Multi-omics analysis reveals drivers of loss of ß-cell function after newly diagnosed autoimmune type 1 diabetes: An INNODIA multicenter study.

AIMS: Heterogeneity in the rate of ß-cell loss in newly diagnosed type 1 diabetes patients is poorly understood and creates a barrier to designing and interpreting disease-modifying clinical trials. Integrative analyses of baseline multi-omics data obtained after the diagnosis of type 1 diabetes may provide mechanistic insight into the diverse rates of disease progression after type 1 diabetes diagnosis. METHODS: We collected samples in a pan-European consortium that enabled the concerted analysis of five different omics modalities in data from 97 newly diagnosed patients. In this study, we used Multi-Omics Factor Analysis to identify molecular signatures correlating with post-diagnosis decline in ß-cell mass measured as fasting C-peptide. RESULTS: Two molecular signatures were significantly correlated with fasting C-peptide levels. One signature showed a correlation to neutrophil degranulation, cytokine signalling, lymphoid and non-lymphoid cell interactions and G-protein coupled receptor signalling events that were inversely associated with a rapid decline in ß-cell function. The second signature was related to translation and viral infection was inversely associated with change in ß-cell function. In addition, the immunomics data revealed a Natural Killer cell signature associated with rapid ß-cell decline. CONCLUSIONS: Features that differ between individuals with slow and rapid decline in ß-cell mass could be valuable in staging and prediction of the rate of disease progression and thus enable smarter (shorter and smaller) trial designs for disease modifying therapies as well as offering biomarkers of therapeutic effect.

A set of circulating microRNAs belonging to the 14q32 chromosome locus identifies two subgroups of individuals with recent-onset type 1 diabetes.

Circulating microRNAs (miRNAs) are linked to the onset and progression of type 1 diabetes mellitus (T1DM), thus representing potential disease biomarkers. In this study, we employed a multiplatform sequencing approach to analyze circulating miRNAs in an extended cohort of prospectively evaluated recent-onset T1DM individuals from the INNODIA consortium. Our findings reveal that a set of miRNAs located within T1DM susceptibility chromosomal locus 14q32 distinguishes two subgroups of individuals. To validate our results, we conducted additional analyses on a second cohort of T1DM individuals, confirming the identification of these subgroups, which we have named cluster A and cluster B. Remarkably, cluster B T1DM individuals, who exhibit increased expression of a set of 14q32 miRNAs, show better glycemic control and display a different blood immunomics profile. Our findings suggest that this set of circulating miRNAs can identify two different T1DM subgroups with distinct blood immunomics at baseline and clinical outcomes during follow-up.

Gut microbiome shifts in people with type 1 diabetes are associated with glycaemic control: an INNODIA study.

AIMS/HYPOTHESIS: The gut microbiome is implicated in the disease process leading to clinical type 1 diabetes, but less is known about potential changes in the gut microbiome after the diagnosis of type 1 diabetes and implications in glucose homeostasis. We aimed to analyse potential associations between the gut microbiome composition and clinical and laboratory data during a 2 year follow-up of people with newly diagnosed type 1 diabetes, recruited to the Innovative approaches to understanding and arresting type 1 diabetes (INNODIA) study. In addition, we analysed the microbiome composition in initially unaffected family members, who progressed to clinical type 1 diabetes during or after their follow-up for 4 years. METHODS: We characterised the gut microbiome composition of 98 individuals with newly diagnosed type 1 diabetes (ND cohort) and 194 autoantibody-positive unaffected family members (UFM cohort), representing a subgroup of the INNODIA Natural History Study, using metagenomic sequencing. Participants from the ND cohort attended study visits within 6 weeks from the diagnosis and 3, 6, 12 and 24 months later for stool sample collection and laboratory tests (HbA1c, C-peptide, diabetes-associated autoantibodies). Participants from the UFM cohort were assessed at baseline and 6, 12, 18, 24 and 36 months later. RESULTS: We observed a longitudinal increase in 21 bacterial species in the ND cohort but not in the UFM cohort. The relative abundance of Faecalibacterium prausnitzii was inversely associated with the HbA1c levels at diagnosis (p=0.0019). The rate of the subsequent disease progression in the ND cohort, as assessed by change in HbA1c, C-peptide levels and insulin dose, was associated with the abundance of several bacterial species. Individuals with rapid decrease in C-peptide levels in the ND cohort had the lowest gut microbiome diversity. Nineteen individuals who were diagnosed with type 1 diabetes in the UFM cohort had increased abundance of Sutterella sp. KLE1602 compared with the undiagnosed UFM individuals (p=1.2 × 10-4). CONCLUSIONS/INTERPRETATION: Our data revealed associations between the gut microbiome composition and the disease progression in individuals with recent-onset type 1 diabetes. Future mechanistic studies as well as animal studies and human trials are needed to further validate the significance and causality of these associations.

