Marc Diedisheim

Cytotoxic and regulatory roles of mucosal-associated invariant T cells in type 1 diabetes

Type 1 diabetes (T1D) is an autoimmune disease that results from the destruction of pancreatic β-cells by the immune system that involves innate and adaptive immune cells. Mucosal-associated invariant T cells (MAIT cells) are innate-like T-cells that recognize derivatives of precursors of bacterial riboflavin presented by the major histocompatibility complex (MHC) class I–related molecule MR1. Since T1D is associated with modification of the gut microbiota, we investigated MAIT cells in this pathology. In patients with T1D and mice of the non-obese diabetic (NOD) strain, we detected alterations in MAIT cells, including increased production of granzyme B, which occurred before the onset of diabetes. Analysis of NOD mice that were deficient in MR1, and therefore lacked MAIT cells, revealed a loss of gut integrity and increased anti-islet responses associated with exacerbated diabetes. Together our data highlight the role of MAIT cells in the maintenance of gut integrity and the control of anti-islet autoimmune responses. Monitoring of MAIT cells might represent a new biomarker of T1D, while manipulation of these cells might open new therapeutic strategies.

β-cell Mass in Nondiabetic Autoantibody-Positive Subjects: An Analysis Based on the Network for Pancreatic Organ Donors Database.

CONTEXT Little information is available about β-cell mass in antibody-positive (Ab+) nondiabetic subjects. OBJECTIVE We have investigated whether the publicly available virtual slides of the Network for Pancreatic Organ Donors with Diabetes (nPOD) project can be used to assess β-cell mass and distribution in nondiabetic antibody-negative (Ab−) and antibody-positive (Ab+) subjects and in patients with recent-onset type 1 diabetes (T1D). SUBJECTS AND METHODS We developed a semi-automated quantification method and applied it to 415 insulin-stained slides from 69 Ab− subjects, 101 slides from 18 Ab+ subjects, and 46 slides from eight recent-onset (<3 y) T1D subjects. Among these subjects, 48, 17, and seven had an available pancreatic mass, respectively, and were used for the quantification of β-cell mass. RESULTS In Ab− subjects, the β-cell and endocrine mass were 0.66 ± 0.42 and 1.0 ± 0.65 g, respectively. Nonexocrine tissue represented 29% of pancreatic area, a proportion that increased with age. Proportional β-cell area relative to total pancreatic area was higher in the tail compared with head (0.83 vs 0.71%; P < .001). In Ab+ subjects, β-cell mass and β-cell area were similar to those of Ab− individuals, whereas these parameters were dramatically decreased in recent-onset T1D patients. CONCLUSION The virtual slides of the nPOD project can be used for quantification projects. In Ab+ nondiabetic subjects, the β-cell mass was not decreased. However, as this cohort is largely composed of donors from the general population, with a single autoantibody, future studies with a larger number of donors with multiple autoantibodies and predisposing human leucocyte antigen genes are required to better define the dynamics of β-cell destruction in the preclinical phases of T1D.

Xenotropic retrovirus Bxv1 in human pancreatic β cell lines

It has been reported that endogenous retroviruses can contaminate human cell lines that have been passaged as xenotransplants in immunocompromised mice. We previously developed and described 2 human pancreatic β cell lines (EndoC-βH1 and EndoC-βH2) that were generated in this way. Here, we have shown that B10 xenotropic virus 1 (Bxv1), a xenotropic endogenous murine leukemia virus (MuLV), is present in these 2 recently described cell lines. We determined that Bxv1 was also present in SCID mice that were used for in vivo propagation of EndoC-βH1/2 cells, suggesting that contamination occurred during xenotransplantation. EndoC-βH1/2 cells released Bxv1 particles that propagated to human 293T and Mus dunni cells. Mobilization assays demonstrated that Bxv1 transcomplements defective MuLV-based retrovectors. In contrast, common rodent β cell lines, rat INS-1E and RIN-5F cells and mouse MIN6 and βTC3 cells, displayed either no or extremely weak xenotropic helper activity toward MuLV-based retrovectors, although xenotropic retrovirus sequences and transcripts were detected in both mouse cell lines. Bxv1 propagation from EndoC-βH1/2 to 293T cells occurred only under optimized conditions and was overall poorly efficient. Thus, although our data imply that MuLV-based retrovectors should be cautiously used in EndoC-βH1/2 cells, our results indicate that an involuntary propagation of Bxv1 from these cells can be easily avoided with good laboratory practices.

Decreased α-cell mass and early structural alterations of the exocrine pancreas in patients with type 1 diabetes: An analysis based on the nPOD repository

Background and aims Abnormal glucagon secretion and functional alterations of the exocrine pancreas have been described in patients with type 1 diabetes (T1D), but their respective anatomical substrata have seldom been investigated. Our aim was to develop an automated morphometric analysis process to characterize the anatomy of α-cell and exocrine pancreas in patients with T1D, using the publicly available slides of the Network for Pancreatic Organ Donors (nPOD). Materials and methods The ratio of β- and α-cell area to total tissue area were quantified in 75 patients with T1D (thereafter patients) and 66 control subjects (thereafter controls), on 2 insulin-stained and 4 glucagon-stained slides from both the head and the tail of the pancreas. The β- and α-cell masses were calculated in the 66 patients and the 50 controls for which the pancreas weight was available. Non-exocrine-non-endocrine tissue area (i.e. non-acinar, non-insular tissue) to total tissue area ratio was evaluated on both insulin- and glucagon-stained slides. Results were expressed as mean ±SD. Results An automated quantification method was set up using the R software and was validated by quantification of β-cell mass, a well characterized parameter. β-cell mass was 29.6±112 mg in patients and 628 ±717 mg in controls (p<0.0001). α-cell mass was 181±176 mg in patients and 349 ±241mg in controls (p<0.0001). Non-exocrine-non-endocrine area to total tissue area ratio was 39±9% in patients and 29± 10% in controls (p<0.0001) and increased with age in both groups, with no correlation with diabetes duration in patients. Conclusion The absolute α-cell mass was lower in patients compared to controls, in proportion to the decrease in pancreas weight observed in patients. Non-exocrine-non-endocrine area to total tissue area ratio increased with age in both groups but was higher in patients at all ages.

