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Browsing by Author "Eizirik, Decio L."
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Item Beta cell extracellular vesicle PD-L1 as a novel regulator of CD8+ T cell activity and biomarker during the evolution of Type 1 Diabetes(bioRxiv, 2024-09-19) Rao, Chaitra; Cater, Daniel T.; Roy, Saptarshi; Xu, Jerry; Olivera, Andre De G.; Evans-Molina, Carmella; Piganelli, Jon D.; Eizirik, Decio L.; Mirmira, Raghavendra G.; Sims, Emily K.; Pediatrics, School of MedicineAims/hypothesis: Surviving beta cells in type 1 diabetes respond to inflammation by upregulating programmed death-ligand 1 (PD-L1) to engage immune cell programmed death-1 (PD-1) and limit destruction by self-reactive immune cells. Extracellular vesicles (EVs) and their cargo can serve as biomarkers of beta cell health and contribute to islet intercellular communication. We hypothesized that the inflammatory milieu of type 1 diabetes increases PD-L1 in beta cell EV cargo and that EV PD-L1 may protect beta cells against immune-mediated cell death. Methods: Beta cell lines and human islets were treated with proinflammatory cytokines to model the proinflammatory type 1 diabetes microenvironment. EVs were isolated using ultracentrifugation or size exclusion chromatography and analysed via immunoblot, flow cytometry, and ELISA. EV PD-L1: PD-1 binding was assessed using a competitive binding assay and in vitro functional assays testing the ability of EV PD-L1 to inhibit NOD CD8 T cells. Plasma EV and soluble PD-L1 were assayed in plasma of individuals with islet autoantibody positivity (Ab+) or recent-onset type 1 diabetes and compared to non-diabetic controls. Results: PD-L1 protein colocalized with tetraspanin-associated proteins intracellularly and was detected on the surface of beta cell EVs. 24-h IFN-α or IFN-γ treatment induced a two-fold increase in EV PD-L1 cargo without a corresponding increase in number of EVs. IFN exposure predominantly increased PD-L1 expression on the surface of beta cell EVs and beta cell EV PD-L1 showed a dose-dependent capacity to bind PD-1. Functional experiments demonstrated specific effects of beta cell EV PD-L1 to suppress proliferation and cytotoxicity of murine CD8 T cells. Plasma EV PD-L1 levels were increased in islet Ab+ individuals, particularly in those with single Ab+, Additionally, in from individuals with either Ab+ or type 1 diabetes, but not in controls, plasma EV PD-L1 positively correlated with circulating C-peptide, suggesting that higher EV-PD-L1 could be protective for residual beta cell function. Conclusions/interpretation: IFN exposure increases PD-L1 on the beta cell EV surface. Beta cell EV PD-L1 binds PD1 and inhibits CD8 T cell proliferation and cytotoxicity. Circulating EV PD-L1 is higher in islet autoantibody positive patients compared to controls. Circulating EV PD-L1 levels correlate with residual C-peptide at different stages in type 1 diabetes progression. These findings suggest that EV PD-L1 could contribute to heterogeneity in type 1 diabetes progression and residual beta cell function and raise the possibility that EV PD-L1 could be exploited as a means to inhibit immune-mediated beta cell death.Item Beta Cell Imaging—From Pre-Clinical Validation to First in Man Testing(MDPI, 2020-10-01) Demine, Stephane; Schulte, Michael L.; Territo, Paul R.; Eizirik, Decio L.; Radiology and Imaging Sciences, School of MedicineThere are presently no reliable ways to quantify human pancreatic beta cell mass (BCM) in vivo, which prevents an accurate understanding of the progressive beta cell loss in diabetes or following islet transplantation. Furthermore, the lack of beta cell imaging hampers the evaluation of the impact of new drugs aiming to prevent beta cell loss or to restore BCM in diabetes. We presently discuss the potential value of BCM determination as a cornerstone for individualized therapies in diabetes, describe the presently available probes for human BCM evaluation, and discuss our approach for the discovery of novel beta cell biomarkers, based on the determination of specific splice variants present in human beta cells. This has already led to the identification of DPP6 and FXYD2γa as two promising targets for human BCM imaging, and is followed by a discussion of potential safety issues, the role for radiochemistry in the improvement of BCM imaging, and concludes with an overview of the different steps from pre-clinical validation to a first-in-man trial for novel tracers.Item Biomarkers of islet beta cell stress and death in type 1 diabetes(Springer Nature, 2018-11) Sims, Emily K.; Evans-Molina, Carmella; Tersey, Sarah A.; Eizirik, Decio L.; Mirmira, Raghavendra G.; Pediatrics, School of MedicineRecent work on the pathogenesis of type 1 diabetes has led to an evolving recognition of the heterogeneity of this disease, both with regards