Pharmaceutical targeting of the cannabinoid type 1 receptor impacts the crosstalk between immune cells and islets to reduce insulitis in humans.

AIMS/HYPOTHESIS: Insulitis, a hallmark of inflammation preceding autoimmune type 1 diabetes, leads to the eventual loss of functional beta cells. However, functional beta cells can persist even in the face of continuous insulitis. Despite advances in immunosuppressive treatments, maintaining functional beta cells to prevent insulitis progression and hyperglycaemia remains a challenge. The cannabinoid type 1 receptor (CB1R), present in immune cells and beta cells, regulates inflammation and beta cell function. Here, we pioneer an ex vivo model mirroring human insulitis to investigate the role of CB1R in this process. METHODS: CD4+ T lymphocytes were isolated from peripheral blood mononuclear cells (PBMCs) from male and female individuals at the onset of type 1 diabetes and from non-diabetic individuals, RNA was extracted and mRNA expression was analysed by real-time PCR. Single beta cell expression from donors with type 1 diabetes was obtained from data mining. Patient-derived human islets from male and female cadaveric donors were 3D-cultured in solubilised extracellular matrix gel in co-culture with the same donor PBMCs, and incubated with cytokines (IL-1ß, TNF-α, IFN-γ) for 24-48 h in the presence of vehicle or increasing concentrations of the CB1R blocker JD-5037. Expression of CNR1 (encoding for CB1R) was ablated using CRISPR/Cas9 technology. Viability, intracellular stress and signalling were assayed by live-cell probing and real-time PCR. The islet function measured as glucose-stimulated insulin secretion was determined in a perifusion system. Infiltration of immune cells into the islets was monitored by microscopy. Non-obese diabetic mice aged 7 weeks were treated for 1 week with JD-5037, then euthanised. Profiling of immune cells infiltrated in the islets was performed by flow cytometry. RESULTS: CNR1 expression was upregulated in circulating CD4+ T cells from individuals at type 1 diabetes onset (6.9-fold higher vs healthy individuals) and in sorted islet beta cells from donors with type 1 diabetes (3.6-fold higher vs healthy counterparts). The peripherally restricted CB1R inverse agonist JD-5037 arrested the initiation of insulitis in humans and mice. Mechanistically, CB1R blockade prevented islet NO production and ameliorated the ATF6 arm of the unfolded protein response. Consequently, cyto/chemokine expression decreased in human islets, leading to sustained islet cell viability and function. CONCLUSIONS/INTERPRETATION: These results suggest that CB1R could be an interesting target for type 1 diabetes while highlighting the regulatory mechanisms of insulitis. Moreover, these findings may apply to type 2 diabetes where islet inflammation is also a pathophysiological factor. DATA AVAILABILITY: Transcriptomic analysis of sorted human beta cells are from Gene Expression Omnibus database, accession no. GSE121863, available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM3448161 .

The extent and magnitude of islet T cell infiltration as powerful tools to define the progression to type 1 diabetes.

