Autoimmune Nonobese Diabetic Mice Research Articles

IL-27 promotes pathogenic Tfh-like CD4 effectors and exhausted-like CD8 T cells in a mouse model of Sjögren’s

Abstract Sjögren’s is an autoimmune disease that targets the exocrine glands leading to profound dryness, extra glandular manifestations, and increased risk of developing lymphoma with no therapy to treat the underlying immune dysregulation. Nonobese diabetic (NOD) mice spontaneously develop T cell mediated inflammation of the lacrimal gland, similar to human disease. IL-27 is elevated in the salvia and serum of people with Sjögren’s and is required for lacrimal gland autoimmunity in NOD mice. Utilizing IL-27Rα KO NOD mice, I have previously found both CD4 and CD8 effector T cells require IL-27 to transfer lacrimal gland autoimmunity to NOD-SCID mice. Here, we utilized an adoptive co-transfer system that promotes infiltration of lacrimal glands by both WT and IL-27Ra KO T cells to define the T cell intrinsic effects of IL-27 signaling in lacrimal gland infiltrating T cells through 28 color spectral flow cytometry and performed unsupervised dimensionality reduction analyses to identify subsets of T cells reliant on intrinsic IL-27 signaling. Among the total concatenated T cell population including both WT and KO T cells, 12 total subsets were identified. Strikingly, when visualizing WT and KO separately, we identified populations of CD4 effector T cells and CD8 T cells that were either heavily skewed or only present in WT. Through phenotyping these populations, I found IL-27 signaling induced Tfh-like PD-1+ICOS+ CD4 effector T cells and PD-1+ICOS+CD39+ exhausted-like CD8 T cells in the glands of recipient mice. Taken together, these data demonstrate a role for IL-27 in promoting pathogenic T cells in the immune dysregulation of Sjögren’s. Supported by grants from NIH(T32 AI007485, R01 EY027731)

Transient Depletion of Foxp3+ Regulatory T Cells Selectively Promotes Aggressive β Cell Autoimmunity in Genetically Susceptible DEREG Mice.

Type 1 diabetes (T1D) represents a hallmark of the fatal multiorgan autoimmune syndrome affecting humans with abrogated Foxp3+ regulatory T (Treg) cell function due to Foxp3 gene mutations, but whether the loss of Foxp3+ Treg cell activity is indeed sufficient to promote β cell autoimmunity requires further scrutiny. As opposed to human Treg cell deficiency, β cell autoimmunity has not been observed in non-autoimmune-prone mice with constitutive Foxp3 deficiency or after diphtheria toxin receptor (DTR)-mediated ablation of Foxp3+ Treg cells. In the spontaneous nonobese diabetic (NOD) mouse model of T1D, constitutive Foxp3 deficiency did not result in invasive insulitis and hyperglycemia, and previous studies on Foxp3+ Treg cell ablation focused on Foxp3DTR NOD mice, in which expression of a transgenic BDC2.5 T cell receptor (TCR) restricted the CD4+ TCR repertoire to a single diabetogenic specificity. Here we revisited the effect of acute Foxp3+ Treg cell ablation on β cell autoimmunity in NOD mice in the context of a polyclonal TCR repertoire. For this, we took advantage of the well-established DTR/GFP transgene of DEREG mice, which allows for specific ablation of Foxp3+ Treg cells without promoting catastrophic autoimmune diseases. We show that the transient loss of Foxp3+ Treg cells in prediabetic NOD.DEREG mice is sufficient to precipitate severe insulitis and persistent hyperglycemia within 5 days after DT administration. Importantly, DT-treated NOD.DEREG mice preserved many clinical features of spontaneous diabetes progression in the NOD model, including a prominent role of diabetogenic CD8+ T cells in terminal β cell destruction. Despite the severity of destructive β cell autoimmunity, anti-CD3 mAb therapy of DT-treated mice interfered with the progression to overt diabetes, indicating that the novel NOD.DEREG model can be exploited for preclinical studies on T1D under experimental conditions of synchronized, advanced β cell autoimmunity. Overall, our studies highlight the continuous requirement of Foxp3+ Treg cell activity for the control of genetically pre-installed autoimmune diabetes.

