Original Article

Immunology and Cell Biology (2010) 88, 734–745; doi:10.1038/icb.2010.29; published online 23 March 2010

New insights into the role of VIP on the ratio of T-cell subsets during the development of autoimmune diabetes

Rebeca Jimeno1, Rosa P Gomariz1, Irene Gutiérrez-Cañas1, Carmen Martínez2, Yasmina Juarranz1,3 and Javier Leceta1,3

  1. 1Departamento de Biología Celular, Facultad de Biología, Universidad Complutense de Madrid, Madrid, Spain
  2. 2Departamento de Biología Celular, Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain

Correspondence: Professor Y Juarranz, Departamento de Biología Celular, Facultad de Biología, Universidad Complutense de Madrid, Madrid 28040, Spain. E-mail: yashina@bio.ucm.es

3These authors contributed equally to this work.

Received 8 September 2009; Revised 10 February 2010; Accepted 11 February 2010; Published online 23 March 2010.

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Abstract

Type I diabetes is an autoimmune T-cell-mediated disease associated with overexpression of inflammatory mediators and the disturbance of different T-cell subsets. Vasoactive intestinal peptide (VIP) is a potent anti-inflammatory agent with regulatory effects on activated T cells. As the equilibrium between different T-cell subsets is involved in the final outcome, leading to tolerance or autoimmunity, we studied the evolution of markers for T cells in nonobese diabetic (NOD) mice. The study of different transcription factors, cytokines or cytokine receptors, shows that VIP interferes with functional phase of T helper 17 (Th17) cells and prevents the increase in the proportion of Th1 to Th17 cells. On the other hand, VIP-treated NOD mice show an increase in the proportion of CD4+CD25+ cells in the spleen. Thus, VIP switches the Tregs/Th17 ratio leading to tolerance in NOD mice. Similarly, VIP reverses the ratio of Th1-/Th2-cell subsets associated with autoimmune pathology. All these effects on the ratio of T-cell subsets and the anti-inflammatory effect of VIP in decreasing proinflammatory mediators result in a reduction of β-cell destruction in pancreas. Taken together, these results show that VIP provides significant protection against spontaneous diabetes by modulating T-cell subsets and counterbalancing tolerance and immunity.

Keywords:

vasoactive intestinal peptide; NOD mice; type I diabetes; Th17; T-cell subsets; regulatory T cell

Human type I diabetes (T1D) is an autoimmune disease that results from the destruction of pancreatic insulin-producing β-cells.1, 2 The autoimmune response is believed to be due to a breakdown of immunological tolerance, resulting in a lack of immunoregulation of autoreactive diabetogenic T cells. The nonobese diabetic (NOD) mouse is a strain that is genetically prone to develop different, organ-specific, autoimmune diseases. It has been a very useful model for studying the mechanisms involved in the initiation and propagation of T1D.3 In NOD mice, islet antigens first appear in the pancreatic lymph nodes at around 2–4 weeks of age; autoreactive effectors T cells are sensitized and then infiltrate the islets.4 Progression to overt disease occurs in 80% of female mice between 10 and 30 weeks and it results from T-cell-mediated islet destruction.

T helper (Th) cells have been classified into different functional subsets, each characterized by its specific cytokine pattern and effector function.5 The Th1 subset is thought to be a crucial player in most organ-specific autoimmunity, including T1D, in which type I cytokines (interferon-γ (IFNγ), interleukin-2 (IL-2) and tumor necrosis factor-α (TNFα)) predominate over type II (Th2) and regulatory (Tregs) cytokines (IL-4 and IL-10). Two important classes of Tregs within the CD4+ T-cell subset are CD4+CD25+ Foxp3+ Tregs and T-regulatory type I (Tr1) cells, that differ in a number of biological features. There are two major categories of CD4+CD25+ Foxp3+ Tregs, the naturally occurring CD4+CD25+ Foxp3+ Tregs from the thymus (nTregs) and the induced CD4+CD25+ Foxp3+ Tregs produced in the periphery (iTregs).6 Type I cytokines initiate a cascade of inflammatory processes in the islets, inducing the production of inflammatory cytokines and both oxygen- and nitrogen-free radicals that are toxic to β-cells.7 Available data suggest that Th1 has a major role in diabetes, driving the development of disease through IFNγ. Conversely, other studies indicate a protective role for IFNγ.8 However, a new subset, the Th17 cells, is believed to orchestrate inflammation and immunity in organ-specific autoimmune diseases.9, 10 It has recently been found that progression from insulitis to diabetes correlates with the expression of IL-17 in the pancreas,11 suggesting that Th17 cells have a pathological role in the development of T1D.8 This subset is dependent on IL-23 for survival and produces IL-17, IL-21 and IL-22. Its differentiation, however, is directed by IL-6, a proinflammatory cytokine, and transforming growth factor-β (TGFβ), a regulatory cytokine. As TGFβ is a key regulator for the differentiation of iTregs, one may hypothesize that the ratio between inflammatory and regulatory cytokines affects the final outcome of tolerance or autoimmunity.9, 12 Inefficient mechanisms of peripheral tolerance have been claimed to contribute to unbalancing the equilibrium between pathogenic and protective T cells, associated with the transition from the pre-diabetic to the diabetic phase. CD4+CD25+ regulatory T cells, acting by distinct mechanisms, prevent the activation of potentially autoreactive effector T cells.6 CD4+CD25+ regulatory T cells may control self-tolerance in NOD mice during the pre-diabetic phase, but their proportion and functional capacity has been shown to be reduced during diabetes development. The progression to irreversible β-cell destruction could be due to the progressive decline in the number and/or the functional capacities of Tregs cells or to the progressive resistance of effector mechanisms to immunoregulation.13

