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Adaptive Immune Responses in the Intestinal Mucosa of
Microscopic Colitis Patients
I would like to dedicate this thesis to my parents
and to microscopic colitis patients
Örebro Studies in Medicine 84
ASHOK KUMAR KUMAWAT
Adaptive Immune Responses in the Intestinal Mucosa of
Microscopic Colitis Patients
© Ashok Kumar Kumawat, 2013
Title: Adaptive Immune Responses in the Intestinal Mucosa
of Microscopic Colitis Patients.
Publisher: Örebro University 2013
www.publications.oru.se
[email protected]
Print: Örebro University, Repro 04/2013
ISSN 1652-4063
ISBN 978-91-7668-929-5
Abstract
Ashok Kumar Kumawat (2013): Adaptive Immune Responses in the Intestinal
Mucosa of Microscopic Colitis Patients, Örebro Studies in Medicine, 84, 83pp.
Microscopic colitis (MC) is a chronic diarrhoeal disease of unknown aetiology,
comprising collagenous colitis (CC) and lymphocytic colitis (LC). The nature of the
adaptive local immune responses in the mucosa of MC patients is however far from
elucidated. The present study investigates phenotypic and functional characteristics
of the adaptive local immune responses in the colonic mucosa of these patients.
Our immunohistochemistry and flow cytometry studies (Paper I & II) demonstrated increased frequencies of CD8+ T cells in the colonic epithelium and lamina propria of both LC and CC patients compared to controls, whereas the frequencies of CD4+ T cells were unaltered or reduced. Our flow cytometry data
revealed increased local activation of both CD4+ and CD8+ T cells in the lamina
propria as well as the intraepithelial compartment of CC and LC patients compared to controls, demonstrated as increased proportions of these cells expressing
the active/memory marker CD45RO and the proliferation marker Ki67.
Analysis of recent thymic emigrants by measuring T cell receptor excision circle (TREC) levels in the colonic mucosa of CC and LC patients revealed reduced
TRECs levels in these patients compared to controls (Paper III). These results
suggests that the observed increased numbers of T cells in the mucosa of CC and
LC patients is due to the expansion of local resident T cells rather than direct
recruitment of recent thymic emigrants to the mucosa.
Molecular analysis of T helper (Th) cell and cytotoxic T lymphocyte (Tc)
mucosal cytokines at messenger and protein levels in the colonic biopsies from
CC and LC patients demonstrated a mixed Th17/Tc17 and Th1/Tc1 mucosal
cytokine profile and revealed significant differences in the mucosal
cytokine levels in CC and LC patients compared to controls (Paper IV).
Finally, we have set up an in vitro model to investigate how the colonic milieu
affects the activation and differentiation of T lymphocytes (Paper V). Our preliminary data indicate increased production of both pro-inflammatory and antiinflammatory cytokines by peripheral blood T cells in the presence of soluble
factors from the inflamed colonic mucosa of CC patients compared to controls.
Keywords: Microscopic colitis, collagenous colitis, lymphocytic colitis, intraepithelial lymphocytes, lamina propria lymphocytes, T cell receptor excision circle,
T helper cells, cytotoxic T lymphocyte and mucosal cytokines.
Ashok Kumar Kumawat, School of Health and Medical Sciences
Örebro University, SE-701 82 Örebro, Sweden, [email protected]
Svensk sammanfattning
Mikroskopisk kolit (MC) är en kronisk diarrésjukdom med okänd etiologi,
uppdelad i diagnoserna kollagen kolit (CC) och lymfocytär kolit (LC).
Kunskapen om det adaptiva immunsvaret lokalt i tarmslemhinnan hos
dessa patienter är fortfarande mycket begränsad. I denna avhandling har vi
fenotypiskt och funktionellt karakteriserat det lokala adaptiva immunsvaret i kolonslemhinnan hos patienter med mikroskopisk kolit.
Våra immunohistokemiska och flödescytometriska studier (delarbete I
& II) visade på ökade frekvenser CD8+ T-lymfocyter i epitelet och i lamina
propria från kolon hos både LC- och CC-patienter jämfört med kontroller,
medan frekvensen CD4+ T-lymfocyter var oförändrad eller minskad. Data
från de immunohistokemiska analyserna visade signifikant ökade mängder
FOXP3+ celler i både epitelet och lamina propria från CC- och LCpatienter jämfört med kontroller. Data från de flödescytometriska analyserna visade på en lokal aktivering av både CD4+ och CD8+ T-lymfocyter
i såväl lamina propria som intraepithelialt i CC- och LC-patienter jämfört
med kontroller, påvisat genom deras ökade uttryck av cellytemarkörerna
CD45RO och Ki67, associerade med aktiverade /minnesceller respektive
prolifererande celler.
Vi analyserade även mängden T-lymfocyter som nyss lämnat
thymus/brässen, så kallade thymusemigranter, genom att mäta mängden
”T cell receptor excision circles (TRECs)” i kolonslemhinnan hos CC- och
LC-patienter. Vi fann då minskade mängder TRECs hos dessa patienter
jämfört med kontroller (delarbete III). Dessa resultat tyder på att de ökade
mängder T-lymfocyter i kolonslemhinnan hos dessa patienter vi observerade i delarbete I och II beror på en lokal expansion av T-lymfocyter i
tarmslemhinnan, snarare än rekrytering av thymusemigranter till slemhinnan.
För att ytterligare karakterisera immunsvaret i slemhinnan hos patienter
med mikroskopisk kolit analyserade vi mängden cytokiner från T-hjälpar
(Th) lymfocyter och cytotoxiska T-lymfocyter (Tc) i biopsier från kolonslemhinnan, både på mRNA och proteinnivå. Vi fann en blandad
Th17/Tc17- och Th1/Tc1- cytokinprofil i slemhinnan, samt även signifikanta skillnader mellan CC- /LC-patienter och kontroller vad gäller mängden cytokiner i slemhinnan (delarbete IV). Då vi i de tidigare delarbetena
visat på ökade frekvenser CD8+ T-lymfocyter, men oförändrade eller minskade frekvenser CD4+ T-lymfocyter, är det sannolikt att den ökade cytokinproduktionen kommer från CD8+ T-lymfocyter i slemhinnan, såväl
som de CD4+ T-hjälpar-lymfocyterna.
För att undersöka hur den lokala miljön i tarmslemhinnan hos patienter
med kollagen kolit påverkar T-lymfocyternas aktivering och differentiering, satte vi i delarbete V upp en in vitro-modell för att undersöka hur
lösliga faktorer i tarmslemhinnan påverkar CD4+ T-lymfocyter från periferblod efter polyklonal aktivering. Våra preliminära resultat visar på ökad
produktion av både proinflammatoriska och antiinflammatoriska cytokiner från perifera T-lymfocyter i närvaro av lösliga faktorer från den inflammerade kolonslemhinnan från patienter med kollagen kolit jämfört
med kontroller.
List of Publications
This thesis is based on the following original papers, which are referred to
in the text by their Roman numerals I-V:
I.
Göranzon C, AK. Kumawat, E. Hultgren-Hörnqvist, C. Tysk, S.
Eriksson, J. Bohr and N. Nyhlin. “Immunohistochemical characterization of lymphocytes in Microscopic Colitis” Journal of
Crohn´s and Colitis;doi;10.1016/j.crohns.2013.02.007
II.
Kumawat AK, H. Strid, K. Elgbratt, C. Tysk, J. Bohr and E. HultgrenHörnquist. ” Microscopic colitis patients have increased proportions
of Ki67+ proliferating and CD45RO+ active/memory CD8+ and
CD4+8+ mucosal Tcells”. Journal of Crohn´s and Colitis;doi;
10.1016/j.crohns.2012.08.014
III.
Ashok Kumar Kumawat, Kristina Elgbratt, Curt Tysk, Johan Bohr
and Elisabeth Hultgren-Hörnquist. "Reduced T cell receptor excision circle (TREC) levels in the colonic mucosa of microscopic colitis patients indicate local proliferation rather than homing of peripheral lymphocytes to the inflamed mucosa" Submitted
IV.
Kumawat AK, H. Strid, C. Tysk, J. Bohr, and E. HultgrenHörnquist. “Microscopic colitis patients demonstrate a mixed
Th17/Tc17 and Th1/Tc1 mucosal cytokine profile”. Molecular
Immunology http://dx.doi.org/10.1016/j.molimm.2013.03.007
V.
Ashok Kumar Kumawat, Curt Tysk, Johan Bohr, Olof Hultgren
and Elisabeth Hultgren-Hörnquist. “An in vitro model for analysis
of the impact of the colonic milieu in collagenous colitis patients
on peripheral T lymphocyte activation and differentiation”. In
Manuscript
Published papers have been reprinted with permission from the publishers.
CONTENTS
LIST OF ABBREVIATIONS .................................................................... 13
INTRODUCTION................................................................................... 15
Microscopic colitis ................................................................................... 15
Epidemiology ........................................................................................... 16
Clinical Features....................................................................................... 16
Diagnosis ................................................................................................. 16
Mechanism(s) of Diarrhoea...................................................................... 18
Treatment ................................................................................................ 18
THE MUCOSAL IMMUNE SYSTEM..................................................... 18
MUCOSAL T CELLS............................................................................... 20
Immunophenotype of lymphocytes .......................................................... 21
T CELL DIFFERENTIATION ................................................................. 22
RECENT THYMIC EMIGRANTS AND T CELL RECEPTOR EXCISION
CIRCLES (TRECS) .................................................................................. 25
INTESTINAL HOMEOSTASIS ............................................................... 26
PATHOGENESIS OF MICROSCOPIC COLITIS.................................... 29
Luminal Factors ....................................................................................... 29
Mucosal Factors....................................................................................... 29
Genetics ................................................................................................... 30
T CELLS AND INFLAMMATORY BOWEL DISEASE .......................... 31
AIMS ....................................................................................................... 33
METHODOLOGICAL CONSIDERATIONS.......................................... 35
Patients (Paper I-V) .................................................................................. 35
Immunohistochemistry (IHC, Paper I) ..................................................... 36
Isolation of intraepithelial lymphocytes and lamina propria lymphocytes
(Papers II and V) ...................................................................................... 37
Flow Cytometric Analysis (Paper II)......................................................... 38
Real-time PCR (Papers III & IV) .............................................................. 39
Luminex (Papers IV & V) ........................................................................ 40
Preparation of conditioned medium from the colonic mucosa (Paper V) . 41
T cell Proliferation and Cytokine Release Assay (Paper V)....................... 42
Statistical analysis .................................................................................... 43
RESULTS AND DISCUSSION................................................................. 45
Phenotypic characterization of lymphocytes in the colonic mucosa of
collagenous colitis and lymphocytic colitis patients (Paper I & Paper II).. 46
Analysis of T cell receptor excision circle (TREC) levels in the CD3+ T cell
compartment in the colonic mucosa of collagenous colitis and lymphocytic
colitis patients (Paper III) ......................................................................... 50
T helper (Th) 1/Th17 and cytotoxic T lymphocyte (Tc) Tc1/Tc17
associated cytokine profile at messenger and protein levels in the colonic
mucosa of collagenous colitis and lymphocytic colitis patients (Paper IV) 51
The role of soluble factors from the colonic mucosa of collagenous patients
in the regulation of effector T cells (Paper V)............................................. 55
Limitations in the studies performed in this thesis.................................... 57
GENERAL DISCUSSION ........................................................................ 59
FUTURE PERSPECTIVES........................................................................ 65
ACKNOWLEDGEMENTS ...................................................................... 67
REFERENCES ......................................................................................... 71
LIST OF ABBREVIATIONS
6-MP – 6-mercaptopurine
APC- antigen presenting cell
AZA – azathioprine
CARD – caspase activation and recruitment domain
CC - collagenous colitis
CCR – chemokine receptor
CCL – chemokine ligand
CD – Crohn’s disease
CTL – cytotoxic T lymphocyte
CM – conditioned medium
DC – dendritic cell
DTT – dithiothretiol
DNB – denuded biopsies
DNA – deoxy-ribonucleic acid
EDTA – ethylenediaminetetraacetic acid
ECM – extracellular matrix
ECP – eosinophilic cationic protein
FAE – follicle-associated epithelium
FOXP3 – forkhead box P3
GALT – gut-associated lymphoid tissue
GATA3 – GATA binding protein
GAPDH – glyceraldehyde phosphate dehydrogenase
GUSB – glucocordinase beta
HLA – human leukocyte antigen
IBD – inflammatory bowel disease
ICOS – inducible co-stimulatory molecule
IFN – interferon
Ig – immunoglobulin
IEL- intraepithelial lymphocyte
ILF – isolated lymphoid follicle
IL – interleukin
iNOS – inducible nitric oxide synthase
LC – lymphocytic colitis
LNSCs – lymph node stromal cells
LPL – lamina propria lymphocyte
LPMCs – lamina propria mononuclear cells
MC – microscopic colitis
MCP – monocyte chemoattractant protein
MHC – major histocompatibility complex
miRNA – micro RNA
MLN – mesenteric lymph node
MMP – matrix metalloproteinase
MФ – macrophage
NF- nuclear factor
NKT – natural killer T cell
NOD – nucleotide binding oligomerization domain
NSAID – nonsteroidal anti-inflammatory drugs
PBL – peripheral blood lymphocyte
PP – Peyer’s patch
RA – retinoic acid
RNA – ribonucleic acid
ROR – retinoic acid receptor-related orphan receptor
RSS – recombination signal sequence
RTE – recent thymic emigrant
STAT – signal transducer and activator of transcription
T-bet – T-box transcription factor
TcR – T cell receptor
TGF – transforming growth factor
Th – T helper
TIMP – tissue inhibitor metalloproteinase
TNF – tumor necrosis factor
Treg – regulatory T cell
TRECs – T cell receptor excision circles
UC – ulcerative colitis
VEGF – vascular endothelial growth factor
INTRODUCTION
Microscopic colitis (MC) is a chronic diarrhoeal disease of unknown
aetiology, and is currently receiving increasing attention in the scientific
community. The colonic mucosa in MC patients is macroscopically normal
or almost normal and the diagnosis relies on microscopic examination of
colonic mucosal biopsies. MC was previously regarded as a rare disease,
but firm epidemiological studies from Europe and North America have
found a dramatic increase in the incidence of MC in the population, most
apparently due to an increased clinical awareness and more frequent
histopathological examination of colonic biopsies from patients with
chronic diarrhoea.
The aetiology of MC is believed to be multi-factorial and mostly
unknown. Although the increasing but still limited pathophysiological data
in microscopic colitis are insufficient to define a firm pathophysiology of
MC, it is postulated that MC is caused by disturbed immune responses to
luminal antigen(s) in predisposed individuals. The nature of the adaptive
local immune responses in the mucosa of MC patients is however far from
elucidated. The present thesis investigates phenotypical and functional
characteristics of the adaptive local immune responses in the colonic
mucosa of these patients.
Microscopic colitis
Microscopic colitis comprises collagenous colitis (CC) and lymphocytic
colitis (LC). Collagenous colitis was first described by the Swedish
pathologist C. Lindström in 1976 (1) where he described a case report of a
middle-aged woman with chronic diarrhoea whose colonic biopsies
showed a sub-epithelial collagen layer comparable to that observed in
collagenous sprue. The term “microscopic colitis” was introduced by Read
el al. in 1980 to describe patients with chronic diarrhoea who had normal
colonoscopy findings but showed mucosal inflammation on microscopic
examination (2). In 1989 Lazenby et al. proposed the term lymphocytic
colitis, where they described chronic diarrhoea patients with a normal
colonoscopy who showed increased infiltration of lymphocytes in the
epithelium upon histopathological examination (3). Both CC and LC have
similar clinical symptoms and share histopathological features except for
epithelial collagen layer in CC. They have therefore been grouped under
the common term “microscopic colitis” (4). However, it is not clear
whether CC and LC are two separate disorders or different manifestations
of the same disease. Transition of LC to CC or vice versa has been reported
(5). Additional subtypes of MC have also been reported, which share
ASHOK KUMAR KUMAWAT Adaptive Immune Responses in the Intestinal ...
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similar clinical features to the classical MC but differ in their
histopathological appearance. These subtypes of MC include MC with
giant cells, paucicellular LC, cryptal LC, incomplete MC,
pseudomembranous CC and MC with granuloma infiltration, as reviewed
in (6-9).
Epidemiology
Population based studies on MC have been performed in several countries
but mostly in Europe and North America. Long-term epidemiological
studies from Sweden and North America since the 1980s demonstrate a
rising incidence in the 1980s and early 1990s followed by a stable plateau
phase in some centres (6-8). In Sweden, the present annual incidence rate
of CC and LC is 5-6 cases per 100,000 individuals for each disorder (6-7).
The reasons for this increase in disease incidence remains unclear, but most
likely it could be due to greater awareness of clinicians and pathologists
when diagnosing MC. Microscopic colitis can occur at any age group but
most commonly affects middle-aged or elderly individuals with a
noticeable female dominance. The epidemiological studies from our group
on patients from Örebro County revealed that the average age at MC
diagnosis was 65 (range 53-74) years and the female:male ratio was 7:1
(6).Williams et al. have reported that patients older than 65 years were
more than five fold more likely to develop MC than the younger
population (8).
Clinical Features
Clinically CC and LC cannot be differentiated from each other. The main
symptom of both conditions is chronic non-bloody, watery diarrhoea, and
is often associated with nocturnal diarrhoea (9). Abdominal pain and
weight loss is significantly common. Furthermore faecal incontinence may
affect these patients, which is one of the major factors for the low quality
of life of these patients (9-11) . The onset of CC can be sudden in about 40
% of patients (11). It has been reported that clinical symptoms in LC are
milder and more likely to disappear than in CC (12). MC is often
associated with other autoimmune diseases such as celiac disease,
rheumatoid arthritis, thyroid disease, or diabetes mellitus (6-7, 13).
Diagnosis
Diagnosis of MC mainly relies on histopathological findings as
colonoscopic examination reveals a colonic mucosa that is mostly normal
or has slight edema or erythema (14). The main histological features of LC
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(Fig. 1B) are increased numbers of intraepithelial lymphocytes (IELs) (≥
20/100 surface epithelial cells) together with surface epithelial cell damage
and infiltration of lymphocytes in the lamina propria, but a normal
collagen layer in contrast to CC, where an increased collagen layer of >10
µm beneath the epithelium is a characteristic feature (7, 12). In uncertain
cases CD3 immunostaining is performed to assess the IEL count to confirm
the diagnosis (7). The histological findings of CC are in addition to
increased numbers of lymphocytes in epithelium and lamina propria,
presented with a deposition of a ≥10 µm thick subepithelial collagen layer
(Fig. 1C). In uncertain cases tenascin immunostaining is used to measure
collagen band thickness (15)
Figure 1. Human colonic biopsies showing (A) normal colonic
mucosa; (B) typical findings of
lymphocytic colitis, with epithelial cell damage with increased
amounts of IELs and infiltration
of lymphocytes in the lamina
propria; (C) typical findings of
collagenous colitis, with an increased sub-epithelial collagen
layer, inflammation of lamina
propria and increased amounts
of IELs with epithelial cell damage with. Photo: Sune Eriksson
Dept. of Pathology, Örebro
University Hospital.
A & B- H&E Staining, C- van
Gieson Staining
The thickness of the collagen layer varies in the colon and is found to be
most prominent in the ascending and transverse colon whereas it may be
absent in the sigmoid colon or rectum (16). Therefore rectal biopsies are
not sufficient for the diagnosis and collection of multiple biopsies from
different parts of the colon is recommended (17).
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Mechanism(s) of Diarrhoea
The precise mechanism for diarrhoea in MC patients is not clearly
understood. However the severity of diarrhoea has been correlated with
the intensity of inflammatory changes in the mucosa but not with the
thickness of the sub-epithelial collagen layer (18-19). Inflammation in the
lamina propria was the first histological change to appear after restoration
of bowel continuity in a CC patient who had undergone a temporary loop
ileostomy (20). These findings suggest that diarrhoea in MC is
inflammatory in origin. In addition, impaired electrolyte absorption and
increased secretion has been reported in MC patients (19, 21).
