Assisting the implementation of screening for type 1 diabetes by using artificial intelligence on publicly available data.

The type 1 diabetes community is coalescing around the benefits and advantages of early screening for disease risk. To be accepted by healthcare providers, regulatory authorities and payers, screening programmes need to show that the testing variables allow accurate risk prediction and that individualised risk-informed monitoring plans are established, as well as operational feasibility, cost-effectiveness and acceptance at population level. Artificial intelligence (AI) has the potential to contribute to solving these issues, starting with the identification and stratification of at-risk individuals. ASSET (AI for Sustainable Prevention of Autoimmunity in the Society; www.asset.healthcare ) is a public/private consortium that was established to contribute to research around screening for type 1 diabetes and particularly to how AI can drive the implementation of a precision medicine approach to disease prevention. ASSET will additionally focus on issues pertaining to operational implementation of screening. The authors of this article, researchers and clinicians active in the field of type 1 diabetes, met in an open forum to independently debate key issues around screening for type 1 diabetes and to advise ASSET. The potential use of AI in the analysis of longitudinal data from observational cohort studies to inform the design of improved, more individualised screening programmes was also discussed. A key issue was whether AI would allow the research community and industry to capitalise on large publicly available data repositories to design screening programmes that allow the early detection of individuals at high risk and enable clinical evaluation of preventive therapies. Overall, AI has the potential to revolutionise type 1 diabetes screening, in particular to help identify individuals who are at increased risk of disease and aid in the design of appropriate follow-up plans. We hope that this initiative will stimulate further research on this very timely topic.

Upregulation of HLA class II in pancreatic beta cells from organ donors with type 1 diabetes.

AIMS/HYPOTHESIS: We aimed to characterise and quantify the expression of HLA class II (HLA-II) in human pancreatic tissue sections and to analyse its induction in human islets. METHODS: We immunostained human pancreatic tissue sections from non-diabetic (n = 5), autoantibody positive (Aab+; n = 5), and type 1 diabetic (n = 5) donors, obtained from the Network of Pancreatic Organ Donors (nPOD), with HLA-II, CD68 and insulin. Each tissue section was acquired with a widefield slide scanner and then analysed with QuPath software. In total, we analysed 7415 islets that contained 338,480 cells. Widefield microscopy was further complemented by high resolution imaging of 301 randomly selected islets, acquired using a Zeiss laser scanning confocal (LSM880) to confirm our findings. Selected beta cells were acquired in enhanced resolution using LSM880 with an Airyscan detector. Further, we cultured healthy isolated human islets and reaggregated human islet microtissues with varying concentrations of proinflammatory cytokines (IFN-γ, TNF-α and IL-1ß). After proinflammatory cytokine culture, islet function was measured by glucose-stimulated insulin secretion, and HLA-I and HLA-II expression was subsequently evaluated with immunostaining or RNA sequencing. RESULTS: Insulin-containing islets (ICIs) of donors with type 1 diabetes had a higher percentage of HLA-II positive area (24.31%) compared with type 1 diabetic insulin-deficient islets (IDIs, 0.67%), non-diabetic (3.80%), and Aab+ (2.31%) donors. In ICIs of type 1 diabetic donors, 45.89% of the total insulin signal co-localised with HLA-II, and 27.65% of the islet beta cells expressed both HLA-II and insulin, while in non-diabetic and Aab+ donors 0.96% and 0.59% of the islet beta cells, respectively, expressed both markers. In the beta cells of donors with type 1 diabetes, HLA-II was mostly present in the cell cytoplasm, co-localising with insulin. In the experiments with human isolated islets and reaggregated human islets, we observed changes in insulin secretion upon stimulation with proinflammatory cytokines, as well as higher expression of HLA-II and HLA-I when compared with controls cultured with media, and an upregulation of HLA-I and HLA-II RNA transcripts. CONCLUSIONS/INTERPRETATION: After a long-standing controversy, we provide definitive evidence that HLA-II can be expressed by pancreatic beta cells from patients with type 1 diabetes. Furthermore, this upregulation can be induced in vitro in healthy isolated human islets or reaggregated human islets by treatment with proinflammatory cytokines. Our findings support a role for HLA-II in type 1 diabetes pathogenesis since HLA-II expressing beta cells can potentially become a direct target of autoreactive CD4+ lymphocytes.

