This project aims to integrate knowledge of genetic susceptibility, immune cell function, and environmental modifiers in determining risk for type 1 diabetes (T1D) in the non-obese diabetic (NOD) mouse model. Similar to human T1D, disease risk in the NOD mouse is polygenic and has been mapped to multiple Idd loci. We have fine-mapped the Idd4.1 locus and identified Nlrp1b as its candidate gene. We report an alternatively spliced isoform in the diabetes-resistant Nrlp1b allele, resulting in a truncated NLRP1b protein that is unable to activate release of IL-1β. In another aspect of this project, we have characterized the critical contribution to T1D pathogenesis by γδ T cells. We report that CD27- γδT cells infiltrate islets of pre-diabetic NOD mice. Adoptive transfer of T1D to lymphocyte-deficient NOD.SCID recipients was potentiated when CD27- γδ T cells were transferred, compared to transfer of αβ T cells alone. Antibody-mediated blockade of IL-17 prevented T1D transfer in this setting. Moreover, introgression of genetic Tcrd deficiency onto the NOD background provided robust T1D protection. Finally, we report novel relationships between the gut microbiome, host sex hormones and metabolism, and T1D pathogenesis in the NOD mouse. Using germ-free, specific pathogen free, and microbiome-transplanted NOD mice, we show that colonization by commensal microbes elevated serum testosterone and protected NOD males from T1D. Transfer of gut microbiota from adult males to immature females altered the recipients’ microbiota, resulting in elevated testosterone and metabolomic changes, reduced islet inflammation and autoantibody production, and endowed robust T1D protection. Collectively, the data presented in this thesis describe a novel genetic lesion involved in T1D risk and its immunological consequences, demonstrate a potent role for IL-17-producing γδ T cells in NOD mouse model, and uncover a novel relationship between the gut microbiome, host hormonal and metabolic phenotypes, and autoimmunity risk.

This project aims to integrate knowledge of genetic susceptibility, immune cell function, and environmental modifiers in determining risk for type 1 diabetes (T1D) in the non-obese diabetic (NOD) mouse model. Similar to human T1D, disease risk in the NOD mouse is polygenic and has been mapped to multiple Idd loci. We have fine-mapped the Idd4.1 locus and identified Nlrp1b as its candidate gene. We report an alternatively spliced isoform in the diabetes-resistant Nrlp1b allele, resulting in a truncated NLRP1b protein that is unable to activate release of IL-1β. In another aspect of this project, we have characterized the critical contribution to T1D pathogenesis by γδ T cells. We report that CD27- γδT cells infiltrate islets of pre-diabetic NOD mice. Adoptive transfer of T1D to lymphocyte-deficient NOD.SCID recipients was potentiated when CD27- γδ T cells were transferred, compared to transfer of αβ T cells alone. Antibody-mediated blockade of IL-17 prevented T1D transfer in this setting. Moreover, introgression of genetic Tcrd deficiency onto the NOD background provided robust T1D protection. Finally, we report novel relationships between the gut microbiome, host sex hormones and metabolism, and T1D pathogenesis in the NOD mouse. Using germ-free, specific pathogen free, and microbiome-transplanted NOD mice, we show that colonization by commensal microbes elevated serum testosterone and protected NOD males from T1D. Transfer of gut microbiota from adult males to immature females altered the recipients’ microbiota, resulting in elevated testosterone and metabolomic changes, reduced islet inflammation and autoantibody production, and endowed robust T1D protection. Collectively, the data presented in this thesis describe a novel genetic lesion involved in T1D risk and its immunological consequences, demonstrate a potent role for IL-17-producing γδ T cells in NOD mouse model, and uncover a novel relationship between the gut microbiome, host hormonal and metabolic phenotypes, and autoimmunity risk.

