköping, Sweden Linköping 2007
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Linköping University Medical Dissertation No. 1006
Barrier Function of the Follicle‐Associated Epithelium in Stress and Crohn’s disease
Åsa Keita
Division of Surgery, Department of Biomedicine and Surgery,
Copyright Åsa Keita pages 1‐105, Paper III and IV. Paper I and II have been
reprinted with the permission from the respective journal. All drawings and
photos are made by the author.
The studies in this thesis were supported by The Swedish Research Council ‐
Medicine (Project 12618), The Swedish Society for Medical Research, The Broad
Medical Research Program of the Eli and Edythe L. Broad Foundation, The Åke
Wiberg Foundation and The ʺLions Forskingsfond mot folksjukdomarʺ.
Printed by LiU‐Tryck, Linköping, Sweden, 2007.
ISBN: 978‐91‐85831‐79‐1
ISSN: 0345‐0082
Även en tusenmilafärd börjar med ett steg
Kinesiskt ordspråk
Till Alpha och Mathilda
ABSTRACT The earliest observable signs of Crohn’s disease are microscopic erosions in the follicle‐associated epithelium (FAE) covering the Peyer’s patches. The FAE, which contains M cells, is specialised in sampling of luminal content and delivery to underlying immune cells. This sampling is crucial for induction of protective immune responses, but it also provides a route of entry for microorganisms into the mucosa. Crohn’s disease is associated with an increased immune response to bacteria, and the disease course can be altered by stress. The overall aim of this thesis was to study the effects of stress on the FAE and elucidate the role of FAE in the development of intestinal inflammation, specifically Crohn’s disease. Initially, rats were submitted to acute and chronic water avoidance stress to study the effects of psychological stress on the FAE. Stressed rats showed enhanced antigen and bacterial passage, and the passage was higher in FAE than in regular villus epithelium (VE). Further, stress gave rise to ultrastructural changes. Subsequent experiments revealed the stress‐induced increase in permeability to be regulated by corticotropin‐releasing hormone and mast cells. Furthermore, vasoactive intestinal peptide (VIP) mimicked the stress effects on permeability, and the VIP effects were inhibited by a mast cell stabiliser. Human studies of ileal mucosa from patients with non‐inflammatory disease and healthy controls showed a higher antigen and bacterial passage in FAE than in VE. In patients with Crohn’s disease, the bacterial passage across the FAE was significantly increased compared to non‐inflammatory and inflammatory controls (ulcerative colitis). Furthermore, there was an enhanced uptake of bacteria into dendritic cells, and augmented TNF‐α release in Crohn’s disease mucosa. Taken together this thesis shows that stress can modulate the uptake of luminal antigens and bacteria via the FAE, through mechanisms involving CRH and mast cells. It further shows that human ileal FAE is functionally distinct from VE, and that Crohn’s disease patients exhibit enhanced FAE permeability compared to inflammatory and non‐inflammatory controls. This thesis presents novel insights into regulation of the FAE barrier, as well as into the pathophysiology of Crohn’s disease by demonstrating a previously unrecognised defect of the FAE barrier function in ileal Crohn’s disease. Keywords: Corticotropin‐releasing hormone, Crohn’s disease, 51Cr‐ EDTA, Escherichia coli, follicle‐associated epithelium, horseradish peroxidase, human, ileum, inflammatory bowel disease, intestinal mucosa, mast cell, M cell, permeability, Peyer’s patches, rat, Ussing chamber, vasoactive intestinal peptide, villus epithelium
LIST OF PAPERS
This thesis is based on the following papers, which are referred to by
their Roman numerals.
I. Increased antigen and bacterial uptake in follicle‐
associated epithelium induced by chronic psychological stress in rats.
Åsa K Velin, Ann‐Charlott Ericson, Ylva Braaf, Conny
Wallon and Johan D Söderholm. Gut 2004; 53:494‐500.
II. Characterization of antigen and bacterial transport in the
follicle‐associated epithelium of human ileum.
Åsa V Keita, Elisabet Gullberg, Ann‐Charlott Ericson,
Sa’ad Y Salim, Conny Wallon, Anders Kald, Per Artursson
and Johan D Söderholm. Lab. Invest. 2006;86:504–516.
III: Increased uptake of non‐pathogenic E. coli via the
follicle‐associated epithelium in ileal Crohn’s disease.
Åsa V Keita, Sa’ad Y Salim, Tieshan Jiang, Ping‐Chang
Yang, Lennart Franzén, Peter Söderkvist, Karl‐Eric
Magnusson and Johan D Söderholm. Submitted
manuscript 2007.
IV. Stress‐induced barrier disruption of the follicle‐
hormone, vasoactive intestinal peptide and mast cells.
Åsa V Keita, Johan D Söderholm and Ann‐Charlott
Ericson. Manuscript 2007.
