Interferons are key cytokines acting on pancreatic islets in type 1 diabetes.

AIMS/HYPOTHESIS: The proinflammatory cytokines IFN-α, IFN-γ, IL-1ß and TNF-α may contribute to innate and adaptive immune responses during insulitis in type 1 diabetes and therefore represent attractive therapeutic targets to protect beta cells. However, the specific role of each of these cytokines individually on pancreatic beta cells remains unknown. METHODS: We used deep RNA-seq analysis, followed by extensive confirmation experiments based on reverse transcription-quantitative PCR (RT-qPCR), western blot, histology and use of siRNAs, to characterise the response of human pancreatic beta cells to each cytokine individually and compared the signatures obtained with those present in islets of individuals affected by type 1 diabetes. RESULTS: IFN-α and IFN-γ had a greater impact on the beta cell transcriptome when compared with IL-1ß and TNF-α. The IFN-induced gene signatures have a strong correlation with those observed in beta cells from individuals with type 1 diabetes, and the level of expression of specific IFN-stimulated genes is positively correlated with proteins present in islets of these individuals, regulating beta cell responses to 'danger signals' such as viral infections. Zinc finger NFX1-type containing 1 (ZNFX1), a double-stranded RNA sensor, was identified as highly induced by IFNs and shown to play a key role in the antiviral response in beta cells. CONCLUSIONS/INTERPRETATION: These data suggest that IFN-α and IFN-γ are key cytokines at the islet level in human type 1 diabetes, contributing to the triggering and amplification of autoimmunity.

Insulitis in human type 1 diabetes: lessons from an enigmatic lesion.

Type 1 diabetes is caused by a deficiency of insulin secretion which has been considered traditionally as the outcome of a precipitous decline in the viability of ß-cells in the islets of Langerhans, brought about by autoimmune-mediated attack. Consistent with this, various classes of lymphocyte, as well as cells of the innate immune system have been found in association with islets during disease progression. However, analysis of human pancreas from subjects with type 1 diabetes has revealed that insulitis is often less intense than in equivalent animal models of the disease and can affect many fewer islets than expected, at disease onset. This is especially true in subjects developing type 1 diabetes in, or beyond, their teenage years. Such studies imply that both the phenotype and the number of immune cells present within insulitic lesions can vary among individuals in an age-dependent manner. Additionally, the influent lymphocytes are often mainly arrayed peripherally around islets rather than gaining direct access to the endocrine cell core. Thus, insulitis remains an enigmatic phenomenon in human pancreas and this review seeks to explore the current understanding of its likely role in the progression of type 1 diabetes.

The role of the interferon/JAK-STAT axis in driving islet HLA-I hyperexpression in type 1 diabetes.

The hyperexpression of human leukocyte antigen class I (HLA-I) molecules on pancreatic beta-cells is widely accepted as a hallmark feature of type 1 diabetes pathogenesis. This response is important clinically since it may increase the visibility of beta-cells to autoreactive CD8+ T-cells, thereby accelerating disease progression. In this review, key factors which drive HLA-I hyperexpression will be explored, and their clinical significance examined. It is established that the presence of residual beta-cells is essential for HLA-I hyperexpression by islet cells at all stages of the disease. We suggest that the most likely drivers of this process are interferons released from beta-cells (type I or III interferon; possibly in response to viral infection) or those elaborated from influent, autoreactive immune cells (type II interferon). In both cases, Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathways will be activated to induce the downstream expression of interferon stimulated genes. A variety of models have highlighted that HLA-I expression is enhanced in beta-cells in response to interferons, and that STAT1, STAT2 and interferon regulatory factor 9 (IRF9) play key roles in mediating these effects (depending on the species of interferon involved). Importantly, STAT1 expression is elevated in the beta-cells of donors with recent-onset type I diabetes, and this correlates with HLA-I hyperexpression on an islet-by-islet basis. These responses can be replicated in vitro, and we consider that chronically elevated STAT1 may have a role in maintaining HLA-I hyperexpression. However, other data have highlighted that STAT2-IRF9 may also be critical to this process. Thus, a better understanding of how these factors regulate HLA-I under chronically stimulated conditions needs to be gathered. Finally, JAK inhibitors can target interferon signaling pathways to diminish HLA-I expression in mouse models. It seems probable that these agents may also be effective in patients; diminishing HLA-I hyperexpression on islets, reducing the visibility of beta-cells to the immune system and ultimately slowing disease progression. The first clinical trials of selective JAK inhibitors are underway, and the outcomes should have important implications for type 1 diabetes clinical management.

