Figure 3. In vivo AG490 treated splenocytes did not transfer diabetes to NOD.Scid mice. Prediabetic NOD mice (4- weeks of age) were treated with AG490 or DMSO for five consecutive week (n = 5 per group), three times per week and mice were sacrificed one week after the last injection at week 10. Splenocyte cell suspension of pooled mice immediately prepared and dead cells and red blood cells were removed by Histopacque gradient separation method and freshly isolated splenocytes containing .99% viable cells were transferred (56106, i.p) into NOD.Scid mice. Blood glucose in recipient mice was monitored at least once a week as described earlier (*: p = 0.02, Kaplan-Meier test). doi:10.1371/journal.pone.0036079.g003 pancrease of AG490 and DMSO treated mice (n = 5 mice/group) was subjected to TaqMan Real-time RT-PCR. Level of Foxp3 and TGF-b1 expression were very low but detectable in AG940 treated NOD mice but we failed to detect expression of Foxp3 in three independent RT-PCR in DMSO treated NOD mice (figure 4C). This data suggests that although administration of AG490 was unable to block infiltration of the leukocytes in the islets or improve the C-peptide concentration; however, it changed composition of the infiltrated leukocytes in the islets. Administration of AG490 does not block expression of Jak-1 and Stat-1 in leukocytes infiltrating the islets Expression of the Jak and Stat family members in the islets of NOD mice treated with AG490 or DMSO were screened by immunofluorescen histochemistry technique. Expression of Jak3, Stat3 and Stat5 was very low/undetectable in AG490 treated mice. Expression of Jak2 was undetectable or it was very low even in DMSO treated NOD mice. AG490 had no effect on constitutive expression of the Jak1 and Stat1(figure S1). Higher expression of Jak1 and Stat1 is of importance because it is known that IFNc uses JAK-1 for activation of Stat1 [19,21,40]. Additionally, since adminsitration of AG490 might inhibit expression of other kinases than members of the JAK family, we screened expression of EGFR, c-Kit, PDGFR and c-Able by western blot in splenocytes of NOD mice treated with AG490 or DMSO (n = 5). In several independent western blots, all detected bands did not match with expected molecular weights of those protein suggesting that expression of these kinases was very low and AG490 likely does not have any impact on expression of these kinases. Administration of AG490 significantly increases expression of Foxp3 To further understand as to whether AG490 prevented and reversed diabetes in treated NOD mice, we measured expression of Foxp3 and other regulatory markers by flow cytometry analysis in different organs of the immune system in NOD mice that were treated with either AG490 or DMSO. As shown in figure 5A–D AG490 Prevents and Reverses Diabetes significant higher expression of CD25+Foxp3+ T-cells (p,0.004) is evident in all organs tested in AG490 treated mice. Further- more, AG490 treatment also increased expression of Foxp3 in CD25+ T-cells in peripheral blood of the same groups of NOD (Fig. 5E). AG490 is not a negative regulator of the immune system; screening of the mouse immune response genes array In a separate experiment, pooled total RNA extracted from purified CD4+ T-cells from spleen of the AG490 or DMSO treated NOD mice (treated 3x/week for 5 weeks and euthanized at week 10, n = 5/group) were used to screen the mouse immune response genes array (Applied Biosystems) by real-time TaqMan Gene Expression RT-PCR. As shown in figure 5F, AG490 did not negatively regulate overall gene expression of the mouse immune response genes (see Table S2). Interestingly, in vivo treatment of NOD mice with AG490 increased (2.15 fold) expression of co- stimulatory Ctla4 in CD4+ T-cell. The Ctla4 gene is a diabetes candidate gene for T1D and its association is well documented in the literature [41–44]. Global gene expression screening from different cellular component of the immune system of NOD mice treated with AG490 is warranted to better understand mechanism underlying modulatory function of AG490 and that may lead to identify its target gene(s)/pathway(s). In vivo and in vitro administration of AG490 modulates phenotype and function of dendritic cells Bone marrow of AG490 and DMSO treated NOD mice (4 weeks/3x/week, treated for 5 weeks) were harvested at week 10 and yeilded the same number of cells in each group (p = not significant). We did not, however, notice any remarkable difference in morphology, such as the size, shape and clonoy formation, between the two groups in the course of differentiation in cell culture. As shown in figure 6A and B, BMDCs (bone marrow derived dendritic cells)of mice treated by AG490 had markdly lower expression of co-stimualtory moelcules CD80 and CD86 and adhesion molecule CD62L. Additionally, our real-time TaqMan Gene expression Asssays (Assays-on-Demand) (Life technologies, Applied Biosystems) indicates that AG490 signifi- cantly increased expression of regulatory cytokine Tgf-b1 in mature and immature DC (dendritic cell) when it was compared with the sham treated control NOD mice (figure 6C, n = 5/group, p = 0.02). Furthermore, BMDCs treated with AG490 (overnight, 20 mM) or DMSO suppressed proliferation of purified responding CD4+CD252 T-cells in vitro (figure 6D). Discussion Presence of proinflammatory cytokines due to ongoing inflam- mation in NOD mice, leads to autoactivation of different members of the Jak and Stat family followed by activation of downstream events as it has been reported by us [37,45] and others [46]. Recently, it has been shown that a transcriptomic analysis of glomerular and tubulointerstitial tissues from diabetic subjects with early and progressive diabetic nephropathy revealed involvement of multiple members of the Jak/Stat signaling pathway [47]. Interestingly, higher expression of the Stat5B in progressive diabetic nephropathy patients has been shown [47]. Tyrosine kinase inhibitors have been reported to decrease insulin resistance [48] and increase insulin secretion in vitro [49,50]. In our study, AG490 decreased hyperglycemia in chemically induced diabetic mice and in naturally developed diabetic NOD mice. In chemically induced study, since the treatment with AG490 was started one week after diabetes onset, beta-cell PLoS ONE | www.plosone.org 5 May 2012 | Volume 7 | Issue 5 | e36079