957 research outputs found
Investigation into the molecular genetics of the inflammatory bowel diseases
The aims of this thesis were firstly to investigate gene expression profiles in human
colonic and terminal ileal biopsies using microarray technology in a well phenotyped
cohort of patients with Crohn's disease, ulcerative colitis and a control cohort. The
role in disease pathogenesis of differentially expressed genes was investigated along
with the expression of candidate genes identified by genome wide association study
and cell lineage analysis. Parallel studies attempted to replicate the Nature Genetics
publications of Peltekova and Stoll and colleagues who investigated the role the IBD5
locus and the DLG5 gene respectively in patients with inflammatory bowel disease.In the healthy adult colon cluster analysis showed differences in gene expression
between the right and left colon. (x²=25.1, p<0.0001). Developmental genes
HOXA13, (p=2.3x10⁻¹⁶, HOXB13 (p <1xl0⁻⁴⁵), GL11 (p=4.0xl0⁻²⁴), and GLI3
(p=2.1x10⁻²⁸) primarily drove this separation.Upregulated genes in the Crohn's disease biopsies compared to the controls included
SAA1 (Fold change (FC) +7.5, p=1.47xl0⁻⁴¹) and REGL (FC +7.3, p=2.3xl0⁻¹⁶).
Cellular detoxification genes including-SLC14A2 (FC -2.49, p= 0.00002) were
downregulated. In the Crohn's disease terminal ileal biopsies diubiquitin (FC+11.3,
p<1x10⁻⁴⁵), MMP3 (FC +7.4, p=1.3x10⁻¹¹) and IRTA1 (FC -11.4, p= 4.7xl0⁻¹²) were
differentially expressed compared to controls. In the colon SAA1 (FC +6.3, p=
5.3xl0⁻⁸) was upregulated and TSLP (FC -2.3, p= 2.7xl0⁻⁶) was downregulated
comparing non-inflamed Crohn's disease and control biopsies.Of the Crohn's disease susceptibility genes identified by genome wide association
scan IL-23A, JAK2 and STAT3 were upregulated in Crohn's disease confirming
dysregulation of Thl7 signalling. Modest differential expression was also observed in
a number of the autophagy genes, notably ATG16L1. When clustering analysis was
undertaken, terminal ileal Crohn's disease and terminal ileal control biopsies
separated from colonic Crohn's disease and colonic control biopsies. Further
clustering analysis of the terminal ileal biopsies showed separation between the
terminal ileal Crohn's disease and control biopsies.When the ulcerative colitis biopsies and control biopsies were compared,
differentially upregulated genes in ulcerative colitis included SAA1 (p<10⁻⁴⁵) the
alpha defensins, DEFA5&6 (p=0.00003 and p=6.95xl0⁻⁷ respectively), MMP3
(p=5.6x10⁻¹⁰) and MMP7 (p=2.3x10⁻⁷). Increased DEFA5&6 expression was further
characterized to Paneth cell metaplasia by immunohistochemistry and in-situ
hybridization.Variants in all the examined IBD5 SNPs were associated with Crohn's disease
(p<0.003). The IBD5 locus was also associated more severe Crohn's disease
behaviour. In the absence of the IBD5 risk haplotype, no association of OCTN1/2
variants with Crohn's disease was detected. The analysis of the DLG5 variant 113A
showed there were no associations with inflammatory bowel disease, Crohn's disease
or ulcerative colitis.Overall these data emphasise the key role of a number of inflammatory molecules and
pathways in pathogenesis of Crohn's disease and ulcerative colitis, and their potential
for translation to therapeutic targets. The results also add considerably to the recent
genome wide association studies in providing complimentary human colonic and ileal
expression data along with detailed analysis of the IL-23 and autophagy pathways
Probiotics and inflammatory bowel disease
The original publication is available at www.springerlink.com © Springer 2006Gordon S. Howarthhttp://www.springer.com/medicine/internal/book/978-1-4020-5701-
Predicting outcome in ulcerative colitis: clinical and genetic determinants of disease susceptibility and behaviour
Ulcerative colitis (UC) and Crohn's disease (CD), collectively known as inflammatory
bowel disease (IBD) are common, chronic inflammatory disorders of the intestines. The
pathogenesis and subsequent clinical course of IBD remain unclear; although, inter¬
relating factors such as environment triggers, dysregulated immune response, defective
gut barrier defence, variability of drug response and genetic susceptibility all contribute.
The current work described in this thesis involves a series of clinical and genetic studies
investigating their respective roles in determining susceptibility and course in UC.
