Diurnal Variation of Islet Autoantibody Titers in Established Type 1 Diabetes Suggests Restricted-Time Sampling Improves Aab Measurement and Detection Diurnal islet autoantibody (Aab) variation in type 1 diabetes (T1D) remains poorly understood and could hinder efforts to develop, test and refine new therapies. We sought to describe the extent and pattern of diurnal variation in islet autoantibodies and, having found significant variation, translate to the clinical setting. We conducted two studies in human subjects with established T1D: (1) a prospective study (n = 10) of the range of islet autoantibody and immunoglobulin daily variation within humans and (2) an independent retrospective, cross-sectional study (n = 705) of the effect of time of sample collection on Aab titer and detection in clinical settings. We found that some individuals have wide Aab variations during the day and which can exceed expected levels of inter-assay variation. For some Aab, this variation followed a circadian pattern. We also found that, in clinical settings, time-restricted sampling can lead to increased IA-2A and ZnT8A detection within specific age-groups. We conclude that time-restriction can potentially improve the use of Radiobinding-measured Aabs as biomarkers for the development and monitoring of disease-modifying therapies and in developing islet transplantation strategies in established T1D. Investigation of diurnal Aab variation and time restriction is needed in early-stage disease. .

Objective: Type 1 diabetes (T1D) is characterised by destruction of pancreatic beta cells by islet-infiltrating cytotoxic lymphocytes, and elevated intra-islet secretion of pro-inflammatory cytokines. However, the underlying pathophysiological mechanisms remain incompletely understood. We hypothesise that abnormal elevation of islet NAD, via activation of NAMPT, plays a key role in driving islet autoimmune processes in T1D, and that conversely, NAMPT inhibition may be an attractive therapeutic approach for T1D. Methods: Islets were isolated from non-diabetic CD1 mice or obtained from human islet donors and exposed to pro-inflammatory cytokine cocktail (IL-1β, TNFα and IFNγ) +/- NAMPT inhibitor. Beta-cell function was determined by static glucose-stimulated insulin secretion. Islet apoptosis was determined by caspase 3/7 activity. Islet cell differential gene expression was assessed via bulk-RNA sequencing. Diabetes was induced in CD1 mice via multiple low dose streptozotocin (MLDS) injection. MLDS mice were then administered FK866 (10 mg/kg; IP) or saline equivalent for 16 days. Ambient blood glucose was measured every 2-3 days. Beta-cell function was assessed by measurement of serum insulin and c-peptide levels. Insulin content was assessed by flow cytometry. Intra-islet immune cell infiltration, as well as immune cell proliferation and cytokine production were assessed via flow cytometry. For migration assays, islets were isolated from BALB/c mice dispersed and reaggregated into reformed islets. CD8+ cells were isolated from splenocytes derived from NY8.3 NOD mice migration of immune cells was assessed using a transwell culture protocol. Results: NAMPT inhibition with FK866 or compound 17 protected mouse and human islets against cytokine-mediated beta cell dysfunction and death. RNAseq revealed that inhibiting NAMPT blocked pro-inflammatory cytokine-mediated gene expression linked to pro-inflammatory responses and leukocyte migration. FK866 improved glycaemic control and beta-cell function in MLDS mice. FK866 also reduced proportions of islet-residing TNFα producing CD4+T-cells and F4/80+macrophages, proliferation of spleen-derived CD4+ and CD8+T-cells, and proliferation of islet-derived CD4+T-cells and F4/80+macrophages. Finally, FK866 was able to block pro-inflammatory cytokine-mediated migration of cytotoxic CD8+T-cells into reformed islets. Conclusions: This data supports a key immunomodulatory role for NAMPT in islet autoimmunity. NAMPT inhibition may represent a novel therapeutic approach for T1D. ### Competing Interest Statement The authors have declared no competing interest.

