Gene Regulation in The Human Immune System. Gene Expression Signatures of Th2 Cell Differentiation, Type 1 Diabetes, and Intrauterine Immune Adaptation

The human immune system is constantly interacting with the surrounding stimuli and microorganisms. However, when directed against self or harmless antigens, these vital defense mechanisms can cause great damage. In addition, the understanding the underlying mechanism of several human diseases caused by aberrant immune cell functions, for instance type 1 diabetes and allergies, remains far from being complete. In this Ph.D. study these questions were addressed using genome-wide transcriptomic analyses. Asthma and allergies are characterized by a hyperactive response of the T helper 2 (Th2) immune cells. In this study, the target genes of the STAT6 transcription factor in na��ve human T cells were identified with RNAi for the first time. STAT6 was shown to act as a central activator of the genes expression upon IL-4 signaling, with both direct and indirect effects on Th2 cell transcriptome. The core transcription factor network induced by IL-4 was identified from a kinetic analysis of the transcriptome. Type 1 diabetes is an autoimmune disease influenced by both the genetic susceptibility of an individual and the disease-triggering environmental factors. To improve understanding of the autoimmune processes driving pathogenesis in the prediabetic phase in humans, a unique series of prospective whole-blood RNA samples collected from HLA-susceptible children in the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study was studied. Changes in different timewindows of the pathogenesis process were identified, and especially the type 1 interferon response was activated early and throughout the preclinical T1D. The hygiene hypothesis states that allergic diseases, and lately also autoimmune diseases, could be prevented by infections and other microbial contacts acquired in early childhood, or even prenatally. To study the effects of the standard of hygiene on the development of neonatal immune system, cord blood samples from children born in Finland (high standard of living), Estonia (rapid economic growth) and Russian Karelia (low standard of living) were compared. Children born in Russian Karelia deviated from Finnish and Estonian children in many aspects of the neonatal immune system, which was developmentally more mature in Karelia, resembling that of older infants. The results of this thesis offer significant new information on the regulatory networks associated with immune-mediated diseases in human. The results will facilitate understanding and further research on the role of the identified target genes and mechanisms driving the allergic inflammation and type 1 diabetes, hopefully leading to a new era of drug development.

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.

CLEVER-1 as an immune suppressive molecule

The immune response and immune suppression are equally essential for the immune system to protect the host against an infection and to protect self-molecules in different pathophysiological conditions. Pregnancy is one of the conditions where the maternal immune system remains resistant against microbes and yet attains tolerance to protect the fetus, whose genetic material differs partially from the mother���s. However, if the balance of immune suppression is not precise in the host it can favor conditions which lead to diseases, such as cancer and autoimmune disorders. This study was initiated to investigate the expression and functions of CLEVER-1/Stabilin-1, a multifunctional protein expressed on subsets of endothelial cells and type II macrophages, as an immune suppressive molecule. Firstly, the expression of CLEVER-1/stabilin-1 and its function in human placental macrophages were examined. Secondly, the expression profile and functional significance of stabilin-1 on healthy human monocytes was investigated. The results clarified the expression of CLEVER-1/stabilin-1 on placental macrophages, and verified that CLEVER-1/stabilin-1 functions as an adhesion and scavenging molecule on these cells. The data from normal monocytes revealed that the monocytes with low stabilin-1 expression carried a pro-inflammatory gene signature, and that stabilin-1 can directly or indirectly regulate pro-inflammatory genes in monocytes. Finally, it was shown that monocyte CLEVER-1/stabilin-1 dampens IFN��� production by T cells. To conclude, CLEVER-1/stabilin-1 is defined as an immune suppressive molecule on monocytes and macrophages. Strikingly, anti-stabilin-1 antibodies may have the potential to promote the Th1 dependent inflammatory response and counteract the tumor induced immune suppression.

