Vedran Brezar

Beyond the Hormone: Insulin as an Autoimmune Target in Type 1 Diabetes

Insulin is not only the hormone produced by pancreatic β-cells but also a key target antigen of the autoimmune islet destruction leading to type 1 diabetes. Despite cultural biases between the fields of endocrinology and immunology, these two facets should not be regarded separately, but rather harmonized in a unifying picture of diabetes pathogenesis. There is increasing evidence suggesting that metabolic factors (β-cell dysfunction, insulin resistance) and immunological components (inflammation and β-cell-directed adaptive immune responses) may synergize toward islet destruction, with insulin standing at the crossroad of these pathways. This concept further calls for a revision of the classical dichotomy between type 1 and type 2 diabetes because metabolic and immune mechanisms may both contribute to different extents to the development of different forms of diabetes. After providing a background on the mechanisms of β-cell autoimmunity, we will explain the role of insulin and its precursors as target antigens expressed not only by β-cells but also in the thymus. Available knowledge on the autoimmune antibody and T-cell responses against insulin will be summarized. A unifying scheme will be proposed to show how different aspects of insulin biology may lead to β-cell destruction and may be therapeutically exploited. We will argue about possible reasons why insulin remains the mainstay of metabolic control in type 1 diabetes but has so far failed to prevent or halt β-cell autoimmunity as an immune modulatory reagent.

Antibodies Recognizing Mycobacterium avium paratuberculosis Epitopes Cross-React with the Beta-Cell Antigen ZnT8 in Sardinian Type 1 Diabetic Patients

The environmental factors at play in the pathogenesis of type 1 diabetes (T1D) remain enigmatic. Mycobacterium avium subspecies paratuberculosis (MAP) is transmitted from dairy herds to humans through food contamination. MAP causes an asymptomatic infection that is highly prevalent in Sardinian T1D patients compared with type 2 diabetes (T2D) and healthy controls. Moreover, MAP elicits humoral responses against several mycobacterial proteins. We asked whether antibodies (Abs) against one of these proteins, namely MAP3865c, which displays a sequence homology with the β-cell protein zinc transporter 8 (ZnT8) could be cross-reactive with ZnT8 epitopes. To this end, Ab responses against MAP3865c were analyzed in Sardinian T1D, T2D and healthy subjects using an enzymatic immunoassay. Abs against MAP3865c recognized two immunodominant transmembrane epitopes in 52–65% of T1D patients, but only in 5–7% of T2D and 3–5% of healthy controls. There was a linear correlation between titers of anti-MAP3865c and anti-ZnT8 Abs targeting these two homologous epitopes, and pre-incubation of sera with ZnT8 epitope peptides blocked binding to the corresponding MAP3865c peptides. These results demonstrate that Abs recognizing MAP3865c epitopes cross-react with ZnT8, possibly underlying a molecular mimicry mechanism, which may precipitate T1D in MAP-infected individuals.

T Cell Recognition of Autoantigens in Human Type 1 Diabetes: Clinical Perspectives

Type 1 diabetes (T1D) is an autoimmune disease driven by the activation of lymphocytes against pancreatic β-cells. Among β-cell autoantigens, preproinsulin has been ascribed a key role in the T1D process. The successive steps that control the activation of autoreactive lymphocytes have been extensively studied in animal models of T1D, but remains ill defined in man. In man, T lymphocytes, especially CD8+ T cells, are predominant within insulitis. Developing T-cell assays in diabetes autoimmunity is, thus, a major challenge. It is expected to help defining autoantigens and epitopes that drive the disease process, to pinpoint key functional features of epitope-specific T lymphocytes along the natural history of diabetes and to pave the way towards therapeutic strategies to induce immune tolerance to β-cells. New T-cell technologies will allow defining autoreactive T-cell differentiation programs and characterizing autoimmune responses in comparison with physiologically appropriate immune responses. This may prove instrumental in the discovery of immune correlates of efficacy in clinical trials.