Firstly, the importance of corticosteroid resistance/dependence as a co-factor in disease
progression was studied in a detailed 5-year inception cohort (1998-2003). This study
demonstrated remarkable concordance with older series and paediatric cohorts suggesting
a defined phenomenon within the innate response to corticosteroids. Secondly, a risk
score to stratify the likelihood of response to standard medical therapy (high dose
corticosteroids) identifying 3 distinct risk groups - low, intermediate and high risk; based
on 167 consecutive patients (largest series studied) with acute severe UC was developed.
A novel robust model was formulated with a sensitivity/specificity of 85% and 75%
respectively to predict failure of medical therapy. These initial studies have critically
provided the assimilation of highly accurate phenotypic and follow-up data, permitting
very detailed statistical analyses to be performed in subsequent genetic studies. The
multidrug resistance gene (MDR1 gene-encoding P-glycoprotein 170, an epithelial efflux
transporter protein) was studied in detail due to its potential role in conferring
susceptibility (based on knock-out animal models and genomic position within IBD locus
of susceptibility) and its function in corticosteroid resistance. In this study, allelic variations of the MDR1 gene were found to be implicated in disease susceptibility in UC,
in particular extensive and severe disease subphenotype (p=0.003, OR 2.64 and T-allele,
p=0.009, OR 1.70. In this study, bi-directional haplotypic contribution to susceptibility in
UC (with protective and susceptible haplotypes) was observed. This led to a more
rigorous analysis of this gene by the application of the novel 'gene-wide' haplotype
tagging approach, confirming a more significant association with UC (p=4.22xl0"7) but
not CD (p=0.22). The strongest association was with the sub-phenotype; extensive UC
(p= 1.7 X 10"7), and critically dependent on one tSNP rs3789243 (p= 3.2 x 10"7- 3.6 x 10"
12). This catalysed further rational approaches to study the genetic variations of the genes
involved in xenobiotic-metabolism and epithelial transport such as the ATP-binding
cassette (ABC) efflux transporters and respective transcriptional regulators in our cohort.
Further significant and replicable association of haplotypic variations of the
ABCC3/MRP3 gene (also involved in epithelial barrier defence) with IBD (p=0.00004,
1200 IBD and 700 controls) was also shown. The characterisation of these genes in a
hitherto unknown pathway regulated by key transcriptional regulators such as PregnaneX receptor (PXR) has now implicated this novel class of transport proteins as important
regulators of mucosal defence and as critical in determining susceptibility to
inflammatory bowel disease
The anti-colitic properties of hookworm protein Na-AIP-1
Geraldine Buitrago tested the anti-inflammatory properties of hookworm secreted protein Na-AIP-1. She found that this protein was able to suppress colon inflammation in mice. It is hoped this protein can be developed as a new treatment for inflammatory bowel diseases
Exploring the utility of metabolic profiling in stratifying patient groups in Inflammatory Bowel Disease
The pathogenesis of IBD, involving dynamic interactions between the microbiome, innate and adaptive immune systems, genetics and environmental factors, is a major focus of academic interest, in order to reveal more about the heterogeneous clinical course of the disease and in pursuit of improved therapeutic targets.
Metabonomics has been previously used with a variety of biofluids to successfully distinguish IBD from controls, but the complex metabolic data also have potential to unlock insights into pathogenesis and better understand how to better stratify patients for personalised clinical care.
In the largest urinary metabonomics IBD study to date, changes in the white European cohort confirmed previous published findings, highlighting discriminatory metabolites of gut microbial and inflammatory pathway sources. Significant metabolic differences were seen when comparing IBD patients and controls from South Asia to white North Europeans, demonstrating the influence of ethnicity on the metabolic profile and showing metabolite changes related to host-nutrition-microbiome interactions.
Results from longitudinal measurements of the IBD metabolome in the same individuals over several years indicate relative stability despite the relapsing-remitting course of the disease and different treatments. This early finding suggests clinical outcomes may only have subtly discernible changes on metabolic profiles, potentially limiting its application as a disease-monitoring tool.
16S rRNA profiling, employed to characterise the microbiome, showed reduced microbial diversity in IBD and 4 key bacterial genera - Veillonella, Acidaminococcus, Lactobacillus and Streptococcus - associated with disease. Significant urinary and faecal metabolites in the same patients were correlated with these bacteria to demonstrate the feasibility of multi-omic integration in IBD.