Type 1 Diabetes (T1D) is an autoimmune disease where the immune system destroys the insulin-producing beta cells within the pancreas. Due to the difficulty of obtaining relevant tissue samples from patients at risk of disease, many researchers have utilized the nonobese diabetic (NOD) mouse as a model of T1D due to their natural high susceptibility for this disease which shares many characteristics with human patients. This model has been critical for uncovering many mechanisms involved in the pathogenesis of T1D including the key roles played by autoreactive T cells in the destruction of beta cells. More recently, NOD mice have shown that self-reactive B cells act as important antigen presenting cells for activating and amplifying the T cell response against beta cells. In order to identify faulty self-tolerance mechanisms causing production and activation of B cells recognizing beta cell proteins, we have developed a transgenic mouse model whereby elevated numbers of B cells are made specific for a neo-self antigen whose expression is restricted to beta cells on the T1D-prone NOD genetic background and compared it to that of transgenic mice of the non-autoimmune prone C57BL/6 (B6) genetic background. These studies revealed that NOD and B6 B cells can both be effectively tolerized to the model beta cell-restricted antigen. However, provision of help from activated T cells readily overturned this tolerance on the NOD but not the B6 background. Prior evidence has associated Idd5 (chromosome 1) and Idd9/11 (chromosome 4) diabetes susceptibility loci in NOD mice with the development of self reactive B cells contributing to T1D. The gene encoding CTLA-4 has been identified as the major candidate susceptibility gene within Idd5, thus leading to our studies comparing B cell expression of this molecule in NOD and diabetes-resistant strains. Although almost always associated with down-modulating T cells responses, our studies and that of others confirm expression of CTLA-4 by activated B cells. We encountered B cell expression of CTLA-4 to vary from that of T cells, being expressed earlier and predominantly on the cell surface rather than within intracellular vesicles. Our studies also showed aberrant expression of different splice variants of CTLA-4 by NOD B cells compared to diabetes-resistant mice controlled by genes within and outside the Idd5 genetic locus. Hence, these studies raise the possibility that CTLA-4 may contribute to T1D through its actions on both T and B cells. Given the large nature of the Idd9/11 susceptibility locus in NOD mice and the absence of any strong candidate genes that may influence the diabetogenic capacity of B cells in this strain, we resorted to microarray technology to reveal putative genes within this genomic region with the potential to control the B cell phenotype. We focused our microarray studies on the first transitional (T1) B cell population in the spleen given that it is an important stage of tolerance to peripherally expressed self-antigens which have been found to possess various defects in NOD mice. Comparing gene expression profiles of NOD T1 B cells that expressed susceptibility or resistance alleles at the Idd9/11 locus identified 20 differentially expressed genes with the potential to contribute to development of diabetogenic B cells. Overall, data presented in this thesis provides a greater understanding of the molecular and cellular mechanisms underlying B cell contribution to T1D in NOD mice. These data are hoped to eventually lead to the development of selective strategies for removing or inhibiting only those B cells that contribute to development of T1D while ensuring that humoral immunity to foreign pathogens remains intact in human patients at risk of developing disease.

Class of 2017 Abstract / Objectives: The purpose of the study was to identify the types of motivation that promote sustained physical activity among adolescents between the ages of 11-17 who are diagnosed with type 1 diabetes (T1D) to prevent diabetes related complications. Methods: Questionnaires were distributed and collected among the Juvenile Diabetes Research Foundation’s (JDRF) listserv, Facebook page, and events in Phoenix and Tucson, Arizona on motivations for managing diabetes and exercise and confidence in diabetes management and performing physical activity. Demographic data was collected on age, gender, and race/ethnicity. Physical activity, levels of activity intensity, weight, height, health- related risk behaviors, chronic health conditions, and use of preventative services were also included in this study. Results: 11 adolescents completed questionnaires, categorized by participants who exercise less than 60 minutes daily (Group below recommended exercise level, GBRE) and participants who exercise more or equal to 60 minutes daily (Group meeting recommended exercise level, GMRE). GBRE’s average mean age was 15.75 and GMRE’s average mean age was 13.92. GMRE was associated with higher intensity physical activity (42.85% versus 0%). GBRE had a relative autonomy index (RAI) of 1.67 on the Treatment Self-regulation Questionnaire (TSRQ) compared to GMRE with a RAI of 3.81 (Mann-Whitney U 19, p-value 0.412). GBRE scored 73.75 on the Diabetes Self-efficacy Scale (DSES) and GMRE scored 78.71 (Mann-Whitney U 7, p-value 0.23). Conclusions: Adolescents who exercised ≥ 60 minutes daily were observed to be self-motivated in managing their diabetes, especially maintaining exercise recommendations to decrease diabetes related complications.

