The main forms of inflammatory bowel diseases (IBD) are Crohns disease and ulcerative colitis. These are chronic diseases, with periods of progression and remission. They are mostly characterized by digestive symptoms such as diarrhea, abdominal pain and weight loss. They affect young individuals and their frequencies have increased for the last decades. The etiology of these pathologies is not well understood, however genetic and environmental factors are involved. The treatment of IBD aims to control the inflammation and to extend periods of clinical remission. Infliximab is an anti-TNF-α antibody, leading to a clear improvement of the symptomatology. However, about 30 % of the patients do not response to this treatment. Genetic factors are certainly involved in these inter-individual differences. The purpose of our work was to find: 1- genetic factors implicated in the response to Infliximab in Crohns disease and 2- genetic factors predisposing to IBD. First we could show that both genes LTA and TNF, which are closely related, are not associated with the answer to Infliximab in Crohns disease. However, different polymorphisms of the ADAM17 gene were associated with a response to the treatment in our Belgian cohort. Second, we could demonstrate an association between an insertion/deletion in the IRF5 gene and IBD. The insertion allele, predisposing to IBD, is expected to create a new binding site for the SP1 transcription factor.

La malaltia de Crohn és una malaltia inflamatòria intestinal de base immunitària. L’etiologia de la malaltia és desconeguda, encara que en general s’admet la següent definició: la malaltia és conseqüència d’una desregulació de la resposta immunitària en front als antígens comuns de la flora bacteriana intestinal en individus genèticament susceptibles. Un component important de la desregulació immunitària es la hiperactivació dels limfòcits T que es dóna en aquesta i altres malalties de tipus autoimmunitari, i en les que tradicionalment s’ha atribuït una major presència i producció de citocines per part de la població limfocitària Th1 en el teixit inflamat. Recentment, amb la descripció de la citocina IL-23 i la població Th17 se ha posat en dubte el paper principal de la població Th1 en les malalties inflamatòries de base immunitària. De fet, en algunes d’aquestes malalties s’ha descrit un augment de las cèl•lules Th17 així com de les citocines produïdes per aquesta població. L’objectiu d’aquesta tesi ha sigut caracteritzar el paper relatiu de les poblacions Th1 i Th17 en la mucosa intestinal inflamada i en la circulació perifèrica de pacients amb malaltia de Crohn activa o en remissió així com avaluar el paper i l’evolució d’aquestes poblacions limfocitàries en els estadis inicials (primers brots de la malaltia) i avançats de la malaltia. En aquest estudi observem que els períodes d’activitat inflamatòria en la malaltia de Crohn s’associen a una resposta sistèmica exacerbada de la població Th17, essent la producció de IL-17 en sobrenedant del cultiu de sang total, el percentatge de limfòcits CD4+IL-17+ i la producció de IL-17 per part d’aquestes cèl•lules més elevada que en els pacients amb malaltia inactiva i que en els controls sans. Paral•lelament hem descrit que la població Th1 circulant no presenta durant un brot d’activitat un augment tan generalitzat com la població Th17, sinó que només observem un augment en el percentatge de limfòcits CD4+IFN-γ+ circulants i en els dobles productors de IL-17 y IFN-γ. Els pacients que denominem debut (pateixen el primer brot de la malaltia) no presenten a nivell sistèmic un augment de la resposta Th17 ni Th1. A diferència del que observem en circulació perifèrica, en la mucosa intestinal inflamada trobem un augment dels trànscrits de les citocines característiques de la població Th17, tant en els pacients debut com en els que es troben en un estat més avançat de la malaltia. A més, en la mucosa inflamada d’ambdós grups de pacients trobem una major infiltració de cèl•lules IL-17+. A partir d’aquests resultats hipotetitzem un model per a la fisiopatologia de la malaltia de Crohn en el que la generació de limfòcits Th17 memòria podrien estar implicats en la cronicitat i recurrència de la malaltia. Així en les primeres fases de la malaltia només detectem un augment d’aquesta població en la mucosa intestinal, com seria d’esperar d’una resposta immunitària local. A conseqüència d’aquesta es generarà una població limfocitària de memòria immunitària que només detectem en circulació en aquells malalts que es troben ja en estadis més crònics de la malaltia. Amb aquesta hipòtesi podem explicar mitjançant mecanismes immunitaris diferents el fet que, en general, la malaltia de Crohn no es manifesta fins la segona o tercera dècada de vida d’un individu, mentre que un cop s’ha manifestat, la recurrència d’activitat inflamatòria és molt més freqüent (degut a la participació dels limfòcits memòria). / One component of the immune deregulation observed in Crohn’s disease is the massive infiltration of T lymphocytes in the inflamed tissue. Classically these hyperreactive lymphocytes have been attributed to the Th1 subpopulation. Recently the description of IL-23 cytokine and Th17 population has questioned the main