RESUMEN
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. Ly6C(hi) and Ly6C(low) 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.
Asunto(s)
Células Dendríticas/inmunología , Diabetes Mellitus/inmunología , Células Progenitoras Mieloides/inmunología , Páncreas/inmunología , Estado Prediabético/inmunología , Animales , Antígenos Ly/inmunología , Antígeno CD11b/inmunología , Diferenciación Celular/inmunología , Células Dendríticas/citología , Femenino , Citometría de Flujo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Microscopía Fluorescente , Células Progenitoras Mieloides/citología , Páncreas/citología , Embarazo , Organismos Libres de Patógenos EspecíficosRESUMEN
Two major dendritic cell (DC) subsets have been described in the pancreas of mice: The CD11c+ CD8α- DCs (strong CD4+ T cell proliferation inducers) and the CD8α+ CD103+ DCs (T cell apoptosis inducers). Here we analyzed the larger subset of CD11c+ CD8α- DCs isolated from the pancreas of pre-diabetic NOD mice for genome-wide gene expression (validated by Q-PCR) to elucidate abnormalities in underlying gene expression networks. CD11c+ CD8α- DCs were isolated from 5 week old NOD and control C57BL/6 pancreas. The steady state pancreatic NOD CD11c+ CD8α- DCs showed a reduced expression of several gene networks important for the prime functions of these cells, i.e. for cell renewal, immune tolerance induction, migration and for the provision of growth factors including those for beta cell regeneration. A functional in vivo BrdU incorporation test showed the reduced proliferation of steady state pancreatic DC. The reduced expression of tolerance induction genes (CD200R, CCR5 and CD24) was supported on the protein level by flow cytometry. Also previously published functional tests on maturation, immune stimulation and migration confirm the molecular deficits of NOD steady state DC. Despite these deficiencies NOD pancreas CD11c+ CD8α- DCs showed a hyperreactivity to LPS, which resulted in an enhanced pro-inflammatory state characterized by a gene profile of an enhanced expression of a number of classical inflammatory cytokines. The enhanced up-regulation of inflammatory genes was supported by the in vitro cytokine production profile of the DCs. In conclusion, our data show that NOD pancreatic CD11c+ CD8α- DCs show various deficiencies in steady state, while hyperreactive when encountering a danger signal such as LPS.
Asunto(s)
Antígeno CD11c/metabolismo , Antígenos CD8/metabolismo , Células Dendríticas/metabolismo , Páncreas/metabolismo , Estado Prediabético/genética , Animales , Movimiento Celular/genética , Movimiento Celular/inmunología , Células Dendríticas/inmunología , Perfilación de la Expresión Génica , Tolerancia Inmunológica/inmunología , Ratones , Ratones Endogámicos NOD , Páncreas/inmunología , Estado Prediabético/inmunología , Estado Prediabético/metabolismoRESUMEN
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.
Asunto(s)
Células Dendríticas/inmunología , Páncreas/inmunología , Animales , Antígeno B7-H1/metabolismo , Recuento de Células , Quimiocina CXCL10/metabolismo , Quimiocina CXCL12/metabolismo , Células Dendríticas/metabolismo , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/patología , Modelos Animales de Enfermedad , Femenino , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interferón-alfa/metabolismo , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Cinética , Linfocitos/inmunología , Linfocitos/metabolismo , Ratones , Ratones Endogámicos NOD , Páncreas/patologíaRESUMEN
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(+)Langerin(+) DCs expressed reduced levels of CCR5, CLEC9A, and IL-10 as compared with control DCs. These alterations in the CD8α(+)CD103(+)Langerin(+) 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.
Asunto(s)
Células Dendríticas/inmunología , Tolerancia Inmunológica/inmunología , Fenotipo , Estado Prediabético/inmunología , Animales , Antígenos CD/metabolismo , Antígenos de Superficie/metabolismo , Antígenos CD8/metabolismo , Células Dendríticas/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Tolerancia Inmunológica/genética , Inmunofenotipificación , Cadenas alfa de Integrinas/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Lectinas de Unión a Manosa/metabolismo , Ratones , Ratones Endogámicos NOD , Estado Prediabético/genética , Estado Prediabético/metabolismo , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismoRESUMEN
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.