Effect of IL4 and IL10 on a human in vitro type 1 diabetes model.

We defined the effect of the anti-inflammatory cytokines IL4 and IL10 on an in vitro model of human T1D. After preincubation with IL4 or IL10, human islet microtissues were co-cultured with PBMC and proinflammatory cytokines for a few hours or for multiple days to assess acute and chronic effects. This resulted in an immune attack with infiltration of T cells into the islet, a loss of beta cell endocrine function, and an upregulation of HLA-I on the beta cells. HLA-I upregulation was associated with infiltration of T cells and both HLA-I expression and infiltration were associated with impaired insulin secretion. Preincubation with IL4 or IL10 did not preserve beta cell function but decreased infiltration of T cells. Our data support the hypothesis that a loss of beta cell endocrine function mediates an increase in beta cell specific antigen presentation. IL4 and IL10 failed to preserve beta cell endocrine function.

Liraglutide protects ß-cells in novel human islet spheroid models of type 1 diabetes.

To enable accurate, high-throughput and longer-term studies of the immunopathogenesis of type 1 diabetes (T1D), we established three in-vitro islet-immune injury models by culturing spheroids derived from primary human islets with proinflammatory cytokines, activated peripheral blood mononuclear cells or HLA-A2-restricted preproinsulin-specific cytotoxic T lymphocytes. In all models, ß-cell function declined as manifested by increased basal and decreased glucose-stimulated insulin release (GSIS), and decreased intracellular insulin content. Additional hallmarks of T1D progression such as loss of the first-phase insulin response (FFIR), increased proinsulin-to-insulin ratios, HLA-class I expression, and inflammatory cytokine release were also observed. Using these models, we show that liraglutide, a glucagon-like peptide 1 receptor agonist, prevented loss of GSIS under T1D-relevant stress, by preserving the FFIR and decreasing immune cell infiltration and cytokine secretion. Our results corroborate that liraglutide mediates an anti-inflammatory effect that aids in protecting ß-cells from the immune-mediated attack that leads to T1D.

Regulatory Immune Mechanisms beyond Regulatory T Cells.

In autoimmunity, aggressive immune responses are counteracted by suppressive rejoinders. For instance, FOXP3-expressing regulatory T cells (Tregs), have shown remarkable effects in limiting autoimmunity in preclinical models. However, early results from human Treg trials have not been as positive. Here, we highlight questions surrounding Treg transfers as putative treatments for autoimmunity. We discuss whether lack of antigenic recognition might be key to shifting cells from contributing to an aggressive autoresponse, to being part of a regulatory network. Moreover, we argue that identifying the physiological range of immunosuppression of Tregs might help potentiate their efficacy. We propose widening the view on immunoregulation by considering the participation of CD8+ Tregs in this process, which could have major implications in autoimmunity.

Current and future therapies for type 1 diabetes.

In type 1 diabetes, insulin remains the mature therapeutic cornerstone; yet, the increasing number of individuals developing type 1 diabetes (predominantly children and adolescents) still face severe complications. Fortunately, our understanding of type 1 diabetes is continuously being refined, allowing for refocused development of novel prevention and management strategies. Hitherto, attempts based on immune suppression and modulation have been only partly successful in preventing the key pathophysiological feature in type 1 diabetes: the immune-mediated derangement or destruction of beta cells in the pancreatic islets of Langerhans, leading to low or absent insulin secretion and chronic hyperglycaemia. Evidence now warrants a focus on the beta cell itself and how to avoid its dysfunction, which is putatively caused by cytokine-driven inflammation and other stress factors, leading to low insulin-secretory capacity, autoantigen presentation and immune-mediated destruction. Correspondingly, beta cell rescue strategies are being pursued, which include antigen vaccination using, for example, oral insulin or peptides, as well as agents with suggested benefits on beta cell stress, such as verapamil and glucagon-like peptide-1 receptor agonists. Whilst autoimmune-focused prevention approaches are central in type 1 diabetes and will be a requirement in the advent of stem cell-based replacement therapies, managing the primarily cardiometabolic complications of established type 1 diabetes is equally essential. In this review, we outline selected recent and suggested future attempts to address the evolving profile of the person with type 1 diabetes.