CONTENTS_____________________________________
1. INTRODUCTION 9
1.1. Crohn’s disease 9
1.1.1. History 9
1.1.2. Epidemiology and symptoms 9
1.1.3. General treatment 11
1.1.4. Aetiology 11
2. BACKGROUND TO THE STUDY 18
2.1. Structure and function of the small intestine 18
2.1.1. The small intestinal wall covered by VE 19
2.1.2. The Peyer’s patches covered by FAE 21
2.3. Intestinal barrier function 27
2.3.1. Permeability 28
2.3.2. Uptake and transport 28
2.3.3. The junctional complex 30
2.3.4. Endocytosis 33
2.3.5. Transcytosis 35
2.3.6. Regulation of endocytosis and transcytosis 37
2.4. Studies of intestinal permeability 38
2.4.1. In vivo 38
2.4.2. In vitro 38
2.4.3. The Ussing chamber 39
2.5. Stress 41
2.5.1. The stress concept 41
2.5.2. Stress and intestinal disease 43
2.5.3. Animal stress models 44
3. AIMS OF THE THESIS 47
4. SUBJECTS AND METHODOLOGY 48
4.1. Animals 48
4.2. Patients 48
4.3. Stress protocol 49
4.4. Permeability studies 50
4.4.1. Tissue preparation 50
4.4.2. Ussing chamber experiments 53
4.4.3. Permeability markers 54
4.5. In vitro co‐culture model of FAE 56
4.6. Immunohistochemistry 57
4.7. Microscopy 58
5. RESULTS 60
6. DISCUSSION 68
7. CONCLUSIONS 77
8. TACK 79
9. SVENSK SAMMANFATTNING 81
10. REFERENCES 82
ABBREVIATIONS 51Cr‐EDTA 51chromium‐EDTA CRH corticotropin‐releasing hormone CRH‐R corticotropin‐releasing hormone receptor E. coli Esherichia. coli FAE follicle‐associated epithelium HRP horseradish peroxidase IBD inflammatory bowel disease Isc short circuit current M cell membranous or microfold cell NK‐1R neurokinin‐receptor 1 PD transepithelial potential difference SED subepithelial dome TER transepithelial electrical resistance VE villus epithelium VIP vasoactive intestinal peptide VIPR vasoactive intestinal peptide receptor WAS water avoidance stress
SUPERVISORS
Johan Dabrosin Söderholm, Associate Professor of Surgery, Division of Surgery, Department of Biomedicine and Surgery, Faculty of Health Sciences, SE‐581 85, Linköping, Sweden Ann‐Charlott Ericson, Associate Professor of Cellbiology, Division of Cellbiology, Department of Biomedicine and Surgery, Faculty of Health Sciences, SE‐581 85, Linköping, Sweden
1. INTRODUCTION
1.1. Crohn’s disease
1.1.1. History
Clinical descriptions of gastrointestinal disease resembling Crohn’s
disease go back to the 16th century 1, when G. F. Hildenus noted during
an autopsy of a boy suffering from abdominal pain and diarrhoea, that
the ulcerated cecum was contracted and ivaginated into the ileum.
Similar reports during the 16th‐19th centuries indicated the appearance of
a unique intestinal inflammatory disease that today would be identified
as Crohn’s disease. Although Dalziel already published a paper in 1913
of a series of patients with granulomatous small bowel inflammation 2, it
is the report by Crohn, Ginzburg and Oppenheimer at Mt Sinai Hospital
in New York that is considered as the original description 3. In this classic
paper, Crohn and his colleagues describe a condition of abdominal pain,
emaciation, diarrhoea and fever. Originally, Crohn himself named the
disease terminal ileitis since it was believed to be strictly localised to the
small bowel. However, criticism was raised that the disease could also
occur at other locations, and the name was changed to regional enteritis
in the publication 3. Today it is well established that Crohn’s disease is a
chronic episodic, inflammatory disease that can affect the entire
gastrointestinal tract, from the mouth to the anus, however, the ileocaecal
region of the bowel is most commonly affected.
1.1.2. Epidemiology and symptoms
Crohn’s disease is a Western world disease with the highest incidence
rates in Scandinavia, Great Britain and North America 4. The disease has
a slightly female predominance and onset at young adulthood with a
peak incidence between 15‐30 years. In 1991, the incidence in Sweden
9
was 6.1 in 100 000 per year and the prevalence was 146 in 100 000 5. Since
this date, no further epidemiological studies in Sweden in general have
been reported, however, a recent study showed that the incidence in
Stockholm between 1990 and 2001 was 8.3 in 100 000 per year and the
prevalence in the 1st of Jan 2002 was 213 in 100 000 6. Together with
ulcerative colitis, Crohn’s disease constitutes the main condition of
inflammatory bowel diseases (IBD).
The symptoms of Crohn’s disease are dependent on the location of the
inflammation but abdominal pain, diarrhoea, weight loss, fever and
vomiting are common features. The presence of abdominal and perianal
fistulae are typical for the disease.
It is not fully understood how Crohn’s disease is initiated, however,
studies have shown that the first observable signs of the disease are ileal
aphtoid lesions, well recognised by endoscopy 7. These lesions have
shown to progress over time to larger ulcerations and stricturing of the
lumen 8. Initially, it was observed that the lesions mainly occur at the
lymphoid follicles 7. It has been shown that they can vary in size from
barely visible to 3 mm in diameter. They are found in 70 % of the Crohn’s
disease patients 9, and most commonly in the clusters of lymphoid
follicles called the Peyer’s patches of the distal ileum 8‐10. Further,
magnifying endoscopy and scanning electron microscopy have been used
to demonstrate that the aphtoid lesions of Crohn’s disease are preceded
by 150‐200 μm sized ultra‐structural erosions of the epithelium covering
the Peyer’s patches, the so called follicle‐associated epithelium (FAE) 11.
The early inflammation in Crohn’s disease is often located at the distal
ileum 7, where Peyer’s patches are more frequent 12. Taken together, these
observations suggest that the lymphoid follicles are the sites of initial
inflammation in ileal Crohn’s disease, where the ulcerations originate
10
from small erosions over the FAE. The FAE and Peyer’s patches are
further discussed in paragraph 2.1.2.
1.1.3. General treatment
In the report from 1932, Crohn et al. proposed resection of the diseased
segment as a cure, and for a long time, radical bowel resection was the
only treatment. However, the development of anti‐inflammatory drugs in
the 1950’s, and increased knowledge about the disease as a panenteric
disorder has lead to a more restrictive surgical approach 13.
The treatment of Crohn’s disease is unsatisfactory, since none of the
existing treatments such as 5‐aminosalicylates, corticosteroids,
immunomodulators (e.g. azathioprine and methotrexate) or surgery, are
curative. Although these treatments have a positive effect on most
patients, the occurrence of relapse is high.
An example of a newer biological medication is infliximab (Remicade®)
that induce remission of the disease by antibodies against TNF‐α 14.
Infliximab and other new immunomodulators are utilised with the goal
of keeping the disease in remission and there is very little evidence that
these treatments alter the natural history and disease course 15.