Vasoactive intestinal peptide (VIP) is a 28 aminoacid neuropeptide first isolated by Said and Mutt14 that belongs to the secretin family. Its action is mediated by three heterotrimeric G-protein-coupled receptors that also interact with pituitary adenylate cyclase-activating peptide, a peptide of the same family which is 68% identical.15, 16 Although VIP is widely distributed in the central and peripheral nervous systems, it has subsequently been found to be produced by different endocrine and immune cells.17, 18, 19 It is capable of eliciting a broad spectrum of biological actions, being a potent neuroendocrine mediator of physiological responses that influence the development of endocrine pancreas and insulin release.20, 21, 22 Moreover, VIP is one of the best-studied immunoregulatory neuropeptides. These studies revealed that VIP can modulate both innate and adaptive immunity, showing a predominantly anti-inflammatory action. It decreases the Th1/Th2 cytokine ratio and promotes T regulatory functions.17, 18, 19, 23 An increasing body of in vivo data indicates that administration of VIP or pituitary adenylate cyclase-activating peptide may have promising outcomes in the treatment of inflammatory and autoimmune diseases, in particular Th1-associated pathologies such as multiple sclerosis, rheumatoid arthritis, Sjogren's syndrome and Crohn's disease.24, 25, 26, 27, 28 It has recently been described that VIP regulates both in vivo Th17 and IL-17 production in autoimmunity,29, 30 and in vitro Th17 differentiation.31, 32 Previous reports indicate that VIP also prevents diabetes development in NOD mice.33, 34 The protective mechanism is associated with reduced circulating levels of Th1 cytokines, increased levels of IL-10 and upregulation of markers for regulatory T cells.27, 33, 35

As the equilibrium between different T-cell subsets is involved in the final outcome leading to tolerance or autoimmunity, the aim of the present work is to examine the evolution of T-cell marker functions during the development of autoimmune diabetes and the effect of VIP treatment, showing a time–course study of its effect on molecular and immunological mechanisms and analyzing the ratio of Th17/Tregs, Th17/Th1 or Th2/Th1 cells.

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Results

VIP delays onset and insulitis of autoimmune diabetes in mice

First evidence of diabetes appeared in vehicle-treated mice at 16–18 weeks of age, the incidence increasing gradually to 70% diabetic mice at 30 weeks. Vehicle-injected mice presented infiltration in 80% of the pancreatic islets, even in pre-diabetic states, at 10 weeks of age, with evidence of islet destruction affecting 60% of the islets during overt diabetes (Figures 1a and b). Islet infiltration in NOD mice treated with VIP was negligible at 10 weeks (lesser 10%) and progressed slowly, leaving 50% of islets intact at 30 weeks. Even in the infiltrated islets, the accumulation of inflammatory cells was restricted to the periphery (Figures 1a and b). We also analyzed two important mediators of inflammation in pancreas from nontreated and VIP-treated NOD mice by real-time PCR. TNFα is a principal proinflammatory cytokine that mediates apoptosis in pancreas acinar cells36, 37 and it has previously been shown that VIP inhibits this TNFα effect in acinar cells from NOD mouse submandibular glands.36 On the other hand, inducible nitric oxide synthase (iNOS)/nitric oxide have been implicated in the pathogenesis of this autoimmune disease, as well as in inflammatory conditions and apoptosis of β-cells in pancreas.38, 39 VIP is able to decrease local mRNA expression of iNOS in other animal models of autoimmune disease, such as rheumatoid arthritis.29 Figure 1c shows that mRNA expression of both proinflammatory mediators increased with the evolution of the disease in nontreated NOD mice. VIP treatment decreased the mRNA expression of TNFα and iNOS significantly in pancreas of NOD mice. With these results, we confirm the anti-inflammatory role of VIP during autoimmune diabetes.

