Effects And Mechanisms Of Transcriptional Factor Foxo1 And Natural Product PL-C On NK Cell Development And Function

Natural killer(NK) cell is a critical component of innate immune system, which represents 5~10% of the total peripheral lymphocytes. NK cells are the first line of immune defence, which provide early surveillance against malignant transformation, viral/fungus clearance. The capacity of NK cells to directly lyse target cells is determined by the balance of activating and inhibitory receptors. Another mechanism by which NK cells eliminate their target cells is through secretion of cytokines, such as the pro-inflammatory cytokine interferon-γ(IFN-γ), which participate in shaping the adaptive immune response.Murine NK cells develop in the bone marrow from common lymphoid progenitor cells through three continuous stages defined as CD122+NK1.1-DX5- precursor NK(p NK) cells, CD122+NK1.1+DX5- immature NK(iNK) cells, and CD122+NK1.1+DX5+ mature NK(mNK) cells, respectively. After acquisition of NK1.1 surface expression, murine NK cells can be further classified into three stages based on surface expression of CD11 b and CD27: CD11b-CD27+â†' CD11b+CD27+â†' CD11b+CD27-.NK cell development, maturation and effector functions depend on extrinsic and intrinsic factors, and are controlled by both positive and negative regulators. For extrinsic factors, we and others previously showed that interleukin-15(IL-15) plays a critical role in promoting NK cell development and effector functions, while transforming growth factor-β(TGF-β) has an important inhibitory role in this respect. Intrinsic factors, including the transcription factors Ikaros, Pu.1, Ets1, and VDUP-1, regulate the early stages of NK development by promoting the generation of p NK, while E4BP4, Id2 and MEF function during the i NK stage to promote early NK maturation, and Gata-3, Tox, Tbx21, Eomes and Irf2 mainly modulate late-stage NK cell maturation. However, most of these reported transcriptional factors play positive roles in NK cell development and function while little is known about the negative ones. The key role of Tbx21 in regulating late-stage maturation of NK cells is supported by the fact that the final stage of CD11b+CD27- subpopulation is almost absent in Tbx21-deficient(Tbx21-/-) mice. Also, little is known about its upstream signal pathway.IFN-γ has an important role in the activation of both innate and adaptive immunity a nd NK cells represent one of major sources of IFN-γ in our bodies. IFN-γ not only displays antiviral activity, but also regulates various cells of the immune system and performs a crucial role in tumor immunosurveillance, through enhancing tumor immunogeni city and antigen presentation, as well as inducing tumor cell apoptosis. NK cell defected in IFN-γ secretion is positively correlated with tumorigenesis and infection. Exogenous recombinant IFN-γ or cytokines-induced endogenous IFN-γ has been used in various cancer immunotherapy trials,however; outcomes have been disappointing due to its toxicity. Therefore, it is of great clinical significance to search new effective and safety melocules that promote IFN- γ production by NK cells.Based on the two above-mentioned critical scientific questions in field of NK cell developmental and functional modulation, we explored the transcriptional factor, Foxo1, the negative regulator of NK cell development and the natural products, phyllanthusmin C(PL-C), a positive activator of NK cell INF-γ production.Methods and Results:1. The roles and mechanisms of Foxo1 on NK cell development and functionTranscriptional factor Foxo1 plays critical roles in regulation of cell cycle, apoptosis, autophage and anti-oxidative stress. Recent studies show that Foxo1 participates in the genesis of hematopoietic stem cell and common lymphocyte progenitor and the regulation of T, B cell development and function in manner of highly cellular specific contex. However, little is known for its role in NK cell development and function. Through in vivo and in vitro models, our findings are as follows:1.1 Foxo1 deficiency impairs NK cells homing to periphery lymphonodes through decreasing CD62 L expressionTo explore the effect of Foxo1 on NK cell development and function, we crossed Ncr1 i Cre mice with mice carrying floxed Foxo1 alleles(Foxo1fl/fl) to generate NK cell-specific Foxo1 conditional knock-out model, referred as to Foxo1?NK. Foxo1?NK mice showed no significant changes of NK cell proportions and cell