1.1.4. Aetiology
The exact cause of Crohn’s disease is unknown but evidence shows that
genetic, immunological and environmental factors all contribute to the
pathogenesis of the disease 14;16 (Fig. 1).
11
Fig. 1. Pathogenesis of Crohn’s disease.
Genetics
Epidemiological studies have shown that ethnic background and family
history are of importance in the susceptibility of Crohn’s disease. First
degree relatives of patients with Crohn’s disease show a 10 to 30 times
increased risk of acquiring the disease 17. A number of studies have
reported that 10‐15 % of first degree relatives have increased intestinal
permeability in the absence of clinical symptoms 18‐20, and approximately
30 % have increased intestinal permeability compared to controls, after
ingestion of acetylsalicylic acid 21. Furthermore, twin studies have
demonstrated a higher pair concordance rate in monozygotic twins with
Crohn’s disease, than in dizygotic twins 22.
In 2001, the first susceptibility gene for Crohn’s disease, CARD15/NOD2,
was identified on chromosome 16 (IBD1) 23;24, and since then, the results
have been widely replicated 25. Whether the Crohnʹs‐associated CARD15
mutations lead to a loss or gain of function of the NOD2 receptor is
subject to controversy 25, and by which mechanisms this change in
function might increase the susceptibility to Crohn’s disease is still under
investigation 26. A recent study showed that high mucosal permeability in
12
healthy first degree relatives is associated with the presence of CARD15
3020insC mutation, indicating that genetic factors may be involved in
impairment of intestinal barrier function in families with IBD 27. Since
CARD15/NOD2 variants only seem to account for 10‐30 % of Crohn’s
disease patients 28, several groups have focused on finding other
candidate susceptibility genes associated with the disease. Additional
putative loci have been mapped to chromosome 5 (OCTN gene), 6 (IBD3),
Fig. 9. Uptake of extracellular material via different types of endocytosis.
R = receptor.
33
Endocytosis occurs in enterocytes of both VE and FAE, however, it is well
known that endocytosis of bacteria and particles primarily occur via the
M cells in the FAE 147.
The first route, present in both enterocytes and M cells, is via clathrin‐
mediated endocytosis 148‐150, a highly specific receptor‐mediated process,
utilised mainly by immunoglobulins, viruses and growth factors from
breast milk. The clathrin‐coated vesicles seldom become larger than 150
nm in diameter 151. In this special type of endocytosis the cells synthesise
receptors and internalise molecules that have bound specifically to them 152.
Larger (up to several μm in size) bacteria, viruses, and particles are taken
up via an adsorptive endocytosis, or phagocytosis 153, involving binding
of molecules to the cell membrane via receptors. Phagocytosis is relevant
for the non‐specific uptake of luminal dietary and bacterial antigens, and
the process is triggered by secreted solubles from the invading bacterium 154. Phagocytosis is a more common process in M cells than in enterocytes 109;155‐157.
Both enterocytes and M cells are capable of actin‐dependent non‐specific
fluid‐phase endocytosis, or macropinocytosis, where substances in the
luminal fluid are internalised 153. The process resembles phagocytosis, but
is not receptor‐mediated 152. For example, the protein antigen horse‐
radish peroxidase (HRP) is known to be taken up via macropinocytosis,
preferentely via M cells 158;159, but the exact way how HRP is sorted and
transported after endocytosis is not fully elucidated.
In recent years, attention has been paid to a fourth mechanism, referred
to as lipid rafts / caveolae. This endocytotic event involves a flask‐shaped
34
invagination of cholesterol‐enriched microdomains within the plasma
membrane that may contain a coat protein, caveolin 160. Endocytosis via
lipid rafts / caveolae is most common in endothelial cells but occurs also
in enterocytes, although this type of endocytosis is rare in M cells as they
contain few to no caveolae 161. Studies have shown that for example
certain enterotoxins and viruses are endocytosed via rafts /caveolae. In
addition, the endocytosis of occludin discussed in paragraph 2.3.3 has
shown to occur via caveolae‐mediated endocytosis 135.
2.3.5. Transcytosis
Following endocytosis, uptaken molecules must be transported through
the cells via transcytosis. For this, enterocytes and M cells have different
systems.
Enterocytes
Enterocytes have apical and basolateral sorting compartments, so called
“apical early endosomes” and “basolateral early endosomes”, that share
a “common recycling compartment” 162. These compartments are used
during clathrin‐mediated endocytosis and phagocytosis. In enterocytes,
transcytosis can occur in three ways.
1) When vesicles bud off from the apical membrane, they can merge with
the apical early endosomes and then be recycled back to the apical
membrane, with or without cytoplasmic release of their content. The
content (protein, virus or particle) bound to the internalised receptor is
most often released into the cytoplasm upon acidification of the vesicles,
while the receptor is delivered back to the cell membrane. However,
large molecules like peptides and proteins may be degraded on their way
to the basolateral membrane since they diffuse rather slowly through the
cytoplasm.
35
2) The vesicle can join with the common recycling compartment and from
there, the content (protein, virus or particle) is often directed into
pathways leading to lysosomal degradation in lysosomes.
3) The vesicle can be transported from the apical to the basolateral side
for subsequent merging with vesicles from the basolateral early
endosomes, although this is a quite rare process compared to the others
described 152;162;163.
Transport of intact proteins or carrier‐mediated systems across
enterocytes is not easy to achieve, however, studies have demonstrated
that the endosomal sorting mechanisms can be modified in order to
decrease apical vesicle recycling, thereby increasing the transcytosis of
proteins across the epithelium 164.
It is known that enterocytes not only transport internalised antigen, in
addition they can, during chronic inflammatory diseases such as IBD and
celiac disease, act as non‐professional antigen‐presenting cells and
promote inflammation 165‐167.