Figure 1.
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VIP delays onset and insulitis of autoimmune diabetes in mice. (a) Histological pancreatic sections marked with Gomori stain of nontreated and VIP-treated NOD mice at 10 or 30 weeks of age. A representative experiment is shown. (b) Scoring of histological grade of insulitis was performed on pancreatic sections marked with Gomori stain. Islets were scored for no-insulitis, peri-insulitis, moderate insulitis and severe insulitis (see Methods). (c) mRNA expression of iNOS and TNFα was determined by real-time PCR at 10, 15, 20 and 25 weeks of age in pancreas and spleen from NOD and VIP-treated NOD mice. Values for the relative expression of each gene were determined as indicated under Methods. Data are means±s.e. of six mice per parameter checked, differences between untreated and VIP-treated NOD mice for each parameter studied were statistically significant; *P<0.05, **P<0.01.

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VIP delays onset of autoimmune diabetes in NOD-severe combined immunodeficiency mice during cell transfer experiments

To evaluate whether splenocytes from VIP-treated mice suppress the diabetogenic potential of diabetic splenocytes, we transferred spleen cells from diabetic NOD mice into female NOD-severe combined immunodeficiency (SCID) mice recipients, alone or cotransferred with spleen cells from age-matched VIP-treated mice (Figure 2a). Blood glucose levels in mice that only received cells from diabetic animals began to rise 3 weeks after transfer and were close to 600mg per 100ml at 4 weeks (Figure 2b). At 5 weeks after transfer, 100% of NOD-SCID mice that had only received cells from NOD mice died. NOD-SCID mice coprovided with cells of VIP-treated mice showed a higher viability, 100% of them being viable after 5 weeks. Increased blood glucose levels were also delayed by 2 weeks and developed more slowly. These results indicate that splenocytes from VIP-treated animals are able to delay the onset of diabetes transfer induced by cells from NOD mice, which is symptomatic of T-cell tolerance, by 15 days.

Figure 2.
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VIP delays onset of autoimmune diabetes in NOD-SCID mice during cell transfer experiments. (a) Cell transfer scheme. Two different groups of NOD-SCID mice were transferred, the first with splenocytes from NOD mice (20 weeks) and the second with the same cells plus splenocytes from VIP-treated NOD mice (20 weeks). (b) Circulating levels of glucose in the animals after cell transfer. Data are means±s.e. of two experiments with five animals per group. Differences between NOD-SCID mice that only received cells transferred from NOD mice and NOD-SCID mice that received cells transferred from NOD mice plus VIP-treated NOD mice were statistically significant; ***P<0.001.

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The VIP effect on the stabilization and functionality of Th17 cell subset in NOD mice

Previous reports using a collagen-induced mouse model of arthritis have shown that VIP treatment resulted in the suppression of IL-17 and IL-23, cytokines produced by Th17 cells.33, 34 To investigate the in vivo VIP effect on Th17 cells more deeply, we checked the mRNA expression of different cytokines related to the functionality (Figure 3a) and stabilization (Figure 3b) of Th17 cell subset.9, 40 NOD female mice received 2.5nmol of VIP intraperitoneally every other day from 4 to 30 weeks of age, and pancreas mRNA expression was evaluated by quantitative real-time PCR. The profile of IL-17, IL-17R, IL-22, IL-23p19, IL-12p40, IL-23R and IL-12Rβ1 was studied during the development of diabetes, from 10 to 25 weeks of age (Figure 3). IL-17, IL-23p19, IL-12p40 and IL-22 profiles of mRNA expression were very similar in NOD mice, all of which showed a peak in pancreas expression between 15 and 20 weeks of age. Treatment with VIP significantly decreased the mRNA expression of IL-17 at 25 weeks and of IL-22 at 20 weeks. IL-17 and IL-23 receptors showed a comparable profile to their ligands in NOD mice. However, no significant differences were observed in mRNA expression of the different receptors tested after VIP treatment. These results suggest that VIP decreases IL-17 and IL-22 expression in the pancreas of NOD mice at the end of diabetes evolution, which is likely to interfere with the functionality of Th17 cells.

Figure 3.
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VIP effect on the stabilization and functionality of Th17 cell subset. Pancreas mRNA expression of cytokines and cytokine receptors of both functional (a) and stabilization phases of Th17 (b) was measured by quantitative real-time PCR. Values for the relative expression of each gene were determined as indicated under Methods. Data are means±s.e. of six mice per parameter checked, differences between untreated and VIP-treated NOD mice for each parameter studied were statistically significant; *P<0.05, ***P< 0.001.

