Development of Type 1 Diabetes: Monocytes and dendritic cells in the pancreas

This thesis focuses on the presence of precursors for dendritic cells and the characterization of dendritic cell subsets in the normal pancreas in mice and humans as well as in the pancreas of the NOD mouse, a type 1 diabetes mouse model. Therefore, we give a short introduction to dendritic cells, the NOD mouse and type 1 diabetes

Abnormalities of dendritic cell precursors in the pancreas of the NOD mouse model of diabetes

The non-obese diabetic (NOD) mouse is a widely used animal model for the study of human diabetes. Before the start of lymphocytic insulitis, DC accumulation around islets of Langerhans is a hallmark for autoimmune diabetes development in this model. Previous experiments indicated that an inflammatory influx of these DCs in the pancreas is less plausible. Here, we investigated whether the pancreas contains DC precursors and whether these precursors contribute to DC accumulation in the NOD pancreas. Fetal pancreases of NOD and control mice were isolated followed by FACS using ER-MP58, Ly6G, CD11b and Ly6C. Sorted fetal pancreatic ER-MP58+ cells were cultured with GM-CSF and tested for DC markers and antigen processing. CFSE labeling and Ki-67 staining were used to determine cell proliferation in cultures and tissues. Ly6Chi and Ly6Clow precursors were present in fetal pancreases of NOD and control mice. These precursors developed into CD11c+MHCII+CD86+ DCs capable of processing DQ-OVA. ER-MP58+ cells in the embryonic and pre-diabetic NOD pancreas had a higher proliferation capacity. Our observations support a novel concept that pre-diabetic DC accumulation in the NOD pancreas is due to aberrant enhanced proliferation of local precursors, rather than to aberrant inflammatory infiltration from the circulation

Plasmacytoid dendritic cells in autoimmune diabetes - Potential tools for immunotherapy

Type 1 diabetes (T1D) is an autoimmune disease in which a T-cell-mediated attack destroys the insulin-producing cells of the pancreatic islets. Despite insulin supplementation severe complications ask For novel treatments that aim at cure or delay of the onset of the disease. In spontaneous animal models for diabetes like the nonobese diabetic (NOD) mouse, distinct steps in the pathogenesis of the disease can be distinguished. In the past 10 years it became evident that DC and macrophages play an important role in all three phases of the pathogenesis of T1D. In phase 1, dendritic cells (DC) and macrophages accumulate at the islet edges. In phase 2, DC and macrophages are involved in the activation of autoreactive T cells that accumulate in the pancreas. In the third phase the islets are invaded by macrophages, DC and NK cells followed by the destruction of the beta-cells. Recent data suggest a role for a new member of the DC family: the plasmacytoid DC (pDC). pDC have been found to induce tolerance in experimental models of asthma. Several studies in humans and the NOD mouse support a similar role for pDC in diabetes. Mechanisms found to be involved in tolerance induction by pDC are inhibition of effector T cells, induction of regulatory T cells, production of cytokines and indoleamine 2,3-dioxygenase (IDO). The exact mechanism of tolerance induction by pDC in diabetes remains to be established but the intrinsic tolerogenic properties of pDC provide a promising, yet underestimated target for therapeutic intervention. (C) 2009 Elsevier GmbH. All rights reserved

NOD mice have a severly impaired ability to recruit leukocytes into sites of inflammation

The accumulation of macrophages (MPhi) and dendritic cells (DC) in the pancreas plays a crucial role in the pathogenesis of autoimmune diabetes. We studied the recruitment of monocytes, MPhi and DC to sites of inflammation, i.e. the peritoneal cavity and a subcutaneously elicited air pouch in the NOD mouse model of autoimmune diabetes. The leukocyte recruitment was studied from 1 to 7 days after injection of thioglycollate (peritoneum), C5a (peritoneum, air pouch), CCL2 and CCL3 (air pouch). C57BL/6 and BALB/c mice served as controls. Morphological and flow cytometric analysis of the recruited cells was performed, IL-1beta, TNF-alpha, IL-6, IL-12 and IL-10 in exudates measured, and in vitro CCL2-chemotaxis of exudate MPhi (Boyden chamber) determined. NOD mice were strongly impaired in the recruitment of MPhi, DC, monocytes, and granulocytes. Chemokine-injected air pouches of NOD mice showed an increased IL-10 and a decreased IL-1beta level, while the other cytokines were normally or very lowly expressed. In addition, NOD exudate MPhi displayed an impaired in vitro CCL2-induced migration. Our data show that NOD mice have an impaired ability to recruit leukocytes into sites of inflammation elicited in the peritoneum and the air pouch. A raised IL-10/ IL-1beta ratio at these sites and a deficient migratory capacity of NOD monocytes are important determinants in this impairment

