Single-cell analysis of the human pancreas in type 2 diabetes using multi-spectral imaging mass cytometry.

Type 2 diabetes mellitus (T2D) is a chronic age-related disorder characterized by hyperglycemia due to the failure of pancreatic beta cells to compensate for increased insulin demand. Despite decades of research, the pathogenic mechanisms underlying T2D remain poorly defined. Here, we use imaging mass cytometry (IMC) with a panel of 34 antibodies to simultaneously quantify markers of pancreatic exocrine, islet, and immune cells and stromal components. We analyze over 2 million cells from 16 pancreata obtained from donors with T2D and 13 pancreata from age-similar non-diabetic controls. In the T2D pancreata, we observe significant alterations in islet architecture, endocrine cell composition, and immune cell constituents. Thus, both HLA-DR-positive CD8 T cells and macrophages are enriched intra-islet in the T2D pancreas. These efforts demonstrate the utility of IMC for investigating complex events at the cellular level in order to provide insights into the pathophysiology of T2D.

IL-17 is expressed on beta and alpha cells of donors with type 1 and type 2 diabetes.

PURPOSE: IL-17 is an important effector cytokine driving immune-mediated destruction in autoimmune diseases such as psoriasis. Blockade of the IL-17 pathway after the initiation of insulitis was effective in delaying or preventing the onset of type 1 diabetes (T1D) in rodent models. Expression of IL-17 transcripts in islets from a donor with recent-onset T1D has been reported, however, studies regarding IL-17 protein expression are lacking. We aimed to study whether IL-17 is being expressed in the islets of diabetic donors. METHODS: We stained human pancreatic tissues from non-diabetic (n = 5), auto-antibody positive (aab+) (n = 5), T1D (n = 6) and T2D (n = 5) donors for IL-17, Insulin, and Glucagon, and for CD45 in selected cases. High resolution images were acquired with Zeiss laser scanning confocal microscope LSM780 and analyzed with Zen blue 2.3 software. Cases stained for CD45 were also acquired with widefield slide scanner and analyzed with QuPath software. RESULTS: We observed a clear cytoplasmic staining for IL-17 in insulin-containing islets of donors with T1D and T2D, accounting for an average of 7.8 ± 8.4% and 14.9 ± 16.8% of total islet area, respectively. Both beta and alpha cells were sources of IL-17, but CD45+ cells were not a major source of IL-17 in those donors. Expression of IL-17 was reduced in islets of non-diabetic donors, aab+ donors and in insulin-deficient islets of donors with T1D. CONCLUSION: Our finding that IL-17 is expressed in islets of donors with T1D or T2D is quite intriguing and warrants further mechanistic studies in human islets to understand the role of IL-17 in the context of metabolic and immune stress in beta cells.

Purification of pancreatic endocrine subsets reveals increased iron metabolism in beta cells.

OBJECTIVES: The main endocrine cell types in pancreatic islets are alpha, beta, and delta cells. Although these cell types have distinct roles in the regulation of glucose homeostasis, inadequate purification methods preclude the study of cell type-specific effects. We developed a reliable approach that enables simultaneous sorting of live alpha, beta, and delta cells from mouse islets for downstream analyses. METHODS: We developed an antibody panel against cell surface antigens to enable isolation of highly purified endocrine subsets from mouse islets based on the specific differential expression of CD71 on beta cells and CD24 on delta cells. We rigorously demonstrated the reliability and validity of our approach using bulk and single cell qPCR, immunocytochemistry, reporter mice, and transcriptomics. RESULTS: Pancreatic alpha, beta, and delta cells can be separated based on beta cell-specific CD71 surface expression and high expression of CD24 on delta cells. We applied our new sorting strategy to demonstrate that CD71, which is the transferrin receptor mediating the uptake of transferrin-bound iron, is upregulated in beta cells during early postnatal weeks. We found that beta cells express higher levels of several other genes implicated in iron metabolism and iron deprivation significantly impaired beta cell function. In human beta cells, CD71 is similarly required for iron uptake and CD71 surface expression is regulated in a glucose-dependent manner. CONCLUSIONS: This study provides a novel and efficient purification method for murine alpha, beta, and delta cells, identifies for the first time CD71 as a postnatal beta cell-specific marker, and demonstrates a central role of iron metabolism in beta cell function.

IL-6 is present in beta and alpha cells in human pancreatic islets: Expression is reduced in subjects with type 1 diabetes.