Animal models of human type 1 diabetes.

Type 1 diabetes is an immune-mediated disease in which pancreatic insulin-producing beta cells are damaged and destroyed. Animal models have served a prominent function in the development of the present ideas of pathogenesis and approaches to therapy. This commentary addresses the utility and limitations of these models for facilitating the 'translation' of immunology research into clinical applications.

HLA class I hyper-expression unmasks beta cells but not alpha cells to the immune system in pre-diabetes.

Human leukocyte antigens of class-I (HLA-I) molecules are hyper-expressed in insulin-containing islets (ICI) of type 1 diabetic (T1D) donors. This study investigated the HLA-I expression in autoantibody positive (AAB+) donors and defined its intra-islet and intracellular localization as well as proximity to infiltrating CD8 T cells with high-resolution confocal microscopy. We found HLA-I hyper-expression had already occurred prior to clinical diagnosis of T1D in islets of AAB+ donors. Interestingly, throughout all stages of disease, HLA-I was mostly expressed by alpha cells. Hyper-expression in AAB+ and T1D donors was associated with intra-cellular accumulation in the Golgi. Proximity analysis showed a moderate but significant correlation between HLA-I and infiltrating CD8 T cells only in ICI of T1D donors, but not in AAB+ donors. These observations not only demonstrate a very early, islet-intrinsic immune-independent increase of HLA-I during diabetes pathogenesis, but also point towards a role for alpha cells in T1D.

The effect of Toll-like receptor stimulation on the motility of regulatory T cells.

Regulatory T cells (Tregs) have suppressive functions and play an important role in controlling inflammation and autoimmunity. The migratory capacity of Tregs determines their location and their location determines whether they inhibit the priming of naïve lymphocytes in lymphoid tissues or the effector phase of immune responses at inflamed sites. Tregs generated or expanded in vitro are currently being tested in clinics for the treatment of autoimmune disorders, however, little is known about the factors controlling their migration towards therapeutically relevant locations. In this study, we have modulated Treg dynamics using Toll-like receptor (TLR) agonists. Dynamic imaging with confocal and two-photon microscopy revealed that Tregs generated in vitro and stimulated with P3C (a TLR2 agonist) but not with R848 (a TLR7 agonist) or LPS (a TLR4 agonist) showed enhanced cell migration within splenic white pulp or draining lymph node when transferred into mice intravenously or into the footpad, respectively. In summary, our data demonstrate that Tregs are more motile in response to direct TLR stimulation in particular towards TLR2 signals. This may have implications for efficient clinical Treg induction protocols.

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.

Interleukin-21 receptor-mediated signals control autoreactive T cell infiltration in pancreatic islets.

It remains unclear how interleukin-21 receptor (IL-21R) contributes to type 1 diabetes. Here we have shown that dendritic cells (DCs) in the pancreas required IL-21R not for antigen uptake, but to acquire the chemokine receptor CCR7 and migrate into the draining lymph node. Consequently, less antigen, major histocompatibility complex (MHC) class II, and CD86 was provided to autoreactive effector cells in Il21r(-/-) mice, impairing CD4(+) T cell activation, CD40:CD40L interactions, and pancreatic infiltration by autoreactive T cells. CD40 crosslinking restored defective CD4(+) cell expansion and CD4 independently expanded autoreactive CD8(+) cells, but CD8(+) cells still required CD4(+) cells to reach the pancreas and induce diabetes. Diabetes induction by transferred T cells required IL-21R-sufficient host antigen-presenting cells. Transferring IL-21R-sufficient DCs broke diabetes resistance in Il21r(-/-) mice. We conclude that IL-21R controls both antigen transport by DCs and the crucial beacon function of CD4(+) cells for autoreactive CD8(+) cells to reach the islets.

Efficacy and safety of liraglutide in type 1 diabetes by baseline characteristics in the ADJUNCT ONE and ADJUNCT TWO randomized controlled trials.

