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The TULA-family consists of two proteins implicated in cellular regulation. TULA-1 is expressed in T-cells and is involved in apoptosis. TULA-2 is a ubiquitously expressed phosphatase that suppresses receptor-mediated signaling. T cells from mice lacking TULA-1 and 2 (double knockout, or dKO) are hypersensitive to TCR stimulation. This may be due to these proteins having a similar function working synergistically or dissimilar functions having a convergent effect. To understand functional interaction of these proteins we have characterized TULA-family knockout mice without and during an immune challenge. We show that CD4+ T cells of dKO mice have a characteristic CD45RB distribution, and that within the CD45RBlow subset effector/memory T cells are expanded only in dKO, but not in single knockouts (sKO) of either TULA-1 or TULA-2. However, CD4+ T cells of sKO and wild-type (WT) mice respond differently to TCR stimulation as seen using signaling and responses in vitro. To evaluate consequences of TULA deficiency in vivo, we utilized two mouse models of inflammatory bowel disease: TNBS-induced colitis and colitis induced by the adoptive transfer of CD45RBhigh CD4+ T cells. Studies utilizing TNBS indicate that deficiency of any TULA-family protein exacerbates TNBS-induced colitis. Likewise, dKO CD45RBhigh CD4+ T cells were significantly more colitogenic than cells from WT mice in the transfer model. Taken together, our data indicate that TULA-family proteins are key to the physiological regulation of T-cell reactivity that drives intestinal inflammation. / Microbiology and Immunology

Serotonin (5-hydroxytryptamine [5-HT]) is a key enteric signaling molecule that is implicated in many gastrointestinal (GI) disorders, including inflammatory bowel disease (IBD). Enterochromaffin (EC) cells are a key subgroup of enteric endocrine cells and produce the majority of 5-HT via tryptophan hydroxylase 1 (Tph1) in the gut. Recently, we have identified a pivotal role of 5-HT in the pathogenesis of experimental colitis, whereby 5-HT plays as a pro-inflammatory molecule. Gut function as well as pathology rely on interactions with gut microbiota. The intestinal epithelial cells produce antimicrobial peptides (AMPs), maintaining the mucosal barrier by shaping gut microbiota composition. Among the AMPs, β-defensins are the most well investigated subtype in the colon. Aberrant β-defensin expression has been reported in association with various GI disease pathogenesis including IBD. As EC cells are dispersed throughout the intestinal epithelium, it seems possible that 5-HT can modify β-defensin production which can regulate gut inflammation by influencing gut microbial composition. Colitis was induced with dextran sulfate sodium (DSS) in Tph1+/+ and Tph1-/- (which have lower amounts of 5-HT in gut). Tph1-/- mice exhibited higher levels of β-defensin in the colon, compared with wild-type littermates post-DSS. In addition, increased expression of β-defensin in Tph1-/- mice was suppressed by 5-hydroxytryptophan (5-HTP; precursor of 5-HT) treatment. 5-HT treatment resulted in decreased human β-defensin (hBD) 1 and hBD-2 expression in HT-29 cells. Peroxisome proliferator-activated receptor gamma (PPAR-γ) is essential for maintaining β-defensin expression in the colon. GW-9662, PPAR-γ antagonist, reduced mouse β-defensin (mBD) 1 and mBD-3 (orthologue of hBD-2). Furthermore, disrupting 5-HT7 receptors, but not 5-HT3 or 5-HT4, led to enhanced expression of PPAR-γ via ERK1/2-dependent mechanism. These observations provide us with novel information on pivotal role of gut-derived 5-HT in innate immune response and highlight the potential benefits of targeting 5-HT signaling in various GI disorders such as IBD. / Thesis / Master of Science (MSc)

Irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) are two of the most commonly diagnosed chronic digestive disorders in Western countries with increasing prevalence among Canadians. However, the etiology of IBS and IBD remain poorly understood due to a complex interplay of genetic, psychosocial and environmental factors, which hampers efforts at early detection/screening, accurate diagnosis and effective treatments notably in children. This thesis aims to reveal new biochemical insights into the pathophysiology underlying IBS and IBD when using an untargeted metabolite profiling (i.e., metabolomics) approach on urine and stool specimens based on multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Chapter I reviews brief history and current challenges in diagnosis and treatment, as well as current metabolomics literature of IBS and IBD. Chapter II first develops a robust method for high throughput profiling of anionic metabolites in human urine samples when using MSI-CE-MS. For the first time, we demonstrate that incidental capillary fractures are caused by irreversible aminolysis of the outer polyimide coating due to the frequent use of volatile ammonia based buffers under alkaline conditions (pH > 9) in electrospray ionization-MS. Chapter III subsequently applies this validated method to investigate differentially excreted urinary metabolites between adult IBS patients and healthy controls, which indicated significantly accelerated rates of collagen degradation and cell turn-over in IBS patients. Chapter IV later develops a novel stool extraction protocol for characterization of the fecal metabolome together with meta-genomic data for elucidating complex host-gut microflora interactions from a cohort of pediatric IBD patients, including Crohn’s disease and ulcerative colitis. In this pilot study, a panel of discriminating metabolites in urine is shown to allow for differential diagnosis of major pediatric IBD sub-types as an alternative to colonoscopy and histopathology that are invasive, expensive and prone to ambiguous test results. Finally, Chapter V involves a longitudinal metabolomics study that aims to identify metabolic trajectories that predict treatment responses of a cohort of pediatric Crohn’s disease patients following initiation of exclusive enteral nutrition (EEN) therapy. In the end, Chapter VI highlights major outcomes of thesis and future direction of metabolomics in IBS and IBD with a specific focus on improved stool specimen collection and validation of biomarker specificity relative to other related gastrointestinal disorders. In summary, this thesis has demonstrated metabolic processes that are associated with exacerbation of symptoms or remission in subset of IBS and pediatric IBD patients. With follow up studies with larger cohort of patients, potential biomarkers identified in this thesis will contribute the development of more accurate and non-invasive decision making process for diagnosis and treatment, resulting in long-lasting remission and improved quality of life of patients suffering from chronic digestive disorders. / Thesis / Doctor of Science (PhD)

Yes / Our aim is to provide an understanding of the experience of women with IBD who have made the transition to motherhood. Twenty-two mothers with IBD were recruited from around the UK. Semi-structured interviews were conducted and analyzed using thematic analysis. The central concept – Blurred Lines – offers a novel frame for understanding the transition to motherhood with IBD through identifying parallels between having IBD and becoming, and being, a mother. Parallels clustered into three main themes: Need for Readiness, Lifestyle Changes, and Monitoring Personal and Physical Development. Hence, women with IBD are in some ways well prepared for the challenges of motherhood even though, as a group, they tend to restrict their reproductive choices. We recommend health professionals initiate conversations about reproduction early and provide a multidisciplinary approach to pregnancy and IBD in which women have confidence that their on-going treatment will be integrated successfully with their maternity care. / Crohn’s & Colitis UK [grant number SP2013/2].

Bakgrund: Kroniska inflammatoriska tarmsjukdomar (IBD) innefattar i första hand Ulcerös kolit (UC) och Crohns sjukdom (CD) vilka är de vanligast förekommande inflammatoriska tarmsjukdomarna. IBD karaktäriseras av symtom som smärta i mag-tarmkanalen, diarré, blod i avföringen och att symtomen går i skov. Syfte: Syftet med denna litteraturstudie var att belysa patienters upplevelser av att leva med en kronisk inflammatorisk tarmsjukdom. Metod: Metoden som användes i studien var allmän litteraturstudie. Vidare användes en induktiv ansats i denna litteraturstudie. Resultat: Resultatet visade att IBD påverkar patienter på flera nivåer. Nivåerna delades in i fyra kategorier: Psykisk hälsa vid IBD, Fysisk aktivitet och energinivåer vid IBD, Samverkan med sjukvårdspersonal vid IBD samt Kosthållning och nutrition vid IBD. Konklusion: Patienter som led av IBD upplevde att sjukdomen har en negativ påverkan på den psykiska hälsan. Dessutom påverkade IBD patienters energinivåer till att patienter hade svårt att utföra vardagliga aktiviteter, främst när de genomgick skov. De patienter som fick möjligheten att vara delaktiga i sin vård upplevde i högre grad minskad ångest relaterat till vald behandlingsmetod. Vidare upplevde patienter att sjukvården inte gav den hjälp gällande kostrådgivning som de själva kände sig behöva. / Background: Inflammatory bowel disease (IBD) primarily includes ulcerative colitis (UC) and crohn's disease (CD), which are the most common forms of inflammatory bowel diseases. IBD are characterized by symptoms such as bowel pain, diarrhea, blood in the stool, and that the symptoms often manifest in flares. Aim: The aim of the study was to illustrate patients experiences of living with inflammatory bowel disease. Method: The method used in this study was a general literature study. Furthermore an inductive approach was used in the study. Results: The result showed that IBD affects patients on different levels. The levels were divided into four categories: Mental health and IBD, Physical health and energy levels and IBD, Collaboration with healthcare personnel and IBD, and Diet and nutrition and IBD. Conclusion: Patients that suffered from IBD experienced a negative affect on their mental health. Additionally, the diagnosis affected the patients energy levels, which led to struggles in the day to day life, foremost when they went through a flare. The patients that participated in their own treatment, experienced lower anxiety in relation to their treatment. Moreover, the patients described that they did not get the help they required when it came to their diet changes.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13601号 / 論医博第2311号 / 新制||医||1072(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊藤 能永, 教授 椛島 健治, 教授 松田 秀一 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25168号 / 医博第5054号 / 新制||医||1071(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 上野 英樹, 教授 波多野 悦朗, 教授 伊藤 能永 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

