Initiation Of Insulitis Research Articles

IL-17 is expressed on beta and alpha cells of donors with type 1 and type 2 diabetes

PurposeIL-17 is an important effector cytokine driving immune-mediated destruction in autoimmune diseases such as psoriasis. Blockade of the IL-17 pathway after the initiation of insulitis was effective in delaying or preventing the onset of type 1 diabetes (T1D) in rodent models. Expression of IL-17 transcripts in islets from a donor with recent-onset T1D has been reported, however, studies regarding IL-17 protein expression are lacking. We aimed to study whether IL-17 is being expressed in the islets of diabetic donors. MethodsWe stained human pancreatic tissues from non-diabetic (n = 5), auto-antibody positive (aab+) (n = 5), T1D (n = 6) and T2D (n = 5) donors for IL-17, Insulin, and Glucagon, and for CD45 in selected cases. High resolution images were acquired with Zeiss laser scanning confocal microscope LSM780 and analyzed with Zen blue 2.3 software. Cases stained for CD45 were also acquired with widefield slide scanner and analyzed with QuPath software. ResultsWe observed a clear cytoplasmic staining for IL-17 in insulin-containing islets of donors with T1D and T2D, accounting for an average of 7.8 ± 8.4% and 14.9 ± 16.8% of total islet area, respectively. Both beta and alpha cells were sources of IL-17, but CD45+ cells were not a major source of IL-17 in those donors. Expression of IL-17 was reduced in islets of non-diabetic donors, aab+ donors and in insulin-deficient islets of donors with T1D. ConclusionOur finding that IL-17 is expressed in islets of donors with T1D or T2D is quite intriguing and warrants further mechanistic studies in human islets to understand the role of IL-17 in the context of metabolic and immune stress in beta cells.

CD8+ CD122+ PD-1- effector cells promote the development of diabetes in NOD mice.

It is well established that CD4 and CD8 T cells are required for the initiation of autoimmune diabetes in NOD mice. However, different subsets of CD4 or CD8 cells may play different roles in the initiation of insulitis. In this study, we evaluated the role of the previously described CD8(+) CD122(+) in this process. We found that prediabetic NOD mice have an almost 50% reduction of CD8(+) CD122(+) T cells in their secondary lymphoid organs compared with BL/6 or Balb/c mouse strains. This reduction is explained by the lack of the regulatory CD8(+) CD122(+) PD-1(+) cell population in the NOD mice, as we found that all CD8(+) CD122(+) T cells from prediabetic NOD mice lack PD-1 expression and regulatory function. Depletion of CD8(+) CD122(+) PD-1(-) cells through injection of anti-CD122 mAb in prediabetic female NOD mice reduced the infiltration of mononuclear cells into the Langerhans islets and delayed the onset and decreased the incidence of overt diabetes. In addition, we found that transfer of highly purified and activated CD8(+) CD122(+) PD-1(-) cells, together with diabetogenic splenocytes from NOD donors to NOD SCID recipients, accelerates the diabetes development in these mice. Together, these results demonstrate that CD8(+) CD122(+) PD-1(-) T cells from NOD mice are effector cells that are involved in the pathogenesis of autoimmune diabetes.

A key role for Fas ligand on B cells in unleashing T cell diabetogenicity by suppression of IL-10-mediated islet tolerance (143.34)

Abstract A key role for the Fas pathway in promoting organ-specific-autoimmunity is indicated by resistance of mice bearing the lpr mutation of Fas or gld mutation of Fas ligand (FasL) to several organ-specific autoimmune diseases, including autoimmune diabetes and multiple sclerosis. However, the underlying mechanism remains elusive. Here we show that FasL expressed on B cells plays a key role in facilitating activation of diabetogenic T cells in pancreatic lymph nodes (PLNs) and pancreatic islet accumulation resulting in initiation of insulitis in the autoimmune diabetes-prone NOD mice. Expression of FasL on diabetogenic T cells themselves is not sufficient to mediate its diabetogenic role. By contrast, expression of FasL on B cells leads to deletion of localized IL-10-producing B cells, the presence of which in PLNs and pancreatic islets is associated with suppression of diabetogenic T cell priming and islet accumulation, respectively. Consequently, FasL inactivation by gld mutation or antibody blockade preserves the local IL-10-producing B cells and protects islets from diabetogenic T cells by a mechanism that is reversed by IL-10R blockade in vivo. The results provide novel insights into how FasL orchestrates the pathogenic relationship among B cell subsets in its implications for diabetogenic T cells.