Heterogeneity and endotypes in type 1 diabetes mellitus.

Despite major advances over the past decade, prevention and treatment of type 1 diabetes mellitus (T1DM) remain suboptimal, with large and unexplained variations in individual responses to interventions. The current classification schema for diabetes mellitus does not capture the complexity of this disease or guide clinical management effectively. One of the approaches to achieve the goal of applying precision medicine in diabetes mellitus is to identify endotypes (that is, well-defined subtypes) of the disease each of which has a distinct aetiopathogenesis that might be amenable to specific interventions. Here, we describe epidemiological, clinical, genetic, immunological, histological and metabolic differences within T1DM that, together, suggest heterogeneity in its aetiology and pathogenesis. We then present the emerging endotypes and their impact on T1DM prediction, prevention and treatment.

Differential expression of genes controlling lymphocyte differentiation and migration in two distinct endotypes of type 1 diabetes.

AIMS: Morphological studies of pancreas samples obtained from young people with recent-onset type 1 diabetes have revealed distinct patterns of immune cell infiltration of the pancreatic islets suggestive of two age-associated type 1 diabetes endotypes that differ by inflammatory responses and rates of disease progression. The objective of this study was to investigate whether these proposed disease endotypes are associated with pathological differences in immune cell activation and cytokine secretion by applying multiplexed gene expression analysis to pancreatic tissue from recent-onset type 1 diabetes cases. METHODS: RNA was extracted from samples of fixed, paraffin-embedded pancreas tissue from type 1 diabetes cases characterised by endotype and from controls without diabetes. Expression levels of 750 genes associated with autoimmune inflammation were determined by hybridisation to a panel of capture and reporter probes and these were counted as a measure of gene expression. Normalised counts were analysed for differences in expression between 29 type 1 diabetes cases and 7 controls without diabetes, and between the two type 1 diabetes endotypes. RESULTS: Ten inflammation-associated genes, including INS, were significantly under-expressed in both endotypes and 48 genes were more highly expressed. A different set of 13 genes associated with the development, activation and migration of lymphocytes was uniquely overexpressed in the pancreas of people developing diabetes at younger age. CONCLUSIONS: The results provide evidence that histologically defined type 1 diabetes endotypes differ in their immunopathology and identify inflammatory pathways specifically involved in disease developing at a young age, essential for a better understanding of disease heterogeneity.

Gene expression profiling in NOD mice reveals that B cells are highly educated by the pancreatic environment during autoimmune diabetes.

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.

Non-coding variants disrupting a tissue-specific regulatory element in HK1 cause congenital hyperinsulinism.

Gene expression is tightly regulated, with many genes exhibiting cell-specific silencing when their protein product would disrupt normal cellular function1. This silencing is largely controlled by non-coding elements, and their disruption might cause human disease2. We performed gene-agnostic screening of the non-coding regions to discover new molecular causes of congenital hyperinsulinism. This identified 14 non-coding de novo variants affecting a 42-bp conserved region encompassed by a regulatory element in intron 2 of the hexokinase 1 gene (HK1). HK1 is widely expressed across all tissues except in the liver and pancreatic beta cells and is thus termed a 'disallowed gene' in these specific tissues. We demonstrated that the variants result in a loss of repression of HK1 in pancreatic beta cells, thereby causing insulin secretion and congenital hyperinsulinism. Using epigenomic data accessed from public repositories, we demonstrated that these variants reside within a regulatory region that we determine to be critical for cell-specific silencing. Importantly, this has revealed a disease mechanism for non-coding variants that cause inappropriate expression of a disallowed gene.

Detection of Antiviral Tissue Responses and Increased Cell Stress in the Pancreatic Islets of Newly Diagnosed Type 1 Diabetes Patients: Results From the DiViD Study.