numbers in bone marrow, spleen and liver, but lowered NK cells propoportion and cell number in periphery lymphonodes. This is correlated with obviously decreased CD62 L expression, a critical factor for lymphocyte homing.1.2 Foxo1 represses late stage of NK cell maturation in a stem cell intrinsic mannerFoxo1 defeciency NK cells showed an overall increase in the frequency of the most mature CD11b+CD27- population and CD43+KLRG+ NK cells. Through bone marrow transplation of wild type mice or Foxo1?NK cells to CD45.1 mice, respectively, or to the same Rag2-/-Il2rg-/- mice for the establishment of bone marrow chimeras, we confirmed that Foxo1 inhibits NK cell development in a stem cell intrinsic manner.1.3 Foxo1 inhibits IFN-γ production and direct tumor cell lysis by NK cellsUnder the stimulation of IL-12 and IL-18, Foxo1?NK splenic NK cells showed unchanged IFN-γ+ NK cell ratio but increased ?per-cell‘ response of IFN-γ secretion. In vitro overexpressing or knockdown of Foxo1 in NKL cells showed the similar manner on the effector function as that in mouse NK cells.Foxo1 defeciency significantly increased NK cell direct lysis of Yac-1 cell ex vivo, whereas Foxo1 overexpressed in NKL cells inhibited its direct lysis of K562. Further in vivo study by using the model of B16F10 cell metastasis to lung, we confirmed the inhibitory role of Foxo1 on NK cell effector function.1.4 Proinflammatory cytokines or tumor cells inactivate Foxo1 by promoting Foxo1 phosphorylation in NK cellsPhosphorylation of Foxo1 may interfere with their DNA binding activity and lead to its inactivation. Critical cytokines to NK cell development and function, such as IL-2, IL-12 and IL-15 promoted Foxo1 phosphorylation immediately in both NK92 cell and mouse NK cells. Increased Foxo1 phosphorylation in lung NK cells was also observed 1 week after B16F10 in vivo challenge.1.5 Differential mechanisms of Foxo1 suppressing Tbx21 expression in human and mouse NK cellsThe mRNA and protein expression levels of Foxo1 decreased during NK cell maturation, whereas Tbx21 had the opposite expression pattern. Foxo1 deficiency mouse NK cells showed increased m RNA and protein levels of Foxo1, while overexpression or knockdown of Foxo1 in NKL cells negatively affected TBX21 expression.Through Ch IP and IP experiments, we observed a direct Foxo1 binding on the forkhead concensus binding site of TBX21 promoter, but with an indirect binding of Foxo1 to Tbx21 promoter in the putative Sp1 binding site.1.6 Foxo1 requires Tbx21 to exert its effect on NK cell maturationWe next undertook a genetic approach to further confirm that Foxo1 acts upstream of Tbx21 to negatively regulate NK cell development and function. We generated Tbx21-/- and Tbx21+/- Foxo1-deficient mice, i.e. Foxo1?NKTbx21-/- and Foxo1?NKTbx21+/-. Parallel analysis of NK cells from these mice by flow cytometry indeed indicated that the function of Foxo1 in NK cell maturation relied on the existence of Tbx21. When Tbx21 was absent in Foxo1?NKTbx21-/- mice, compared to control Tbx21-/- mice, the loss of Foxo1 in Foxo1?NK Tbx21-/- mice had no further effect on late-stage NK cell maturation. When only a single allele of Tbx21 was present(i.e. Tbx21+/-), the loss of Foxo1 in Foxo1?NK Tbx21+/- mice rescued the defect in NK cell maturation that occurred in Tbx21+/- mice.2. Mechanisms of Enhanced IFN-γ Production by Natural Product PL-CTo explore more ways for NK cell activation and improve the side effects of in vivo IFN-γ therapy, we screened more than 50 kinds of natural products for their abil ity to enhance NK cell production of IFN-γ. We found that PL-C, a small molecule enriched in lignans of plants, can promote human primary NK cell IFN-γ production.2.1 PL-C promotes IFN-γ transcription and secretion by human NK cellsIn the presence or absence of IL-12 or IL-15, PL-C enhanced IFN-g transcription and secretion by human NK cells in PBMC, enriched NK or purified NK cells. PL-C also synergized with IL-12, even at the low cytokine concentration of 0.1 mg/ml, to promote IFN-g production in both human CD56 bright and CD56 dim NK cell subsets.2.2 No effect of PL-C on both NK cell cytotoxicity and T cell IFN-γ productionTo further explore whether PL-C activates NK cell cytotoxicity, we performed the direct cytotoxicity of NK cells by using high sensitive target cell K562 cell line or low sensitive target cell ARH-77 multimyeloma cell line. We didn‘t observe any significant changes of NK direct lysis on both K562 and