Expression Of TIPE2Contributes To The Immunosuppressive Property Of CD4~+CD25~+Regulatory T Cells

BACKGROUND AND OBJECTIVERegulatory T cells (Tregs) have unique immunological characteristics compared with other regulatory or suppressor T cells. For example, they constitutively express many cell surface molecules, including glucocorticoid-induced tumor necrosis factor (TNF) receptor (GITR) and intracellular cytotoxic T-lymphocyte-associated antigen (CTLA)-4, while still other factors contribute to the development and activity of Tregs, such as forkhead/winged helix transcription factor p3(Foxp3), and Toll-like receptors (TLRs), which recognize pathogen associated molecule patterns or can augment Treg function or proliferation. In addition, immunosuppressive cytokines produced by Tregs, such as transforming growth factor (TGF)-β and interleukin (IL)-10migtht also play important roles in the suppression of effector T cells by Tregs.Tumor necrosis factor-α induced protein8like-2(TNFAIP8L2, TIPE2), a lately discovered negative regulator of innate immunity and cellular immunity, shares considerable sequence homology with members of the tumor necrosis factor-α induced protein8(TNFAIP8) family. It is preferentially expressed in lymphoid-derived and marrow-derived cells. Experiments in vitro demonstrate that TIPE2could inhibit activation of activator protein (AP)-1and nuclear factor (NF)-κB, and moreover, it is an essential negative regulator of TLRs and T cell receptor signal activation. Furthermore, experimental data had shown that lipopolysaccharide (LPS)-induced sepsis in mice that were injected with a low dose of LPS, septic shock was dramatically accelerated and excerbated in TIPE2-/-mice compared to wild type controls, the continuous activation of lymphocytes due to down-regulation of TIPE2expression might result in elevated expression of Fas and increase in apoptosis of lymphocytes in patients suffering from systemic lupus erythematosus, and its deficiency in mice could give rise to an elevation of serum levels of inflammatory cytokines, including IL-4, IL-6, IL-12, and interferon (IFN)-y. Considering normal expression of TIPE2gene in the immune system is essential to prevent hyperresponsiveness and maintains immune homeostasis, we hypothesized that the TIPE2gene maybe related to the immune regulation mediated by Tregs.The present study was designed to examine whether naturally occurring CD4+CD25+Tregs isolated from murine spleens expressed TIPE2by the use of Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Based on the expression of cell surface molecules, including CTLA-4and Foxp3on CD4+CD25+Tregs, and cytokines including IL-10as well as TGF-β were analyzed to investigate the functional role of TIPE2in controlling suppressive activity of CD4+CD25+Tregs. Meanwhile, IL-2release, the ratio of IFN-γ/IL-4in CD4+CD25+Treg/CD4+CD25-T cell coculture supernatants in T lymphocytes were determined to examine the effects of TIPE2on the T-cell proliferation and differentiation induced by CD4+CD25+Tregs. An understanding of the potential role together with the precise mechanism of TIPE2in the suppressive activity of CD4+CD25+Tregs may provide us insights to further investigate inflammatory response as well as immune homeostasis secondary to acute insults.METHODS1. CD4+CD25+Tregs were isolated from murine spleens with a CD4+CD25+Regulatory T Cell Isolation kit (Miltenyi Biotec, Germany). The purity of isolated CD4+CD25+Tregs was analyzed by flow cytometry.2. The present study was designed to examine whether naturally occurring CD4+CD25+Tregs were expressed TIPE2by the use of laser scanning confocal microscope, Western blot, and RT-PCR, respectively. Laser scanning confocal microscope and Western blot were performed to determine the portein expression of TIPE2in CD4+CD25+Tregs. Total RNA was extracted from CD4+CD25+Tregs and CD4+T cells using TRIZOL, the mRNA levels for target genes were analyzed by RT-PCR, and the level of β-actin mRNA was also designed as an internal control for each sample.3. To evaluate the potential role of TIPE2gene on CTLA-4and Foxp3expression in CD4+CD25+Tregs. In the current experiment, we detected the expression of CTLA-4and Foxp3by flow cytometry after CD4+CD25+Tregs were cultured for24hours. To analyze the secretion of cytokines, purified CD4+CD25+Treg and CD4+CD25-T cells (1:1) were stimulated with both anti-CD3and anti-CD28antibodies for68hours. These cytokines including IL-10as well as TGF-β were analyzed to evaluate the functional role of TIPE2in controlling suppressive activity of CD4+CD25+Tregs. 