The INNODIA Type 1 Diabetes Natural History Study: a European cohort of newly diagnosed children, adolescents and adults.

AIMS/HYPOTHESIS: Type 1 diabetes is an heterogenous condition. Characterising factors explaining differences in an individual's clinical course and treatment response will have important clinical and research implications. Our aim was to explore type 1 diabetes heterogeneity, as assessed by clinical characteristics, autoantibodies, beta cell function and glycaemic outcomes, during the first 12 months from diagnosis, and how it relates to age at diagnosis. METHODS: Data were collected from the large INNODIA cohort of individuals (aged 1.0-45.0 years) newly diagnosed with type 1 diabetes, followed 3 monthly, to assess clinical characteristics, C-peptide, HbA1c and diabetes-associated antibodies, and their changes, during the first 12 months from diagnosis, across three age groups: <10 years; 10-17 years; and ≥18 years. RESULTS: The study population included 649 individuals (57.3% male; age 12.1±8.3 years), 96.9% of whom were positive for one or more diabetes-related antibodies. Baseline (IQR) fasting C-peptide was 242.0 (139.0-382.0) pmol/l (AUC 749.3 [466.2-1106.1] pmol/l × min), with levels increasing with age (p<0.001). Over time, C-peptide remained lower in participants aged <10 years but it declined in all age groups. In parallel, glucose levels progressively increased. Lower baseline fasting C-peptide, BMI SD score and presence of diabetic ketoacidosis at diagnosis were associated with lower stimulated C-peptide over time. HbA1c decreased during the first 3 months (p<0.001), whereas insulin requirement increased from 3 months post diagnosis (p<0.001). CONCLUSIONS/INTERPRETATION: In this large cohort with newly diagnosed type 1 diabetes, we identified age-related differences in clinical and biochemical variables. Of note, C-peptide was lower in younger children but there were no main age differences in its rate of decline.

CD4+ T Cells From Individuals With Type 1 Diabetes Respond to a Novel Class of Deamidated Peptides Formed in Pancreatic Islets.

The ß-cell plays a crucial role in the pathogenesis of type 1 diabetes, in part through the posttranslational modification of self-proteins by biochemical processes such as deamidation. These neoantigens are potential triggers for breaking immune tolerance. We report the detection by LC-MS/MS of 16 novel Gln and 27 novel Asn deamidations in 14 disease-related proteins within inflammatory cytokine-stressed human islets of Langerhans. T-cell clones responsive against one Gln- and three Asn-deamidated peptides could be isolated from peripheral blood of individuals with type 1 diabetes. Ex vivo HLA class II tetramer staining detected higher T-cell frequencies in individuals with the disease compared with control individuals. Furthermore, there was a positive correlation between the frequencies of T cells specific for deamidated peptides, insulin antibody levels at diagnosis, and duration of disease. These results highlight that stressed human islets are prone to enzymatic and biochemical deamidation and suggest that both Gln- and Asn-deamidated peptides can promote the activation and expansion of autoreactive CD4+ T cells. These findings add to the growing evidence that posttranslational modifications undermine tolerance and may open the road for the development of new diagnostic and therapeutic applications for individuals living with type 1 diabetes.