ARH-77 cells after PL-C treatment in the present of IL-12 or IL-15.To further explore whether PL-C activates NK cell IFN-γ production in a cell specific context, we also tested the effect of PL-C on IFN-γ production by CD4+ or CD8+ T cells. Data showed no significant increased IFN-γ production in CD4+ or CD8+ T cells after PL-C treatment in the presence of IL-12 or IL-15.2.3 Induction of IFN-g production by PL-C in human NK cells is correlated with activation of NF-kB signalingTo explore the mechanisms of PL-C on enhanced IFN-γ production by NK cells, we systemically evaluated the main signal pathway that participating in IFN-γ transcription and secretion. We found no significantly changes of IL-12 or IL-15 receptors and their related downstream signal pathways, such as TBX21 and STATs family after PL-C treatment in the presence of IL-12 or IL-15. Further researches showed that PL-C signicantly increased the phosphorylation of NF-κB p65 whether in the presence or absence of IL-12 or Il-15. NF-κB inhibitor, N-tosyl-L-phenylalanine chloromethyl ketone(TPCK), could inhibit the enhanced IFN-γ production by PL-C in both human primary NK cells and NKL cell line. By both electrophoretic mobility shift assay(EMSA) and Chromatin immunoprecipitation(Ch IP) assay, we confirmed our finding that PL-C obviously promoted p65 binding to IFNG promoter C3-3 3P κB site.2.4 PL-C promotes IFN-γ production through augmenting TLRs-NF-κB signaling in human NK cellsActivation of TLRs-NF-κB axis is critical to the robust induction of cytokine expression in immune cells, and human NK cells mainly express TLR1, TLR3, and TLR6. We thus next tested the role of TLRs mediated NF-κB activation in PL-C-induced IFN-γ production by using TLRs blocking antibodies, TLRs ligands, luciferase assay and TLR sh RNA. Through blocking TLR1, TLR3, and TLR6 signaling by blocking antibodies, we found TLR1 and TLR6 blocking inhibited PL-C-induced IFN-γ production together with NF-κB inactivation. PL-C also enhanced IFN-γ production by TLR1 and TLR6 ligand at the presence of IL-12. Through coexpression of TLR1 or TLR6, pGL-3κB-Luc and control plasmid p RL-TK renilla-luciferase plasmids, PL-C treatment was found to increase NF-κB binding to the κB binding sites in a dose-dependent fashion. Through sh RNA retrovirus, we found that knockdown of TLR1 in NKL cells abolished the increased IFN-γ production by PL-C treatment.Conclusions:1. Roles and mechanisms of Foxo1 on NK cell development and function1) Foxo1 deficiency impairs NK cell homing to peripheral lymphonodes through decreased CD62 L expression;2) Foxo1 negatively controls NK cell late stage maturation without affecting p NK and iNK development;3) Foxo1 represses NK cell IFN-γ secretion and direct tumor lysis in vivo and in vitro;4) Pro-inflammatory cytokines and in vivo tumor challenge inactivate Foxo1 through promoting Foxo1 phosphorylation;5) Through the animimal model of Foxo1 and Tbx21 double knockout in NK cells, we demonstrated that promotion of late-stage NK cell maturation by loss of Foxo1 requires Tbx21 expression.6) Foxo1 inhibits Tbx21 expression in human NK cells through direct promoter binding and in murine NK cells through recruitment by Sp1 to the proximal prom oter region.2. The natural product PL-C enhances IFN-γ production by human natural killer cells through upregulation of TLR-mediated NF-κB signaling1) PL-C promotes both CD56 bright and CD56 dim NK cell IFN- γ secretion with no effects on NK cell cytotoxicity and T cell IFN-γ production;2) PL-C could not affect IL-12 or IL-15 receptor and its related downstream signal pathway;3) PL-C enhances NK cell IFN- γ production through directly activating TLR-NF-κB signal axis.To summarize, our current work is to explore the regulation of NK cell development and function from two aspects: transcriptional factor Foxo1 and natural activator PL-C. We for the first time found that transcriptional factor Foxo1 negatively control s late stage of NK maturation and NK function, which is mainly through its inhibition on the expr ession of the key transcriptional factor-Tbx21. This finding greatly enriched the negative network of NK cell development and function. On the other hand, we found a natural product-PL-C selectively enhanced IFN-γ production by NK cells, which is mainly through the activation of TLR-NF-κB axis. This selectivity of PL-C in immune activation should make it more suitable for development of clinically useful immune modulator in the future.