CD8+ T cells specific for β cells encounter their cognate antigens in the islets of NOD mice

CD8(+) T cells play a key role in the initiation of insulitis. However, the site(s) where naive CD8(+) T cells encounter beta-cell antigens and the mechanism(s) by which beta-cell autoimmunity is initiated remain to be determined. In the current study, an adoptive transfer model was employed assessing the initial site of priming and the nature of antigen recognition by naive beta-cell-specific CD8(+) T cells. Temporal analysis demonstrated that unlike CD4(+) T cells that are primed in the draining pancreatic lymph nodes, initial proliferation of transferred CD8(+) T cells was detected in the islets. These results indicate that in our model, naive beta-cell-specific CD8(+) T cells encounter beta-cell antigens in the islets. Furthermore, ectopic expression of CD80 by beta cells accelerated the onset of insulitis mediated by beta-cell-specific CD8(+) T cells, but had no effect on CD4(+) T-cell-mediated diabetes, suggesting an antigenic interaction between beta cells and naive CD8(+) T cells. However, it remains to be determined whether the initiation of insulitis in spontaneous diabetes is the result of a cognate interaction between naive CD8(+) T cells and islet beta cells.

Equivalent Specificity of Peripheral Blood and Islet-Infiltrating CD8+ T Lymphocytes in Spontaneously Diabetic HLA-A2 Transgenic NOD Mice

CD8(+) T cells play an important role in the initiation of insulitis and in the destructive stage leading to insulin-dependent diabetes mellitus. A string of recent studies has led to the identification of numerous HLA-A2-restricted epitopes derived from pancreatic beta cell Ags. It is hoped that assays detecting responses of patient PBMC to such epitopes might be instrumental for early diagnosis of beta cell-directed autoimmunity and for monitoring trials of immunointervention. However, it remains unclear whether the results of assays studying PBMC reflect responses of islet-infiltrating lymphocytes, and to what extent they correlate with disease risk and/or activity. We have used female and male humanized NOD mice expressing HLA-A2 in addition to murine MHC class I molecules to study spontaneous responses of islet-infiltrating blood, spleen, and lymph node lymphocytes of various age groups to a panel of 16 epitopes. Twelve of these are restricted by HLA-A2, have previously been shown to be recognized by patient CTL, and have identical sequences in human and murine autoantigens. Using an IFN-gamma ELISPOT assay, we find highly similar hierarchies of epitope immunodominance in the different T cell compartments, including peripheral blood and pancreatic islets. Moreover, we demonstrate that most of the epitopes eliciting dominant responses in humans display similar status in the mouse model. These results emphasize the potential of humanized mice as tools for studying spontaneous autoimmune CTL responses, and they provide a strong rationale for the development and use of assays monitoring responses of CD8(+) PBMC in human type 1 diabetes.

Rotavirus Infection of Infant and Young Adult Nonobese Diabetic Mice Involves Extraintestinal Spread and Delays Diabetes Onset

Rotaviruses have been implicated as a possible viral trigger for exacerbations in islet autoimmunity, suggesting they might modulate type 1 diabetes development. In this study, the ability of rotavirus strain RRV to infect the pancreas and affect insulitis and diabetes was examined in nonobese diabetic (NOD) mice, an experimental model of type 1 diabetes. Mice were inoculated either orally or intraperitoneally as infants or young adults. In infant mice inoculated orally, rotavirus antigen was detected in pancreatic macrophages outside islets and infectious virus was found in blood cells, pancreas, spleen, and liver. Extraintestinal RRV spread and pancreatic presence of infectious virus also occurred in intraperitoneally inoculated infant and adult mice. The initiation of insulitis was unaltered by infection. The onset of diabetes was delayed in infant mice inoculated orally and infant and adult mice inoculated intraperitoneally. In contrast, adult mice inoculated orally showed no evidence of pancreatic RRV, the lowest rate of detectable RRV replication, and no diabetes modulation. Thus, the ability of RRV infection to modulate diabetes development in infant and young adult NOD mice was related to the overall extent of detectable virus replication and the presence of infectious virus extraintestinally, including in the pancreas. These studies show that RRV infection of infant and young adult NOD mice provides significant protection against diabetes. As these findings do not support the hypothesis that rotavirus triggers autoimmunity related to type 1 diabetes, further research is needed to resolve this issue.