AIMS/HYPOTHESIS: Insulitis is not present in all islets, and it is elusive in humans. Although earlier studies focused on islets that fulfilled certain criteria (e.g. ≥15 CD45+ cells or ≥6 CD3+ cells), there is a fundamental lack of understanding of the infiltration dynamics in terms of its magnitude (i.e. how much) and extent (i.e. where). Here, we aimed to perform an in-depth characterisation of T cell infiltration by investigating islets with moderate (1-5 CD3+ cells) and high (≥6 CD3+ cells) infiltration in individuals with and without type 1 diabetes. METHODS: Pancreatic tissue sections from 15 non-diabetic, eight double autoantibody-positive and ten type 1 diabetic (0-2 years of disease duration) organ donors were obtained from the Network for Pancreatic Organ Donors with Diabetes, and stained for insulin, glucagon, CD3 and CD8 by immunofluorescence. T cell infiltration was quantified in a total of 8661 islets using the software QuPath. The percentage of infiltrated islets and islet T cell density were calculated. To help standardise the analysis of T cell infiltration, we used cell density data to develop a new T cell density threshold capable of differentiating non-diabetic and type 1 diabetic donors. RESULTS: Our analysis revealed that 17.1% of islets in non-diabetic donors, 33% of islets in autoantibody-positive and 32.5% of islets in type 1 diabetic donors were infiltrated by 1 to 5 CD3+ cells. Islets infiltrated by ≥6 CD3+ cells were rare in non-diabetic donors (0.4%) but could be found in autoantibody-positive (4.5%) and type 1 diabetic donors (8.2%). CD8+ and CD8- populations followed similar patterns. Likewise, T cell density was significantly higher in the islets of autoantibody-positive donors (55.4 CD3+ cells/mm2) and type 1 diabetic donors (74.8 CD3+ cells/mm2) compared with non-diabetic individuals (17.3 CD3+ cells/mm2), which was accompanied by higher exocrine T cell density in type 1 diabetic individuals. Furthermore, we showed that the analysis of a minimum of 30 islets and the use of a reference mean value for T cell density of 30 CD3+ cells/mm2 (the 30-30 rule) can differentiate between non-diabetic and type 1 diabetic donors with high specificity and sensitivity. In addition, it can classify autoantibody-positive individuals as non-diabetic or type 1 diabetic-like. CONCLUSIONS/INTERPRETATION: Our data indicates that the proportion of infiltrated islets and T cell density change dramatically during the course of type 1 diabetes, and these changes can be already observed in double autoantibody-positive individuals. This suggests that, as disease progresses, T cell infiltration extends throughout the pancreas, reaching the islets and exocrine compartment. While it predominantly targets insulin-containing islets, large accumulations of cells are rare. Our study fulfils the need to further understand T cell infiltration, not only after diagnosis but also in individuals with diabetes-related autoantibodies. Furthermore, the development and application of new analytical tools based on T cell infiltration, like the 30-30 rule, will allow us to correlate islet infiltration with demographic and clinical variables with the aim of identifying individuals at the very early stages of the disease.

Gut dysbiosis promotes islet-autoimmunity by increasing T-cell attraction in islets via CXCL10 chemokine.

CXCL10 is an IFNγ-inducible chemokine implicated in the pathogenesis of type 1 diabetes. T-cells attracted to pancreatic islets produce IFNγ, but it is unclear what attracts the first IFNγ -producing T-cells in islets. Gut dysbiosis following administration of pathobionts induced CXCL10 expression in pancreatic islets of healthy non-diabetes-prone (C57BL/6) mice and depended on TLR4-signaling, and in non-obese diabetic (NOD) mice, gut dysbiosis induced also CXCR3 chemokine receptor in IGRP-reactive islet-specific T-cells in pancreatic lymph node. In amounts typical to low-grade endotoxemia, bacterial lipopolysaccharide induced CXCL10 production in isolated islets of wild type and RAG1 or IFNG-receptor-deficient but not type-I-IFN-receptor-deficient NOD mice, dissociating lipopolysaccharide-induced CXCL10 production from T-cells and IFNγ. Although mostly myeloid-cell dependent, also ß-cells showed activation of innate immune signaling pathways and Cxcl10 expression in response to lipopolysaccharide indicating their independent sensitivity to dysbiosis. Thus, CXCL10 induction in response to low levels of lipopolysaccharide may allow islet-specific T-cells imprinted in pancreatic lymph node to enter in healthy islets independently of IFN-g, and thus link gut dysbiosis to early islet-autoimmunity via dysbiosis-associated low-grade endotoxemia.

Beneficial effects of a T. monococcum wheat cultivar on diabetes incidence evaluated in non-obese diabetic mice and after in vitro simulated gastroduodenal digestion.

Wheat consumption can represent one of the nutritional factors involved in the onset of diabetes. We specifically investigated the potential diabetogenic effects of Hammurabi, a T. monococcum wheat cultivar, in non-obese diabetic (NOD) mice and analysed the levels of resistant starch in pasta manufactured with Hammurabi after in vitro gastroduodenal digestion. NOD mice were fed with Hammurabi, bread wheat or rice flour to evaluate diabetes incidence and insulitis score. An enzymatic method was applied to compare the content of resistant starch in Hammurabi pasta and durum wheat pasta (control). In NOD mice, the Hammurabi-based diet significantly delayed diabetes onset (p = 0.0042) and reduced insulitis score compared to rice or wheat-based diet. Furthermore, the resistant starch value following in vitro digestion of Hammurabi pasta was significantly higher (4.08%) than that of durum wheat pasta (2.28%). Taken together, these results highlighted the potential positive effects of the Hammurabi-based diet on diabetes incidence.