Aims/hypothesis: The Diabetes Virus Detection (DiViD) study has suggested the presence of low-grade enteroviral infection in pancreatic tissue collected from six of six live adult patients newly diagnosed with type 1 diabetes. The present study aimed to compare the gene and protein expression of selected virally induced pathogen recognition receptors and interferon stimulated genes in islets from these newly diagnosed type 1 diabetes (DiViD) subjects vs age-matched non-diabetic (ND) controls. Methods: RNA was extracted from laser-captured islets and Affymetrix Human Gene 2.0 ST arrays used to obtain gene expression profiles. Lists of differentially expressed genes were subjected to a data-mining pipeline searching for enrichment of canonical pathways, KEGG pathways, Gene Ontologies, transcription factor binding sites and other upstream regulators. In addition, the presence and localisation of specific viral response proteins (PKR, MxA and MDA5) were examined by combined immunofluorescent labelling in sections of pancreatic tissue. Results: The data analysis and data mining process revealed a significant enrichment of gene ontologies covering viral reproduction and infectious cycles; peptide translation, elongation and initiation, as well as oxidoreductase activity. Enrichment was identified in the KEGG pathways for oxidative phosphorylation; ribosomal and metabolic activity; antigen processing and presentation and in canonical pathways for mitochondrial dysfunction, oxidative phosphorylation and EIF2 signaling. Protein Kinase R (PKR) expression did not differ between newly diagnosed type 1 diabetes and ND islets at the level of total RNA, but a small subset of ß-cells displayed markedly increased PKR protein levels. These PKR+ ß-cells correspond to those previously shown to contain the viral protein, VP1. RNA encoding MDA5 was increased significantly in newly diagnosed type 1 diabetes islets, and immunostaining of MDA5 protein was seen in α- and certain ß-cells in both newly diagnosed type 1 diabetes and ND islets, but the expression was increased in ß-cells in type 1 diabetes. In addition, an uncharacterised subset of synaptophysin positive, but islet hormone negative, cells expressed intense MDA5 staining and these were more prevalent in DiViD cases. MxA RNA was upregulated in newly diagnosed type 1 diabetes vs ND islets and MxA protein was detected exclusively in newly diagnosed type 1 diabetes ß-cells. Conclusion/interpretation: The gene expression signatures reveal that pathways associated with cellular stress and increased immunological activity are enhanced in islets from newly diagnosed type 1 diabetes patients compared to controls. The increases in viral response proteins seen in ß-cells in newly diagnosed type 1 diabetes provide clear evidence for the activation of IFN signalling pathways. As such, these data strengthen the hypothesis that an enteroviral infection of islet ß-cells contributes to the pathogenesis of type 1 diabetes.

Detection of enterovirus RNA in peripheral blood mononuclear cells correlates with the presence of the predisposing allele of the type 1 diabetes risk gene IFIH1 and with disease stage.

AIMS/HYPOTHESIS: Enteroviral infection has been implicated consistently as a key environmental factor correlating with the appearance of autoimmunity and/or the presence of overt type 1 diabetes, in which pancreatic insulin-producing beta cells are destroyed by an autoimmune response. Genetic predisposition through variation in the type 1 diabetes risk gene IFIH1 (interferon induced with helicase C domain 1), which encodes the viral pattern-recognition receptor melanoma differentiation-associated protein 5 (MDA5), supports a potential link between enterovirus infection and type 1 diabetes. METHODS: We used molecular techniques to detect enterovirus RNA in peripheral blood samples (in separated cellular compartments or plasma) from two cohorts comprising 79 children or 72 adults that include individuals with and without type 1 diabetes who had multiple autoantibodies. We also used immunohistochemistry to detect the enteroviral protein VP1 in the pancreatic islets of post-mortem donors (n=43) with type 1 diabetes. RESULTS: We observed enhanced detection sensitivity when sampling the cellular compartment compared with the non-cellular compartment of peripheral blood (OR 21.69; 95% CI 3.64, 229.20; p<0.0001). In addition, we show that children with autoimmunity are more likely to test positive for enterovirus RNA than those without autoimmunity (OR 11.60; 95% CI 1.89, 126.90; p=0.0065). Furthermore, we found that individuals carrying the predisposing allele (946Thr) of the common variant in IFIH1 (rs1990760, Thr946Ala) are more likely to test positive for enterovirus in peripheral blood (OR 3.07; 95% CI 1.02, 8.58; p=0.045). In contrast, using immunohistochemistry, there was no correlation between the common variant in IFIH1 and detection of enteroviral VP1 protein in the pancreatic islets of donors with type 1 diabetes. CONCLUSIONS/INTERPRETATION: Our data indicate that, in peripheral blood, antigen-presenting cells are the predominant source of enterovirus infection, and that infection is correlated with disease stage and genetic predisposition, thereby supporting a role for enterovirus infection prior to disease onset.