NOTE: The first page of text has been automatically extracted and included below in lieu of an abstract Session 2323 An Interdisciplinary Junior Level Team Design Experience in Engineering Dr. James V. Pearson Division of Engineering and Technology John Brown University Abstract The course (EN3222, Design Laboratory, two semester hours, spring semester) described in this paper was initiated in the spring of 1992 at John Brown University to provide students with design-cycle experience and interdisciplinary team activities. Typically the teams of this course are formed with three students of engineering and two students of graphic design. Each team forms a company which is then asked to respond to a Request For Proposal from "investors" for a consumer product design appropriate to one of five consumer markets. The engineers on the team work on the technical design, computer drawings, Failure Mode and Effects Analysis, reliability studies, economic analysis, testing; and consulted on the case design and technical manual content. The graphic designers work on the company identity, advertising layouts, marketing plan, web-page design, case design, manual design and packaging. Five design seminars are presented by the faculty team. Four design reviews are conducted with each team during the semester. The final presentation by the team before the " investors" includes a demonstration of the working prototype and the presentation of all documentation and marketing elements. Team interaction in the course has been effective though sometimes frustrating to the student. Student response to the course has been positive. The course has provided a good preparation for the full-year senior design project. The paper also discusses creativity issues, the use of computer tools, the application of reliability factors, student evaluation techniques, and some of the product designs. "The mind is not a vessel to be filled but a fire to be kindled." Plutarch I. Introduction The engineering faculty at John Brown University began discussing a junior-level design laboratory in 1990. Students were spending extensive amounts of creative time in the computer room but not in the electrical or mechanical laboratories. One factor was that the labs were not open for them to do this. Another factor was that the standard lab experiments were not open-ended enough to make the students think through the project. It was necessary for the faculty to design some lab experiences that would stimulate real thought and draw students to the lab to confirm their ideas. It was also necessary for the labs to be open when students wanted to work there.

AbstractToll-like receptor 9 (TLR9) recognizes bacterial, viral and self DNA and play an important role in immunity and inflammation. However, the role of TLR9 in obesity is less well-studied. Here, we generate B-cell-specific Tlr9-deficient (Tlr9fl/fl/Cd19Cre+/-, KO) B6 mice and model obesity using a high-fat diet. Compared with control mice, B-cell-specific-Tlr9-deficient mice exhibited increased fat tissue inflammation, weight gain, and impaired glucose and insulin tolerance. Furthermore, the frequencies of IL-10-producing-B cells and marginal zone B cells were reduced, and those of follicular and germinal center B cells were increased. This was associated with increased frequencies of IFNγ-producing-T cells and increased follicular helper cells. In addition, gut microbiota from the KO mice induced a pro-inflammatory state leading to immunological and metabolic dysregulation when transferred to germ-free mice. Using 16 S rRNA gene sequencing, we identify altered gut microbial communities including reduced Lachnospiraceae, which may play a role in altered metabolism in KO mice. We identify an important network involving Tlr9, Irf4 and Il-10 interconnecting metabolic homeostasis, with the function of B and T cells, and gut microbiota in obesity.

IntroductionThe incidence of the autoimmune disease, type 1 diabetes (T1D), has been increasing worldwide and recent studies have shown that the gut microbiota are associated with modulating susceptibility to T1D. Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and is widely expressed on many cells, including dendritic cells (DCs), which are potent antigen-presenting cells (APCs). TLR5 modulates susceptibility to obesity and alters metabolism through gut microbiota; however, little is known about the role TLR5 plays in autoimmunity, especially in T1D.MethodsTo fill this knowledge gap, we generated a TLR5-deficient non-obese diabetic (NOD) mouse, an animal model of human T1D, for study.ResultsWe found that TLR5-deficiency led to a reduction in CD11c+ DC development in utero, prior to microbial colonization, which was maintained into adulthood. This was associated with a bias in the DC populations expressing CD103, with or without CD8α co-expression, and hyper-secretion of different cytokines, both in vitro (after stimulation) and directly ex vivo. We also found that TLR5-deficient DCs were able to promote polyclonal and islet antigen-specific CD4+ T cell proliferation and proinflammatory cytokine secretion. Interestingly, only older TLR5-deficient NOD mice had a greater risk of developing spontaneous T1D compared to wild-type mice.DiscussionIn summary, our data show that TLR5 modulates DC development and enhances cytokine secretion and diabetogenic CD4+ T cell responses. Further investigation into the role of TLR5 in DC development and autoimmune diabetes may give additional insights into the pathogenesis of Type 1 diabetes.

