Aberrant expression of transglutaminase 2 in pancreas and thymus of NOD mice underscores the importance of deamidation in neoantigen generation

Post-translational modifications can lead to a break in immune tolerance in autoimmune diseases such as type 1 diabetes (T1D). Deamidation, the conversion of glutamine to glutamic acid by transglutaminase (TGM) enzymes, is a post-translational modification of interest, with deamidated peptides being reported as autoantigens in T1D. However, little is known about how Tgm2, the most ubiquitously expressed Tgm isoform, is regulated and how tolerance against deamidated peptides is lost. Here, we report on the aberrant expression and regulation of Tgm2 in the pancreas and thymus of NOD mice. We demonstrate that Tgm2 expression is induced by the inflammatory cytokines IL1β and IFNγ in a synergistic manner and that murine pancreatic islets of NOD mice have higher Tgm2 levels, while Tgm2 levels in medullary thymic epithelial cells are reduced. We thus provide the first direct evidence to our knowledge that central tolerance establishment against deamidated peptides might be impaired due to lower Tgm2 expression in NOD medullary thymic epithelial cells, which together with the aberrantly high levels of deamidated peptides in NOD β-cells underscores the role of deamidation in amplifying T-cell reactivity

Peptidylarginine Deiminase Inhibition Prevents Diabetes Development in NOD Mice

Protein citrullination plays a role in several autoimmune diseases. Its involvement in murine and human type 1 diabetes has recently been recognized through the discovery of antibodies and T-cell reactivity against citrullinated peptides. In the current study, we demonstrate that systemic inhibition of peptidylarginine deiminases (PADs), the enzymes mediating citrullination, through BB-Cl-amidine treatment, prevents diabetes development in NOD mice. This prevention was associated with reduced levels of citrullination in the pancreas, decreased circulating autoantibody titers against citrullinated GRP78 and reduced spontaneous NETosis of bone marrow-derived neutrophils. Moreover, BB-Cl-amidine treatment induced a shift from Th1 to Th2 cytokines in the serum and an increase in the frequency of regulatory T cells in the blood and spleen. In the pancreas, BB-Cl-amidine treatment preserved insulin production and was associated with a less destructive immune infiltrate, characterized by reduced frequencies of effector memory CD4+ T cells and a modest reduction in the frequency of IFNγ-producing CD4+ and CD8+ T cells. Our results point to a role of citrullination in the pathogenesis of autoimmune diabetes, with PAD inhibition leading to disease prevention through modulation of immune pathways. These findings provide insight in the potential of PAD inhibition for treating autoimmune diseases like type 1 diabetes

Ga-68-NODAGA-Exendin-4 PET/CT Improves the Detection of Focal Congenital Hyperinsulinism

Surgery with curative intent can be offered to congenital hyperinsulinism (CHI) patients, provided that the lesion is focal. Radiolabeled exendin-4 specifically binds the glucagonlike peptide 1 receptor on pancreatic beta-cells. In this study, we compared the performance of F-18-DOPA PET/CT, the current standard imaging method for CHI, and PET/CT with the new tracer Ga-68-NODAGA-exendin-4 in the preoperative detection of focal CHI. Methods: Nineteen CHI patients underwent both F-18-DOPA PET/CT and Ga-68-NODAGA-exendin-4 PET/CT before surgery. The images were evaluated in 3 settings: a standard clinical reading, a masked expert reading, and a joint reading. The target (lesion)-to-nontarget (normal pancreas) ratio was determined using SUVmax. Image quality was rated by pediatric surgeons in a questionnaire. Results: Fourteen of 19 patients having focal lesions underwent surgery. On the basis of clinical readings, the sensitivity of Ga-68-NODAGA-exendin-4 PET/CT (100%; 95% CI, 77%-100%) was higher than that of F-18-DOPA PET/CT (71%; 95% CI, 42%-92%). Interobserver agreement between readings was higher for Ga-68-NODAGA-exendin-4 than for F-18-DOPA PET/CT (Fleiss kappa = 0.91 vs. 0.56). Ga-68-NODAGA-exendin-4 PET/CT provided significantly (P = 0.021) higher target-to-nontarget ratios (2.02 +/- 0.65) than did F-18-DOPA PET/CT (1.40 +/- 0.40). On a 5-point scale, pediatric surgeons rated Ga-68-NODAGA-exendin-4 PET/CT as superior to F-18-DOPA PET/CT. Conclusion: For the detection of focal CHI, Ga-68-NODAGA-exendin-4 PET/CT has higher clinical sensitivity and better interobserver correlation than F-18-DOPA PET/CT. Better contrast and image quality make Ga-68-NODAGA-exendin-4 PET/CT superior to F-18-DOPA PET/CT in surgeons' intraoperative quest for lesion localization.Peer reviewe