Treatment
Based on the currently available data from clinical trials for the treatment
of MC, oral budesonide is the best documented treatment that markedly
ameliorates the clinical symptoms and improves the patient’s quality of life
(22-23). Budesonide is effective in both CC and LC patients for inducing
clinical remission and as long-term maintenance therapy (24-25). However
upon withholding the budesonide treatment symptom relapse occurred in
40-80 % of patients within six months (22).
Immunomodulators that decrease inflammatory responses, such as
azathioprine (AZA) or 6 mercaptopurine (6-MP) or methotrexate have
occasionally have been used in steroid dependent severe case of MC (2627). Antidiarrhoeals, such as loperamide or cholestyramine, have not been
formally studied in randomised controlled trials, but are generally
recommended as the first step of treatment in the patient with mild
symptoms. (7) Other options include aminosalicylates, bismuth
subsalicylate, antibiotics, probiotics and Boswellia serrata extract but the
evidence for these alternatives is limited. Two case reports have
demonstrated that anti-TNF therapy may be effective in severe MC
patients (28-29). The surgical treatment in MC is very rare, but for severe
and therapy resistant cases ileostomy may be an ultimate option.
The Mucosal Immune System
The small and large intestine in humans have distinct function, where
absorption of nutrition occurs in small intestine, whereas the large intestine
plays vital role in the absorption of water and salt. Human intestinal
mucosa is home to a vast number of commensal bacteria, and at times also
pathogens. The intestinal immune system plays a major role by responding
to harmful pathogens while tolerating dietary antigens and beneficial
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bacteria. The intestinal immune system contains the largest amount of
lymphocytes in the whole body. These lymphocytes are spread in the
epithelium, the intraepithelial lymphocytes (IELs) and in the lamina
propria (LP), and in organized lymphoid tissues such as the Peyers patches
(PP) located predominantly in the small intestine and isolated lymphoid
follicles (ILFs) in both the small and large intestine, the latter with similar
structure and function as Peyers patches and in mesenteric lymph node
(MLN) (30-32). In addition to the immune cells, LP also contains e.g
fibroblasts, mesenchymal stromal cells and mucosal nerves.
The intestinal epithelium acts as a physical barrier, with a single layer of
epithelial cells folding into villi and crypts (small intestine) or crypts (large
intestine) that covers a surface area of approximately 400 m2. This physical
barrier is selective, allowing regulated passage of fluids and antigens. The
function of the physical barrier is achieved by complex interactions
between different cellular components. The epithelium has structures
named tight junctions between epithelial cells, which are made up of
complex proteins such as claudin, occludin, ZO-1, ZO-2, ZO-3, cingulin
and 7H6, regulating permeability between the cells (33). The mucus
produced by goblet cells prevents the bacteria to reach the epithelial
surface and the Paneth cells located at the base of the crypts secretes
antimicrobial proteins (e.g. defensins) (34).
A specialized follicle-associated epithelium (FAE) containing M cells is
overlying the PP and ILFs. These specialized areas support the transport of
antigens into the lamina propria for antigen presentation by dendritic cells
(DC) or macrophages. The antigens are processed by DCs, which then
either activate naïve T and B cells within the Peyer’s patches (PP) or
migrate to MLN to activate naïve T cells (Fig. 2).
Activated B cells undergo maturation and differentiate into plasma cells
that produce large amounts of immunoglobulin A (IgA) that is transported
to the lumen. IgA produced by plasma cells is a dimer and is linked via a
joining (J) chain. During transepithelial transport of IgA, the J chain binds
to the poly-Ig receptor (pIgR) expressed by the intestinal epithelial cells.
This complex is then actively transported to the intestinal lumen. In the
lumen the receptor is cleaved by proteolysis, releasing the secretory
component still attached to the IgA molecule, which is now termed
secretory IgA (sIgA) (35). In the lumen, IgA binds to microbes and toxins
and neutralize them by blocking their entry into the host.
The antigen experienced T cells undergo maturation and starts to
express the activation/memory marker CD45RO. Mucosal DCs induce
expression of the integrin α4β7 and CCR7 on lymphocytes in MLN (a
hallmark of LP migrating and -resident lymphocytes). These activated
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lymphocytes leave the MLN through the efferent lymph via the thoracic
duct into the bloodstream and enter the lamina propria.
Figure 2. Antigen uptake in the small intestinal mucosa.
The epithelium acts as a physical barrier to entry of luminal antigens into the
lamina propria (LP), but it also has specialized cells like follicle-associated
epithelium (FAE) and M cells, both with a less pronounced brush border. The
antigen enters via M cells and FAE into the PP. The antigens are processed by
dendritic cells (DCs) which then either activate naïve T cells in Peyer’s patches (PP)
or migrate to mesenteric lymph nodes (MLN) to activate naïve T cells. Activated T
cells leave the MLN through the draining lymphatics via the thoracic duct and into
blood stream and finally back to lamina propria (LP)
Mucosal T cells
In humans, mucosal T cells reside in the gut associated lymphoid tissues
(GALT), such as the MLN, PP, the LP and the epithelium. Whereas MLN
and PP contains naïve T cells, most T cells in the LP and especially in the
epithelium have an effector/memory phenotype.
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Mucosal T cells are divided into two major groups based on TcR and
co-receptor expression (36), which are sometimes referred as conventional
and non-conventional T cells. Conventional T cells consist of TcRαβ+
major histocompatibility complex (MHC) class II-restricted CD4+ and
MHC class I-restricted CD8αβ+ T cells. These conventional T cells have
previously been primed in response to an antigen in the periphery and have
migrated to the intestine, where they reside in the LP as effector-memory T
cells. The non-conventional T cells are either CD8αα+ or CD8αα- and
express TcRαβ or TcRγδ, or are “double negative” (DN) TcRαβ+ cells that
lack expression of both CD4 and CD8αβ (32). Generally, conventional T
cells dominate the LP compartment, whereas non-conventional cells are
predominant in the epithelium. I
ELs that reside within the epithelium are mostly T cells and their
frequency varies along the intestine. In humans, there is approximately one
IEL per 5-10 enterocytes in the small intestine compared to one IEL per 40
enterocytes in the large intestine (32). The majority of IELs in human is
dominated by TcRαβ+ T cells with only a minority being TcRγδ+ IELs. Of
the TcRαβ+ T cells, the majority of them are conventional CD8αβ+ T cells
with only few CD8αα+ and CD4+ T cells normally present.
The TcRγδ+ T cells in humans is believed to help in elimination of
stressed epithelial cells by recognizing the stress induced polymorphic
antigens MICA and MICB (37). In mice, the non-conventional TcRαβ+ DN
cells and CD8αα+ cells are believed to contain self reactive T cell receptors
(38), however their role in the human gut is yet to be fully elucidated.
T cells in the human LP are mainly conventional CD4+ T cells with
lower frequencies of CD8αβ+ T cells. In addition, there are also invariant
natural killer T (NKT) cells and mucosa associated invariant T cells that
interact with the non-classical MHC molecules CD1d and MR1
respectively (39-40).
Despite the effector/memory phenotype of these frontline T cells in the
mucosa, it is evident that LPLs have reduced proliferation rate in response
to TcR/CD3 mediated stimulation compared to peripheral T cells (41).
This hyporesponsiveness response seems to be selective for TcR/CD3
mediated signalling, as CD2 or CD28-driven activation resulted in strong
proliferation (42).
Immunophenotype of lymphocytes
The T cell population can be grouped into specific subsets based on their
phenotype that is defined by the expression of diverse cell surface
receptors. In humans, T cell activation markers such as CD45RA or
CD45RO define naïve and memory/effector T cells, respectively, together
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with different co-stimulatory molecules such as CD28. The naïve or resting
T cells can be defined as CCR7+CD45RA+CD27+CD28+, whereas antigen
experienced T cells are defined as CCR7-CD45RA+/-CD27-CD28- (43).
Upon activation T cells express CD69 in the early stage whereas in the late
stage they express CD45RO.
The B cells are most commonly characterized by expression of surface
markers including CD19, CD20, CD27 and CD38. The naïve B cells are
defined as CD19+CD20+CD27-CD38- and memory B cells are defined as
CD19+CD20+CD27+CD38- whereas the plasma cells are defined as
CD19+CD20-CD27++CD38++CD138+ (44-46). Generally in textbooks plasma cells are described as CD19 negative. However, many studies report
that CD19 is indeed expressed on plasma cells (45, 47-48). Yet, Harada et
al reported that malignant plasma cells are negative for CD19 expression.
T cell differentiation
T lymphocytes mostly consist of CD4+ and CD8+ T cells. The CD4+ T cells
are the main conductors of immune responses through the production of
specific cytokines. They carry out various immunological processes
including promotion of maturation of B cells into plasma cells and
activation of cytotoxic T lymphocytes (CTLs), macrophages and nonimmune cells, but they also play a critical role in suppression of immune
responses. The differentiation of different CD4+ T helper (Th) subsets is
orchestrated by a complex network of transcription factors and specific
cytokines.
T cells undergo differentiation when their TcR interacts with antigens
bound to the MHC molecules expressed by DCs or other antigen
presenting cells (APCs). Co-stimulatory molecules such as CD28 and the
inducible co-stimulatory molecule (ICOS), amplifies TCR signalling,
thereby promoting T cell proliferation and differentiation.
When activated, naïve CD4+ T cells differentiate into effector cells
including the Th1, Th2 and Th17 subsets and induced regulatory T cells
(iTreg) (see below) (Fig. 3). Each subset has specific roles in promoting
immune responses towards particular pathogens. Th1 cells mainly produce
interferon (IFN)-γ, lymphotoxin and tumor necrosis factor (TNF) and
activate macrophages and neutrophils to better combat intracellular
pathogens, but they are also implicated in autoimmune inflammation. Th2
cells, producing e.g. interleukin (IL)-4, IL-5, IL-10 and IL-13 promote
clearance of extracellular parasitic infections and they are also involved in
allergic inflammation.
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The Th17 cells produce mainly IL-17A but also IL-17F, IL-21, IL-22
and IL-26, and are abundant at the intestinal mucosa. Th17 cells protect
the host against pathogenic bacteria and fungal infections, partly by
maintaining the intestinal barrier function by inducing the tight junction
protein claudin as well as promoting mucus production (49). The Th17
cells have however been implicated as important mediators in mucosal
inflammation and other various inflammatory diseases, by their promotion
of pro-inflammatory cytokine production as well as recruitment and
activation of neutrophils.
The Treg cells suppress T lymphocytes, DCs and NK cell via cell-to-cell
contact. In addition they secrete anti-inflammatory cytokines such as IL10, transforming growth factor (TGF)-β and IL-35 that play important
roles in suppression of immune responses (50-53).
The T-box transcription factor (T-bet) promotes development of the
Th1 lineage, but also suppresses the development of other cell lineages
(54). Both IFN-γ and IL-12 play a vital role in maturation of Th1 cells.
Augmented levels of IL-12, secreted by APCs, ensure expansion of Th1
cells. T-bet induces IFN-γ production via the signal transducer and
activator of transcription 1 (STAT1), whereas IL-12 induced STAT4
promotes IFN-γ production that further ensures expansion of Th1 cells
(52).
The differentiation of Th2 cells is driven mainly by IL-4. IL-4 induces
STAT6 that up regulates the expression of the main Th2 lineage regulator
GATA binding protein (GATA3) (55). In addition GATA3 suppress Th1
differentiation by downregulating STAT4 (56).
The Th17 differentiation is mainly orchestrated by the transcription
factor retinoic acid-related orphan receptor (ROR)C2 in humans and
RORγt in mice as well as RORα and STAT3 (51, 57). The Th17 cells
differentiates in response to the STAT3 activating cytokines IL-6, IL-21
and IL-23 together with IL-1β and TGF–β (58).
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Figure 3. Development of T helper (Th) 1, Th2, Th17 and induced regulatory T
cells (iTreg cells) from naive CD4+ T cells. Cytokines inducing the development of
Th1, Th2, Th17 and iTreg cells are marked in red. The main effector cytokines of
these four cell subsets are marked in blue. ICOS, inducible co-stimulatory molecule;
T-bet, T-box transcription factor; GATA3, GATA binding protein; RORC2, retinoic acid receptor-related orphan receptor C2; FOXP3, forkhead box P3.
The natural Tregs are formed in the thymus as a distinct lineage, whereas
induced Treg (iTreg) are induced in peripheral organs after antigen priming. The iTreg lineage is regulated by the transcription factor forkhead box
P3 (FOXP3) (59). TGF–β is the master regulator of iTreg/Th17 lineage
commitment (60). Abundant amounts of TGF–β induces FOXP3 specific
iTreg differentiation, whereas at low concentration and in the presence of
IL-6, TGF-β induces Th17 cell differentiation (52, 60). STAT5 induces IL-2
downstream signalling and enhances FOXP3 expression ensuring selective
iTreg differentiation (52).
The Th subsets were previously believed to be terminally differentiated,
but it has now become apparent that some Th subsets display plasticity
between them. Whereas Th1 and Th2 have relatively stable phenotypes,
iTreg and Th17 can readily switch to other Th subsets. iTreg cells can
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switch to Th17 cells under the influence of IL-6 and Th17 cells can switch
into Th1 cells in the presence of IL-12 (61-62). Recently a subset of Th17
cells has been identified that co-produces IL-17 and IFN-γ possibly
representing an intermediate Th17/Th1 phenotype (63). These IL-17/ IFN-γ
producing T cells have been implicated in the pathogenesis of
inflammatory bowel disease (64).
CD8+ T cells differentiate into cytotoxic T cells (CTLs) upon activation
by antigen. These CTLs play an important role in inducing FasLigand- or
Granzyme mediated apoptosis in cells infected with intracellular bacteria
or viruses, as well as (tumor) transformed cells. Different subsets of CD8+
CTLs (Tc) have also been described, such as IFN-γ producing Tc1 and IL4, IL-5 and IL-10 producing Tc2. Tc1 and Tc2 cell development is driven
by the transcription factors T-bet and GATA3 respectively (65-67). The
Tc1 maturation is stimulated by IL-12 and IFN-γ while Tc2 maturation is
driven by IL-4 (65-67). More recently IL-17 producing CD8+ T cells, so
called Tc17 cells, have also been described (67-70). The Tc17 cells produce
IL17, IL-21, IL-22, IFN-γ and TNF-α and express the IL-23R, the
chemokine receptor CCR6 and the transcription factor RORC2 (humans)
or RORγt (mice) (67-71).
Recent Thymic Emigrants and T cell Receptor
Excision Circles (TRECs)
The thymus provides a specialized microenvironment for the maturation
and selection of the vast majority of functional T cells. The T cell receptor
gene rearrangement is a critical step in the development of mature T cells.
The T cell receptors are associated with the CD3 subunits γ, δ, ε, and ζ.
Generation of the T cell repertoires with diverse antigen specificities is
achieved by random rearrangement of TcR gene segments termed V, D and
J for variable, diversity and joining, respectively, in the thymus. TcR α and
γ chains consist of V and J gene segments, whereas TcR β and δ chains
consists of V, D and J gene segments (72). This process is initiated by
recognition of recombination signal sequences (RSS) that flank the coding
sequences on DNA and during this process the two signal ends are
circularized, forming an extrachromosomal circular excision product,
which are known as a T Cell Receptor Excision Circles – TRECs (72).
In the formation of TcR αβ, the β chain is rearranged first and then pairs
with a pTα (also termed pre-T-α) chain. During the gene rearrangement for
formation of a functional TcR α chain two TREC variants, one single joint
TREC (sjTREC) and one coding joint TREC (cjTREC) are formed,
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simultaneously resulting in the deletion of the TcR δ locus which is situated
within the TcR α locus (73).
The TRECs are relatively stable but are not replicated during mitosis,
and consequently they are diluted in each cell division (74). Therefore,
TRECs levels are a direct reflection of the amount of recent thymic
emigrants (RTE) in the periphery. The newly exported RTEs in the
periphery contain high amounts of TRECs compared with the peripheral T
cells that have undergone various rounds of cell division resulting in
diluted TREC content. The cell division rate can be analyzed by evaluating
the expression of proliferation marker Ki67, a nuclear antigen that is
expressed in all dividing cells and absent in resting cells (75). Analysis of
both T cell proliferation and TREC content in peripheral lymphocytes for
quantification of T cells that have recently left thymus is a good
measurement of thymic output.
TRECs measurements are used extensively to document T cell
reconstitution following treatment of HIV infection and in patients who
have undergone haematopoietic stem cell transplantation (75-76). Previous
studies by our group have used TRECs measurements to quantify the
amount of recently matured T cells in the periphery in both IBD patients
and mouse models of colitis (77-78).
Intestinal Homeostasis
The normal appearance of the gastrointestinal mucosa is in state of “physiological inflammation” as it contains large number of leukocytes in the
epithelium and subepithelial compartments (79). This is mostly due to an
immune response to harmful pathogens while dietary antigens and beneficial bacteria are normally tolerated. At the cellular level the mucosa contains a network of complementary regulatory interactions between different types of immune and non-immune cells as well as the microbiota to
maintain mucosal homeostasis in the gut (Fig 4).
The human gut is home to the largest collection of microbes. The distal
gut is densely populated with approximately 1012 organisms per milliliter
or gram of luminal contents (80). This microbial community is mainly
dominated by bacteria and among the 100 known phyla, members of
Firmicutes and Bacteroidetes are the most prevalent in the distal gut
community (81). Therefore the synergistic relationship between the host
immune system and its micobiota should be seen as a functional entity.
Pattern recognition receptors, e.g. Toll like receptors and NOD-like
receptors that are expressed by epithelial cells, M cells and DCs can
recognize conserved structures on microbes. There is a complex crosstalk
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between the intestinal epithelial cells, the microbiota and the intestinal
immune cells, where the microbiota defines the development and choices of
immune cells, suggesting a coevolution of the immune system with the
microbiota in vertebrates (50, 80). In addition, studies in mice have
revealed that mice raised under germ free conditions have an
underdeveloped intestinal immune system and that reconstitution of the
gut microbial flora from conventionally raised mice restores the mucosal
immune system (82). In addition, the commensal flora protects the host
from pathogen colonization and also provides extra nutrients to the host
by metabolizing a broader range of dietary components such as complex
carbohydrates.
Intestinal stromal cells, important in tissue regeneration and wound
repair can communicate with the epithelial and immune cells (83-84). A
recent study reported that direct presentation of intestinal self antigens by
lymph node stromal cells (LNSCs) resulted in activation and deletion of
CD8+ T cells but not CD4+ T cells, suggesting a role of LNSCs in the
regulation of CD8+ T cell mediated peripheral tolerance in the intestine.
(85). Human intestinal mucosal microvascular endothelial cells from
patients with inflammatory bowel disease (IBD) were shown to have
increased adhesiveness for leukocytes compared to the microvascular
endothelial cells from normal mucosa (86).
It is now believed that the regulatory interactions in the gut mucosa
occur in the midst of a complex mixture of proteins, known as the
extracellular matrix (ECM). The major constituents of ECM include
collagens, laminin, fibronectin, glycoproteins, tenascin, proteoglycans,
elastin and others (79, 87). The ECM together with the mucosal soluble
mediators such as cytokines and growth factors derived from mesenchymal
cells, immune cells and epithelial cells regulate cell differentiation,
proliferation and apoptotic processes (87). It has been suggested that the
composition of ECM determines which type of cell surface receptors are
expressed on leukocytes, thereby controlling the quantity of and state of
activation of the leukocytes in local tissues (88). ECM components are
degraded by proteolytic activity of matrix metalloproteinases (MMPs).
Recent studies have implicated the involvement of human gut microbiota
derived proteolytic activity with the capacity to engage in degradation of
ECM components, adding a new branch to the network of complex
interactions in intestinal homeostasis (89).
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Figure 4. Components of Intestinal
Homeostasis. Different types of immune
and
non-immune
cells as well as the
microbiota interact
with each other to
maintain
intestinal
homeostasis.
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Pathogenesis of Microscopic colitis
The aetiology of MC is mostly unknown but believed to be multi-factorial.
However it is postulated that MC is caused by disturbed immune responses
to luminal antigen(s) in predisposed individuals.
Luminal Factors
The involvement of luminal agents in MC is best demonstrated by the fact
that diversion of the faecal stream via loop ileostomy leads to a normalized
clinical and histopathological response (20, 90). Upon restoration of bowel
continuity, both clinical symptoms and histopathological findings of MC
recurred (20, 90).