role given to Th1 population. In this regard, an increase in the frequency of Th17 cells and in the production of their signature cytokines has been already described in some inflammatory diseases. In this study we have observed that active inflammation in Crohn’s disease associates to an increased systemic response of Th17 cells described as overproduction of IL-17 in supernatants of whole blood cultures, increased percentage of CD4+IL-17+ lymphocytes and increased production of IL-17 from these cells related to remission periods of disease or healthy controls. Early patients (patients who suffer their first flare of the disease) do not present a systemic increase of Th1 or Th17 cells despite suffering of active inflammation. Otherwise, in inflamed intestinal mucosa there is an increase in the expression of Th17 cytokines both in early as well as late chronic patients. Moreover we found a significant higher number of Th17 cells infiltrating the mucosa of both groups of patients. Taking these results into account we hypothesize a model for the physiopathology of Crohn’s disease in which the generation of memory Th17 lymphocytes could be involved in the chronicity and recurrence of the disease. Therefore, in early phases of disease we only detect an increase of this population in the intestinal mucosa, as we could expect from a local immune response. As a consequence of this activation there would be a generation of memory lymphocytes that we can detect in peripheral circulation only in that patients that have already suffered repeated flares of the disease. This hypothesis allows us to explain the different immune mechanisms that could be acting during the evolution of Crohn’s disease, as it is the first appearance of disease, generally occurring during the second or third decade of the individual life, and the subsequent appearance of recurrences that are much more frequent and life-long lasting.

Inflammatory bowel disease (including Crohn’s disease and ulcerative colitis) and coeliac disease are debilitating gastrointestinal diseases that seriously affect the quality of life of those affected. Under normal circumstances, the intestinal immune system is maintained in a state of controlled inflammation, whereby balance exists between protective immunity, mediated by effector cells, and tolerance mediated by cells with regulatory function. However, an aberrant immune response is believed to contribute to the intestinal inflammation present in individuals afflicted by these diseases. This thesis investigated the involvement of CD4⁺ CD25[superscript]high Foxp3⁺ Regulatory T cells (Treg) and Th17 Effector cells in both inflammatory bowel disease (IBD) and coeliac disease. The reciprocal relationship between Treg and Th17 cells under certain cytokine conditions, has prompted the exploration of these two cell types in IBD and coeliac disease. Previous studies have examined these factors individually in a range of diseases, however, to our knowledge the study of both Treg and Th17 in IBD and coeliac disease subjects represents a novel area of research. Crohn’s disease (CD), ulcerative colitis (UC) and coeliac disease subjects were recruited through the Department of Gastroenterology and Hepatology at The Queen Elizabeth Hospital (QEH) in Adelaide, South Australia. In total, one-hundred and seventeen subjects were enlisted in this study to donate blood samples. In addtion, intestinal biopsy samples were collected from fifty-six subjects undergoing colonoscopy at the QEH Department of Gastroenterology and Hepatology. All subjects participated, with informed consent and ethics approval. Treg and Th17 cell numbers were investigated in the peripheral blood of Crohn’s disease, ulcerative colitis, coeliac disease and control subjects using multi-colour, intracellular flow cytometry. A decrease in Treg cell numbers and an increase in Th17 cell numbers was observed in IBD, but not in coeliac disease. Closer investigation into the ratio of Treg and Th17 cells within patients identified a near 1:1 Treg/Th17 ratio in control subjects, but a lower Treg/Th17 ratio in IBD patients. This suggested a disturbance in regulatory and effector cell equilibrium. Furthermore, the excess of Th17 cells and deficiency of Tregs could contribute to the pathologies observed in IBD. The discovery of an imbalance in Treg and Th17 cell numbers in IBD prompted further investigation of these cells in intestinal biopsies collected from IBD, coeliac and control subjects. Real time RT-PCR of intestinal biopsy samples demonstrated increased expression of the Th17 cytokine, IL-17a, in both IBD and coeliac disease. Elevated levels of the Treg transcription factor Foxp3 were also identified in intestinal biopsies from IBD subjects. It was therefore hypothesised that Treg cells may have been actively recruited from the periphery in an attempt to control inflammation in the gut; however, the intestinal cytokine microenvironment may have restricted the regulatory function of these cells. Cytokines known to promote human Th17 differentiation, namely IL-1β, IL-6, TGF-β, IL-21 and IL-23, were explored in intestinal biopsy samples from IBD, coeliac and control subjects. High levels of IL-1β and IL-6 were detected in IBD