Furthermore, the breath VOC profiles of IBD patients obtained by SIFT-MS were distinct from those of heathy controls, with the significant compounds originating from microbial sources, and inflammatory pathways, demonstrating the potential of this technology and another facet to metabolic profiling in IBD.Open Acces
Modulation of Intestinal Dendritic Cells by Manipulation of Enteric Bacteria in Intestinal Inflammation
Inflammatory bowel disease (IBD) involves dysregulated immune responses to intestinal
microbiota. Intestinal dendritic cells (DC) play a pivotal role in bacterial recognition,
tolerance induction, T cell homing and differentiation.
We hypothesized that alterations in human colonic DC are central to the inflammatory
process, lymphocyte homing, and therapeutic responses in patients with IBD. Colonic
CD11c+ cells have been shown to be activated in IBD but CD11c- cells have not been
characterised. We identified, in ulcerative colitis (UC), a CD11c- population that had
morphological features of DC, expressed MHC class II and Natural Killer cell marker
CD56, expressed less activation markers and produced less cytokine, and were weakly
stimulatory. Few were plasmacytoid DC. Their number increased in UC and Crohn’s
disease (CD) but decreased after inflammation resolved.
We explored function and homing properties of colonic CD11c+ DC, and their relationship
with intestinal microbiota in IBD. In acute UC, IL-10+ and IL-12p40+ CD11c+ DC
increased, and fewer CD11c+ DC expressed the homing molecule α-E (CD103). In active
CD, IL-12p40+ DC increased and the ratio of pro:anti-inflammatory bacteria, namely
bacteroides:bifidobacteria, correlated positively with IL-12p40+ DC; IL-6+ DC also
increased and correlated with increased C-reactive protein, but negatively with antiinflammatory
Faecalibacterium praustnitzii.
In IBD probiotics and corticosteroids may work, in part, by modulating DC function. In
UC patients treated with the probiotic mixture VSL#3, TLR-2+ DC and IL-12p40+ DC decreased while IL-10+ DC increased. In patients on corticosteroids similar changes were
seen. Such effects were however not seen in patients on placebo.
In conclusion, intestinal inflammation in IBD is associated with novel human colonic cells
that share features of DC and NK cells. Intestinal DC function is influenced by
composition of the commensal microbiota. Probiotics and corticosteroids are associated
with altered “favourable” DC function; these effects may contribute to therapeutic benefit
in patients with IBD
Intestinal macrophages in health and inflammation
The healthy large intestinal mucosa contains a vast pool of macrophages (mφ) that are close to the local bacterial flora and have several unique phenotypic and functional properties compared with other mφ populations. Although human colonic mφ retain some of the hallmark functions of mφ, such as the ability to phagocytose particulate material and exert bactericidal activity, they are unable to produce pro-inflammatory mediators. Thus it has been suggested that intestinal mφ are functionally adapted to the microbe-rich, immunostimulatory environment of the gut, where strong inflammatory responses to harmless commensal bacteria would lead to continuous inflammation and ultimately tissue pathology. Indeed, there is mounting evidence that mφ play an essential role in maintaining homeostasis and epithelial renewal in the normal intestine. In contrast, mφ from the intestine of patients with inflammatory bowel disease (IBD) differ markedly from those present physiologically, exhibiting heightened inflammatory and bactericidal activities, and contributing to the tissue damage. How these differing properties of colonic mφ are controlled and how this potentially dangerous population is kept quiescent under physiological conditions are important questions. Most existing information comes from either simple, observational studies of human tissue, or from work on cell culture systems which aim to reproduce the unusual phenotype of resident intestinal mφ in vitro. Importantly analogous experiments of resident and inflammatory mφ have not been carried out in murine systems, where it would be possible to characterise the cells fully and explore their origin, function and role in inflammatory processes.
Therefore, the aims of this thesis were first to characterise mφ in the resting murine colon both functionally and phenotypically, focussing particularly on their expression of toll-like receptors (TLR) and responsiveness to TLR ligation and on their population dynamics in vivo. By comparing colonic mφ with other tissue mφ populations, I hoped to gain an understanding of how resident gut mφ might have adapted to their local environment. In the second part of my work, I examined how the properties of colonic mφ altered in inflammation, employing a well-established experimental model of colitis, with the aim of determining how resident and inflammatory mφ might relate to each other. Lastly, I explored the effects of the ES-62 parasite product, known to have potent anti-inflammatory effects on mφ, on experimental colitis in vivo.