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VIP shifts the Th17/Th1 ratio in NOD mice

In NOD mice, a generalized intrinsic T-cell defect results in a deregulated cytokine effector function. The increase in circulating levels of Th1 cytokines, as well as an enhanced accumulation of Th1 cells in the pancreas, are markers of the progression of diabetes.7 However, increasing evidence points to the role of Th17 cells in the development of NOD mice, although Th17 cells transferred into NOD mice have shown to acquire a Th1-pathogenic phenotype.41, 42 There are no current studies related to the Th17/Th1 ratio in NOD mice; therefore, we considered the profile of this ratio during diabetes development and the effect of VIP treatment. The ratio between the expression of the transcription factors that commit to Th17 (RORγt) and Th1 (T-bet, encoded by Tbx21) cell lineages in both pancreas and spleen was determined. In general, this ratio decreased in pancreas and spleen from NOD mice with the onset of the diabetes (Figure 4). Treatment with VIP increased it in both tissues. IL-12 is the differentiation factor for Th1 cells and it is comprised of IL-12p35 and IL-12p40 subunits. It shares the latter with IL-23 cytokine, the stabilization factor for Th17 cells. In this respect, the IL-23p19/IL-12p35 mRNA ratio indicates the prevalence of Th1 or Th17 differentiation factors. This ratio decreased with age in NOD mice pancreas and the treatment with VIP increased it (Figure 4a). IL-23R, the specific receptor for IL-23, is comprised of IL-23R and IL-12Rβ1 subunits. The specific receptor for IL-12 is formed by IL-12Rβ1 and IL-12Rβ2 subunits; hence, IL-23R/IL-12Rβ2 ratio indicates the proportion of cells sensitive to Th1 or Th17 differentiation. VIP was also able to significantly increase the mRNA IL-23R/IL-12Rβ2 ratio in pancreas from NOD mice (Figure 4a). The ratio between IL-17 and IFNγ, the signature cytokines for Th17 and Th1, respectively, significantly switched in the spleen of VIP-treated NOD mice at 15 weeks of age (Figure 4b). In summary, NOD mice show declining values of this ratio with the progression of the disease that could be due to an increase of the Th1 cell subset or a decrease of the Th17 cell subset as the disease develops. Treatment with VIP reverts this ratio, increasing Th17 cells relative to the Th1-cell subset.

Figure 4.
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VIP effect on the Th17/Th1 ratio in NOD mice. mRNA expression of Tbx21, RORγt, IL-23p19, IL-12p35, IL-23R, IL-12Rβ2, IL-17 and IFNγ was determined by real-time PCR at 10, 15, 20 and 25 weeks of age in pancreas (a) and spleen (b) from NOD and VIP-treated NOD mice. Values for the relative expression of each gene were determined as indicated under Methods. The ratios between mRNA expression of RORγt/Tbx21, IL-23p19/IL-12p35, IL-23R/IL-12Rβ2 and IL-17/IFNγ are shown. Data are means±s.e. of six mice per parameter checked, differences between untreated and VIP-treated NOD mice for each parameter studied were statistically significant; *P<0.05, **P<0.01, ***P< 0.001.

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VIP modulates the presence of CD4+CD25+ T cells in the spleens of NOD mice

CD4+CD25+ Tregs cells are a significant regulatory subset involved in control of diabetes in NOD mice.43, 44 Previous results indicate that VIP restores tolerance to pancreatic islets from NOD mice by promoting the local differentiation and function of Tregs.33 However, there are no data related to VIP effect at systemic level in this model. We therefore studied the effect of VIP treatment on the evolution of this cell population in the spleen by flow cytometry. Figure 5 shows that the proportion of CD4+CD25+ T cells progressively decreased as the disease progressed. However, in mice treated with VIP, the percentage of this cell population was significantly higher than in nontreated mice between 20 and 30 weeks of age. These results confirm that VIP modulates CD4+CD25+ T cells development and function, and may override the breakdown of self-tolerance during autoimmune diabetes in mice.

Figure 5.
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VIP modulates the presence of CD4+CD25+ T cells in the spleen of NOD mice. Flow cytometry analysis of splenic CD4+CD25+ cells was performed on spleen cell suspensions stained with PE-CD4 and FITC-CD25 monoclonal antibodies and analyzed on a FACSCalibur flow cytometer (above). The indicated proportion of double-positive cells was determined in the gated lymphocyte subpopulation based on forward and side-scatter parameters. A representative experiment is shown for 10 and 30 weeks of age (below). Data are means±s.e. of six mice per parameter checked, differences between untreated and VIP-treated NOD mice for each parameter studied were statistically significant; *P<0.05.

