Ivan Gerling

The decline in first-phase insulin response (FPIR) during the presymptomatic period of type 1 diabetes (T1D) is well established. In-situ functional studies with pancreas tissue slices showed that β-cell loss of glucose-responsiveness was independent of T-cell infiltration into islets in recent-onset T1D cases. However, the mechanisms driving β-cell dysfunction before the onset of T1D remain unclear. In pancreas tissue from donors across the natural history of T1D, we utilized an in-situ, whole - islet phenotypical and transcriptomic approach to unravel novel targets in the glucose-stimulus coupled secretion pathway that are similarly impaired in T-cell infiltrated and non-infiltrated islets. Specifically, we observed that islets from autoantibody positive (single(s) or multiple(m) AAb+) donors exhibited activation of post-transcriptional gene regulation along with reduced protein translation, processing in the endoplasmic reticulum (ER), and ER stress. Disrupted mitochondrial metabolism and bioenergetics were prominent in islets from multiple AAb+ and T1D donors with disease durations ≤7 years. In addition, T1D islets presented reduced mitochondrial protein import, quality control, and dynamics, together with downregulated genes in insulin secretory pathways. During infiltration, these pathways remain dysregulated while immune/inflammatory transcripts were increased. These studies identified novel mechanisms of β-cell dysregulation before symptomatic onset and independent of T-cell infiltration in T1D pathogenesis.

Aims/hypothesis: The nPOD-Virus group collaboratively applied innovative technologies to detect and sequence viral RNA in pancreas and other tissues from organ donors with type 1 diabetes. These analyses involved the largest number of pancreas samples collected to date. The aim of the current work was to examine the presence of enterovirus RNA in pancreas and lymphoid tissues of organ donors with and without type 1 diabetes. Methods: We analysed pancreas, spleen, pancreatic lymph nodes and duodenum samples from the following groups: (1) donors with type 1 diabetes (n=71) with (n=35) or without (n=36) insulin-containing islets; (2) donors with single or double islet autoantibody positivity without diabetes (n=22); and (3) autoantibody-negative donors without diabetes (control donors) (n=74). Five research laboratories participated in this collaborative effort using approaches for unbiased discovery of RNA viruses (two RNA-Seq platforms), targeted detection of Enterovirus A–D species using RT-PCR, and tests for virus growth in cell culture. Results: Direct RNA-Seq did not detect virus signal in pancreas samples, whereas RT-PCR detected enterovirus RNA confirmed by sequencing in low amounts in pancreas samples in three of the five donor groups: donors with type 1 diabetes with insulin-containing islets, 16% (5/32) being positive; donors with single islet autoantibody positivity, 53% (8/15) being positive; and non-diabetic donors, 8% (4/49) being positive. Detection of enterovirus RNA was significantly more frequent in single islet autoantibody-positive donors compared with donors with type 1 diabetes with insulin-deficient islets (pp=0.004). In some donors, pancreatic lymph nodes were also positive. RT-PCR detected enterovirus RNA also in the spleen of a small number of donors and virus enrichment in susceptible cell lines before RT-PCR resulted in much higher rate in spleen positivity, particularly in donors with type 1 diabetes. Interestingly, the enterovirus strains detected did not cause a typical lytic infection, possibly reflecting their persistence-prone nature. Conclusions/interpretation: This was the largest coordinated effort to examine the presence of enterovirus RNA in the pancreas of organ donors with type 1 diabetes, using a multitude of assays. These findings are consistent with the notion that donors with type 1 diabetes and donors with islet autoantibodies may carry a low-grade enterovirus infection in the pancreas and lymphoid tissues. Graphical

Summary: The loss of insulin secretory function associated with type 1 diabetes (T1D) is attributed to the immune-mediated destruction of beta cells. Yet, at onset of T1D, patients often have a significant beta cell mass remaining while T cell infiltration of pancreatic islets is sporadic. Thus, we investigated the hypothesis that the remaining beta cells in T1D are largely dysfunctional using live human pancreas tissue slices prepared from organ donors with recently diagnosed T1D. Beta cells in slices from donors with T1D had significantly diminished Ca ²⁺ mobilization and insulin secretion responses to glucose. Beta cell function was equally impaired in T cell-infiltrated and non-infiltrated islets. Fixed tissue staining and gene expression profiling of laser-capture microdissected islets revealed significant decreases of proteins and genes in the glucose stimulus secretion coupling pathway. From these data, we posit that functional defects occur in the remaining mass of beta cells during human T1D pathogenesis.