An Insulin-Chromogranin A Hybrid Peptide Activates DR11-Restricted T Cells in Human Type 1 Diabetes.

Hybrid insulin peptides (HIPs) formed through covalent cross-linking of proinsulin fragments to secretory granule peptides are detectable within murine and human islets. The 2.5HIP (C-peptide-chromogranin A [CgA] HIP), recognized by the diabetogenic BDC-2.5 clone, is a major autoantigen in the nonobese diabetic mouse. However, the relevance of this epitope in human disease is currently unclear. A recent study probed T-cell reactivity toward HIPs in patients with type 1 diabetes, documenting responses in one-third of the patients and isolating several HIP-reactive T-cell clones. In this study, we isolated a novel T-cell clone and showed that it responds vigorously to the human equivalent of the 2.5HIP (designated HIP9). Although the responding patient carried the risk-associated DRB1*04:01/DQ8 haplotype, the response was restricted by DRB1*11:03 (DR11). HLA class II tetramer staining revealed higher frequencies of HIP9-reactive T cells in individuals with diabetes than in control participants. Furthermore, in DR11+ participants carrying the DRB4 allele, HIP9-reactive T-cell frequencies were higher than observed frequencies for the immunodominant proinsulin 9-28 epitope. Finally, there was a negative correlation between HIP9-reactive T-cell frequency and age at diagnosis. These results provide direct evidence that this C-peptide-CgA HIP is relevant in human type 1 diabetes and suggest a mechanism by which nonrisk HLA haplotypes may contribute to the development of ß-cell autoimmunity.

Targeted serum proteomics of longitudinal samples from newly diagnosed youth with type 1 diabetes distinguishes markers of disease and C-peptide trajectory.

AIMS/HYPOTHESIS: There is a growing need for markers that could help indicate the decline in beta cell function and recognise the need and efficacy of intervention in type 1 diabetes. Measurements of suitably selected serum markers could potentially provide a non-invasive and easily applicable solution to this challenge. Accordingly, we evaluated a broad panel of proteins previously associated with type 1 diabetes in serum from newly diagnosed individuals during the first year from diagnosis. To uncover associations with beta cell function, comparisons were made between these targeted proteomics measurements and changes in fasting C-peptide levels. To further distinguish proteins linked with the disease status, comparisons were made with measurements of the protein targets in age- and sex-matched autoantibody-negative unaffected family members (UFMs). METHODS: Selected reaction monitoring (SRM) mass spectrometry analyses of serum, targeting 85 type 1 diabetes-associated proteins, were made. Sera from individuals diagnosed under 18 years (n=86) were drawn within 6 weeks of diagnosis and at 3, 6 and 12 months afterwards (288 samples in total). The SRM data were compared with fasting C-peptide/glucose data, which was interpreted as a measure of beta cell function. The protein data were further compared with cross-sectional SRM measurements from UFMs (n=194). RESULTS: Eleven proteins had statistically significant associations with fasting C-peptide/glucose. Of these, apolipoprotein L1 and glutathione peroxidase 3 (GPX3) displayed the strongest positive and inverse associations, respectively. Changes in GPX3 levels during the first year after diagnosis indicated future fasting C-peptide/glucose levels. In addition, differences in the levels of 13 proteins were observed between the individuals with type 1 diabetes and the matched UFMs. These included GPX3, transthyretin, prothrombin, apolipoprotein C1 and members of the IGF family. CONCLUSIONS/INTERPRETATION: The association of several targeted proteins with fasting C-peptide/glucose levels in the first year after diagnosis suggests their connection with the underlying changes accompanying alterations in beta cell function in type 1 diabetes. Moreover, the direction of change in GPX3 during the first year was indicative of subsequent fasting C-peptide/glucose levels, and supports further investigation of this and other serum protein measurements in future studies of beta cell function in type 1 diabetes.