Circulating C-Peptide Levels in Living Children and Young People and Pancreatic ß-Cell Loss in Pancreas Donors Across Type 1 Diabetes Disease Duration.

C-peptide declines in type 1 diabetes, although many long-duration patients retain low, but detectable levels. Histological analyses confirm that ß-cells can remain following type 1 diabetes onset. We explored the trends observed in C-peptide decline in the UK Genetic Resource Investigating Diabetes (UK GRID) cohort (N = 4,079), with ß-cell loss in pancreas donors from the network for Pancreatic Organ donors with Diabetes (nPOD) biobank and the Exeter Archival Diabetes Biobank (EADB) (combined N = 235), stratified by recently reported age at diagnosis endotypes (<7, 7-12, ≥13 years) across increasing diabetes durations. The proportion of individuals with detectable C-peptide declined beyond the first year after diagnosis, but this was most marked in the youngest age group (<1-year duration: age <7 years: 18 of 20 [90%], 7-12 years: 107 of 110 [97%], ≥13 years: 58 of 61 [95%] vs. 1-5 years postdiagnosis: <7 years: 172 of 522 [33%], 7-12 years: 604 of 995 [61%], ≥13 years: 225 of 289 [78%]). A similar profile was observed in ß-cell loss, with those diagnosed at younger ages experiencing more rapid loss of islets containing insulin-positive (insulin+) ß-cells <1 year postdiagnosis: age <7 years: 23 of 26 (88%), 7-12 years: 32 of 33 (97%), ≥13 years: 22 of 25 (88%) vs. 1-5 years postdiagnosis: <7 years: 1 of 12 (8.3%), 7-12 years: 7 of 13 (54%), ≥13 years: 7 of 8 (88%). These data should be considered in the planning and interpretation of intervention trials designed to promote ß-cell retention and function.

Temporal regulation of interferon signalling in human EndoC-ßH1 cells.

During the development of type 1 diabetes, interferons (IFN) are elaborated from islet-infiltrating immune cells and/or from virally infected ß-cells. They act via specific receptors to increase, acutely, the phosphorylation of the transcription factors STAT1 and 2. However, the longer-term impacts of chronic IFN stimulation are poorly understood and were investigated in the current study. Human EndoC-ßH1 cells were treated with IFNα, IFNγ or IFNλ either acutely (<2 h) or chronically (≥24 h) and STAT phosphorylation, expression and activity were assessed by Western blotting and transcriptional reporter assays. Exposure of ß-cells to IFNα or IFNλ induced a swift increase in the phosphorylation of both STAT1 and STAT2, whereas IFNγ increased only pSTAT1. Over more extended periods (≥24 h), STAT phosphorylation declined but STAT1 and STAT2 expression were enhanced in a sustained manner. All IFNs stimulated ISRE transcriptional activity (but with different time courses), whereas GAS activity was responsive only to IFNγ. The re-addition of a second bolus of IFNα, 24 h after an initial dose, failed to cause renewed STAT1/2 phosphorylation. By contrast, when IFNγ was added 24 h after exposure to IFNα, rapid STAT1 phosphorylation was re-initiated. Exposure of ß-cells to IFNs leads to rapid, transient, STAT phosphorylation and to slower and more sustained increases in total STAT1/2 levels. The initial phosphorylation response is accompanied by marked desensitisation to the cognate agonist. Together, the results reveal that the response of ß-cells to IFNs is regulated both temporally and quantitatively to achieve effective signal integration.