patient samples, however, no change in levels of IL-21 or IL-23 were observed in IBD or coeliac disease subjects. Elevated levels of TGF-β were only identified in UC. No changes in cytokine expression were observed between control and coeliac subjects, except a significant decrease in IL-6 levels was identified in coeliac disease sufferers. The pro-inflammatory microenvironment identified in intestinal biopsies from IBD subjects may have promoted the continual differentiation and development of Th17 cells, whilst restricting Treg activity. Moreover, the observed deficiency of Treg in IBD patients may have impaired the ability of the immune system to limit excessive pathogenic Th17 driven immune responses in the intestinal mucosa. Therefore, therapeutic approaches that aim to re-establish regulatory and effector cell homeostasis by increasing Treg numbers in IBD patients, and specifically targeting Th17 cells, may prove effective in the treatment of IBD. Approaches such as these could provide greater focus to treatment strategies for IBD management compared to current broad-spectrum immunosuppressive therapies that could increase susceptibility to cancer or infection in IBD patients. In addition, the imbalance of regulatory and effector cells demonstrated in the peripheral blood of IBD patients may potentially provide new options for a noninvasive diagnostic tool. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1457580 / Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2009

Inflammatory bowel disease (including Crohn’s disease and ulcerative colitis) and coeliac disease are debilitating gastrointestinal diseases that seriously affect the quality of life of those affected. Under normal circumstances, the intestinal immune system is maintained in a state of controlled inflammation, whereby balance exists between protective immunity, mediated by effector cells, and tolerance mediated by cells with regulatory function. However, an aberrant immune response is believed to contribute to the intestinal inflammation present in individuals afflicted by these diseases. This thesis investigated the involvement of CD4⁺ CD25[superscript]high Foxp3⁺ Regulatory T cells (Treg) and Th17 Effector cells in both inflammatory bowel disease (IBD) and coeliac disease. The reciprocal relationship between Treg and Th17 cells under certain cytokine conditions, has prompted the exploration of these two cell types in IBD and coeliac disease. Previous studies have examined these factors individually in a range of diseases, however, to our knowledge the study of both Treg and Th17 in IBD and coeliac disease subjects represents a novel area of research. Crohn’s disease (CD), ulcerative colitis (UC) and coeliac disease subjects were recruited through the Department of Gastroenterology and Hepatology at The Queen Elizabeth Hospital (QEH) in Adelaide, South Australia. In total, one-hundred and seventeen subjects were enlisted in this study to donate blood samples. In addtion, intestinal biopsy samples were collected from fifty-six subjects undergoing colonoscopy at the QEH Department of Gastroenterology and Hepatology. All subjects participated, with informed consent and ethics approval. Treg and Th17 cell numbers were investigated in the peripheral blood of Crohn’s disease, ulcerative colitis, coeliac disease and control subjects using multi-colour, intracellular flow cytometry. A decrease in Treg cell numbers and an increase in Th17 cell numbers was observed in IBD, but not in coeliac disease. Closer investigation into the ratio of Treg and Th17 cells within patients identified a near 1:1 Treg/Th17 ratio in control subjects, but a lower Treg/Th17 ratio in IBD patients. This suggested a disturbance in regulatory and effector cell equilibrium. Furthermore, the excess of Th17 cells and deficiency of Tregs could contribute to the pathologies observed in IBD. The discovery of an imbalance in Treg and Th17 cell numbers in IBD prompted further investigation of these cells in intestinal biopsies collected from IBD, coeliac and control subjects. Real time RT-PCR of intestinal biopsy samples demonstrated increased expression of the Th17 cytokine, IL-17a, in both IBD and coeliac disease. Elevated levels of the Treg transcription factor Foxp3 were also identified in intestinal biopsies from IBD subjects. It was therefore hypothesised that Treg cells may have been actively recruited from the periphery in an attempt to control inflammation in the gut; however, the intestinal cytokine microenvironment may have restricted the regulatory function of these cells. Cytokines known to promote human Th17 differentiation, namely IL-1β, IL-6, TGF-β, IL-21 and IL-23, were explored in intestinal biopsy samples from IBD, coeliac and control subjects. High levels of IL-1β and IL-6 were detected in IBD patient samples, however, no change in levels of IL-21 or IL-23 were observed in IBD or coeliac disease subjects. Elevated levels of TGF-β were only identified in UC. No changes in cytokine expression were observed between control and coeliac subjects, except a significant decrease in IL-6 levels was identified in coeliac disease sufferers. The pro-inflammatory microenvironment identified in intestinal biopsies from IBD subjects