Probiotic interventions in infancy: Benefit and Safety Assessment of Extended Applications

Immaturity of the gut barrier system in the newborn has been seen to underlie a number of chronic diseases originating in infancy and manifesting later in life. The gut microbiota and breast milk provide the most important maturing signals for the gut-related immune system and reinforcement of the gut mucosal barrier function. Recently, the composition of the gut microbiota has been proposed to be instrumental in control of host body weight and metabolism as well as the inflammatory state characterizing overweight and obesity. On this basis, inflammatory Western lifestyle diseases, including overweight development, may represent a potential target for probiotic interventions beyond the well documented clinical applications. The purpose of the present undertaking was to study the efficacy and safety of perinatal probiotic intervention. The material comprised two ongoing, prospective, double-blind NAMI (Nutrition, Allergy, Mucosal immunology and Intestinal microbiota) probiotic interventions. In the mother-infant nutrition and probiotic study altogether 256 women were randomized at their first trimester of pregnancy into a dietary intervention and a control group. The intervention group received intensive dietary counselling provided by a nutritionist, and were further randomized at baseline, double-blind, to receive probiotics <i>(Lactobacillus rhamnosus</i> GG and <i>Bifidobacterium lactis)</i> or placebo. The intervention period extended from the first trimester of pregnancy to the end of exclusive breastfeeding. In the allergy prevention study altogether 159 women were randomized, double-blind, to receive probiotics (<i>Lactobacillus rhamnosus</i> GG) or placebo 4 weeks before expected delivery, the intervention extending for 6 months postnatally. Additionally, patient data on all premature infants with very low birth weight (VLBW) treated in the Department of Paediatrics, Turku University Hospital, during the years 1997 - 2008 were utilized. The perinatal probiotic intervention reduced the risk of gestational diabetes mellitus (GDM) in the mothers and perinatal dietary counselling reduced that of fetal overgrowth in GDM-affected pregnancies. Early gut microbiota modulation with probiotics modified the growth pattern of the child by restraining excessive weight gain during the first years of life. The colostrum adiponectin concentration was demonstrated to be dependent on maternal diet and nutritional status during pregnancy. It was also higher in the colostrum received by normal-weight compared to overweight children at the age of 10 years. The early perinatal probiotic intervention and the postnatal probiotic intervention in VLBW infants were shown to be safe. To conclude, the findings in this study provided clinical evidence supporting the involvement of the initial microbial and nutritional environment in metabolic programming of the child. The manipulation of early gut microbial communities with probiotics might offer an applicable strategy to impact individual energy homeostasis and thus to prevent excessive body-weight gain. The results add weight to the hypothesis that interventions aiming to prevent obesity and its metabolic consequences later in life should be initiated as early as during the perinatal period.

System-wide approaches to uncover Th2 cell lineage commitment

T helper (Th) cells are vital regulators of the adaptive immune system. When activated by presentation of cognate antigen, Th cells demonstrate capacity to differentiate into functionally distinct effector cell subsets. The Th2 subset is required for protection against extracellular parasites, such as helminths, but is also closely linked to pathogenesis of asthma and allergies. The intracellular molecular signal transduction pathways regulating T helper cell subset differentiation are still incompletely known. Moreover, great majority of studies regarding Th2 differentiation have been conducted with mice models, while studies with human cells have been fewer in comparison. The goal of this thesis was to characterize molecular mechanisms promoting the development of Th2 phenotype, focusing specifically on human umbilical cord blood T cells as an experimental model. These primary cells, activated and differentiated to Th2 cells in vitro, were investigated by complementary system-wide approaches, targeting levels of mRNA, proteins, and lipid molecules. Specifically, the results indicated IL4-regulated recruitment of nuclear protein, and described novel components of the Th2-promoting STAT6 enhanceosome complex. Furthermore, the development of the activated effector cell phenotype was found to correlate with remodeling of the cellular lipidome. These findings will hopefully advance the understanding of human Th2 cell lineage commitment and development of Th2-associated disease states.