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VIP shifts the Tregs/Th17 ratio in NOD mice

To study whether the protective effect of VIP on diabetes outcome in NOD mice33 involves a switch of this ratio, we studied the evolution of several markers related to these T-cell subsets in VIP-treated and nontreated NOD mice. Pancreas and spleen mRNA was tested at different weeks of age by quantitative real-time PCR for Foxp3, TGFβ, IL-10 or IL-27p28 as markers for Tregs, and RORγt, IL-6, IL-17 and IL-23p19 as markers for Th17 cells. The ratio between the expression of the transcription factor that corresponds to Tregs (Foxp3) and Th17 (RORγt) cell lineages in both pancreas and spleen was determined (Figure 6). At local level, in pancreas, the treatment with VIP significantly augmented the Foxp3/RORγt ratio at 10, 15 and 25 weeks of age, whereas it decreased at 20 weeks of age. Treatment with VIP in spleen also showed an increase in this ratio at 25 weeks of age. Two cytokines with opposing effects, TGFβ and IL-6, cooperate to induce the differentiation of Th17 cells. However, TGFβ alone drives the conversion of naïve T cells into Foxp3+ Tregs. We therefore also checked the ratio between TGFβ and IL-6. VIP treatment also notably increased the TGFβ/IL-6 ratio in pancreas at 10, 15 and 25 weeks (Figure 6a). Two other cytokines involved in the Tregs-Th17 dichotomy, IL-10 and IL-17, were also analyzed by the means of their mRNA expressions in pancreas and spleen. VIP increased IL-10 expression versus IL-17 at 20 and 25 weeks of age in pancreas (Figure 6a), whereas there were no significant differences in spleen (Figure 6b). IL-23, comprised of IL-12p40 plus IL-23p19 subunits, is the stabilization factor for Th17 cells. IL-27, comprised of IL-12p40 plus IL-27p28, inhibits Th17 response or, together with TGFβ, might be the differentiation factor for IL-10-producing T cells, Tr1.9, 45, 46 We thus studied the evolution of the IL-27p28/IL-23p19 ratio during the onset of diabetes in NOD mice. Results showed a decreased ratio between 15 and 20 weeks of age, which was increased at 10 and 25 weeks (Figure 6a). The treatment with VIP only increased this ratio significantly at 10 weeks of age (Figure 6a). All these results indicate that VIP reverses the ratio between Tregs/Th17 cell subsets, leading to tolerance in NOD mice.

Figure 6.
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VIP shifts the Tregs/Th17 ratio in NOD mice. mRNA expression of Foxp3, RORγt, TGFβ, IL-6, IL-10, IL-17, IL-27p28, IL-23p19 was determined by real-time PCR at 10, 15, 20 and 25 weeks of age in pancreas (a) and spleen (b) from NOD and VIP-treated NOD mice. Values for the relative expression for each gene were determined as indicated under Methods. The ratios between mRNA expression of Foxp3/RORγt, TGFβ/IL-6, IL-10/IL-17 and IL-27p28/IL-23p19 are shown. Data are means±s.e. of six mice per parameter checked, differences between untreated and VIP-treated NOD mice for each parameter studied were statistically significant; *P<0.05, **P<0.01, ***P<0.001.

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VIP modifies the Th1/Th2 ratio in NOD mice

Th2 cells are associated with a beneficial effect in NOD mice.47 It has been generally shown that VIP increases Th2 and decreases Th1 cells in different animal models of autoimmune disease.17, 18 In humans, VIP can differentially modify the functional capacity of human lymphocytes by inducing Th2 differentiation.48 In addition, previous results have shown that VIP increased GATA-3 (the transcription factor for Th2 cells) and decreased Tbx21 mRNA expression in pancreas from NOD mice at 15 weeks.33 To monitor the effect of VIP on the Th1/Th2 ratio during the evolution of diabetes in NOD mice, we tested the mRNA expression of Tbx21 and GATA-3 in pancreas and spleen from nontreated and VIP-treated NOD mice at 10, 15, 20 and 25 weeks. Pancreas and spleen Tbx21/GATA-3 ratios were significantly decreased in VIP-treated NOD mice at 10, 15 and 25 weeks of age, compared with nontreated NOD mice (Figure 7). This means that NOD mice show an increased Th1 cell subset during the development of the autoimmune pathology. However, treatment with VIP reverses this ratio, increasing Th2 relative to Th1 cell subsets.

Figure 7.
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VIP changes Th1/Th2 ratio in NOD mice. mRNA expression of Tbx21 and GATA-3 was determined by real-time PCR at 10, 15, 20 and 25 weeks of age in pancreas and spleen from NOD and VIP-treated NOD mice. Values for the relative expression of each gene were determined as indicated under Methods. The ratios between mRNA expression of Tbx21/GATA-3 are shown (left). The individual mRNA expression values are shown (right). Data are means±s.e. of six mice per parameter checked, differences between untreated and VIP-treated NOD mice for each parameter studied were statistically significant; *P<0.05, **P<0.01, ***P<0.001.