Presumption of guilt for T cells in type 1 diabetes: lead culprits or partners in crime depending on age of onset?

Available evidence provides arguments both for and against a primary pathogenic role for T cells in human type 1 diabetes. Genetic susceptibility linked to HLA Class II lends strong support. Histopathology documents HLA Class I hyperexpression and islet infiltrates dominated by CD8+ T cells. While both hallmarks are near absent in autoantibody-positive donors, the variable insulitis and residual beta cells of recent-onset donors suggests the existence of a younger-onset endotype with more aggressive autoimmunity and an older-onset endotype with more vulnerable beta cells. Functional arguments from ex vivo and in vitro human studies and in vivo 'humanised' mouse models are instead neutral or against a T cell role. Clinical support is provided by the appearance of islet autoantibodies before disease onset. The faster C-peptide loss and superior benefits of immunotherapies in individuals with younger-onset type 1 diabetes reinforce the view of age-related endotypes. Clarifying the relative role of T cells will require technical advances in the identification of their target antigens, in their detection and phenotyping in the blood and pancreas, and in the study of the T cell/beta cell crosstalk. Critical steps toward this goal include the understanding of the link with environmental triggers, the description of T cell changes along the natural history of disease, and their relationship with age and the 'benign' islet autoimmunity of healthy individuals. Graphical abstract.

Robo4-mediated pancreatic endothelial integrity decreases inflammation and islet destruction in autoimmune diabetes.

In Type 1 Diabetes Mellitus (T1DM), leukocyte infiltration of the pancreatic islets and the resulting immune-mediated destruction of beta cells precede hyperglycemia and clinical disease symptoms. In this context, the role of the pancreatic endothelium as a barrier for autoimmunity- and inflammation-related destruction of the islets is not well studied. Here, we identified Robo4, expressed on endothelial cells, as a regulator of pancreatic vascular endothelial permeability during autoimmune diabetes. Circulating levels of Robo4 were upregulated in mice subjected to the Multiple Low-Dose Streptozotocin (MLDS) model of diabetes. Upon MLDS induction, Robo4-deficiency resulted in increased pancreatic vascular permeability, leukocyte infiltration to the islets and islet apoptosis, associated with reduced insulin levels and faster diabetes development. On the contrary, in vivo administration of Slit2 in mice modestly delayed the emergence of hyperglycaemia and ameliorated islet inflammation in MLDS-induced diabetes. Thus, Robo4-mediated endothelial barrier integrity reduces insulitis and islet destruction in autoimmune diabetes. Our findings highlight the importance of the endothelium as gatekeeper of pancreatic inflammation during T1DM development and may pave the way for novel Robo4-related therapeutic approaches for autoimmune diabetes.

Administration of metrnl delays the onset of diabetes in non-obese diabetic mice.

Type 1 diabetes is a chronic metabolic disease characterized by hyperglycemia due to progressive destruction of pancreatic beta cells via autoimmune attack. Meteorin-like protein (metrnl) is a secreted protein homologous to the neurotrophin metrn and it is induced after exercise in the skeletal muscle. In our paper published previously, we showed that the serum level of metrnl was significantly correlated with the lipid profile, glucose profile and insulin resistance. In this experiment, we asked whether intravenous administration of metrnl could delay the onset of diabetes in non-obese diabetic (NOD) mice. 4-week-old NOD mice were injected intravenously with metrnl. Blood glucose levels were measured weekly. Insulitis scoring, intraperitoneal glucose tolerance test, adoptive T cell transfer, flow cytometry analysis and real-time PCR were performed to investigate the underlying mechanism. The results showed that intravenous administration of metrnl delayed the onset of diabetes in NOD mice. Histology of pancreas showed a decreased infiltration of leukocytes, which was in association with augmentation of regulatory T cells, suppression of autoreactive T cells and altered cytokine secretion. To sum up, the present study showed that intravenous administration of metrnl ameliorated islet lymphocyte infiltration and modulated immune cell responses, raising the possibility that it might be beneficial in improving islet function clinically.

In vitro-derived insulin-producing cells modulate Th1 immune responses and induce IL-10 in streptozotocin-induced mouse model of pancreatic insulitis.