Type 1 diabetes is an autoimmune disease that involves destruction of pancreatic cells that produce insulin. The disease typically presents in children and adolescents. The burden of disease management, fear of complications, and disruption of normal childhood that the disease causes place youth with type 1 diabetes at increased risk for developing depression compared to peers without the disease. The presence and severity of depression correlates with disease outcomes. Use of continuous subcutaneous insulin infusion pumps has been shown to improve youth’s quality of life compared to use of multiple daily insulin injections. Although quality of life measures are associated with the risk of developing depression, no studies have compared depression symptomatology in youth using insulin pumps to those using multiple daily insulin injections. The proposed project will assess relative depression symptomatology in youth ages 10-17 using insulin pumps and multiple daily insulin injections. The results of this proposed project could help inform clinicians’ decisions about whether to initiate type 1 diabetes therapy in youth with either insulin pumps or insulin injections. Given the financial burden of depression, it could also potentially encourage insurance companies to increase coverage of insulin pumps.

There is strong evidence that skeletal muscle health is compromised in persons with type 1 diabetes mellitus (T1D). These impairments include reduced strength, mitochondrial dysfunction, and decreased satellite cell (SC) content. Maintaining healthy muscle requires successful muscle repair. Preclinical models of T1D consistently show impaired muscle regeneration. To date, the impact of T1D on human skeletal muscle repair has not been established; however, attenuated repair would account for the reduced functional capacity and premature institutionalization that often characterizes those with diabetes. The purpose of this study was to determine the impact of T1D on the recovery of skeletal muscle function, morphology, and ultrastructure after 300 unilateral eccentric contractions (90°/s) of the knee extensors. Eighteen men and women (18-30 years old) with (n=9) and without (n=9) T1D performed the exercise protocol. Pre-damage, and at 48- and 96-hours post-damage, subjects gave a blood sample and vastus lateralis biopsy, and performed a maximal isometric knee extension. Given the sex-specific differences in muscle damage, control and T1D men and women were analyzed together and separately. Force production and recovery were comparable between control and T1D men and women. Exercise-related increases in creatine kinase activity and ultrastructural damage were also comparable between groups. There was a trend towards T1D men having more type 2 fast-twitch muscle fibers than T1D women (p=0.055). While baseline SC content was not different between groups, proliferating SC content was trending lower at 48-, and higher at 96-hours post-damage in T1D women compared to controls (p=0.07). In those with T1D, there was no correlation between muscle damage and HbA1c, but HbA1c was strongly correlated with vigorous physical activity (r=0.881, p=0.002). Contrary to preclinical studies, our data is the first to show that skeletal muscle repair is largely unaltered in otherwise healthy young adults with T1D. We attribute these differences to glycemic control and speculate that muscle repair is unaffected if individuals are optimally managing their diabetes. Considering the exercise-related dysglycemia seen in T1D, our results emphasize a need to define the dose of physical activity required for those with diabetes to properly regulate their blood glucose levels. We expect that this would in turn, improve skeletal muscle health and ultimately, extend the healthy lifespan of those living with T1D. / Thesis / Master of Science in Medical Sciences (MSMS) / Type 1 diabetes mellitus (T1D) is a chronic disease where the body does not make enough insulin to control blood glucose levels. Overtime, unstable blood glucose levels can damage major organ systems, including skeletal muscle. Skeletal muscle plays a pivotal role in regulating our physical and metabolic capacities. In those with T1D, exercise-mediated improvements in muscle health have been shown to delay health complications. However, we do not know how diabetic skeletal muscle repairs from exercise in humans. In this thesis, we investigated the ability of skeletal muscle to recover from damaging exercise in young adults with T1D. For the first time, we showed that skeletal muscle repair was similar between otherwise healthy young adults with T1D and those without diabetes. Our findings suggest that persons with T1D can engage in high levels of physical activity without compromising their muscle health. Further studies are needed to understand how exercise type, intensity, and duration impact glycemic control in men and women with T1D.