to clinical phenotype and responses to therapies to prevent or revert diabetes. This heterogeneity not only limits efforts to accurately predict clinical disease but also is reflected in differing responses to immunomodulatory therapeutics. Thus, there is a need for robust biomarkers of beta cell health, which could provide insight into pathophysiological differences in disease course, improve disease prediction, increase the understanding of therapeutic responses to immunomodulatory interventions and identify individuals most likely to benefit from these therapies. In this review, we outline current literature, limitations and future directions for promising circulating markers of beta cell stress and death in type 1 diabetes, including markers indicating abnormal prohormone processing, circulating RNAs and circulating DNAs.Item Circulating unmethylated CHTOP and INS DNA fragments provide evidence of possible islet cell death in youth with obesity and diabetes(BMC, 2020-07-31) Syed, Farooq; Tersey, Sarah A.; Turatsinze, Jean-Valery; Felton, Jamie L.; Kang, Nicole Jiyun; Nelson, Jennifer B.; Sims, Emily K.; Defrance, Mathieu; Bizet, Martin; Fuks, Francois; Cnop, Miriam; Bugliani, Marco; Marchetti, Piero; Ziegler, Anette-Gabriele; Bonifacio, Ezio; Webb-Robertson, Bobbie-Jo; Balamurugan, Appakalai N.; Evans-Molina, Carmella; Eizirik, Decio L.; Mather, Kieren J.; Arslanian, Silva; Mirmira, Raghavendra G.; Pediatrics, School of MedicineBackground Identification of islet β cell death prior to the onset of type 1 diabetes (T1D) or type 2 diabetes (T2D) might allow for interventions to protect β cells and reduce diabetes risk. Circulating unmethylated DNA fragments arising from the human INS gene have been proposed as biomarkers of β cell death, but this gene alone may not be sufficiently specific to report β cell death. Results To identify new candidate genes whose CpG sites may show greater specificity for β cells, we performed unbiased DNA methylation analysis using the Infinium HumanMethylation 450 array on 64 human islet preparations and 27 non-islet human tissues. For verification of array results, bisulfite DNA sequencing of human β cells and 11 non-β cell tissues was performed on 5 of the top 10 CpG sites that were found to be differentially methylated. We identified the CHTOP gene as a candidate whose CpGs show a greater frequency of unmethylation in human islets. A digital PCR strategy was used to determine the methylation pattern of CHTOP and INS CpG sites in primary human tissues. Although both INS and CHTOP contained unmethylated CpG sites in non-islet tissues, they occurred in a non-overlapping pattern. Based on Naïve Bayes classifier analysis, the two genes together report 100% specificity for islet damage. Digital PCR was then performed on cell-free DNA from serum from human subjects. Compared to healthy controls (N = 10), differentially methylated CHTOP and INS levels were higher in youth with new onset T1D (N = 43) and, unexpectedly, in healthy autoantibody-negative youth who have first-degree relatives with T1D (N = 23). When tested in lean (N = 32) and obese (N = 118) youth, increased levels of unmethylated INS and CHTOP were observed in obese individuals. Conclusion Our data suggest that concurrent measurement of circulating unmethylated INS and CHTOP has the potential to detect islet death in youth at risk for both T1D and T2D. Our data also support the use of multiple parameters to increase the confidence of detecting islet damage in individuals at risk for developing diabetes.Item Comprehensive Proteomics Analysis of Stressed Human Islets Identifies GDF15 as a Target for Type 1 Diabetes Intervention(Elsevier, 2020-02-04) Nakayasu, Ernesto S.; Syed, Farooq; Tersey, Sarah A.; Gritsenko, Marina A.; Mitchell, Hugh D.; Chan, Chi Yuet; Dirice, Ercument; Turatsinze, Jean-Valery; Cui, Yi; Kulkarni, Rohit N.; Eizirik, Decio L.; Qian, Wei-Jun; Webb-Robertson, Bobbie-Jo M.; Evans-Molina, Carmella; Mirmira., Raghavendra G.; Metz, Thomas O.; Pediatrics, School of MedicineType 1 diabetes (T1D) results from the progressive loss of β cells, a process propagated by pro-inflammatory cytokine signaling that disrupts the balance between pro- and anti-apoptotic proteins. To identify proteins involved in this process, we performed comprehensive proteomics of human pancreatic islets treated with interleukin-1β and interferon-γ, leading to the identification of 11,324 proteins, of which 387 were significantly regulated by treatment. We then tested the function of growth/differentiation factor 15 (GDF15), which was repressed by the treatment. We found that GDF15 translation was blocked during inflammation, and it was depleted in islets from individuals with T1D. The addition of exogenous GDF15 inhibited interleukin-1β+interferon-γ-induced apoptosis of human islets. Administration of GDF15 reduced by 53% the incidence