Effect Of Indirubin On CD4+CD25+ Regulatory T Cells In Mice And Experimental Transient Immune Thrombocytopenia Mice Model

Immunoregulatory CD4~+CD25~+ T cells play an important role in the induction and maintenance of peripheral self-tolerance. These professional regulatory cells prevent the activation and proliferation of potentially autoreactive T cells that have escaped thymic deletion. Therefore, CD4~+CD25~+ T cells are believed to possibly play an important role in pathogenic autoimmune diseases.Naturally arising CD4~+CD25~+ T cells, which regulate autoreactive T cells, have been described in rodent models. These professional regulatory cells prevent the activation and proliferation of potentially autoreactive T cells that have escaped thymic deletion. In animal models, the elimination or inactivation of CD4~+CD25~+ T cells is known to result in various severe autoimmune diseases. In humans, CD4~+CD25~+ T cells have also been reported to have a similar regulatory function to that of the murine population as they were anergic to stimulation by T cell receptor cross-linking (TCR) in the absence of exogenous IL-2 and also regarding their ability to suppress the activation of other T cells in a cell-contact dependent manner that could not be inhibited by blocking such cytokines as IL-10.Some autoimmune diseases such as Grave's disease, myasthenia gravis and immune cytopenias have been recognized to be mediated by pathogenic autoantibodies. It is evident that the autoantibody production by B cells requires the presence of autoantigens-specific T cells .Indeed, autoreactive T cells have been identified in patients with various systemic or organ-specific autoimmune diseases . On the other hand, autoreactive T cells are also present in peripheral blood of healthy individual without any evidence of autoimmune disease. These findings thus suggest that some mechanisms exist which regulate these autoreactive T cells in order to thereby prevent autoimmune disease. Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disease characterized by an increased platelet clearance caused by anti-platelet autoantibodies, which bind to circulating platelets, thus resulting in the destruction of platelet autoantibody complex by the reticuloendothelial system and. The major targets of anti-platelet antibodies have been reported to be platelet membrane GPs, including GPIIb-IIIa, GPIb-IX and GPIV . However, the mechanisms of onset and maintenance of the disease are still unclear. In 1998, Kuwana et al. reported that CD4~+ T cells to GPIIb-llla are involved in production of anti-platelet autoantibody in ITP patients and are related to the pathogenic process in chronic ITP. From these findings, it is speculated that a disorder of the regulatory T cells which regulate autoreactive T cells thus exists regarding the onset and maintenance of disease in ITP as other autoimmune diseases. And new evidence proved that the count of CD4~+CD25~+ T cells in patients with idiopathic thrombocytopenic purpura (ITP) significantly decreased , and the level of CD4~+CD25~+ T cells recovered when patients in remission.Indirubin is a traditional Chinese medicine used in the treatment of chronic myelocytic leukemia and many non-cancer conditions. It has recently been shown to inhibit numerous protein kinases including cyclin-dependent protein kinases (CDKs), a family of key cell cycle regulators,and glycogen symthetase kinase 3, a major kinases involved in tau hyperphosphorylation, c-Src kinase, and c-Jun NH2-terminal kinase and so on. Treatment with indirubin derivatives also showed potent antiproliferative activity on various human cancer cells, whether suppression of all other kinases and cancer cells occurs through the same mechanism is not yet clear. Further studies showed that inhibition of c-Src by indirubin through blocking STAT3 activation, and suppression of the modulation of NF-KB and NF-KB-regulated gene products, contributes to inducing apoptotic effects of indirubin. Therefore Indirubin constitutes a promising family of lead compounds, which deserve to be evaluated as therapeutic agents in various human cancer and neurodegenerative disorders such as Alzheimer's disease. It has also been indicated that indirubin could inhibit replication of HIV-1 by blocking viral gene expression mediated by the cellular factor P-TEFb against wild-type and drug-resistant strains of HIV-1, which provides evidence that indirubin pay potencial effect on antiviral immunity. Indirubin and its derivatives have also been reported to exert an anti-inflammtory effect by suppressing the release of cytokine, including IFN-γ and IL-6 which indicates that indirubin may suppress not only delayed-type hypersensitivity reactions but possibly also the onset of T helper 1 type autoimmune diseasesIn this study we intend to elucidate the potential action mechanisms of indirubins on CD4~+CD25~+Treg cells in mice and to better provide evidence of indirubin as therapeutic strategy in autoimmune disease.Therefore we investigate whether indirubin made it by influencing CD4~+CD25~+ Treg in mice and effect of this drug on phenotypes and function of CD4~+CD25~+ Treg. Treatment with indirubin significantly enhanced the ratios of CD4~+CD25~+ Treg cells or CD4~+CD25~+ Foxp3~+ Treg to CD4~+ T cells in PBMCs, lymph nodes and spleens (P<0.01). Furthermore, splenic CD4~+CD25~+ Treg cells in indirubin-treated mice showed immunosuppressive ability on the immune response of T effector cells to alloantigens or mitogenas efficiently as the control CD4~+CD25~+ Treg cells in vitro. It has been well known that Foxp3 is predominantly expressed in the CD4~+CD25~+Treg cells and Foxp3 expression in naive T cells convert these cells to a regulatory T cell phenotype functionally similar to naturally occurring CD4~+CD25~+Treg cells(1-3) while none of the markers described above is exclusively expressed in CD4~+CD25~+Treg cells because once activated CD4~+CD25" T cells also express CD25, CD69, CD44, CTLA-4, GITR and OX-40 (4). So Foxp3 is recognized as to be a specific marker for CD4~+CD25~+Treg cells, we detected the expression of Foxp3 in CD4~+CD25~+T cells in LNs .spleens and thymi of indirubin-treated mice. Significant changes of Foxp3 in CD4~+CD25~+Treg cells were observed in the periphery after the treatment with indirubin in ITP model as determined by FCM. It was possible that indirubin not only inhibited the development of CD4~+CD25~+Treg cells in the thymus but also influenced the homeostasis of them in the periphery. Owing to CD4~+CD25~+Treg cells were more resistant to the blockade of CDKs induced by indirubin than CD4~+CD25T cells, so the percentage of peripheral Foxp3~+CD4~+CD25~+Treg cells was increased.At the same time, we founded that indirubin significantly enhanced the percentages of CD4~+CD25~+FoxP3~+Treg while their absolute cell number was decreased. As indirubin inhibits the CDKs or NF-kB, subsequently induces T cell apoptosis, it seems to induce less cell death of CD4~+CD25~+Treg cells than CD4~+CD25~- T cells. To decide Whether indirubin expand the group of CD4~+CD25~+Foxp3~+Treg cells, we need more evidences. Meanwhile our results indirectly indicated that CDKs pathway might play an important role in the development, survival and activity of CD4~+CD25~+Treg cells.And similar results were found in ITP mice , even stronger.We confirmed indirubin may have impact on immune response through influenced the relative percentage of CD4~+CD25~+Foxp3~+Treg cells in the periphery and central immune organs. The present studies indicated that indirubin may interfere with immune response by influencing the balance between CD4~+CD25~+ Treg cells and effector cells, which possibly provides a new alternative therapy for autoimmune disease. As far as we know this is the first report demonstrating that indirubin can be effective on regulating CD4~+CD25~+ Treg cells, and we believe further study of application with indirubin on autoimmune immunity would be worthwhile.