IL-4 and IL-17 Are Required for House Dust Mite-Driven Airway Hyperresponsiveness in Autoimmune Diabetes-Prone Non-Obese Diabetic Mice.

Allergic asthma is characterized by airway inflammation with a Th2-type cytokine profile, hyper-IgE production, mucus hypersecretion, and airway hyperreactivity (AHR). It is increasingly recognized that asthma is a heterogeneous disease implicating complex immune mechanisms resulting in distinct endotypes observed in patients. In this study, we showed that non-obese diabetic (NOD) mice, which spontaneously develop autoimmune diabetes, undergo more severe allergic asthma airway inflammation and AHR than pro-Th2 BALB/c mice upon house dust mite (HDM) sensitization and challenge. The use of IL-4-deficient NOD mice and the in vivo neutralization of IL-17 demonstrated that both IL-4 and IL-17 are responsible by the exacerbated airway inflammation and AHR observed in NOD mice. Overall, our findings indicate that autoimmune diabetes-prone NOD mice might become useful as a new HDM-induced asthma model to elucidate allergic dysimmune mechanisms involving Th2 and Th17 responses that could better mimic some asthmatic endoytpes.

MiRNA142-3p targets Tet2 and impairs Treg differentiation and stability in models of type 1 diabetes

In type 1 diabetes, the appearance of islet autoantibodies indicates the onset of islet autoimmunity, often many years before clinical symptoms arise. While T cells play a major role in the destruction of pancreatic beta cells, molecular underpinnings promoting aberrant T cell activation remain poorly understood. Here, we show that during islet autoimmunity an miR142-3p/Tet2/Foxp3 axis interferes with the efficient induction of regulatory T (Treg) cells, resulting in impaired Treg stability in mouse and human. Specifically, we demonstrate that miR142-3p is induced in islet autoimmunity and that its inhibition enhances Treg induction and stability, leading to reduced islet autoimmunity in non-obese diabetic mice. Using various cellular and molecular approaches we identify Tet2 as a direct target of miR142-3p, thereby linking high miR142-3p levels to epigenetic remodeling in Tregs. These findings offer a mechanistic model where during islet autoimmunity miR142-3p/Tet2-mediated Treg instability contributes to autoimmune activation and progression.

Interleukin-27 Is Essential for Type 1 Diabetes Development and Sjögren Syndrome-like Inflammation

SUMMARYHuman genetic studies implicate interleukin-27 (IL-27) in the pathogenesis of type 1 diabetes (T1D), but the underlying mechanisms remain largely unexplored. To further define the role of IL-27 in T1D, we generated non-obese diabetic (NOD) mice deficient in IL-27 or IL-27Rα. In contrast to wild-type NOD mice, both NOD.Il27−/− and NOD.Il27ra−/− strains are completely resistant to T1D. IL-27 from myeloid cells and IL-27 signaling in T cells are critical for T1D development. IL-27 directly alters the balance of regulatory T cells (Tregs) and T helper 1 (Th1) cells in pancreatic islets, which in turn modulates the diabetogenic activity of CD8 T cells. IL-27 also directly enhances the effector function of CD8 T cells within pancreatic islets. In addition to T1D, IL-27 signaling in T cells is also required for lacrimal and salivary gland inflammation in NOD mice. Our study reveals that IL-27 contributes to autoimmunity in NOD mice through multiple mechanisms and provides substantial evidence to support its pathogenic role in human T1D.

Transplantation of MHC-mismatched mouse embryonic stem cell-derived thymic epithelial progenitors and MHC-matched bone marrow prevents autoimmune diabetes