MECHANISMS IN ENDOCRINOLOGY: Insulin and type 1 diabetes: immune connections

Insulin is the hormone produced by pancreatic β-cells, with a central role in carbohydrate and fat metabolism. Together with its precursors preproinsulin and proinsulin, insulin is also a key target antigen (Ag) of the autoimmune islet destruction leading to type 1 diabetes. Being recognized by both autoantibodies (aAbs) and autoreactive T cells, insulin plays a triggering role, at least in rodent models, in diabetes pathogenesis. It is expressed not only by β-cells but also in the thymus, where it plays a major role in central tolerance mechanisms. We will summarize current knowledge concerning insulin, its role in β-cell autoimmunity as initial target Ag, its recognition by aAbs and autoreactive T cells, and the detection of these immune responses to provide biomarkers for clinical trials employing insulin as an immune modulatory agent.

Short-term subcutaneous insulin treatment delays but does not prevent diabetes in NOD mice.

Despite encouraging results in the NOD mouse, type 1 diabetes prevention trials using subcutaneous insulin have been unsuccessful. To explain these discrepancies, 3‐week‐old NOD mice were treated for 7 weeks with subcutaneous insulin at two different doses: a high dose (0.5 U/mouse) used in previous mouse studies; and a low dose (0.005 U/mouse) equivalent to that used in human trials. Effects on insulitis and diabetes were monitored along with immune and metabolic modifications. Low‐dose insulin did not have any effect on disease incidence. High‐dose treatment delayed but did not prevent diabetes, with reduced insulitis reappearing once insulin discontinued. This effect was not associated with significant immune changes in islet infiltrates, either in terms of cell composition or frequency and IFN‐γ secretion of islet‐reactive CD8+ T cells recognizing the immunodominant epitopes insulin B15‐23 and islet‐specific glucose‐6‐phosphatase catalytic subunit‐related protein (IGRP)206‐214. Delayed diabetes and insulitis were assoc‐iated with lower blood glucose and endogenous C‐peptide levels, which rapidly returned to normal upon treatment discontinuation. In conclusion, high‐ but not low‐dose prophylactic insulin treatment delays diabetes onset and is associated with metabolic changes suggestive of β‐cell “rest” which do not persist beyond treatment. These findings have important implications for designing insulin‐based prevention trials.

To B or not to B: (anti)bodies of evidence on the crime scene of type 1 diabetes?

Although autoantibodies (auto-Abs) against β-cell antigens helped in defining type 1 diabetes as an autoimmune disease and are invaluable biomarkers, their pathogenic role is unclear. Studies in nonobese diabetic (NOD) mice devoid of B cells ( Igμ null or treated with anti-μ Abs) suggest that B cells are necessary for the disease to develop (1,2). The critical role of B cells in this process is thought to be linked to their antigen-presenting function through major histocompatibility class II molecules, as NOD mice harboring I-Ag7–deficient B cells are also protected from diabetes (3). The capacity of B cells to efficiently uptake β-cell antigens through surface Ig is critical to this function, as inhibiting this Ig-mediated uptake abolishes the β-cell antigen-presenting function of B cells in vitro (4), while transgenic manipulation of the Ig specificity in NOD mice impacts on diabetes incidence (5). Thus, autoreactive B cells may be exquisitely efficient in capturing and presenting self antigens, leading to autoimmune T-cell activation. In a therapeutic perspective, treatment with depleting anti-CD20 Abs delays and reduces diabetes onset in NOD mice and is even capable of reversing established disease (6). These findings have been successfully translated into human clinical trials (7). In this scenario, the role of B cell–secreted auto-Abs has been controversial. On one hand, NOD embryos implanted into nonautoimmune foster mothers are diabetes-protected compared with embryos implanted into NOD females, suggesting that maternally transmitted factors (but not necessarily Abs) play a role (8). Moreover, passive transfer of Abs against islet-expressed …

T Cells Recognizing a Peptide Contaminant Undetectable by Mass Spectrometry

Synthetic peptides are widely used in immunological research as epitopes to stimulate their cognate T cells. These preparations are never completely pure, but trace contaminants are commonly revealed by mass spectrometry quality controls. In an effort to characterize novel major histocompatibility complex (MHC) Class I-restricted β-cell epitopes in non-obese diabetic (NOD) mice, we identified islet-infiltrating CD8+ T cells recognizing a contaminating peptide. The amount of this contaminant was so small to be undetectable by direct mass spectrometry. Only after concentration by liquid chromatography, we observed a mass peak corresponding to an immunodominant islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206-214 epitope described in the literature. Generation of CD8+ T-cell clones recognizing IGRP206-214 using a novel method confirmed the identity of the contaminant, further underlining the immunodominance of IGRP206-214. If left undetected, minute impurities in synthetic peptide preparations may thus give spurious results.

