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伴随着凛冽的寒风，小编又感冒了，喉咙肿痛，高烧不退，扁桃体肿的好象个鸡蛋，吃嘛嘛不香，扁桃体发炎了。打针挂水不能断，医生说：“哎哎哎~药不能停呀”。炎症真是让人又爱又恨的小妖精，可以保护人体不受侵害，但是也会引起人体过敏反应，比如类风湿性关节炎和红班狼疮症等免疫系统过敏病症疾病。拖着“病弱”的身躯，小编我对炎症是被什么调控的产生了莫大的兴趣。下边就让小编在本期的经典文献回顾中，跟随作者的脚步探究一下影响炎症的其中一个重要因子吧~ 药不能停[1]
Th17 说道免疫细胞，大家首先想到的一定是免疫T细胞，那么咱们一定要说道一下Th17，作为一种能够分泌白介素17(interleukin 17, IL-17)的T 细胞亚群，Th17在自身免疫性疾病和机体防御反应中具有重要的意义[2]。 Th17是宿主防御细胞外病原体所必需的细胞，当调节失调时，可促进人类炎症疾病，包括多发性硬化、类风湿关节炎、炎症性肠病、银屑病和哮喘[3-5]。白细胞介素23 (IL-23)是T helper 17 (Th17)细胞致病性所需的促炎细胞因子，但其调控这一过程的分子机制尚不清楚。研究者发现转录因子Blimp-1 (Prdm1)是IL-23诱导的驱动Th17细胞炎症功能的关键因子，与RORγt协同作用，激活Th17炎症程序。今天咱们就一步一步来探讨一下，那么Blimp-1 (Prdm1)是如何调节Th17的致炎性的吧。
Prdm1的表达 既然咱们认为Prdm1可以调节Th17细胞的致炎性，那么咱们肯定要了解一下Prdm1在Th17细胞中是怎样诱导表达的啦。 很多数据显示，IL-23是Th17细胞获得效应功能的关键。为了研究IL-23R信号缺失和存在时的基因表达差异，我们将带有Il23r-/-同源标记的和野生型(WT) OT-II T细胞转入C57BL/6小鼠体内，并进行分析（图1 A）。结果显示，WT和Il23r-/- 中产生IL-17的细胞转录Il17a和Rorc 的mRNA均高表达，而IL-23R缺陷型中产生IL-17的细胞转录Il23r的 mRNA表达减少（图1 A）。与IL-17-细胞相比，WT IL-17 + OT-II细胞中转录Prdm1（Blimp-1）、Csf2和Tbx21的mRNA表达量更高，并且在没有IL-23信号传导的情况下该表达显着降低（图1A）。Th17信号基因如Il17a、Il23r、Rorc的表达在Th17细胞(RORγt+IL-17+ IFNγ-)中升高，而真正的Th1基因Ifng、Tbx21在Th1细胞(RORγt-IFNγ+IL-17-)中转录增强(图1B)。与Th1细胞相比，Th17细胞以及同时表达IL-17和IFN-γ (RORγt+ IL-17+ IFN-γ+)的炎性Th17细胞中，Prdm1的转录水平显著升高(4- 8倍)(图1B)。并且Blimp-1表达仅局限于实验性自身免疫性脑脊髓炎(EAE)模型中的Th17细胞中，而不在Th1细胞(图1C)。此外，Blimp-1+ Th17细胞下调IL-2，说明Th17群体更加成熟。这些结果显示IL-23信号可以促进Prdm1的表达，进而促进Th17细胞的功能成熟。 Figure 1. IL-23 Signaling Induces Prdm1 Expression in Th17 Cells (A) Differential gene-expression analysis of intracellularly stained IL-17+ and IL-17_ wild-type or Il23r–/– CD45.1+CD4+ OT-II cells from draining lymph nodes of CD45.2+ mice at day 7 post immunization with OVA(323–339) in CFA. (B) Differential gene-expression analysis of intracellularly stained IL-17+ and IFN-γ+ cells from draining lymph nodes of C57BL/6J mice at day 7 post immunization with MOG35–55 in CFA. (C) Intracellular staining for IL-17, IFN-γ, and IL-2 expression by CD4+Blimp-1-YFP+ cells from draining lymph nodes of MOG35–55 immunized Blimp-1-YFP reporter mice (n = 5 per group). Data are representative of three independent experiments. Error bars in all graphs are SEM ns, not significant; *p < 0.05,="" **p="">< 0.01,and="" ***p="">< 0.001="" (student’s="" t=""> (D) STAT binding sites on Prdm1 locus as assessed by chromatin immunoprecipitation (ChIP) and massive parallel sequencing of Th17 cells differentiated in the presence of TGF-β, IL-6, and IL-23. (E) Naive CD4+ T cells from STAT-3 CKO (Stat3fl/flCd4cre), STAT-1 KO (Stat1_/_), or wild-type control mice were cultured under Th17 condition (plate bound anti- CD3, anti-CD28, IL-1b, IL-6, IL-23, anti-TGF-b1, anti-TGF-b2, anti-IFN-g, and anti-IL-4) for 3 days. Prdm1 and Il23r mRNA expression were assessed by quantitative RT-PCR. See also Figure S1。