IL-6 is a pro-inflammatory cytokine upregulated in some autoimmune diseases. The role of IL-6 in the development of type 1 diabetes (T1D) is unclear. Clinical studies are investigating whether tocilizumab (anti-IL-6 receptor) can help preserve beta cell function in patients recently diagnosed with T1D. However, in some rodent models and isolated human islets, IL-6 has been found to have a protective role for beta cells by reducing oxidative stress. Hence, we systematically investigated local tissue expression of IL-6 in human pancreas from non-diabetic, auto-antibody positive donors and donors with T1D and T2D. IL-6 was constitutively expressed by beta and alpha cells regardless of the disease state. However, expression of IL-6 was highly reduced in insulin-deficient islets of donors with T1D, and the expression was then mostly restricted to alpha cells. Our findings suggest that the implication of IL-6 in T1D pathogenesis might be more complex than previously assumed.

Antibodies against H1N1 influenza virus cross-react with α-cells of pancreatic islets.

Epidemiological studies have documented that the incidence of human type 1 diabetes was significantly increased after H1N1 epidemic. However, a direct link between human type 1 diabetes and virus infection remains elusive. We generated 84 clones of murine monoclonal antibodies against the H1N1, and carried out immunohistochemistry in normal human tissue microarray. The results showed that two clones specifically cross-reacted with human α-cells of pancreatic islets. Reverse transcription polymerase chain reaction and deoxyribonucleic acid sequencing showed that the amino acid sequences of light and heavy chains of these clones were different. Importantly, the expression profiles of two monoclonal antibodies were individual different. For the first time, we provide direct evidence that monoclonal antibodies against H1N1 can cross-react with human pancreas α-cells, another source of ß-cells, suggesting α-cells might be a novel target to be investigated in diabetes research.

Signals in the pancreatic islet microenvironment influence ß-cell proliferation.

The progressive loss of pancreatic ß-cell mass that occurs in both type 1 and type 2 diabetes is a primary factor driving efforts to identify strategies for effectively increasing, enhancing or restoring ß-cell mass. While factors that seem to influence ß-cell proliferation in specific contexts have been described, reliable stimulation of human ß-cell proliferation has remained a challenge. Importantly, ß-cells exist in the context of a complex, integrated pancreatic islet microenvironment where they interact with other endocrine cells, vascular endothelial cells, extracellular matrix, neuronal projections and islet macrophages. This review highlights different components of the pancreatic microenvironment, and reviews what is known about how signaling that occurs between ß-cells and these other components influences ß-cell proliferation. Future efforts to further define the role of the pancreatic islet microenvironment on ß-cell proliferation may lead to the development of successful approaches to increase or restore ß-cell mass in diabetes.

Expression-Based Genome-Wide Association Study Links Vitamin D-Binding Protein With Autoantigenicity in Type 1 Diabetes.

Type 1 diabetes (T1D) is caused by autoreactive T cells that recognize pancreatic islet antigens and destroy insulin-producing ß-cells. This attack results from a breakdown in tolerance for self-antigens, which is controlled by ectopic antigen expression in the thymus and pancreatic lymph nodes (PLNs). The autoantigens known to be involved include a set of islet proteins, such as insulin, GAD65, IA-2, and ZnT8. In an attempt to identify additional antigenic proteins, we performed an expression-based genome-wide association study using microarray data from 118 arrays of the thymus and PLNs of T1D mice. We ranked all 16,089 protein-coding genes by the likelihood of finding repeated differential expression and the degree of tissue specificity for pancreatic islets. The top autoantigen candidate was vitamin D-binding protein (VDBP). T-cell proliferation assays showed stronger T-cell reactivity to VDBP compared with control stimulations. Higher levels and frequencies of serum anti-VDBP autoantibodies (VDBP-Abs) were identified in patients with T1D (n = 331) than in healthy control subjects (n = 77). Serum vitamin D levels were negatively correlated with VDBP-Ab levels in patients in whom T1D developed during the winter. Immunohistochemical localization revealed that VDBP was specifically expressed in α-cells of pancreatic islets. We propose that VDBP could be an autoantigen in T1D.

Loss of Peripheral Protection in Pancreatic Islets by Proteolysis-Driven Impairment of VTCN1 (B7-H4) Presentation Is Associated with the Development of Autoimmune Diabetes.

