| Regulatory T cells(Tregs)as a group of immunosuppressive lymphocytes have been well-recognized for their vital role in maintaining immune homeostasis.Tregs are also broadly involved in the pathogenesis of tumor and autoimmune diseases.We previously demonstrated that there is a unique T cell subpopulation been significantly increased in pCD86:RAE-1? transgenic mice as well as colorectal tumor-bearing(MC38)mice,the phenotype of which is NK1.1-CD4+NKG2D+.On the fundamental basis,this group of cells is referred to nonclassical Tregs as to express high level of TGF-?1 and IL-10,low level of IFN-y,negatively express IL-17A as well as Foxp3,ROR-?t,and GATA-3.Meanwhile,these cells show the immunosuppressive capacity by repressing the activation and proliferation of CD8+T cells,natural killer cells,and dendritic cells rely on TGF-?1 production,and consequently induce tumor invasion.However,the mechanism underlying the transcriptional regulation of TGF-?1 within NK1.1-CD4+NKG2D+T cells remains unclear.In addition,our previous study showed that NK1.1-CD4+NKG2D+T cells are able to protect against DSS-induced murine colitis by expressing TGF-?1 as well.Astilbin,a natural flavonoid compound,could be extracted from plants including Rhizoma smilacis glabrae that shows significant anti-oxidative and anti-inflammatory effects over multiple autoimmune diseases and experimental models.Nevertheless,whether astilbin could regulate NK1.1-CD4+NKG2D+T cells contributing to the protection against DSS-induced murine colitis remains elusive.Therefore,we take our sight into NK1.1-CD4+NKG2D+T cells to clarify the machinery underlying the transcriptional regulation of TGF-?1 and figure out that if astilbin is able to ameliorate murine colitis by inducing regulatory NK1.1-CD4+NKG2D+T cells and its underlying mechanisms.Part 1 Study on the machinery underlying PI3K p85,JNK/AP-1,STAT3,and NF-?B signals-induced TGF-?1 transcription within NK1.1-CD4+NKG2D+T cellsWe isolated NK1.1-CD4+NKG2D+T cells by magnetic cell sorting and treated them with different stimulations,namely,anti-CD3,soluble RAE(sRAE),anti-CD3 plus sRAE,as well as anti-CD3 plus anti-CD28,then the expressions of TGF-?1 were observed at different time points.We found that TGF-?1 in NK1.1-CD4+NKG2D+T cells was significantly induced among protein level,transcriptional level as well as secretion level following 8-hour stimulation of sRAE or anti-CD3/sRAE.However,anti-CD3/anti-CD28 failed to induce TGF-?1 expression over all time points.Subsequently,we observed that NKG2D engagement or TCR/NKG2D co-engagement were able to induce the expression of PI3K p85 in NK1.1-CD4+NKG2D+T cells.Meanwhile,PI3K inhibitor profoundly suppressed the TCR/NKG2D-induced expression of TGF-?1 in these cells.Furthermore,PI3K p85 could subsequently initiate JNK/AP-1 signaling pathways and result in the transcription of TGF-?1 in this group of cells.In turn,the expressions of TGF-?1 were reduced as a result of JNK inhibitor or AP-1 inhibitor treatment.Likewise,TCR/NKG2D co-engagement was able to activate NF-?B p65 and STAT3 signals and further regulate TGF-?1 transcription.STAT3 could be enriched in the promoter region of TGF-?1 to directly regulate gene transcription accordingly,as confirmed by chromatin immunoprecipitation(ChIP)assay.These results illustrated the outline of the transcription machinery underlying the TCR/NKG2D-dependent induction of TGF-?1 within NK1.1-CD4+NKG2D+T cells and emphasized the significances of AP-1,STAT3,and NF-?B p65 as transcription factors towards the immunosuppressive capacity over these cells.Part 2 Study on the mechanism whereby Egr2/3 regulate the transcription of TGF-?1 in NK1.1-CD4+NKG2D+T cellsBased on the conclusions we achieved from Part 1,continuous efforts were made to evaluate the contributions of early growth response genes 2(Egr2)and Egr3 towards TGF-?1 transcription in NK1.1-CD4+NKG2D+T cells.We,in the first place,demonstrated that Egr2 and