Introduction:Gut microbiota have been linked to modulating susceptibility to Type 1 diabetes; however, there are many ways in which the microbiota interact with host cells, including through microbial ligand binding to intracellular inflammasomes (large multi-subunit proteins) to initiate immune responses. NLRP6, a microbe-recognizing inflammasome protein, is highly expressed by intestinal epithelial cells and can alter susceptibility to cancer, obesity and Crohn's disease; however, the role of NLRP6 in modulating susceptibility to autoimmune diabetes, was previously unknown.Methods:We generated NLRP6-deficient Non-obese diabetic (NOD) mice to study the effect of NLRP6-deficiency on the immune cells and susceptibility to Type 1 diabetes development.Results:NLRP6-deficient mice exhibited an expansion of CD103+ B cells and were protected from type 1 diabetes. Moreover, NLRP6-deficient CD103+ B cells express regulatory markers, secreted higher concentrations of IL-10 and TGFb1 cytokines and suppressed diabetogenic T cell proliferation, compared to NLRP6-sufficient CD103+ B cells. Microarray analysis of NLRP6-sufficient and -deficient CD103+ B cells identified 79 significantly different genes including genes regulated by lipopolysaccharide (LPS), tretinoin, IL-10 and TGFb, which was confirmed in vitro following LPS stimulation. Furthermore, microbiota from NLRP6-deficient mice induced CD103+ B cells in colonized NLRP6-sufficient germ-free mice; however, the long-term maintenance of the CD103+ B cells required the absence of NLRP6 in the hosts, or continued exposure to microbiota from NLRP6-deficient mice.Discussion:Together, our data indicate that NLRP6 deficiency promotes expansion and maintenance of a novel TGF -dependent CD103+ Breg population. Thus, targeting NLRP6 therapeutically may prove clinically useful.

Aims/hypothesis IgM is the primary antibody produced by B cells and we hypothesise that IgM antibodies to gut microbiota may play a role in immunometabolism in obesity and type 2 diabetes. To test our hypothesis, we used B6 mice deficient in activation-induced cytidine deaminase ( Aid −/− [also known as Aicda −/− ]) which secrete only IgM antibodies, and human faecal samples. Methods We studied the immunometabolic effects and gut microbial changes in high-fat-diet-induced obesity (HFDIO) in Aid −/− B6 mice compared with wild-type mice. To determine similarities between mice and humans, human stool samples were collected from children and adolescents who were obese with normal glucose tolerance (NGT), obese with glucose intolerance (IGT), or obese and newly diagnosed with type 2 diabetes, for faecal microbiota transplant (FMT) into germ-free (GF) B6 mice and we assessed IgM-bound bacteria and immune responses. Results Compared with wild-type mice, Aid −/− B6 mice developed exacerbated HFDIO due to abundant levels of IgM. FMT from Aid −/− B6 to GF B6 mice promoted greater weight gain in recipient mice compared with FMT using wild-type mouse faecal microbiota. Obese youth with type 2 diabetes had more IgM-bound gut bacteria. Using the stools from the obese youth with type 2 diabetes for FMT to GF B6 mice, we observed that the gut microbiota promoted body weight gain and impaired glucose tolerance in the recipient GF B6 mice. Importantly, some clinical features of these obese young individuals were mirrored in the GF B6 mice following FMT. Conclusions/interpretation Our results suggest that IgM-bound gut microbiota may play an important role in the immuno-pathogenesis of obesity and type 2 diabetes, and provide a novel link between IgM in obesity and type 2 diabetes in both mice and humans. Data availability The 16s rRNA sequencing datasets supporting the current study have been deposited in the NCBI SRA public repository ( https://www.ncbi.nlm.nih.gov/sra ; accession no. SAMN18796639). Graphical abstract

Type 2 diabetes mellitus, obesity and metabolic syndrome are becoming more prevalent worldwide and will present an increasingly challenging burden on healthcare systems. These interlinked metabolic abnormalities predispose affected individuals to a plethora of complications and comorbidities. Furthermore, diabetes is estimated by the World Health Organization to have caused 1.5 million deaths in 2019, with this figure projected to rise in coming years. This highlights the need for further research into the management of metabolic diseases and their complications. Studies on circadian rhythms, referring to physiological and behavioral changes which repeat approximately every 24 hours, may provide important insight into managing metabolic disease. Epidemiological studies show that populations who are at risk of circadian disruption such as night shift workers and regular long-haul flyers are also at an elevated risk of metabolic abnormalities such as insulin resistance and obesity. Aberrant expression of circadian genes appears to contribute to the dysregulation of metabolic functions such as insulin secretion, glucose homeostasis and energy expenditure. The potential clinical implications of these findings have been highlighted in animal studies and pilot studies in humans giving rise to the development of circadian interventions strategies including chronotherapy (time-specific therapy), time-restricted feeding, and circadian molecule stabilizers/analogues. Research into these areas will provide insights into the future of circadian medicine in metabolic diseases. In this review, we discuss the physiology of metabolism and the role of circadian timing in regulating these metabolic functions. Also, we review the clinical aspects of circadian physiology and the impact that ongoing and future research may have on the management of metabolic disease.