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Discussion

Type I diabetes is a chronic autoimmune disease, in which different phases can be recognized. During the induction and pre-diabetic stage, innate immune mechanisms contribute to the development of insulitis, a self-amplification process ultimately leading to an adaptive immune response against pancreatic antigens.2 During this process, islets are infiltrated and damaged in such a way that, by 20 weeks of age, 80% of islets show a high degree of destruction, coinciding with diabetes onset. The late stage of diabetes is characterized by stabilization of insulitis. In this stage, there is no further destruction of islets and possibly some kind of resolution may occur, as proposed by some authors who consider T1D as a relapsing–remitting disease.49 On the other hand, in VIP-treated NOD mice insulitis is always mild, develops slowly and by 30 weeks, 75% of islets show a well-preserved architecture. Different components of innate immunity contribute to the development of insulitis, and its defective resolution contributes to progressive loss of β-cells. Inflammatory mediators, such as IL-1, TNFα and nitric oxide are toxic to β-cells and induce their apoptosis,50 mediated, at least in part, by activation of the NF-κB signaling pathway. These mediators are also responsible for the recruitment of cells participating in the adaptive immune reaction that amplifies and maintains the inflammatory response in this manner. However, under the influence of VIP, there is reduced sensitivity of cells to inflammatory stimuli that produce a reduced amount of chemokines, inflammatory cytokines and inflammatory mediators such as nitric oxide.28, 35, 51 This effect is related to the property of VIP to downregulate the expression and signaling of different Toll-like receptors,23, 52, 53 receptors recognizing pattern-recognition motifs that participate in the induction of T1D.2 These properties of VIP may be responsible for the low expression of TNFα and iNOS in the pancreas of treated NOD mice, which may explain the preservation of islet structure preventing diabetes onset.

In addition to the importance of these inflammatory mediators, different T cells have a critical role in the development of diabetes in NOD mice. For this reason, the present report describes the evolution of markers associated with the activity of different T-cell subsets and/or functions implicated in diabetes evolution in NOD mice.

T helper 1 autoreactive subsets that mediate delayed-type hypersensitivity reactions have been implicated in several autoimmune disorders, including T1D. This assumption is based on the predominant production of the Th1 cytokine IFNγ in the draining lymph node and target organs after appropriate immunization and the transfer of the disease by antigen-specific Th1 clones. Moreover, in relapsing–remitting diseases, a decrease in the level of IFNγ-producing cells was observed in the recovery phase of the disease. Moreover, genetic deficiency of IFNγ led to increased susceptibility in experimental autoimmune encephalomyelitis,54 and IFNγ has been shown to have a protective role in joint damage during rheumatoid arthritis.55 On the other hand, mice deficient in IL-12p35 show exacerbated diseases and IL-23p19 deficiency protects from developing experimental autoimmune encephalomyelitis.56 However, IL-23-deficient mice show IFNγ-secreting Th1 cells infiltrating the nervous tissue after MOG immunization, but are protected from experimental autoimmune encephalomyelitis development.57 These unexpected findings provide evidence against a critical pathogenic role for Th1 cells in autoimmunity and point to the importance of Th17 in certain autoimmune disorders. There are also studies that show that IL-17 is not absolutely required for autoimmune arthritis.58 Studies performed in the NOD mouse model indicate that both Th1 and Th17 antigen-specific cells are diabetogenic.42

Results presented here indicate that Th17 activity, as evidenced by the expression of RORγt, IL-17, IL-17R, IL-22, IL23p19 or IL-23R, gradually increases in the pancreas of NOD mice, peaking at the onset of diabetes (20 weeks) and then declining. On the other hand, Th1 activity, indicated by the expression of Tbx21, IL-12p35 and IL-12Rβ2, also increases as diabetes develops and, although there is a reduction at the time of maximal incidence, their expression is still high. As a consequence, the Th17/Th1 ratio gradually decreases during development of diabetes, indicating a preponderance of the Th1 response. Although increasing evidence points to the role of Th17 cells in NOD mice,41 it seems that the conversion of this cell subset into Th142, 59 is more important. In VIP-treated mice, the Th17 evolution is similar, although at the time of maximal diabetes incidence, the expression of the reported factors is lower than in nontreated mice. Besides, the expression of the Th1 markers is significantly lower in VIP-treated mice. Our results from examining the RORγt/Tbx21, IL-23p19/IL-12p35 and IL-23R/IL-12Rβ2 ratios, reveal that this process may be impaired by VIP treatment as this ratio in VIP-treated mice increases gradually, indicating a reduction in Th1 activity that could be protective. However, the ratio of the effector cytokines IL-17 and IFNγ is similar in both groups; hence, these observations indicate that other subsets with different functions may be the source of IFNγ.8, 60