M cells
Endocytosis via the FAE and M cells is well characterised, however, the
transcytosis and fate of internalised content have not been very well
studied 168. M cells only contain few lysosomes 156 and can not express
MHC‐II, consequently they can not function as true antigen‐presenting
cells 166. It is known that the apical part of the M cell cytoplasm contains
several endosomes and vesicles with lysosomal markers on the surface 149;156. Ultrastructural studies have demonstrated that soluble tracer
proteins infused into the lumen are incorporated into the membrane
vesicles of the M cells and rapidly transported across the narrow bridge
36
of the apical cytoplasm, and released by exocytosis into the sequestered
intraepithelial space 158.
2.3.6. Regulation of endocytosis and transcytosis
Both endocytosis and transcytosis can be influenced by numerous factors.
Although the mechanisms are not fully elucidated, bacterial exposure in
one way or another leads to enhanced uptake and transport across the
intestinal epithelium 169. Bacterial stimulation also leads to the production
of pro‐inflammatory cytokines, increasing endocytosis and transcytosis.
For example, TNF‐α has shown to induce HRP endocytosis in intestinal
epithelial cell culture 170;171, and increased transcytosis of HRP could be
correlated to TNF‐α mRNA levels in the underlying mucosal tissue 170.
Another factor affecting epithelial uptake is intestinal disease, in which
dysfunctional intestinal motility can prolong the exposure time to
luminal bacteria. Furthermore, studies have shown that antigen‐binding
speeds up the transcytosis. For example, when conjugating HRP to IgE,
the protein was carried across the epithelial membrane into the lamina
propria within three minutes compared with hours for unconjugated
HRP 165.
In FAE, the size and number of Peyer’s patches and M cells are of
importance for endocytosis and subsequent transcytosis. Smith et al
reported that the number of M cells increased after transfer of germ‐free
mice to normal housing conditions 172. Subsequently, several other
studies have shown an increased number of M cells and enhanced
particle uptake after bacterial stimulation 99;101;173;174. However, Gebert et al 169 recently found that the enhanced uptake seen after bacterial
stimulation depends on increased transport capacity of the M cells
already present in the FAE, and not an increase in numbers. In addition,
intestinal inflammation may increase the M cell numbers. For example,
indomethacin‐induced ileitis in rats increases the M cell number and
37
apoptosis, which may alter the intestinal barrier function 90. Moreover, an
increased number of M cells has been found in ileal mucosa of patients
with spondylarthropathy, and the observation of M cell cytoplasm
disruption, with lymphocytes entering the gut lumen at these sites,
suggest a possible mechanism for the development of aphtoid ulcers 175.
2.4. Studies of intestinal permeability
2.4.1. In vivo
Intestinal permeability in vivo in humans was first studied using
intestinal infusion of solutes 176. Today, intestinal barrier function is
mainly studied as the urinary or blood excretion of orally ingested
markers. Obviously, the characteristics of the permeability probes are of
high importance and the ideal probe should be water soluble, non‐toxic,
nondegradable, and not be metabolised. Moreover, the probe should
ensure complete urinary excretion and the analysis should be sensitive,
accurate and uncomplicated. There is, of course, no probe that fulfils all
these criteria, but some probes are close and consequently used for
permeability studies. The most commonly used small pore markers (5‐8
Å) are monosaccharides (mannitol, rhamnose), and polyethylene glycols
(PEG), with molecular weight around 400 Da. The most frequently used
large pore markers (9.5‐11 Å) are disaccharides (lactulose, cellobiose), 51chromium‐EDTA (51Cr‐EDTA), and PEG, with molecular weight around
1000 Da. Permeability is usually presented as the ratio between the large
pore and small pore marker.
2.4.2. In vitro
The current in vivo methods for permeability studies of the human
intestinal mucosa cannot elucidate passage routes and mechanisms
involved in barrier function in IBD. In addition, there are considerable
38
difficulties with in vivo studies of intestinal uptake of intact protein 177. In
vitro techniques offer possibilities to study processes in human tissue that
would be impossible to study in vivo, and much of the basic knowledge
of gastrointestinal physiology has been achieved through in vitro
techniques. Several techniques for in vitro studies of intestinal mucosa
have been developed, and one of them is mucosal sheets in Ussing
chambers 178;179. The good viability‐supporting possibilities with
oxygenation and effective circulation of the fluid on both sides of the
tissue, combined with the possibility to monitor membrane
electrophysiological parameters, provide the Ussing chamber technique
with important advantages compared to other in vitro techniques for
intestinal tissue experiments 180.
2.4.3. The Ussing chamber
The Ussing chamber was first described in 1951 by the Danish
physiologists Ussing and Zerhan 181. The Ussing chamber technique has
many applications, but mostly it is used to study ion transport, drug
absorption, protein absorption, and studies of several pathophysiological
processes in both animals and humans 179;180;182;183. The initial methodology
of the Ussing chambers, that was rather complicated, was modified and
simplified by Grass et al in 1988 184. The principle is that a flat sheet of
mucosa is mounted between two half‐chambers filled with continuously
oxygenated buffer (Fig. 10). The arrangement of the gas ports provide
buffer circulation, which gives efficient mixing of the fluid and reduces
the thickness of the unstirred water layer to physiological levels 185. The
chambers are kept at 37°C and two pairs of electrodes enable the
monitoring of electrophysiological parameters during the experiment.
The marker solution is added to the mucosal buffer, and at defined time
intervals samples are redrawn from the serosal buffer as a measurement
of passage.
39
A B C
D
IPt
PDAg/AgCl
Mucosa
Gasinlet
BuffersolutionMarker
solution
Fluidcirculation
Platinum-electrodes
Ag/AgCl-electrodes
Samplecollection
A B C
D
IPt
PDAg/AgCl
Mucosa
Gasinlet
BuffersolutionMarker
solution
Fluidcirculation
Platinum-electrodes
Ag/AgCl-electrodes
Samplecollection
Fig. 10. The Ussing chamber. (A‐B) Schematic drawing and photograph of the
Ussing chamber. Tissue is mounted between the two chamber halves and is
continuously oxygenated. One pair of Ag/AgCl‐electrodes enables measurement
of potential difference and one pair of platinum‐electrodes gives current to the
system. (C) Tissue is carefully mounted so it covers the entire surface area of the
opening that connects the two chamber halves. (D) After mounting, chambers
are filled with buffer and put in the 37°C Ussing chamber system.