Ag-specific activation of T cells is an essential process in the control of effector immune responses. Defects in T cell activation, particularly in the costimulation step, have been associated with many autoimmune conditions, including type 1 diabetes (T1D). Recently, we demonstrated that the phenotype of impaired negative costimulation, due to reduced levels of V-set domain-containing T cell activation inhibitor 1 (VTCN1) protein on APCs, is shared between diabetes-susceptible NOD mice and human T1D patients. In this study, we show that a similar process takes place in the target organ, as both α and ß cells within pancreatic islets gradually lose their VTCN1 protein during autoimmune diabetes development despite upregulation of the VTCN1 gene. Diminishment of functional islet cells' VTCN1 is caused by the active proteolysis by metalloproteinase N-arginine dibasic convertase 1 (NRD1) and leads to the significant induction of proliferation and cytokine production by diabetogenic T cells. Inhibition of NRD1 activity, alternatively, stabilizes VTCN1 and dulls the anti-islet T cell responses. Therefore, we suggest a general endogenous mechanism of defective VTCN1 negative costimulation, which affects both lymphoid and peripheral target tissues during T1D progression and results in aggressive anti-islet T cell responses. This mechanism is tied to upregulation of NRD1 expression and likely acts in two synergistic proteolytic modes: cell-intrinsic intracellular and cell-extrinsic systemic. Our results highlight an importance of VTCN1 stabilization on cell surfaces for the restoration of altered balance of immune control during T1D.

Insulitis and ß-Cell Mass in the Natural History of Type 1 Diabetes.

Descriptions of insulitis in human islets throughout the natural history of type 1 diabetes are limited. We determined insulitis frequency (the percent of islets displaying insulitis to total islets), infiltrating leukocyte subtypes, and ß-cell and α-cell mass in pancreata recovered from organ donors with type 1 diabetes (n = 80), as well as from donors without diabetes, both with islet autoantibodies (AAb(+), n = 18) and without islet autoantibodies (AAb(-), n = 61). Insulitis was observed in four of four donors (100%) with type 1 diabetes duration of ≤1 year and two AAb(+) donors (2 of 18 donors, 11%). Insulitis frequency showed a significant but limited inverse correlation with diabetes duration (r = -0.58, P = 0.01) but not with age at disease onset. Residual ß-cells were observed in all type 1 diabetes donors with insulitis, while ß-cell area and mass were significantly higher in type 1 diabetes donors with insulitis compared with those without insulitis. Insulitis affected 33% of insulin(+) islets compared with 2% of insulin(-) islets in donors with type 1 diabetes. A significant correlation was observed between insulitis frequency and CD45(+), CD3(+), CD4(+), CD8(+), and CD20(+) cell numbers within the insulitis (r = 0.53-0.73, P = 0.004-0.04), but not CD68(+) or CD11c(+) cells. The presence of ß-cells as well as insulitis several years after diagnosis in children and young adults suggests that the chronicity of islet autoimmunity extends well into the postdiagnosis period. This information should aid considerations of therapeutic strategies seeking type 1 diabetes prevention and reversal.

Differential cell autonomous responses determine the outcome of coxsackievirus infections in murine pancreatic α and ß cells.

Type 1 diabetes (T1D) is an autoimmune disease caused by loss of pancreatic ß cells via apoptosis while neighboring α cells are preserved. Viral infections by coxsackieviruses (CVB) may contribute to trigger autoimmunity in T1D. Cellular permissiveness to viral infection is modulated by innate antiviral responses, which vary among different cell types. We presently describe that global gene expression is similar in cytokine-treated and virus-infected human islet cells, with up-regulation of gene networks involved in cell autonomous immune responses. Comparison between the responses of rat pancreatic α and ß cells to infection by CVB5 and 4 indicate that α cells trigger a more efficient antiviral response than ß cells, including higher basal and induced expression of STAT1-regulated genes, and are thus better able to clear viral infections than ß cells. These differences may explain why pancreatic ß cells, but not α cells, are targeted by an autoimmune response during T1D.

Influence of in vitro and in vivo oxygen modulation on ß cell differentiation from human embryonic stem cells.

The possibility of using human embryonic stem (hES) cell-derived ß cells as an alternative to cadaveric islets for the treatment of type 1 diabetes is now widely acknowledged. However, current differentiation methods consistently fail to generate meaningful numbers of mature, functional ß cells. In order to address this issue, we set out to explore the role of oxygen modulation in the maturation of pancreatic progenitor (PP) cells differentiated from hES cells. We have previously determined that oxygenation is a powerful driver of murine PP differentiation along the endocrine lineage of the pancreas. We hypothesized that targeting physiological oxygen partial pressure (pO2) levels seen in mature islets would help the differentiation of PP cells along the ß-cell lineage. This hypothesis was tested both in vivo (by exposing PP-transplanted immunodeficient mice to a daily hyperbaric oxygen regimen) and in vitro (by allowing PP cells to mature in a perfluorocarbon-based culture device designed to carefully adjust pO2 to a desired range). Our results show that oxygen modulation does indeed contribute to enhanced maturation of PP cells, as evidenced by improved engraftment, segregation of α and ß cells, body weight maintenance, and rate of diabetes reversal in vivo, and by elevated expression of pancreatic endocrine makers, ß-cell differentiation yield, and insulin production in vitro. Our studies confirm the importance of oxygen modulation as a key variable to consider in the design of ß-cell differentiation protocols and open the door to future strategies for the transplantation of fully mature ß cells.