Drug consumption has been implicated in inducing MC. (7, 91).
Nonsteroidal anti-inflammatory drugs (NSAIDs), aspirin, acarbose,
cyclo3fort, lansoprazole, ranitidine, ticlopidine and sertraline have all been
suggested to be strongly associated with microscopic colitis (91).
Involvement of microbes in MC pathogenesis has been discussed (9294). Case reports on associations between MC and Clostridium difficile
and Yersinia enterocolitica have been reported (92-93), but there are no
consistent data available on specific pathogens or bacterial products in the
MC pathogenesis.
Bile acid malabsorption can contribute to MC pathogenesis and
aggravates clinical symptoms. It has been associated with MC in 27-44%
of CC patients and in 9-60% of LC patients (95).
Mucosal Factors
Immunohistochemical data have revealed increased infiltration of CD3+ T
cells in the lamina propria (LP) and the intraepithelial compartment of
both CC and LC patients (96-97). Both these studies also reported that the
infiltration in the epithelium is dominated by CD8+ T cells expressing the
αβ T cell receptor (96-97). Furthermore, a Th1 cytokine profile has been
reported, with elevated mucosal mRNA levels of TNF-α, IFN-γ and IL-15
but not IL-2 or IL-4 in the mucosa of both LC and CC patients (98).
However, immunohistochemical analysis of the mucosa of LC patients
showed that the majority of lamina propria CD4+ T cells expressed the Th2
transcription factor GATA-3, whereas CD8+ T cells expressed both the
Th1 transcription factor, T-bet and GATA-3 at similar levels (99). In
contrast, in the epithelium the majority of CD8+ IELs expressed T-bet (99).
Recently it was demonstrated that CD4+ and CD8+ intestinal T cells had
reduced expression of the activation marker CD69 in patients with active
CC (100).
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Active nuclear factor (NF) κB levels are increased in mucosal epithelial
cells of CC patients (101). Significantly increased concentrations of luminal
nitric oxide have been observed, with an increase in inducible nitric oxide
synthase (iNOS) (98, 102). Infusion of the iNOS inhibitor NGmonomethyl-L-arginine in the colon of CC patients decreased fluid
secretion, suggesting that NO is involved in the diarrhoea in CC patients
(103).
Apart from lymphocytic infiltration, increased numbers of functionally
active eosinophils have been detected in the mucosa of CC patients (100),
as well as augmented luminal levels of eosinophilic cationic protein (ECP)
in perfusion fluids from the colon (104).
Several mechanisms have been proposed to explain the altered collagen
deposition in CC patients, but no single mechanism has emerged. Increased
numbers or activity of myofibroblasts could result in an increased collagen
synthesis in CC (105). Reduced mRNA levels of the matrix degrading
enzymes MMP1 and 13, and increased expression of tissue inhibitor of
metalloproteinase (TIMP)-1, suggests impaired collagen degradation in CC
(106).
In CC patients infiltrating eosinophilic granulocytes have increased
mRNA expression levels of TGF-β1 and this may affect the connective
tissue remodelling (107). Furthermore, enhanced luminal levels of the
potent fibrosis enhancing vascular endothelial growth factor (VEGF) has
been found in the colonic mucosa of CC patients (108).
Genetics
The role of genetic factors in MC still remains unclear. Occurrence of MC
within families has been reported (109). An increased prevalence of human
leukocyte antigen (HLA)-DQ2 has been reported in both CC and LC
patients (110-111). In addition, an increased frequency of HLA-A1 and a
decreased frequency of HLA-A3 have been found in LC but not in CC
patients (112). Polymorphism in the gene encoding TNF-α has been
reported in both CC and LC patients (111). In addition polymorphism in
the MMP-9 gene has been associated with CC (113). In contrast to
Crohn’s disease, no association with NOD2/CARD15 has been observed in
CC patients (114). Koskela et al. analyzed the frequency of polymorphism
in the IL-6, IL-1β, IL-1RA, IL-10 and CD14 genes in MC, and showed that
the IL-6-174-GG allele was more prevalent in both CC and LC patients
compared to the controls, but did not find any significant association
between polymorphisms in the other cytokine genes and MC (115).
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T cells and Inflammatory Bowel Disease
Dysregulated immune responses in the intestine can lead to chronic
inflammatory diseases such as inflammatory bowel disease (IBD). IBD is
mainly divided into two entities; Crohn’s disease (CD) and Ulcerative
colitis (UC) and causes a chronic inflammation characterized by acute
flares followed by remission. CD can affect any part of the gastrointestinal
tract in a discontinuous manner, whereas UC is primarily affecting the
colon and rectum. The hallmark of active inflammatory bowel disease is a
pronounced infiltration of innate immune cells; neutrophils, macrophages,
dendritic cells (DCs) and natural killer T (NKT) cells as well as T and B
lymphocytes in the lamina propria (116). The disease is found globally, but
a dramatically increased prevalence is found in Europe and North America.
Although major insights into the nature of IBD have been offered by
human as well as animals studies, the etiology of IBD is still not clearly
understood. Accumulating evidences suggests that the pathogenesis of IBD
is characterized by exaggerated immune responses to intestinal microbes in
a genetically susceptible host (116-117). Several studies of inflammatory
bowel disease in mice and humans have demonstrated immune
disturbances by subsets of CD4+ T-helper (Th) cells as part of the
pathogenesis. Generally CD is affiliated with Th1 and Th17 cells, whereas
UC display a mixed Th1/Th2/Th17 cytokine profile (118-119).
The IL-23 produced by antigen presenting cells stimulated with bacteria
has been implicated in the pathogenesis of IBD (120-121), as it promotes
the production of both Th1 and Th17 cytokines in the inflamed gut
mucosa of CD patients and Th17 cytokines in UC patients (119, 121).
Th17 cells and increased levels of the Th17 cytokines IL-17A, IL-17F, IL21 and IL-22 have been detected in the inflamed mucosa of both human
IBD and experimental models of colitis (49). Increasing evidence from T
cell mediated colitis in mice shows augmented levels of proinflammatory
cytokines and chemokines in the intestinal mucosa, including IL-1β,
monocyte chemoattractant protein (MCP)-1, IL-6, IFN-γ, tumor necrosis
factor (TNF)-α and IL-17A that were significantly attenuated in the
absence of IL-23, as reviewed in (122). A recently identified subset of Th17
cells that coproduces the Th1 cytokine IFN-γ was prominent in the
intestinal mucosa of active CD patients (64).
Increased numbers of CD4+CD25+ FOXP3+ regulatory T cells have been
detected in the inflamed mucosa of both UC and CD patients compared to
controls (123-124). However, increased apoptosis of both LP and
peripheral blood regulatory T cells have also been observed in both UC
and CD patients compared to controls (124).
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AIMS
The overall aim of the present thesis was to investigate phenotypical and
functional characteristics of the adaptive local immune responses in the
colonic mucosa of microscopic colitis patients.
The specific aims for the papers were the following:
•
To characterize mucosal lymphocytes in the colonic mucosa of
microscopic colitis patients by immunohistochemical analysis.
(Paper I)
•
To characterize phenotypes of lamina propria and intraepithelial
lymphocytes separately in microscopic colitis patients using flow
cytometry. (Paper II)
•
To investigate the amount of the recent thymic emigrants by
measuring T cell receptor excision circle (TREC) levels in the
colonic mucosa of microscopic colitis patients. (Paper III)
•
To investigate the T helper (Th) cell and cytotoxic T lymphocyte
(Tc) mucosal cytokine profile at messenger and protein levels in
microscopic colitis patients. (Paper IV)
•
To setup an in vitro model for analysis of the impact of the colonic
milieu in collagenous colitis patients on peripheral T lymphocyte
activation and differentiation. (Paper V)
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METHODOLOGICAL CONSIDERATIONS
The following section gives a brief overview of the experimental approaches used in this thesis, as well as reasons for and comments on the
choice of material and methods used. For a more detailed description of
specific methods, see paper I-V.
Patients (Paper I-V)
Colon biopsy specimens were obtained from previously diagnosed CC, LC
and UC patients with an ongoing clinically active disease. Biopsies from
patients with diarrhoea, but with histologically normal mucosa and no
earlier diagnosis of MC or IBD were also investigated. Finally, controls
without diarrhoeal symptoms were recruited among patients undergoing
examination for rectal bleeding or suspicion of malignancy; with a normal
mucosal appearance and histology. Patients with a previous history of
Crohn’s disease, clinical signs of gastrointestinal infection, ischemic colitis
or neoplastic disease were excluded from the studies.
In study I previously collected, formalin fixed, paraffin-embedded
colonic biopsies were obtained from patients with LC, CC and controls
from the archive at the Dept of Pathology, Örebro University Hospital,
Sweden.
In study II, III, IV & V the biopsy specimens from MC patients,
diarrhoea patients and controls were taken from the right flexure, whereas
in UC patients they were taken from macroscopically affected areas of the
colon. The colonoscopies were performed at the division of
Gastroenterology, Örebro University Hospital, Sweden.
In Study V blood samples were collected from healthy blood donors at
Örebro University Hospital, Sweden.
All patients in these five studies had provided written informed consent.
The studies were approved by the regional ethical committee of ÖrebroUppsala County, Sweden, with the ethical approval ID #2008/278;
081015.
Comments:
The composition of well-defined patient populations is important in
clinical studies. The recruitment of microscopic colitis patients in these
studies took place from October 2008 till January 2013, still resulting in a
small cohort of patients. There are several difficulties in recruiting MC
patients. The MC patients can only be diagnosed following a
histopathological examination and patients with a confirmed diagnosis are
not normally undergoing repeated colonoscopy. Most often the patients
with a confirmed diagnosis are treated with immunomodulating agents,
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e.g. budesonide which will most likely influence the immune responses, and
therefore these patients are most of the times excluded.
Another important clinical issue encountered during the work with this
thesis is a subgroup of MC patients with a previous diagnosis of LC or CC
that at time of biopsy collection for this study showed no histological signs
of inflammation despite clinical symptoms of active LC/CC. These patients
were grouped separately, and were referred to as LC-histopathological
remission (LC-HR) and CC-histopathological remission (CC-HR).
In the second and fifth study where the experiments had to be performed
on fresh mucosal specimens the recruitment was further obstructed as a
fraction of the new patients, recruited based on symptoms of clinically
active MC but not previously diagnosed with MC, did not fulfil
histopathological criteria for MC. They were therefore grouped separately
as diarrhoea patients.
We chose ulcerative colitis patients as a positive control for colonic
inflammation, as immunopathological data on UC is well documented. The
UC patients included in this study were recruited from those referred to the
outpatient Gastroenterology clinic, Örebro University Hospital, Sweden.
Immunohistochemistry (IHC, Paper I)
To characterize the inflammatory cells, we investigated expression of
different proteins on colonic mucosal lymphocytes by imunohistochemical
staining. Four µm thick sections of formalin-fixed, paraffin-embedded
tissues from proximal colon were mounted and deparaffinized according to
standard laboratory procedures. The stainings were performed in a routine
laboratory (Department of Pathology, Örebro University Hospital) using
an automated immunostaining instrument. The images were captured using
a microscope with a x20 objective lens, Leica DMRXA 2 equipped with a
digital camera; Leica DFC 330 FX and analysed by the macro program
Leica QUIPS (Leica Microsystems, Wetzlar, Germany). This program
interfaces with the Leica Qwin software; and a sequence of instructions for
repetitive image analyses was set up.
Comments:
Immunohistochemistry is widely used to determine protein expression and
to localize it within a tissue or cell. One of the major advantages with IHC
is that it can be performed on preserved tissues. We had access to the
archive at the Dept. of Pathology, Örebro University Hospital, and
therefore we chose this technique to characterize the inflammatory cells.
Traditionally, observer-dependent, semi-quantitative methods such as point
counting are used for immunohistochemical evaluation. This method is
time consuming and it is hard to define borders between different areas, as
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there are more chances of overlap between different areas and requires
highly trained individuals.
We chose the computerized image analysis that enables you to obtain
results in shorter period of time compared to point counting analysis, as
the computer calculates the positively stained areas based on predefined
limits or thresholds. It also gives the possibility to divide the colonic
mucosa in separate areas of interest without any overlap between them. In
addition the images can be saved and reanalyzed by a second evaluator
within the same fields of vision. However, this approach still requires
manual definition of thresholds for colour intensity to set background
areas and stained lymphocyte areas. It also requires manual definition of
different areas of subsections of colonic mucosa for the computer to
calculate positively stained areas as well as exclusion of falsely marked
areas.
Isolation of intraepithelial lymphocytes and lamina propria
lymphocytes (Papers II and V)
Fresh mucosal biopsies were treated with ethylenediaminetetraacetic acid
(EDTA) to break down the epithelial layer and thus release the
intraepithelial lymphocytes (IELs). The denuded biopsies were further
digested with collagenase to break down peptide bonds in collagen in the
lamina propria to release lamina propria lymphocytes (LPLs), with the
addition of DNAse to degrade DNA, as DNA released from dead cells into
the medium can cause cells to clump together resulting in clogging of
released lymphocytes.
The colonic biopsies were thoroughly washed with PBS. IELs were
isolated by incubation in EDTA-containing medium with constant stirring,
4 times 15 min, with collection of released cells from the first three
incubations. The cells released during the fourth incubation were discarded
to minimize contamination by LPLs. Isolated cells were passed twice
through a 100 µm and once through a 30 µm pore size nylon mesh strainer
in order to reduce mucus remnants and cell debris. They were kept on ice
while the denuded biopsies were further digested with complete collagenase
type VIII and DNAse I type IV for 1-1.5 hrs. Isolated LPLs were passed
through strainers as described above for IELs, and both LPLs and IELs
were washed twice, resuspended in PBS and kept on ice until further
analysis.
Comments:
In general, the cell yield was lower for IELs compared to LPLs, probably at
least partly due to mucus remnants in the cell suspension. Mucus remnants
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in the LPL and IEL cell suspensions also likely reduced surface staining, as
mucus might block the binding sites on the surface of cells for antibodies.
Several strategies were employed to reduce mucus and increase cell yield as
well as to obtain less viscous cell suspensions; (1) Dithiothreitol (DTT) and
different types of collagenase enzymes were used to degrade mucus.
However DTT and less pure collagenase markedly reduced cell surface
staining. DTT and less pure collagenase containing protease contaminants
might disrupt the cell surface binding sites for antibodies. Thus, DTT was
excluded and more pure collagenase containing less protease contaminants
was used during the IEL and LPL isolations. (2) Different cell strainers
were found to reduce the mucus levels in cell suspension, so they were used
during both the IEL and LPL isolations. The calcium chelating agent EDTA
was used to detach the epithelial cells from the biopsies, but it also disrupts
mucus. However, EDTA concentrations exceeding 1 mM inhibited the
enzymatic activity of the collagenase during the LPL isolation and we were
therefore restricted to use EDTA during the IEL isolation.
Flow Cytometric Analysis (Paper II)
The flow cytometry technique can determine the different characteristics of
cells within a mixed population by quantifying cell size, granularity or
irregularity, using fluorochrome labelled antibodies bound to the surface of
or within the cells. Thus we choose this technique to determine different
surface and intracellular markers and to characterize the
immunophenotype of IELs and LPLs separately.
Cells were incubated together with different fluorochrome labelled
monoclonal antibodies that binds to the protein of interest. Subsequently
the cells were passed through a laser that excite the fluorochrome and
emits fluorescence of specific wavelengths, which gives information on the
expression of particular proteins on the surface or within each cell. We
have performed 4-colour staining to identify different markers. Isolated
LPLs and IELs were incubated with an anti-Fc-receptor reagent to block
unspecific binding, followed by incubation with the monoclonal antibodies
or their respective isotype control antibodies. The isotype-matched controls
were used to estimate non-specific staining, and the unstained cells were
used to predict the gating of cells negative for the staining.
Stained cells were acquired on a Coulter Epics AltraTM flow cytometer
(Beckman Coulter, Fullerton, CA, USA) and analyzed using Kaluza
software (Beckman Coulter).
Comments:
Exclusion of DTT and use of more pure collagenase resulted in a normal
staining, that is, staining intensities comparable to peripheral blood
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lymphocytes. As mentioned above, different cell strainers were found to
reduce the mucus levels in the cell suspension, resulting in a much cleaner
cell suspension where antibodies could bind to cell surface markers much
more efficiently.
Due to technical difficulties some samples were excluded: During IELs
isolation and surface staining, some stained samples had to be excluded
from the analysis, due to < 1000 events for a particular marker, or samples
with mucus remnants that clogged the flow cytometer during acquisition.
To prevent clogging in the flow cytometer the machine was cleaned after
acquisition of every two stained samples. This was due to the impure mucosal cell suspension, which is not the case while acquiring more pure cell
suspensions such as from a cell line or peripheral blood lymphocytes.
Flow cytometric analysis gives information about the size and
granularity of the cells, which are defined by forward scatter (FS) and side
scatter (SS) respectively. During analysis the particular cell population is
gated in FS and SS and analyzed subsequently for expression of other
markers. Unlike peripheral blood lymphocytes, the mucosal cell suspension
contains a mixture of several cell populations including T- and B
lymphocytes but also plasma cells, NKT cells, epithelial cells and other
innate immune cells as well as cell debris that makes it difficult to locate a
specific lymphocyte population directly by FS and SS.
We excluded cellular debris based on FS and SS characteristics and the
lymphocyte gates were set by “back-gating” on CD3+ lymphocytes in
forward and side scatter. Analysis was performed on gated lymphocytes.
With the exception of the B lymphocytes and plasma cells, all lymphocyte
subpopulation characteristics were expressed as percentage of a gated, cell
surface marker defined population.
Real-time PCR (Papers III & IV)
Real-time reverse transcription PCR analysis quantifies the amount of
RNA being transcribed from the genome for protein production. Due to its
sensitivity to degradation, it is convenient to reverse transcribe RNA into
the complementary DNA (cDNA) which is a more stable form.
Subsequently cDNA is amplified by a PCR reaction to measure the
transcript levels of the genes.
In paper III, the level of T cell receptor excision circles (TRECs) was
analyzed in the colonic mucosa of CC and LC patients as a measurement
of recent thymic emigrants (RTE). Purified genomic DNA from colonic
biopsies was analyzed for TREC content by real-time PCR. As analysis of
TREC content is based on detection of genomic DNA that is expressed in
all cells, and the analysis was performed on total DNA from biopsies,
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rather than from purified T lymphocytes, we had to use a marker specific
for T cells to relate the TRECs amount. We therefore analyzed expression
of the CD3 gamma gene (i.e. CD3 gamma mRNA) to estimate the total
amount of T cells in the colonic biopsies, and this was used as a reference
for TREC analysis. The results were presented as relative quantification of
each sample as we were interested to compare TREC content in colonic
tissues from MC patients and controls. Real-Time PCR was performed
using the thermal cycler TaqMan 7900 Fast Real-time PCR System
(Applied Biosystems) with 7900 Fast Sequence Detection and Relative
Quantification software packages.
In paper IV, real-time reverse transcription PCR was used to investigate
the T helper (Th) cell and cytotoxic T lymphocyte (Tc) mucosal cytokine
profiles at the transcript level. The mRNA expression of the following
cytokines and transcription factors was determined: IL-1β, IL-4, IL-5, IL-6,
IL-10, IL-12p35, IL-17A, IL-21, IL-22, IL-23A, IFN-γ, TNF-α, T-bet,
RORC2, GAPDH and GUSB.
Real-time PCR was performed using the thermal cycler TaqMan 7500
Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA)
with 7500 Fast Sequence Detection and Relative Quantification software
packages.
Luminex (Papers IV & V)
A common method to analyze specific protein levels is the enzyme-linked
immunosorbant assay (ELISA), where 50-100 µL of sample is required for
analysis of each protein. Each mucosal biopsy homogenate yielded
approximately 200-250 µL of supernatant and due to the limited amount
of colonic tissues from MC patients we chose to use Luminex analysis,
which only requires 50 µL of sample for analysis for several proteins.