The preparation techniques of intestinal tissue for the use in Ussing
chambers depend on the species and bowel segment being studied 180.
Generally, the intestinal specimens are dissected and immediately
transported to the laboratory, in oxygenated buffer without circulation.
Tissues are either unstripped, or stripped, which means dissection of the
muscle layers. In unstripped bowel, the whole bowel wall is intact, and it
is possible to evaluate effects of the enteric nerves on intestinal tissue.
However, unstripped segments are not that often used in permeability
studies because of the longer diffusion distance for the probe molecules.
In stripped tissues, the external muscle layers and myenteric plexus are
removed and the mucosa mounted thus consists of the epithelium, the
underlying lamina propria and the muscularis mucosae. After mounting
40
in the chambers, tissues are equilibrated for 20‐60 minutes to achieve
steady state conditions in transepithelial potential difference (PD).
Electrophysiology
A characteristic for all transporting epithelia is the ability to maintain a
PD. The ability depends on all the electrogenic ion pumps activity in the
epithelial cell membrane, mainly Na+ / K+‐ATPase, and on the epithelial
barrier function 186. Theoretically these two can be separated into the
short circuit current (Isc) and TER. The Isc represents the current needed
to nullify the PD and is a function of the ion pumps activity. The TER
reflects the electrical resistance of the paracellular routes, mainly via the
tight junctions. The electrodes enabling the monitoring of
electrophysiological parameters during the experiments consist of one
pair of Ag / AgCl‐electrodes with agar‐salt bridges and one pair of
current‐giving platinum electrodes. Since active ion transport requires
energy production, the basal PD or Isc can be used as a measure of tissue
viability. By passing the current (I) through the epithelium, the change in
PD can determine the TER by Ohm’s law, PD = I x R. However, this
calculation relies on a simplified epithelial model, viewing the epithelium
as a parallel circuit consisting of parcellular and transcellular pathways.
2.5. Stress
2.5.1. The stress concept
Stress is a normal part of life and has been defined in many different
ways 187, for example as “any threat to the homeostasis of an organism” 188 or “the range of tensions of modern life” 189. An adequate stress
response is essential for survival, but the ways of coping with stress is
highly individual 190. Under normal circumstances, physiological systems
are turned on and off in response to stress, matching the duration and
41
severity of the stressors, a so‐called adaptive response. However, in some
individuals stress may become harmful and cause damage to the
organism, a so‐called maladaptive response, also referred to as
pathologic stress. Maladaptive responses are often associated with
chronic daily life stressors such as losses, financial problems,
unemployment, and have been linked to exacerbations of irritable bowel
syndrome symptoms 191.
Regardless if the threat is real (physical), perceived (physiological) or
environmental, the principal stress responses triggered to maintain
homeostasis are quite similar 192. Normally, the stress response
constitutes of a behavioural response (e.g. anxiety), an autonomic
response (e.g. raised heart rate), and a hypothalamic‐pituitary‐adrenal
mithochondrial swelling and initiation of inflammation 202‐206;208;210;211. With
this in mind, we decided to submit rats to acute and chronic
physiological stress to evaluate the effects on FAE. Our results revealed
that the FAE barrier was sensitive to stress, shown by changes in
electrophysiology and increased uptake of luminal antigens and bacteria.
Hereby we could, for the first time, demonstrate an enhanced
permeability in FAE after stress, and also higher permeability increase in
FAE compared to VE.
In a follow‐up study we wanted to confirm the increased FAE
permeability in humans. Results showed a considerably more
pronounced bacterial and antigen uptake also in human FAE, compared
to VE. Since we could show that stress increased FAE permeability, and
68
stress is a contributing factor to IBD and Crohn’s disease 63;64, we
speculated that there could be an altered FAE function in patients with
Crohn’s disease. Our results showed that patients with Crohn’s disease
displayed an enhanced bacterial passage in FAE compared to non‐
inflammatory and inflammatory controls.
In the present studies, we found an enhanced antigen and bacterial
permeability in FAE compared to VE in both rats and humans. In
addition, we found that stress further increased the permeability in FAE.
The HRP passage was increased almost four times in FAE after stress
compared to 1.8 times in VE, and bacterial uptake increased 30 times in
FAE after stress, as compared to only 1.6 times in VE. This suggests that
FAE is more vulnerable to stress than VE, and it might be speculated that
a major part of the bacterial translocation that occurs in stress may be due
to invasion across the FAE. When comparing the permeability in Crohn’s
patients with that in non‐inflammatory controls, a higher bacterial
permeability in FAE was seen. However, the HRP passage was similar,
suggesting different mechanisms of the FAE barrier dysfunction during
stress and inflammation.
The higher passage in FAE compared to VE in the normal situation
probably refers to the different surface characteristics 81, making FAE
more accessible for luminal antigen and bacteria. In addition, the
presence of M cells within the FAE contributes to the increased
permeability 80. However, the fact that the FAE permeability increases
after stress, and is enhanced in Crohn’s disease, indicates that something
occurs in the epithelium that further facilitates for luminal antigens and
bacteria to cross the barrier. Since a higher M cell number has been found
during inflammation 90;175, the increased permeability in Crohn’s disease
could, at least partly, be due to an increased M cell number within the
FAE. One could further speculate that the numbers are also increased
69
after chronic stress, which could explain the enhanced permeability.
Thus, it would have been highly interesting to verify the M cells number
in that FAE tissue investigated in our studies, but as already mentioned,
there is no reliable rat or human M cell marker, which hampers the
possibility to identify the M cells present in our FAE tissue investigated.