Role of endogenous IL-6 in the neonatal expansion and functionality of Wistar rat pancreatic alpha cells.

AIMS/HYPOTHESIS: Plasma glucagon concentrations rise sharply during the early postnatal period. This condition is associated with increased alpha cell mass. However, the trophic factors that regulate alpha cell turnover during the perinatal period have not been studied. Macrophage infiltrations are present in the neonatal pancreas, and this cell type releases cytokines such as IL-6. Alpha cells have been identified as a primary target of IL-6 actions. We therefore investigated the physiological relevance of IL-6 to neonatal pancreatic alpha cell maturation. METHODS: Histochemical analyses were performed to quantify alpha cell mass, replication and apoptosis. Pancreatic Il6 expression was determined by quantitative RT-PCR. The biological effect of IL-6 was tested in two in vivo rat models of IL-6 blockade and chronic undernutrition. RESULTS: Alpha cell mass increased sharply shortly after birth but decreased significantly after weaning. Pancreatic alpha cell proliferation was as high as 2.5% at the beginning of suckling but diminished with time to 1.2% in adulthood. Similarly, alpha cell neoformation was remarkably high on postnatal day (PN) 4, whereas alpha cell apoptosis was low throughout the neonatal period. Moreover, Il6 mRNA exhibited developmental upregulation in the pancreas of suckling rats, with the highest expression on PN2. Neutralisation of IL-6 reduced alpha cell mass expansion and glucagon production. IL-6 staining was detected within the islets, mainly in the alpha cells. Finally, undernourished neonates showed altered alpha cell number and function and delayed appearance of IL-6 in the pancreas. CONCLUSIONS/INTERPRETATION: These data point to a potential role for local IL-6 in the regulation of alpha cell growth and function during suckling.

Toll-like receptor 4 on islet ß cells senses expression changes in high-mobility group box 1 and contributes to the initiation of type 1 diabetes.

Type 1 diabetes mellitus is caused by the autoimmune destruction of ß cells within the islets. In recent years, innate immunity has been proposed to play a key role in this process. High-mobility group box 1 (HMGB1), an inflammatory trigger in a number of autoimmune diseases, activates proinflammatory responses following its release from necrotic cells. Our aim was to determine the significance of HMGB1 in the natural history of diabetes in non-obese diabetic (NOD) mice. We observed that the rate of HMGB1 expression in the cytoplasm of islets was much greater in diabetic mice compared with non-diabetic mice. The majority of cells positively stained for toll-like receptor 4 (TLR4) were ß cells; few α cells were stained for TLR4. Thus, we examined the effects of anti-TLR4 antibodies on HMGB1 cell surface binding, which confirmed that HMGB1 interacts with TLR4 in isolated islets. Expression changes in HMGB1 and TLR4 were detected throughout the course of diabetes. Our findings indicate that TLR4 is the main receptor on ß cells and that HMGB1 may signal via TLR4 to selectively damage ß cells rather than α cells during the development of type 1 diabetes mellitus.

Toll-like receptor 4 on islet beta cells senses expression changes in high-mobility group box 1 and contributes to the initiation of type 1 diabetes

Type 1 diabetes mellitus is caused by the autoimmune destruction of beta cells within the islets. In recent years, innate immunity has been proposed to play a key role in this process. High-mobility group box 1 (HMGB1), an inflammatory trigger in a number of autoimmune diseases, activates proinflammatory responses following its release from necrotic cells. Our aim was to determine the significance of HMGB1 in the natural history of diabetes in non-obese diabetic (NOD) mice. We observed that the rate of HMGB1 expression in the cytoplasm of islets was much greater in diabetic mice compared with non-diabetic mice. The majority of cells positively stained for toll-like receptor 4 (TLR4) were beta cells; few alpha cells were stained for TLR4. Thus, we examined the effects of anti-TLR4 antibodies on HMGB1 cell surface binding, which confirmed that HMGB1 interacts with TLR4 in isolated islets. Expression changes in HMGB1 and TLR4 were detected throughout the course of diabetes. Our findings indicate that TLR4 is the main receptor on beta cells and that HMGB1 may signal via TLR4 to selectively damage beta cells rather than alpha cells during the development of type 1 diabetes mellitus.