Luminex is a fluorescent-bead based method that allows the detection of
several analytes within a single sample at the same time. Color-coded
polystyrene or magnetic beads are used and each bead is precoated with an
antibody towards the specific protein that is analyzed in a sample. The
sample is passed through two lasers in a Luminex analyzer, where one laser
identifies each bead and the second laser identifies any of the reporter dyes
captured during assay.
In paper IV, to complement the transcription level data on T helper (Th)
cell and cytotoxic T lymphocyte (Tc) mucosal cytokine profiles, the
mucosal tissue protein levels of IL-1β, IL-5, IL-6, IL-10, IL-12p70, IL-17A,
IL-21, IL-23, IFN-γ and TNF-α were analysed using the xMAP technology
developed by Luminex® (Austin, TX, USA).
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In paper V, we were interested to investigate the possible effects of
soluble factors derived from the intestinal mucosa on T cell differentiation.
The cytokines related to different T cell subsets (e.g. Th1, Th2 and Th17)
were analyzed in supernatants of CD4+ PBL cultures, incubated with
soluble factors from colonic mucosa of MC and controls. The cytokines
analyzed were IL-1β, IL-4, IL-6, IL-10, IL-17A, IFN-γ and TNF-α.
All protein concentrations were determined using the Milliplex® Map
Kit according to the manufacturer’s instructions (Millipore, MA, USA).
Samples were read and analysed using a Luminex 200 TM with xPONENT
software.
Comments:
During our optimization of the method we found that the beads were not
detected while reading the samples in the Luminex analyser. We found that
this was due to the viscosity of the supernatant from mucosal tissues, as
centrifugation of the tissue homogenates at 10,000 rpm for 5 minutes as
well as a two fold dilution of sample supernatants with assay buffer during
incubation with beads resulted in appropriate bead count and analyte
detection.
Compared to 1 hr incubation at room temperature, overnight incubation
of the samples with beads at 4°C resulted in increased assay sensitivity for
analytes and an appropriate bead count.
Although the Luminex analysis detects several analytes at the same time,
there is limitation to the method as the combinatorial possibilities of
different cytokines within a certain panel is restricted due to the cross
reactivity of antibody with other cytokines as well as overlap in
wavelengths in the different fluorescences of the different beads Because of
this we were not able to mix the desired cytokines in a single panel.
According to the manufacturers, 100 different analytes can be analyzed
simultaneously by Luminex technology. However, we found difficulties to
get an appropriate bead count in multiplex bioassays analysing for more
than 10 analytes. The insufficient identification of beads by the Luminex
analyzer could be due to the complexity of multiplexing of large number of
cytokines.
Preparation of conditioned medium from the colonic mucosa
(Paper V)
In this study we were interested to investigate how the soluble factors
derived from the local colonic milieu from mucosa of CC patients and
controls affects peripheral T cells. Twelve fresh colonic biopsies were
treated with EDTA to remove the epithelial layer and IELs. Six of these
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denuded biopsies (DNB) were kept in serum free culture medium (RPMI1640, streptomycin, gentamycin, penicillin and HEPES) on ice until further
use, and the remaining six denuded biopsies were further digested with
collagenase and DNAse to extract lamina propria mononuclear cells
(LPMCs). Denuded biopsies and LPMCs were cultured in culture medium
overnight at 37°C, 5% CO2, to generate conditioned medium (CM). The
CM was tested for endotoxin and total protein levels, and stored at -80°C
until further use.
Comments:
We were interested to investigate soluble factors released by extracellular
matrix (ECM) from the colonic mucosa of CC patients and controls.
However we were unable to obtain cell free ECM from the colonic biopsies
and we therefore chose to investigate the soluble factors released by two
different preparations of the mucosa, the denuded biopsies and the LPMC
fraction. Due to the fact that only two CC patients could be recruited
within the time frame for this study, and thereby enabling generation of
only a fairly low volume of conditioned medium, we were unable to
analyze the conditioned medium separately from each individual.
Therefore the conditioned medium from the DNB and LPMC fractions
from CC patients and non-inflamed controls, respectively, were pooled.
We tested different concentrations of total protein amounts in CM from
DNB as well as LPMC from CC patients and controls to analyze the
correlation between the different amounts of total proteins in CM and T
cell responses.
T cell Proliferation and Cytokine Release Assay (Paper V)
A central point to this study was to recapitulate the in vivo situation as
closely as possible. To determine the influence of the local intestinal milieu
on newly recruited peripheral T cells into the colonic mucosa, we cultured
normal CD4+ peripheral blood lymphocytes (PBLs) with conditioned
medium derived from culture of denuded colonic biopsies and lamina
propria mononuclear cells.
In order to determine the influence of soluble factors from the colonic
mucosa, CD4+ PBLs were isolated by the Human CD4+ T cell Enrichment
Cocktail kit (STEMCELL Technologies, Grenoble, France). Subsequently,
CD4+ PBLs were stimulated with α-CD3/α-CD28 antibodies and were
incubated in a culture medium (RPMI-1640, HEPES, L-Glutamine, AB
Serum, streptomycin, gentamycin and penicillin) in either the absence or
presence of CM derived from DNB and LPMC fractions from colonic
mucosa of MC patients and non-inflamed controls. The cells were cultured
for 3 days at 37°C, 5% CO2, whereafter supernatants were harvested and
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stored at -80°C until determination of cytokine content by Luminex.
Similarly, T cell proliferation was measured after 3 days using the CellTiter
96® AQueous One Solution Cell Proliferation Assay (Promega, Madison,
WI, USA).
Comments:
According to the manufacturer, the Human CD4+ T cell Enrichment
Cocktail kit can be used for any volume of whole blood for CD4+ PBL
isolation. However we found that although the yield of CD4+ PBLs for
small volumes of whole blood (up to 4 mL) was according to the
manufacturer, larger volumes of whole blood (10-15 mL) did not fulfill
this pledge. In future experiments, we plan to use a buffy coat; a leukocyteenriched fraction of whole blood. This will convert the larger whole blood
volume into a smaller volume of buffy coat that can be used for CD4+ PBLs
enrichment. The approach for CD4+ PBLs enrichment from buffy coat will
still be the same as the one used for whole blood.
Statistical analysis
In general all data were presented as median values, as they were not
normally distributed. In paper I and II, data were also expressed as median
and interquartile range (IQR). The Mann-Whitney two-tailed nonparametrical test was used for analysis of statistical significances between
two groups (Paper I-IV). The Kruskal-Wallis one-way ANOVA test was
used to calculate the presence/absence of statistically significant differences
between more than two groups (Paper I). Statistical outliers were defined
by the Grubbs test (Paper III). The correlation between two variables was
calculated with the Spearman correlation analysis: semi-automated image
analysis and point counting (Paper I), different phenotype of lymphocytes
and age (Paper II), and degree of diarrhoea and cytokine mRNA expression
(Paper IV). Values of p≤0.05 were regarded as statistically significant.
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RESULTS AND DISCUSSION
This section provides a brief overview of the main findings in this thesis
(Fig. 5). As three of the papers are accepted but not yet published, and two
are not yet accepted for publication, the results are not shown in detail, but
rather generally discussed. Detailed descriptions of the results are presented
in the respective papers, and are referred to by their figures within the
manuscripts.
Figure 5. Schematic overview of the results in this thesis. The respective papers
are indicated with roman numerals. Results from immunohistochemistry and
flow cytometry analysis to characterize phenotype of mucosal lymphocytes are
presented in teal coloured text (Paper I and II). Analysis of T cell receptor excision circle (TREC) levels in the CD3+ T cell compartment in the colonic mucosa
is presented with blue text (Paper III). Results from analysis of Th1/Th2/Th17
and Tc1/Tc2/Tc17 associated mucosal cytokine profile are shown in black text
(Paper IV). Findings of the impact of the mucosal microenvironment on differentiation of peripheral CD4+ T cells are shown in red text (Paper V).
All the data presented here is in comparison to non-inflamed controls.
Increase; Decrease; = no change
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Phenotypic characterization of lymphocytes in the colonic mucosa
of collagenous colitis and lymphocytic colitis patients (Paper I &
Paper II)
In paper one; we characterized lymphocytes in the mucosa of CC and LC
patients using immunohistochemistry. There was a significant increase in
the amount of CD8+ T cells in the epithelium in both LC and CC patients
compared to controls, with the most pronounced rise seen in LC patients
(Paper I Fig. 3), whereas the amount of CD4+ T cells were markedly
reduced in the lamina propria (LP) of both LC and CC patients compared
to controls (Paper I Fig. 4).
The expression of the active/memory marker CD45RO and the
transcription factor Foxp3 that is involved in regulatory T cells (Treg)
differentiation, was more abundant in both the epithelium and LP in both
patient groups compared to controls (Paper I, Table III). Likewise,
abundant amounts of the CD30 marker, expressed on activated but not
resting lymphocytes, were observed in the LP of both patient groups
compared to controls (Paper I, Table III). Expression of the proliferation
marker Ki67 was significantly increased, but in contrast to CD45RO, this
was only seen in the epithelium of both CC and LC patients compared to
controls
In this study we only performed single antibody staining and therefore
we cannot say whether the increase in expression of Ki67 and CD45RO is
solely due to an increase in activated T cells or by other cells such as
monocytes, macrophages and granulocytes.
We therefore performed a detailed phenotypic characterization of freshly
isolated lamina propria and intraepithelial lymphocytes from colonic
biopsies from CC and LC patients compared to controls, using four-colour
flow cytometry, in Paper II. This allows determination of the expression of
particular markers on the surface or within each specific cell type. As
immunopathological data on UC is well documented, we used colonic
biopsies from UC patients as positive controls in this study.
The intraepithelial CD8+ T cells were markedly increased in LC patients
compared to controls and UC patients (Paper II, Fig. 1A). An increase was
also noted in CC and CC-HR patients (see Materials and Methods for
definition of the latter patient group) compared to controls, albeit not
statistically significant. The CD8+ population was also significantly
augmented in LC-HR patients compared to controls (Paper II, Fig. 1A). We
next analyzed the expression of the CD45RO marker within the CD8+ T
cells. The proportion of CD45RO+CD8+ T cells was not significantly
increased in any of the groups of MC patients compared to controls (Paper
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II, Fig. 1B), but a trend towards increased proportions were noted in CC
patients (Paper II, Fig. 1B). The proportion of Ki67+ cells within the CD8+
T cell population was significantly increased in CC patients compared to
controls (Paper II, Fig. 1D).
The proportions of CD4+ IELs in MC patients were not significantly
changed from controls (Paper II Fig. 2A), whereas UC patients
demonstrated a significantly increased proportion compared to controls as
well as to LC and CC patients.
In contrast to IELs, we did not find any significant differences in the
CD8+ lamina propria T cells between the different colitis groups and
controls (Paper II Fig. 4A). However, the proportions of CD45RO+CD8+
LP T cells were significantly increased in CC patients compared to controls
and UC patients (Paper II Fig. 4B). CC patients also had markedly
increased proportions of Ki67+CD8+ LP T cells compared to controls.
There was a trend towards increased proportions of Ki67+CD8+ T cells in
LC, LC-HR and UC patients, but it did not reach statistical significance
(Paper II Fig. 4D).
A trend towards reduced proportions of CD4+ T cells in lamina propria
were observed in CC, CC-HR and LC patients compared to controls
(Paper II Fig. 5A). However the proportions of CD45RO+CD4+ LP T cells
were markedly increased in CC patients compared to controls (Paper II Fig.
5B). The proportions of Ki67+CD4+ LP T cells were significantly increased
in CC, LC and LC-HR patients, but this proportion was even higher in UC
patients compared to controls (Paper II Fig. 5B).
Furthermore, we observed normalized proportions of CD45RO+ and
Ki67+ expression within CD4+ and CD8+ IEL and LPL populations in CC
patients in histological remission compared to CC patients, but not in the
LC-HR patients compared to LC patients.
Discussion:
Generally both UC and CD are believed to be driven by aberrant CD4+ IEL
and LPL responses (116, 120). In accordance, we also noted heavy
infiltration of CD4+ IELs in UC patients but not in MC patients. Instead,
the MC patients are presented with heavy infiltration of CD8+ IELs. Our
findings of increased proportions of CD8+ IELs in CC and especially LC
patients compared to controls in paper I and paper II are in agreement with
previous immunohistochemical studies (96-97, 125). However,
immunohistochemical studies by Mosnier et al and Armes et al state that
CD4+ T cells were more numerous than CD8+ T cells in the lamina propria
of MC patients; although none of these two studies present these data in
their results (96, 125). In contrast we found reduced or unaltered amounts
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of CD4+ T cells in the epithelium and lamina propria compartment of MC
patients compared to controls in our immunohistochemistry and flow
cytometry analysis. Our data on CD4+ T cells also differ from the findings
in CC patients by flow cytometric analysis by Wagner et al., but they did
not analyze IELs and LPLs separately (100). The reduced proportions
CD4+ IELs in LC and CD4+ LPLs in both CC and LC patients suggest that
these cells might undergo apoptosis. However, in CC patients this can also
partly be explained by the space occupied by the thick collagen layer.
Along the same thoughts, the increased numbers of locally expanded CD8+
T cells in MC patients could be a result of decreased apoptosis of these
cells. Whether there is an aberrant apoptosis of mucosal T cells in MC
patients needs to be investigated, and such information is relevant to
explain the disturbed T cell responses in mucosa of MC patients.
Furthermore we found increased proportions of CD8+ IELs and LPLs
expressing CD45RO and the proliferation marker Ki67 in both CC and LC
patients compared to controls, which was not the case in UC patients. MC
patients had increased proportions of Ki67+ CD4+ IELs and CD4+ LPLs
compared to controls, but theses populations were much larger in UC
patients. Collectively these data suggests that UC and MC are driven by
different T cell subsets.
Generally the proportion of active/memory T cells gradually increases
with age. As the MC patients are mostly of old age, we were concerned
that the observed increased proportions of CD45RO+ and Ki67+ T cells
was due to older age in the MC patient groups compared to the controls.
Our correlation analysis between CD45RO+ and Ki67+ T cell frequencies
and age in the control group, consisting of individuals with a wider age
span, demonstrated a significant correlation between higher proportions of
CD45RO+ within the CD4+ T cell population and increasing age (Paper II,
Fig. 2C, 5C), but importantly, this was not found in the CD8+CD45RO+ or
CD8+ Ki67+T cells nor in the CD4+Ki67+ cells (Paper II, Fig. 1C, 4C),
suggesting that the changes observed in the latter are indeed due to the
disease itself. These findings collectively
suggest that the
immunopathological responses in MC are driven by CD8+ T cells. It has
been demonstrated that diversion of the faecal stream via a loop ileostomy
leads to a normalized clinical and histopathological response in CC
patients, suggesting that luminal agent might be involved in MC
pathogenesis (20, 90). Increased amounts of antibodies against the Yersinia
bacterium have been detected in CC patients compared to controls (92). In
addition, increased uptake and altered mucosal reactivity to nonpathogenic bacteria in CC patients compared to controls (94), suggest the
involvement of microbes in MC pathology.
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The cytotoxic activity of CD8+ T cells directed against epithelial cells
containing endogenous bacteria or luminal agents may promote
proinflammatory responses leading to initiation and/or exacerbation of
inflammatory conditions in the intestine, as seen in MC. It is possible that
these luminal agents are taken up by intestinal epithelial cells and thereby
these exogenous antigens would be presented by MHC class II molecules
and recognized by CD4+ rather than CD8+ T cells. The effector CD4+ T
cells could then target these antigens as well as activate the CD8+ T cells
through their cytokine production.
The regulatory T cells (Tregs) are key suppressors of the active immune
responses. Increased frequencies of CD4+CD25+ Foxp3+ Tregs are found in
the inflamed mucosa of UC patients and their numbers increases with the
disease activity in UC patients (123-124, 126). In accordance to these
findings, our immunohistochemistry data (paper I) as well as others show
significantly increased amounts of Foxp3+ cells in both the epithelium and
lamina propria compartment of CC and LC patients compared to controls
(127). However there are no data showing whether the increase in Foxp3
expression is from CD4+ or CD8+ T cells in MC patients and warrants
further studies. Recently Veltkamp et al. reported on a significant increase
in apoptosis rate of CD4+Foxp3+ Tregs in MC patients but this was even
greater in both UC and CD patients compared to non-inflamed controls,
resulting in reduced numbers of local Tregs (124). This suggests that
despite the increased frequency of Tregs in the mucosa of IBD patients,
they are relatively ineffective in suppressing the inflammation because of
their much higher rate of apoptosis.
In addition to CC and LC patients, we have identified and
phenotypically characterized two previously not described groups of LC
and CC patients with active clinical disease, but in histopathological
remission. We found clearly reduced proportions of both proliferating and
activated/memory CD4+ and CD8+ IELs and LPLs in CC patients in
histopathological
remission
compared
to
CC
patients
with
histopathological changes, indicating that CC-HR patients display signs of
normalized proportions of mucosal T cells. Thereby other factors seem to
be involved in triggering the clinical symptoms of active CC in patients in
histopathological remission. Interestingly, no significant differences was
noted in the proportions of CD45RO+ or Ki67+ cells between LC-HR and
LC patients, suggesting that in contrast to CC-HR, mucosal T cells in LCHR were as active as in LC patients.
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Analysis of T cell receptor excision circle (TREC) levels in the
CD3+ T cell compartment in the colonic mucosa of collagenous
colitis and lymphocytic colitis patients (Paper III)
After having demonstrated the heavy infiltration of CD8+ IELs in the
mucosa of CC and especially in LC patients by immunohistochemistry and
flow cytometry, we were interested to investigate whether the increased
numbers of CD8+ IELs is due to a greater influx of T cells or a pronounced
expansion of local resident T cells in the mucosa of CC and LC patients.
Such information is a first step toward understanding whether the
activating antigen(s) reside in the mucosa, or is rather transported via e.g.
dendritic cells to the draining lymph nodes to activate naïve T cells there,
thereby adding information on the nature of the chronic colonic
inflammation in the mucosa of MC patients.
In this study we investigated recent thymic emigrants by measuring the
TREC content in the colonic biopsies relative to the amount of the CD3+ T
cells, in CC and LC patients compared to controls, using real-time PCR
analysis.
We noted reduced median TREC levels in CC and LC as well as in LCHR patients compared to controls (Paper III Fig. 1). However, the changes
did not reach statistical significance. There were no differences in the
median TREC levels in either CC-HR or LC-HR patients compared to CC
and LC patients with histopathologically active disease.
Discussion:
Increased infiltration of T cells in the intestinal mucosa is evident in both
MC and UC patients compared to non-inflamed controls. Our previous
study measuring recent thymic emigrants by TREC analysis demonstrated
enhanced TREC levels in the mucosal lymphocytes from UC patients
compared to controls, suggesting that recent thymic emigrants are recruited
to the inflamed mucosa of UC patients (78). Increased TREC levels in
colonic biopsies from UC patients in paper III is in accordance with our
previous findings. In contrast to UC patients, we found reduced level of
TRECs in the colonic mucosa of MC patients. These reduced TREC levels
and our previous observations in paper II on elevated proportions of both
CD8+ and CD4+ LPLs/IELs expressing the Ki67+ phenotype, identifying
proliferating cells, in both CC and LC patients strongly suggest that MC
patients have local expansion of resident T cells rather than migration of
peripheral lymphocytes to the inflamed mucosa. Furthermore, increased
proportions of CD8+CD45RO+ IELs and LPLs in paper II and the
previously shown involvement of luminal agents and bacteria in MC
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pathology (90, 92, 94), suggests that constant exposure of local mucosal or
luminal agents to the activated T cells could lead to proinflammatory
responses resulting in epithelial damage and chronic inflammation in the
intestinal mucosa, as observed in MC.
These results indicate major differences in pathogenesis between UC/CD
patients and MC patients, where the latter would likely not benefit from
treatment with antibodies blocking homing to the intestinal mucosa, where
e.g. Natalizumab have shown good responses in CD patients (128). This
also support our theory that it is indeed a local mucosal antigen triggering
the T cell activation in MC pathology, possibly by one or several
microbiota-derived antigens, or by drugs that may aggravate MC in some
patients (91).