of diabetes in NOD mice. Our approach provides a unique resource for the identification of the human islet proteins regulated by cytokines and was effective in discovering a potential target for T1D therapy.Item Distinct gene expression pathways in islets from individuals with short‐ and long‐duration type 1 diabetes(Wiley, 2018) Mastracci, Teresa L.; Turatsinze, Jean-Valery; Book, Benita K.; Restrepo, Ivan A.; Pugia, Michael J.; Weibke, Eric A.; Pescovitz, Mark D.; Eizirik, Decio L.; Mirmira, Raghavendra G.; Biochemistry and Molecular Biology, School of MedicineAims Our current understanding of the pathogenesis of type 1 diabetes (T1D) arose, in large part, from studies using the non‐obese diabetic (NOD) mouse model. In the present study, we chose a human‐focused method to investigate T1D disease mechanisms and potential targets for therapeutic intervention by directly analysing human donor pancreatic islets from individuals with T1D. Materials and Methods We obtained islets from a young individual with T1D for 3 years and from an older individual with T1D for 27 years and performed unbiased functional genomic analysis by high‐depth RNA sequencing; the T1D islets were compared with islets isolated from 3 non‐diabetic donors. Results The islets procured from these T1D donors represent a unique opportunity to identify gene expression changes in islets after significantly different disease duration. Data analysis identified several inflammatory pathways up‐regulated in short‐duration disease, which notably included many components of innate immunity. As proof of concept for translation, one of the pathways, governed by IL‐23(p19), was selected for further study in NOD mice because of ongoing human trials of biologics against this target for different indications. A mouse monoclonal antibody directed against IL‐23(p19) when administered to NOD mice resulted in a significant reduction in incidence of diabetes. Conclusion While the sample size for this study is small, our data demonstrate that the direct analysis of human islets provides a greater understanding of human disease. These data, together with the analysis of an expanded cohort to be obtained by future collaborative efforts, might result in the identification of promising novel targets for translation into effective therapeutic interventions for human T1D, with the added benefit of repurposing known biologicals for use in different indications.Item IFN-α induces a preferential long-lasting expression of MHC class I in human pancreatic beta cells(Springer, 2018-04) de Brachène, Alexandra Coomans; Dos Santos, Reinaldo Sousa; Marroqui, Laura; Colli, Maikel L.; Marselli, Lorella; Mirmira, Raghavendra G.; Marchetti, Piero; Eizirik, Decio L.; Pediatrics, School of MedicineAims/hypothesis IFN-α, a cytokine expressed in human islets from individuals affected by type 1 diabetes, plays a key role in the pathogenesis of diabetes by upregulating inflammation, endoplasmic reticulum (ER) stress and MHC class I overexpression, three hallmarks of islet histology in early type 1 diabetes. We tested whether expression of these mediators of beta cell loss is reversible upon IFN-α withdrawal or IFN-α pathway inhibition. Methods IFN-α-induced MHC class I overexpression, ER stress and inflammation were evaluated by flow cytometry, immunofluorescence and real-time PCR in human EndoC-βH1 cells or human islets exposed to IFN-α with or without the presence of Janus kinase (JAK) inhibitors. Protein expression was evaluated by western blot. Results IFN-α-induced expression of inflammatory and ER stress markers returned to baseline after 24–48 h following cytokine removal. In contrast, MHC class I overexpression at the cell surface persisted for at least 7 days. Treatment with JAK inhibitors, when added with IFN-α, prevented MHC class I overexpression, but when added 24 h after IFN-α exposure these inhibitors failed to accelerate MHC class I return to baseline. Conclusions/interpretation IFN-α mediates a long-lasting and preferential MHC class I overexpression in human beta cells, which is not affected by the subsequent addition of JAK inhibitors. These observations suggest that IFN-α-stimulated long-lasting MHC class I expression may amplify beta cell antigen presentation during the early phase of type 1 diabetes and that IFN-α inhibitors might need to be used at very early stages of the disease to be effective.Item Impact of Proinflammatory Cytokines on Alternative Splicing Patterns in Human Islets(American Diabetes Association, 2021) Wu, Wenting; Syed, Farooq; Simpson, Edward; Lee, Chih-Chun; Liu, Jing; Chang, Garrick; Dong, Chuanpeng; Seitz, Clayton; Eizirik, Decio L.; Mirmira, Raghavendra G.; Liu, Yunlong; Evans-Molina, Carmella; Medical and Molecular Genetics, School of MedicineAlternative splicing (AS) within the β-cell has been proposed as one potential pathway that may