BACKGROUND: Insulitis is defined by the presence of immune cells infiltrating in the pancreatic islets that might progress into the complete ß-cell loss. The immunomodulatory properties of bone marrow-derived mesenchymal stem cells (BM-MSCs) have attracted much attention. This study aimed to evaluate the possible immunomodulatory effects of rat BM-MSCs and MSCs-derived insulin-producing cells (IPCs) in a mouse model of pancreatic insulitis. METHODS: Insulitis was induced in BALB/c mice using five consecutive doses of streptozotocin. MSCs or IPCs were directly injected into the pancreas of mice and their effects on the expression of Th subsets-related genes were evaluated. RESULTS: Both BM-MSCs and IPCs significantly reduced the expression of pancreatic Th1-related IFN-γ (P < 0.001 and P < 0.05, respectively) and T-bet genes (both P < 0.001). Moreover, the expression of IL-10 gene was significantly increased in IPC-treated compared to BM-MSC- or PBS-treated mice (P < 0.001 both comparisons). CONCLUSIONS: BM-MSCs and IPCs could successfully suppress pathologic Th1 immune responses in the mouse model of insulitis. However, the marked increase in IL-10 gene expression by IPCs compared to BM-MSCs suggests that their simultaneous use at the initial phase of autoimmune diabetes might be a better option to reduce inflammation but these results need to be verified by further experiments.

Hyaluronan deposition in islets may precede and direct the location of islet immune-cell infiltrates.

AIMS/HYPOTHESIS: Substantial deposition of the extracellular matrix component hyaluronan (HA) is characteristic of insulitis in overt type 1 diabetes. We investigated whether HA accumulation is detectable in islets early in disease pathogenesis and how this affects the development of insulitis and beta cell mass. METHODS: Pancreas tissue from 15 non-diabetic organ donors who were positive for islet autoantibodies (aAbs) and from 14 similarly aged aAb- control donors were examined for the amount of islet HA staining and the presence of insulitis. The kinetics of HA deposition in islets, along with the onset and progression of insulitis and changes in beta cell mass, were investigated in BioBreeding DRLyp/Lyp rats (a model of spontaneous autoimmune diabetes) from 40 days of age until diabetes onset. RESULTS: Abundant islet HA deposits were observed in pancreas tissues from n = 3 single- and n = 4 double-aAb+ donors (aAb+HAhigh). In these seven tissues, the HA-stained areas in islets measured 1000 ± 240 µm2 (mean ± SEM) and were fourfold larger than those from aAb- control tissues. The aAb+HAhigh tissues also had a greater prevalence of islets that were highly rich in HA (21% of the islets in these tissues contained the largest HA-stained areas [>2000 µm2] vs less than 1% in tissues from aAb- control donors). The amount of HA staining in islets was associated with the number of aAbs (i.e. single- or double-aAb positivity) but not with HLA genotype or changes in beta cell mass. Among the seven aAb+HAhigh tissues, three from single- and one from double-aAb+ donors did not show any islet immune-cell infiltrates, indicating that HA accumulates in aAb+ donors independently of insulitis. The three aAb+HAhigh tissues that exhibited insulitis had the largest HA-stained areas and, in these tissues, islet-infiltrating immune cells co-localised with the most prominent HA deposits (i.e. with HA-stained areas >2000 µm2). Accumulation of HA in islets was evident prior to insulitis in 7-8-week-old presymptomatic DRLyp/Lyp rats, in which the islet HA-stained area measured 2370 ± 170 µm2 (mean ± SEM), which was threefold larger than in 6-week-old rats. This initial islet HA deposition was not concurrent with beta cell loss. Insulitis was first detected in 9-10-week-old rats, in which the HA-stained areas were 4980 ± 500 µm2. At this age, the rats also exhibited a 44% reduction in beta cell mass. Further enlargement of the HA-positive areas (mean ± SEM: 7220 ± 880 µm2) was associated with invasive insulitis. HA deposits remained abundant in the islets of rats with destructive insulitis, which had lost 85% of their beta cells. CONCLUSIONS/INTERPRETATION: This study indicates that HA deposition in islets occurs early in type 1 diabetes and prior to insulitis, and points to a potential role of HA in triggering islet immune-cell infiltration and the promotion of insulitis.

Studies of insulin and proinsulin in pancreas and serum support the existence of aetiopathological endotypes of type 1 diabetes associated with age at diagnosis.