Sotilaslent��j��n fyysinen suorituskyky sek�� ty��per��iset tuki- ja liikuntaelinoireet

Kirjallisuudesta v��littyy useitten vuosikymmenten ajalta tiet��mys sotilaslent��misen fyysisest�� kuormittavuudesta. G-voimista aiheutuva kuormittuminen n��ytt���� johtavan joko akuutisti tai pitk��aikaisesti lent��j��n tuki- ja liikuntaelimist��n toimintakyky�� alentaviin ongelmiin. Erityisesti on selvitetty niskan alueen ty��per��isten ongelmien synty��, jolloin on havaittu lentotoiminnan fyysisen kuormittavuuden johtavan ennenaikaiseen rakenteelliseen rappeumaan, haittaa aiheuttavan oireen lis��ksi. Kansainv��lisen kirjallisuuden mukaan ammatista johtuvista eli ty��per��isist�� oireista k��rsii v��hint����n 2/3 kaikista sotilaslent��jist��. Tietyin edellytyksin lent��jien kaularangan alueen rappeuma on Suomessa hyv��ksytty ammattitaudiksi vuodesta 1995 alkaen. On arveltu, ett�� hyv��st�� fyysisest�� suorituskyvyst�� olisi apua tuki- ja liikuntaelin (TULE)-oireilun ennaltaehk��isemisess�� ja toimintakyvyn yll��pit��misess��. Tutkimusn��ytt�� t��st�� on lent��jien osalta ollut toistaiseksi eritt��in niukkaa. T��m��n tutkimuksen tavoitteena oli selvitt���� suomalaisten sotilaslent��jien ty��per��isen TULE-oireilun esiintyvyytt��, oireista koetun haitan tasoa, lent��jien fyysisen kunnon tasoja virkauran aikana ja n��itten kaikkien v��lisi�� yhteyksi�� sek�� ty��per��isen TULE-oireen merkityst�� sotilaan toimintakykyyn. Tutkimus jakautui kahteen osaan. Poikkileikkauksena lentotoimintaper��isi�� TULE-oireita kartoitettiin kyselytutkimuksella, johon vastasi vuositarkastuksen yhteydess�� 267 lent��j���� vuosina 2004-2005. Joukosta poimittiin ne 195 lent��j����, jotka olivat suorittaneet yleissotilaalliset kuntotestit puolen vuoden sis��ll�� kyselyyn vastaamisesta, ja mitatut testitulokset yhdistettiin kyselytutkimusaineistoon. T��ss�� aineistossa toteutettiin fyysisesti erilailla kuormittuvien lent��j��ryhmien v��lisi�� vertailuja fyysisen kunnon, TULE-esiintyvyyden ja koetun haitan suhteen. Poikkileikkausosassa tutkittiin my��s lent��jien virkauran aikaisia tasoeroja yleissotilaallisissa kuntotesteiss�� (n=195) verrattuna muihin suomalaisiin sotilaisiin. Lis��ksi (N=289) selvitettiin ilmailul����ketieteellisen tarkastuksen yhteydess�� mitattuja, ns. ammatillisia fyysisi�� erityisominaisuuksia eri ik��luokissa. Pitkitt��isosassa seurattiin 67:n Hawk-suihkuharjoituskoneella aloittaneen Ilmavoimien sotilaslent��jien lentouran aikaista lentotoimintaper��isten TULE-oireitten esiintyvyytt�� vuosien 1996 ja 2008 v��lill��. Lis��ksi tutkittiin lent��jien kontakteja ty��terveyshuoltoon, oireen aiheuttamaa lentokelvottomuusaikaa, ty��n kuormituksen kumulatiivista kertym���� lentotuntien lis����ntyess�� ja TULE-oireiden esiintyvyyden kannalta kriittisi�� ajankohtia lentouran aikana. Tulokset osoittivat, ett�� kaikki seurannassa olleet suomalaiset sotilaslent��j��t kokivat jonkinasteisen lentotoimintaper��isen TULE-oireen uransa aikana. Niskan ammattitautiluokituksen tasoisen ongelman esiintyvyys oli 4 % koko lent��j��populaatiosta ja 10 % suihkuharjoituskonevaiheen jo l��p��isseist��, mutta vastaavanlaisia TULE-ongelmia, ilman riitt��v���� n��ytt���� ammattitaudista, esiintyi l��hes joka kolmannella sotilaslent��j��ll��. Alasel��n osalta lent��j��t oireilivat l��hes samassa m����rin, mutta n��it�� oireita ei toistaiseksi ole mahdollista m����ritt���� ammattitaudiksi. Lent��j��t k��viv��t varsin v��h��n valittamassa oireistaan ty��terveyshuoltoon, jossa k��yt��neen vasta silloin, kun oire jo selv��sti heikent���� ty��teht��viss�� vaadittavaa toimintakyky��. Merkitt��vin lentotoimintaper��isten oireitten esiintymisen kasvu ajoittui 200 Hawk-lentotunnin kohdalle, jolloin koneella saavutetaan er����nlainen optimaalinen G-indeksi eli taktisen liikehtelyn G-tasoylitysten vaihtelu. T��m��n j��lkeen lent��j��t ovat erityisen alttiina akuuteille lennonaikaisille TULE-ongelmille. Oireitten esiintyminen kasvoi eksponentiaalisesti noin 600 lentotuntiin asti. Monimuuttujamallien mukaan ty��per��isen TULE-oireen esiintyvyysriski�� v��hensiv��t alaraajojen hyv�� motoriikka, korkeat valintapisteet ja korkea kaulan fleksion voimataso maksimaalisessa isometrisess�� testiss��. Yleissotilaallisilla kuntotasoilla ei ollut yhteytt�� oireiluun, mutta lihaskunnoltaan voimakkaimmat lent��j��t k��rsiv��t tilastollisesti merkitt��v��sti v��hemm��n haittaa lentotoimintaper��isist�� TULE-oireistaan. Yleissotilaallisissa kuntotesteiss�� lent��j��t olivat parempia kuin muut suomalaiset sotilaat. Aktiivisimman lentouran aikana, 30-40-vuotiaina, lent��jien fyysinen suorituskyky oli normaaliv��est����n n��hden vain keskim����r��inen ja urheilijoihin n��hden keskim����r��ist�� heikompi. K��yt��nn��ss�� lent��j��t eiv��t kyenneet yll��pit��m����n valintavaiheen fyysist�� suorituskyky����n edes kadettivaiheen loppuun asti. Huomattavaa oli lis��ksi, ett�� aktiivisen lentouran p����tytty�� fyysinen kunto n��ytti jossain m����rin palautuvan kohti l��ht��tasoa lent��jien ik����ntymisest�� huolimatta. Lent��jien valintavaiheen aikana mitatun fyysisen suorituskyvyn tason s��ilyminen aktiivisen lentopalveluksen loppuun asti vaatisi lent��jien fyysisen toimintakyvyn yll��pidon ja kehitt��misen tehostamista koulutuksen ja ty��uran eri vaihessa. T��h��n tavoitteeseen n��hden Ilmavoimien fyysisen kasvatuksen j��rjestelyt vaikuttivat alimitoitetuilta. Operatiivisesti huolestuttavaa oli Ilmavoimien ohjaajien fyysisen suorituskyvyn heikentyminen silloin, kun heid��n taitojensa puolesta olisi pit��nyt olla suorituskykyisimpi�� taisteluteht��viins��. My��s lent��j��n terveytt�� ja toimintakyky�� pit��isi pysty�� reaaliaikaisemmin seuraamaan koko lentouran aikana. Ilmavoimille suositellaan moniammatillista l��hestymist�� sotilaslent��jien toimintakyvyn yll��pit��miseen ja terveysriskien hallintaan yhdess�� liikunnan, ty��terveyshuollon, lentoturvallisuusalan ja operatiivisen suunnittelun asiantuntijoitten kanssa. Lis��ksi suositellaan avoimempaa ja eettisesti kest��v��mp���� suhtautumista ammattiin liittyvien terveysongelmien kuvaamiseen sek�� fyysisen kunnon kysymyksiin jo lent��jien rekrytointivaiheessa.