BackgroundType 1 diabetes (T1D) is an autoimmune disease resulting from the destruction of insulin-secreting islet β cells by autoreactive T cells. Non-obese diabetic (NOD) mice are the widely used animal model for human T1D. Autoimmunity in NOD mice is associated with particular major histocompatibility complex (MHC) loci and impaired islet autoantigen expression and/or presentation in the thymus, which results in defects in both central and peripheral tolerance. It has been reported that induction of mixed chimerism with MHC-mismatched, but not MHC-matched donor bone marrow (BM) transplants prevents the development T1D in NOD mice. We have reported that mouse embryonic stem cells (mESCs) can be selectively induced in vitro to generate thymic epithelial progenitors (TEPs) that further develop into thymic epithelial cells (TECs) in vivo to support T cell development.MethodsTo determine whether transplantation of MHC-mismatched mESC-TEPs could prevent the development of insulitis and T1D, NOD mice were conditioned and injected with MHC-mismatched B6 mESC-TEPs and MHC-matched BM from H-2g7 B6 mice. The mice were monitored for T1D development. The pancreas, spleen, BM, and thymus were then harvested from the mice for evaluation of T1D, insulitis, chimerism levels, and T cells.ResultsTransplantation of MHC-mismatched mESC-TEPs and MHC-matched donor BM prevented insulitis and T1D development in NOD mice. This was associated with higher expression of proinsulin 2, a key islet autoantigen in the mESC-TECs, and an increased number of regulatory T cells.ConclusionsOur results suggest that embryonic stem cell-derived TEPs may offer a new approach to control T1D.

Type I Interferon Signaling Is Required for Dacryoadenitis in the Nonobese Diabetic Mouse Model of Sjögren Syndrome.

Nonobese diabetic (NOD) mice spontaneously develop lacrimal and salivary gland autoimmunity similar to human Sjögren syndrome. In both humans and NOD mice, the early immune response that drives T-cell infiltration into lacrimal and salivary glands is poorly understood. In NOD mice, lacrimal gland autoimmunity spontaneously occurs only in males with testosterone playing a role in promoting lacrimal gland inflammation, while female lacrimal glands are protected by regulatory T cells (Tregs). The mechanisms of this male-specific lacrimal gland autoimmunity are not known. Here, we studied the effects of Treg depletion in hormone-manipulated NOD mice and lacrimal gland gene expression to determine early signals required for lacrimal gland inflammation. While Treg-depletion was not sufficient to drive dacryoadenitis in castrated male NOD mice, chemokines (Cxcl9, Ccl19) and other potentially disease-relevant genes (Epsti1, Ubd) were upregulated in male lacrimal glands. Expression of Cxcl9 and Ccl19, in particular, remained significantly upregulated in the lacrimal glands of lymphocyte-deficient NOD-severe combined immunodeficiency (SCID) mice and their expression was modulated by type I interferon signaling. Notably, Ifnar1-deficient NOD mice did not develop dacryoadenitis. Together these data identify disease-relevant genes upregulated in the context of male-specific dacryoadenitis and demonstrate a requisite role for type I interferon signaling in lacrimal gland autoimmunity in NOD mice.

Endogenous Reprogramming of Alpha Cells into Beta Cells, Induced by Viral Gene Therapy, Reverses Autoimmune Diabetes

Successful strategies for treating type 1 diabetes need to restore the function of pancreatic beta cells that are destroyed by the immune system and overcome further destruction of insulin-producing cells. Here, we infused adeno-associated virus carrying Pdx1 and MafA expression cassettes through the pancreatic duct to reprogram alpha cells into functional beta cells and normalized blood glucose in both beta cell-toxin-induced diabetic mice and in autoimmune non-obese diabetic (NOD) mice. The euglycemia in toxin-induced diabetic mice and new insulin+ cells persisted in the autoimmune NOD mice for 4months prior to reestablishment of autoimmune diabetes. This gene therapy strategy also induced alpha to beta cell conversion in toxin-treated human islets, which restored blood glucose levels inNOD/SCID mice upon transplantation. Hence, this strategy could represent a new therapeutic approach, perhaps complemented by immunosuppression, to bolster endogenous insulin production. Our study thus provides a potential basis for further investigation in human type 1 diabetes.