Chloroform methanol (CM) soluble extract of a cereal-based diet has been suggested to induce type 1 diabetes in an experimental animal model of type 1 diabetes. However, the individual constituent of this extract responsible for the disease induction and its disease pathogenesis mechanism remained unexplored. A previous study in our laboratory failed to show that the sphingolipid enriched fraction of CM soluble extract of wheat gluten triggers type 1 diabetes. Therefore, to study the involvement of CM soluble gliadin proteins in type 1 diabetes, we retrospectively analyzed proteins from sphingolipid enriched extract. SDS-PAGE analysis of CM soluble extract of wheat gluten and sphingolipid enriched fraction exhibited protein bands corresponding to the masses of the wheat gliadins, suggesting the presence of gliadin proteins in the CM soluble sphingolipid enriched extract. We studied the effect of five different dietary treatments on the histopathology of pancreatic tissue from BBdp rats includes insulitis scores i.e. lymphocytic infiltration in islet of Langerhans in order to test gliadin specific sphingolipid enriched extract (GSLEE) as possible a diabetogen. However, there were no significant differences in pancreatic insulitis scores and lymphoid tissue content due to addition of (GSLEE) to the hydrolyzed casein (HC) diet. We also investigated changes in jejunal histopathology and sera IFN-γ, TNF-α cytokine concentration in type 1 diabetes, induced by GSLEE. A decrease in jejunal CD4+ and γδ TCR + cell counts and inflammatory cell infiltrate were observed due to presence of CM soluble GSLEE in the HC diet, although this decrease was not statistically significant. A significant increase in sera IFN-γ cytokine concentration was found in BBdp rats fed the HC + GSLEE diet as compared to rats on HC diet. A numerical decrease in sera TNF-α concentration was also observed in BBdp rats fed the HC + GSLEE diet, when compared to BBdp rats on the HC diet. In contrast, a significant increase in serum IFN-γ concentrations in BBdp rats were observed after removing the CM soluble GSLEE from the wheat gluten based diet (WG) when compared to the WG diet alone. Removing GSLEE from WG diet resulted in insignificant increase in serum TNF-α concentration in BBdp rats when compared to WG dietary treatment group's BBdp rats. However, there were no significant differences in jejunal enteropathy parameters (i.e. lymphocytic infiltration, mucosal thickness, epithelial erosion, jejunal villi flattenings), jejunal CD4+ and jejunal γδ TCR+ cell counts; pancreatic insulitis scores, lymphoid tissue content after removing the CM soluble GSLEE from the WG diet when compared to the WG diet. Since overall findings regarding the CM soluble GSLEE's potential to induce type 1 diabetes by changing pancreatic and jejunal histopathology and elevating serum IFN-γ, TNF-α cytokine levels largely remained inconclusive, further investigations are warranted regarding immune suppression potential of the CM soluble sphingolipids in type 1 diabetes and the search of diabetogenic agents remaining in the residue after CM extraction. / Master of Science

