Immunomodulation of allergic immune response during specific immunotherapy

Atopic, IgE-mediated allergies are one of the major public health problems in Finland and other Western countries. These diseases are characterized by type 2 T helper (Th2) cell predominated immune responses (interleukin-4 (IL-4), IL-5) against ubiquitous environmental allergens. Despite of adequate pharmacological treatment, more than 20% of the patients with allergic rhinitis develop asthma. Allergen specific immunotherapy (SIT) is the only treatment currently available to affect to the natural course of allergic diseases. This treatment involves repeated administration of allergens to the patients either via sublingual route (sublingual immunotherapy, SLIT) or by subcutaneous injections (subcutaneous immunotherapy, SCIT). Successful treatment with SCIT or SLIT has been shown to provide long-term remission in symptoms, and prevent disease progression to asthma, but the immunological mechanisms behind these beneficial effects are not yet completely understood. Increased knowledge of such mechanisms could not only help to improve SIT efficacy, but also provide tools to monitor the development of clinical response to SIT in individual patients, and possibly also, predict the ultimate therapeutic outcome. The aim of this work was to clarify the immunological mechanisms associated with SIT by investigating the specific allergen-induced immune responses in peripheral blood mononuclear cells (PBMC) of allergic rhinitis patients during the course of SLIT and SCIT. The results of this work demonstrate that both therapies induced increases in the protective, Th2-balancing Th1 type immune responses in PBMC, e.g. by up-regulating signaling lymphocytic activation molecule (SLAM) and interferon gamma (IFN-γ) expression, and augmented tolerogenic T regulatory (Treg) cell type responses against the specific allergens, e.g. by increasing IL-10 or Forkhead box P3 (FOXP3) expression. The induction of allergen-specific Th1 and Treg type responses during SLIT were dependent on the treatment dose, favoring high allergen dose SLIT. During SCIT, the early decrease in Th2 type cytokine production - in particular of IL-4 mRNA and IL-4/IFN-γ expression ratio - was associated with the development of good therapeutic outcome. Conversely, increases in both Th2 (IL-5) and Th1 (IFN-γ, SLAM) type responses and IL-10 mRNA production were seen in the patients with less effective outcome. In addition, increase in Th17 type cytokine (IL-17) mRNA production was found in the PBMC of patients with less effective outcome during both SLIT and SCIT. These data strengthen the current hypothesis that immunomodulation of allergen-specific immune responses from the prevailing Th2-biased responses towards a more Th1 type, and induction of tolerogenic Treg cells producing IL-10 represent the two key mechanisms behind the beneficial effects of SIT. The data also give novel insight into the mechanisms why SIT may fail to be effective in some patients by demonstrating a positive correlation between the proinflammatory IL-17 responses, Th2 type IL-5 production and clinical symptoms. Taken together, these data indicate that the analysis of Th1, Th2, Treg ja Th17-associated immune markers such as IL-10, SLAM, IL-4, IL-5 and IL-17 could provide tools to monitor the development of clinical response to SIT, and thereby, predict the ultimate clinical outcome already in the early course of the treatment.

The Influence of Diet and Microbes on Colonic Immune Regulation and their Implications on Type 1 Diabetes

Dietary and microbial factors are thought to contribute to the rapidly increasing prevalence of T1D in many countries worldwide. The impact of these factors on immune regulation and diabetes development in non-obese diabetic (NOD) mice are investigated in this thesis. Diabetes can be prevented in NOD mice through dietary manipulation. Diet affects the composition of intestinal microbiota, which may subsequently influence intestinal immune homeostasis. However, the specific effects of anti-diabetogenic diets on gut immunity and the explicit associations between intestinal immune disruption and type 1 diabetes onset remain unclear. The research presented herein demonstrates that newly weaned NOD mice suffer from a mild level of colitis, which shifts the colonic immune cell balance towards a proinflammatory status. Several aberrations can also be observed in the peritoneal B cells of NOD mice; an increase in activation marker expression, increased trafficking to the pancreatic lymph nodes and significantly higher antigen presenting cell (APC) efficiency towards insulin-specific T cells. A shift towards inflammation is likewise observed in the colon of germ-free NOD mice, but signs of peritoneal B cell activation are lacking in these mice. Remarkably, most of the abnormalities in the colon, peritoneal macrophages and the peritoneal B cell APC activity of NOD mice are abrogated when NOD mice are maintained on a diabetes-preventive, soy-based diet (ProSobee) from the time of weaning. Dietary and microbial factors hence have a significant impact on colonic immune regulation and peritoneal B cell activation and it is suggested that these factors influence diabetes development in NOD mice.