Differential routing and disposition of the long-chain saturated fatty acid palmitate in rodent vs human beta-cells.

BACKGROUND: Rodent and human ß-cells are differentially susceptible to the "lipotoxic" effects of long-chain saturated fatty acids (LC-SFA) but the factors accounting for this are unclear. Here, we have studied the intracellular disposition of the LC-SFA palmitate in human vs rodent ß-cells and present data that reveal new insights into the factors regulating ß-cell lipotoxicity. METHODS: The subcellular distribution of the LC-SFA palmitate was studied in rodent (INS-1E and INS-1 823/13 cells) and human (EndoC-ßH1) ß-cells using confocal fluorescence and electron microscopy (EM). Protein expression was assessed by Western blotting and cell viability, by vital dye staining. RESULTS: Exposure of INS-1 cells to palmitate for 24 h led to loss of viability, whereas EndoC-ßH1 cells remained viable even after 72 h of treatment with a high concentration (1 mM) of palmitate. Use of the fluorescent palmitate analogue BODIPY FL C16 revealed an early localisation of the LC-SFA to the Golgi apparatus in INS-1 cells and this correlated with distention of intracellular membranes, visualised under the EM. Despite this, the PERK-dependent ER stress pathway was not activated under these conditions. By contrast, BODIPY FL C16 did not accumulate in the Golgi apparatus in EndoC-ßH1 cells but, rather, co-localised with the lipid droplet-associated protein, PLIN2, suggesting preferential routing into lipid droplets. When INS-1 cells were treated with a combination of palmitate plus oleate, the toxic effects of palmitate were attenuated and BODIPY FL C16 localised primarily with PLIN2 but not with a Golgi marker. CONCLUSION: In rodent ß-cells, palmitate accumulates in the Golgi apparatus at early time points whereas, in EndoC- ßH1 cells, it is routed preferentially into lipid droplets. This may account for the differential sensitivity of rodent vs human ß-cells to "lipotoxicity" since manoeuvres leading to the incorporation of palmitate into lipid droplets is associated with the maintenance of cell viability in both cell types.

Expression of CD47 in the pancreatic ß-cells of people with recent-onset type 1 diabetes varies according to disease endotype.

AIMS: We are studying the dialogue between ß-cells and the immune system in type 1 diabetes and have identified a cell surface receptor, signal regulatory protein-alpha (SIRPα) as an important component in the regulation of ß-cell survival. SIRPα interacts with another protein, CD47, to mediate signalling. In the present work, we have studied the expression and role of CD47 in human islet cells in type 1 diabetes. METHODS: Clonal EndoC-ßH1 cells were employed for functional studies. Cells were exposed to pro-inflammatory cytokines and their viability monitored by flow cytometry after staining with propidium iodide. Targeted knockdown of CD47 or SIRPα was achieved with small interference RNA molecules and the expression of relevant proteins studied by Western blotting or immunocytochemistry. Human pancreas sections were selected from the Exeter Archival Diabetes Biobank and used to examine the expression of CD47 by immunofluorescence labelling. Image analysis was employed to quantify expression. RESULTS: CD47 is abundantly expressed in both α and ß cells in human pancreas. In type 1 diabetes, the levels of CD47 are increased in α cells across all age groups, whereas the expression in ß-cells varies according to disease endotype. Knockdown of either CD47 or SIRPα in EndoC-ßH1 cells resulted in a loss of viability. CONCLUSIONS: We conclude that the CD47 plays a previously unrecognised role in the regulation of ß-cell viability. This system is dysregulated in type 1 diabetes suggesting that it may be targeted therapeutically to slow disease progression.

Investigation of the utility of the 1.1B4 cell as a model human beta cell line for study of persistent enteroviral infection.