AAV-mediated pancreatic overexpression of Igf1 counteracts progression to autoimmune diabetes in mice

ObjectiveType 1 diabetes is characterized by autoimmune destruction of β-cells leading to severe insulin deficiency. Although many improvements have been made in recent years, exogenous insulin therapy is still imperfect; new therapeutic approaches, focusing on preserving/expanding β-cell mass and/or blocking the autoimmune process that destroys islets, should be developed. The main objective of this work was to test in non-obese diabetic (NOD) mice, which spontaneously develop autoimmune diabetes, the effects of local expression of Insulin-like growth factor 1 (IGF1), a potent mitogenic and pro-survival factor for β-cells with immunomodulatory properties. MethodsTransgenic NOD mice overexpressing IGF1 specifically in β-cells (NOD-IGF1) were generated and phenotyped. In addition, miRT-containing, IGF1-encoding adeno-associated viruses (AAV) of serotype 8 (AAV8-IGF1-dmiRT) were produced and administered to 4- or 11-week-old non-transgenic NOD females through intraductal delivery. Several histological, immunological, and metabolic parameters were measured to monitor disease over a period of 28–30 weeks. ResultsIn transgenic mice, local IGF1 expression led to long-term suppression of diabetes onset and robust protection of β-cell mass from the autoimmune insult. AAV-mediated pancreatic-specific overexpression of IGF1 in adult animals also dramatically reduced diabetes incidence, both when vectors were delivered before pathology onset or once insulitis was established. Transgenic NOD-IGF1 and AAV8-IGF1-dmiRT-treated NOD animals had much less islet infiltration than controls, preserved β-cell mass, and normal insulinemia. Transgenic and AAV-treated islets showed less expression of antigen-presenting molecules, inflammatory cytokines, and chemokines important for tissue-specific homing of effector T cells, suggesting IGF1 modulated islet autoimmunity in NOD mice. ConclusionsLocal expression of Igf1 by AAV-mediated gene transfer counteracts progression to diabetes in NOD mice. This study suggests a therapeutic strategy for autoimmune diabetes in humans.

Despite Increased Type 1 IFN, Autoimmune Nonobese Diabetic Mice Display Impaired Dendritic Cell Response to CpG and Decreased Nuclear Localization of IFN-Activated STAT1.

Innate immune signals help break self-tolerance to initiate autoimmune diseases such as type 1 diabetes, but innate contributions to subsequent regulation of disease progression are less clear. Most studies have measured in vitro innate responses of GM-CSF dendritic cells (DCs) that are functionally distinct from conventional DCs (cDCs) and do not reflect in vivo DC subsets. To determine whether autoimmune NOD mice have alterations in type 1 IFN innate responsiveness, we compared cDCs from prediabetic NOD and control C57BL/6 (B6) mice stimulated in vivo with the TLR9 ligand CpG, a strong type 1 IFN inducer. In response to CpG, NOD mice produce more type 1 IFN and express higher levels of CD40, and NOD monocyte DCs make more TNF. However, the overall CpG-induced transcriptional response is muted in NOD cDCs. Of relevance the costimulatory proteins CD80/CD86, signals needed for regulatory T cell homeostasis, are upregulated less on NOD cDCs. Interestingly, NOD Rag1(-/-) mice also display a defect in CpG-induced CD86 upregulation compared with B6 Rag1(-/-), indicating this particular innate alteration precedes adaptive autoimmunity. The impaired response in NOD DCs is likely downstream of the IFN-α/β receptor because DCs from NOD and B6 mice show similar CpG-induced CD86 levels when anti-IFN-α/β receptor Ab is added. IFN-α-induced nuclear localization of activated STAT1 is markedly reduced in NOD CD11c(+) cells, consistent with lower type 1 IFN responsiveness. In conclusion, NOD DCs display altered innate responses characterized by enhanced type 1 IFN and activation of monocyte-derived DCs but diminished cDC type 1 IFN response.

Flow cytometric gating for spleen monocyte and DC subsets: differences in autoimmune NOD mice and with acute inflammation

The role of antigen presenting cells (APCs) in the pathogenesis of autoimmune and other inflammatory diseases is now better understood due to advances in multicolor flow cytometry, gene expression analysis of APC populations, and functional correlation of mouse to human APC populations. A simple but informative nomenclature of conventional and plasmacytoid dendritic cell subsets (cDC1, cDC2, pDC) and monocyte-derived populations incorporates these advances, but accurate subset identification is critical. Ambiguous gating schemes and alterations of cell surface markers in inflammatory condition can make comparing results between studies difficult. Both acute inflammation, such as TLR–ligand stimulation, and chronic inflammation as found in mouse models of autoimmunity can alter DC subset gating. Here, we address these issues using in vivo CpG stimulation as an example of acute inflammation and the non-obese diabetic (NOD) mouse as a model of chronic inflammation.We provide a flow cytometric antibody panel and gating scheme that differentiate 2 monocytic and 3DC subsets in the spleen both at steady state and after CpG stimulation. Using this method, we observed differences in the composition of NOD DCs that have been previously reported, and newly identified increases in the number of NOD monocyte-derived DCs. Finally, we established a protocol for DC phosphoflow to measure the phosphorylation state of intracellular proteins, and use it to confirm functional differences in the identified subsets. Therefore, we present optimized methods for distinguishing monocytic and DC populations with and without inflammation and/or autoimmunity associated with NOD mice.