Congenic mapping of the diabetogenic locus Idd4 to a 5.2-cM region of chromosome 11 in NOD mice: identification of two potential candidate subloci.

Twenty diabetes susceptibility loci on 12 mouse chromosomes have been identified to control the development of type 1 diabetes at the level of either initiation of insulitis or progression from insulitis to overt diabetes or both. Previously, we demonstrated that the genetic control of T-cell proliferative unresponsiveness in nonobese diabetic (NOD) mice is linked to Idd4 on mouse chromosome 11. Here, we show by congenic mapping of three newly generated NOD.B6Idd4 diabetes-resistant mouse strains that Idd4 is limited to a 5.2-cM interval of chromosome 11. This B6-derived region expressed in NOD.B6Idd4A mice maps between the D11Nds1 (43.8 cM) and D11Mit38/D11Mit325 (49.0 cM) markers and dramatically reduces the development of both insulitis and type 1 diabetes. NOD.B6Idd4B and NOD.B6Idd4C mice, which carry a smaller B6-derived segment of chromosome 11 that spans <5.2 cM distal to D11Nds1, exhibit protection against type 1 diabetes with the restoration of T-cell proliferation. Our findings suggest that diabetes resistance conferred by Idd4 may be mediated by the Idd4.1 and Idd4.2 subloci. Idd4.1 is localized in the D11Nds1 interval that influences both diabetes and insulitis. Idd4.2 is localized within the D11Mit38/325 interval that mainly influences diabetes incidence and restores T-cell proliferative responsiveness. Three potential candidate genes, platelet activating factor acetylhydrolase Ib1, nitric oxide synthase-2, and CC chemokine genes, are localized in the 5.2-cM interval.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) regulates the unfolding of autoimmune diabetes.

Evidence has been accumulating that shows that insulin-dependent diabetes is subject to immunoregulation. To determine whether cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is involved, we injected anti-CTLA-4 mAb into a TCR transgenic model of diabetes at different stages of disease. When injected into young mice, months before they would normally become diabetic, anti-CTLA-4 induced diabetes rapidly and essentially universally; this was not the result of a global activation of T lymphocytes, but did reflect a much more aggressive T cell infiltrate in the pancreatic islets. These effects were only observed if anti-CTLA-4 was injected during a narrow time window, before the initiation of insulitis. Thus, engagement of CTLA-4 at the time when potentially diabetogenic T cells are first activated is a pivotal event; if engagement is permitted, invasion of the islets occurs, but remains quite innocuous for months, if not, insulitis is much more aggressive, and diabetes quickly ensues.

Excessive production of nitric oxide by macrophages from DP-BB rats is secondary to the T-lymphopenic state of these animals.

Activated macrophages are the first mononuclear cells to migrate to the pancreas of DP-BB rats at the initiation of insulitis. These cells produce an excess of NO, which has been implicated as a mediator of both beta-cell damage and inhibition of T-cell proliferation in this rat strain. Genetic studies have shown that the impaired proliferative response of T-cells segregates with one of the diabetes-susceptibility genes of the DP-BB rat, lyp, which is responsible for a peripheral T-lymphopenia. This observation suggests that the dysregulated expression of inducible NO synthase (iNOS) is under the control of lyp itself or a gene in linkage disequilibrium with lyp. Using two models of hemopoietic chimeras--DP-BB rats reconstituted with isocongenic T-cells and irradiated (WF x DP-BB)F1 animals reconstituted with bone marrow of both parental strains--we demonstrated that the production of NO by DP-BB macrophages is normal when these cells originate from a non-T-lymphopenic environment. Consequently, these macrophages no longer inhibit the stimulation of DNA synthesis in activated T-cells. Macrophages of young WF rats were found to produce high levels of NO, which inhibited T-cell proliferation in vitro. This observation strongly suggests that upregulation of NO synthesis in DP-BB macrophages represents the abnormal persistence of a phenomenon restricted to the first few weeks of life in non-diabetes-prone rats. Taken together, these results demonstrate that the elevated production of NO by DP-BB macrophages results from the lyp mutation and represents a crucial mechanism through which T-lymphopenia contributes to the development of diabetes.