Reactive oxygen species in inflammation

Chronic inflammation is the underlying cause of many common disabling conditions such as rheumatoid arthritis (RA), multiple sclerosis, coeliac disease, type I diabetes and coronary artery disease. NOX2 complex derived reactive oxygen species (ROS) are known to regulate joint inflammation in rats and mice, and additionally recent genetic evidence associates phagocyte ROS and the development RA in humans. Ncf1mutated mice have lost the functionality of their NOX2 complex and thus have no phagocyte ROS production. These mice suffer from exacerbated arthritis. The immune suppressive effect of the NOX2 complex derived ROS is mediated by monocytes/macrophages that downregulate the activation of autoreactive T cells. The aim of this thesis was to study how ROS modulate immune responses in different arthritis models and in tumor development. Additionally, genome wide gene expression profiling was carried out to assess the global effects of NOX2 complex derived ROS. Firstly, these results confirmed the potent anti-inflammatory nature of phagocyte ROS in arthritis models that were driven by the adaptive immune system. Secondly, arthritis models with predominantly innate immunity induced pathophysiology were moderately enhanced by phagocyte, more specifically, neutrophil derived ROS. Thirdly, the ROS induced immune suppression mediated by the adaptive immune system allowed development of bigger implanted tumors, while phagocyte ROS production did not affect the development of spontaneously growing tumors. Lastly, genome wide gene expression analysis revealed that both humans and mice with abrogated phagocyte NOX2 complex ROS production had an enhanced type I interferon signature in blood, reflecting their hyperinflammatory immune status.

Virulence properties of Aggregatibacter actinomycetemcomitans biofilm and characterisation of its putative cytokine exploitation

Biofilms are surface-attached multispecies microbial communities that are embedded by their self-produced extracellular polymeric substances. This lifestyle enhances the survival of the bacteria and plays a major role in many chronic bacterial infections. For instance, periodontitis is initiated by multispecies biofilms. The phases of active periodontal tissue destruction and notably increased levels of proinflammatory mediators, such as the key inflammatory mediator interleukin (IL)-1beta, are typical of the disease. The opportunistic periodontal pathogen Aggregatibacter actinomycetemcomitans is usually abundant at sites of aggressive periodontitis. Despite potent host immune system responses to subgingival invaders, A. actinomycetemcomitans is able to resist clearance attempts. Moreover, some strains of A. actinomycetemcomitans can generate genetic diversity through natural transformation, which may improve the species��� adjustment tothe subgingival environment in the long term. Some biofilm forming species are known to bind and sense human cytokines. As a response to cytokines, bacteria may increase biofilm formation and alter their expression of virulence genes. Specific outer membrane receptors for interferon-�� or IL-1�� have been characterised in two Gram-negative pathogens. Because little is known about periodontal pathogens��� ability to sense cytokines, we used A. actinomycetemcomitans as a model organism to investigate how the species responds to IL-1beta. The main aims of this thesis were to explore cytokine binding on single-species A. actinomycetemcomitans biofilms and to determine the effects of cytokines on the biofilm formation and metabolic activity of the species. Additionally, the cytokine���s putative internalisation and interaction with A. actinomycetemcomitans proteins were studied. The possible impact of biofilm IL-1beta sequestering on the proliferation and apoptosis of gingival keratinocyte cells was evaluated in an organotypic mucosa co-culture model. Finally, the role of the extramembranous domain of the outer membrane protein HofQ (emHofQ) in DNA binding linked to DNA uptake in A. actinomycetemcomitans was examined. Our main finding revealed that viable A. actinomycetemcomitans biofilms can bind and take up the IL-1�� produced by gingival cells. At the sites of pathogen-host interaction, the proliferation and apoptosis of gingival keratinocytes decreased slightly. Notably, the exposure of biofilms to IL-1beta caused their metabolic activity to drop, which may be linked to the observed interaction of IL-1beta with the conserved intracellular proteins DNA binding protein HU and the trimeric form of ATP synthase subunit beta. A Pasteurellaceaespecific lipoprotein, which had no previously determined function, was characterized as an IL-1beta interacting membrane protein that was expressed in the biofilm cultures of all tested A. actinomycetemcomitans strains. The use of a subcellular localisation tool combined with experimental analyses suggested that the identified lipoprotein, bacterial interleukin receptor I (BilRI), may be associated with the outer membrane with a portion of the protein oriented towards the external milieu. The results of the emHofQ study indicated that emHofQ has both the structural and functional capability to bind DNA. This result implies that emHofQ plays a role in DNA assimilation. The results from the current study also demonstrate that the Gram-negative oral species appears to sense the central proinflammatory mediator IL-1beta.