Paradoxically, Th17 development is induced by factors that also differentiate T-regulatory cells. The latter cell type has been implicated in modulating immune tolerance. TGFβ is required for maintaining the population of naturally occurring T-regulatory cells and also induces de novo generation of the so-called induced regulatory T cells. However, in the presence of IL-6 produced during inflammation, TGFβ generates Th17 cells, indicating a reciprocal relationship in the development of Th17 and Treg cells.12 Several cell populations with immunoregulatory properties influence the progression of diabetes in NOD mice. In fact, their depletion or a deficiency in their number or activity is associated with more aggressive disease, whereas their transfer confers protective effects.61 Some authors have suggested primary defects in the number or function of Tregs in NOD mice, but these results have not been reproduced by other groups. Some discrepancy may arise from the use of different cell markers to identify Treg populations. Different T-cell subsets have been identified with regulatory function. nTregs and iTregs are characterized as CD4+CD25+ cells expressing the committing transcription factor Foxp3. Tr1 cells develop after antigen presentation in the presence of IL-10 and only express CD25 and Foxp3 after activation.62 Its therapeutic importance stems from observations indicating that the development of similar cell subsets differentiates under tolerance induction for diabetogenic antigens and reverses ongoing diabetes, preventing epitope spreading and restoring normoglycemia.8 This cell subset is characterized by the high production of IL-10, but it also produces IFNγ.

The results observed indicate that Foxp3 and TGFβ expression in pancreas, as a marker of Tregs activity, rises gradually in untreated NOD mice. VIP treatment increased their expression at the onset of diabetes. The more obvious change observed in VIP-treated mice is the high expression of IL-10 at 20 weeks, when diabetes appeared in NOD mice, and at this point, there is no reduction of IFNγ production.

Protection against diabetes development in NOD mice achieved by VIP may be mediated by the promotion of the development of Tregs. Other groups have found that the major mechanism by which VIP induces tolerance is through the generation of antigen-specific Tregs.63 The data observed in cell transfer to NOD-SCID mice indicate the presence of cells that delay the onset of diabetes in VIP-treated mice. However, although the induced character of this cell population is a well-established fact, a better characterization needs to be achieved. Observations arising from the data presented here indicate that these cells may belong to the Tr1 subset. These regulatory cells present less stable Tregs markers.64 Tr1 cells have been shown to differentiate on TCR stimulation in anti-inflammatory environments. In this context VIP, by their anti-inflammatory properties, may drive the differentiation of Tr1 cells.63

As described above, there are reciprocal relationships between Treg and Th17 cells,9, 10 and the ratio between these two T-cell subsets is important in determining the final outcome leading to tolerance or immunity. Foxp3/RORγt and TGFβ/IL-6 ratios increased with the development of diabetes in NOD mice. Treatment of VIP increased these ratios at 10, 15 and 25 weeks of age. There is also a significant effect of VIP on the IL-10/IL-17 ratio at 20 weeks of age. IL-27 was initially shown to induce Tbx21 expression and to enhance Th1 responses, but it also has dominant anti-inflammatory properties.65 It has also been identified as a differentiation factor for the generation of IL-10-producing Tr1 cells in the presence of TGFβ.9, 45, 46 Treatment with VIP increased the IL-27p28/IL-23p19 ratio during the onset of diabetes in NOD mice, indicating that VIP may be involved in the generation of Tr1 cells.

Initial immunoregulatory strategies to treat T1D used immunosuppressive agents, that were later followed by attempts to skew the immune response to self-antigens toward a nonaggressive Th2 response counteracting the inflammatory pathogenic Th1 response.66 Th2 differentiation was the first-reported immunoregulatory property of VIP.67 Accordingly, in the present report, the ratio between the expression of the specific transcription factors T-bet and GATA3 is skewed toward Th2 in the pancreas of VIP-treated mice. Although cytokine shift has been associated with disease protection and has been ascribed to the Th1/Th2 ratio, this assumption may not be valid as some cytokines considered as markers for the subpopulations are produced by different cell types. In particular, IL-10 is produced by several T-cell subsets and also by macrophages and dendritic cells under certain stimuli. Dendritic cells are especially important in the differentiation of the effector cell type mediating the immune response. They also produce inflammatory mediators. In vitro studies have shown that dendritic cells differentiated in the presence of VIP-induced antigen-specific T cells with Tr1 phenotype.63 In this respect, the effects of VIP on the innate immune system may be especially relevant for the evolution of autoimmune diseases, and particularly in T1D described here for the NOD mouse model.

In conclusion, although additional studies are needed to clarify the interrelation between T-cell subsets during the development of diabetes autoimmunity in NOD mice, our data show that VIP modulates inflammation as well as the Th17/Th1, Tregs/Th17 and Th1/Th2 ratios, providing significant protection against spontaneous diabetes and counterbalancing tolerance and immunity.

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Methods

Mouse and treatment protocol

Female NOD mice (NOD/NHS; Harlan, IN, USA) were injected intraperitoneally every other day from 4 to 30 weeks of age with 2.5nmol of VIP (Neosystem, Strasbourg, France) in 200μl phosphate-buffered saline. Control animals received 200μl of phosphate-buffered saline alone. Blood glucose levels in tail venous blood were quantified once a week using Accutrend Sensor Complete (Roche Diagnostics, Mannheim, Germany). A diagnosis of diabetes was made after two consecutive measurements higher than 240mg per 100ml. The experiments were performed according to rules accepted by the local ethics commission for investigations on live animals.