Contradictory to the hypothesis that the increased permeability is due to
M cells in the FAE, is one paper 236 showing an M cell independent
mechanism. In a co‐culture model of mice dendritic cells and human
epithelial cells, it was shown that dendritic cells directly can sample
luminal antigen by unzipping tight junctions and extending their
dendrites through the intact epithelium. However, this observation has
not yet been reproduced in human tissue.
The importance of FAE in permeability might be questioned due to its
smaller surface area compared to VE. The VE contains numerous folded
villi that increase the surface area considerably compared to FAE.
However, the smaller surface area is highly compensated by the larger
capacity of the FAE cells to sample luminal content, and the importance
of the underlying Peyer’s patches for initiation of immune responses.
When antigen and bacteria enter the FAE and M cells, they are
transported across the cells, and delivered to antigen‐presenting cells in
the Peyer’s patches to induce inflammatory responses. Lymphocyte‐
containing M cell pockets decrease the travelling distance from the apical
to the basolateral side, which further improves the passage in FAE. In VE,
the major part of entering content is degraded on their way through the
cells via for example lysosomal events, and only a minor amount reaches
all the way through intact.
From our results it may be suggested that in Crohn’s disease, antigen and
bacteria enter the FAE barrier at a higher rate due to a disturbed barrier
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function. The question is whether the disturbed barrier is caused by a
primary epithelial defect, or if it is the other way around and the
increased permeability is a consequence of an early immune activation.
In the first theory, it could be speculated that patients with Crohn’s
disease have a genetically driven disrupted barrier function leading to
increased uptake of luminal antigens and bacteria, which are
phagocytosed and presented to immune cells that starts to produce
inflammatory mediators. After secretion, cytokines and chemokines
attract other inflammatory cells that start to produce even more
inflammatory mediators. Finally this could lead to further destruction of
the epithelial cells, by the inflammatory mediators, affecting epithelial
defence and control of permeability. This theory is strengthened by
studies showing increased permeability in healthy relatives of patients
with Crohn’s disease 18;20;21.
The second theory says that immune cell activation and inflammation
precede the barrier disruption. The importance of immune cell activation
in barrier dysfunction has been confirmed by several studies. For
example TNF‐α and IFN‐γ have shown to increase both paracellular and
transcellular permeability 170;237;238 and studies have shown that the barrier
dysfunction in Crohn’s disease is TNF‐α‐dependent 170;239;240. Furthermore,
it has been demonstrated that monocytes from patients with Crohn’s
disease secrete TNF‐α upon bacterial stimulation 140, and mast cells of
Crohn’s disease mucosa are major producers of TNF‐α 241;242. A study
speaking in favour of inflammation coming first is that 54 % of healthy
relatives to Crohn’s disease patients have subclinical inflammation,
measured as levels of calprotectin 243. Moreover, in a rat model of
intestinal inflammation it was shown that the rats developed
inflammation without getting increased permeability 244.
Both of the theories make sense. It could be that in Crohn’s disease the
epithelial barrier is initially to some extent disrupted, for example due to
71
an abnormal response to bacteria. This leads to increased passage,
immune activation and subsequently elevated levels of for example TNF‐
α, IFN‐γ and IL‐4 142;170;237;238 that further destroy the barrier and increase
the permeability.
The importance of the luminal microflora in Crohn’s disease is well
documented 41;42. For example studies have shown increased number of
adherent‐invasive E. coli in Crohn’s disease mucosa 43;44. The significance
of bacteria in intestinal inflammation is further strengthened by the fact
that rats susceptible for intestinal inflammation did not develop IBD
when bread under germfree conditions 49.
The novel findings in Paper III, an increased uptake of non‐pathogenic
bacteria in FAE despite an equal permeability to 51Cr‐EDTA and HRP,
point to the importance of FAE in Crohn’s disease and highlight the
importance of FAE‐bacterial interactions leading to inflammation. Since
the FAE tissues of patients with ulcerative colitis showed no increase in
bacterial passage, the enhanced bacterial transport may be specific to the
FAE of Crohn’s disease. What are the mechanisms behind this increase?
Possible explanations could be the involvement of receptors, referred to
as pattern‐recognition receptors (PRR), that recognise the so called
pathogen‐associated molecular patterns (PAMPS), such as
lipopolysaccharide (LPS) and peptidoglycans, 245 The two major groups
of PRRs are the NOD‐containing proteins and toll‐like receptors (TLRs).
The best characterised NOD proteins relevant to the intestinal physiology
are NOD1 and NOD2, where NOD2 recognises structures in a wider
range than NOD1, and is more restricted to the small intestine. Although
several potential signalling pathways involved in inflammation and
innate immunity linked to NOD2 activation have been demonstrated, the
physiologic functions are less well understood. Under normal conditions
the NOD2 expression is low, however, during inflammation the
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expression increases 246, which could suggest a role for altered microbial
sensing in the increased bacterial uptake in Crohn’s disease. Mutations in
the NOD2 gene lead to inhibited activation of the NOD2 protein,
however, since none of the Crohn’s disease patients included in our
study carried the mutation, one can presume that the NOD2 protein was
unaffected.
TLR‐4 is the best studied TLR and is essential for the recognition of LPS,
and further limiting LPS responsiveness. A TLR‐4 polymorphism and
increased apical expression of TLR‐4 has been found in both Crohn’s
disease and ulcerative colitis 247;248. The overexpression of TLR‐4 could
result in LPS hyper‐responsiveness leading to consecutive
proinflammatory cytokine secretion which in turn stimulates TLR‐4
expression and further inappropriate signalling in the presence of
luminal LPS. It is known that TLR‐4, is upregulated in FAE and M cells of
mice 249, and may be involved in regulation of uptake of bacteria and
microparticles 250. Therefore, the role of TLR‐4 in the FAE dysfunction in
Crohn’s disease should be elucidated in further studies.