Aims/hypothesis B cells play an important role in driving the development of type 1 diabetes; however, it remains unclear how they contribute to local beta cell destruction during disease progression. Here, we use gene expression profiling of B cell subsets identified in inflamed pancreatic tissue to explore their primary functional role during the progression of autoimmune diabetes. Methods Transcriptional profiling was performed on FACS-sorted B cell subsets isolated from pancreatic islets and the pancreatic lymph nodes of NOD mice. Results B cells are highly modified by the inflamed pancreatic tissue and can be distinguished by their transcriptional profile from those in the lymph nodes. We identified both a discrete and a core shared gene expression profile in islet CD19 + CD138 – and CD19 + CD138 + B cell subsets, the latter of which is known to have enriched autoreactivity during diabetes development. On localisation to pancreatic islets, compared with CD138 – B cells, CD138 + B cells overexpress genes associated with adhesion molecules and growth factors. Their shared signature consists of gene expression changes related to the differentiation of antibody-secreting cells and gene regulatory networks associated with IFN signalling pathways, proinflammatory cytokines and Toll-like receptor (TLR) activation. Finally, abundant TLR7 expression was detected in islet B cells and was enhanced specifically in CD138 + B cells. Conclusions/interpretation Our study provides a detailed transcriptional analysis of islet B cells. Specific gene signatures and interaction networks have been identified that point towards a functional role for B cells in driving autoimmune diabetes. Graphical abstract

Lymph node stromal cells (LNSC) are essential for providing and maintaining peripheral self-tolerance of potentially autoreactive cells. In type 1 diabetes, proinsulin-specific CD8+T-cells, escaping central and peripheral tolerance, contribute to beta-cell destruction. Using G9Cα-/-CD8+T-cells specific for proinsulin, we studied the mechanisms by which LNSC regulate low-avidity autoreactive cells in the nonobese diabetic (NOD) mouse model of type 1 diabetes. Whereas MHC-matched NOD-LNSC significantly reduced G9Cα-/-CD8+T-cell cytotoxicity and DC-induced proliferation, they failed to sufficiently regulate T-cells stimulated by anti-CD3/CD28. In contrast, non-MHC matched, control C57BL/6 mouse LNSC suppressed T-cell receptor engagement by anti-CD3/CD28 via MHC-independent mechanisms. This C57BL/6-LNSC suppression was maintained even after removal of the LNSC, demonstrating a direct effect of LNSC on T-cells, modifying antigen sensitivity and effector function. Thus, our results suggest that a loss of NOD-LNSC MHC-independent suppressive mechanisms may contribute to diabetes development.

Latent autoimmune diabetes in adults (LADA) is an autoimmune disease that shares some genetic, immunological and clinical features with both type 1 diabetes and type 2 diabetes. Immune cells including CD4+ T cells, CD8+ T cells, B cells, macrophages and dendritic cells (DCs) have been detected in the pancreas of patients with LADA and a rat model of LADA. Therefore, similar to type 1 diabetes, the pathogenesis of LADA may be caused by interactions between islet β-cells and innate and adaptive immune cells. However, the role of the immunity in the initiation and progression of LADA remains largely unknown. In this review, we have summarized the potential roles of innate immunity and immune-modulators in LADA development. Furthermore, we have examined the evidence and discussed potential innate immunological reasons for the slower development of LADA compared with type 1 diabetes. More in-depth mechanistic studies are needed to fully elucidate the roles of innate immune-associated genes, molecules and cells in their contributions to LADA pathogenesis. Undertaking these studies will greatly enhance the development of new strategies and optimization of current strategies for the diagnosis and treatment of the disease.

Microbiota have been identified as an important modulator of susceptibility in the development of Type 1 diabetes in both animal models and humans. Collectively these studies highlight the association of the microbiota composition with genetic risk, islet autoantibody development and modulation of the immune responses. However, the signaling pathways involved in mediating these changes are less well investigated, particularly in humans. Importantly, understanding the activation of signaling pathways in response to microbial stimulation is vital to enable further development of immunotherapeutics, which may enable enhanced tolerance to the microbiota or prevent the initiation of the autoimmune process. One such signaling pathway that has been poorly studied in the context of Type 1 diabetes is the role of the inflammasomes, which are multiprotein complexes that can initiate immune responses following detection of their microbial ligands. In this review, we discuss the roles of the inflammasomes in modulating Type 1 diabetes susceptibility, from genetic associations to the priming and activation of the inflammasomes. In addition, we also summarize the available inhibitors for therapeutically targeting the inflammasomes, which may be of future use in Type 1 diabetes.