AIM: To evaluate 26 weeks of liraglutide treatment in type 1 diabetes (T1D) by subgroups in the ADJUNCT ONE and ADJUNCT TWO trials. MATERIALS AND METHODS: ADJUNCT ONE and ADJUNCT TWO were randomized controlled phase 3 trials in 1398 and 835 participants with T1D treated with liraglutide (1.8, 1.2, or 0.6 mg) or placebo (adjuncts to insulin). This post hoc analysis evaluated treatment effects by subgroups: HbA1c (< or ≥8.5%), body mass index (BMI; < or ≥27 kg/m2 ), and insulin regimen (basal bolus or continuous subcutaneous insulin infusion). RESULTS: In both trials at week 26, reductions in HbA1c, body weight, and daily insulin dose did not differ significantly (P > .05) by baseline HbA1c or BMI. Risk of clinically significant hypoglycaemia or hyperglycaemia with ketosis did not differ significantly (P > .05) by baseline HbA1c, BMI, or insulin regimen. At week 26 in ADJUNCT ONE, these risks did not differ (P > .05) between treatment groups. Placebo-adjusted reductions in HbA1c, body weight, and insulin dose (-0.30%-points, -5.0 kg, and -12%, respectively, with liraglutide 1.8 mg), were significant (P < .05), greater than at week 52, and similar to those in ADJUNCT TWO (-0.35%, -4.8 kg, and -10%, respectively, with liraglutide 1.8 mg). CONCLUSIONS: In ADJUNCT ONE and ADJUNCT TWO, the efficacy and glycaemic safety of liraglutide did not depend on subgroups, leaving residual beta-cell function as the only identified variable impacting the effect of glucagon-like peptide-1 receptor agonists (GLP-1 RAs) in T1D. These findings support a role for GLP-1 RAs as adjuncts to insulin in T1D, warranting further study.

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.

The urgent need for integrated science to fight COVID-19 pandemic and beyond.

The COVID-19 pandemic has become the leading societal concern. The pandemic has shown that the public health concern is not only a medical problem, but also affects society as a whole; so, it has also become the leading scientific concern. We discuss in this treatise the importance of bringing the world's scientists together to find effective solutions for controlling the pandemic. By applying novel research frameworks, interdisciplinary collaboration promises to manage the pandemic's consequences and prevent recurrences of similar pandemics.

Human herpesvirus-6 is present at higher levels in the pancreatic tissues of donors with type 1 diabetes.

Human herpesvirus-6 (HHV-6) is a ubiquitous pathogen associated with nervous and endocrine autoimmune disorders. The aim of this study was to investigate the presence of HHV-6 in pancreatic tissue sections from non-diabetic, auto-antibody positive (AAB+), and donors with type 1 diabetes (T1D) and explore whether there is any association between HHV-6 and MHC class I hyperexpression and CD8 T cell infiltration. HHV-6 DNA was detected by PCR and its protein was examined by indirect immunofluorescence assay followed by imaging using high-resolution confocal microscopy. Viral DNA (U67) was found in most pancreata of non-diabetic (3 out of 4), AAB+ (3 out of 5) and T1D donors (6 out of 7). Interestingly, HHV-6 glycoprotein B (gB) was more expressed in islets and exocrine pancreas of donors with T1D. However, gB expression was not directly associated with other pathologies. Out of 20 islets with high gB expression, only 3 islets (15%) showed MHC class I hyperexpression. Furthermore, no correlation was found between gB expression and CD8 T cell infiltration on a per-islet basis in any of the groups. Our observations indicate that HHV-6 DNA and protein are present in the pancreas of non-diabetic subjects but gB expression is higher in the pancreas of donors with T1D. The possible role of HHV-6 as a contributory factor for T1D should therefore be further investigated.

Type 1 diabetes: etiology, immunology, and therapeutic strategies.

