AG490 Prevents and Reverses Diabetes Figure 5. AG490 upregulates expression of Foxp3 regulatory CD25+ T-cells in vivo. A- Prediabetic NOD mice (4 week) were treated either with AG490 or DMSO three times per week for 5 consecutive weeks and they were sacrificed one week after the last injection at week 10. Data represents pool cell suspensions of 5 NOD mice per group and is gated on live CD4+ T-cells. AG490 treatment significantly increases number of Foxp3 regulatory T-cells (p,0.004) (A–D). E- CD25+Foxp3+ gated population on CD4+ T-cell of several lymph nodes in AG490 and the sham treated control NOD mice. Bars represent standard deviation of the mean. F- Whole blood of NOD mouse (pool of 5 mice/group) treated as described above was stained directly with antibodies indicated as described in materials and methods. Data of gated CD4+ T-cells are shown. G- Total RNA from purified CD4+ T-cells of splenocytes of NOD mice (10 weeks old) treated either with AG490 or DMSO from 5 weeks were subjected to real-time RT-PCR and expression of the immune response genes was screened as described in materials and methods. Data shown is the mean of fold change expression of 18 genes after they were normalized based on expression of two housekeeping genes and then compared with the expression of the same gene in DMSO treated control NOD mice. Spl = spleen, PLN = pancreatic lymph node, MLN = mesenteric lymph node, PP = Peyer’s patches, PB = peripheral blood. doi:10.1371/journal.pone.0036079.g005 an excellent target for immunotherapy of T1D and may also be effective in type II diabetes, although this remains to be seen [51]. Autoactivation of kinases/transcription factors due to ongoing autoimmunity will generate inflammatory cytokines which subse- quently will lead to further inflammation. The Jak-Stat signaling pathway is the most important signaling pathway for transduction of signals from the cell surface to the nucleus [21,52–54]. This signaling pathway is involved in the generation of pro-inflamma- tory cytokines and is bound to have many more important roles in the pathogenesis of T1D than currently is appreciated. Our study uncovered several novel features of AG490 such as prevention of diabetes, initiation of remission in newly diagnosed NOD mice and in pharmacological induced diabetic mice, increasing number of Foxp3 regulatory T-cells and probably generation of regulatory DC. It is important to mention that significant differences (p,0.01) in incidence of diabetes was observed between the AG490 and DMSO treated mice for up to 52 week when treatment was started at week 4. This data may suggest that appropriate blocking of the Jak-Stat signaling pathway could bring about a durable and long-lasting tolerance and further is supporting the notion of safety and efficacy of AG490 in prevention of autoimmune diabetes in NOD mouse. Our data suggest that administration of AG490 blocks expression of several members of the Jak and Stat families in infiltrated leukocytes in the islets of NOD mice. Lack of the Stat3 expression is fatal perinataly [55], however, none of the treated mice in this study died because of AG490 treatment suggesting that treatment by AG490 was not life threatening. Our microarray real-time TaqMan gene expression data suggests that AG490 may exert its regulatory effect via upregulation of negative co- stimulatory molecule, Ctla4, in CD4+ T-cells; however, additional screening of different cellular component of the immune system is warranted to identify it target gene(s)/pathway(s) and better understand its molecular mechanism. Our data indicate that body mass in AG490 and the sham treated control mice was not significantly different from each other suggesting that AG490 treatment did not cause obesity [56]. In this study, we demonstrate that AG490 initiates remission of diabetes in newly diagnosed and established diabetic mice, no insulin was injected at any time, and sustained euglycemia was evident for several months following drug withdrawal. It has been shown that AG490 increased glucose uptake (10269) in L6 myotubes [48] under high glucose concentration in comparison with vehicle-treated cells [57]. AG490 decreased fibronectin expression; higher expression of fibronectin is associated with nephropathy in diabetic subjects, under high glucose condition in glomerular mesangial cell; therefore, AG490 potentially could be an effective treatment for prevention of diabetic nephropathy [58]. Adoptive transfer of splenocytes isolated from in vivo treated mice with AG490 failed to transfer diabetes to immunodeficient NOD mice supporting the notion of inactivation of autoreactive T-cell or induction of regulatory T-cell by AG490. DC plays a central role in induction and maintenance of the central and peripheral tolerance [59,60] and its development is impaired in NOD mice [61]. As shown in this study, DC treated with AG490 possesses regulatory function and suppress proliferation of responding T- cells in vitro. Recently, Louvet et al have reported successful reversal of diabetes by oral administration of tyrosine kinase inhibitors [5], however, diabetes was transferred from drug and the sham treated control mice into immunedeficient hosts with the same kinetics [5] suggesting that induction of tolerance was achieved by other means than generation of the regulatory T-cells. In summary, this study provides evidence regarding application of AG490 in prevention/reversal of autoimmune T1D in NOD mouse. Administration of AG490 in vivo was very well tolerated and treated NOD mice with AG490 showed no signs of toxicity and they appeared healthier as they had a longer life span and they were significantly diabetes-free when compared to the sham treated or untouched control NOD mice. Additionally, this study provides evidence regarding the crucial role of the Jak-Stat signaling pathway in development of autoimmune diabetes and regulation of blood glucose. Therefore, the Jak-Stat signaling pathway may be an excellent pathway to be targeted for immunotherapy and/or to reverse diabetes. The use of such compounds like AG490, given their extensive safety profiles, offers a solid foundation for their translation to diabetic subjects with or at increased risk for the disease. Materials and Methods Ethics statement All mice were maintained and handled in accordance with the University of Florida Institutional Animal Care and Use Com- mittee (IACUC), who specifically approved this study. Mice Female NOD/LtJ, NOD.Scid, and BALB/c mice were purchased from Jackson Laboratories (Bar Harbor, ME) and housed in ventilated cages in a specific pathogen free facility. Compound AG490 (EMD Chemicals, Gibbstown, NJ), 5 mg, initially was dissolved in sterile DMSO (EMD Chemicals) and brought to final volume by sterile PBS followed by several pipetting to bring the compound into solution. One vial of compound containing 5 mg of AG490 was injected into5 mice (1 mg/mouse) via the i.p route. The control groups were received the same volume of the vehicle under the same regimens and conditions. Administration of AG490 Mice were injected either at 4 or 8 weeks of age, three times per week for 5 consecutive weeks followed by 5 more injections, once a week for 5 additional weeks. Body mass and blood glucose levels were measured at the beginning of the study (baseline) and PLoS ONE | www.plosone.org 8 May 2012 | Volume 7 | Issue 5 | e36079