Preventative role of interleukin-17 producing regulatory T helper type 17 (Treg17) cells in type 1 diabetes in non-obese diabetic mice

T helper type 17 (Th17) cells have been shown to be pathogenic in autoimmune diseases; however, their role in type 1 diabetes (T1D) remains inconclusive. We have found that Th17 differentiation of CD4⁺ T cells from BDC2·5 T cell receptor transgenic non-obese diabetic (NOD) mice can be driven by interleukin (IL)-23 + IL-6 to produce large amounts of IL-22, and these cells induce T1D in young NOD mice upon adoptive transfer. Conversely, polarizing these cells with transforming growth factor (TGF)-β + IL-6 led to non-diabetogenic regulatory Th17 (T_reg17) cells that express high levels of aryl hydrocarbon receptor (AhR) and IL-10 but produced much reduced levels of IL-22. The diabetogenic potential of these Th17 subsets was assessed by adoptive transfer studies in young NOD mice and not NOD.severe combined immunodeficient (SCID) mice to prevent possible transdifferentiation of these cells in vivo. Based upon our results, we suggest that both pathogenic Th17 cells and non-pathogenic regulatory T_reg17 cells can be generated from CD4⁺ T cells under appropriate polarization conditions. This may explain the contradictory role of Th17 cells in T1D. The IL-17 producing T_reg17 cells offer a novel regulatory T cell population for the modulation of autoimmunity.     

CD40‐mediated signalling influences trafficking,T‐cell receptor expression,and T‐cell pathogenesis,in the NOD model of type 1 diabetes

CD40 plays a critical role in the pathogenesis of type 1 diabetes (T1D). The mechanism of action, however, is undetermined, probably because CD40 expression has been grossly underestimated. CD40 is expressed on numerous cell types that now include T cells and pancreatic β cells. CD40⁺ CD4⁺ cells [T helper type 40 (TH40)] prove highly pathogenic in NOD mice and in translational human T1D studies. We generated BDC2.5.CD40^−/− and re‐derived NOD.CD154^−/− mice to better understand the CD40 mechanism of action. Fully functional CD40 expression is required not only for T1D development but also for insulitis. In NOD mice, TH40 cell expansion in pancreatic lymph nodes occurs before insulitis and demonstrates an activated phenotype compared with conventional CD4⁺ cells, apparently regardless of antigen specificity. TH40 T‐cell receptor (TCR) usage demonstrates increases in several Vα and Vβ species, particularly Vα3.2⁺ that arise early and are sustained throughout disease development. TH40 cells isolated from diabetic pancreas demonstrate a relatively broad TCR repertoire rather than restricted clonal expansions. The expansion of the Vα/Vβ species associated with diabetes depends upon CD40 signalling; NOD.CD154^−/− mice do not expand the same TCR species. Finally, CD40‐mediated signals significantly increase pro‐inflammatory Th1‐ and Th17‐associated cytokines whereas CD28 co‐stimulus alternatively promotes regulatory cytokines.     

Interleukin‐17‐producing γδ+ T cells protect NOD mice from type 1 diabetes through a mechanism involving transforming growth factor‐β

Whether interleukin (IL)‐17 promotes a diabetogenic response remains unclear. Here we examined the effects of neutralization of IL‐17 on the progress of adoptively transferred diabetes. IL‐17‐producing cells in non‐obese diabetic (NOD) mice were identified and their role in the pathogenesis of diabetes examined using transfer and co‐transfer assays. Unexpectedly, we found that in vivo neutralization of IL‐17 did not protect NOD–severe combined immunodeficiency (SCID) mice against diabetes transferred by diabetic splenocytes. In NOD mice, γδ⁺ T cells were dominated by IL‐17‐producing cells and were found to be the major source of IL‐17. Interestingly, these IL‐17‐producing γδ T cells did not exacerbate diabetes in an adoptive transfer model, but had a regulatory effect, protecting NOD mice from diabetes by up‐regulating transforming growth factor (TGF)‐β production. Our data suggest that the presence of IL‐17 did not increase the chance of the development of diabetes; γδ T cells protected NOD mice from diabetes in a TGF‐β‐dependent manner, irrespective of their role as major IL‐17 producers.     