T helper (Th) 1/Th17 and cytotoxic T lymphocyte (Tc) Tc1/Tc17
associated cytokine profile at messenger and protein levels in
the colonic mucosa of collagenous colitis and lymphocytic colitis
patients (Paper IV)
Based on our flow cytometric data that revealed increased local T cell
responses in the lamina propria and intraepithelial compartment of CC and
LC patients, demonstrated as elevated expression of CD45RO and Ki67,
we continued to determine the functional profile of Th and Tc in the
colonic mucosa of CC and LC patients. We investigated the expression of
different Th1/Th2/Th17 and Tc1/Tc2/Tc17 cell associated cytokines and
transcription factors at both the messenger and protein levels in the MC
patients compared to controls, with UC patients serving as positive controls,
using real-time reverse transcription PCR and Luminex® analysis of multiple
cytokines in the same colonic biopsies.
We found markedly up regulated mucosal mRNA levels of the Th1/Tc1
associated cytokine IFN-γ in CC, LC as well as UC patients compared to
controls (Paper IV Fig. 1). Likewise, the IL-12/IL-35 subunit p35 was also
significantly up regulated in CC and LC patients, although at lower
magnitudes, but was not significantly altered in UC patients compared to
controls (Paper IV Fig. 1). Interestingly transcript levels of both of these
cytokine were higher in MC patients than in UC patients. In accordance
with the Th1/Tc1 cytokine profile in CC and LC patients, the mRNA levels
of T-bet, transcription factor for Th1/Tc1 maturation was also significantly
upregulated in CC and to lesser extent in LC patients (Paper IV Fig. 1).
However, no significant differences were noted in IFN-γ and IL-12p70
protein levels in patient groups and controls (Paper IV Fig. 2).
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The proinflammatory cytokines IL-6 and IL-1β are involved in
differentiation of human Th17/Tc17 cells. mRNA levels for IL-6 was
significantly up regulated in CC, LC and LC-HR patients but not as much
as in UC patients compared to controls (Paper IV Fig. 3). Increased levels
of IL-1β transcript were noted in CC patients but less than in UC patients.
In contrast, no differences were found in LC patients compared to controls
(Paper IV Fig. 3). IL-23 transcript levels were also up regulated in CC and
LC patients compared to controls, although at lower magnitude (Paper IV
Fig. 3) The transcript levels of one of the principle cytokine produced by
Th17 and Tc17 cells IL-17A, was markedly up regulated in both CC and
LC patients compared to controls, but less than in UC patients (Paper IV
Fig. 3). Furthermore, mucosal mRNA levels of the Th17/Tc17 associated
cytokines IL-21 and IL-22 were also significantly up regulated in both CC
and LC patients compared to controls, albeit less than in UC patients
(Paper IV Fig. 3). In contrast, the Th17 cell attracting chemokine CCL20
was not significantly up regulated in CC and LC patients, but in UC
patients compared to controls. In support of the mRNA data, significantly
enhanced IL-6 protein levels were also observed in CC patients and a trend
towards increased levels was noted in UC but not LC patients compared to
controls (Paper IV Fig. 4). Similarly, significantly increased amounts of IL21 protein was observed in both CC and LC patients as well as a trend
towards increased levels in UC patients compared to controls (Paper IV
Fig. 4).
Although the transcript levels of the pro-inflammatory cytokine TNF-α
was not significantly upregulated in any of the colitis groups compared to
controls, TNF-α protein expression was significantly increased in CC and
UC patients an a trend towards increased levels was noted in LC patients
compared to controls (Paper IV Fig. 5B).
In contrast, neither mRNA nor protein levels of the Th2/Tc2 associated
cytokines IL-4, IL-5 nor IL-10 were significantly altered in any of the
colitis groups compared to the controls.
Furthermore, we investigated the possible correlation between mucosal
mRNA expression of cytokines and clinical activity of the disease in MC
patients. We found significant positive correlations between increased
frequencies of bowel movements and enhanced transcript levels of IFN-γ,
IL-21 and IL-22 in MC patients (Paper IV Fig. 7).
We also noted that LC-HR but especially CC-HR patients had
normalized levels of all cytokines investigated compared to LC and CC
patients respectively.
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Discussion:
Our mucosal mRNA analysis shows that the transcript levels of the Th1
and Tc1 associated cytokines IFN-γ and IL-12 were higher in the colonic
mucosa of MC patients than in UC patients. This is likely due to the
increased number of CD8+ IELs, as well as the increased proportion of
CD8+ IELs and LPLs expressing the active/memory marker CD45RO and
the proliferation marker Ki67 in the mucosa of both CC and LC patients, as
the frequencies of CD4+ T cells were unaltered or reduced in MC patients. On
the other hand, increased mucosal mRNA levels of the Th17/Tc17
associated cytokines IL-17A, IL-21 and IL-22 were more profound in UC
patients than in MC patients. It is likely that Th17 cells secrete large
amounts of IL-17A, IL-21 and IL-22 as seen in UC patients and there could
be low number of Th17 cells in MC as CD4+ T cells are reduced or
unchanged in these patients. However, these cytokine may also be derived
from Tc17 cells, as increased proportions of activated CD8+ T cells are
present in MC patients. So far there are no comparative studies for
secretion of cytokine levels between Th17 cells and Tc17 cells. In addition,
apart from Th17/Tc17 cells there are various other immune cellular
sources for secretion of these cytokines, such as NKT cells, monocytes and
macrophages (49).
The markedly up regulated IFN-γ mRNA levels as well as increased
protein levels of TNF-α in MC patients are in accordance with the study by
Tagkalidis et al. (98). Furthermore our study confirms the correlation
between transcription levels of mucosal IFN-γ and the degree of diarrhoea
in MC patients previously described by Tagkalidis et. al. (98). IFN-γ
activates macrophages to release proinflammatory cytokines like TNF-α,
IL-1β and IL-6, which in turn sustain and increase local inflammatory
responses (129). IFN-γ also plays a vital role in lymphocyte infiltration in
the gut, as it regulates the production of IEL-attracting chemokines like
CXCL10 (IP-10) and CXCL9 (MIG) by intestinal epithelial cells (130).
Significantly increased amounts of CXCL10 (IP-10) and CXCL9 (MIG)
have been reported in the epithelium and lamina propria of LC patients
(130). It has been shown that both these chemokines chemoattracts
activated T lymphocytes (131-132). The common receptor for CXCL10
(IP-10) and CXCL9 (MIG), CXCR3, is highly expressed on IELs and
activated T cells (130, 133), suggesting that IFN-γ indirectly regulates the
lymphocyte infiltration into the gut. TNF-α has been shown to cause an
increase in intestinal epithelial permeability in Crohn’s disease (134).
mRNA and protein levels of IL-21, being enhanced in both CC and LC
patients, are secreted by Th17 and Tc17 cells. IL-21 has pleiotropic effects
on immune cells; for example it enhances IFN-γ production by mucosal T
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cells and NK cells (68, 135) and promotes the lytic activity of CD8+ CTLs
(135). We found a positive correlation mucosal mRNA level of IL-21 and
IL-22 and the number of bowel movements per day in MC patients. In
addition to Th17 cells, IL-22 is also expressed by Th1 lymphocytes, CD8+
Tc17 cells and NK cells as well as CD11+ DCs and innate lymphoid cells
(49, 70, 136). IL-22 induces the production of IL-8 and TNF-α by
epithelial cells and colonic myofibroblasts, and increases the epithelial
barrier function by activation of proliferative and/or anti-apoptotic
programs (136). Increased activity of colonic myofibroblasts by IL-22
might result in excess production and deposition of collagen in the
basement membrane. Whether IL-22 is involved in repairing epithelial cell
damage in CC and LC or in collagen layer deposition in CC remains
unclear, but increased numbers or activity of myofibroblasts has been
reported in the mucosa of CC patients (105), indicating that IL-22 might
promote inflammation rather than protection in the colonic mucosa of CC
patients. Our data on markedly up regulated transcript levels of the
chemokine CCL20, which is induced by Th17 cytokines and attracts
CCR6+ Th17 cells, in UC patients but not in MC patients, suggest that MC
patients have local expansion of resident T cells rather than influx of Th17
cells in the gut mucosa. This is also supported by our findings in the studies
on phenotypic characterization of the mucosal T cells (Papers I and II) as
well as the study on TREC levels (Paper III). Data on whether CCL20 also
attracts Tc17 cells are unknown, but they do express both CCL20 and the
CCL20 receptor CCR6, indicating that they can respond to CCL20 (69,
71).
In line with our flow cytometry data on normalized proportions of
activated mucosal T cells in CC-HR patients but not in LC-HR patients
compared to histopathologically active CC and LC patients respectively,
we found that LC-HR but especially CC-HR patients had normalized levels
of all cytokines investigated. Apparently other factors have to contribute to
the clinical symptoms in MC patients in histological remission: One
possibility is bile acid malabsorption, as it has been shown to be associated
with MC in 27-44% of CC patients and in 9-60% of LC patients (6) and
aggravates clinical symptoms (95).
In this study the cytokine levels were measured in colonic biopsies rather
than on isolated lymphocytes. Therefore it is difficult to draw any firm
conclusions on whether the cytokines analyzed stems mainly from T helper
cells or CTLs. Cytokine analysis on sorted CD4+ and CD8+ IELs and LPLs
from colonic mucosa of MC patients would provide relevant information
on whether the immune responses are mainly derived from CD8+ T cells.
Furthermore, elucidating the cytotoxic activity of CD8+ T cells in the
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colonic mucosa of MC patients by measuring perforin and granzyme
expression would provide further knowledge to understand the nature of
the CD8+ T cells in MC patients.
The role of soluble factors from the colonic mucosa of collagenous
patients in the regulation of effector T cells (Paper V)
We next analyzed the impact of the colonic milieu in collagenous colitis
patients on peripheral T lymphocyte activation and differentiation. We
developed a novel system that mimics the in vivo exposure of newly
recruited peripheral blood T cells to the soluble factors derived from the
colonic milieu of normal individuals compared to CC patients with an
inflamed mucosa. Anti-CD3/anti-CD28 stimulated normal CD4+ peripheral
blood T cells were incubated in the presence or absence of conditioned
medium (CM) derived from the culture of denuded colonic biopsies (DNB)
or isolated colonic lamina propria mononuclear cells (LPMCs). T cell
proliferation as well as secretion of effector T cell cytokines was analysed.
We found reduced proliferation of peripheral CD4+ T cells exposed to
CM from the colonic mucosa of both non-inflamed controls and CC
patients. This inhibition in proliferation was however less pronounced with
DNB-CM derived from CC patients compared to non-inflamed control
mucosa (Paper V Fig. 1). In contrast, LPMC-CM derived from CC patients
was more effective in inhibiting proliferation compared to LPMC-CM
derived from non-inflamed controls (Paper V Fig. 2).
We next analyzed effector T cell cytokine production by α-CD3/α-CD28
stimulated CD4+ peripheral T cells in the presence of soluble factors
derived from the colonic mucosa. We found increased production of the
pro-inflammatory cytokines IL-17A, IFN-γ, TNF-α and IL-6 as well as the
anti-inflammatory cytokines IL-4 and IL-10 by peripheral T cells in the
presence of CM generated from the culture of both DNB and LPMC
fractions from the colonic mucosa of CC patients compared to DNB-CM
and LPMC-CM from non-inflamed controls (Paper V Fig. 3, Fig.4).
Discussion:
Generally normal human intestinal LP T cells show reduced proliferation
upon TcR/CD3 stimulation compared to peripheral T cells, whereas CD2
or CD28 stimulation results in strong proliferation (42, 137). However LP
T cells from patients with UC and CD showed enhanced proliferation
compared with controls upon stimulation with CD3 in the presence of IL-2
(138).
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This behaviour of mucosal T cells is thought to be influenced by the
mucosal microenvironment (137). Our data on reduced inhibition of CD4+
peripheral T cell proliferation by soluble factors derived from denuded
biopsies from the inflamed colonic mucosa of CC patients compared to
controls is in accordance with the findings by Huff et al. on stroma
conditioned medium from inflamed Crohn’s disease mucosa (139).
Similarly, our findings on enhanced production of IFN-γ and IL-17A by
CD4+ peripheral T cell in the presences of soluble factors derived from
denuded biopsies and LPMC fractions from inflamed CC mucosa
compared to controls are in line with the observations in that study (139).
Interestingly, we also observed increased production of anti-inflammatory
cytokines IL-4 and IL-10 by CD4+ peripheral T cells in the presences of
both CMs derived from DNB and LPMCs fractions, from inflamed CC
mucosa compared to controls. No data on the secretion of these cytokines
are available in the Huff study. The increased production of IL-4 and IL-10
can be interpreted as that the local colonic milieu in CC patients makes an
attempt to ameliorate the inflammatory response. IL-10 inhibits T cell
proliferation as well as cytokine production (140). The recorded reduced
proliferation of peripheral T cells could be due to enhanced production of
IL-10 by peripheral T cells in the presence of CC-LPMC-CM but not CCDNB-CM, suggesting that the composition in the two types of CM differ.
Additional factors apart from IL-10 are also likely involved in the
immunoregulatory effects of colonic CM on T cell proliferation and
differentiation. As we found enhanced levels of IFN-γ, IL-17A and TNF-α,
this suggests that in addition to IL-10 and other immunoregulatory
molecules, there are also immune-stimulating molecules that drive the
synthesis of these pro-inflammatory cytokines.
Altogether these data suggests that the local mucosal microenvironment
is impaired in its’ ability to regulate the adaptive immune responses in MC
patients, similarly to what has previously been demonstrated in IBD
patients (139). Nevertheless, in the light of our previous studies identifying
the CD8+ T cells as a more likely pathogenic T cells subset in MC, studies
on the effect of soluble factors from inflamed mucosa of MC patients on
CD8+ T cell activation and differentiation will provide a further
understanding on whether they are the main player in driving the
immunopathology in MC or not
In the present study more patients as well as additional healthy blood
donors needs to be investigated to draw any firm conclusions on the
impact of the mucosal milieu on the differentiation of the peripheral CD4+
and CD8+ T cells, as alluded to above. Our in vitro model can be useful for
analysis of the immunopathogenetic mechanisms behind the findings of
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worsened disease in patients being treated with certain groups of drugs
such as NSAIDS and proton pump inhibitors. In addition, the effect(s) of
existing drugs on T cell differentiation in the intestinal mucosa can be
investigated, thereby aiding in the decision on optimal doses required for
suppression of T cell inflammatory responses in MC patients.
Limitations in the studies performed in this thesis.
The major limitation to the studies performed in this thesis is the small
cohorts of MC patients. The various reasons in difficulties to recruit these
patients are discussed in the materials and methods section. In addition,
most of the studies were restricted due to the small amount of tissues
available as colonic biopsies were used to perform experiments. Unlike
IBD, surgery is very rare in MC patients, and therefore there is no resection
material available for experimental purposes.
Conclusions on CD8+ T cell mediated pathology in MC are mainly
based on the descriptive studies. However, the descriptive mapping of
immune responses were (and are still) incomplete, but necessary to
understand the pathophysiology in MC, Therefore we felt that this type of
information is a very important step to unravel the underlying pathogenic
mechanisms, as well as for planning future more mechanistic studies in
MC.
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GENERAL DISCUSSION
Is MC and IBD a similar disease?
Inflammatory bowel disease and microscopic colitis are both chronic
inflammatory conditions of the intestine. MC is considered a mild form of
colitis as the intestinal inflammation in MC is more subtle compared to
ulcerative colitis and Crohn´s disease. Clinically MC is characterized by
chronic non-bloody, watery diarrhoea, whereas UC is characterized by
bloody diarrhoea. Despite the fact that MC has a subtler inflammation, it
severely reduces the quality of life of these patients as they often have
fatigue, nausea weight loss, abdominal pain and urgency as well as
nocturnal diarrhoea (11).
One important difference is that most of the cases in MC are treatable
(albeit probably not curable) and hardly ever lead to life-threatening
complications as are seen in UC and CD. The age at onset of IBD is
relatively low, with the peak incidence at 20-30 years of age, with an
almost identical disease distribution in males and females (141). In
contrast, MC most commonly affects middle aged or elderly individuals
with a noticeable female dominance, the peak incidence being around 6065 year of age (6). One could think that the female dominance for this age
group in MC is possibly due to the hormonal changes that occur after
menopause in females. Interestingly it has been reported that the main
incidence peak for CC and LC in females is 60-69 years, whereas in males
the incidence peak is around 70-79 years of age. There is also a second
lower incidence peak for CC in females at 80-89 years (142). To get
further insight into the MC pathophysiology, it might be relevant to
understand the nature of the physiological changes in the body taking place
at these ages.
What are the factors involved in MC pathology?
The pathogenesis of IBD is thought to be due to exaggerated immune
responses to intestinal microbes in a genetically susceptible host (116, 120).
However due to the so far restricted pathophysiological data in MC, there
are no firm suggested mechanism(s) on MC pathology. It has been
postulated that MC is caused by disturbed immune responses to various
luminal antigen(s), such as drugs, gluten or infectious agents, in
predisposed individuals (6, 13). Generally, people of older age tend to
consume medications for other complications, and their adverse effects
might lead to diarrhoea. Nonsteroidal anti-inflammatory drugs (NSAIDs),
proton pump inhibitors, aspirin and selective serotonin reuptake inhibitors
have all been shown to be associated with microscopic colitis (91).
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Hepatobiliary complications such as primary sclerosing cholangitis (PSC)
have been associated with IBD (143). The hepatobiliary complications in
MC need to be investigated. However bile acid malapsorption has been
associated with a fraction of CC and LC patients (6), and shown to
aggravate clinical symptoms in MC patients (95).
Are there similar immunopathological responses in MC and Celiac disease?
MC is often associated with celiac disease. It has been reported that
33% of celiac disease patients have histological changes in the colonic
mucosa that are found in microscopic colitis, reviewed in (13). Celiac
disease is a T cell mediated immune response against dietary gluten and
causes inflammation in the small intestine whereas MC affects the large
intestine. Celiac disease patients have heavy infiltration of CD8+ IELs that
destroy the epithelium. In celiac disease, CD8+ IELs destroy the stressed
intestinal epithelial cells expressing the MHC class I polypeptide related
molecules MICA and MICB (144). Furthermore stressed intestinal
enterocytes produce large amounts of IL-15, which up regulates NKG2D
expression on CD8+ IELs. This enables cytotoxic killing of epithelial cells
expressing the NKG2D ligand (144). Our findings in this thesis suggest
that in contrast to IBD but similarly to celiac disease, immunopathological
responses in MC are mainly derived from CD8+ T cells. In MC, CD8+ IELs
may carry out cytotoxic activity as in celiac disease, but in contrast to
celiac disease, most likely they target stressed epithelial cells or epithelial
cells containing endogenous bacteria rather than dietary antigens, as the
uptake of the later occurs in the small intestine.
Are local resident T cells culprits in MC immunopathology?
Although we did not find increased numbers of CD4+ T cells, they had a
phenotype typical of activated lymphocytes, i.e. increased proportions of
CD45RO+ and Ki67+ CD4+ IELs and LPLs were found in MC patients, as
well as increased production of Th1/Th17 associated cytokines in the
mucosa of these patients. In addition, our TRECs data support the notion
that mucosal T cells in MC is locally expanded, rather than a result of
enhanced migration of peripheral lymphocytes to the inflamed mucosa.
Constant exposure of the activated T cells to luminal agents such as
infectious agents, drugs or dietary antigen such as gluten likely leads to
proinflammatory responses resulting in epithelial damage and chronic
inflammation in the intestinal mucosa, as observed in MC. However, as
alluded to above, the antigens in MC are less likely to be derived from
dietary antigens, as the inflammation is limited to the colon. The
previously observed epithelial damage that leads to increased permeability
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in MC (7) could be due to cytotoxic activity of CD8+ T cells or the
cytokines secreted by both CD8+ and CD4+ T cells.
Does context matter for vitamin A to regulate immune responses?
Recently vitamin A and its derivative retinoic acid (RA) and its role in
the immune system have been of interest to several researchers, as RA
seems to be a key inducer of gut homing receptors (145). In addition,
accumulating evidence show that retinoic acid increases the differentiation
of naïve T cells into inducible FOXP3+ regulatory T cells (Treg) and
inhibits their differentiation into pro-inflammatory Th17 cells (146-147).
