The Effect And Mechanism Of GSK-3β On The Maturation And Function Of Murine Myeloid Dendritic Cell

Background & ObjectiveSystemic lupus erythematosus (SLE) is considered to be a prototype of chronic systemic autoimmune disorder which can affect multiple organs such as skin, joints, central nervous system, or kidneys. Even though the etiology and pathogenesis of SLE remains unclear, it is postulated that SLE is based on the interaction of genetic predisposition, hormonal and environmental triggers that result in immune dysfunction. SLE is characterized by dysregulated T- and B-cell activation, a plethora of circulating autoantibodies, immune complexes deposition in organs, disturbed Th/Ts proportion and perturbed cytokine activities. Loss of tolerance to self-antigens or overpresenting some antigen by immune system is essential for SLE initiation and progress, which induced autoreactive T- and B-cell activation leading to production of autoantibodies.Dendritic cells (DCs) have been identified as the most powerful and professional antigen-presenting cells (APCs) with a capacity of inducing either immune tolerance or immune activation, which play a crucial role in regulating immune balance. Studies have show that the functional properties of DC are strictly dependent on their maturity status. Immature DC (iDC) is inclined to induce immunological tolerance while mature DC (mDC) to induce immunological response. Under healthy conditions, iDCs reside in most peripheral tissues, expressing low levels of major histocompatibility complex MHC-Ⅱ molecules and co-stimulatory molecules and exhibiting limited pro-inflammatory cytokine production. IDCs continuously take up a few endogenously expressed antigens and present them to T-cell, which mainly maintains peripheral tolerance by inducing autoreactive T-cell anergy or apoptosis and Tregs differentiation. When stimulated by pathogenic antigens or danger signals, iDCs begin a maturation process with unregulated MHC-Ⅱ and co-stimulatory molecules on their surface and increased flammatory cytokines secretion. The maturing DCs migrate to secondary lymphoid organs where they present antigens to lymphocytes for initiating specific immunity against antigens. DCs from lupus patients shows increased susceptibility to be mature, and most of them exhibit the highly activated phenotypes. Increased immunogenicity of DCs overpresenting self-antigens to autoreactive T cells has been established as essential players in the mechanisms underlying SLE. Therefore, it is considered to be a potential therapeutic approach in the restoration of immune tolerance in SLE to keep DCs in immaturity status and induce tolerogenic DCs.Glycogen synthase kinase-3β (GSK-3β) is a multifunctional serine/threonine kinase which regulates substrate activity and stability by phosphorylation. GSK-3β involves in multiple signaling pathways including NF-κB, PI3K-Akt, Wnt/β-catenin and regulates many physiological and pathological processes. Immunologically, GSK-3β contributes to its powerful ability to regulate the balance between pro-and anti- inflammatory cytokine production and to influence immune cells activation and differentiation, which plays a critical role in both innate and adaptive immune responses. It is already evident that GSK-3β inhibitors can have therapeutic effects in some animal models with autoimmune diseases, such as RA, EAE and SLE, which reveals the therapeutic potential of these dregs in autoimmune diseases. However, the role of GSK-3β in DC remains unclear. In this study, we used selectively inhibitor SB216763 to investigate the effect on DC activation and maturation by inhibiting the activity of GSK-3β. In addition, our previous studies have reported that RelB plays a decisive role in DC maturation process, and DC with inhibited RelB expression remained immature and possessed the capability to induce T cell tolerance. A recent study has reported that GSK-3β is a crucial mediator of signal-induced RelB degradation. We have constructed lentivirus over-expressing vector of mice GSK-3β gene and transfected it into DC2.4 cell lines, for making a further study to elucidate a new mechanism whether GSK-3β modulated DC activation and maturation via RelB, and this would provide some theories data to support that GSK-3β could be a potential new target for the treatment of SLE.Methods1. Culture in vitro and identification of dendritic cells from mouse bone marrow and detection of GSK-3β activityIn vitro, a low dose of rmGM-CSF and rmIL-4 were used to jointly induced mouse bone-marrow precursors to differentiate into DCs. There were two groups here, one was iDC group with DCs cultured by 6 days, and the other was mDC group with DCs stimulated by LPS for 24h before harvested at the 6th day. Both DCs were purified by CD11c+ immunomagnetic beads positive selection, and the expression of specific molecular markers CD11c and costimulatory molecules were detected by flow cytometry. Cytokines were detected by qRT-PCR and the changes of iDCs and mDCs functions was detected by Mixed Lymphocyte Reaction (MLR). The purpose was to establish the large-scale culture of iDCs and mDCs system in vitro, and laid the foundation for further research on the mechanism of activation and maturation of DC. In addition, LPS was used to deal with iDCs for 30min or 60min, and western blotting was used to detect GSK-3β phosphorylation, which revealed the change of GSK-3β activity.2. The effect and mechanism of GSK-3β on the maturation of murine myeloid dendritic cellsBMDCs were handled with SB216763, which is a selective inhibitory agent against GSK-30.24h later, CD40 and CD86 were determined by flow cytometry (FCM), the expression of IL-6, IL-12 and IL-10 mRNA were detected by qRT-PCR and the change of immunogenicity was reflected by Mixed Lymphocyte Reaction (MLR). Lentivirus over-expressing vectors of mice GSK-3β gene were constructed and transfected into DC2.4 cell lines, and the expression of GSK-3β