O16 Des anticorps dirigés contre le Mycobacterium avium paratuberculosis montrent une réactivité croisée avec l’antigène bêta-cellulaire ZnT8 chez les patients diabétiques de type 1 sardes

Introduction Les facteurs environnementaux impliques dans la pathogenese du diabete de type 1 (DT1) demeurent enigmatiques. Le Mycobacterium avium paratuberculosis (MAP) est transmis du betail laitier a l’homme par contamination des aliments. Le MAP provoque une infection asymptomatique qui est tres prevalente chez les patients DT1 par rapport aux sujets DT2 et sains de Sardaigne, une region qui affiche une incidence de DT1 parmi les plus hautes au monde. De plus, le MAP induit une reponse anticorpale contre plusieurs proteines mycobacteriennes. Patients et methodes Une de ces proteines, nommee MAP3865c, montre une homologie de sequence avec la proteine β-cellulaire transporteur de zinc 8 (ZnT8). Nous avons donc recherche si les anticorps (Acs) diriges contre MAP3865c pouvaient reconnaitre de facon croisee les epitopes peptidiques ZnT8 correspondants. A cette fin, les Acs anti-MAP386c ont ete mesures chez des sujets sardes DT1, DT2 et sains a l’aide d’un test Elisa. Resultats Les Acs anti-MAP3865c reconnaissent deux epitopes immuno-dominants dans 52–65 % des patients DT1, mais seulement dans 5–7 % des DT2 et 3–5 % des sains. Il y a une correlation lineaire entre les titres des Acs anti-MAP3865c et les titres des anti-ZnT8 qui ciblent les deux epitopes homologues. L’adsorption des serums par les epitopes ZnT8 inhibe la liaison aux epitopes MAP3865c correspondants. Conclusion Les Acs reconnaissant des epitopes MAP3865c presentent une reactivite croisee avec ZnT8. Cette reactivite croisee pourrait declencher un mecanisme de mimetisme moleculaire accelerant le DT1 chez des individus infectes par le MAP

P140 Un traitement à court terme par l’insuline sous-cutanée retarde mais ne prévient pas le diabète auto-immun chez la souris NOD

Introduction En depit de resultats encourageants obtenus chez la souris NOD, les essais cliniques de prevention du diabete de type 1 par l’insuline sous-cutanee sont restes sans succes. Materiels et methodes Pour expliquer ces divergences, des souris NOD âgees de 3 semaines ont ete traitees pendant 7 semaines par deux differentes doses d’insuline sous-cutanee : une forte dose (0,5 U/souris) utilisee dans des etudes murines anterieures; et une faible dose (0,005 U/souris) equivalente a celle utilisee dans les essais cliniques chez l’homme. Les effets sur l’insulite et le diabete et les modifications immunologiques et metaboliques ont ete suivis pendant et apres traitement. Resultats Le traitement par faible dose d’insuline n’a pas montre d’effet sur l’incidence de la maladie. Le traitement par forte dose retarde mais ne previent pas le diabete, induisant une diminution de l’insulite qui toutefois reapparait apres arret du traitement. Cet effet n’est pas associe a des modifications immunologiques significatives dans l’infiltrat des ilots, ni en terme de composition cellulaire, ni en terme de frequence et de secretion d’IFN-g par les cellules T CD8 + reconnaissant les epitopes immunodominants de l’insuline B15–23 et de la proteine islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) 206–214. Les retards du diabete et de l’insulite sont associes a des niveaux de glucose sanguin et de peptide C endogene diminues, mais qui reviennent rapidement a la normale apres interruption du traitement. Conclusion Un traitement prophylactique par l’insuline a forte dose – mais pas a faible dose – retarde l’apparition du diabete et est associe a des changements metaboliques suggerant une « mise au repos » des cellules beta, qui toutefois ne persistent pas au-dela du traitement. Ces resultats ont d’importantes implications pour la conception d’essais cliniques bases sur une therapie prophylactique par l’insuline.