Undifferentiated Wharton's Jelly Mesenchymal Stem Cell Transplantation Induces Insulin-Producing Cell Differentiation and Suppression of T-Cell-Mediated Autoimmunity in Nonobese Diabetic Mice.

Type 1 diabetes mellitus is caused by T-cell-mediated autoimmune destruction of pancreatic β-cells. Systemic administration of mesenchymal stem cells (MSCs) brings about their incorporation into a variety of tissues with immunosuppressive effects, resulting in regeneration of pancreatic islets. We previously showed that human MSCs isolated from Wharton's jelly (WJ-MSCs) represent a potential cell source to treat diabetes. However, the underlying mechanisms are unclear. The purpose of this study was to discern whether undifferentiated WJ-MSCs can differentiate into pancreatic insulin-producing cells (IPCs) and modify immunological responses in nonobese diabetic (NOD) mice. Undifferentiated WJ-MSCs underwent lentiviral transduction to express green fluorescent protein (GFP) and then were injected into the retro-orbital venous sinus of NOD mice. Seven days after transplantation, fluorescent islet-like cell clusters in the pancreas were apparent. WJ-MSC-GFP-treated NOD mice had significantly lower blood glucose and higher survival rates than saline-treated mice. Systemic and local levels of autoaggressive T-cells, including T helper 1 cells and IL-17-producing T-cells, were reduced, and regulatory T-cell levels were increased. Furthermore, anti-inflammatory cytokine levels were increased, and dendritic cells were decreased. At 23 days, higher human C-peptide and serum insulin levels and improved glucose tolerance were found. Additionally, WJ-MSCs-GFP differentiated into IPCs as shown by colocalization of human C-peptide and GFP in the pancreas. Significantly more intact islets and less severe insulitis were observed. In conclusion, undifferentiated WJ-MSCs can differentiate into IPCs in vivo with immunomodulatory effects and repair the destroyed islets in NOD mice.

Identification of endogenous murine leukemia retrovirus antigens attributing to exosome-induced autoimmunity in NOD mice (BA6P.122)

Abstract Endogenous autoimmune triggers and antigens are required for initiating the innate and cellular immune responses in the islet of non-obese diabetic (NOD) mice. We found that secreted, nano-sized microvesicles or exosomes are potent inflammatory triggers that, importantly, stimulated autoreactive B and T cells. By performing proteomic analysis, we identified endogenous retrovirus (ERV) proteins, highly homologous to murine leukemia retrovirus (MLV), that are enriched in the exosomes. To study whether these ERV antigens attribute to the autoimmune induction, we produced exosomes lacking the ERV Gag protein and virus-like particles (VLPs), which are nano-sized microvesicles overexpressing Gag and Env proteins. We found that NOD mice spontaneously develop autoantibodies binding to the MLV Env, and also accumulate Env-specific B cells in periphery as compared with diabetes-resistant mice. Autoreactive T cells did not respond to the MLV antigens directly; however, immunization with the exosomes or VLPs induced Gag-specific T cells that inhibited the function of diabetogenic T cell clones in the pancreas. Therefore, ERV antigens may be the targets of autoreactive B and T cells in this T1D mouse model. Consistently, we detected endogenous MLV antigens expressed in the pancreas and by the islet stromal/stem cells in NOD mice. These data support that unique ERV antigens and/or altered formation of exosomes in islet cells may trigger tissue-specific autoimmunity in NOD mice.

Periostin conditions the matrix to generate a niche for islet regeneration.