Plan-β: a bioengineering approach against type 1 diabetes

The restoration of euglycemia after intraportal islet transplantation remains short-lived, requires islets from multiple donors, and necessitates life-long immunosuppressive therapy. In this thesis we propose different bioengineering strategies to improve islet transplantation outcome. \ud In chapter 2 and 3 we describe the development and characterization of a microwell scaffold platform for extrahepatic islet transplantation. In chapter 2 the concept of the microwell design is introduced. We have shown that human islets cultured in this platform for seven days exhibit an insulin secretion profile and insulin content comparable to those of free-floating control islets. Chapter 3 explores three different fabrication methods to introduce pores in the microwell scaffold platform: particulate leaching, solvent casting on pillared wafers, and laser drilling. Methods were evaluated for pore size and geometry, reproducibility and flexibility, and the form and stability of the final construct. Transplantation studies in the epididymal fat elucidate the potential of this porous scaffold platform to restore blood glucose levels in a diabetic mouse model. However, we do observe a significant decrease in beta-cell density and a changed vascularization pattern in scaffold-implanted islets compared to native pancreatic islets, which, we hypothesize, are indications for delayed revascularization. Therefore, we explore in chapter 4 the potential of composite islets with different proangiogenic cell types to improve islet revascularization after transplantation. Here, we demonstrate that composite islets with hMSCs and HUVECs exhibit a higher in vivo angiogenic potential compared with uncoated islets or islets combined with hMSCs or pre-conditioned hMSCs in EGM-2 medium. In chapter 5, we propose a method to monitor non-invasively beta-cell survival after islet transplantation. For this, we describe the use of radiolabelled exendin-3, which specifically targets pancreatic β-cells. We demonstrate that the uptake of 111In-exendin-3 in scaffold-implanted islets could be clearly visualized on ex vivo SPECT scans, with histology and autoradiography studies showing vascularized islets with specific tracer uptake in β-cell regions. Provided that tracer uptake can also be determined in vivo, this presents a viable method to monitor the influence of native and engineered transplantation sites on long-term β-cell survival

Ectopic lipid deposition in muscle and liver, quantified by proton magnetic resonance spectroscopy

Advances in the development of noninvasive imaging techniques have spurred investigations into ectopic lipid deposition in the liver and muscle and its implications in the development of metabolic diseases such as type 2 diabetes. Computed tomography and ultrasound have been applied in the past, though magnetic resonance-based methods are currently considered the gold standard as they allow more accurate quantitative detection of ectopic lipid stores. This review focuses on methodological considerations of magnetic resonance-based methods to image hepatic and muscle fat fractions, and it emphasizes anatomical and morphological aspects and how these may influence data acquisition, analysis, and interpretation

High-Throughput Analysis of Neutrophil Extracellular Trap Levels in Subtypes of People with Type 1 Diabetes

Neutrophils might play an important role in the pathogenesis of autoimmune diseases, including type 1 diabetes (T1D), by contributing to immune dysregulation via a highly inflammatory program called neutrophil extracellular trap (NET) formation or NETosis, involving the extrusion of chromatin entangled with anti-microbial proteins. However, numerous studies reported contradictory data on NET formation in T1D. This might in part be due to the inherent heterogeneity of the disease and the influence of the disease developmental stage on neutrophil behavior. Moreover, there is a lack of a standardized method to measure NETosis in an unbiased and robust manner. In this study, we employed the Incucyte® ZOOM live-cell imaging platform to study NETosis levels in various subtypes of adult and pediatric T1D donors compared to healthy controls (HC) at baseline and in response to phorbol–myristate acetate (PMA) and ionomycin. Firstly, we determined that the technique allows for an operator-independent and automated quantification of NET formation across multiple time points, which showed that PMA and ionomycin induced NETosis with distinct kinetic characteristics, confirmed by high-resolution microscopy. NETosis levels also showed a clear dose-response curve to increasing concentrations of both stimuli. Overall, using Incucyte® ZOOM, no aberrant NET formation was observed over time in the different subtypes of T1D populations, irrespective of age, compared to HC. These data were corroborated by the levels of peripheral NET markers in all study participants. The current study showed that live-cell imaging allows for a robust and unbiased analysis and quantification of NET formation in real-time. Peripheral neutrophil measures should be complemented with dynamic quantification of NETing neutrophils to make robust conclusions on NET formation in health and disease