Technical Validation and Utility of an HLA Class II Tetramer Assay for Type 1 Diabetes: A Multicenter Study.

CONTEXT: Validated assays to measure autoantigen-specific T-cell frequency and phenotypes are needed for assessing the risk of developing diabetes, monitoring disease progression, evaluating responses to treatment, and personalizing antigen-based therapies. OBJECTIVE: Toward this end, we performed a technical validation of a tetramer assay for HLA-DRA-DRB1*04:01, a class II allele that is strongly associated with susceptibility to type 1 diabetes (T1D). METHODS: HLA-DRA-DRB1*04:01-restricted T cells specific for immunodominant epitopes from islet cell antigens GAD65, IGRP, preproinsulin, and ZnT8, and a reference influenza epitope, were enumerated and phenotyped in a single staining tube with a tetramer assay. Single and multicenter testing was performed, using a clone-spiked specimen and replicate samples from T1D patients, with a target coefficient of variation (CV) less than 30%. The same assay was applied to an exploratory cross-sectional sample set with 24 T1D patients to evaluate the utility of the assay. RESULTS: Influenza-specific T-cell measurements had mean CVs of 6% for the clone-spiked specimen and 11% for T1D samples in single-center testing, and 20% and 31%, respectively, for multicenter testing. Islet-specific T-cell measurements in these same samples had mean CVs of 14% and 23% for single-center and 23% and 41% for multicenter testing. The cross-sectional study identified relationships between T-cell frequencies and phenotype and disease duration, sex, and autoantibodies. A large fraction of the islet-specific T cells exhibited a naive phenotype. CONCLUSION: Our results demonstrate that the assay is reproducible and useful to characterize islet-specific T cells and identify correlations between T-cell measures and clinical traits.

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.

Gene expression signature predicts rate of type 1 diabetes progression.

BACKGROUND: Type 1 diabetes is a complex heterogenous autoimmune disease without therapeutic interventions available to prevent or reverse the disease. This study aimed to identify transcriptional changes associated with the disease progression in patients with recent-onset type 1 diabetes. METHODS: Whole-blood samples were collected as part of the INNODIA study at baseline and 12 months after diagnosis of type 1 diabetes. We used linear mixed-effects modelling on RNA-seq data to identify genes associated with age, sex, or disease progression. Cell-type proportions were estimated from the RNA-seq data using computational deconvolution. Associations to clinical variables were estimated using Pearson's or point-biserial correlation for continuous and dichotomous variables, respectively, using only complete pairs of observations. FINDINGS: We found that genes and pathways related to innate immunity were downregulated during the first year after diagnosis. Significant associations of the gene expression changes were found with ZnT8A autoantibody positivity. Rate of change in the expression of 16 genes between baseline and 12 months was found to predict the decline in C-peptide at 24 months. Interestingly and consistent with earlier reports, increased B cell levels and decreased neutrophil levels were associated with the rapid progression. INTERPRETATION: There is considerable individual variation in the rate of progression from appearance of type 1 diabetes-specific autoantibodies to clinical disease. Patient stratification and prediction of disease progression can help in developing more personalised therapeutic strategies for different disease endotypes. FUNDING: A full list of funding bodies can be found under Acknowledgments.

2D-DIGE Analysis of Liver Disease in Mice.

Hepatocellular carcinoma (HCC) is the major type of primary liver cancer. In this chapter, we describe our routine two-dimensional difference gel electrophoresis (2D-DIGE) workflow for analysis of mouse liver tissue in physiological conditions, as well as of mouse HCC. 2D-DIGE still constitutes a valuable comparative proteomics technique, not only providing information on global protein expression in a sample but also on potential posttranslational protein modifications, occurrence of protein degradation fragments, and the existence of protein isoforms. Thus, 2D-DIGE analysis provides highly complementary data to non-gel-based shotgun mass spectrometry (MS) methods (e.g., liquid chromatography (LC)-MS/MS)-allowing, for example, identification of novel protein biomarkers for HCC or increasing insights into the molecular mechanisms underlying hepatocarcinogenesis.