Islet replacement via the Edmonton Protocol offers proof of concept that transient insulin independence can be achieved in patients with diabetes (1–4). However, the need for life-long immunosuppression, an extreme shortage of human islets, along with graft failure due to rejection and continued autoimmunity, limits the widespread application of this approach. Although recent progress has been made in the derivation of “ -like” cells from human pluripotent stem cell sources (embryonic stem cells or induced pluripotent stem cells) (5–10), inducing the replication of surviving -cells in situ (11, 12) and/or stimulating new islet formation from progenitor cells within the pancreas (islet neogenesis) (13–16) represents the most direct way to increase -cell mass. Studies in both rodents and humans suggest endogenous islet repair mechanisms may remain intact during diabetes and can be activated by cellor pharmacologically-mediated stimuli. Evidence of ongoing -cell regeneration has been observed in type 1 diabetes (T1D) patients at early onset (17) or many years after diagnosis (18). Indeed, recent analysis of T1D patients with disease duration of more than 50 years (medalists) has uncovered sustained c-peptide and proliferating -cells within the pancreas in the face of ongoing autoimmunity (19). Moreover, the increase in -cell mass in response to pregnancy suggests that the human endocrine pancreas has the capacity to regenerate if we can understand how to “tip the balance” in favor of islet regeneration vs destruction during diabetes. Interestingly, increased -cell replication was first suggested as the mechanism for increased -cell mass during pregnancy, but a recent autopsy study of human pancreases during pregnancy showed an increased proportion of small islets and increased number of insulin cells in the ducts but no change in -cell replication or apoptosis frequency (20). Nonetheless, novel strategies to optimize islet regeneration, either by stimulating -cell replication or by harnessing endogenous islet neogenic processes within the diabetic pancreas, remain under intense investigation for the treatment of diabetes. In initial publications documenting endogenous islet regeneration by stem cells, transplantation of bone marrow-derived c-kit cells reduced hyperglycemia in mice with streptozotocin (STZ)-induced -cell deletion (21). Importantly, transplanted stem cells did not acquire insulin expression as previously thought. In contrast, donor cells surrounded regenerating islets or engrafted in ductal regions and stimulated proliferation within recipient islets via undetermined paracrine activities (21). Subsequently, other preclinical and early clinical reports have shown that both human hematopoietic (22–26) or multipotent stromal cell (25–28), also known as mesenchymal stem cell (MSC) lineages, can enhance islet regeneration. Furthermore, islet regeneration and immune protection may be achieved by coinfusion of murine hematopoietic cells with MSC in autoimmune non-obese diabetic mice (29). Although this concept termed “stem cell-stimulated islet regeneration” has emerged as a central process for pancreas repair, either by stimulating -cell proliferation (11, 24– 26) or by initiating new islet formation from ductal or islet-derived precursors (14–16, 25, 26), the molecular signaling mechanisms by which distinct stem and progenitor cell types mediate islet recovery are not well understood. More importantly, recent controversy of the role of betatrophin in islet regeneration (30, 31) has underscored

The dual role of scavenger receptor class A in development of diabetes in autoimmune NOD mice.

Human type 1 diabetes is an autoimmune disease that results from the autoreactive destruction of pancreatic β cells by T cells. Antigen presenting cells including dendritic cells and macrophages are required to activate and suppress antigen-specific T cells. It has been suggested that antigen uptake from live cells by dendritic cells via scavenger receptor class A (SR-A) may be important. However, the role of SR-A in autoimmune disease is unknown. In this study, SR-A−/− nonobese diabetic (NOD) mice showed significant attenuation of insulitis, lower levels of insulin autoantibodies, and suppression of diabetes development compared with NOD mice. We also found that diabetes progression in SR-A−/− NOD mice treated with low-dose polyinosinic-polycytidylic acid (poly(I∶C)) was significantly accelerated compared with that in disease-resistant NOD mice treated with low-dose poly(I∶C). In addition, injection of high-dose poly(I∶C) to mimic an acute RNA virus infection significantly accelerated diabetes development in young SR-A−/− NOD mice compared with untreated SR-A−/− NOD mice. Pathogenic cells including CD4+CD25+ activated T cells were increased more in SR-A−/− NOD mice treated with poly(I∶C) than in untreated SR-A−/− NOD mice. These results suggested that viral infection might accelerate diabetes development even in diabetes-resistant subjects. In conclusion, our studies demonstrated that diabetes progression was suppressed in SR-A−/− NOD mice and that acceleration of diabetes development could be induced in young mice by poly(I∶C) treatment even in SR-A−/− NOD mice. These results suggest that SR-A on antigen presenting cells such as dendritic cells may play an unfavorable role in the steady state and a protective role in a mild infection. Our findings imply that SR-A may be an important target for improving therapeutic strategies for type 1 diabetes.