AIMS/HYPOTHESIS: It is unclear whether type 1 diabetes is a single disease or if endotypes exist. Our aim was to use a unique collection of pancreas samples recovered soon after disease onset to resolve this issue. METHODS: Immunohistological analysis was used to determine the distribution of proinsulin and insulin in the islets of pancreas samples recovered soon after type 1 diabetes onset (<2 years) from young people diagnosed at age <7 years, 7-12 years and ≥13 years. The patterns were correlated with the insulitis profiles in the inflamed islets of the same groups of individuals. C-peptide levels and the proinsulin:C-peptide ratio were measured in the circulation of a cohort of living patients with longer duration of disease but who were diagnosed in these same age ranges. RESULTS: Distinct patterns of proinsulin localisation were seen in the islets of people with recent-onset type 1 diabetes, which differed markedly between children diagnosed at <7 years and those diagnosed at ≥13 years. Proinsulin processing was aberrant in most residual insulin-containing islets of the younger group but this was much less evident in the group ≥13 years (p < 0.0001). Among all individuals (including children in the middle [7-12 years] range) aberrant proinsulin processing correlated with the assigned immune cell profiles defined by analysis of the lymphocyte composition of islet infiltrates. C-peptide levels were much lower in individuals diagnosed at <7 years than in those diagnosed at ≥13 years (median <3 pmol/l, IQR <3 to <3 vs 34.5 pmol/l, IQR <3-151; p < 0.0001), while the median proinsulin:C-peptide ratio was increased in those with age of onset <7 years compared with people diagnosed aged ≥13 years (0.18, IQR 0.10-0.31) vs 0.01, IQR 0.009-0.10 pmol/l; p < 0.0001). CONCLUSIONS/INTERPRETATION: Among those with type 1 diabetes diagnosed under the age of 30 years, there are histologically distinct endotypes that correlate with age at diagnosis. Recognition of such differences should inform the design of future immunotherapeutic interventions designed to arrest disease progression.

Systemic Klotho therapy protects against insulitis and enhances beta-cell mass in NOD mice.

The levels of the anti-aging protein α-Klotho, in its soluble form (s-Klotho), are depressed in the circulation of patients with type 1 diabetes (T1D) or type 2 diabetes (T2D). Gene transfer experiments have suggested a protective role for ß-cell specific expression of α-Klotho in murine models of T1D and T1D, but these approaches are not easily translatable to clinical therapy. It is unknown whether systemic s-Klotho protein treatment ameliorates disease in T1D, which is characterized by autoimmune destruction of ß cells. We previously reported from in vitro experiments with ß cells that s-Klotho increases insulin secretion, reduces cells death and promotes ß-cell replication. Here, we investigated s-Klotho protein therapy in NOD mice, which have autoimmune T1D. We observed that diabetic NOD mice have significantly lower plasma levels of s-Klotho, compared to their non-diabetic counterparts. To examine in vivo effects of Klotho, we treated NOD mice with s-Klotho protein, or with a Klotho blocking antibody. Systemic treatment with s-Klotho ameliorated diabetes; notably increasing ß-cell replication and total ß-cell mass. Klotho expression was increased locally in the islets. s-Klotho also markedly reduced immune-cell infiltration of islets (insulitis). In contrast, administration of the Klotho antibody was detrimental, and aggravated the loss of ß-cell mass. Thus, s-Klotho has protective effects in this model of T1D, and this appears to depend on a combination of increased ß-cell replication and reduced insulitis. These findings suggest that s-Klotho might be effective as a new therapeutic agent for T1D.

From immunohistological to anatomical alterations of human pancreas in type 1 diabetes: New concepts on the stage.

The histological analysis of human pancreatic samples in type 1 diabetes (T1D) has been proven essential to move forward in the evaluation of in situ events characterizing T1D. Increasing availability of pancreatic tissues collected from diabetic multiorgan donors by centralized biorepositories, which have shared tissues among researchers in the field, has allowed a deeper understanding of T1D pathophysiology, using novel immunohistological and high-throughput methods. In this review, we provide a comprehensive update of the main recent advancements in the characterization of cellular and molecular events involving endocrine and exocrine pancreas as well as the immune system in the onset and progression of T1D. Additionally, we underline novel elements, which provide evidence that T1D pathological changes affect not only islet ß-cells but also the entire pancreas.

Anti-Inflammatory Strategies Targeting Metaflammation in Type 2 Diabetes.