Interestingly, in contrast to this it was recently shown that RA together
with IL-15 suppress Foxp3+ Treg induction and promotes differentiation of
T helper cells to Th1 and Th17 cells as well as induces IL-17 and IFN-γ
production (148). Future studies elucidating the role of IL-15 and vitamin
A in MC are relevant to understand the basic immunopathophysiology of
this disease.
Does the mucosal microenvironment affect the T cell responses?
Our data on increased production of both pro- and anti-inflammatory
cytokines by peripheral CD4+ T cells in the presence of soluble factors
derived from the inflamed mucosa of CC patients compared to controls
suggest that the local colonic milieu in CC patients is in fact attempting to
counteract the inflammatory response. The identity of these regulatory
soluble factors from the colonic mucosa is mostly unknown, and it will be
very valuable to determine the protein profile by proteomics to reveal any
immunoregulatory molecules in MC that keeps the inflammation at bay in
contrast to IBD patients. One obvious immunoregulatory cytokine whose
abundance and activity in the colonic mucosa of MC patients should be
investigated is TGF-β. Apart from proteins, the recently appreciated post
transcriptional regulators in the form of non-coding miroRNAs (miRNAs)
has been implicated as important regulators of inflammatory responses
(149), and their role in MC needs to be investigated.
Is histological remission sufficient to get rid of the clinical symptoms in
MC?
In addition to CC and LC patients, in this thesis work we have identified
previously groups of LC and CC patients with active clinical disease, but in
histopathological remission that were previously not described. Our flow
cytometry data and mucosal cytokine expression data show that LC-HR
but especially CC-HR patients display normalized proportions of activated
mucosal T cells and mucosal cytokine expression compared to LC and CC
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patients with histopathological changes. Apparently there are other factors
contributing to the clinical symptoms in these patients. As discussed above,
bile acid malapsorption could be one of them and therefore bile absorption
would be interesting to investigate in these patient subgroups. Further
studies are clearly required to investigate the mechanisms behind the
clinical symptoms in MC patients with histological remission.
Is CC and LC are two separate entities or different manifestation of same
disease?
As CC and LC have many clinical similarities and share
histopathological features, except for the subepithelial collagen layer found
in CC, it has been discussed whether LC and CC are two separate but
related disorders, or the same disease seen in different stages of
development. Conversion of LC to CC or the opposite has been reported,
but only infrequently (5). Our flow cytometry based phenotypic
characterization of the mucosal lymphocytes support the notion that CC
and LC are two separate entities. It has been reported that CC patients
present with more aggressive clinical symptoms compared to LC patients
and this was also reflected in our flow cytometry data, as CC patients had
higher proportion of CD8+ IELs and LPLs expressing CD45RO and Ki67
compared to LC patients. However we did not find any significant
differences in the expression of mucosal cytokines in CC and LC.
Why is the inflammation in MC subtler than in IBD?
It has been proposed that in IBD the immunopathological response are
mainly orchestrated by CD4+ T cells. In contrast, our findings in this thesis
support that in MC the immunopathological responses are mainly
mediated by CD8+ T cells. A recently identified subset of Th17 cells that
coproduces the Th1 cytokine IFN-γ have been implicated in the
immunopathogenesis of IBD (64). The IL-17 cytokine activates epithelial
cells, macrophages and fibroblasts to release proinflammatory chemokines
and cytokines that leads to recruitment and accumulation of mainly
neutrophilic granulocytes but also lymphocytes and DCs and perpetuates
the inflammation in IBD (51). It is possible that CD8+ T cells do not
produce the large amount of cytokines associated with Th17 and Th1
subsets. As it is believed that in IBD, aberrant immune responses are
directed to intestinal microbiota, innate immune cells such as macrophages
and neutrophils may initiate the immune responses against bacteria. Both
macrophage and neutrophils have been implicated in tissue damage. In the
majority of MC cases there is limited infiltration of neutrophils (150). In
contrast, a common feature of UC is infiltration of neutrophils in the
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epithelium accompanied by cryptitis, crypt abscesses leading to permanent
distortion of crypts (120). This might be one explanation to why the
inflammation in MC is subtler than UC.
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FUTURE PERSPECTIVES
The descriptive mapping of immune responses was missing in the MC and
the work in this thesis has demonstrated aberrant adaptive immune responses in the colonic musoca of MC patients.
Our data on disturbed CD4 + and CD8+ T cell numbers indicates that
this could be due to an aberrant apoptosis of mucosal T cells in MC
patients, and thus this warrants investigation.
We have demonstrated increased expression of Foxp3+ regulatory T cells
in the mucosa of MC patients, but we do not know whether the Foxp3
expression stems from CD4+ or CD8+ T cells and this warrants further
studies. During our flow cytometry analysis, we were restricted in making
choices for immunostaining due to the limited amounts of fresh
lymphocytes, as they are isolated from colonic biopsies. Future studies
analyzing both the frequency and efficiency of CD4+ and CD8+ Tregs, as
well as the regulatability of the mucosal effector T cells, as performed
previously in our group (151)
In addition, perforin and granzyme expression within the CD8+ IELs and
CD8+ LPLs, would be highly interesting to define the Treg profile as well
the pathogenic profile of CD8+ T cells in MC.
Our data on the Th17/Tc17 and Th1/Tc1 mucosal cytokine profiles in
colonic biopsies needs to be validated on sorted CD4+ and CD8+ IELs and
LPLs to draw a firm conclusion whether the immunopathological
responses are mainly derived by T helper subsets or CTL subsets.
In the light of these studies, identifying the CD8+ T cells as a more likely
pathogenic T cell subset in MC, studies on the effect of soluble factors
from the inflamed mucosa of MC patients on peripheral CD8+ T cell
activation and differentiation will provide a further understanding on
whether they are the main players in driving the immunopathology in MC
or not.
In addition, the changes in the inflammation in the colonic mucosa over
time has not been elucidated in the MC and would be highly interesting to
define whether the patients with longer disease duration have different
types of immune responses compared to newly diagnosed MC patients.
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Our in vitro model can be useful for analysis of the immunopathogenetic
mechanisms behind the findings of worsened disease in patients being
treated with certain groups of drugs such as NSAIDS and proton pump
inhibitors. In addition, this model can be utilized to analyze the efficacy of
existing drugs on an individual basis, enabling tailor made therapy of
microscopic colitis.
I believe that my thesis work have added some important pieces of
information to understand the nature of immunopathological responses in
microscopic colitis and hope that the above future studies will add a
valuable information in MC pathophysiology.
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ACKNOWLEDGEMENTS
This has been an exciting journey with a lot of fun and lot of struggle.. ☺
During this journey many people have contributed to make this thesis possible. I have tried my best to remember everyone, my apologies if I forget
someone!!!!
I would like to express my honest gratefulness and warmest gratitude to
my supervisor Elisabeth Hultgren-Hornquist (Bettan) for giving me the
opportunity to perform my PhD studies in the very exciting field of mucosal immunology. I am very grateful for your guidance and constructive
criticism during my research studies as well as preparation of publications
and this thesis, and especially making time to discuss my questions and
your prompt responses on manuscripts. I also appreciate the discussions
outside research area throughout the years. Of course thanks a lot for my
training in Swedish.
I am grateful to my second supervisor Johan Bohr for introducing me to
the clinical parts of microscopic colitis and your comments on manuscripts.
I also appreciate your efforts to recruit MC patients. I also would like to
express my warmest gratitude to Curt Tysk for your enthusiasm, and your
knowledge and guidance through the clinical parts of MC. I also appreciate
your prompt responses and critical comments on my manuscripts.
My sincere thanks and warmest gratitude to my third supervisor Allan
Sirsjo, for introducing me to the research field in biomedicine. Thank you
for taking the risk to inviting me to the Sweden ☺. I appreciate your help,
guidance and discussions within research and outside research field.
I also would like to thank Nils Nyhlin, Anna Wickbom, Jonas Halfvarsson
and all the gastroenterologists at the division of gastroenterology for their
excellent support in collecting the mucosal biopsies.
I am very grateful to all the nursing staff (Ulla Vidmark, Jennette, Ulla,
Anna, Kristin…I am sorry I do not remember all the names!!!) for their
excellent support in collecting the mucosal biopsies. My sincere thanks
geos to Ulla Vidmark for helping to collect the clinical data and in recruitment of patients. I also would like to thank you all for my training in Swedish.
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I also would like to express my warmest gratitude to all the patients for
donating the biopsies to carry out the studies.
I would like to thank Calle and Nils for a really nice collaboration and
teaching me immunohistochemical analysis.
Kristina, thank you for teaching me flow cytometry, and how to handle
and kill a mouse ☺. I enjoyed your company in our group and time in Boston and Reykjavik.
Sezin, thanks for your company and joyful environment in the lab. Be happy always☺.
Hilja, thanks a lot for a great collaboration and helping me in gene and
protein expression studies. I really appreciate the generous support from
you and your family members, since the time I have arrived in Sweden.
You are a really kind person ☺.
Ignacio, a huge thanks to you for your patience while listening my questions in both scientific and non-scientific areas. I really appreciate our discussions ☺ Monks will be explored one day ☺.
Olof, I am grateful to you in helping for all the tricky calculations to understand the data from functional studies. I appreciate your enthusiasm
and encouragement for functional studies. The credit goes to you for introducing me to Chimay ☺, I enjoy it often on weekends!
I would like to express my gratitude to all PhD students and personal at
Clinical Research Centre Orebro University Hospital and School of Health
and Medical Sciences, Orebro University.
Margareta and Elisabeth – for a well running facilities and working
environment. Margareta- for your contribution to my training in Swedish.
Elisabeth and Lena- for your great technical assistance with Luminex,
Lena- for your contribution to my training in Swedish. I really appreciate
it. Ann-Charlotte – for excellent administrative services.
Johanna S, Kristin, Eleonor, Boxy, Kaddy, Breezy, Rashida, Sahida,
Johhana L, Lesslie, Lotta, Sabina, Maria, Anna, Marike, Seta, Anders and
all other people at CRC for creating a nice and friendly environment.
Berolla – for your all the help in MS word issues. Of course for the great
discussions in research as well as on male and female brains☺. Thank you
for everything Penny ☺. I appreciate it.
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Members of Gentlemen Club – Mikael, Robert, Daniel, Hazeem, Isak,
John Peter, Igor and other members – Guys a huge thanks to all of you for
creating this club. Best time of the Friday ☺.
Isak- Thanks a lot for all the help and for your great company in Paris ☺.
John Peter and Rajan Ji- thank you very much for introducing me to the
volleyball and innebundy games on Friday evenings. I also would like to
thank all the members who come to play these games, and having great
laughter moments ☺.
I also would like to thank all my teachers from primary and secondary
schools and colleges in Jaipur and Bangalore.
I also would like to thank all the friends but specially the fantastic four in
Bangalore city, Monu, Brijesh bhai, Titu bhai and Vinu da for a great time
of my life we spent together.
Rakesh Ji, Adinath Ji, Sudhir, Viraja, Neha and Shanti for wonderful moments on our gatherings in Orebro.
Sisir and Ravi- for nice discussions and great time on Sundays!!!!
Sanja and her family members for their support for everything. Sanja- for
always encouraging me in rough times, for enormous support, and believing in me. Thank you for being with me always ☺.
From the bottom of my heart I would like to express my gratitude to my
mother, my father, my brother and other family members. Especially I
would like to thank my parents for their belief in me and their endless support and for teaching me to never give up. I am blessed to have you as my
parents.
This work was supported by grants from Swedish Society of Medicine
(Bengt Ihre Foundation), Örebro University Hospital Research Foundation,
the Research Council Sweden (VR), the Karl Jeppsson Foundation, the
Lars Hierta and the Sigurd and Elsa Goljes Foundation.
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REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Lindstrom CG. 'Collagenous colitis' with watery diarrhoea--a new
entity? Pathol Eur. 1976;11(1):87-9.
Read NW, Krejs GJ, Read MG, Santa Ana CA, Morawski SG,
Fordtran JS. Chronic diarrhea of unknown origin. Gastroenterology.
1980 Feb;78(2):264-71.
Lazenby AJ, Yardley JH, Giardiello FM, Jessurun J, Bayless TM.
Lymphocytic ("microscopic") colitis: a comparative histopathologic
study with particular reference to collagenous colitis. Hum Pathol.
1989 Jan;20(1):18-28.
Levison DA, Lazenby AJ, Yardley JH. Microscopic colitis cases
revisited. Gastroenterology. 1993 Nov;105(5):1594-6.
Vigren L, Olesen M, Benoni C, Sjoberg K. Are collagenous and
lymphocytic colitis different aspects of the same disease? Scand J
Gastroenterol. 2012 Sep 28.
Tysk C, Bohr J, Nyhlin N, Wickbom A, Eriksson S. Diagnosis and
management of microscopic colitis. World J Gastroenterol. 2008 Dec
28;14(48):7280-8.
Munch A, Aust D, Bohr J, Bonderup O, Fernandez Banares F,
Hjortswang H, Madisch A, Munck LK, Strom M, Tysk C, Miehlke
S. Microscopic colitis: Current status, present and future challenges:
Statements of the European Microscopic Colitis Group. J Crohns
Colitis. 2012 Oct;6(9):932-45.
Williams JJ, Kaplan GG, Makhija S, Urbanski SJ, Dupre M,
Panaccione R, Beck PL. Microscopic colitis-defining incidence rates
and risk factors: a population-based study. Clin Gastroenterol
Hepatol. 2008 Jan;6(1):35-40.
Bohr J, Tysk C, Eriksson S, Abrahamsson H, Jarnerot G.
Collagenous colitis: a retrospective study of clinical presentation and
treatment in 163 patients. Gut. 1996 Dec;39(6):846-51.
Madisch A, Heymer P, Voss C, Wigginghaus B, Bastlein E,
Bayerdorffer E, Meier E, Schimming W, Bethke B, Stolte M, Miehlke
S. Oral budesonide therapy improves quality of life in patients with
collagenous colitis. Int J Colorectal Dis. 2005 Jul;20(4):312-6.
Hjortswang H, Tysk C, Bohr J, Benoni C, Vigren L, Kilander A,
Larsson L, Taha Y, Strom M. Health-related quality of life is
impaired in active collagenous colitis. Dig Liver Dis. 2011
Feb;43(2):102-9.
ASHOK KUMAR KUMAWAT Adaptive Immune Responses in the Intestinal ...
I
71
12.
Olesen M, Eriksson S, Bohr J, Jarnerot G, Tysk C. Lymphocytic
colitis: a retrospective clinical study of 199 Swedish patients. Gut.
2004 Apr;53(4):536-41.
Pardi DS, Kelly CP. Microscopic colitis. Gastroenterology. 2011
Apr;140(4):1155-65.
Warren BF, Edwards CM, Travis SP. 'Microscopic colitis':
classification and terminology. Histopathology. 2002 Apr;40(4):3746.
Muller S, Neureiter D, Stolte M, Verbeke C, Heuschmann P,
Kirchner T, Aigner T. Tenascin: a sensitive and specific diagnostic
marker of minimal collagenous colitis. Virchows Arch. 2001
May;438(5):435-41.
Tanaka M, Mazzoleni G, Riddell RH. Distribution of collagenous
colitis: utility of flexible sigmoidoscopy. Gut. 1992 Jan;33(1):65-70.
Yantiss RK, Odze RD. Optimal approach to obtaining mucosal
biopsies for assessment of inflammatory disorders of the
gastrointestinal tract. Am J Gastroenterol. 2009 Mar;104(3):774-83.
Lee E, Schiller LR, Vendrell D, Santa Ana CA, Fordtran JS.
Subepithelial collagen table thickness in colon specimens from
patients with microscopic colitis and collagenous colitis.
Gastroenterology. 1992 Dec;103(6):1790-6.
Burgel N, Bojarski C, Mankertz J, Zeitz M, Fromm M, Schulzke JD.
Mechanisms of diarrhea in collagenous colitis. Gastroenterology.
2002 Aug;123(2):433-43.
Munch A, Soderholm JD, Wallon C, Ost A, Olaison G, Strom M.
Dynamics of mucosal permeability and inflammation in collagenous
colitis before, during, and after loop ileostomy. Gut. 2005
Aug;54(8):1126-8.
Rask-Madsen J, Grove O, Hansen MG, Bukhave K, Scient C,
Henrik-Nielsen R. Colonic transport of water and electrolytes in a
patient with secretory diarrhea due to collagenous colitis. Dig Dis
Sci. 1983 Dec;28(12):1141-6.
Stewart MJ, Seow CH, Storr MA. Prednisolone and budesonide for
short- and long-term treatment of microscopic colitis: systematic
review and meta-analysis. Clin Gastroenterol Hepatol. 2011
Oct;9(10):881-90.
Chande N, McDonald JW, Macdonald JK. Interventions for treating
collagenous
colitis.
Cochrane
Database
Syst
Rev.
2008(2):CD003575.
Miehlke S, Madisch A, Karimi D, Wonschik S, Kuhlisch E,
Beckmann R, Morgner A, Mueller R, Greinwald R, Seitz G, Baretton
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
72
I
ASHOK KUMAR KUMAWAT Adaptive Immune Responses in the Intestinal ...
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
G, Stolte M. Budesonide is effective in treating lymphocytic colitis: a
randomized
double-blind
placebo-controlled
study.
Gastroenterology. 2009 Jun;136(7):2092-100.
Bonderup OK, Hansen JB, Teglbjaerg PS, Christensen LA,
Fallingborg JF. Long-term budesonide treatment of collagenous
colitis: a randomised, double-blind, placebo-controlled trial. Gut.
2009 Jan;58(1):68-72.
Pardi DS, Loftus EV, Jr., Tremaine WJ, Sandborn WJ. Treatment of
refractory microscopic colitis with azathioprine and 6mercaptopurine. Gastroenterology. 2001 May;120(6):1483-4.
Riddell J, Hillman L, Chiragakis L, Clarke A. Collagenous colitis:
oral low-dose methotrexate for patients with difficult symptoms:
long-term
outcomes.
J
Gastroenterol
Hepatol.
2007
Oct;22(10):1589-93.
Munch A, Ignatova S, Strom M. Adalimumab in budesonide and
methotrexate refractory collagenous colitis. Scand J Gastroenterol.
2012 Jan;47(1):59-63.
Esteve M, Mahadevan U, Sainz E, Rodriguez E, Salas A, FernandezBanares F. Efficacy of anti-TNF therapies in refractory severe
microscopic colitis. J Crohns Colitis. 2011 Dec;5(6):612-8.
Mannon P. Normal Gut Mucosal Immunity: A Dynamic Balance of
Tolerance and Defense. Gastroenterology & Hepatology.
2005;1(1):50-6.
Mowat AM. Anatomical basis of tolerance and immunity to
intestinal antigens. Nat Rev Immunol. 2003 Apr;3(4):331-41.
Gibbons DL, Spencer J. Mouse and human intestinal immunity:
same ballpark, different players; different rules, same score. Mucosal
Immunol. 2011 Mar;4(2):148-57.
Nusrat A, Turner JR, Madara JL. Molecular physiology and
pathophysiology of tight junctions. IV. Regulation of tight junctions
by extracellular stimuli: nutrients, cytokines, and immune cells. Am J
Physiol Gastrointest Liver Physiol. 2000 Nov;279(5):G851-7.
Lievin-Le Moal V, Servin AL. The front line of enteric host defense
against unwelcome intrusion of harmful microorganisms: mucins,
antimicrobial peptides, and microbiota. Clin Microbiol Rev. 2006
Apr;19(2):315-37.
Johansen FE, Brandtzaeg P. Transcriptional regulation of the
mucosal IgA system. Trends Immunol. 2004 Mar;25(3):150-7.
Hayday A, Theodoridis E, Ramsburg E, Shires J. Intraepithelial
lymphocytes: exploring the Third Way in immunology. Nat
Immunol. 2001 Nov;2(11):997-1003.
ASHOK KUMAR KUMAWAT Adaptive Immune Responses in the Intestinal ...
I
73
37.
Groh V, Steinle A, Bauer S, Spies T. Recognition of stress-induced
MHC molecules by intestinal epithelial gammadelta T cells. Science.
1998 Mar 13;279(5357):1737-40.
Ishikawa H, Naito T, Iwanaga T, Takahashi-Iwanaga H, Suematsu
M, Hibi T, Nanno M. Curriculum vitae of intestinal intraepithelial T
cells: their developmental and behavioral characteristics. Immunol
Rev. 2007 Feb;215:154-65.