Type 1 diabetes (T1D) is a chronic autoimmune disease in which destruction or damaging of the beta-cells in the islets of Langerhans results in insulin deficiency and hyperglycemia. We only know for sure that autoimmunity is the predominant effector mechanism of T1D, but may not be its primary cause. T1D precipitates in genetically susceptible individuals, very likely as a result of an environmental trigger. Current genetic data point towards the following genes as susceptibility genes: HLA, insulin, PTPN22, IL2Ra, and CTLA4. Epidemiological and other studies suggest a triggering role for enteroviruses, while other microorganisms might provide protection. Efficacious prevention of T1D will require detection of the earliest events in the process. So far, autoantibodies are most widely used as serum biomarker, but T-cell readouts and metabolome studies might strengthen and bring forward diagnosis. Current preventive clinical trials mostly focus on environmental triggers. Therapeutic trials test the efficacy of antigen-specific and antigen-nonspecific immune interventions, but also include restoration of the affected beta-cell mass by islet transplantation, neogenesis and regeneration, and combinations thereof. In this comprehensive review, we explain the genetic, environmental, and immunological data underlying the prevention and intervention strategies to constrain T1D.

Low HLA binding of diabetes-associated CD8+ T-cell epitopes is increased by post translational modifications.

BACKGROUND: Type 1 diabetes (T1D) is thought to be an autoimmune disease driven by anti-islet antigen responses and mediated by T-cells. Recent published data suggests that T-cell reactivity to modified peptides, effectively neoantigens, may promote T1D. These findings have given more credence to the concept that T1D may not be solely an error of immune recognition but may be propagated by errors in protein processing or in modifications to endogenous peptides occurring as result of hyperglycemia, endoplasmic reticulum (ER) stress, or general beta cell dysfunction. In the current study, we hypothesized that diabetes-associated epitopes bound human leukocyte antigen (HLA) class I poorly and that post-translational modifications (PTM) to key sequences within the insulin-B chain enhanced peptide binding to HLA class I, conferring the CD8+ T-cell reactivity associated with T1D. RESULTS: We first identified, through the Immune Epitope Database (IEDB; www.iedb.org ), 138 published HLA class I-restricted diabetes-associated epitopes reported to elicit positive T-cell responses in humans. The peptide binding affinity for their respective restricting allele(s) was evaluated in vitro. Overall, 75% of the epitopes bound with a half maximal inhibitory concentration (IC50) of 8250 nM or better, establishing a reference affinity threshold for HLA class I-restricted diabetes epitopes. These studies demonstrated that epitopes from diabetes-associated antigens bound HLA with a lower affinity than those of microbial origin (binding threshold of 500 nM for 85% of the epitopes). Further predictions suggested that diabetes epitopes also bind HLA class I with lower affinity than epitopes associated with other autoimmune diseases. Therefore, we measured the effect of common PTM (citrullination, chlorination, deamidation, and oxidation) on HLA-A*02:01 binding of insulin-B-derived peptides, compared to native peptides. We found that these modifications increased binding for 44% of the insulin-B epitopes, but only 15% of the control peptides. CONCLUSIONS: These results demonstrate that insulin-derived epitopes, commonly associated with T1D, generally bind HLA class I poorly, but can be subject to PTM that improve their binding capacity and may, in part, be responsible for T-cell activation in T1D and subsequent beta cell death.

Oral insulin does not alter gut microbiota composition of NOD mice.

BACKGROUND: Oral insulin as a preventive strategy and/or treatment of type 1 diabetes has been the target of much research. Producing oral insulins is a complex and challenging task, with numerous pitfalls, due to physiological, physical, and biochemical barriers. Our aim was to determine the impact of oral insulin on the delicate gut microbiota composition. METHODS: Female nonobese diabetic mice were given oral porcine insulin 2 times a week from 5 weeks of age for 4 weeks, and then subsequently once a week for 21 weeks, or until euthanized. The mice were divided into groups on a gluten-reduced diet or a standard diet. Gut microbiota composition was analysed based on faecal samples, and the type 1 diabetes incidence of the mice was monitored. RESULTS: We observed no influence of the oral porcine insulin on the gut microbiota composition of mice on a gluten-reduced or a standard diet at 9 weeks of age. Also, the administration of oral insulin did not influence the incidence of type 1 diabetes at 30 weeks of age. CONCLUSIONS: Oral porcine insulin does not alter the gut microbiota composition of nonobese diabetic mice on either a gluten-reduced diet or standard diet. Also, the oral porcine insulin did not influence the incidence of type 1 diabetes in the groups.