One of the concepts explaining the coincidence of obesity and type 2 diabetes (T2D) is the metaflammation theory. This chronic, low-grade inflammatory state originating from metabolic cells in response to excess nutrients, contributes to the development of T2D by increasing insulin resistance in peripheral tissues (mainly in the liver, muscles, and adipose tissue) and by targeting pancreatic islets and in this way impairing insulin secretion. Given the role of this not related to infection inflammation in the development of both: insulin resistance and insulitis, anti-inflammatory strategies could be helpful not only to control T2D symptoms but also to treat its causes. This review presents current concepts regarding the role of metaflammation in the development of T2D in obese individuals as well as data concerning possible application of different anti-inflammatory strategies (including lifestyle interventions, the extra-glycemic potential of classical antidiabetic compounds, nonsteroidal anti-inflammatory drugs, immunomodulatory therapies, and bariatric surgery) in the management of T2D.

Targeting gut microbiota and barrier function with prebiotics to alleviate autoimmune manifestations in NOD mice.

AIMS/HYPOTHESIS: Adopting a diet containing indigestible fibre compounds such as prebiotics to fuel advantageous bacteria has proven beneficial for alleviating inflammation. The effect of the microbial changes on autoimmunity, however, remains unknown. We studied the effects of prebiotic xylooligosaccharides (XOS) on pancreatic islet and salivary gland inflammation in NOD mice and tested whether these were mediated by the gut microbiota. METHODS: Mother and offspring mice were fed an XOS-supplemented diet until diabetes onset or weaning and were compared with a control-fed group. Diabetes incidence was monitored, insulitis and sialadenitis were scored in histological sections from adult mice, and several metabolic and immune variables were analysed in mice before the development of diabetes. Gut barrier function was assessed using an in vivo FITC-dextran permeability test. The importance of XOS-mediated gut microbial changes were evaluated in antibiotic-treated mice fed either XOS or control diet or given a faecal microbiota transplant from test animals. RESULTS: Diabetes onset was delayed in the XOS-fed mice, which also had fewer cellular infiltrations in their pancreatic islets and salivary glands. Interestingly, insulitis was most reduced in the XOS-fed groups when the mice were also treated with an antibiotic cocktail. There was no difference in sialadenitis between the dietary groups treated with antibiotics; the mice were protected by microbiota depletion regardless of diet. Faecal microbiota transplantation was not able to transfer protection. No major differences in glucose-insulin regulation, glucagon-like peptide-1, or short-chain fatty acid production were related to the XOS diet. The XOS diet did, however, reduce gut permeability markers in the small and large intestine. This was accompanied by a more anti-inflammatory environment locally and systemically, dominated by a shift from M1 to M2 macrophages, a higher abundance of activated regulatory T cells, and lower levels of induction of natural killer T cells and cytotoxic T cells. CONCLUSIONS/INTERPRETATION: Prebiotic XOS have microbiota-dependent effects on salivary gland inflammation and microbiota-independent effects on pancreatic islet pathology that are accompanied by an improved gut barrier that seems able to heighten control of intestinal diabetogenic antigens that have the potential to penetrate the mucosa to activate autoreactive immune responses.

B cell depletion reduces T cell activation in pancreatic islets in a murine autoimmune diabetes model.

AIMS/HYPOTHESIS: Type 1 diabetes is a T cell-mediated autoimmune disease characterised by the destruction of beta cells in the islets of Langerhans, resulting in deficient insulin production. B cell depletion therapy has proved successful in preventing diabetes and restoring euglycaemia in animal models of diabetes, as well as in preserving beta cell function in clinical trials in the short term. We aimed to report a full characterisation of B cell kinetics post B cell depletion, with a focus on pancreatic islets. METHODS: Transgenic NOD mice with a human CD20 transgene expressed on B cells were injected with an anti-CD20 depleting antibody. B cells were analysed using multivariable flow cytometry. RESULTS: There was a 10 week delay in the onset of diabetes when comparing control and experimental groups, although the final difference in the diabetes incidence, following prolonged observation, was not statistically significant (p = 0.07). The co-stimulatory molecules CD80 and CD86 were reduced on stimulation of B cells during B cell depletion and repopulation. IL-10-producing regulatory B cells were not induced in repopulated B cells in the periphery, post anti-CD20 depletion. However, the early depletion of B cells had a marked effect on T cells in the local islet infiltrate. We demonstrated a lack of T cell activation, specifically with reduced CD44 expression and effector function, including IFN-γ production from both CD4+ and CD8+ T cells. These CD8+ T cells remained altered in the pancreatic islets long after B cell depletion and repopulation. CONCLUSIONS/INTERPRETATION: Our findings suggest that B cell depletion can have an impact on T cell regulation, inducing a durable effect that is present long after repopulation. We suggest that this local effect of reducing autoimmune T cell activity contributes to delay in the onset of autoimmune diabetes.