Inhibition of type 1 diabetes in filaria‐infected non‐obese diabetic mice is associated with a T helper type 2 shift and induction of FoxP3+ regulatory T cells

We sought to determine whether Litomosoides sigmodontis, a filarial infection of rodents, protects against type 1 diabetes in non‐obese diabetic (NOD) mice. Six‐week‐old NOD mice were sham‐infected or infected with either L3 larvae, adult male worms, or adult female worms. Whereas 82% of uninfected NOD mice developed diabetes by 25 weeks of age, no L. sigmodontis‐infected mice developed disease. Although all mice had evidence of ongoing islet cell inflammation by histology, L. sigmodontis‐infected mice had greater numbers of total islets and non‐infiltrated islets than control mice. Protection against diabetes was associated with a T helper type 2 (Th2) shift, as interleukin‐4 (IL‐4) and IL‐5 release from α‐CD3/α‐CD28‐stimulated splenocytes was greater in L. sigmodontis‐infected mice than in uninfected mice. Increased circulating levels of insulin‐specific immunoglobulin G1, showed that this Th2 shift occurs in response to one of the main autoantigens in diabetes. Multicolour flow cytometry studies demonstrated that protection against diabetes in L. sigmodontis‐infected NOD mice was associated with significantly increased numbers of splenic CD4⁺ CD25⁺ FoxP3⁺ regulatory T cells. Interestingly, injection of crude worm antigen into NOD mice also resulted in protection against type 1 diabetes, though to a lesser degree than infection with live L. sigmodontis worms. In conclusion, these studies demonstrate that filarial worms can protect against the onset of type 1 diabetes in NOD mice. This protection is associated with a Th2 shift, as demonstrated by cytokine and antibody production, and with an increase in CD4⁺ CD25⁺ FoxP3⁺ regulatory T cells.     

Detection of vasostatin‐1‐specific CD8+ T cells in non‐obese diabetic mice that contribute to diabetes pathogenesis

Chromogranin A (ChgA) is an antigenic target of pathogenic CD4⁺ T cells in a non‐obese diabetic (NOD) mouse model of type 1 diabetes (T1D). Vasostatin‐1 is a naturally processed fragment of ChgA. We have now identified a novel H2‐K^d‐restricted epitope of vasostatin‐1, ChgA 36‐44, which elicits CD8⁺ T cell responses in NOD mice. By using ChgA 36‐44/K^d tetramers we have determined the frequency of vasostatin‐1‐specific CD8⁺ T cells in pancreatic islets and draining lymph nodes of NOD mice. We also demonstrate that vasostatin‐1‐specific CD4⁺ and CD8⁺ T cells constitute a significant fraction of islet‐infiltrating T cells in diabetic NOD mice. Adoptive transfer of T cells from ChgA 36‐44 peptide‐primed NOD mice into NOD/severe combined immunodeficiency (SCID) mice led to T1D development. These findings indicate that vasostatin‐1‐specific CD8⁺ T cells contribute to the pathogenesis of type 1 diabetes in NOD mice.     

Pathogenicity of T helper 2 T-cell clones from T-cell receptor transgenic non-obese diabetic mice is determined by tumour necrosis factor-alpha

Autoimmune diabetes is predominated by a T helper 1 (Th1) response at the expense of an impaired Th2 response. Although T cells producing Th2 cytokines are generally thought to counter a Th1 response, there have been reports of Th2 T-cell clones with pathogenic activity, including one previously reported by us in which the Th2 T-cell clone was derived from a T-cell receptor transgenic (TCR-Tg) mouse bearing pathogenic TCR. In this study, our goal was to determine whether Th2 T-cell clones derived from a TCR-Tg in which the autoantigen was absent would be pathogenic and if so, to investigate possible mechanisms by which the Th2 T-cell clone could promote disease. We found that a Th2 T-cell clone derived from the 6.9 TCR-Tg/non-obese diabetic (NOD).C6 mouse in which 6.9 T cells do not encounter autoantigen, produced Th2 cytokines but not interferon-gamma. This Th2 T-cell clone, like the previous one we had isolated from the 2.5 TCR-Tg/NOD mouse, also turned out to be pathogenic. Intracellular staining revealed that these Th2 T-cell clones produce low levels of tumour necrosis factor-alpha (TNF-alpha) in vitro, and after adoptive transfer, they migrate to the pancreas where they produce TNF-alpha as well as Th2 cytokines (interleukin (IL)-4, IL-10). Induction of disease was prevented by administration of soluble TNF-alpha receptor to recipient mice, suggesting that the diabetogenicity of these Th2 T-cell clones is caused by their low level production of TNF-alpha.     