Molecular pathway alterations in CD4 T-cells of nonobese diabetic (NOD) mice in the preinsulitis phase of autoimmune diabetes

Type 1 diabetes (T1D) is a multigenic disease caused by T-cell mediated destruction of the insulin producing pancreatic islet β-cells. The earliest sign of islet autoimmunity in NOD mice, islet leukocytic infiltration or insulitis, is obvious at around 5 weeks of age. The molecular alterations that occur in T cells prior to insulitis and that may contribute to T1D development are poorly understood. Since CD4 T-cells are essential to T1D development, we tested the hypothesis that multiple genes/molecular pathways are altered in these cells prior to insulitis. We performed a genome-wide transcriptome and pathway analysis of whole, untreated CD4 T-cells from 2, 3, and 4 week-old NOD mice in comparison to two control strains (NOR and C57BL/6). We identified many differentially expressed genes in the NOD mice at each time point. Many of these genes (herein referred to as NOD altered genes) lie within known diabetes susceptibility (insulin-dependent diabetes, Idd) regions, e.g. two diabetes resistant loci, Idd27 (tripartite motif-containing family genes) and Idd13 (several genes), and the CD4 T-cell diabetogenic activity locus, Idd9/11 (2 genes, KH domain containing, RNA binding, signal transduction associated 1 and protein tyrosine phosphatase 4a2). The biological processes associated with these altered genes included, apoptosis/cell proliferation and metabolic pathways (predominant at 2 weeks); inflammation and cell signaling/activation (predominant at 3 weeks); and innate and adaptive immune responses (predominant at 4 weeks). Pathway analysis identified several factors that may regulate these abnormalities: eight, common to all 3 ages (interferon regulatory factor 1, hepatic nuclear factor 4, alpha, transformation related protein 53, BCL2-like 1 (lies within Idd13), interferon gamma, interleukin 4, interleukin 15, and prostaglandin E2); and two each, common to 2 and 4 weeks (androgen receptor and interleukin 6); and to 3 and 4 weeks (interferon alpha and interferon regulatory factor 7). Others were unique to the various ages, e.g. myelocytomatosis oncogene, jun oncogene, and amyloid beta (A4) to 2 weeks; tumor necrosis factor, transforming growth factor, beta 1, NFκB, ERK, and p38MAPK to 3 weeks; and interleukin 12 and signal transducer and activator of transcription 4 to 4 weeks. Thus, our study demonstrated that expression of many genes that lie within several Idds (e.g. Idd27, Idd13 and Idd9/11) was altered in CD4 T-cells in the early induction phase of autoimmune diabetes and identified their associated molecular pathways. These data offer the opportunity to test hypotheses on the roles played by the altered genes/molecular pathways, to understand better the mechanisms of CD4 T-cell diabetogenesis, and to develop new therapeutic strategies for T1D.

Attenuation of Donor-Reactive T Cells Allows Effective Control of Allograft Rejection Using Regulatory T Cell Therapy

Regulatory T cells (Tregs) are essential for the establishment and maintenance of immune tolerance, suggesting a potential therapeutic role for Tregs in transplantation. However, Treg administration alone is insufficient in inducing long-term allograft survival in normal hosts, likely due to the high frequency of alloreactive T cells. We hypothesized that a targeted reduction of alloreactive T effector cells would allow a therapeutic window for Treg efficacy. Here we show that preconditioning recipient mice with donor-specific transfusion followed by cyclophosphamide treatment deleted 70–80% donor-reactive T cells, but failed to prolong islet allograft survival. However, infusion of either 5 × 106 Tregs with direct donor reactivity or 25 × 106 polyclonal Tregs led to indefinite survival of BALB/c islets in more than 70% of preconditioned C57BL/6 recipients. Notably, protection of C3H islets in autoimmune nonobese diabetic mice required islet autoantigen-specific Tregs together with polyclonal Tregs. Treg therapy led to significant reduction of CD8+ T cells and concomitant increase in endogenous Tregs among graft-infiltrating cells early after transplantation. Together, these results demonstrate that reduction of the donor-reactive T cells will be an important component of Treg-based therapies in transplantation.