Dutronc Y, Porcelli SA. The CD1 family and T cell recognition of
lipid antigens. Tissue Antigens. 2002 Nov;60(5):337-53.
Shimamura M, Huang YY, Okamoto N, Suzuki N, Yasuoka J,
Morita K, Nishiyama A, Amano Y, Mishina T. Modulation of
Valpha19 NKT cell immune responses by alpha-mannosyl ceramide
derivatives consisting of a series of modified sphingosines. Eur J
Immunol. 2007 Jul;37(7):1836-44.
De Maria R, Fais S, Silvestri M, Frati L, Pallone F, Santoni A, Testi
R. Continuous in vivo activation and transient hyporesponsiveness
to TcR/CD3 triggering of human gut lamina propria lymphocytes.
Eur J Immunol. 1993 Dec;23(12):3104-8.
Pirzer UC, Schurmann G, Post S, Betzler M, Meuer SC. Differential
responsiveness to CD3-Ti vs. CD2-dependent activation of human
intestinal T lymphocytes. Eur J Immunol. 1990 Oct;20(10):2339-42.
Appay V, van Lier RA, Sallusto F, Roederer M. Phenotype and
function of human T lymphocyte subsets: consensus and issues.
Cytometry A. 2008 Nov;73(11):975-83.
Shapiro-Shelef M, Calame K. Regulation of plasma-cell
development. Nat Rev Immunol. 2005 Mar;5(3):230-42.
Caraux A, Klein B, Paiva B, Bret C, Schmitz A, Fuhler GM, Bos NA,
Johnsen HE, Orfao A, Perez-Andres M. Circulating human B and
plasma cells. Age-associated changes in counts and detailed
characterization of circulating normal CD138- and CD138+ plasma
cells. Haematologica. 2010 Jun;95(6):1016-20.
Mesin L, Di Niro R, Thompson KM, Lundin KE, Sollid LM. Longlived plasma cells from human small intestine biopsies secrete
immunoglobulins for many weeks in vitro. J Immunol. 2011 Sep
15;187(6):2867-74.
Mei HE, Yoshida T, Sime W, Hiepe F, Thiele K, Manz RA,
Radbruch A, Dorner T. Blood-borne human plasma cells in steady
state are derived from mucosal immune responses. Blood. 2009 Mar
12;113(11):2461-9.
Harada H, Kawano MM, Huang N, Harada Y, Iwato K, Tanabe O,
Tanaka H, Sakai A, Asaoku H, Kuramoto A. Phenotypic difference
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
74
I
ASHOK KUMAR KUMAWAT Adaptive Immune Responses in the Intestinal ...
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
of normal plasma cells from mature myeloma cells. Blood. 1993
May 15;81(10):2658-63.
Morrison PJ, Ballantyne SJ, Kullberg MC. Interleukin-23 and T
helper 17-type responses in intestinal inflammation: from cytokines
to T-cell plasticity. Immunology. 2011 Aug;133(4):397-408.
Littman DR, Rudensky AY. Th17 and regulatory T cells in
mediating and restraining inflammation. Cell. 2010 Mar
19;140(6):845-58.
Brand S. Crohn's disease: Th1, Th17 or both? The change of a
paradigm: new immunological and genetic insights implicate Th17
cells in the pathogenesis of Crohn's disease. Gut. 2009
Aug;58(8):1152-67.
Luckheeram RV, Zhou R, Verma AD, Xia B. CD4(+)T cells:
differentiation
and
functions.
Clin
Dev
Immunol.
2012;2012:925135.
Chaturvedi V, Collison LW, Guy CS, Workman CJ, Vignali DA.
Cutting edge: Human regulatory T cells require IL-35 to mediate
suppression and infectious tolerance. J Immunol. 2011 Jun
15;186(12):6661-6.
Djuretic IM, Levanon D, Negreanu V, Groner Y, Rao A, Ansel KM.
Transcription factors T-bet and Runx3 cooperate to activate Ifng
and silence Il4 in T helper type 1 cells. Nat Immunol. 2007
Feb;8(2):145-53.
Glimcher LH, Murphy KM. Lineage commitment in the immune
system: the T helper lymphocyte grows up. Genes Dev. 2000 Jul
15;14(14):1693-711.
Usui T, Nishikomori R, Kitani A, Strober W. GATA-3 suppresses
Th1 development by downregulation of Stat4 and not through
effects on IL-12Rbeta2 chain or T-bet. Immunity. 2003
Mar;18(3):415-28.
Unutmaz D. RORC2: the master of human Th17 cell programming.
Eur J Immunol. 2009 Jun;39(6):1452-5.
Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 Cells.
Annu Rev Immunol. 2009;27:485-517.
Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G,
Wahl SM. Conversion of peripheral CD4+CD25- naive T cells to
CD4+CD25+ regulatory T cells by TGF-beta induction of
transcription factor Foxp3. J Exp Med. 2003 Dec 15;198(12):187586.
ASHOK KUMAR KUMAWAT Adaptive Immune Responses in the Intestinal ...
I
75
60.
Torchinsky MB, Blander JM. T helper 17 cells: discovery, function,
and physiological trigger. Cell Mol Life Sci. 2010 May;67(9):140721.
Rovedatti L, Kudo T, Biancheri P, Sarra M, Knowles CH, Rampton
DS, Corazza GR, Monteleone G, Di Sabatino A, Macdonald TT.
Differential regulation of interleukin 17 and interferon gamma
production in inflammatory bowel disease. Gut. 2009
Dec;58(12):1629-36.
Xu L, Kitani A, Fuss I, Strober W. Cutting edge: regulatory T cells
induce CD4+CD25-Foxp3- T cells or are self-induced to become
Th17 cells in the absence of exogenous TGF-beta. J Immunol. 2007
Jun 1;178(11):6725-9.
Boniface K, Blumenschein WM, Brovont-Porth K, McGeachy MJ,
Basham B, Desai B, Pierce R, McClanahan TK, Sadekova S, de Waal
Malefyt R. Human Th17 cells comprise heterogeneous subsets
including IFN-gamma-producing cells with distinct properties from
the Th1 lineage. J Immunol. 2010 Jul 1;185(1):679-87.
Annunziato F, Cosmi L, Santarlasci V, Maggi L, Liotta F, Mazzinghi
B, Parente E, Fili L, Ferri S, Frosali F, Giudici F, Romagnani P,
Parronchi P, Tonelli F, Maggi E, Romagnani S. Phenotypic and
functional features of human Th17 cells. J Exp Med. 2007 Aug
6;204(8):1849-61.
Cerwenka A, Carter LL, Reome JB, Swain SL, Dutton RW. In vivo
persistence of CD8 polarized T cell subsets producing type 1 or type
2 cytokines. J Immunol. 1998 Jul 1;161(1):97-105.
Chamoto K, Kosaka A, Tsuji T, Matsuzaki J, Sato T, Takeshima T,
Iwakabe K, Togashi Y, Koda T, Nishimura T. Critical role of the
Th1/Tc1 circuit for the generation of tumor-specific CTL during
tumor eradication in vivo by Th1-cell therapy. Cancer Sci. 2003
Oct;94(10):924-8.
Hamada H, Garcia-Hernandez Mde L, Reome JB, Misra SK, Strutt
TM, McKinstry KK, Cooper AM, Swain SL, Dutton RW. Tc17, a
unique subset of CD8 T cells that can protect against lethal influenza
challenge. J Immunol. 2009 Mar 15;182(6):3469-81.
Huber M, Heink S, Grothe H, Guralnik A, Reinhard K, Elflein K,
Hunig T, Mittrucker HW, Brustle A, Kamradt T, Lohoff M. A
Th17-like developmental process leads to CD8(+) Tc17 cells with
reduced cytotoxic activity. Eur J Immunol. 2009 Jul;39(7):1716-25.
Kondo T, Takata H, Matsuki F, Takiguchi M. Cutting edge:
Phenotypic characterization and differentiation of human CD8+ T
cells producing IL-17. J Immunol. 2009 Feb 15;182(4):1794-8.
61.
62.
63.
64.
65.
66.
67.
68.
69.
76
I
ASHOK KUMAR KUMAWAT Adaptive Immune Responses in the Intestinal ...
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
Kuang DM, Peng C, Zhao Q, Wu Y, Zhu LY, Wang J, Yin XY, Li L,
Zheng L. Tumor-activated monocytes promote expansion of IL-17producing CD8+ T cells in hepatocellular carcinoma patients. J
Immunol. 2010 Aug 1;185(3):1544-9.
Guillot-Delost M, Le Gouvello S, Mesel-Lemoine M, Cherai M,
Baillou C, Simon A, Levy Y, Weiss L, Louafi S, Chaput N, Berrehar
F, Kerbrat S, Klatzmann D, Lemoine FM. Human CD90 identifies
Th17/Tc17 T cell subsets that are depleted in HIV-infected patients.
J Immunol. 2012 Feb 1;188(3):981-91.
Hazenberg MD, Verschuren MC, Hamann D, Miedema F, van
Dongen JJ. T cell receptor excision circles as markers for recent
thymic emigrants: basic aspects, technical approach, and guidelines
for interpretation. J Mol Med (Berl). 2001 Nov;79(11):631-40.
Ribeiro RM, Perelson AS. Determining thymic output quantitatively:
using models to interpret experimental T-cell receptor excision circle
(TREC) data. Immunol Rev. 2007 Apr;216:21-34.
Livak F, Schatz DG. T-cell receptor alpha locus V(D)J recombination
by-products are abundant in thymocytes and mature T cells. Mol
Cell Biol. 1996 Feb;16(2):609-18.
Hazenberg MD, Otto SA, Cohen Stuart JW, Verschuren MC,
Borleffs JC, Boucher CA, Coutinho RA, Lange JM, Rinke de Wit TF,
Tsegaye A, van Dongen JJ, Hamann D, de Boer RJ, Miedema F.
Increased cell division but not thymic dysfunction rapidly affects the
T-cell receptor excision circle content of the naive T cell population
in HIV-1 infection. Nat Med. 2000 Sep;6(9):1036-42.
Somech R. T-cell receptor excision circles in primary
immunodeficiencies and other T-cell immune disorders. Curr Opin
Allergy Clin Immunol. 2011 Dec;11(6):517-24.
Jansson A, Pernestig AK, Nilsson P, Jirstrand M, Hultgren
Hornquist E. Toward quantifying the thymic dysfunctional state in
mouse models of inflammatory bowel disease. Inflamm Bowel Dis.
2013 Mar;19(4):881-8.
Elgbratt K, Kurlberg G, Hahn-Zohric M, Hornquist EH. Rapid
migration of thymic emigrants to the colonic mucosa in ulcerative
colitis patients. Clin Exp Immunol. 2010 Nov;162(2):325-36.
Fiocchi C. Intestinal inflammation: a complex interplay of immune
and nonimmune cell interactions. Am J Physiol. 1997 Oct;273(4 Pt
1):G769-75.
Garrett WS, Gordon JI, Glimcher LH. Homeostasis and
inflammation in the intestine. Cell. 2010 Mar 19;140(6):859-70.
ASHOK KUMAR KUMAWAT Adaptive Immune Responses in the Intestinal ...
I
77
81.
Ley RE, Hamady M, Lozupone C, Turnbaugh PJ, Ramey RR,
Bircher JS, Schlegel ML, Tucker TA, Schrenzel MD, Knight R,
Gordon JI. Evolution of mammals and their gut microbes. Science.
2008 Jun 20;320(5883):1647-51.
Macpherson AJ, Harris NL. Interactions between commensal
intestinal bacteria and the immune system. Nat Rev Immunol. 2004
Jun;4(6):478-85.
Stappenbeck TS, Miyoshi H. The role of stromal stem cells in tissue
regeneration
and
wound
repair.
Science.
2009
Jun
26;324(5935):1666-9.
Owens BM, Simmons A. Intestinal stromal cells in mucosal
immunity and homeostasis. Mucosal Immunol. 2013 Mar;6(2):22434.
Magnusson FC, Liblau RS, von Boehmer H, Pittet MJ, Lee JW,
Turley SJ, Khazaie K. Direct presentation of antigen by lymph node
stromal cells protects against CD8 T-cell-mediated intestinal
autoimmunity. Gastroenterology. 2008 Apr;134(4):1028-37.
Binion DG, West GA, Ina K, Ziats NP, Emancipator SN, Fiocchi C.
Enhanced leukocyte binding by intestinal microvascular endothelial
cells in inflammatory bowel disease. Gastroenterology. 1997
Jun;112(6):1895-907.
Schonherr E, Hausser HJ. Extracellular matrix and cytokines: a
functional unit. Dev Immunol. 2000;7(2-4):89-101.
de Sousa M, Tilney NL, Kupiec-Weglinski JW. Recognition of self
within self: specific lymphocyte positioning and the extracellular
matrix. Immunol Today. 1991 Aug;12(8):262-6.
Pruteanu M, Hyland NP, Clarke DJ, Kiely B, Shanahan F.
Degradation of the extracellular matrix components by bacterialderived metalloproteases: implications for inflammatory bowel
diseases. Inflamm Bowel Dis. 2011 May;17(5):1189-200.
Jarnerot G, Tysk C, Bohr J, Eriksson S. Collagenous colitis and fecal
stream diversion. Gastroenterology. 1995 Aug;109(2):449-55.
Beaugerie L, Pardi DS. Review article: drug-induced microscopic
colitis - proposal for a scoring system and review of the literature.
Aliment Pharmacol Ther. 2005 Aug 15;22(4):277-84.
Bohr J, Nordfelth R, Jarnerot G, Tysk C. Yersinia species in
collagenous colitis: a serologic study. Scand J Gastroenterol. 2002
Jun;37(6):711-4.
Erim T, Alazmi WM, O'Loughlin CJ, Barkin JS. Collagenous colitis
associated with Clostridium difficile: a cause effect? Dig Dis Sci.
2003 Jul;48(7):1374-5.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
78
I
ASHOK KUMAR KUMAWAT Adaptive Immune Responses in the Intestinal ...
94.
95.
96.
97.
98.
99.
100.
101.
102.
103.
104.
105.
Munch A, Soderholm JD, Ost A, Strom M. Increased transmucosal
uptake of E. coli K12 in collagenous colitis persists after budesonide
treatment. Am J Gastroenterol. 2009 Mar;104(3):679-85.
Ung KA, Gillberg R, Kilander A, Abrahamsson H. Role of bile acids
and bile acid binding agents in patients with collagenous colitis. Gut.
2000 Feb;46(2):170-5.
Mosnier JF, Larvol L, Barge J, Dubois S, De La Bigne G, Henin D,
Cerf
M.
Lymphocytic
and
collagenous
colitis:
an
immunohistochemical study. Am J Gastroenterol. 1996
Apr;91(4):709-13.
Lazenby AJ AR, Fox WM, Giardiello FM, Yardley JH. T cell
receptors in collagenous and lymphocytic colitis. Gastroenterology.
[Abstract]. 1990;98:A459.
Tagkalidis PP, Gibson PR, Bhathal PS. Microscopic colitis
demonstrates a T helper cell type 1 mucosal cytokine profile. J Clin
Pathol. 2007 Apr;60(4):382-7.
Johrens K, Grunbaum M, Anagnostopoulos I. Differences in the Tbet and GATA-3 expression patterns between lymphocytic colitis
and coeliac disease. Virchows Arch. 2010 Oct;457(4):451-6.
Wagner M, Lampinen M, Sangfelt P, Agnarsdottir M, Carlson M.
Budesonide treatment of patients with collagenous colitis restores
normal eosinophil and T-cell activity in the colon. Inflamm Bowel
Dis. 2010 Jul;16(7):1118-26.
Andresen L, Jorgensen VL, Perner A, Hansen A, Eugen-Olsen J,
Rask-Madsen J. Activation of nuclear factor kappaB in colonic
mucosa from patients with collagenous and ulcerative colitis. Gut.
2005 Apr;54(4):503-9.
Olesen M, Middelveld R, Bohr J, Tysk C, Lundberg JO, Eriksson S,
Alving K, Jarnerot G. Luminal nitric oxide and epithelial expression
of inducible and endothelial nitric oxide synthase in collagenous and
lymphocytic colitis. Scand J Gastroenterol. 2003 Jan;38(1):66-72.
Perner A, Andresen L, Normark M, Fischer-Hansen B, Sorensen S,
Eugen-Olsen J, Rask-Madsen J. Expression of nitric oxide synthases
and effects of L-arginine and L-NMMA on nitric oxide production
and fluid transport in collagenous colitis. Gut. 2001 Sep;49(3):38794.
Taha Y, Carlson M, Thorn M, Loof L, Raab Y. Evidence of local
eosinophil activation and altered mucosal permeability in
collagenous colitis. Dig Dis Sci. 2001 Apr;46(4):888-97.
Salas A, Fernandez-Banares F, Casalots J, Gonzalez C, Tarroch X,
Forcada P, Gonzalez G. Subepithelial myofibroblasts and tenascin
ASHOK KUMAR KUMAWAT Adaptive Immune Responses in the Intestinal ...
I
79
expression in microscopic colitis. Histopathology. 2003 Jul;43(1):4854.
Gunther U, Schuppan D, Bauer M, Matthes H, Stallmach A,
Schmitt-Graff A, Riecken EO, Herbst H. Fibrogenesis and fibrolysis
in collagenous colitis. Patterns of procollagen types I and IV, matrixmetalloproteinase-1 and -13, and TIMP-1 gene expression. Am J
Pathol. 1999 Aug;155(2):493-503.
Stahle-Backdahl M, Maim J, Veress B, Benoni C, Bruce K, Egesten
A. Increased presence of eosinophilic granulocytes expressing
transforming growth factor-beta1 in collagenous colitis. Scand J
Gastroenterol. 2000 Jul;35(7):742-6.
Taha Y, Raab Y, Larsson A, Carlson M, Loof L, Gerdin B, Thorn
M. Vascular endothelial growth factor (VEGF)--a possible mediator
of inflammation and mucosal permeability in patients with
collagenous colitis. Dig Dis Sci. 2004 Jan;49(1):109-15.
Jarnerot G, Hertervig E, Granno C, Thorhallsson E, Eriksson S, Tysk
C, Hansson I, Bjorknas H, Bohr J, Olesen M, Willen R, Kagevi I,
Danielsson A. Familial occurrence of microscopic colitis: a report on
five families. Scand J Gastroenterol. 2001 Sep;36(9):959-62.
Fine KD, Do K, Schulte K, Ogunji F, Guerra R, Osowski L,
McCormack J. High prevalence of celiac sprue-like HLA-DQ genes
and enteropathy in patients with the microscopic colitis syndrome.
Am J Gastroenterol. 2000 Aug;95(8):1974-82.
Koskela RM, Karttunen TJ, Niemela SE, Lehtola JK, Ilonen J,
Karttunen RA. Human leucocyte antigen and TNFalpha
polymorphism association in microscopic colitis. Eur J Gastroenterol
Hepatol. 2008 Apr;20(4):276-82.
Giardiello FM, Lazenby AJ, Yardley JH, Bias WB, Johnson J,
Alianiello RG, Bedine MS, Bayless TM. Increased HLA A1 and
diminished HLA A3 in lymphocytic colitis compared to controls and
patients with collagenous colitis. Dig Dis Sci. 1992 Apr;37(4):496-9.
Madisch A, Hellmig S, Schreiber S, Bethke B, Stolte M, Miehlke S.
Allelic variation of the matrix metalloproteinase-9 gene is associated
with
collagenous
colitis.
Inflamm
Bowel
Dis.
2011
Nov;17(11):2295-8.
Madisch A, Hellmig S, Schreiber S, Bethke B, Stolte M, Miehlke S.
NOD2/CARD15 gene polymorphisms are not associated with
collagenous colitis. Int J Colorectal Dis. 2007 Apr;22(4):425-8.
Koskela RM, Karttunen TJ, Niemela SE, Lehtola JK, Bloigu RS,
Karttunen RA. Cytokine gene polymorphism in microscopic colitis
106.
107.
108.
109.
110.
111.
112.
113.
114.
115.
80
I
ASHOK KUMAR KUMAWAT Adaptive Immune Responses in the Intestinal ...