exacerbate autoimmunity and unveil novel immunogenic epitopes in type 1 diabetes (T1D). We used a computational strategy to prioritize pathogenic splicing events in human islets treated with interleukin-1β plus interferon-γ as an ex vivo model of T1D and coupled this analysis with a k-mer–based approach to predict RNA-binding proteins involved in AS. In total, 969 AS events were identified in cytokine-treated islets, with a majority (44.8%) involving a skipped exon. ExonImpact identified 129 events predicted to affect protein structure. AS occurred with high frequency in MHC class II–related mRNAs, and targeted quantitative PCR validated reduced inclusion of exon 5 in the MHC class II gene HLA-DMB. Single-molecule RNA fluorescence in situ hybridization confirmed increased HLA-DMB splicing in β-cells from human donors with established T1D and autoantibody positivity. Serine/arginine-rich splicing factor 2 was implicated in 37.2% of potentially pathogenic events, including exon 5 exclusion in HLA-DMB. Together, these data suggest that dynamic control of AS plays a role in the β-cell response to inflammatory signals during T1D evolution.Item Inhibition of the Eukaryotic Initiation Factor-2-α Kinase PERK Decreases Risk of Autoimmune Diabetes in Mice(bioRxiv, 2024-06-03) Muralidharan, Charanya; Huang, Fei; Enriquez, Jacob R.; Wang, Jiayi E.; Nelson, Jennifer B.; Nargis, Titli; May, Sarah C.; Chakraborty, Advaita; Figatner, Kayla T.; Navitskaya, Svetlana; Anderson, Cara M.; Calvo, Veronica; Surguladze, David; Mulvihill, Mark J.; Yi, Xiaoyan; Sarkar, Soumyadeep; Oakes, Scott A.; Webb-Robertson, Bobbie-Jo M.; Sims, Emily K.; Staschke, Kirk A.; Eizirik, Decio L.; Nakayasu, Ernesto S.; Stokes, Michael E.; Tersey, Sarah A.; Mirmira, Raghavendra G.; Pediatrics, School of MedicinePreventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress-responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eIF2α. In T1D, maladaptive unfolded protein response (UPR) in insulin-producing β cells renders these cells susceptible to autoimmunity. We show that inhibition of the eIF2α kinase PERK, a common component of the UPR and ISR, reverses the mRNA translation block in stressed human islets and delays the onset of diabetes, reduces islet inflammation, and preserves β cell mass in T1D-susceptible mice. Single-cell RNA sequencing of islets from PERK-inhibited mice shows reductions in the UPR and PERK signaling pathways and alterations in antigen processing and presentation pathways in β cells. Spatial proteomics of islets from these mice shows an increase in the immune checkpoint protein PD-L1 in β cells. Golgi membrane protein 1, whose levels increase following PERK inhibition in human islets and EndoC-βH1 human β cells, interacts with and stabilizes PD-L1. Collectively, our studies show that PERK activity enhances β cell immunogenicity, and inhibition of PERK may offer a strategy to prevent or delay the development of T1D.Item Inhibition of the eukaryotic initiation factor-2α kinase PERK decreases risk of autoimmune diabetes in mice(American Society for Clinical Investigation, 2024-06-18) Muralidharan, Charanya; Huang, Fei; Enriquez, Jacob R.; Wang, Jiayi E.; Nelson, Jennifer B.; Nargis, Titli; May, Sarah C.; Chakraborty, Advaita; Figatner, Kayla T.; Navitskaya, Svetlana; Anderson, Cara M.; Calvo, Veronica; Surguladze, David; Mulvihill, Mark J.; Yi, Xiaoyan; Sarkar, Soumyadeep; Oakes, Scott A.; Webb-Robertson, Bobbie-Jo M.; Sims, Emily K.; Staschke, Kirk A.; Eizirik, Decio L.; Nakayasu, Ernesto S.; Stokes, Michael E.; Tersey, Sarah A.; Mirmira, Raghavendra G.; Pediatrics, School of MedicinePreventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress–responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eukaryotic translation initiation factor-2α (eIF2α). In T1D, maladaptive unfolded protein response (UPR) in insulin-producing β cells renders these cells susceptible to autoimmunity. We found that inhibition of the eIF2α kinase PKR-like ER kinase (PERK), a common component of the UPR and ISR, reversed the mRNA translation block in stressed human islets and delayed the onset of diabetes, reduced islet inflammation, and preserved β cell mass in T1D-susceptible mice. Single-cell RNA-Seq of islets from PERK-inhibited mice showed reductions in the UPR and PERK signaling pathways and alterations in antigen-processing and presentation pathways in β cells. Spatial proteomics of islets from these mice showed an increase in the immune checkpoint protein programmed death-ligand 1 (PD-L1) in β cells. Golgi membrane protein 1, whose levels increased following PERK inhibition in human islets and EndoC-βH1 human β cells, interacted with and stabilized PD-L1. Collectively, our studies show that PERK activity enhances β cell immunogenicity and that inhibition of PERK may offer a strategy for preventing or delaying the development of T1D.