Standardized bovine colostrum derivative impedes development of type 1 diabetes in rodents

Bovine colostrum is a rich source of nutrients and immunologically active components that play a role in conveying passive immunity to the offspring, protection and maturation of new-born’s gastrointestinal tract. Colostrum has exerted positive effects in diseases affecting gastrointestinal tract, as well as type 2 diabetes (T2D). However, health-promoting effects in type 1 diabetes have not been reported. The aim of this study was to investigate therapeutic value of oral administration of standardized bovine colostrum derivative (SBCD) in three models of type 1 diabetes (T1D): spontaneously developed T1D in NOD mice and BB-DP rats, and in chemically induced T1D in C57BL/6 mice with multiple low doses of streptozotocin (MLDS). SBCD was administered per os and the disease development was evaluated by weekly measurement of blood glucose and by histological analyses of the pancreas. SBCD administration prevented diabetes development in all three models, as indicated by euglicaemia. Ex vivo analysis of cytokine expression and production in the spleen and mesenteric lymph nodes (MLN) in MLDS challenged mice revealed a strong modulation of the immune response. In the MLN cells SBCD disrupted harmful Th17 response induced by MLDS. Expression of Th1 signature cytokine IFN-γ was down-regulated in MLN cells of SBCD-treated mice, while IL-4 secretion (Th2 cytokine) was up-regulated in comparison to diabetic group. Modulation of the immune response seen in the MLN protruded to the spleen, giving overall less infiltration of immune cells to the pancreas. SBCD acted on immune cells and halted (auto) aggression towards pancreatic beta cells. Moreover, SBCD induced beta cell proliferation. Hence, this derivative could be tested in diabetes and other similar diseases with aberrant immune response.     

Regulatory T cells enter the pancreas during suppression of type 1 diabetes and inhibit effector T cells and macrophages in a TGF‐β‐dependent manner

Treg can suppress autoimmune diseases such as type 1 diabetes, but their in vivo activity during suppression remains poorly characterized. In type 1 diabetes, Treg activity has been demonstrated in the pancreatic lymph node, but little has been studied in the pancreas, the site of autoimmune islet destruction. In this study we induced islet‐specific Treg from the BDC‐6.9 TCR transgenic mouse by activation of T cells in the presence of TGF‐β. These Treg can suppress spontaneous diabetes as well as transfer of diabetes into NOD.scid mice by diabetic NOD spleen cells or activated BDC‐2.5 TCR transgenic Th1 effector T cells. In the latter transfer model, we observed infiltration of the pancreas by both effector T cells and Treg, suggesting that Treg are active in the inflammatory site and are not just restricted to the draining lymph node. Within the pancreas, we demonstrate that Treg transfer causes a reduction in the number of effector Th1 T cells and macrophages, and also inhibits effector T‐cell cytokine and chemokine production. Although we found no role for TGF‐β in vitro, transfection of effector T cells with a dominant‐negative TGF‐β receptor demonstrated that in vivo suppression of diabetes by TGF‐β‐induced Treg is TGF‐β‐dependent.     

The choice of adjuvant determines the cytokine profile of T cells in proteoglycan‐induced arthritis but does not influence disease severity

Rheumatoid arthritis (RA) is a debilitating autoimmune disease characterized by chronic inflammation of the synovial joints. Collagen‐induced arthritis (CIA) and proteoglycan‐induced arthritis (PGIA) are mouse models of inflammatory arthritis; CIA is a T helper type 17 (Th17) ‐dependent disease that is induced with antigen in complete Freund's adjuvant, whereas PGIA is Th1‐mediated and is induced using antigen in dimethyldioctadecyl‐ammonium bromide (DDA) as an adjuvant. To investigate whether the type of adjuvant determines the cytokine profile of the pathogenic T cells, we have compared the effect of CFA and DDA on T‐cell responses in a single arthritis model. No differences in incidence or disease severity between aggrecan‐T‐cell receptor transgenic mice immunized with aggrecan in either CFA or DDA were observed. Immunization with CFA resulted in a higher proportion of Th17 cells, whereas DDA induced more Th1 cells. However, the levels of interleukin‐17 (IL‐17) produced by T cells isolated from CFA‐immunized mice after antigen‐specific stimulation were not significantly different from those found in DDA‐immunized mice, indicating that the increased proportion of Th17 cells did not result in significantly higher ex vivo IL‐17 levels. Hence, the choice of adjuvant can affect the overall proportions of Th1 and Th17 cells, without necessarily affecting the level of cytokine production or disease incidence and severity.     