Supplementary Figure 1 哦，原来IL-23是prdm1在Th17细胞的一个关键因素啊，但是prdm1在Th17细胞中难道只被IL-23诱导？就没有其他的隐藏BOSS了吗？
当然还有啦，在IL-23驱动的Th17细胞中，全基因组的STAT-3占用研究(ChIP-Seq)证实了STAT-3与Prdm1位点在启动子、内含子和3端直接结合(图1D)。为了进一步探讨IL -23介导的STAT-3或STAT-1激活信号是不是Th17细胞中Prdm1的表达的必要因素。我们将STAT-3 cKO (Stat3fl/flCd4cre)和STAT-1 KO (STAT1-/-) CD4+ T细胞极化至Th17细胞，并测试Prdm1的表达(图1E)。结果表明STAT-3在Th17分化中十分重要，IL-23通过STAT-3对Prdm1进行诱导。作为对照，我们还研究了同样依赖于STAT-3的Il23r和Il17a的诱导情况，最近的研究发现的IL-23靶基因Tbx21是依赖STAT-1 表达，而不是STAT-3[6.7]。这些结果表明IL-23通过STAT-3可以直接诱导Blimp-1在Th17细胞中表达。 Th17细胞缺失Blimp-1说了这么多表达Blimp-1的情况，那么要是Th17细胞中缺乏Blimp-1，Th17细胞还能不能健康快乐的成长呢？
为了检测Blimp-1缺失是否影响Th17分化，我们检测了CFA免疫小鼠的dLNs中活化的T细胞产生细胞因子的情况。在没有Prdm1的情况下，YFP+ CD4 T细胞产生IL-17的比例略有降低(图2A）。但更重要的是，缺乏prdm1的IL-17细胞不能共同表达GM-CSF和IFN-γ(图2B），些因子已被证明是Th17细胞炎症功能的关键因子[6.8-10]。在没有Blimp-1的情况下，Th17细胞无法达到完全的功能成熟。另一个与Th17细胞成熟相关的因素是IL-2的下调[11]。我们发现约60%的Th17细胞是IL-17和IL-2共同产生的 (图2C）。在没有Blimp-1的情况下，Th17细胞均在早期活化阶段被抑制，无法下调IL-2的表达。这些结果表明Blimp-1在Th17细胞的全效应分化中起着关键作用。 果然，没有Blimp-1的Th17细胞再也不能快乐的长大了。 Figure 2. Blimp-1 Deficiency Blocks Development of Inflammatory Th17 Cells Co-expressing IL-17, GM-CSF, and IFN-γ (A)Frequency and absolute number of IL-17-producing CD4+YFP+ T cells from d7 draining lymph nodes of MOG35–55 immunized Blimp-1 CKO (Prdm1fl/flRosa-YFPflDlckcre) and heterozygous (Prdm1fl/WTrosa-YFPflDlckcre) mice (n = 5 per group). (B) Frequency and absolute number of GM-CSF and IFN-γ-producing IL-17+ cells from (A). (C)Blimp-1 represses IL-2 expression by Th17 cells. Error bars in all graphs are SEM ns, not significant; *p < 0.05,="" **p="">< 0.01="" and="" ***p="">< 0.001="" (student’s="" t="">
Blimp-1 cKO小鼠EAE的发生率降低听说Blimp-1条件性敲除的小鼠，不容易得EAE呢，所以某些时候缺乏Blimp-1难道也是有好处的？ 为了研究Blimp-1基因与EAE的关系，作者又选用了一种Th17细胞依赖性的人多发性硬化症的Blimp-1 cKO小鼠模型，来研究EAE的发生率和严重程度。Blimp-1 cKO小鼠的EAE降低(图3A，表1)。在Blimp-1 cKO小鼠中枢神经系统(CNS)(脊髓和大脑)中的CD4+ T细胞明显少于杂合型和野生型小鼠(图3B)。CD4 T细胞的绝对数量减少(减少90%)进一步证实该能力缺陷的Blimp-1 cKO CD4+ T细胞影响中枢神经系统 (图3B)。此外，少数能够影响中枢神经系统的Blimp-1 cKO YFP+ CD4+ T细胞功能缺陷，IL-17产量降低(图3C)。更重要的是，blimp-1缺失的Th17细胞共表达GM-CSF和IFN-γ的能力存在显著缺陷(图3D)。已知IFN-γ生成的RORγt+ Th17细胞存在于EAE的病变组织中，在多发性硬化患者的中枢神经系统中优先积累[12]，证实其对炎症的促进作用。 