Genetic Control of Diabetes Progression

Autoimmune diabetes in both the human and the nonobese diabetic mouse has elaborate genetics; in the latter case, the disease is influenced by at least 15–20 loci. We anticipated that the genetics would be simpler in the BDC2.5 T cell receptor transgenic mouse model of diabetes, wherein many T cells express a particular diabetogenic specificity. Initiation of insulitis in this model was the same on the two genetic backgrounds analyzed, but the kinetics and penetrance of diabetes were strikingly different, permitting us to focus on genetic influences during a defined window of disease progression. The differences correlated with variations in five genomic intervals, certain ones of which have been previously implicated in susceptibility to autoimmune disease. This reductionist approach indeed simplified the analysis of diabetes susceptibility loci.

Progression from insulitis to beta-cell destruction in NOD mouse requires L3T4+ T-lymphocytes.

The identity of the cells responsible for beta-cell destruction in type I (insulin-dependent) diabetes is still uncertain. L3T4+ T-lymphocytes have a role in the initiation of insulitis and in damaging transplanted allogeneic islets in nonobese diabetic (NOD) mice. The role of L3T4+ T-lymphocytes in destruction of beta-cells of the NOD mouse was studied in cyclophosphamide (CY)-induced diabetic NOD mice with a rat anti-L3T4 monoclonal antibody (MoAb). After administration of CY, most untreated animals became diabetic, whereas all antibody-treated animals remained normoglycemic. Insulitis was still present in MoAb-treated animals, but immunocytochemical staining showed rat antibody blocking the L3T4 antigen on T-lymphocytes. This study provides further evidence that L3T4+ T-lymphocytes are critical to the process of beta-cell destruction in NOD mice. The means by which L3T4+ cells exert their effect remains to be clarified.

Evidence for initial involvement of macrophage in development of insulitis in NOD mice.

Macrophages have been shown to be the major population of infiltrated immunocytes at the early stage of insulitis in diabetes-prone BB rats. This study was undertaken to investigate the role of macrophages in the development of insulitis in nonobese diabetic (NOD) mice. Administration of cyclophosphamide to NOD mice resulted in a significant increase in the incidence of overt diabetes and severity of insulitis compared with that in untreated NOD mice. Intraperitoneal injections of silica completely prevented the development of diabetes and insulitis in both cyclophosphamide-treated and untreated animals. Because silica is selectively toxic to macrophages, the results suggest that macrophages play an important role in the initiation of insulitis in NOD mice.

Administration of silica particles or anti-Lyt2 antibody prevents beta-cell destruction in NOD mice given cyclophosphamide.

The cellular pathway of beta-cell destruction in type I (insulin-dependent) diabetes is still undefined. L3T4+ T-lymphocytes have a role in both the initiation of insulitis and in recurrent disease in transplanted allogeneic islets in nonobese diabetic (NOD) mice. The roles of macrophages and Lyt2+ T-lymphocytes in beta-cell destruction were studied in cyclophosphamide-induced diabetic NOD mice with silica particles and a rat anti-Lyt2 monoclonal antibody. After administration of cyclophosphamide, 10 of 26 untreated mice and 1 of 21 anti-Lyt2-treated mice became diabetic. Insulitis was significantly reduced in anti-Lyt2-treated mice, and immunocytochemical staining showed a lack of Lyt2+ cells. Only 1 of 19 silica-treated mice became diabetic, compared to 8 of 19 control mice. This study demonstrates that both Lyt2+ T-lymphocytes and macrophages are necessary, but not sufficient, for beta-cell destruction in NOD mice. Therefore, we propose that macrophages present beta-cell antigen to L3T4+ cells, which induce cytotoxic Lyt2+ cells to specifically destroy beta-cells.