Reduced numbers of dendritic cells with a tolerogenic phenotype in the prediabetic pancreas of NOD mice

The NOD mouse is a widely used animal model of autoimmune diabetes. Prior to the onset of lymphocytic insulitis, DCs accumulate at the islet edges. Our recent work indicated that these DCs may derive from aberrantly proliferating local precursor cells. As CD8α DCs play a role in tolerance induction in steady-state conditions, we hypothesized that the autoimmune phenotype might associate with deficiencies in CD8α +DCs in the prediabetic NOD mouse pancreas. We studied CD8α +DCs in the pancreas and pLNs of NOD and control mice, focusing on molecules associated with tolerance induction (CD103, Langerin, CLEC9A, CCR5). mRNA expression levels of tolerance-modulating cytokines were studied in pancreatic CD8α +DCs of NOD and control mice. In the NOD pancreas, the frequency of CD8α +CD103+Langerin+ cells was reduced significantly compared with control mice. NOD pancreatic CD8α +CD103+Langerinα DCs expressed reduced levels of CCR5, CLEC9A, and IL-10 as compared with control DCs. These alterations in the CD8α +CD103+ Langerinα DC population were not present in pLNs. We demonstrate local abnormalities in the CD8α +DC population in the prediabetic NOD pancreas. These data suggest that abnormal differentiation of pancreatic DCs contributes to loss of tolerance, hallmarking the development of autoimmune diabetes

The kinetics of plasmacytoid dendritic cell accumulation in the pancreas of the NOD mouse during the early phases of insulitis.

In non-obese diabetic (NOD) mice that spontaneously develop autoimmune diabetes, plasmacytoid dendritic cells (pDCs) have a diabetes-promoting role through IFN-α production on one hand, while a diabetes-inhibiting role through indoleamine 2,3-dioxygenase (IDO) production on the other. Little is known about the kinetics and phenotype of pDCs in the NOD pancreas during the development of autoimmune diabetes. While para/peri-insular accumulation of conventional dendritic cells (cDCs) could be observed from 4 weeks of age onwards in NOD mice, pDCs only started to accumulate around the islets of Langerhans from 10 weeks onwards, which is concomitant with the influx of lymphocytes. NOD pancreatic pDCs showed a tolerogenic phenotype as assessed by their high expression of IDO and non-detectable levels of IFN-α and MxA. Furthermore, expression of the pDC-attracting chemokines CXCL10 and CXCL12 was significantly increased in the NOD pancreas at 10 weeks and the circulating pDC numbers were increased at 4 and 10 weeks. Our data suggest that a simultaneous accumulation of IDO(+) pDCs and lymphocytes in the pancreas in 10 weeks old NOD mice, which may reflect both an immunogenic influx of T cells as well as a tolerogenic attempt to control these immunogenic T cells

Accumulation of mDCs and pDCs in the NOD pancreas.

<p>The localization of mDCs and pDCs in the C57BL/6, NOR and NOD pancreas at the age of 4, 10 and 20 weeks was determined by immunohistochemical detection. Pictures show CD11c and Siglec-H expression in the pancreas of mice from 4, 10 and 20 weeks of age, magnification 200x (A). Bar graphs represent the mean insulitis score of CD11c⁺ (B) and Siglec-H⁺ cells (C) in the pancreas. Data are presented as average+SEM, n = 5 mice, * p<0.04, ** p<0.01, *** p<0.001 as determined by the unpaired Mann-Whitney U test.</p