Figure 3. Blimp-1 Deficiency Reduces EAE Incidence and Severity (A) Clinical scores of Blimp-1 CKO mice after EAE induction. (n = 9 per group). (B) Frequency and absolute number of CD4+ T cells in the CNS of Prdm1 conditional knockout mice at 16 days post immunization (n = 10 for het, n = 5 for ko). (C) Frequency and absolute number of IL-17-producing YFP+CD4+ T cells from the CNS of mice in (B). (D) Frequency and absolute number of GM-CSF and IFN-g-producing IL-17+YFP+CD4+ T cells from (B). Data are representative of three independent experiments.Error bars in all graphs are SEM ns; **p < 0.01="" (student’s="" t="">
Blimp -1介导Th17致炎性的分子机制Blimp -1对Th17致炎性影响这么大，咱们必须研究一下是啥分子机制啦。 为鉴定Blimp-1在Th17细胞中的转录靶点，对纯化的IL-17进行全基因组Blimp-1占用率研究(ChIP-Seq)。EGFP+细胞在TGF-β、IL-6、IL-23存在下分化(图4)。本研究发现Blimp-1直接与Il23r、Il17和Csf2调控区域结合(图4）此外，Blimp-1在p300、STAT-3和RORγt附近共定位(图4)。推测Blimp-1调控IL-23介导的Th17效应子功能主要是通过诱导Il23r表达来扩大IL-23信号，同时增强Il17和Csf2的转录。为了验证这一点，我们在活化的Th17细胞中异位表达Blimp-1，并在Th17细胞中表达Blimp-1强烈上调Il23r表达(图5A)。为了进一步证实blimp -1介导的Il23r调控，用Blimp-1对IL-23R荧光探针标记的小鼠进行转导。Blimp-1过表达显著增强IL-23R荧光探针在Th17细胞中的表达(图5B)。与既往研究一致，Blimp-1过表达并未改变IL-17的表达，说明Blimp-1不能直接抑制IL-17 [13]。异位表达blimp1但是不表达rorγt,强烈诱导Th17细胞产生GM-CSF(图5C)。综上所述，这些结果验证了Blimp-1是IL-23调控Th17的主要影响因子。最近的一篇论文表明，Blimp-1和IRF4可以促进Treg细胞中IL-10的产生[14]. 然而，过表达Blimp-1并没有促进Th17细胞产生IL-10 (图5E)。 Figure 4. Blimp-1 Directly Binds Th17 Signature Genes Blimp-1, STAT-3, co-activator p300, and RORγt binding on Il23r, Csf2, and Il17f loci as assessed by chromatin immunoprecipitation and massive parallel sequencing of purified IL-17.EGFP+ cells differentiated in the presence of TGF-β, IL-6, and IL-23. The RORγt data is extracted from Ciofani et al. (2012)[14] (GEO# GSM1004855 WT Rorg ChIP;GSM1004854 RORγKO RORγ ChIP). Blimp-1异位表达仅在RORγt+ Th17细胞中驱动Th17标记基因，而在Th0细胞（CD4+ Th细胞）中不驱动Th17标记基因，说明RORγt的存在对Th17细胞中Blimp-1调节的功能至关重要(图5D)。为了进一步证实这一点，利用RORγ特异性敲除的RORγ cKO（Rorcfl/flRosacre/ERT2)）小鼠进行研究。IL-23激活的Th17细胞中GM-CSF的稳健表达(图5D top panel)。在活化阶段，Rorc的缺失不仅降低了IL-17的表达，并且完全抑制了Th17细胞对GM-CSF的表达(图5D top panel)。即使在没有添加IL-23的情况下，Blimp-1过表达也足以诱导Th17细胞产生最佳GM-CSF(图5D bottom panel)。最重要的是，Blimp-1中Rorc的缺失导致Th17细胞完全阻断GM-CSF的表达，抑制Th17的致病分化。这些数据表明Blimp1和RORγt协同作用稳定Th17炎性分化程序。 