Fifty years of pancreatic islet pathology in human type 1 diabetes: insights gained and progress made.

Type 1 diabetes is increasing in incidence in many parts of the world and it might be imagined that the pathological processes that underlie disease progression are firmly understood. However, this is not the case; rather, our collective understanding is still surprisingly rudimentary. There are various reasons for this but one of the most important is that the target organ (the pancreas) has been examined at, or soon after, diagnosis in only a small number of cases worldwide over the past half a century. This review provides a summary of some of the insights gained from these studies and highlights areas of ongoing uncertainty. In particular, it considers the process of insulitis (a form of islet inflammation that occurs characteristically in type 1 diabetes) and discusses the factors that may influence the access of immune cells to the beta cells. Attention is also drawn to recent evidence implying that two distinct profiles of insulitis exist, which occur differentially in people who develop type 1 diabetes at increasing ages. Emphasis is also placed on the emerging (and somewhat surprising) consensus that the extent of beta cell loss is variable among people with type 1 diabetes and that many (especially those who are older at onset) retain significant numbers of insulin-producing cells long after diagnosis. We conclude by emphasising the importance of renewed efforts to study the human pancreas at disease onset and consider how the current insights may inform the design of future strategies to slow or halt the rate of beta cell loss.

Pancreas Pathology During the Natural History of Type 1 Diabetes.

PURPOSE OF REVIEW: We provide an overview of pancreas pathology in type 1 diabetes (T1D) in the context of its clinical stages. RECENT FINDINGS: Recent studies of pancreata from organ donors with T1D and non-diabetic donors expressing T1D-associated autoantibodies reveal pathological changes/disease mechanisms beyond the well-known loss of ß cells and lymphocytic infiltrates of the islets (insulitis), including ß-cell stress, dysfunction, and viral infections. Pancreas pathology evolves through disease stages, is asynchronous, and demonstrates a chronic disease that remains active years after diagnosis. Critically, ß-cell loss is not complete at onset, although young age is associated with increased severity. The recognition of multiple pathogenic alterations and the chronic nature of disease mechanisms during and after the development of T1D inform improved clinical trial design and reveal additional targets for therapeutic manipulation, in the context of an expanded time window for intervention.

Noninvasive mapping of pancreatic inflammation in recent-onset type-1 diabetes patients.

The inability to visualize the initiation and progression of type-1 diabetes (T1D) noninvasively in humans is a major research and clinical stumbling block. We describe an advanced, exportable method for imaging the pancreatic inflammation underlying T1D, based on MRI of the clinically approved magnetic nanoparticle (MNP) ferumoxytol. The MNP-MRI approach, which reflects nanoparticle uptake by macrophages in the inflamed pancreatic lesion, has been validated extensively in mouse models of T1D and in a pilot human study. The methodological advances reported here were enabled by extensive optimization of image acquisition at 3T, as well as by the development of improved MRI registration and visualization technologies. A proof-of-principle study on patients recently diagnosed with T1D versus healthy controls yielded two major findings: First, there was a clear difference in whole-pancreas nanoparticle accumulation in patients and controls; second, the patients with T1D exhibited pronounced inter- and intrapancreatic heterogeneity in signal intensity. The ability to generate noninvasive, 3D, high-resolution maps of pancreatic inflammation in autoimmune diabetes should prove invaluable in assessing disease initiation and progression and as an indicator of response to emerging therapies.

Insulitis in human diabetes: a histological evaluation of donor pancreases.