Aberrant expression of transglutaminase 2 in pancreas and thymus of NOD mice underscores the importance of deamidation in neoantigen generation

International audiencePost-translational modifications can lead to a break in immune tolerance in autoimmune diseases such as type 1 diabetes (T1D). Deamidation, the conversion of glutamine to glutamic acid by transglutaminase (TGM) enzymes, is a post-translational modification of interest, with deamidated peptides being reported as autoantigens in T1D. However, little is known about how Tgm2, the most ubiquitously expressed Tgm isoform, is regulated and how tolerance against deamidated peptides is lost. Here, we report on the aberrant expression and regulation of Tgm2 in the pancreas and thymus of NOD mice. We demonstrate that Tgm2 expression is induced by the inflammatory cytokines IL1b and IFNg in a synergistic manner and that murine pancreatic islets of NOD mice have higher Tgm2 levels, while Tgm2 levels in medullary thymic epithelial cells are reduced. We thus provide the first direct evidence to our knowledge that central tolerance establishment against deamidated peptides might be impaired due to lower Tgm2 expression in NOD medullary thymic epithelial cells, which together with the aberrantly high levels of deamidated peptides in NOD b-cells underscores the role of deamidation in amplifying T-cell reactivity

Aberrant expression of transglutaminase 2 in pancreas and thymus of NOD mice underscores the importance of deamidation in neoantigen generation

Post-translational modifications can lead to a break in immune tolerance in autoimmune diseases such as type 1 diabetes (T1D). Deamidation, the conversion of glutamine to glutamic acid by transglutaminase (TGM) enzymes, is a post-translational modification of interest, with deamidated peptides being reported as autoantigens in T1D. However, little is known about how Tgm2 , the most ubiquitously expressed Tgm isoform, is regulated and how tolerance against deamidated peptides is lost. Here, we report on the aberrant expression and regulation of Tgm2 in the pancreas and thymus of NOD mice. We demonstrate that Tgm2 expression is induced by the inflammatory cytokines IL1β and IFNγ in a synergistic manner and that murine pancreatic islets of NOD mice have higher Tgm2 levels, while Tgm2 levels in medullary thymic epithelial cells are reduced. We thus provide the first direct evidence to our knowledge that central tolerance establishment against deamidated peptides might be impaired due to lower Tgm2 expression in NOD medullary thymic epithelial cells, which together with the aberrantly high levels of deamidated peptides in NOD β-cells underscores the role of deamidation in amplifying T-cell reactivity

NET Proteome in Established Type 1 Diabetes Is Enriched in Metabolic Proteins

Background and aims: Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by a T-cell-mediated destruction of the pancreatic insulin-producing beta cells. A growing body of evidence suggests that abnormalities in neutrophils and neutrophil extracellular trap (NET) formation (NETosis) are associated with T1D pathophysiology. However, little information is available on whether these changes are primary neutrophil defects or related to the environmental signals encountered during active disease. Methods: In the present work, the NET proteome (NETome) of phorbol 12-myristate 13-acetate (PMA)- and ionomycin-stimulated neutrophils from people with established T1D compared to healthy controls (HC) was studied by proteomic analysis. Results: Levels of NETosis, in addition to plasma levels of pro-inflammatory cytokines and NET markers, were comparable between T1D and HC subjects. However, the T1D NETome was distinct from that of HC in response to both stimuli. Quantitative analysis revealed that the T1D NETome was enriched in proteins belonging to metabolic pathways (i.e., phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, and UTP-glucose-1-phosphate uridylyltransferase). Complementary metabolic profiling revealed that the rate of extracellular acidification, an approximate measure for glycolysis, and mitochondrial respiration were similar between T1D and HC neutrophils in response to both stimuli. Conclusion: The NETome of people with established T1D was enriched in metabolic proteins without an apparent alteration in the bio-energetic profile or dysregulated NETosis. This may reflect an adaptation mechanism employed by activated T1D neutrophils to avoid impaired glycolysis and consequently excessive or suboptimal NETosis, pivotal in innate immune defence and the resolution of inflammation