Players of Th-cell differentiation and immune dysregulation – focus on GIMAP family genes

The balance of T helper (Th) cell differentiation is the fundamental process that ensures that the immune system functions correctly and effectively. The differentiation is a fine tuned event, the outcome of which is driven by activation of the T-cell in response to recognition of the specific antigen presented. The co-stimulatory signals from the surrounding cytokine milieu help to determine the outcome. An impairment in the differentiation processes may lead to an imbalance in immune responses and lead to immune-mediated pathologies. An over-representation of Th1 type cytokine producing cells leads to tissue-specific inflammation and autoimmunity, and excessive Th2 response is causative for atopy, asthma and allergy. The major factors of Th-cell differentiation and in the related disease mechanisms have been extensively studied, but the fine tuning of these processes by the other factors cannot be discarded. In the work presented in this thesis, the association of T-cell receptor costimulatory molecules CTLA4 and ICOS with autoimmune diabetes were studied. The underlying aspect of the study was to explore the polymorphism in these genes with the different disease rates observed in two geographically close populations. The main focus of this thesis was set on a GTPase of the immunity associated protein (GIMAP) family of small GTPases. GIMAP genes and proteins are differentially regulated during human Th-cell differentiation and have been linked to immune-mediated disorders. GIMAP4 is believed to contribute to the immunological balance via its role in T-cell survival. To elucidate the function of GIMAP4 and GIMAP5 and their role in human immunity, a study combining genetic association in different immunological diseases and complementing functional analyses was conducted. The study revealed interesting connections with the high susceptibility risk genes. In addition, the role of GIMAP4 during Th1-cell differentiation was investigated. A novel function of GIMAP4 in relation to cytokine secretion was discovered. Further assessment of GIMAP4 and GIMAP5 effect for the transcriptomic profile of differentiating Th1-cells revealed new insights for GIMAP4 and GIMAP5 function.

Diet, microbes and gut immune responses in the pathogenesis of experimental type 1 diabetes

Type 1diabetes (T1D) is an autoimmune disease, which is influenced by a variety of environmental factors including diet and microbes. These factors affect the homeostasis and the immune system of the gut. This thesis explored the altered regulation of the immune system and the development of diabetes in non-obese diabetic (NOD) mice. Inflammation in the entire intestine of diabetes-prone NOD mice was studied using a novel ex-vivo imaging system of reactive oxygen and nitrogen species (RONS), in relation to two feeding regimens. In parallel, gut barrier integrity and intestinal T-cell activation were assessed. Extra-intestinal manifestations of inflammation and decreased barrier integrity were sought for by studying peritoneal leukocytes. In addition, the role of pectin and xylan as dietary factors involved in diabetes development in NOD mice was explored. NOD mice showed expression of RONS especially in the distal small intestine, which coincided with T-cell activation and increased permeability to macromolecules. The introduction of a casein hydrolysate (hydrolysed milk protein) diet reduced these phenomena, altered the gut microbiota and reduced the incidence of T1D. Extra-intestinally, macrophages appeared in large numbers in the peritoneum of NOD mice after weaning. Peritoneal macrophages (PM) expressed high levels of interleukin-1 receptor associated kinase M (IRAK-M), which was indicative of exposure to ligands of toll-like receptor 4 (TLR-4) such as bacterial lipopolysaccharide (LPS). Intraperitoneal LPS injections activated T cells in the pancreatic lymph nodes (PaLN) and thus, therefore potentially could activate islet-specific T cells. Addition of pectin and xylan to an otherwise diabetes-retarding semisynthetic diet affected microbial colonization of newly-weaned NOD mice, disturbed gut homeostasis and promoted diabetes development. These results help us to understand how diet and microbiota impact the regulation of the gut immune system in a way that might promote T1D in NOD mice.