Experiments detailing my initial characterisation of the myeloid cells expressing the F4/80 mφ marker in the colon of normal mice are described in Chapter 3. These revealed that the F4/80+ population in the gut is extremely heterogeneous compared with other mφ populations in the body. Virtually all in vitro-differentiated BM mφ (BMM) and mφ from the resting peritoneum (PEC mφ) exhibited the conventional F4/80+CD11b+CD11c- phenotype of classical mφ and upregulated costimulatory molecules in response to TLR ligation. In stark contrast, the colon contained three F4/80+ subsets, one F4/80+CD11b+CD11cint, one F4/80+CD11b+CD11c- and a smaller population of F4/80+CD11b-CD11c- cells. None of these subsets expressed co-stimulatory molecules, even after LPS stimulation, but unlike other mφ, the majority of colonic mφ expressed high levels of class II MHC without stimulation. BMM and PEC mφ also produced several pro-inflammatory cytokines and chemokines following stimulation, whereas colonic mφ showed no mediator production under these conditions. Nevertheless, colonic mφ did retain avid endocytic and phagocytic activities, indicating that colonic mφ may engulf bacteria without initiating inflammation.
In Chapter 4, I explored the unresponsiveness of resting colonic mφ to microbial stimuli in more detail and found that the TLR refractoriness is associated with reduced expression of TLR2, 3, 4 and 9. Apart from a small proportion of mφ that retained TLR2 expression, TLR expression was downregulated both at the protein level and to some extent also at the mRNA level; TLRs were not re-expressed following ex vivo culture of purified mφ. This global downregulation of TLRs could not be reproduced in BMM by treatment with TLR ligands, and was also present in colonic mφ taken from mice unable to signal via TLR2 or TLR4, suggesting it was not simply a form of endotoxin “tolerance”. However, the mechanism seemed to involve IL-10, as colonic mφ from IL-10-deficient animals displayed a heightened level of TLR expression and responsiveness, even prior to the onset of intestinal inflammation.
In Chapter 5, I examined the phenotype and function of mφ during the experimental colitis induced by feeding dextran sodium sulphate (DSS). During inflammation, the absolute number of F4/80+ mφ increased 6-fold, the majority of which now expressed TLR, CD11b and low levels of CD11c. This new population of colitic mφ also expressed class II MHC, low levels of co-stimulatory molecules and produced large amounts of TNFα. In Chapter 6, I went on to examine the population dynamics of colonic mφ under resting conditions and during inflammation, showing that the overall turnover rate of the total mφ population was increased during colitis, as assessed by uptake of BrdU in vivo. The increased turnover was mainly due to the TLR-expressing, TNFα+ population of mφ and more detailed analysis showed that the small number of these cells present in resting colon had identical turnover rates to those found in colitis. In contrast, the TLR negative mφ had much lower turnover rates in resting and inflamed colon, suggesting that the TLR+ and TLR- subsets may represent distinct mφ populations with different population dynamics, and that during intestinal inflammation, the TLR+ subset may display a preferential recruitment into the gut. Indeed, proliferation in situ was minimal, indicating that the recently divided, TLR-expressing mφ proliferated outside the intestine before being recruited into the gut. My subsequent experiments suggested that this recruitment may involve the CCR2 chemokine receptor, which was expressed at high levels specifically by the TLR+ subset of mφ both in resting and inflamed colon.
Finally in Chapter 7, I treated colitic mice with ES-62, a phosphorylcholine (PC)-containing glycoprotein secreted by the filarial nematode, Acanthocheilonema viteae, which has been shown to modulate pro-inflammatory cytokine production by mφ in vitro. ES-62 treatment had no significant effect on weight loss or pro-inflammatory cytokine production in the colon of mice with DSS colitis, although it slightly delayed the onset of the clinical signs of disease. Thus further studies of ES-62 as a modulator of mφ-dependent intestinal inflammation may be warranted.
Taken together, my data suggest that under resting conditions, intestinal mφ are heterogeneous and adapt to their microenvironment by being non-inflammatory via active downregulation of TLR expression and function, which may be partly dependent on IL-10. During inflammation, large numbers of TLR expressing, fully responsive mφ appear, probably via CCR2-dependent recruitment of recently divided blood-derived monocytes. Interestingly, small numbers of these TLR-expressing, rapidly turning over mφ are also present in normal colon and my data suggest that these pro-inflammatory mφ may be quite distinct from the more sessile mφ which are the dominant “resident” population in normal gut. A delicate balance between these two mφ populations must ensure homeostasis and appropriate responses to inflammation
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