AIMS/HYPOTHESIS: According to the consensus criteria developed for type 1 diabetes, an individual can be diagnosed with insulitis when ≥ 15 CD45+ cells are found within the parenchyma or in the islet-exocrine interface in ≥ 3 islets. The aim of this study was to determine the frequency of individuals with type 2 diabetes fulfilling these criteria with reference to non-diabetic and type 1 diabetic individuals. METHODS: Insulitis was determined by examining CD45+ cells in the pancreases of 50, 13 and 44 organ donors with type 2 diabetes, type 1 diabetes and no diabetes, respectively. CD3+ cells (T cells) infiltrating the islets were evaluated in insulitic donors. In insulitic donors with type 2 diabetes, the pancreases were characterised according to the presence of CD68 (macrophages), myeloperoxidase (MPO; neutrophils), CD3, CD20 (B cells) and HLA class I hyperstained islets. In all type 2 diabetic donors, potential correlations of insulitis with dynamic glucose-stimulated insulin secretion in vitro or age, BMI, HbA1c or autoantibody positivity were examined. RESULTS: Overall, 28% of the type 2 diabetic donors fulfilled the consensus criteria for insulitis developed for type 1 diabetes. Of the type 1 diabetic donors, 31% fulfilled the criteria. None of the non-diabetic donors met the criteria. Only type 1 diabetic donors had ≥ 15 CD3+ cells in ≥ 3 islets. Type 2 diabetic donors with insulitis also had a substantial number of CD45+ cells in the exocrine parenchyma. Macrophages constituted the largest fraction of CD45+ cells, followed by neutrophils and T cells. Of type 2 diabetic pancreases with insulitis, 36% contained islets that hyperstained for HLA class I. Isolated islets from type 2 diabetic donors secreted less insulin than controls, although with preserved dynamics. Insulitis in the type 2 diabetic donors did not correlate with glucose-stimulated insulin secretion, the presence of autoantibodies, BMI or HbA1c. CONCLUSIONS/INTERPRETATION: The current definition of insulitis cannot be used to distinguish pancreases retrieved from individuals with type 1 diabetes from those with type 2 diabetes. On the basis of our findings, we propose a revised definition of insulitis, with a positive diagnosis when ≥ 15 CD3+ cells, not CD45+ cells, are found in ≥ 3 islets.

ERK1 is dispensable for mouse pancreatic beta cell function but is necessary for glucose-induced full activation of MSK1 and CREB.

AIMS/HYPOTHESIS: Insufficient insulin secretion from pancreatic beta cells, which is associated with a decrease in beta cell mass, is a characteristic of type 2 diabetes. Extracellular signal-related kinase 1 and 2 (ERK1/2) inhibition in beta cells has been reported to affect insulin secretion, gene transcription and survival, although whether ERK1 and ERK2 play distinct roles is unknown. The aim of this study was to assess the individual roles of ERK1 and ERK2 in beta cells using ERK1 (also known as Mapk3)-knockout mice (Erk1 -/- mice) and pharmacological approaches. METHODS: NAD(P)H, free cytosolic Ca2+ concentration and insulin secretion were determined in islets. ERK1 and ERK2 subplasmalemmal translocation and activity was monitored using total internal reflection fluorescence microscopy. ERK1/2, mitogen and stress-activated kinase1 (MSK1) and cAMP-responsive element-binding protein (CREB) activation were evaluated by western blot and/or immunocytochemistry. The islet mass was determined from pancreatic sections. RESULTS: Glucose induced rapid subplasmalemmal recruitment of ERK1 and ERK2. When both ERK1 and ERK2 were inhibited simultaneously, the rapid transient peak of the first phase of glucose-induced insulin secretion was reduced by 40% (p < 0.01), although ERK1 did not appear to be involved in this process. By contrast, ERK1 was required for glucose-induced full activation of several targets involved in beta cell survival; MSK1 and CREB were less active in Erk1 -/- mouse beta cells (p < 0.01) compared with Erk1 +/+ mouse beta cells, and their phosphorylation could only be restored when ERK1 was re-expressed and not when ERK2 was overexpressed. Finally, the islet mass of Erk1 -/- mice was slightly increased in young animals (4-month-old mice) vs Erk1 +/+ mice (section occupied by islets [mean ± SEM]: 0.74% ± 0.03% vs 0.62% ± 0.04%; p < 0.05), while older mice (10 months old) were less prone to age-associated pancreatic peri-insulitis (infiltrated islets [mean ± SEM]: 7.51% ± 1.34% vs 2.03% ± 0.51%; p < 0.001). CONCLUSIONS/INTERPRETATION: ERK1 and ERK2 play specific roles in beta cells. ERK2 cannot always compensate for the lack of ERK1 but the absence of a clear-cut phenotype in Erk1 -/- mice shows that ERK1 is dispensable in normal conditions.