RNA extraction and quantitative real-time reverse transcriptase-PCR

A tissue tearer was used to homogenize pancreas or spleen tissue and total RNA was extracted with the Ultraspec RNA reagent, as recommended by the manufacturer (Biotecx, Houston, TX, USA). RNA was resuspended in diethylpyrocarbonate water and quantitated at 260 or 280nm. RNA (2μg) was used for reverse transcription. cDNA was obtained by SuperScript reverse transcriptase (Invitrogen, Barcelona, Spain) or High Capacity cDNA reverse transcription (Applied Biosystems, Foster City, CA, USA) (Table 1). Then, cDNA was amplified by PCR analysis, using SYBR Green (Applied Biosystems) as a marker for DNA content. In brief, reactions were performed in 20μl, with 2μl cDNA, 10μl 2 × SYBR Green PCR Master Mix, together with the 0.3μM primers. The sequences of primers used and the accession numbers of the analyzed genes are summarized in Table 2. Amplification was performed in an 7900 HT Fast Real-Time PCR Systems apparatus (Applied Biosystems, Carlsbad, CA, USA) under the following conditions: 2min at 50°C, 10min at 95°C, 40 cycles of denaturation at 95°C for 15s and annealing/extension at 60°C for 1min.



For relative quantification, we used a method that compares the amount of target normalized to an endogenous reference. The formula used was Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author, representing the n-fold differential expression of a specific gene in a treated sample compared with the control sample, where Ct is the mean of the threshold cycle (the cycle at which the amplification of the PCR product is initially detected). ΔCt was the difference in the Ct values for the target gene and the reference gene and β-actin (in each sample assayed), and ΔΔCt represents the difference between the Ct from IL-12p40 gene expression of control Balb/c and each datum. Therefore, the final results do not correspond to particular quantitative measures, but to relative measures of each gene compared with the established common pattern, the IL-12p40 cytokine. Previously, we performed a validation experiment comparing the standard curve of the reference and the target, to show that efficiencies were approximately equal.

Flow cytometry

Spleens were removed and gently minced in a stainless steel sieve. Cell suspensions were rendered free of red blood cells by exposure to a solution containing 0.83% NH4Cl. The splenocytes were stained with a phycoerythrin-labeled anti-CD4 antibody and a fluorescein isothiocyanate-labeled anti-CD25 antibody (BD Bioscience, Pharmingen, San Diego, CA, USA). Incubation with the antibodies was performed at 4°C for 30min. Aliquots of splenocytes were incubated with a single fluorochrome-conjugated antibody and with isotype-matched control antibodies, to compensate for fluorescence emission overlap and nonspecific fluorescence, respectively. Cells were analyzed on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA).

Histopathology

Pancreases were removed, embedded in Tissue-Tek OTC compound (Miles Laboratories Inc, Clifton, NJ, USA) and snap-frozen on liquid nitrogen; 8-μm cryostat sections were stained according to the Gomori–Bergman protocol (chromic-hematoxylin floxin). The incidence and severity of insulitis was scored for at least 20 islets from each specimen by two independent, treatment-blind observers according to the following criteria: no lymphocytic infiltration (no-insulitis); lymphocytic infiltrations surrounding islets and ducts, but no infiltration of the islet architecture (peri-insulitis); lymphocytic infiltrations between 25 and 50% of the islet area (moderate insulitis); lymphocytic infiltrations >50% of the islet area and/or loss of islet architecture (severe insulitis).

Transfer of diabetes to NOD-SCID mice

Spleen cells were obtained from 20-week-old female NOD mice or VIP-treated NOD mice by mechanical dissociation using a stainless steel screen and isolated by centrifugation on Histopaque gradients (Sigma, St Louis, MO, USA). Splenocytes from VIP-treated and nontreated NOD mice were pooled separately and transferred to 8-week-old NOD-SCID mice. Recipients were divided into two groups and cells were injected intravenously through the tail vein in 200μl RPMI. One group received a cell suspension of 20 × 106 spleen cells of NOD mouse and a second group was cotransferred with 20 × 106 spleen cells from NOD mouse plus 20 × 106 spleen cells from VIP-treated NOD mouse. Blood glucose levels were determined once a week.

Statistical analysis

All values are expressed as the mean±s.e. of data obtained from at least four mice. Comparison between groups was made using the Student's t-test, Mann–Whitney W-test and ANOVA (analysis of variance) test.

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Acknowledgements

This work was supported by grants PI080025 from the Instituto de Salud Carlos III (ISCIII), GR58/08 from UCM-BSCH and by grants from the Instituto de Salud Carlos III (ISCIII) to IGC and RJ. This work was partially supported by RETICS Program, RD08/0075 (RIER) from the Instituto de Salud Carlos III (ISCIII), within the VI PN de I+D+I 2008-2011.