Virus-like infection induces human β cell dedifferentiation

Type 1 diabetes (T1D) is a chronic disease characterized by an autoimmune-mediated destruction of insulin-producing pancreatic β cells. Environmental factors such as viruses play an important role in the onset of T1D and interact with predisposing genes. Recent data suggest that viral infection of human islets leads to a decrease in insulin production rather than β cell death, suggesting loss of β cell identity. We undertook this study to examine whether viral infection could induce human β cell dedifferentiation. Using the functional human β cell line EndoC-βH1, we demonstrate that polyinosinic-polycytidylic acid (PolyI:C), a synthetic double-stranded RNA that mimics a byproduct of viral replication, induces a decrease in β cell-specific gene expression. In parallel with this loss, the expression of progenitor-like genes such as SOX9 was activated following PolyI:C treatment or enteroviral infection. SOX9 was induced by the NF-κB pathway and also in a paracrine non-cell-autonomous fashion through the secretion of IFN-α. Lastly, we identified SOX9 targets in human β cells as potentially new markers of dedifferentiation in T1D. These findings reveal that inflammatory signaling has clear implications in human β cell dedifferentiation.

Modeling human pancreatic beta cell dedifferentiation

Objective Dedifferentiation could explain reduced functional pancreatic β-cell mass in type 2 diabetes (T2D). Methods Here we model human β-cell dedifferentiation using growth factor stimulation in the human β-cell line, EndoC-βH1, and human pancreatic islets. Results Fibroblast growth factor 2 (FGF2) treatment reduced expression of β-cell markers, (INS, MAFB, SLC2A2, SLC30A8, and GCK) and activated ectopic expression of MYC, HES1, SOX9, and NEUROG3. FGF2-induced dedifferentiation was time- and dose-dependent and reversible upon wash-out. Furthermore, FGF2 treatment induced expression of TNFRSF11B, a decoy receptor for RANKL and protected β-cells against RANKL signaling. Finally, analyses of transcriptomic data revealed increased FGF2 expression in ductal, endothelial, and stellate cells in pancreas from T2D patients, whereas FGFR1, SOX,9 and HES1 expression increased in islets from T2D patients. Conclusions We thus developed an FGF2-induced model of human β-cell dedifferentiation, identified new markers of dedifferentiation, and found evidence for increased pancreatic FGF2, FGFR1, and β-cell dedifferentiation in T2D.

O79 Distribution des cellules B chez l’homme : une analyse de 69 individus non diabétiques de la base nPOD

Objectif La base nPOD, network for pancreatic organ donors, sous l’egide de la JDRF offre l’opportunite d’une analyse a grande echelle de pancreas humains de haute qualite. Nous avons utilise cette base pour l’etude de la distribution des ilots dans le pancreas humain. Materiels et methodes Nous avons telecharge dans la base nPOD toutes les lames virtuelles disponibles au 01/07/2013, marquees par un anti-insuline au fast-red, provenant d’individus non diabetiques au moment du prelevement. Les lames virtuelles permettent d’analyser l’integralite de chaque coupe a un agrandissement x100. Nous avons developpe une methode de quantification semi-automatisee des surfaces immunomarquees a l’insuline, des ilots endocrines et du tissu non-pancreatique (tissu adipeux, tissu conjonctif, vaisseaux). Resultats Nous avons analyse 415 lames virtuelles provenant de 69 individus. Notre protocole de mesure des surfaces pancreatiques est valide par des resultats conformes aux donnees de la litterature : masse insulaire moyenne de 1 g chez les adultes, correlee a l’âge et a l’IMC, et augmentee de 65 % chez les adultes obeses compares aux adultes normo-ponderaux. Le tissu non pancreatique represente 29 % de la surface de chaque coupe de pancreas, cette proportion augmente avec l’âge. C’est un parametre important a prendre en compte dans de telles analyses morphometriques, ce qui n’a pas toujours ete le cas dans la litterature. Ainsi, selon le type d’analyse, la fraction cellulaire beta est plus importante dans la queue comparee a la tete du pancreas lorsqu’elle est exprimee en proportion de la surface de coupe (0,83 vs 0,71 % dans la tete), mais pas lorsque l’analyse exclut le tissu non pancreatique. Conclusion Notre travail fournit un protocole automatise d’analyse du pancreas humain a partir de la base nPOD qui inclut a ce jour plus de 250 individus diabetiques et non diabetiques. Cette automatisation de l’analyse permet l’etude de grands echantillons. A partir de cette base nous ne retrouvons pas d’heterogeneite de distribution des ilots entre la tete et la queue sur un echantillon de 69 individus non diabetiques.