116.
117.
118.
119.
120.
121.
122.
123.
124.
125.
126.
association with the IL-6-174 GG genotype. Eur J Gastroenterol
Hepatol. 2011 Jul;23(7):607-13.
Abraham C, Cho JH. Inflammatory bowel disease. N Engl J Med.
2009 Nov 19;361(21):2066-78.
Sartor RB. Mechanisms of disease: pathogenesis of Crohn's disease
and ulcerative colitis. Nat Clin Pract Gastroenterol Hepatol. 2006
Jul;3(7):390-407.
Fuss IJ. Is the Th1/Th2 paradigm of immune regulation applicable to
IBD? Inflamm Bowel Dis. 2008 Oct;14 Suppl 2:S110-2.
Kobayashi T, Okamoto S, Hisamatsu T, Kamada N, Chinen H, Saito
R, Kitazume MT, Nakazawa A, Sugita A, Koganei K, Isobe K, Hibi
T. IL23 differentially regulates the Th1/Th17 balance in ulcerative
colitis and Crohn's disease. Gut. 2008 Dec;57(12):1682-9.
Xavier RJ, Podolsky DK. Unravelling the pathogenesis of
inflammatory bowel disease. Nature. 2007 Jul 26;448(7152):427-34.
Kamada N, Hisamatsu T, Honda H, Kobayashi T, Chinen H,
Takayama T, Kitazume MT, Okamoto S, Koganei K, Sugita A,
Kanai T, Hibi T. TL1A produced by lamina propria macrophages
induces Th1 and Th17 immune responses in cooperation with IL-23
in patients with Crohn's disease. Inflamm Bowel Dis. 2010
Apr;16(4):568-75.
Maloy KJ, Kullberg MC. IL-23 and Th17 cytokines in intestinal
homeostasis. Mucosal Immunol. 2008 Sep;1(5):339-49.
Holmen N, Lundgren A, Lundin S, Bergin AM, Rudin A, Sjovall H,
Ohman L. Functional CD4+CD25high regulatory T cells are
enriched in the colonic mucosa of patients with active ulcerative
colitis and increase with disease activity. Inflamm Bowel Dis. 2006
Jun;12(6):447-56.
Veltkamp C, Anstaett M, Wahl K, Moller S, Gangl S, Bachmann O,
Hardtke-Wolenski M, Langer F, Stremmel W, Manns MP, SchulzeOsthoff K, Bantel H. Apoptosis of regulatory T lymphocytes is
increased in chronic inflammatory bowel disease and reversed by
anti-TNFalpha treatment. Gut. 2011 Oct;60(10):1345-53.
Armes J, Gee DC, Macrae FA, Schroeder W, Bhathal PS.
Collagenous colitis: jejunal and colorectal pathology. J Clin Pathol.
1992 Sep;45(9):784-7.
Yu QT, Saruta M, Avanesyan A, Fleshner PR, Banham AH,
Papadakis KA. Expression and functional characterization of
FOXP3+ CD4+ regulatory T cells in ulcerative colitis. Inflamm
Bowel Dis. 2007 Feb;13(2):191-9.
ASHOK KUMAR KUMAWAT Adaptive Immune Responses in the Intestinal ...
I
81
127. Bai S, Siegal GP, Jhala NC. Foxp3 expression patterns in
microscopic colitides: a clinicopathologic study of 69 patients. Am J
Clin Pathol. 2012 Jun;137(6):931-6.
128. Targan SR, Feagan BG, Fedorak RN, Lashner BA, Panaccione R,
Present DH, Spehlmann ME, Rutgeerts PJ, Tulassay Z, Volfova M,
Wolf DC, Hernandez C, Bornstein J, Sandborn WJ. Natalizumab for
the treatment of active Crohn's disease: results of the ENCORE
Trial. Gastroenterology. 2007 May;132(5):1672-83.
129. Abbas AK, Murphy KM, Sher A. Functional diversity of helper T
lymphocytes. Nature. 1996 Oct 31;383(6603):787-93.
130. Shibahara T, Wilcox JN, Couse T, Madara JL. Characterization of
epithelial chemoattractants for human intestinal intraepithelial
lymphocytes. Gastroenterology. 2001 Jan;120(1):60-70.
131. Farber JM. Mig and IP-10: CXC chemokines that target
lymphocytes. J Leukoc Biol. 1997 Mar;61(3):246-57.
132. Taub DD, Lloyd AR, Conlon K, Wang JM, Ortaldo JR, Harada A,
Matsushima K, Kelvin DJ, Oppenheim JJ. Recombinant human
interferon-inducible protein 10 is a chemoattractant for human
monocytes and T lymphocytes and promotes T cell adhesion to
endothelial cells. J Exp Med. 1993 Jun 1;177(6):1809-14.
133. Loetscher M, Gerber B, Loetscher P, Jones SA, Piali L, Clark-Lewis I,
Baggiolini M, Moser B. Chemokine receptor specific for IP10 and
mig: structure, function, and expression in activated T-lymphocytes.
J Exp Med. 1996 Sep 1;184(3):963-9.
134. Ma TY, Iwamoto GK, Hoa NT, Akotia V, Pedram A, Boivin MA,
Said HM. TNF-alpha-induced increase in intestinal epithelial tight
junction permeability requires NF-kappa B activation. Am J Physiol
Gastrointest Liver Physiol. 2004 Mar;286(3):G367-76.
135. Monteleone G, Pallone F, Macdonald TT. Interleukin-21 as a new
therapeutic target for immune-mediated diseases. Trends Pharmacol
Sci. 2009 Aug;30(8):441-7.
136. Sonnenberg GF, Fouser LA, Artis D. Border patrol: regulation of
immunity, inflammation and tissue homeostasis at barrier surfaces
by IL-22. Nat Immunol. 2011 May;12(5):383-90.
137. Qiao L, Schurmann G, Betzler M, Meuer SC. Activation and
signaling status of human lamina propria T lymphocytes.
Gastroenterology. 1991 Dec;101(6):1529-36.
138. Qiao L, Golling M, Autschbach F, Schurmann G, Meuer SC. T cell
receptor repertoire and mitotic responses of lamina propria T
lymphocytes in inflammatory bowel disease. Clin Exp Immunol.
1994 Aug;97(2):303-8.
82
I
ASHOK KUMAR KUMAWAT Adaptive Immune Responses in the Intestinal ...
139. Huff KR, Akhtar LN, Fox AL, Cannon JA, Smith PD, Smythies LE.
Extracellular matrix-associated cytokines regulate CD4+ effector Tcell responses in the human intestinal mucosa. Mucosal Immunol.
2011 Jul;4(4):420-7.
140. Taga K, Mostowski H, Tosato G. Human interleukin-10 can directly
inhibit T-cell growth. Blood. 1993 Jun 1;81(11):2964-71.
141. Karlinger K, Gyorke T, Mako E, Mester A, Tarjan Z. The
epidemiology and the pathogenesis of inflammatory bowel disease.
Eur J Radiol. 2000 Sep;35(3):154-67.
142. Olesen M, Eriksson S, Bohr J, Jarnerot G, Tysk C. Microscopic
colitis: a common diarrhoeal disease. An epidemiological study in
Orebro, Sweden, 1993-1998. Gut. 2004 Mar;53(3):346-50.
143. Lichtenstein DR. Hepatobiliary complications of inflammatory
bowel disease. Curr Gastroenterol Rep. 2011 Oct;13(5):495-505.
144. Abadie V, Discepolo V, Jabri B. Intraepithelial lymphocytes in celiac
disease
immunopathology.
Semin
Immunopathol.
2012
Jul;34(4):551-66.
145. Iwata M, Hirakiyama A, Eshima Y, Kagechika H, Kato C, Song SY.
Retinoic acid imprints gut-homing specificity on T cells. Immunity.
2004 Oct;21(4):527-38.
146. Sun CM, Hall JA, Blank RB, Bouladoux N, Oukka M, Mora JR,
Belkaid Y. Small intestine lamina propria dendritic cells promote de
novo generation of Foxp3 T reg cells via retinoic acid. J Exp Med.
2007 Aug 6;204(8):1775-85.
147. Kang SG, Lim HW, Andrisani OM, Broxmeyer HE, Kim CH.
Vitamin A metabolites induce gut-homing FoxP3+ regulatory T cells.
J Immunol. 2007 Sep 15;179(6):3724-33.
148. DePaolo RW, Abadie V, Tang F, Fehlner-Peach H, Hall JA, Wang
W, Marietta EV, Kasarda DD, Waldmann TA, Murray JA, Semrad
C, Kupfer SS, Belkaid Y, Guandalini S, Jabri B. Co-adjuvant effects
of retinoic acid and IL-15 induce inflammatory immunity to dietary
antigens. Nature. 2011 Mar 10;471(7337):220-4.
149. O'Connell RM, Rao DS, Baltimore D. microRNA regulation of
inflammatory responses. Annu Rev Immunol. 2012;30:295-312.
150. Mahajan D, Goldblum JR, Xiao SY, Shen B, Liu X. Lymphocytic
colitis and collagenous colitis: a review of clinicopathologic features
and immunologic abnormalities. Adv Anat Pathol. 2012
Jan;19(1):28-38.
151. Gotlind YY, Raghavan S, Bland PW, Hornquist EH. CD4+FoxP3+
regulatory T cells from Galphai2-/- mice are functionally active in
vitro, but do not prevent colitis. PLoS One. 2011;6(9):e25073.
ASHOK KUMAR KUMAWAT Adaptive Immune Responses in the Intestinal ...
I
83
Publications in the series
Örebro Studies in Medicine
1.
Bergemalm, Per-Olof (2004). Audiologic and cognitive long-term
sequelae from closed head injury.
2.
Jansson, Kjell (2004). Intraperitoneal Microdialysis.
Technique and Results.
3.
Windahl, Torgny (2004). Clinical aspects of laser treatment of
lichen sclerosus and squamous cell carcinoma of the penis.
4.
Carlsson, Per-Inge (2004). Hearing impairment and deafness.
Genetic and environmental factors – interactions – consequences.
A clinical audiological approach.
5.
Wågsäter, Dick (2005). CXCL16 and CD137 in Atherosclerosis.
6.
Jatta, Ken (2006). Inflammation in Atherosclerosis.
7.
Dreifaldt, Ann Charlotte (2006). Epidemiological Aspects on
Malignant Diseases in Childhood.
8.
Jurstrand, Margaretha (2006). Detection of Chlamydia trachomatis
and Mycoplasma genitalium by genetic and serological methods.
9.
Norén, Torbjörn (2006). Clostridium difficile, epidemiology and
antibiotic resistance.
10. Anderzén Carlsson, Agneta (2007). Children with Cancer – Focusing
on their Fear and on how their Fear is Handled.
11. Ocaya, Pauline (2007). Retinoid metabolism and signalling in
vascular smooth muscle cells.
12. Nilsson, Andreas (2008). Physical activity assessed by accelerometry
in children.
13. Eliasson, Henrik (2008). Tularemia – epidemiological, clinical and
diagnostic aspects.
14. Walldén, Jakob (2008). The influence of opioids on gastric function:
experimental and clinical studies.
15. Andrén, Ove (2008). Natural history and prognostic factors in
localized prostate cancer.
16. Svantesson, Mia (2008). Postpone death? Nurse-physician
perspectives and ethics rounds.
17. Björk, Tabita (2008). Measuring Eating Disorder Outcome
– Definitions, dropouts and patients’ perspectives.
18. Ahlsson, Anders (2008). Atrial Fibrillation in Cardiac Surgery.
19. Parihar, Vishal Singh (2008). Human Listeriosis – Sources and Routes.
20. Berglund, Carolina (2008). Molecular Epidemiology of MethicillinResistant Staphylococcus aureus. Epidemiological aspects of MRSA
and the dissemination in the community and in hospitals.
21. Nilsagård, Ylva (2008). Walking ability, balance and accidental falls in
persons with Multiple Sclerosis.
22. Johansson, Ann-Christin (2008). Psychosocial factors in patients
with lumbar disc herniation: Enhancing postoperative outcome by
the identification of predictive factors and optimised physiotherapy.
23. Larsson, Matz (2008). Secondary exposure to inhaled tobacco
products.
24. Hahn-Strömberg, Victoria (2008). Cell adhesion proteins in different
invasive patterns of colon carcinoma: A morphometric and molecular
genetic study.
25. Böttiger, Anna (2008). Genetic Variation in the Folate Receptor-α
and Methylenetetrahydrofolate Reductase Genes as Determinants
of Plasma Homocysteine Concentrations.
26. Andersson, Gunnel (2009). Urinary incontinence. Prevalence,
treatment seeking behaviour, experiences and perceptions among
persons with and without urinary leakage.
27. Elfström, Peter (2009). Associated disorders in celiac disease.
28. Skårberg, Kurt (2009). Anabolic-androgenic steroid users in treatment:
Social background, drug use patterns and criminality.
29. de Man Lapidoth, Joakim (2009). Binge Eating and Obesity Treatment
– Prevalence, Measurement and Long-term Outcome.
30. Vumma, Ravi (2009). Functional Characterization of Tyrosine
and Tryptophan Transport in Fibroblasts from Healthy Controls,
Patients with Schizophrenia and Bipolar Disorder.
31. Jacobsson, Susanne (2009). Characterisation of Neisseria meningitidis
from a virulence and immunogenic perspective that includes variations
in novel vaccine antigens.
32. Allvin, Renée (2009). Postoperative Recovery. Development of a
Multi-Dimensional Questionnaire for Assessment of Recovery.
33. Hagnelius, Nils-Olof (2009). Vascular Mechanisms in Dementia
with Special Reference to Folate and Fibrinolysis.
34. Duberg, Ann-Sofi (2009). Hepatitis C virus infection. A nationwide
study of assiciated morbidity and mortality.
35. Söderqvist, Fredrik (2009). Health symptoms and potential effects on
the blood-brain and blood-cerebrospinal fluid barriers associated with
use of wireless telephones.
36. Neander, Kerstin (2009). Indispensable Interaction. Parents’ perspectives
on parent–child interaction interventions and beneficial meetings.
37. Ekwall, Eva (2009). Women’s Experiences of Gynecological Cancer
and Interaction with the Health Care System through Different Phases
of the Disease.
38. Thulin Hedberg, Sara (2009). Antibiotic susceptibility and resistance
in Neisseria meningitidis – phenotypic and genotypic characteristics.
39. Hammer, Ann (2010). Forced use on arm function after stroke.
Clinically rated and self-reported outcome and measurement during
the sub-acute phase.
40. Westman, Anders (2010). Musculoskeletal pain in primary health
care: A biopsychosocial perspective for assessment and treatment.
41. Gustafsson, Sanna Aila (2010). The importance of being thin
– Perceived expectations from self and others and the effect on
self-evaluation in girls with disordered eating.
42. Johansson, Bengt (2010). Long-term outcome research on PDR
brachytherapy with focus on breast, base of tongue and lip cancer.
43. Tina, Elisabet (2010). Biological markers in breast cancer and acute
leukaemia with focus on drug resistance.
44. Overmeer, Thomas (2010). Implementing psychosocial factors in
physical therapy treatment for patients with musculoskeletal pain
in primary care.
45. Prenkert, Malin (2010). On mechanisms of drug resistance in
acute myloid leukemia.
46. de Leon, Alex (2010). Effects of Anesthesia on Esophageal Sphincters
in Obese Patients.
47. Josefson, Anna (2010). Nickel allergy and hand eczema – epidemiological
aspects.
48. Almon, Ricardo (2010). Lactase Persistence and Lactase Non-Persistence.
Prevalence, influence on body fat, body height, and relation to the metabolic
syndrome.
49. Ohlin, Andreas (2010). Aspects on early diagnosis of neonatal sepsis.
50. Oliynyk, Igor (2010). Advances in Pharmacological Treatment of Cystic
Fibrosis.
51. Franzén, Karin (2011). Interventions for Urinary Incontinence in Women.
Survey and effects on population and patient level.
52. Loiske, Karin (2011). Echocardiographic measurements of the heart. With
focus on the right ventricle.
53. Hellmark, Bengt (2011). Genotypic and phenotypic characterisation of
Staphylococcus epidermidis isolated from prosthetic joint infections.
54. Eriksson Crommert, Martin (2011). On the role of transversus abdominis
in trunk motor control.
55. Ahlstrand, Rebecca (2011). Effects of Anesthesia on Esophageal Sphincters.
56. Holländare, Fredrik (2011). Managing Depression via the Internet
– self-report measures, treatment & relapse prevention.
57. Johansson, Jessica (2011). Amino Acid Transport and Receptor Binding
Properties in Neuropsychiatric Disorders using the Fibroblast Cell Model.
58. Vidlund, Mårten (2011). Glutamate for Metabolic Intervention in Coronary
Surgery with special reference to the GLUTAMICS-trial.
59. Zakrisson, Ann-Britt (2011). Management of patients with Chronic
Obstructive Pulmonary Disease in Primary Health Care. A study of a
nurse-led multidisciplinary programme of pulmonary rehabilitation.
60. Lindgren, Rickard (2011). Aspects of anastomotic leakage, anorectal
function and defunctioning stoma in Low Anterior Resection of the
rectum for cancer.
61. Karlsson, Christina (2011). Biomarkers in non-small cell lung carcinoma.
Methodological aspects and influence of gender, histology and smoking
habits on estrogen receptor and epidermal growth factor family receptor
signalling.
62. Varelogianni, Georgia (2011). Chloride Transport and Inflammation in
Cystic Fibrosis Airways.
63. Makdoumi, Karim (2011). Ultraviolet Light A (UVA) Photoactivation of
Riboflavin as a Potential Therapy for Infectious Keratitis.
64. Nordin Olsson, Inger (2012). Rational drug treatment in the elderly: ”To
treat or not to treat”.
65. Fadl, Helena (2012). Gestational diabetes mellitus in Sweden: screening,
outcomes, and consequences.
66. Essving, Per (2012). Local Infiltration Analgesia in Knee Arthroplasty.
67. Thuresson, Marie (2012). The Initial Phase of an Acute Coronary Syndrome.
Symptoms, patients’ response to symptoms and opportunity to reduce time to
seek care and to increase ambulance use.
68. Mårild, Karl (2012). Risk Factors and Associated Disorders of Celiac
Disease.
69. Fant, Federica (2012). Optimization of the Perioperative Anaesthetic Care
for Prostate Cancer Surgery. Clinical studies on Pain, Stress Response and
Immunomodulation.
70. Almroth, Henrik (2012). Atrial Fibrillation: Inflammatory and
pharmacological studies.
71. Elmabsout, Ali Ateia (2012). CYP26B1 as regulator of retinoic acid in
vascular cells and atherosclerotic lesions.
72. Stenberg, Reidun (2012). Dietary antibodies and gluten related seromarkers
in children and young adults with cerebral palsy.
73. Skeppner, Elisabeth (2012). Penile Carcinoma: From First Symptom to
Sexual Function and Life Satisfaction. Following Organ-Sparing Laser
Treatment.
74. Carlsson, Jessica (2012). Identification of miRNA expression profiles for
diagnosis and prognosis of prostate cancer.
75. Gustavsson, Anders (2012): Therapy in Inflammatory Bowel Disease.
76. Paulson Karlsson, Gunilla (2012): Anorexia nervosa – treatment expectations,
outcome and satisfaction.
77. Larzon, Thomas (2012): Aspects of endovascular treatment of abdominal
aortic aneurysms.
78. Magnusson, Niklas (2012): Postoperative aspects of inguinal hernia surgery
– pain and recurrences.
79. Khalili, Payam (2012): Risk factors for cardiovascular events and incident
hospital-treated diabetes in the population.
80. Gabrielson, Marike (2013): The mitochondrial protein SLC25A43 and its
possible role in HER2-positive breast cancer.
81. Falck, Eva (2013): Genomic and genetic alterations in endometrial
adenocarcinoma.
82. Svensson, Maria A (2013): Assessing the ERG rearrangement for clinical
use in patients with prostate cancer.
83. Lönn, Johanna (2013): The role of periodontitis and hepatocyte growth
factor in systemic inflammation.
84. Kumawat, Ashok Kumar (2013): Adaptive Immune Responses in the
Intestinal Mucosa of Microscopic Colitis Patients.