may have promoted the continual differentiation and development of Th17 cells, whilst restricting Treg activity. Moreover, the observed deficiency of Treg in IBD patients may have impaired the ability of the immune system to limit excessive pathogenic Th17 driven immune responses in the intestinal mucosa. Therefore, therapeutic approaches that aim to re-establish regulatory and effector cell homeostasis by increasing Treg numbers in IBD patients, and specifically targeting Th17 cells, may prove effective in the treatment of IBD. Approaches such as these could provide greater focus to treatment strategies for IBD management compared to current broad-spectrum immunosuppressive therapies that could increase susceptibility to cancer or infection in IBD patients. In addition, the imbalance of regulatory and effector cells demonstrated in the peripheral blood of IBD patients may potentially provide new options for a noninvasive diagnostic tool. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1457580 / Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2009

Inflammatory bowel disease (including Crohn’s disease and ulcerative colitis) and coeliac disease are debilitating gastrointestinal diseases that seriously affect the quality of life of those affected. Under normal circumstances, the intestinal immune system is maintained in a state of controlled inflammation, whereby balance exists between protective immunity, mediated by effector cells, and tolerance mediated by cells with regulatory function. However, an aberrant immune response is believed to contribute to the intestinal inflammation present in individuals afflicted by these diseases. This thesis investigated the involvement of CD4⁺ CD25[superscript]high Foxp3⁺ Regulatory T cells (Treg) and Th17 Effector cells in both inflammatory bowel disease (IBD) and coeliac disease. The reciprocal relationship between Treg and Th17 cells under certain cytokine conditions, has prompted the exploration of these two cell types in IBD and coeliac disease. Previous studies have examined these factors individually in a range of diseases, however, to our knowledge the study of both Treg and Th17 in IBD and coeliac disease subjects represents a novel area of research. Crohn’s disease (CD), ulcerative colitis (UC) and coeliac disease subjects were recruited through the Department of Gastroenterology and Hepatology at The Queen Elizabeth Hospital (QEH) in Adelaide, South Australia. In total, one-hundred and seventeen subjects were enlisted in this study to donate blood samples. In addtion, intestinal biopsy samples were collected from fifty-six subjects undergoing colonoscopy at the QEH Department of Gastroenterology and Hepatology. All subjects participated, with informed consent and ethics approval. Treg and Th17 cell numbers were investigated in the peripheral blood of Crohn’s disease, ulcerative colitis, coeliac disease and control subjects using multi-colour, intracellular flow cytometry. A decrease in Treg cell numbers and an increase in Th17 cell numbers was observed in IBD, but not in coeliac disease. Closer investigation into the ratio of Treg and Th17 cells within patients identified a near 1:1 Treg/Th17 ratio in control subjects, but a lower Treg/Th17 ratio in IBD patients. This suggested a disturbance in regulatory and effector cell equilibrium. Furthermore, the excess of Th17 cells and deficiency of Tregs could contribute to the pathologies observed in IBD. The discovery of an imbalance in Treg and Th17 cell numbers in IBD prompted further investigation of these cells in intestinal biopsies collected from IBD, coeliac and control subjects. Real time RT-PCR of intestinal biopsy samples demonstrated increased expression of the Th17 cytokine, IL-17a, in both IBD and coeliac disease. Elevated levels of the Treg transcription factor Foxp3 were also identified in intestinal biopsies from IBD subjects. It was therefore hypothesised that Treg cells may have been actively recruited from the periphery in an attempt to control inflammation in the gut; however, the intestinal cytokine microenvironment may have restricted the regulatory function of these cells. Cytokines known to promote human Th17 differentiation, namely IL-1β, IL-6, TGF-β, IL-21 and IL-23, were explored in intestinal biopsy samples from IBD, coeliac and control subjects. High levels of IL-1β and IL-6 were detected in IBD patient samples, however, no change in levels of IL-21 or IL-23 were observed in IBD or coeliac disease subjects. Elevated levels of TGF-β were only identified in UC. No changes in cytokine expression were observed between control and coeliac subjects, except a significant decrease in IL-6 levels was identified in coeliac disease sufferers. The pro-inflammatory microenvironment identified in intestinal biopsies from IBD subjects may have promoted the continual differentiation and development of Th17 cells, whilst restricting Treg activity. Moreover, the observed deficiency of Treg in IBD patients may have impaired the ability of the immune system to limit excessive pathogenic Th17 driven immune responses in the intestinal mucosa. Therefore, therapeutic approaches that aim to re-establish regulatory and effector cell homeostasis by increasing Treg numbers in IBD patients, and specifically targeting Th17 cells, may prove effective in the treatment of IBD. Approaches such as these could provide greater focus to treatment strategies for IBD management compared to current broad-spectrum immunosuppressive therapies that could increase susceptibility to cancer or infection in IBD patients. In addition, the imbalance of regulatory and effector cells demonstrated in the peripheral blood of IBD patients may potentially provide new options for a noninvasive diagnostic tool. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1457580 / Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2009

