Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of ...cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Between Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.
For more than 35 years, the NOD mouse has been the primary animal model for studying autoimmune diabetes. During this time, striking similarities to the human disease have been uncovered. In both ...species, unusual polymorphisms in a major histocompatibility complex (MHC) class II molecule confer the most disease risk, disease is caused by perturbations by the same genes or different genes in the same biological pathways and that diabetes onset is preceded by the presence of circulating autoreactive T cells and autoantibodies that recognize many of the same islet antigens. However, the relevance of the NOD model is frequently challenged due to past failures translating therapies from NOD mice to humans and because the appearance of insulitis in mice and some patients is different. Nevertheless, the NOD mouse remains a pillar of autoimmune diabetes research for its usefulness as a preclinical model and because it provides access to invasive procedures as well as tissues that are rarely procured from patients or controls. The current article is focused on approaches to improve the NOD mouse by addressing reasons why immune therapies have failed to translate from mice to humans. We also propose new strategies for mixing and editing the NOD genome to improve the model in ways that will better advance our understanding of human diabetes. As proof of concept, we report that diabetes is completely suppressed in a knock-in NOD strain with a serine to aspartic acid substitution at position 57 in the MHC class II Aβ. This supports that similar non-aspartic acid substitutions at residue 57 of variants of the human class II HLA-DQβ homolog confer diabetes risk.
The complex etiology of type 1 diabetes (T1D) is the outcome of failures in regulating immunity in combination with beta cell perturbations. Mitochondrial dysfunction in beta cells and immune cells ...may be involved in T1D pathogenesis. Mitochondrial energy production is essential for the major task of beta cells (the secretion of insulin in response to glucose). Mitochondria are a major site of reactive oxygen species (ROS) production. Under immune attack, mitochondrial ROS (mtROS) participate in beta cell damage. Similarly, T cell fate during immune responses is tightly regulated by mitochondrial physiology, morphology, and metabolism. Production of mtROS is essential for signaling in antigen-specific T cell activation. Mitochondrial dysfunction in T cells has been noted as a feature of some human autoimmune diseases. Recent Advances: Preclinical and clinical studies indicate that mitochondrial dysfunction in beta cells sensitizes these cells to immune-mediated destruction via direct or indirect mechanisms. Sensitivity of beta cells to mtROS is associated with genetic T1D risk loci in human and the T1D-prone nonobese diabetic (NOD) mouse. Mitochondrial dysfunction and altered metabolism have also been observed in immune cells of NOD mice and patients with T1D. This immune cell mitochondrial dysfunction has been linked to deleterious functional changes.
It remains unclear how mitochondria control T cell receptor signaling and downstream events, including calcium flux and activation of transcription factors during autoimmunity.
Mechanistic studies are needed to investigate the mitochondrial pathways involved in autoimmunity, including T1D. These studies should seek to identify the role of mitochondria in regulating innate and adaptive immune cell activity and beta cell failure.
Protein tyrosine phosphatase, non-receptor type 22 (PTPN22) regulates a panoply of leukocyte signaling pathways. A single nucleotide polymorphism (SNP) in
,
, is associated with increased risk of ...Type 1 Diabetes (T1D) and other autoimmune diseases. Over the past decade PTPN22 has been studied intensely in T cell receptor (TCR) and B cell receptor (BCR) signaling. However, the effect of the minor allele on PTPN22 function in TCR signaling is controversial with some reports concluding it has enhanced function and blunts TCR signaling and others reporting it has reduced function and increases TCR signaling. More recently, the core function of PTPN22 as well as functional derangements imparted by the autoimmunity-associated variant allele of PTPN22 have been examined in monocytes, macrophages, dendritic cells, and neutrophils. In this review we will discuss the known functions of PTPN22 in human cells, and we will elaborate on how autoimmunity-associated variants influence these functions across the panoply of immune cells that express PTPN22. Further, we consider currently unresolved questions that require clarification on the role of PTPN22 in immune cell function.
Aims/hypothesis
Human pancreatic beta cells may be complicit in their own demise in type 1 diabetes, but how this occurs remains unclear. One potentially contributing factor is hyperexpression of HLA ...class I antigens. This was first described approximately 30 years ago, but has never been fully characterised and was recently challenged as artefactual. Therefore, we investigated HLA class I expression at the protein and RNA levels in pancreases from three cohorts of patients with type 1 diabetes. The principal aims were to consider whether HLA class I hyperexpression is artefactual and, if not, to determine the factors driving it.