The Black Death caused by Yersinia pestis is the single largest human mortality event in recorded history. That pandemic killed up to 30-50% of the Afro-Eurasia population. As such, it is predicted to have had selective effects for immune response genes that could help protect against fatal outcomes. Similarly, many autoimmune diseases, including type 1 diabetes (at least before modern medicine), were under strong selective pressure because of their fatal outcomes. This study used ancient DNA extracts from two different populations before, during, and after Black Death to pinpoint immune loci that appeared to have been under positive selection. Four of those were found in both cohorts. One of those is associated with production of full length ERAP2 transcripts and the ability to produce cytokines and control intracellular levels of Yersinia pestis in macrophages. Several of the genes that appeared to have been positively selected by the pandemic are also associated with autoimmune/autoinflammatory diseases in present-day populations. This provides data and potential mechanisms for how a genetic variation that may increase risk of fatal outcomes from one immune-related disease can be “viable” long-term in that same population because it protects against fatal outcomes of another disease.

In article cited above, the cell description “CD44−CD62L− (naive)” mistakenly appeared throughout the legend of Fig. 4; the correct term is CD44−CD62L+ (naive).

Interferon response sensors are expressed in the islet of Langerhans during the course of type 1 diabetes.

Enterovirus (EV) infection of insulin-producing pancreatic beta cells is associated with type 1 diabetes (T1D), but little is known about the mechanisms that lead the virus to cause a persistent infection and, possibly, to induce beta cell autoimmunity. A cell line susceptible to most enterovirus types was infected with EV isolates from cases of T1D and, for comparison, with a replication-competent strain of coxsackievirus B3. The transcription of immune-related genes and secretion of cytokines was evaluated in infected vs. uninfected cells. Acutely infected cells showed the preserved transcription of type I interferon (IFN) pathways and the enhanced transcription/secretion of IL6, IL8, LIF, MCP1, and TGFB1. On the other hand, infection by defective EV strains obtained from diabetic subjects suppressed IFN pathways and the transcription of most cytokines, while enhancing the expression of IL8, IL18, IL32, and MCP1. IL18 and IL32 are known for their pathogenic role in autoimmune diabetes. Thus, the cytokine profile of AV3 cells infected by diabetes-derived EV strains closely matches that observed in patients at the early stages of T1D. The concordance of our results with clinically verified information reinforces the hypothesis that the immune changes observed in type 1 diabetic patients are due to a hardly noticeable virus infection.

This paper reports on a study undertaken within The Environmental Determinants of Diabetes in the Young (TEDDY). Longitudinal stool samples were analyzed for the presence of virus from the time of birth in individuals who later developed autoantibodies to islets of Langerhans or type 1 diabetes, and compared to matched controls. A number of interesting observations were made possible by the frequent stool sampling and sequencing of viromes from each participant. Positive correlations were found between islet autoimmunity and the following parameters: 1.) a single-nucleotide polymorphism (SNP) in the coxsackie and adenovirus receptor CXADR; 2.) prolonged shedding of what (by sequence similarity) appeared to be the the same Coxsackievirus B4 (likely due to failure of rapidly clearing such infections); and 3.) repeated infection with the adenovirus HAdV-F. Interestingly, infection early in life with another adenovirus (HAdV-C) was inversely correlated with development of islet autoantibodies. Although the observational nature of the study precludes any mechanistic conclusions, it does raise some very interesting questions of the potential roles of the timing and nature of viral infections on the development of islet autoimmunity.

Cells that die may release some or all of their DNA content into the circulation. This DNA is likely to be unmethylated in regions with genes that are expressed in those cells. It has been suggested that the presense of unmethylated DNA from genes that are exclusively expressed in specific cells, could serve as a marker of death of those cells. This paper is specifically investigating whether the presence of unmethylated insulin gene DNA sequences in serum could serve as a marker for death of the insulin-producing beta cells (located in the islets of Langerhans) – and more broadly as a marker of progression of autoimmune type 1 diabetes. This study made use of a serum collected from a group of well characterized at-risk individuals that had been followed longitudinally (TrialNet). There is no gold standard for loss of beta cell mass with which to compare this potential new biomarker. However, the results demonstrate that unmethylated insulin DNA is increased in the period just before diagnosis of diabetes – a time when other studies have suggested there is intense beta cell destruction and loss of beta cell mass. Although no firm conclusions can be made, this work would encourage further investigation of this potential biomarker of beta cell destruction.

The transcription factor MafA is well know for its role in development and function of insulin producing beta-cells in the islets of Langerhans. Those same cells are the target of autoimmunity in type 1 diabetes (T1D). Polymorphisms in the MafA gene have been associated with T1D susceptibility. This paper demonstrates that MafA-deficient mouse islets had elevated interferon-beta-1 levels and a stronger antiviral response, leaving them less susceptible to infection by coxackievirus B3 in culture. This would suggest that low MafA expression provides an advantage in the context of antiviral defenses even as it may have detrimental effects on beta cell development and function. Yet the same proinflammatory antiviral response that helps mount an effective anti-viral defense is also involved in initiation of autoimmune responses in T1D. This paper puts into focus the many, and at times counteracting, roles of this diabetes susceptibility gene in regulating beta cell function and vulnerability to viral infections as well as autoimmunity.