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 IL1ß 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 ß-cells underscores the role of deamidation in amplifying T-cell reactivity.

Citrullination of glucokinase is linked to autoimmune diabetes.

Inflammation, including reactive oxygen species and inflammatory cytokines in tissues amplify various post-translational modifications of self-proteins. A number of post-translational modifications have been identified as autoimmune biomarkers in the initiation and progression of Type 1 diabetes. Here we show the citrullination of pancreatic glucokinase as a result of inflammation, triggering autoimmunity and affecting glucokinase biological functions. Glucokinase is expressed in hepatocytes to regulate glycogen synthesis, and in pancreatic beta cells as a glucose sensor to initiate glycolysis and insulin signaling. We identify autoantibodies and autoreactive CD4+ T cells to glucokinase epitopes in the circulation of Type 1 diabetes patients and NOD mice. Finally, citrullination alters glucokinase biologic activity and suppresses glucose-stimulated insulin secretion. Our study define glucokinase as a Type 1 diabetes biomarker, providing new insights of how inflammation drives post-translational modifications to create both neoautoantigens and affect beta cell metabolism.

Study protocol: Minimum effective low dose: anti-human thymocyte globulin (MELD-ATG): phase II, dose ranging, efficacy study of antithymocyte globulin (ATG) within 6 weeks of diagnosis of type 1 diabetes.

INTRODUCTION: Type 1 diabetes (T1D) is a chronic autoimmune disease, characterised by progressive destruction of the insulin-producing ß cells of the pancreas. One immunosuppressive agent that has recently shown promise in the treatment of new-onset T1D subjects aged 12-45 years is antithymocyte globulin (ATG), Thymoglobuline, encouraging further exploration in lower age groups. METHODS AND ANALYSIS: Minimal effective low dose (MELD)-ATG is a phase 2, multicentre, randomised, double-blind, placebo-controlled, multiarm parallel-group trial in participants 5-25 years diagnosed with T1D within 3-9 weeks of planned treatment day 1. A total of 114 participants will be recruited sequentially into seven different cohorts with the first cohort of 30 participants being randomised to placebo, 2.5 mg/kg, 1.5 mg/kg, 0.5 mg/kg and 0.1 mg/kg ATG total dose in a 1:1:1:1:1 allocation ratio. The next six cohorts of 12-15 participants will be randomised to placebo, 2.5 mg/kg, and one or two selected middle ATG total doses in a 1:1:1:1 or 1:1:1 allocation ratio, as dependent on the number of middle doses, given intravenously over two consecutive days. The primary objective will be to determine the changes in stimulated C-peptide response over the first 2 hours of a mixed meal tolerance test at 12 months for 2.5 mg/kg ATG arm vs the placebo. Conditional on finding a significant difference at 2.5 mg/kg, a minimally effective dose will be sought. Secondary objectives include the determination of the effects of a particular ATG treatment dose on (1) stimulated C-peptide, (2) glycated haemoglobin, (3) daily insulin dose, (4) time in range by intermittent continuous glucose monitoring measures, (5) fasting and stimulated dry blood spot (DBS) C-peptide measurements. ETHICS AND DISSEMINATION: MELD-ATG received first regulatory and ethical approvals in Belgium in September 2020 and from the German and UK regulators as of February 2021. The publication policy is set in the INNODIA (An innovative approach towards understanding and arresting Type 1 diabetes consortium) grant agreement (www.innodia.eu). TRIAL REGISTRATION NUMBER: NCT03936634; Pre-results.

CD8+ T Cells Variably Recognize Native Versus Citrullinated GRP78 Epitopes in Type 1 Diabetes.