Calcineurin/nuclear factor of activated T cells and MAPK signaling induce TNF-{alpha} gene expression in pancreatic islet endocrine cells.

Cytokines contribute to pancreatic islet inflammation, leading to impaired glucose homeostasis and diabetic diseases. A plethora of data shows that proinflammatory cytokines are produced in pancreatic islets by infiltrating mononuclear immune cells. Here, we show that pancreatic islet α cells and ß cells express tumor necrosis factor-α (TNF-α) and other cytokines capable of promoting islet inflammation when exposed to interleukin-1ß (IL-1ß). Cytokine expression by ß cells was dependent on calcineurin (CN)/nuclear factor of activated T cells (NFAT) and MAPK signaling. NFAT associated with the TNF-α promoter in response to stimuli and synergistically activated promoter activity with ATF2 and c-Jun. In contrast, the ß-cell-specific transcriptional activator MafA could repress NFAT-mediated TNF-α gene expression whenever C/EBP-ß was bound to the promoter. NFAT differentially regulated the TNF-α gene depending upon the expression and MAPK-dependent activation of interacting basic leucine zipper partners in ß cells. Both p38 and JNK were required for induction of TNF-α mRNA and protein expression. Collectively, the data show that glucose and IL-1ß can activate signaling pathways, which control induction and repression of cytokines in pancreatic endocrine cells. Thus, by these mechanisms, pancreatic ß cells themselves may contribute to islet inflammation and their own immunological destruction in the pathogenesis of diabetes.

Generation of novel single-chain antibodies by phage-display technology to direct imaging agents highly selective to pancreatic beta- or alpha-cells in vivo.

OBJECTIVE: Noninvasive determination of pancreatic beta-cell mass in vivo has been hampered by the lack of suitable beta-cell-specific imaging agents. This report outlines an approach for the development of novel ligands homing selectively to islet cells in vivo. RESEARCH DESIGN AND METHODS: To generate agents specifically binding to pancreatic islets, a phage library was screened for single-chain antibodies (SCAs) on rat islets using two different approaches. 1) The library was injected into rats in vivo, and islets were isolated after a circulation time of 5 min. 2) Pancreatic islets were directly isolated, and the library was panned in the islets in vitro. Subsequently, the identified SCAs were extensively characterized in vitro and in vivo. RESULTS: We report the generation of SCAs that bind highly selective to either beta- or alpha-cells. These SCAs are internalized by target cells, disappear rapidly from the vasculature, and exert no toxicity in vivo. Specific binding to beta- or alpha-cells was detected in cell lines in vitro, in rats in vivo, and in human tissue in situ. Electron microscopy demonstrated binding of SCAs to the endoplasmatic reticulum and the secretory granules. Finally, in a biodistribution study the labeling intensity derived from [(125)I]-labeled SCAs after intravenous administration in rats strongly predicted the beta-cell mass and was inversely related to the glucose excursions during an intraperitoneal glucose tolerance test. CONCLUSIONS: Our data provide strong evidence that the presented SCAs are highly specific for pancreatic beta-cells and enable imaging and quantification in vivo.

Evidence for Induced Expression of HLA Class II on Human Islets: Possible Mechanism for HLA Sensitization in Transplant Recipients.

Recent reports have shown that islet transplant recipients develop antibodies against donor human leukocyte antigen (HLA) class I and II. Because human islets do not express HLA class II under normal conditions, mechanisms underlying induction of the anti-class II response are unclear. We hypothesized that under inflammatory conditions, islets will have induced expression of HLA class II leading to sensitization. Isolated human islets were divided into two groups. Group 1 was cultured at 37 degrees C as control; group 2 was cultured similarly in presence of tumor necrosis factor alpha and interferon gamma. After treatment, islets were analyzed for expression of HLA class II using real-time polymerase chain reaction, immunofluorescence and flow cytometry. Furthermore, serum from an islet transplant recipient who developed anti-class II antibody was tested by flow cytometry for immunoglobulin (Ig) binding to cytokine-stimulated islets. Real-time polymerase chain reaction analysis for gene transcripts of class II transactivator, HLA-DRagr;, and HLA-DRbeta1 showed maximum 9.38-, 18.95-, and 46.5-fold increase, respectively in group 2 when compared with control at 24 hr. Cytokine treatment increased HLA class II expression markedly on both alpha and beta cells in islets as evidenced by fluorescent imaging and flow cytometric analysis. When patient serum was analyzed by flow cytometry, both IgM and IgG binding was observed in cytokine-treated, HLA class II matched islet cells alone. We conclude that inflammation leads to induced expression of HLA class II on transplanted islet cells potentially causing antidonor sensitization and adversely impacting islet transplant outcomes.