Diss. (sammanfattning) Linköping : Linköpings universitet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Microbiota colonisation of the intestinal tract makes it difficult for pattern recognition receptors (PRR) to discriminate between beneficial microbes and harmful pathogens. We aim to define the roles of cytosolic Nod-like receptors (NLR) in intestinal immunity and homeostasis. Upon activation, some NLR form inflammasomes that mediate the release of inflammatory cytokines and pyroptosis, an inflammatory form of cell death. NLR activation in the non-hematopoietic compartment was shown to be protective during acute intestinal infection. To identify the cell type responsible for this protection, we generated transgenic mice in which the key inflammasome adaptor molecule Asc is selectively ablated in intestinal epithelial cells (IEC) (Asc^ΔVC) and observed that inflammasomes are important for controlling Citrobacter rodentium clearance in these mice. To further dissect the importance of pathogen clearance by IEC inflammasome, ex vivo cultures of primary IEC organoids were established. Thus far this system has revealed profound differences in inflammasome regulation between IEC organoids and bone marrow-derived macrophages (BMDM). This research will inform our understanding of cell type-specific regulation of inflammasomes.

Les espèces réactives de l'oxygène (ROS) sont produites en continu dans tous les organismes aérobies et sont impliquées dans la signalisation cellulaire, les défenses contre les pathogènes, mais aussi le stress oxydant. Ce dernier correspond à un déséquilibre entre la production des ROS et leur prise en charge par les défenses anti-oxydantes de la cellule. Le stress oxydant est associé à de nombreuses pathologies, notamment les maladies inflammatoires chroniques de l'intestin (MICI). Parmi les métallo-enzymes qui contrôlent la concentration en ROS, les superoxide dismutases (SOD) jouent un rôle essentiel. Ces enzymes sont responsables de la régulation du superoxyde le premier ROS produit lors de la réduction du dioxygène. Dans ces travaux, des complexes de Mn(II) mimant l'activité de la Mn-SOD (SODm) ont été conçus en utilisant une approche biomimétique. Leur intérêt pour limiter le stress oxydant et l'inflammation dans un modèle cellulaire des MICI a été examiné. En particulier, leur activité biologique a été étudiée au vu de leurs propriétés physico-chimiques et de leur biodisponibilité. Les résultats obtenus avec un complexe parent ont mené à la conception d'une deuxième génération de SODm couplés à une sonde multimodale, à des peptides pénétrants, ou à des peptides adressant aux mitochondries. L'étude du complexe parent fonctionnalisé par des peptides polyarginines a démontré l'influence de charges positives portées par le ligand sur la constante de vitesse. Dans la continuité de l'approche biomimétique développée ici, la conception de SODm de novo est présentée et constitue un premier pas vers l'imitation de l'influence de la seconde sphère de coordination. / Reactive oxygen species (ROS) are produced continuously in all aerobic organisms and are involved in cell signaling, defenses against pathogens, but also oxidative stress. This latter corresponds to an imbalance between ROS production and their consumption by the antioxidant defenses of the cell. Oxidative stress is associated with numerous pathologies, such as inflammatory bowel diseases (IBD). Among the metalloenzymes controlling the concentration of ROS, superoxide dismutases (SOD) play a crucial role. These enzymes are responsibles for the regulation of superoxide, the first ROS produced by the reduction of oxygen. In this work, Mn(II) complexes mimicking the activity of the Mn-SOD (SODm) were designed using a biomimetic approach. Their relevance to limit oxidative stress and inflammation in a cellular model of IBD was investigated. In particular, their biological activity was studied in light of their physico-chemical properties and of their bioavailability. The results obtained with a parent complex led to the design of a second generation of SOD mimics conjugated with a single core multimodal probe, cell-penetrating peptides, or mitochondria-penetrating peptides. An effect of electrostatic interactions on the catalytic rate constant of the parent complex functionalized with polyarginines peptides was demonstrated, similarly to what is observed for the enzyme. In the continuity of the biomimetic approach envisioned here, the design of de novo SOD mimics is presented and constitutes a first step toward the mimicry of second sphere influence.