Methods
Pancreas samples from type 1 diabetes patients with residual insulin-containing islets (
n
= 26) from the Network for Pancreatic Organ donors with Diabetes (nPOD), Diabetes Virus Detection study (DiViD) and UK recent-onset type 1 diabetes collections were immunostained for HLA class I isoforms, signal transducer and activator of transcription 1 (STAT1), NLR family CARD domain containing 5 (NLRC5) and islet hormones. RNA was extracted from islets isolated by laser-capture microdissection from nPOD and DiViD samples and analysed using gene-expression arrays.
Results
Hyperexpression of HLA class I was observed in the insulin-containing islets of type 1 diabetes patients from all three tissue collections, and was confirmed at both the RNA and protein levels. The expression of β2-microglobulin (a second component required for the generation of functional HLA class I complexes) was also elevated. Both ‘classical’ HLA class I isoforms (i.e. HLA-ABC) as well as a ‘non-classical’ HLA molecule, HLA-F, were hyperexpressed in insulin-containing islets. This hyperexpression did not correlate with detectable upregulation of the transcriptional regulator NLRC5. However, it was strongly associated with increased STAT1 expression in all three cohorts. Islet hyperexpression of HLA class I molecules occurred in the insulin-containing islets of patients with recent-onset type 1 diabetes and was also detectable in many patients with disease duration of up to 11 years, declining thereafter.
Conclusions/interpretation
Islet cell HLA class I hyperexpression is not an artefact, but is a hallmark in the immunopathogenesis of type 1 diabetes. The response is closely associated with elevated expression of STAT1 and, together, these occur uniquely in patients with type 1 diabetes, thereby contributing to their selective susceptibility to autoimmune-mediated destruction.
A detailed understanding of the molecular pathways and cellular interactions that result in islet beta cell (β cell) destruction is essential for the development and implementation of effective ...therapies for prevention or reversal of type 1 diabetes (T1D). However, events that define the pathogenesis of human T1D have remained elusive. This gap in our knowledge results from the complex interaction between genetics, the immune system, and environmental factors that precipitate T1D in humans. A link between genetics, the immune system, and environmental factors are type 1 interferons (T1-IFNs). These cytokines are well known for inducing antiviral factors that limit infection by regulating innate and adaptive immune responses. Further, several T1D genetic risk loci are within genes that link innate and adaptive immune cell responses to T1-IFN. An additional clue that links T1-IFN to T1D is that these cytokines are a known constituent of the autoinflammatory milieu within the pancreas of patients with T1D. The presence of IFNα/β is correlated with characteristic MHC class I (MHC-I) hyperexpression found in the islets of patients with T1D, suggesting that T1-IFNs modulate the cross-talk between autoreactive cytotoxic CD8
T lymphocytes and insulin-producing pancreatic β cells. Here, we review the evidence supporting the diabetogenic potential of T1-IFN in the islet microenvironment.
Type 1 Diabetes (T1D) is an autoimmune disease characterized by destruction of pancreatic β cells. Genetics and environmental factors play crucial roles in T1D development. Four single nucleotide ...polymorphisms in IFIH1, encoding the viral sensor MDA5 have been linked to T1D. MDA5 promotes anti-viral responses and may link genetics with environmental factors to promote or suppress T1D. A stop-gain mutation in IFIH1 (rs35744605) results in a non-functional MDA5 protein (MDA5627X). We hypothesized that MDA5627X protects against T1D by dampening inflammatory anti-viral responses resulting in reduced autoimmunity. We gene edited human induced pluripotent stem cells at rs35744605 using a CRISPR/Cas9 strategy to generate iPSC encoding the stop-gain variant (MDA5627X). Macrophages (Mφ) have been demonstrated to be indispensable for T1D-initiation and regulate anti-viral responses. We differentiated iPSCs into Mφ and studies responses to high molecular weight Poly I:C to mimic viral infection. The outcome measures were Type 1 Interferon (IFN1) production, antigen presentation, and immune activation markers. Treatment of MDA5 intact Mφ with Poly I:C resulted in 60-fold increase in IFN1 production and a 3-fold elevation in human leukocyte antigen Class I (HLA-I) expression. In contrast, treated MDA5627X Mφ failed to increase IFN1 or HLA-I at any Poly I:C dose. While Poly I:C did not increase HLA-I expression, untreated MDA5627X Mφ had elevated HLA-1 compared to MDA5 intact Mφ (P=0.0306). Also, we assessed the expression of CD80, CD86, CD40, and MHC-II, however there were no differences in the expression between Mφ with intact MDA5 or MDA5627X. In contrast, IFNg and LPS treatment elevated expression of CD80, CD86, CD40, and MHC-II in both Mφ populations. In conclusion, our data point to a protective effect of the MDA5627X variant in T1D through decreased inflammatory IFN1 levels and T cell activation by Mφ.