Egr3 were expressed at a high level in quiescent NK1.1-CD4+NKG2D+T cells rather than NKG2D-counterparts.Moreover,TCR or NKG2D engagement and co-engagement could lead to a significant induction of Egr2 and Egr3 in NK1.1-CD4+NKG2D+T cells.Then the expressions of Egr2 or/and Egr3 were manipulated in these cells by lentivirus transfection,and we observed that knockdown of Egr2 or/and Egr3 resulted in the downregulation of TGF-?1 in NK1.1-CD4+NKG2D+T cells.In contrary,ectopic expressions of Egr2 or/and Egr3 led to the overexpression of TGF-?1.However,the synergistic effect had not occurred between Egr2 and Egr3 with regards to the transcriptional regulation of TGF-?1 in these cells.In addition,we confirmed two potential binding sites in the TGF-?1 promoter region that could be bound by both Egr2 and Egr3 and promote the gene transcription as referred to electrophoretic mobility shift assay(EMSA)and dual-luciferase gene reporter assay.On the molecular basis,Egr2 and Egr3 could be induced by NKG2D-initiated PI3K signal following the activation of NF-?B p65 and JNK/AP-1.By applying ChIP assay,we also demonstrated that NF-?B p65 and AP-1 were able to enrich in the promoter region of both Egr2 and Egr3 and consequently induce gene transcription.However,PI3K-initiated STAT3 signal,as well as TCR-engaged NFAT signal,may not induce the induction of Egr2 and Egr3 in NK1.1-CD4+NKG2D+T cells.Thus,we arrived at our conclusion that TCR/NKG2D-induced Egr2 and Egr3 play important roles in regulating the immunosuppressive activity in NK1.1-CD4+NKG2D+T cells.Part 3 Study on astilbin protecting against DSS-induced murine colitis by inducing NK1.1-CD4+NKG2D+T cells and its underlying mechanism.Firstly,splenic CD4+T cells from wildtype mice as well as human acute T cell leukemia(Jurkat)cells were treated with astilbin in different concentrations in vitro.A pronounced dose-dependent increase of NKG2D expression was observed upon CD4+T cells as well as upon Jurkat cells.However,CD69 and CD44,T cell activated receptors,were not affected by astilbin treatment.Meanwhile,astilbin was able to enhance the proliferative capacity in NK1.1-CD4+NKG2D+T cells both by low dose and high dose.Phenotypically,astilbin-induced NK1.1-CD4+NKG2D+T cells express TGF-?1 and IL-10 at a high level and highly express CCR6 and CCR9 in a dose-dependent manner.Moreover,astilbin-induced NK1.1-CD4+NKG2D+T cells could inhibit the activation of CD8+T cells and macrophages by producing TGF-?1 and IL-10,indicating the regulatory effects of these cells.For in vivo study,we found that astilbin remarkably ameliorated the DSS-induced colitis in mice that accompanied by the enhanced frequencies of NK1.1-CD4+NKG2D+T cells in spleens and colons.Subsequently,regulatory NK1.1-CD4+NKG2D+T cells were induced by astilbin ex vivo,then been adoptively transferred into DSS-induced colitis mice.Astilbin-induced NK1.1-CD4+NKG2D+T cells were able to recruit to the inflammatory sections of colons and ameliorated colitis in DSS-administrated mice.On the molecular basis,astilbin could initiate the activation of PI3K,STAT3,and MAPK signals and contribute to NKG2D induction in CD4+T cells.Thus,we pointed out the significant role of astilbin that protects against DSS-induced murine colitis by inducing regulatory NK1.1-CD4+NKG2D+T cells and its underlying mechanisms.Taken together,this study elucidated the mechanisms whereby TCR/NKG2D co-engagement induces TGF-?1 transcription in NK1.1-CD4+NKG2D+T cells,and underscored the NKG2D-determined regulatory capacities in these cells.In addition,we demonstrated the immunosuppressive abilities of astilbin that protect against DSS-induced murine colitis through the induction of regulatory NK1.1-CD4+NKG2D+T cells.These findings provide unique perspectives for astilbin as a therapeutic candidate protects against inflammatory bowel disease as well as other inflammatory diseases. |
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