4. Purified CD4+CD25+Tregs with CD4+CD25-T cells (1:1ratio) were seeded on96-well cell culture plate, stimulated with a combination of soluble anti-CD3and1μg/ml of soluble anti-CD28monoclonal antibody. All the samples of T-cell proliferative respose were tested with thiazolyl blue (MTT).5. CD4+CD25+Tregs stimulated with anti-CD3/CD28antibodies were co-cultured with CD4+CD25-T cells. Three days later, supernatants were harvested for analysis of cytokines production in CD4+CD25-T cells. IL-2is a potent T-cell growth factor that acts upon itself in an autocrine fashion. We measured the production of of IL-2after co-cultured with small interference RNA (siRNA)-TIPE2transfected CD4+CD25+Tregs. Subsequently the co-culture supernatants were harvested for analysis of the changes in Thl and Th2cytokines. The ratio of IFN-γ/IL-4in CD4+CD25+Treg/CD4+CD25-T cell coculture supernatant and nuclear factor of activated T cells (NF-AT) activation in T lymphocytes were determined to examine the effects of TIPE2on the IL-2release and T-cell differentiation induced by CD4+CD25+Tregs.6. Statistical MethodsData were expressed as means±standard deviation (SD), with the number of respective experiment run in triplicate. The significance of the differences in mean values between and within multiple groups was examined by independent-samples T test and one-way ANOVA, respectively. The level of significance was set up at P<0.05.RESULTS AND CONCLUSIONS1. The purity of CD4+CD25+Tregs was above92%after isolated twice by magnetic beads, and the activity of CD4+CD25+Tregs exceeded98%. 2. It was found that TIPE2was a cytoplasmic protein expressed in CD4+CD25+Tregs by fluorescent confocal microscopy. Meanwhile, a clear band of TIPE2with a molecular mass of approximately21kD was found from CD4+CD25+Tregs by Western blot analysis, and TIPE2mRNA was expressed with the expected size of147bp from CD4+CD25+Tregs by RT-PCR.3. In this study, we detected the expression of CTLA-4and Foxp3by flow cytometry after CD4+CD25+Tregs were cultured for24hours. Compared with the normal control group, gene silence of TIPE2significantly down-regulated the CTLA-4expression on surface of CD4+CD25+Tregs (t=13.596, P=0.000). In addition, this siRNA-TIPE2also had effect on Foxp3expression levels, which had similar effect on CTLA-4expression (t=28.833, P=0.000).4. Upon anti-CD3/CD28antibodies costimulation, we detected high levels of TGF-P and IL-10, which was produced by CD4+CD25+Tregs. By contrast, IL-10and TGF-P produced by siRNA-TIPE2transfected CD4+CD25+Tregs were reduced as compared to those normal control cells (t=8.054, P=0.000; t=6.454, P=0.000). The results suggested that TIPE2could influence the release of anti-inflammatory cytokines from CD4+CD25+Tregs.5. CD4+CD25+Tregs were mixed with CD4+CD25-T cells at a ratio of1:1in the presence of anti-CD3/CD28antibodies. Indeed, we found that there was a decrease in proliferation capacity when cocultured with CD4+CD25+Tregs, but the proliferation significantly increased after TIPE2gene silencing by siRNA (P=0.000). The results indicate that TIPE2augmented the ability of CD4+CD25+Tregs to suppress T cells, and in further to enhance the suppressive activity of CD4+CD25+Tregs. 6. CD4+CD25+Tregs stimulated with anti-CD3/CD28antibodies were co-cultured with CD4+CD25-T cells. Three days later, supernatants were harvested for analysis of cytokine production in CD4+CD25-T cells. IL-2is a potent T-cell growth factor that acts upon itself in an autocrine fashion. We measured the expression of IL-2after co-cultured with siRNA-TIPE2transfected CD4+CD25+Tregs. The level of IL-2release in CD4+CD25-T cells were low when co-cultured with CD4+CD25+Tregs. When TIPE2gene silencing by siRNA pretreated CD4+CD25+Tregs, IL-2expression in T cells was significantly enhanced (P=0.000), indicating that down-regulation of TIPE2expression maybe an important cause of the increase of IL-2production and in further leads to an decreased CD4+CD25+Treg-mediated suppression of CD4+T lymphocytes.7. Subsequently the co-culture supernatants were harvested for analysis of the changes in Thl and Th2cytokines. It was noted that CD4+CD25+Tregs could significantly elevate expression of IL-4and reduce expression of IFN-γ when mixed with CD4+CD25-T cells. However, we found a significant increase of IFN-γ/IL-4ratio after TIPE2gene silencing by siRNA in CD4+CD25+Tregs (P=0.000). Taken together, these results indicate that CD4+CD25+Tregs might be efficiently induce typel T-cell polarization after TIPE2gene silencing.8. Activation of transcription factors by receptor mediated is an essential step for T lymphocyte effector function. To further characterize the signal pathway of T cell activation influenced by CD4+CD25+Tregs, we performed a experiment to assess NF-AT activation, which regulates transcription of cytokine including IL-2. NF-AT activation of CD4+CD25-T cells in the presence of anti-CD3/CD28antibodies were significantly increased after72hours co-cultured with siRNA-TIPE2transfected CD4+CD25+Tregs (P=0.000). The results suggested that TIPE2appeared to be a critical immunoregulatory molecule involved in immunosuppressive function of CD4+CD25+Tregs.