Disclosure
O.Iwaloye: None. L.H.Armitage: None. A.M.Meacham: None. C.E.Mathews: None.
Funding
National Institutes of Health (R01DK127497, P01AI042288, UH3DK122638)
Pancreatic β-cells are prone to endoplasmic reticulum (ER) stress due to their role in insulin secretion. They require sustainable and efficient adaptive stress responses to cope with this stress. ...Whether episodes of chronic stress directly compromise β-cell identity is unknown. We show here under reversible, chronic stress conditions β-cells undergo transcriptional and translational reprogramming associated with impaired expression of regulators of β-cell function and identity. Upon recovery from stress, β-cells regain their identity and function, indicating a high degree of adaptive plasticity. Remarkably, while β-cells show resilience to episodic ER stress, when episodes exceed a threshold, β-cell identity is gradually lost. Single cell RNA-sequencing analysis of islets from type 1 diabetes patients indicates severe deregulation of the chronic stress-adaptation program and reveals novel biomarkers of diabetes progression. Our results suggest β-cell adaptive exhaustion contributes to diabetes pathogenesis.
Aims/hypothesis
Sphingolipids play important roles in beta cell physiology, by regulating proinsulin folding and insulin secretion and in controlling apoptosis, as studied in animal models and cell ...cultures. Here we investigate whether sphingolipid metabolism may contribute to the pathogenesis of human type 1 diabetes and whether increasing the levels of the sphingolipid sulfatide would prevent models of diabetes in NOD mice.
Methods
We examined the amount and distribution of sulfatide in human pancreatic islets by immunohistochemistry, immunofluorescence and electron microscopy. Transcriptional analysis was used to evaluate expression of sphingolipid-related genes in isolated human islets. Genome-wide association studies (GWAS) and a T cell proliferation assay were used to identify type 1 diabetes related polymorphisms and test how these affect cellular islet autoimmunity. Finally, we treated NOD mice with fenofibrate, a known activator of sulfatide biosynthesis, to evaluate the effect on experimental autoimmune diabetes development.
Results
We found reduced amounts of sulfatide, 23% of the levels in control participants, in pancreatic islets of individuals with newly diagnosed type 1 diabetes, which were associated with reduced expression of enzymes involved in sphingolipid metabolism. Next, we discovered eight gene polymorphisms (
ORMDL3
,
SPHK2
,
B4GALNT1
,
SLC1A5
,
GALC
,
PPARD
,
PPARG
and
B4GALT1
) involved in sphingolipid metabolism that contribute to the genetic predisposition to type 1 diabetes. These gene polymorphisms correlated with the degree of cellular islet autoimmunity in a cohort of individuals with type 1 diabetes. Finally, using fenofibrate, which activates sulfatide biosynthesis, we completely prevented diabetes in NOD mice and even reversed the disease in half of otherwise diabetic animals.
Conclusions/interpretation
These results indicate that islet sphingolipid metabolism is abnormal in type 1 diabetes and suggest that modulation may represent a novel therapeutic approach.
Data availability
The RNA expression data is available online at
https://www.dropbox.com/s/93mk5tzl5fdyo6b/Abnormal%20islet%20sphingolipid%20metabolism%20in%20type%201%20diabetes%2C%20RNA%20expression.xlsx?dl=0
. A list of SNPs identified is available at
https://www.dropbox.com/s/yfojma9xanpp2ju/Abnormal%20islet%20sphingolipid%20metabolism%20in%20type%201%20diabetes%20SNP.xlsx?dl=0
.
Type 1 diabetes results from chronic autoimmune destruction of insulin-producing β-cells within pancreatic islets. Although insulin is a critical self-antigen in animal models of autoimmune diabetes, ...due to extremely limited access to pancreas samples, little is known about human antigenic targets for islet-infiltrating T cells. Here we show that proinsulin peptides are targeted by islet-infiltrating T cells from patients with type 1 diabetes. We identified hundreds of T cells from inflamed pancreatic islets of three young organ donors with type 1 diabetes with a short disease duration with high-risk HLA genes using a direct T-cell receptor (TCR) sequencing approach without long-term cell culture. Among 85 selected CD4 TCRs tested for reactivity to preproinsulin peptides presented by diabetes-susceptible HLA-DQ and HLA-DR molecules, one T cell recognized C-peptide amino acids 19-35, and two clones from separate donors responded to insulin B-chain amino acids 9-23 (B:9-23), which are known to be a critical self-antigen-driving disease progress in animal models of autoimmune diabetes. These B:9-23-specific T cells from islets responded to whole proinsulin and islets, whereas previously identified B:9-23 responsive clones from peripheral blood did not, highlighting the importance of proinsulin-specific T cells in the islet microenvironment.
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