and RelB was determined by western blotting. The relationship between GSK-3β and RelB was analysis then.Results1. Morphology and functional identification of mouse bone marrow-derived dendritic cells and detection of GSK-3β activity1.1 Morphology observation of BMDCs in vitroOn the 6th day, the iDCs colony gradually increased, but still presenting a growth of loose adherence. The volume of iDCs increased and some of them begined to stretch small stings on the surface. After LPS stimulation, the number and volume of suspension cells increased, and significantly lengthened and enlarged dendritic-like structure could be seen on the cell surface, relatively conforming to the typical cell morphology of mature DC.1.2 Purity and phenotype identification of BMDCsCD11c is a relative specific surface marker of classical DC, which used for DC purification and identification. After immunomagnetic beads positive selection, the positive ratio of CD11c in DCs was beyond 90%, which had met the requirements of molecular biology experiments. The results from flow cytometry showed that CD40 and CD86 expressed at low level on the surface of iDCs while expressed at high level on the surface of mDCs, which conformed to the phenotypic characteristics of surface molecules with low expression of imDCs and high expression of mDCs.1.3 Cytokine IL-6 and IL-12 mRNA levels in BMDCsThe purified BMDCs were harvested, and cytokine IL-6 and IL-12 mRNA levels were assayed by qRT-PCR. The results showed IL-6(P<0.05) and IL-12(P<0.05) mRNA expression in iDCs were significantly lower than that in mDCs.1.4 Proliferation capacity of T cells evaluated by MLRThe purified BMDCs were cocultured with splenic T lymphocyte for 72h, CCK-8 was added to assay the proliferation capacity of T cells. The result was as follows:the relative growth rate of iDCs was 0.51±0.06, and that of mDCs was 1.14 ±1.20, with a significant difference (P<0.01)。1.5 Changes of GSK-3β activity in BMDCs activated by LPSGSK-3β was constitutively active in iDCs as shown by high Tyr216 phosphorylation and low Ser9 phosphorylation of GSK-3p. After stimulated by LPS, GSK-3β activity in iDCs was reduced as demonstrated by increased Ser9 phosphorylation and reduced Tyr216 phosphorylation compared with unstimulated groups.2. The effect and mechanism of GSK-3β on the maturation of murine myeloid dendritic cells2.1 Effect of GSK-3β inhibition on maturation and function of BMDCsSurface molecules CD40 (P<0.01) and CD86 (P<0.01) on BMDCs with GSK-3β inhibition were 2.5 and 2.4 fold higher than untreated iDCs. After stimulated by GSK-3β inhibitor, IL-6 (P<0.05) and IL-12 (P<0.05) mRNA levels were reduced, and anti-inflammatory cytokine IL-10 mRNA undergo a notable upregulation (P<0.01). MLR results showed that stimulation ability of iDCs treated with SB216763 to activate T cells had no significantly difference with that of untreated iDCs. In other words, after GSK-3p inhibition, DCs became phenotypically mature with increased surface expression of CD40 and CD86. However, GSK-3β inhibition did not result in increased pro-inflammatory cytokines and stimulation ability to activate T cells in DCs. In addition, a modest increased expression of anti-inflammatory cytokine IL-10 could be seen in these DCs. This implied that GSK-3β inhibition was essential but not sufficient to induce full DC maturation. In addition, SB216763 inhibited LPS-induced IL-12 (P<0.01) and IL-6 (P<0.05) mRNA expression while enhanced IL-10 (P<0.01) mRNA expression, and it also inhibited the stimulation ability of iDCs treated with LPS to activate T cells. This implied GSK-3P acquired a proinflammatory task in the context of DC activation.2.2 RelB expression after overexpression of GSK-3βAfter transfection into DC2.4 cell, GSK-3β was unregulated while RelB level was reduced, suggesting that the overexpression of GSK-3β might attribute to the reduction of RelB protein.Conclusions1. Under the low dose rmGM-CSF and rmIL-4, a large number of iDCs was differentiated and proliferated from the mouse bone marrow precursors. The purity of DCs was up to 90% after immunomagnetic beads selecting. With LPS stimulation, iDCs differentiated to mDCs with typical morphological characteristics. The flow cytometry, qRT-PCR and mixed lymphocyte reaction results respectively demonstrated that the expression of iDC costimulatory molecules and proinflammatory cytokines mRNA expression in iDC were significantly lower than those of mDC, which conformed to the characteristics of iDC or mDC.2. GSK-3β is constitutively active in iDCs; After stimulated by LPS, GSK-3β activity was reduced notably, which suggested that deactivation of GSK-3P induced by LPS might mediate activation and maturation of DCs.3. GSK-3p is a crucial enzyme involved in differentiation and maintenance of an immature phenotype of DCs. On the one hand, GSK-3 was constitutively active in iDCs and inhibited their spontaneous maturation. After GSK-3β inhibition, DCs become phenotypically mature with increased surface expression of CD40 and CD 86. However, GSK-3β inhibition did not result in increased pro-inflammatory cytokines and stimulation ability to activate T cells in DCs. In addition, a modest increased expression of anti-inflammatory cytokine IL-10 can be seen in these DCs. This implied that GSK-3p inhibition was essential but not sufficient to induce full DC maturation. On the other hand, GSK-3p acquired a proinflammatory task in the context of DC activation. Following activation of DCs induced by LPS, GSK-3β activity contributed to enhanced IL-6 and IL-12 expression and inhibited IL-10 expression, and GSK-3β inhibition block ed the MRL of LPS-induced DCs.4. Overexpression of GSK-3P resulted in reduced RelB protein levels, This demonstrated a new mechanism that phenotypically maturation of DC might be regulated by GSK-3β through RelB. And it has provided some theories data to support that GSK-3β might be a target for inducing tolerogenic DC to treat SLE.