Imaging Neuroinflammation in Progressive Multiple Sclerosis

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system CNS), where inflammation and neurodegeneration lead to irreversible neuronal damage. In MS, a dysfunctional immune system causes auto���reactive lymphocytes to migrate into CNS where they initiate an inflammatory cascade leading to focal demyelination, axonal degeneration and neuronal loss. One of the hallmarks of neuronal injury and neuroinflammation is the activation of microglia. Activated microglia are found not only in the focal inflammatory lesions, but also diffusely in the normal���appearing white matter (NAWM), especially in progressive MS. The purine base, adenosine is a ubiquitous neuromodulator in the CNS and also participates in the regulation of inflammation. The effect of adenosine mediated via adenosine A2A receptors has been linked to microglial activation, whereas modulating A2A receptors may exert neuroprotective effects. In the majority of patients, MS presents with a relapsing disease course, later advancing to a progressive phase characterised by a worsening, irreversible disability. Disease modifying treatments can reduce the severity and progression in relapsing MS, but no efficient treatment exists for progressive MS. The aim of this research was to investigate the prevalence of adenosine A2A receptors and activated microglia in progressive MS by using in vivo positron emission tomography (PET) imaging and [11C]TMSX and [11C](R)���PK11195 radioligands. Magnetic resonance imaging (MRI) with diffusion tensor imaging (DTI) was performed to evaluate structural brain damage. Non���invasive input function methods were also developed for the analyses of [11C]TMSX PET data. Finally, histopathological correlates of [11C](R)���PK11195 radioligand binding related to chronic MS lesions were investigated in post���mortem samples of progressive MS brain using autoradiography and immunohistochemistry. [11C]TMSX binding to A2A receptors was increased in NAWM of secondary progressive MS (SPMS) patients when compared to healthy controls, and this correlated to more severe atrophy in MRI and white matter disintegration (reduced fractional anisotropy, FA) in DTI. The non���invasive input function methods appeared as feasible options for brain [11C]TMSX images obviating arterial blood sampling. [11C](R)���PK11195 uptake was increased in the NAWM of SPMS patients when compared to patients with relapsing MS and healthy controls. Higher [11C](R)���PK11195 binding in NAWM and total perilesional area of T1 hypointense lesions was associated with more severe clinical disability, increased brain atrophy, higher lesion load and reduced FA in NAWM in the MS patients. In autoradiography, increased perilesional [11C](R)���PK11195 uptake was associated with increased microglial activation identified using immunohistochemistry. In conclusion, brain [11C]TMSX PET imaging holds promise in the evaluation of diffuse neuroinflammation in progressive MS. Being a marker of microglial activation, [11C](R)��� PK11195 PET imaging could possibly be used as a surrogate biomarker in the evaluation of the neuroinflammatory burden and clinical disease severity in progressive MS.

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.