Another way in which TLRs are involved in regulation of tolerance to
commensal bacteria is through their effect on dendritic cells. After
sampling of luminal content, dendritic cells migrate across the epithelium
and by stimuli from bacteria via TLR signalling, maturation is induced 236;251. Dendritic cells are located in the SED for phagocytosis and
presenting of antigen 94;252. It has been shown that they can squeeze in
between epithelial cells through the tight junctions to sense and sample
luminal microbes 236. Our findings in Paper III of increased bacterial
uptake into dendritic cells in Crohn’s disease mucosa suggest that
dendritic cells may play an active role in the immune cell‐bacterial
interaction leading to inflammation in Crohn’s disease.
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Our results, demonstrating a disrupted FAE barrier function after stress
in rats gave rise to Paper IV where we wanted to elucidate the role of
mast cells and neuropeptides in the increased uptake. The significance of
mast cells in stress‐induced mucosal dysfunction of VE has been clearly
demonstrated by several groups 202;204;211;212, and we could also confirm
this in Paper I, where we observed a 3‐fold increase in mast cell number
in stressed rats compared to controls. Numerous studies have
highlighted the role of CRH in stress 207;215;216, and some of them have
demonstrated inhibited effects of CRH when blocking the mast cells,
however, the importance of mast cells and CRH in stress‐induced FAE
permeability has not been reported. Our results showed inhibited stress
effects when blocking the receptors for CRH, and mimicked stress‐effects
on permeability when exposing FAE to CRH peptide in Ussing chambers.
In addition, when stabilising the mast cells with doxantrazole, the effects
of both stress and CRH were inhibited, which points to that CRH is
released during stress and increases permeability via activation of mast
cells.
In addition, we could, in Paper IV, for the first time, identify VIP as a
regulator of mucosal permeability. FAE exposure to VIP in Ussing
chambers resulted in increased paracellular permeability and
transcellular passage to antigen and bacteria. Furthermore, we found VIP
peptide and VIP receptors both in and close to mast cells at follicle
margins and in adjacent villi. The involvement of VIP in permeability has
previously only been described briefly. In a co‐culture model of human
submucosa containing the submucosal neuronal network and human
polarised colonic monolayers, the paracellular permeability was
increased by electrical stimulation of submucosal neurons 218. The effects
of the neuron activation were blocked by a VIP receptor antagonist, and
reproduced by VIP peptide. This, together with our results regarding
74
VIP‐induced increased permeability, suggest an important modulatory
role for VIP in the regulation barrier function.
In addition to the importance of mast cells and neurons during stress,
their roles in IBD have also been highlighted. Degranulation of mast cells,
and increased numbers, have been found in mucosa from patients with
ulcerative colitis 253 as well as Crohn’s disease 254. Moreover, elevated
levels of histamine have been measured in gut lumen of Crohn’s disease
patients 255.
There are only a few studies present regarding the consequences of
neuronal changes on mucosal function in IBD. However, changes in
neuropeptide innervation 256, and altered VIP 257;258 and substance P 259;260
expressions, have been found in mucosa from patients with ulcerative
colitis and Crohn’s disease. There are no reports on immunoreactivity to
CRH in Crohn’s disease, however, in mucosal inflammatory cells of
ulcerative colitis, increased CRH expressions have been observed 261.
Even if our results demonstrate a role of VIP in FAE barrier function
during stress, it can not be directly applied to the permeability changes
seen in FAE of Crohn’s disease. However, several studies have shown a
link between stress and IBD 191;194;199;200, and the fact that a disrupted
barrier function can be induced in healthy rats, by submitting them to
stress, suggests that stress may be implicated in the initiation,
perpetuation, or exacerbation of the inflammation seen in IBD.
Consequently, it could be speculated that VIP is important also in
regulating the permeability in Crohn’s disease, not at least considering
the increased VIP expression found in Crohn’s disease mucosa. However,
further studies are needed to elucidate the role of VIP in FAE barrier
function of Crohn’s disease.
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In conclusion, this thesis presents novel insights into regulation of the
FAE barrier, as well as into the pathophysiology of Crohn’s disease by
demonstrating a previously unrecognised defect of the FAE barrier
function in ileal Crohn’s disease. Further studies in a rat IBD model
would be highly valuable to better understand the mechanisms involved,
and the role of FAE in the interplay between stress and intestinal
inflammation.
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7. CONCLUSIONS
• The FAE barrier can be modulated by stress as illustrated by
enhanced antigen and bacterial passage and ultrastructural
changes after acute and chronic stress in rats.
• The more pronounced increase of transmucosal passage in FAE
compared to VE after stress suggests that FAE is more stress‐
reactive and prone to interact with antigen and bacteria
• Human FAE in biopsies and surgical specimens can be identified
by transilluminating the mucosa from below in dissection
microscope, and the techniques can be considered equal
regarding permeability measurements in Ussing chambers.
• Human FAE exhibits a substantially higher antigen and bacterial
passage compared to VE. In vitro experiments revealed an actin‐
dependent passage of E. coli and HRP uptake via
macropinocytosis.
• Patients with Crohn’s disease demonstrate a higher transmucosal
uptake of E. coli K‐12 and HB101 compared to non‐IBD controls
and patients with ulcerative colitis, suggesting that the FAE
barrier is altered in Crohn’s disease by disease‐specific
mechanisms.
• Following bacterial uptake, patients with Crohn’s disease reveal
a higher percentage of E. coli internalised by dendritic cells
compared to non‐IBD controls.
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• The stress‐induced increase in FAE permeability seen in rats is
regulated by CRH and mast cells. Further stress effects on
permeability can be mimicked in vitro by VIP, and the effects are
abolished by blocking the mast cells.
• In rats, mast cells are present mainly in the adjacent villi, but also
in follicles and SED. The neuropeptides CRH and VIP and their
receptors are expressed in mast cells within these regions.