IL-23 is an essential mediator of chronic intestinal inflammation in experimental models of colitis. Polymorphisms in the IL23R locus are associated with IBD susceptibility in humans. The biological activity of IL-23 has been linked to Th17 cells but little is known about the cellular and molecular mechanism by which IL-23 drives intestinal inflammation. The work presented herein has identified that direct IL-23 signalling into CD4+ T cells was not only required for the accumulation of Th17 cells in the intestine but also modulated their phenotype. Through direct cell intrinsic effects on T cells, IL-23 drove the emergence of an IL-17A+IFN-γ+ population of T cells that co-expressed RORγ and T-bet. Interestingly, we found that expression of RORγ but not T-bet by T cells was required for the development of intestinal inflammation. Furthermore, colitis induced by T-bet deficient T cells was dependent on IL-17A, and showed a unique inflammatory phenotype, thus demonstrating that pathogenic intestinal Th17 responses can develop independently of T-bet. In addition, using transcriptional profiling we identified a core set of genes that is regulated by direct cell-intrinsic IL-23 signals into intestinal CD4+ T cells. This revealed a previously unrecognised role for IL-23 in suppressing Th2 associated genes, such as GATA3 and IL-33R. Functional experiments demonstrated that expression of GATA3 in CD4+ T cells limited their colitogenic potential, suggesting that IL-23-mediated inhibition of GATA3 might contribute to the development of intestinal inflammation. Finally, we described a novel function for IL-33 as a factor that promotes Foxp3+ iTreg differentiation in vitro and in vivo through direct effects on T cells. This activity of IL-33 was inhibited in the presence of IL-23, providing a mechanistic link for the known role of IL-23 in restraining iTreg generation. Collectively, these data suggest that IL-23 promotes acquisition of a pathogenic effector T cell phenotype through multiple mechanisms. This indicates that therapeutic blockade of IL-23 is likely to reduce pro-inflammatory mediators while also facilitating the expansion of regulatory pathways that might help to re-establish intestinal homeostasis.

Leukocyte extravasation is mediated in part by tissue specific chemotactic cytokines (chemokines) and specific chemokine receptors expressed on the surface of circulating cells. C-C chemokine ligand CCL25 is expressed exclusively in the intestine and thymus and mediates chemotaxis by cells expressing receptor CCR9. This chemokine and receptor pair may be relevant in the pathogenesis of intestinal inflammation, in diseases such as Crohn’s disease (CD) and coeliac disease. In this thesis I investigated CCR9 expression in situ, in tissues affected by intestinal inflammation, and also examined the effects of CCR9 antagonist treatment in patients. In vitro I investigated CCR9 function using human peripheral blood T cells enriched for CCR9 by cell sorting or all-trans retinoic acid treatment. Using tissues collected as part of a clinical trial in CD testing CCR9 antagonist, CCX282-B, I investigated ways of measuring if treatment reduced the number of CCR9 expressing cells in the intestinal mucosa. However, in situ staining for CCR9 by immunohistochemistry was unsuccessful, and in this thesis, I explored reasons why this might be the case. Treatment with CCX282-B did however, show a tendency to reduce T cell density in the intestinal mucosa, although results were highly variable between individuals. In an examination of human CCR9 function in vitro I demonstrate for the first time that CCL25 stimulates CCR9 surface internalization. These data clarify the observation that CCR9 staining by IHC produces poor results in tissues where ligand is abundant, such as the intestine and thymus. I describe a novel technique for measuring calcium flux in two populations simultaneously by flow cytometry, which confirmed that in a heterogeneous population of cells, only CCR9 expressing cells respond to CCL25 by calcium flux. Variability in clinical trials is partly created by the use of concomitant medications, and in CD, corticosteroids are widely used. For the first time I show that glucocorticoids (GC) impair CCR9 mediated chemotaxis, calcium flux and intracellular signalling without changes to CCR9 mRNA and surface protein expression. Reduced CCR9 mediated signalling was accompanied by an enhanced expression and function of co-expressed CXCR4, demonstrating that the effects of GC were receptor-specific and not mediated by non-specific toxicity or inhibition of cell signalling. In a second study CCX282-B was tested in patients with coeliac disease, and in this trial, there was no reported concomitant use of GCs. It was confirmed that dietary gluten stimulates significant T cell recruitment to the intestinal mucosa with a pronounced accumulation of intraepithelial lymphocytes (IEL) and a rise in the frequency of FoxP3 expressing cells. Patients on CCX282-B had lower IEL counts, and an equivalent proportion of FoxP3 expressing T cells, suggesting that CCR9 blockade restricted the recruitment of effector T cell subsets. This thesis confirms that the accumulation of T cells is central to inflammation in the intestine and that modulating chemokine receptor function may affect this. Furthermore, this thesis demonstrates that the function of CCR9 is suppressed by GCs, which are widely used therapeutically and therefore could identify a novel mechanistic basis for their activity in CD.