In type 1 diabetes, autoimmune ß-cell destruction may be favored by neoantigens harboring posttranslational modifications (PTMs) such as citrullination. We studied the recognition of native and citrullinated glucose-regulated protein (GRP)78 peptides by CD8+ T cells. Citrullination modulated T-cell recognition and, to a lesser extent, HLA-A2 binding. GRP78-reactive CD8+ T cells circulated at similar frequencies in healthy donors and donors with type 1 diabetes and preferentially recognized either native or citrullinated versions, without cross-reactivity. Rather, the preference for native GRP78 epitopes was associated with CD8+ T cells cross-reactive with bacterial mimotopes. In the pancreas, a dominant GRP78 peptide was instead preferentially recognized when citrullinated. To further clarify these recognition patterns, we considered the possibility of citrullination in the thymus. Citrullinating peptidylarginine deiminase (Padi) enzymes were expressed in murine and human medullary epithelial cells (mTECs), with citrullinated proteins detected in murine mTECs. However, Padi2 and Padi4 expression was diminished in mature mTECs from NOD mice versus C57BL/6 mice. We conclude that, on one hand, the CD8+ T cell preference for native GRP78 peptides may be shaped by cross-reactivity with bacterial mimotopes. On the other hand, PTMs may not invariably favor loss of tolerance because thymic citrullination, although impaired in NOD mice, may drive deletion of citrulline-reactive T cells.

Citrullination and PAD Enzyme Biology in Type 1 Diabetes - Regulators of Inflammation, Autoimmunity, and Pathology.

The generation of post-translational modifications (PTMs) in human proteins is a physiological process leading to structural and immunologic variety in proteins, with potentially altered biological functions. PTMs often arise through normal responses to cellular stress, including general oxidative changes in the tissue microenvironment and intracellular stress to the endoplasmic reticulum or immune-mediated inflammatory stresses. Many studies have now illustrated the presence of 'neoepitopes' consisting of PTM self-proteins that induce robust autoimmune responses. These pathways of inflammatory neoepitope generation are commonly observed in many autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and type 1 diabetes (T1D), among others. This review will focus on one specific PTM to self-proteins known as citrullination. Citrullination is mediated by calcium-dependent peptidylarginine deiminase (PAD) enzymes, which catalyze deimination, the conversion of arginine into the non-classical amino acid citrulline. PADs and citrullinated peptides have been associated with different autoimmune diseases, notably with a prominent role in the diagnosis and pathology of rheumatoid arthritis. More recently, an important role for PADs and citrullinated self-proteins has emerged in T1D. In this review we will provide a comprehensive overview on the pathogenic role for PADs and citrullination in inflammation and autoimmunity, with specific focus on evidence for their role in T1D. The general role of PADs in epigenetic and transcriptional processes, as well as their crucial role in histone citrullination, neutrophil biology and neutrophil extracellular trap (NET) formation will be discussed. The latter is important in view of increasing evidence for a role of neutrophils and NETosis in the pathogenesis of T1D. Further, we will discuss the underlying processes leading to citrullination, the genetic susceptibility factors for increased recognition of citrullinated epitopes by T1D HLA-susceptibility types and provide an overview of reported autoreactive responses against citrullinated epitopes, both of T cells and autoantibodies in T1D patients. Finally, we will discuss recent observations obtained in NOD mice, pointing to prevention of diabetes development through PAD inhibition, and the potential role of PAD inhibitors as novel therapeutic strategy in autoimmunity and in T1D in particular.

Neoepitopes in Type 1 Diabetes: Etiological Insights, Biomarkers and Therapeutic Targets.