Figure5 Ectopic Blimp-1 Expression Upregulates Th17 Signature Genes (A) Naive OT-II cells were activated under Th0 or Th17 conditions, retrovirally transduced on day 2 with pMIG (Empty) or pMIG-Blimp-1 RV, rested, and reactivated for 72 hr followed by gene-expression analysis of sorted GFP+ cells. (B) IL-23R-mCherry cells were activated under Th17 conditions, retrovirally transduced on day 2 with pMIG (Empty) or pMIG-Blimp-1 RV, rested, and reactivated for 72 hr followed by surface analysis of IL-23R-mCherry on bulk live cells (top) or transduced GFP+CD4+ cells (bottom). (C) Naive OT-II cells were activated under Th17 conditions, retrovirally transduced on day 2 with pMIG (Empty), pMIG-RORγt or pMIG-Blimp-1 RV, rested, and reactivated for 72 hr followed by GM-CSF analysis by ELISA of transduced culture supernatants. (D) Differential gene-expression analysis of retrovirally transduced and sorted GFP+ Th0 or Th17 cells at 72 hr post-secondary stimulation. Heat map represents fold differences compared to the control Th0-pMIG-GFP. Error bars in all graphs are SEM. **p < 0.01="" (student’s="" t=""> (E) IL-10 expression analysis of retrovirally transduced and sorted GFP+ cells from (D). 结论Becher及其同事报道，RORγt的存在对于GM-CSF Th17细胞的产生是至关重要的[7]。这可能是由于RORγt是直接参与Csf2增强的因子。然而，仅仅强制表达RORγt并不会增加Th17细胞中GM-CSF的表达，还需要额外的增强因子。Blimp-1是Th17的转录调控因子之一，该转录调控因子是Th17细胞致病功能的控制基因。而Blimp-1作为单一因子不控制任何T细胞效应表型。相反，根据由BATF、IRF4、c-MAF、STAT-3、STAT-5、RORγt或Foxp3组成的多因子转录复合物所产生的炎症或抗炎增强因子，Blimp-1可能具有独特的基因调控活性。 哦哦哦~到这里小编就明白啦，原来炎症的调控机制如此复杂，不是单一因子可以调控的。今天的经典文献就到这里啦，以后有机会小编在和大家一起探究身体防御机制更深的奥秘吧~文章还有很多精彩实验没有呈现，大家有兴趣一定要去看原文哦[15]。 百奥赛图非常荣幸在此次研究中提供了IL-17-EGFP敲除小鼠，了解更多模式小鼠请关注我们哦~ 参考文献 [1] http://pic.sogou.com/dquery=%D2%A9%B2% BB%C4%DC%CD%A3%B1%ED%C7%E9%B0%FC& mode=1&did=201#did200 [2] https://baike.sogou.com/v58886253.htm?fromTitle=Th17%E7%BB%86%E8%83%9E [3] Bettelli, E., Carrier, Y., Gao, W., Korn, T., Strom, T.B., Oukka, M., Weiner, H.L.,and Kuchroo, V.K. (2006). Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441, 235–238. [4] Mangan, P.R., Harrington, L.E., O’Quinn, D.B., Helms, W.S., Bullard, D.C.,Elson, C.O., Hatton, R.D., Wahl, S.M., Schoeb, T.R., and Weaver, C.T.(2006). Transforming growth factor-beta induces development of the T(H)17 lineage. Nature 441, 231–234. [5] Veldhoen, M., Hocking, R.J., Atkins, C.J., Locksley, R.M., and Stockinger, B.(2006). TGFbeta in the context of an inflammatory cytokine milieu supports denovo differentiation of IL-17-producing T cells. Immunity 24, 179–189. [6] Hirota, K., Duarte, J.H., Veldhoen, M., Hornsby, E., Li, Y., Cua, D.J., Ahlfors, H.,Wilhelm, C., Tolaini, M., Menzel, U., et al. (2011). Fate mapping of IL-17-producing T cells in inflammatory responses. Nat. Immunol. 