Type 1A diabetes mellitus or T1D is a chronic disease characterized by T cell mediated destruction of the insulin producing β cells in the islets of Langerhans. The classical symptoms include high glucose levels in urine and blood, polyuria, and polydipsia. Complications associated with T1D include blindness, amputations, and end-stage renal disease, and premature death. The non-obese diabetic (NOD) mouse, first described in 1980, is widely used as a model organism for T1D. T1D disease in the NOD mouse shares a number of similarities to human T1D including dependence on genetic and environmental factors. More than 30 disease associated gene regions or loci (termed insulin dependent diabetes, or Idd, loci) have been associated with T1D development in NOD. For some of these Idds, the corresponding region in human has been linked to the development of T1D in human. T1D, both in humans and mice, is recognized as a T cell mediated disease. However, many studies have shown the importance of both the metabolome and the immune system in the pathogenesis of the disease. Appearance of autoantibodies in the serum of patients is the first sign of pathogenesis. However, molecular and cellular events precede the immune attack on the β-cell immunity. It has been shown that patients who developed T1D have an altered metabolome prior to the appearance of autoantibodies. Although much is known about the pathogenesis of T1D, the contribution of the environment/immune factors triggering the disease is still to be revealed.  In the present study both metabolic and immune deviations observed in the NOD mouse was analyzed. Serum metabolome analysis of the NOD mouse revealed striking resemblance to the human metabolic profile, with many metabolites in the TCA cycle significantly different from the non-diabetic control B6 mice. In addition, an increased level of glutamic acid was of the most distinguishing metabolite. A detailed bioinformatics analysis revealed various genes/enzymes to be present in the Idd regions. Compared to B6 mice, many of the genes correlated to the metabolic pathways, showed single nucleotide polymorphism (SNP), which can eventually affect the functionality of the protein. A genetic analysis of the increased glutamic acid revealed several Idd regions to be involved in this phenotype. The regions mapped in the genetic analysis harbor important enzymes and transporters related to glutamic acid. In-vitro islet culture with glutamic acid led to increased beta cell death indicating a toxic role of glutamic acid specifically towards insulin producing beta cells. In the analysis of the immune system, B cells from NOD mice, which are known to express high levels of TACI, were stimulated with APRIL, a TACI ligand. This resulted in enhanced plasma cell differentiation accompanied with increased class switching and IgG production. NOD mice have previously been shown to react vigorously to T-dependent antigens upon immunization. In this study we confirmed this as NOD mice showed an enhanced and prolonged immune response to hen egg lysozyme. Thus, serum IgG levels were significantly increased in the NOD mice and were predominantly of the IgG1 subtype. Immunofluorescence analysis revealed increased number of germinal centers in the NOD mice. Transfer of purified B and T cells from NOD to an immune deficient mouse could reproduce the original phenotype as seen in the NOD mice.     Collectively, this thesis has analyzed the metabolomics and immune deviations observed in the NOD mice.