The mechanisms underlying type 1 diabetes (T1D) pathogenesis remain largely unknown. While autoantibodies to pancreatic beta-cell antigens are often the first biological response and thereby a useful biomarker for identifying individuals in early stages of T1D, their role in T1D pathogenesis is not well understood. Recognition of these antigenic targets by autoreactive T-cells plays a pathological role in T1D development. Recently, several beta-cell neoantigens have been described, indicating that both neoantigens and known T1D antigens escape central or peripheral tolerance. Several questions regarding the mechanisms by which tolerance is broken in T1D remain unanswered. Further delineating the timing and nature of antigenic responses could allow their use as biomarkers to improve staging, as targets for therapeutic intervention, and lead to a better understanding of the mechanisms leading to loss of tolerance. Multiple factors that contribute to cellular stress may result in the generation of beta-cell derived neoepitopes and contribute to autoimmunity. Understanding the cellular mechanisms that induce beta-cells to produce neoantigens has direct implications on development of therapies to intercept T1D disease progression. In this perspective, we will discuss evidence for the role of neoantigens in the pathogenesis of T1D, including antigenic responses and cellular mechanisms. We will additionally discuss the pathways leading to neoepitope formation and the cross talk between the immune system and the beta-cells in this regard. Ultimately, delineating the timing of neoepitope generation in T1D pathogenesis will determine their role as biomarkers as well as therapeutic targets.

Targeting citrullination in autoimmunity: insights learned from preclinical mouse models.

INTRODUCTION: Aberrant citrullination and excessive peptidylarginine deiminase (PAD) activity are detected in numerous challenging autoimmune diseases such as rheumatoid arthritis, inflammatory bowel diseases, systemic lupus erythematosus, multiple sclerosis, and type 1 diabetes. Because excessive PAD activity is a common denominator in these diseases, PADs are interesting potential therapeutic targets for future therapies. AREAS COVERED: This review summarizes the advances made in the design of PAD inhibitors, their utilization and therapeutic potential in preclinical mouse models of autoimmunity. Relevant literature encompasses studies from 1994 to 2021 that are available on PubMed.gov. EXPERT OPINION: Pan-PAD inhibition is a promising therapeutic strategy for autoimmune diseases. Drugs achieving pan-PAD inhibition were capable of ameliorating, reversing, and preventing clinical symptoms in preclinical mouse models. However, the implications for PADs in key biological processes potentially present a high risk for clinical complications and could hamper the translation of PAD inhibitors to the clinic. We envisage that PAD isozyme-specific inhibitors will improve the understanding the role of PAD isozymes in disease pathology, reduce the risk of side-effects and enhance prospects for future clinical translation.

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 glucose-regulated protein 78, and reduced spontaneous neutrophil extracellular trap formation 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 interferon-γ-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.

Identification of Deamidated Peptides in Cytokine-Exposed MIN6 Cells through LC-MS/MS Using a Shortened Digestion Time and Inspection of MS2 Spectra.

Enzymatic deamidation, the conversion of glutamine (Gln) into glutamic acid (Glu) residues, mediated by tissue transglutaminase enzymes, can provoke autoimmunity by generating altered self-epitopes, a process well-known in celiac disease and more recently also described in type 1 diabetes (T1D). To identify deamidated proteins, liquid chromatography-tandem mass spectrometry is the method of choice. However, as nonenzymatic deamidations on asparagine (Asn) and to a minor extent on Gln are frequently induced in vitro during proteomics sample preparation, the accurate detection of in vivo deamidation can be hampered. Here we report on the optimization of a method to reduce in vitro generated deamidation by 70% using improved trypsin digestion conditions (90 min/pH 8). We also point to the critical importance of manual inspection of MS2 spectra, considering that only 55% of the high quality peptides with Gln deamidation were assigned correctly using an automated search algorithm. As proof of principal, using these criteria, we showed a significant increase in levels of both Asn and Gln deamidation in cytokine-exposed murine MIN6 ß-cells, paralleled by an increase in tissue transglutaminase activity. These findings add evidence to the hypothesis that deamidation is occurring in stressed ß-cell proteins and can be involved in the autoimmune process in T1D.