Preventive effects of andrographolide on the development of diabetes in autoimmune diabetic NOD mice by inducing immune tolerance

Andrographolide, an active component in traditional anti-diabetic herbal plants, is a diterpenoid lactone isolated from Andrographis paniculata because of its potent anti-inflammatory and hypoglycemic effects. However, the effect of andrographolide on the development of diabetes in autoimmune non-obese diabetic (NOD) mice remains unknown. This study aimed to investigate the protective effects of andrographolide on the development of autoimmune diabetes and clarify the underlying mechanism. NOD mice were randomly divided into four groups and administered with water and andrographolide at 50, 100, and 150mg/kg body weight for four weeks. ICR mice were also selected as the control group. Oral glucose tolerance and histopathological insulitis were examined. Th1/Th2/Th17 cytokine secretion was determined by ELISA. The transcriptional profiles of T-bet, GATA3, and RORγt in the pancreatic lymphatic node samples derived from the NOD mice were detected by RT-PCR. After four weeks of oral supplementation, andrographolide significantly inhibited insulitis, delayed the onset, and suppressed the development of diabetes in 30-week-old NOD mice in a dose dependent manner. This protective status was correlated with a substantially decreased production of interferon (IFN)-γ and interleukin (IL)-2, increased IL-10 and transforming growth factor (TGF)-β, and a reduced IL-17. Andrographolide also increased GATA3 mRNA expression but decreased T-bet and RORγt mRNA expressions. Our results suggested that andrographolide prevented type 1 diabetes by maintaining Th1/Th2/Th17 homeostasis.

MHC Class II Protein Turnover In vivo and Its Relevance for Autoimmunity in Non-Obese Diabetic Mice.

Major histocompatibility complex class II (MHCII) proteins are loaded with endosomal peptides and reside at the surface of antigen-presenting cells (APCs) for a time before being degraded. In vitro, MHCII protein levels and turnover are affected by peptide loading and by rates of ubiquitin-dependent internalization from the cell surface, which is in turn affected by APC type and activation state. Prior work suggested that fast turnover of disease-associated MHCII alleles may contribute to autoimmunity. We recently developed novel stable isotope tracer techniques to test this hypothesis in vivo. In non-obese diabetic (NOD) mice, a model of type 1 diabetes (T1D), MHCII turnover was affected by APC type, but unaffected by disease-associated structural polymorphism. Differences in MHCII turnover were observed between NOD colonies with high and low T1D incidence, but fast turnover was dispensable for autoimmunity. Moreover, NOD mice with gene knockouts of peptide loading cofactors do not develop T1D. Thus, fast turnover does not appear pathogenic, and conventional antigen presentation is critical for autoimmunity in NOD mice. However, shared environmental factors may underpin colony differences in MHCII protein turnover, immune regulation, and pathogenesis.

Insulin as a key autoantigen in the development of type 1 diabetes

Type 1 diabetes is a T-cell-mediated autoimmune disease against pancreatic beta cells. T cells target various antigens such as insulin, chromogranin A, glutamic acid decarboxylase and islet-specific glucose-6-phosphatase catalytic subunit-related protein. Elimination of insulin dramatically prevents diabetes in the non-obese diabetic (NOD) mouse model and response to insulin occurs prior to that to other antigens. These findings suggest that insulin is a target antigen at the early stage of the disease and is likely to be essential to cause anti-islet autoimmunity in NOD mice. In this review, we discuss whether insulin is truly essential and is only the single essential autoantigen for NOD mice and potentially for man. Although the ultimate principle is still being addressed, it is certain that T-cell response to insulin is a major check point to develop type 1 diabetes in NOD mice. Given multiple similarities between diabetes of NOD mice and man, targeting insulin and insulin-reactive T cells may provide opportunities to develop robust immunotherapies.