The generation of a human pancreatic beta cell line which reproduces the responses seen in primary beta cells, but is amenable to propagation in culture, has long been an important goal in diabetes research. This is particularly true for studies focussing on the role of enteroviral infection as a potential cause of beta-cell autoimmunity in type 1 diabetes. In the present work we made use of a clonal beta cell line (1.1B4) available from the European Collection of Authenticated Cell Cultures, which had been generated by the fusion of primary human beta-cells with a pancreatic ductal carcinoma cell, PANC-1. Our goal was to study the factors allowing the development and persistence of a chronic enteroviral infection in human beta-cells. Since PANC-1 cells have been reported to support persistent enteroviral infection, the hybrid 1.1B4 cells appeared to offer an ideal vehicle for our studies. In support of this, infection of the cells with a Coxsackie virus isolated originally from the pancreas of a child with type 1 diabetes, CVB4.E2, at a low multiplicity of infection, resulted in the development of a state of persistent infection. Investigation of the molecular mechanisms suggested that this response was facilitated by a number of unexpected outcomes including an apparent failure of the cells to up-regulate certain anti-viral response gene products in response to interferons. However, more detailed exploration revealed that this lack of response was restricted to molecular targets that were either activated by, or detected with, human-selective reagents. By contrast, and to our surprise, the cells were much more responsive to rodent-selective reagents. Using multiple approaches, we then established that populations of 1.1B4 cells are not homogeneous but that they contain a mixture of rodent and human cells. This was true both of our own cell stocks and those held by the European Collection of Authenticated Cell Cultures. In view of this unexpected finding, we developed a strategy to harvest, isolate and expand single cell clones from the heterogeneous population, which allowed us to establish colonies of 1.1B4 cells that were uniquely human (h1.1.B4). However, extensive analysis of the gene expression profiles, immunoreactive insulin content, regulated secretory pathways and the electrophysiological properties of these cells demonstrated that they did not retain the principal characteristics expected of human beta cells. Our data suggest that stocks of 1.1B4 cells should be evaluated carefully prior to their use as a model human beta-cell since they may not retain the phenotype expected of human beta-cells.

Altered ß-Cell Prohormone Processing and Secretion in Type 1 Diabetes.

Analysis of data from clinical cohorts, and more recently from human pancreatic tissue, indicates that reduced prohormone processing is an early and persistent finding in type 1 diabetes. In this article, we review the current state of knowledge regarding alterations in islet prohormone expression and processing in type 1 diabetes and consider the clinical impact of these findings. Lingering questions, including pathologic etiologies and consequences of altered prohormone expression and secretion in type 1 diabetes, and the natural history of circulating prohormone production in health and disease, are considered. Finally, key next steps required to move forward in this area are outlined, including longitudinal testing of relevant clinical populations, studies that probe the genetics of altered prohormone processing, the need for combined functional and histologic testing of human pancreatic tissues, continued interrogation of the intersection between prohormone processing and autoimmunity, and optimal approaches for analysis. Successful resolution of these questions may offer the potential to use altered prohormone processing as a biomarker to inform therapeutic strategies aimed at personalized intervention during the natural history of type 1 diabetes and as a pathogenic anchor for identification of potential disease-specific endotypes.

Identification and characterisation of tertiary lymphoid organs in human type 1 diabetes.

AIMS/HYPOTHESIS: We and others previously reported the presence of tertiary lymphoid organs (TLOs) in the pancreas of NOD mice, where they play a role in the development of type 1 diabetes. Our aims here are to investigate whether TLOs are present in the pancreas of individuals with type 1 diabetes and to characterise their distinctive features, in comparison with TLOs present in NOD mouse pancreases, in order to interpret their functional significance. METHODS: Using immunofluorescence confocal microscopy, we examined the extracellular matrix (ECM) and cellular constituents of pancreatic TLOs from individuals with ongoing islet autoimmunity in three distinct clinical settings of type 1 diabetes: at risk of diabetes; at/after diagnosis; and in the transplanted pancreas with recurrent diabetes. Comparisons were made with TLOs from 14-week-old NOD mice, which contain islets exhibiting mild to heavy leucocyte infiltration. We determined the frequency of the TLOs in human type 1diabetes with insulitis and investigated the presence of TLOs in relation to age of onset, disease duration and disease severity. RESULTS: TLOs were identified in preclinical and clinical settings of human type 1 diabetes. The main characteristics of these TLOs, including the cellular and ECM composition of reticular fibres (RFs), the presence of high endothelial venules and immune cell subtypes detected, were similar to those observed for TLOs from NOD mouse pancreases. Among 21 donors with clinical type 1 diabetes who exhibited insulitis, 12 had TLOs and had developed disease at younger age compared with those lacking TLOs. Compartmentalised TLOs with distinct T cell and B cell zones were detected in donors with short disease duration. Overall, TLOs were mainly associated with insulin-containing islets and their frequency decreased with increasing severity of beta cell loss. Parallel studies in NOD mice further revealed some differences in so far as regulatory T cells were essentially absent from human pancreatic TLOs and CCL21 was not associated with RFs. CONCLUSIONS/INTERPRETATION: We demonstrate a novel feature of pancreas pathology in type 1 diabetes. TLOs represent a potential site of autoreactive effector T cell generation in islet autoimmunity and our data from mouse and human tissues suggest that they disappear once the destructive process has run its course. Thus, TLOs may be important for type 1 diabetes progression.