Introduction: Key studies have found an association between attachment style and poor diabetes outcomes in the adult diabetic populations. Specifically insecure attachment has been found to predict elevated glycated haemoglobin levels (HbA1c). Further studies have indicated that substance use and mental health difficulties also influence HbA1c. These factors have been looked at individually making it difficult to directly assess the overall effect of attachment on HbA1c and the potential mediating effects of substance use and mental health. The adolescent population has not been considered in studies examining these relationships. This study compares attachment security, level of substance use, interpersonal problems, anxiety and depression in relation to their role in blood glucose control in an adolescent population with Type 1 diabetes. Method: A quantitative, cross sectional, questionnaire design was employed to examine the role of the aforementioned factors in relation to HbA1c level. The target population included all patients aged 14 years to 18 years, inclusive, who attended for review at Diabetes Clinics across Lothian. Participants had a diagnosis of Type 1 Diabetes for at least one year and no additional diagnoses of mental health disorder or other chronic condition. At the clinic patients were approached and asked to complete a set of self report questionnaires. Measures of attachment were adapted versions of the Relationship Questionnaire (RQ) and the Relationship Scales Questionnaire (RSQ). Interpersonal problems were assessed using the short version of the Inventory of Interpersonal Problems (IIP-32). The Hospital Anxiety and Depression Scale (HADS) assessed levels of anxiety and depression. The Adolescent Substance Abuse Subtle Screening Inventory- A2 (SASSI-A2) was used to measure substance use. Blood glucose levels (HbA1c%) were obtained from clinic staff. A total of 88 participants returned completed questionnaires (response rate 79.3%). Results: When all correlations between predictors and HbA1c were examined, a negative correlation was found between attachment and HbA1c level. A positive correlation was found between anxiety and HbA1c level. Multiple regression analyses examined the relationship between attachment security and HbA1c before analysing additional predictors in the same model. No significant relationships emerged however the multiple regression model was not a significant fit for the data. Path Analysis considered all relationships between variables simultaneously while also providing information on how the model fits the data. Attachment security directly related to HbA1c levels when the contributions of gender, interpersonal problems and substance use were considered. Anxiety and depression did not predict HbA1c nor did they contribute to any other relationships with HbA1c. Interpersonal problems had a direct relationship with HbA1c when the contribution of substance use and attachment were considered. Conclusion: Attachment predicts HbA1c. The nature of this relationship is further understood when the contribution of additional psychological variables are considered. Methodological issues, clinical implications and directions for future research are discussed.

The non-obese diabetic (NOD) mouse is widely used model Type 1 diabetes (T1D), a chronic inflammatory disease characterized by destruction of the insulin producing β cells in the islets of Langerhans by immune cells. The classical symptoms include increased glucose levels in urine and blood, frequent urination and enhanced thirst. The disease has a strong genetic component and is also influenced by the environment. NOD mice develop T1D spontaneously. The disease occurs in two phases; insulitis - the infiltration of immune cells in the islets of Langerhans and overt diabetes caused by the destruction of insulin producing β cells. Several disease associated gene regions or loci [termed insulin dependent diabetes (Idd) loci] have been associated with T1D development. Although, T1D is recognized as a T cell mediated disease in both mouse and man, many studies have shown the importance of B cells in the pathogenesis of the disease. Autoantibodies appear prior to islet infiltration and several molecular and cellular events precede this beta-cell autoimmunity. Although the pathogenesis of T1D is well characterized, less is known about the environmental and immunological factors that trigger the disease. In this thesis, we studied the contribution of B cell anomalies to the skewed immune response observed in the NOD mouse. In our studies covered in the thesis we observed that NOD mice display enhanced IgE in the serum already at one week of age. In addition, upon treatment of pre-diabetic NOD mice with anti-IgE antibodies, diabetes incidence was delayed. We hypothesize that the presence of IgE in the system may be explained due to enhanced class switching. Antibody feedback however, is an essential component of the immune response and can lead to either enhanced or dampened responses. Thus, increased IgE may provide positive feedback that might sustain an immune response. We also aimed to analyze the biological consequence of this feature. In vitro stimulation of B cells by the TACI ligand APRIL resulted in enhanced plasma cell differentiation accompanied with increased class switching and IgG production. In addition, TACI+ cells were observed in NOD germinal centers facilitating increased BAFF uptake and subsequent escape of low affinity antibody producing clones. NOD mice elicited an enhanced and prolonged immune response towards T-dependent antigens such as hen-egg lysozyme (HEL). Serum HEL-specific IgG level was significantly increased and was predominantly of the IgG1 isotype. Immunofluorescence analysis of NOD spleen revealed the presence of spontaneous germinal centers which others have perceived to provide a ready niche for the entry of naïve B cells that encountered novel antigen. Adoptive transfer experiments of purified B and T cells from NOD into NOD.Rag2-/- (NOD-RAG) mice illustrated the importance of B cell intrinsic defects in the reproduction of the original phenotype as observed in NOD.