Effects of combination therapy with dipeptidyl peptidase‐IV and histone deacetylase inhibitors in the non‐obese diabetic mouse model of type 1 diabetes

Type 1 diabetes (T1D) results from T helper type 1 (Th1)‐mediated autoimmune destruction of insulin‐producing β cells. Novel experimental therapies for T1D target immunomodulation, β cell survival and inflammation. We examined combination therapy with the dipeptidyl peptidase‐IV inhibitor MK‐626 and the histone deacetylase inhibitor vorinostat in the non‐obese diabetic (NOD) mouse model of T1D. We hypothesized that combination therapy would ameliorate T1D by providing protection from β cell inflammatory destruction while simultaneously shifting the immune response towards immune‐tolerizing regulatory T cells (T_regs). Although neither mono‐ nor combination therapies with MK‐626 and vorinostat caused disease remission in diabetic NOD mice, the combination of MK‐626 and vorinostat increased β cell area and reduced the mean insulitis score compared to diabetic control mice. In prediabetic NOD mice, MK‐626 monotherapy resulted in improved glucose tolerance, a reduction in mean insulitis score and an increase in pancreatic lymph node T_reg percentage, and combination therapy with MK‐626 and vorinostat increased pancreatic lymph node T_reg percentage. We conclude that neither single nor combination therapies using MK‐626 and vorinostat induce diabetes remission in NOD mice, but combination therapy appears to have beneficial effects on β cell area, insulitis and T_reg populations. Combinations of vorinostat and MK‐626 may serve as beneficial adjunctive therapy in clinical trials for T1D prevention or remission.     

Endogenous interleukin‐22 protects against inflammatory bowel disease but not autoimmune cholangitis in dominant negative form of transforming growth factor beta receptor type II mice

During chronic inflammation, interleukin (IL)‐22 expression is up‐regulated in both CD4 and CD8 T cells, exerting a protective role in infections. However, in autoimmunity, IL‐22 appears to have either a protective or a pathogenic role in a variety of murine models of autoimmunity and, by extrapolation, in humans. It is not clear whether IL‐22 itself mediates inflammation or is a by‐product of inflammation. We have taken advantage of the dominant negative form of transforming growth factor beta receptor type II (dnTGF‐βRII) mice that develop both inflammatory bowel disease and autoimmune cholangitis and studied the role and the biological function of IL‐22 by generating IL‐22^–/– dnTGF‐βRII mice. Our data suggest that the influence of IL‐22 on autoimmunity is determined in part by the local microenvironment. In particular, IL‐22 deficiency exacerbates tissue injury in inflammatory bowel disease, but has no influence on either the hepatocytes or cholangiocytes in the same model. These data take on particular significance in the previously defined effects of IL‐17A, IL‐12p40 and IL‐23p19 deficiency and emphasize that, in colitis, there is a dominant role of IL‐23/T helper type 17 (Th17) signalling. Furthermore, the levels of IL‐22 are IL‐23‐dependent. The use of cytokine therapy in patients with autoimmune disease has significant potential, but must take into account the overlapping and often promiscuous effects that can theoretically exacerbate inflammation.     

NK cells modulate the lung dendritic cell‐mediated Th1/Th17 immunity during intracellular bacterial infection

The impact of the interaction between NK cells and lung dendritic cells (LDCs) on the outcome of respiratory infections is poorly understood. In this study, we investigated the effect and mechanism of NK cells on the function of LDCs during intracellular bacterial lung infection of Chlamydia muridarum in mice. We found that the naive mice receiving LDCs from C. muridarum‐infected NK‐cell‐depleted mice (NK‐LDCs) showed more serious body weight loss, bacterial burden, and pathology upon chlamydial challenge when compared with the recipients of LDCs from infected sham‐treated mice (NK+LDCs). Cytokine analysis of the local tissues of the former compared with the latter exhibited lower levels of Th1 (IFN‐γ) and Th17 (IL‐17), but higher levels of Th2 (IL‐4), cytokines. Consistently, NK‐LDCs were less efficient in directing C. muridarum‐specific Th1 and Th17 responses than NK+LDCs when cocultured with CD4⁺ T cells. In NK cell/LDC coculture experiments, the blockade of NKG2D receptor reduced the production of IL‐12p70, IL‐6, and IL‐23 by LDCs. The neutralization of IFN‐γ in the culture decreased the production of IL‐12p70 by LDCs, whereas the blockade of TNF‐α resulted in diminished IL‐6 production. Our findings demonstrate that NK cells modulate LDC function to elicit Th1/Th17 immunity during intracellular bacterial infection.     