Footprints of Immune Cells in the Pancreas in Type 1 Diabetes; to "B" or Not to "B": Is That Still the Question?

Significant progress has been made in understanding the phenotypes of circulating immune cell sub-populations in human type 1 diabetes but much less is known about the equivalent populations that infiltrate the islets to cause beta-cell loss. In particular, considerable uncertainties remain about the phenotype and role of B-lymphocytes in the pancreas. This gap in understanding reflects both the difficulty in accessing the gland to study islet inflammation during disease progression and the fact that the number and proportion of islet-associated B-lymphocytes varies significantly according to the disease endotype. In very young children (especially those <7 years at onset) pancreatic islets are infiltrated by both CD8+ T- and CD20+ B-lymphocytes in roughly equal proportions but it is widely held that the CD8+ T-lymphocytes are responsible for driving beta-cell toxicity. By contrast, the role played by B-lymphocytes remains enigmatic. This is compounded by the fact that, in older children and teenagers (those ≥13 years at diagnosis) the proportion of B-lymphocytes found in association with inflamed islets is much reduced by comparison with those who are younger at diagnosis (reflecting two endotypes of disease) whereas CD8+ T-lymphocytes form the predominant population in both groups. In the present paper, we review the current state of understanding and develop a proposal to stimulate further discussion of the roles played by islet-associated B-lymphocytes in human type 1 diabetes. We cite evidence indicating that sites of direct contact can be found between CD8+ and CD20+-lymphocytes in and around inflamed islets and propose that such interactions may be important in determining the efficiency of beta cell killing.

Long-chain saturated fatty acid species are not toxic to human pancreatic ß-cells and may offer protection against pro-inflammatory cytokine induced ß-cell death.

Obesity is a major risk factor for type 2 diabetes (T2D) although the causal links remain unclear. A feature shared by both conditions however is systemic inflammation and raised levels of circulating fatty acids (FFA). It is widely believed that in obese individuals genetically prone to T2D, elevated levels of plasma FFA may contribute towards the death and dysfunction of insulin-producing pancreatic ß-cells in a process of (gluco)lipotoxicity. In support of this, in vitro studies have shown consistently that long-chain saturated fatty acids (LC-SFA) are toxic to rodent ß-cells during chronic exposure (> 24 h). Conversely, shorter chain SFA and unsaturated species are well tolerated, suggesting that toxicity is dependent on carbon chain length and/or double bond configuration. Despite the wealth of evidence implicating lipotoxicity as a means of ß-cell death in rodents, the evidence that a similar process occurs in humans is much less substantial. Therefore, the present study has evaluated the effects of chronic exposure to fatty acids of varying chain length and degree of saturation, on the viability of human ß-cells in culture. We have also studied the effects of a combination of fatty acids and pro-inflammatory cytokines. Strikingly, we find that LC-FFA do not readily promote the demise of human ß-cells and that they may even offer a measure of protection against the toxic effects of pro-inflammatory cytokines. Therefore, these findings imply that a model in which elevated circulating LC-FFA play a direct role in mediating ß-cell dysfunction and death in humans, may be overly simplistic.