Innate immunity mediated by Toll-like receptors (TLRs), which can recognize pathogen molecular patterns, plays a critical role in type 1 diabetes development. TLR7 is a pattern recognition receptor that senses single-stranded RNAs from viruses and host tissue cells; however, its role in type 1 diabetes development remains unclear. In our study, we discovered that Tlr7-deficient (Tlr7−/−) nonobese diabetic (NOD) mice, a model of human type 1 diabetes, exhibited a significantly delayed onset and reduced incidence of type 1 diabetes compared with Tlr7-sufficient (Tlr7+/+) NOD mice. Mechanistic investigations showed that Tlr7 deficiency significantly altered B-cell differentiation and immunoglobulin production. Moreover, Tlr7−/− NOD B cells were found to suppress diabetogenic CD4+ T-cell responses and protect immunodeficient NOD mice from developing diabetes induced by diabetogenic T cells. In addition, we found that Tlr7 deficiency suppressed the antigen-presenting functions of B cells and inhibited cytotoxic CD8+ T-cell activation by downregulating the expression of both nonclassical and classical MHC class I (MHC-I) molecules on B cells. Our data suggest that TLR7 contributes to type 1 diabetes development by regulating B-cell functions and subsequent interactions with T cells. Therefore, therapeutically targeting TLR7 may prove beneficial for disease protection.

Type 1 diabetes is an autoimmune disease caused by T cell-mediated destruction of insulin-producing β cells.BDC2.5T cells inBDC2.5CD4+T cell receptor transgenic Non-Obese Diabetic (NOD) mice (BDC2.5+NOD mice) can abruptly invade the pancreatic islets resulting in severe insulitis that progresses rapidly but rarely leads to spontaneous diabetes. This prevention of diabetes is mediated by T regulatory (Treg) cells in these mice. In this study, we investigated the role of interleukin 10 (IL-10) in the inhibition of diabetes inBDC2.5+NOD mice by generatingIl-10-deficientBDC2.5+NOD mice (BDC2.5+Il-10-/-NOD mice). Our results showed thatBDC2.5+Il-10-/-NOD mice displayed robust and accelerated diabetes development.Il-10deficiency inBDC2.5+NOD mice promoted the generation of neutrophils in the bone marrow and increased the proportions of neutrophils in the periphery (blood, spleen, and islets), accompanied by altered intestinal immunity and gut microbiota composition.In vitrostudies showed that the gut microbiota fromBDC2.5+Il-10-/-NOD mice can expand neutrophil populations. Moreover,in vivostudies demonstrated that the depletion of endogenous gut microbiota by antibiotic treatment decreased the proportion of neutrophils. AlthoughIl-10deficiency inBDC2.5+NOD mice had no obvious effects on the proportion and function of Treg cells, it affected the immune response and activation of CD4+T cells. Moreover, the pathogenicity of CD4+T cells was much increased, and this significantly accelerated the development of diabetes when these CD4+T cells were transferred into immune-deficient NOD mice. Our study provides novel insights into the role of IL-10 in the modulation of neutrophils and CD4+T cells inBDC2.5+NOD mice, and suggests important crosstalk between gut microbiota and neutrophils in type 1 diabetes development.

Circadian rhythms, referring to 24-h daily oscillations in biological and physiological processes, can significantly regulate host immunity to pathogens, as well as commensals, resulting in altered susceptibility to disease development. Furthermore, vaccination responses to microbes have also shown time-of-day-dependent changes in the magnitude of protective immune responses elicited in the host. Thus, understanding host circadian rhythm effects on both gut bacteria and viruses during infection is important to minimize adverse effects on health and identify optimal times for therapeutic administration to maximize therapeutic success. In this review, we summarize the circadian modulations of gut bacteria, viruses and their interactions, both in health and during infection. We also discuss the importance of chronotherapy (i.e., time-specific therapy) as a plausible therapeutic administration strategy to enhance beneficial therapeutic responses.

SummaryType 1 diabetes (T1D) is an autoimmune disease characterised by T cell-mediated destruction of the insulin-producing β cells in the pancreas. Similar to other autoimmune diseases, the incidence of T1D is increasing globally. The discovery of insulin 100 years ago dramatically changed the outlook for people with T1D, preventing this from being a fatal condition. As we celebrate the centenary of this milestone, therapeutic options for T1D are once more at a turning point. Years of effort directed at developing immunotherapies are finally starting to pay off, with signs of progress in new onset and even preventative settings. Here, we review a selection of immunotherapies that have shown promise in preserving β cell function and highlight future considerations for immunotherapy in the T1D setting.