Th17 cells promote pancreatic inflammation but only induce diabetes efficiently in lymphopenic hosts after conversion into Th1 cells

IDDM is characterized by leukocyte invasion to the pancreatic tissues followed by immune destruction of the islets. Despite the important function of Th17 cells in other autoimmune disease models, their function in IDDM is relatively unclear. In this study, we found association of elevated Th17 cytokine expression with diabetes in NOD mice. To understand the function of Th17 cells in IDDM, we differentiated islet‐reactive BDC2.5 TcR transgenic CD4⁺ cells in vitro into Th17 cells and transferred them into NOD.scid and neonate NOD mice. NOD.scid recipient mice developed rapid onset of diabetes with extensive insulitic lesions, whereas in newborn NOD mice, despite extensive insulitis, most recipient mice did not develop diabetes. Surprisingly, BDC2.5⁺ cells recovered from diabetic NOD.scid mice, in comparison with those from neonate NOD mice, showed predominant IFN‐γ over IL‐17 expression, indicating conversion of donor cells into Th1 cells. Moreover, diabetes progression in NOD.scid recipients was dependent on IFN‐γ while anti‐IL‐17 treatment reduced insulitic inflammation. These results indicate that islet‐reactive Th17 cells promote pancreatic inflammation, but only induce IDDM upon conversion into IFN‐γ producers.     

IL‐17‐producing CD4+ T cells contribute to the loss of B‐cell tolerance in experimental autoimmune myasthenia gravis

The role of Th17 cells in the pathogenesis of autoantibody‐mediated diseases is unclear. Here, we assessed the contribution of Th17 cells to the pathogenesis of experimental autoimmune myasthenia gravis (EAMG), which is induced by repetitive immunizations with Torpedo californica acetylcholine receptor (tAChR). We show that a significant fraction of tAChR‐specific CD4⁺ T cells is producing IL‐17. IL‐17^ko mice developed fewer or no EAMG symptoms, although the frequencies of tAChR‐specific CD4⁺ T cells secreting IL‐2, IFN‐γ, or IL‐21, and the percentage of FoxP3⁺ T_reg cells were similar to WT mice. Even though the total anti‐tAChR antibody levels were equal, the complement fixating IgG2b subtype was reduced in IL‐17^ko as compared to WT mice. Most importantly, pathogenic anti‐murine AChR antibodies were significantly lower in IL‐17^ko mice. Furthermore, we confirmed the role of Th17 cells in EAMG pathogenesis by the reconstitution of TCR β/δ^ko mice with WT or IL‐17^ko CD4⁺ T cells. In conclusion, we show that the level of IgG2b and the loss of B‐cell tolerance, which results in pathogenic anti‐murine AChR‐specific antibodies, are dependent on IL‐17 production by CD4⁺ T cells. Thus, we describe here for the first time how Th17 cells are involved in the induction of classical antibody‐mediated autoimmunity.     

The S. mansoni glycoprotein ω-1 induces Foxp3 expression in NOD mouse CD4⁺ T cells

Immunization with Schistosoma mansoni soluble antigen preparations protects non‐obese diabetic (NOD) mice against the development of type 1 diabetes. These preparations have long been known to induce Th2 responses in vitro and in vivo. Recently, two separate groups have reported that ω‐1, a well‐characterized glycoprotein in S. mansoni soluble egg antigens (SEA), which with IL‐4 inducing principle of S. mansoni eggs (IPSE/α‐1) is one of the two major glycoproteins secreted by live eggs, is a major SEA component responsible for this effect. We found that ω‐1 induces Foxp3 as well as IL‐4 expression when injected in vivo. We confirmed that ω‐1 conditions DCs to drive Th2 responses and further demonstrated that ω‐1 induces Foxp3⁺ T cells from NOD mouse naïve T cells. In contrast, IPSE/α‐1 did not drive Foxp3 responses. The in vitro development of Foxp3‐expressing T cells by ω‐1 was TGF‐β‐ and retinoic acid‐dependent. Our work, therefore, identifies ω‐1 as an important factor for the induction of Foxp3⁺ T cells by SEA in NOD mice.