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8. TACK Det finns många personer som på ett eller annat sätt hjälpt och stöttat mig i mitt avhandlingsarbete. Av dem vill jag framförallt tacka; Johan D Söderholm, min huvudhandledare, som gav mig chansen att göra detta. Ditt positiva tänkande och uppmuntran har gjort att det hela tiden har känts roligt att forska! Ann‐Charlott ʺLottaʺ Ericson, min biträdande handledare, som enträget har suttit med mig vid de olika mikroskopen trots att det ibland känts hopplöst. Men Murphy’s lag till trots så löste vi alla tekniska problem och hade riktigt roligt på kuppen. Alla medförfattare, speciellt tack till Elisabet Gullberg och Per Artursson för trevligt och givande samarbete. Ylva Braaf, för ovärderlig hjälp i Ussinglabbet, ibland kan man undra vem som blev mest stressade, vi eller råttorna? Eva Sjödahl för mycket värdefull hjälp med histologi. Conny Wallon för att du hjälpte mig så mycket i början och för hjälp med insamling av patientmaterial. Ida Schoultz, min kollega och med tiden mycket goda vän, för ditt stora stöd och uppmuntran. Utan dig på jobbet hade allt varit mycket tråkigare! Tack också för att du släpat ut mig till fikarummet ibland när jag inte kunnat slita mig från datorn, de avbrotten har nog varit mer än välbehövliga... Sa’ad Y Salim, my colleague and dear friend, for always helping me out whenever I need it, especially during my maternity leave. Thanks also for your encouragement and for always being such a good friend. Femke Lutgendorff och Johan Junker, mina rumskompisar under senare delen av avhandlingsarbetet, och mina rumskompisar under den tidigare delen, Henrik Blomqvist och Hanna Olausson, för att ni bidragit till trevliga stunder på kontoret och diskussioner både vad gäller forskning och fritid. Lena Trulsson, min kollega och goda vän, för intressanta diskussioner och goda råd både vad gäller forskning och annat i livet. Tack även till Staffan Smeds för att du inspirerade mig till att själv börja forska och ge mig in i den här världen.
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Alla andra på KEF som hjälpt mig, framförallt Pia Karlsson som sett till att jag kan köra mina försök dag efter dag utan att behöva lägga tid på disk och beställningar, samt Håkan Wiktander, Iréne Cavalli‐Björkman och Kerstin Hagersten för all god service! All personal på Djuravdelningen, särskilt tack till Dan Linghammar och Jan Szymanowski för praktisk hjälp och goda råd. All personal på Kirurgen som på olika sätt har hjälpt mig i mitt forskningsarbete. Framförallt alla kirurger, ingen nämnd ingen glömd, för hjälp med insamling av operationspreparat, och sköterskor för assistans vid biopsiprovtaging, och sist men inte minst Britt‐Marie Johansson och Ulla Svensson Bater för hjälp med många praktiska saker genom åren. Tack även till alla patienter och friska frivilliga för att ni har ställt upp med material till min forskning! Personal på Patologen som har tagit sig tid att hjälpa till vid utskärning av operationsmaterial. Rakel Martinsson, Markus Jansson, Robert Berglund, och Tina Betmark, mina studenter, som under examensarbeten har hjälpt till med delar av det laborativa. Alla mina vänner för att ni har stått ut med mig under våren. Jag lovar att ta igen alla inställda luncher och träffar när detta är över :o) Alpha, min själsfrände, för allt ditt tålamod med mig senaste tiden då jag bara jobbat, jobbat och jobbat. Utan all din hjälp och ditt stöd hade jag aldrig orkat. Det här är din avhandling lika mycket som min. Tack även till dig Mathilda, min lilla solstråle, för att du finns, och ger mig perspektiv på livet. Min övriga familj, framförallt mamma, pappa och svärmor för all hjälp med Mathilda när inte tiden räckt till. Det är inte helt lätt att skriva en avhandling med en liten i knäet, även om det faktiskt tidvis gått det också….
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9. SVENSK SAMMANFATTNING Crohns sjukdom är en kronisk inflammatorisk tarmsjukdom av okänd orsak. Det tidigaste tecknet på Crohns sjukdom är mikroskopiska sår i det s.k. follikelassocierade epitelet (FAE) som täcker ansamlingar av immunceller i tarmen. FAE är specialiserat för att fånga innehåll från tarmen och transportera det till underliggande immunvävnad. Denna funktion är viktig för att inducera skyddande immunsvar, men den utgör också en ingångsväg för sjukdomsalstrande bakterier. Crohns sjukdom är associerat med ett kraftigt ökat immunsvar mot bakterier, och sjukdomsförloppet kan ändras av stress. Det övergripande syftet med avhandlingen var att studera effekterna av stress på FAE samt att undersöka rollen av FAE vid utvecklingen av tarminflammation, särskilt vid Crohns sjukdom. Inledningsvis studerades effekterna av psykologisk stress på FAE. Stressade råttor uppvisade ökad genomsläpplighet av bakterier efter stress, och passagen var högre i FAE än i vanligt epitel. Efterföljande experiment visade att stressförändringarna i slemhinnan regleras via kortikotropinfrisättande hormon och mastceller. Vidare visade det sig att vasoaktiv intestinal peptid kunde efterlikna stressens effekter på genomsläppligheten, och att detta kunde förhindras genom att blockera mastcellerna. Studier av tunntarmsslemhinna från patienter med icke‐inflammatorisk tarmsjukdom och friska kontroller visade en högre passage av bakterier i FAE än i vanligt epitel. Hos patienter med Crohns sjukdom var bakteriepassagen genom FAE betydligt ökad jämfört med kontroller. Resultaten från detta avhandlingsarbete visar att stress kan förändra upptaget av bakterier från tarmen via FAE, med mekanismer som innefattar kortikotropinfrisättande hormon och mastceller. Detta har gett nya kunskaper kring regleringen av slemhinnebarriären. Vidare presenterar denna avhandling nya insikter i sjukdomsuppkomsten vid Crohns sjukdom genom att påvisa en tidigare okänd defekt i barriärfunktionen i FAE.
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