12, 255–263. [7] Lee, Y., Awasthi, A., Yosef, N., Quintana, F.J., Xiao, S., Peters, A., Wu, C.,Kleinewietfeld, M., Kunder, S., Hafler, D.A., et al. (2012). Induction and molecular signature of pathogenic TH17 cells. Nat. Immunol. 13, 991–999. [8] Codarri, L., Gyu¨ lve´ szi, G., Tosevski, V., Hesske, L., Fontana, A., Magnenat, L.,Suter, T., and Becher, B. (2011). RORgt drives production of the cytokine GMCSF in helper T cells, which is essential for the effector phase of autoimmune neuroinflammation. Nat. Immunol. 12, 560–567. [9] El-Behi, M., Ciric, B., Dai, H., Yan, Y., Cullimore, M., Safavi, F., Zhang, G.-X.,Dittel, B.N., and Rostami, A. (2011). The encephalitogenicity of T(H)17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GMCSF.Nat. Immunol. 12, 568–575. [10] Ghoreschi, K., Laurence, A., Yang, X.-P., Tato, C.M., McGeachy, M.J., Konkel,J.E., Ramos, H.L., Wei, L., Davidson, T.S., Bouladoux, N., et al. (2010).Generation of pathogenic T(H)17 cells in the absence of TGF-b signalling.Nature 467, 967–971. [11] McGeachy, M.J., Chen, Y., Tato, C.M., Laurence, A., Joyce-Shaikh, B.,Blumenschein, W.M., McClanahan, T.K., O’Shea, J.J., and Cua, D.J. (2009).The interleukin 23 receptor is essential for the terminal differentiation of interleukin 17-producing effector T helper cells in vivo. Nat. Immunol. 10, 314–324. [12] Kebir, H., Ifergan, I., Alvarez, J.I., Bernard, M., Poirier, J., Arbour, N., Duquette,P., and Prat, A. (2009). Preferential recruitment of interferon-gamma-expressing TH17 cells in multiple sclerosis. Ann. Neurol. 66, 390–402. [13] Salehi, S., Bankoti, R., Benevides, L., Willen, J., Couse, M., Silva, J.S., Dhall,D., Meffre, E., Targan, S., and Martins, G.A. (2012). B lymphocyte-induced maturation protein-1 contributes to intestinal mucosa homeostasis by limiting the number of IL-17-producing CD4+ T cells. J. Immunol. 189, 5682–5693. [14] Cretney, E., Xin, A., Shi, W., Minnich, M., Masson, F., Miasari, M., Belz, G.T.,Smyth, G.K., Busslinger, M., Nutt, S.L., and Kallies, A. (2011). The transcription factors Blimp-1 and IRF4 jointly control the differentiation and function of effector regulatory T cells. Nat. Immunol. 12, 304–311. [15] Renu J.,Yi C.,Yuka K., Barbara JS., Golnaz V., Kiyoshi H.,Wendy M.B.,et al.(2015). Interleukin-23-Induced Transcription Factor Blimp-1 Promotes Pathogenicity of T Helper 17 Cells.J. Immunity.Volume 44,Issue 1,19 January 2016, Pages 131-142.
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