Transcriptional Regulation of Immune Cell Development, Activation and Functions

免疫细胞发育、激活和功能的转录调控

• 批准号: 9566743
• 负责人: Jinfang Zhu
• 金额: $ 147.66万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9566743
• 关键词: Address; Affect; Allergic; Anti-Inflammatory Agents; Anti-inflammatory; Area; Autoantigens; Autoimmune Diseases; B-Cell Lymphomas; Binding; Biological Process; CCR6 gene; CD4 Positive T Lymphocytes; CRISPR/Cas technology; Cell Differentiation process; Cell Lineage; Cells; Cessation of life; ChIP-seq; Characteristics; Chronic; Collaborations; Complex; Data; Development; Disease; Enzymes; Eosinophilia; Exhibits; FOXP3 gene; GATA3 gene; Gene Activation; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Generations; Genes; Genetic Transcription; Helper-Inducer T-Lymphocyte; Heterogeneity; Homeostasis; Immune; Immune Tolerance; Immune response; Immunity; Immunology; In Vitro; Infection; Inflammation; Inflammatory; Interferon Type II; Interferon-alpha; Interleukin 2 Receptor; Interleukin 2 Receptor Gamma; Interleukin-10; Interleukin-13; Interleukin-17; Interleukin-4; Interleukin-5; Interleukins; Intestines; Knock-in Mouse; Knock-out; Lead; Liver; Location; Lung; Lymphocyte; Lymphoid; Lymphoid Cell; Lymphoid Tissue; Mediating; Methylation; Molecular; Mouse Strains; Mus; National Heart, Lung, and Blood Institute; Natural Killer Cells; Nature; Neutrophilia; Organ; Pathogenesis; Pattern; Play; Population; Production; Property; Regulation; Regulatory Element; Regulatory T-Lymphocyte; Reporter; Reporting; Role; Signal Transduction; Stem cells; T-Cell Receptor; T-Lymphocyte; Technology; Th1 Cells; Th2 Cells; Tissues; Toxoplasmosis; Transcriptional Regulation; Up-Regulation; Work; ZNF145 gene; adaptive immune response; cytokine; eosinophil; gene repression; genome-wide analysis; histone demethylase; immunopathology; immunoregulation; in vivo; interleukin-22; member; microorganism; neutrophil; novel; organ growth; pathogen; progenitor; protein protein interaction; response; transcription factor; transcriptome sequencing

CD4 T cells play a central role in orchestrating adaptive immune responses. After being activated through their T cell receptor (TCR) in a particular cytokine milieu, naive CD4 T cells differentiate into distinct T helper (Th) lineages, including Th1, Th2 and Th17 cells that produce interferon (IFN)-gamma, interleukin (IL)-4 and IL-17, respectively, as their signature effector cytokines. These cells are indispensable for different types of immunity to various microorganisms. Inappropriate Th responses to pathogens may lead to chronic infection and/or tissue damage to the host. Similarly, unnecessary activation of Th1, Th17 or Th2 cells by harmless environmental- or self-antigens can cause organ-specific autoimmune diseases or allergic inflammatory diseases. The activation, differentiation and expansion of Th cells are tightly regulated by specific transcription factors. Among the lineage-specific transcription factors, T-bet, GATA3, RORgt and Foxp3 are deterministic for the differentiation and functions of Th1, Th2, Th17 and Treg cells, respectively. These transcription factors have been referred as to master regulators. Innate counterparts of Th cells are innate lymphoid cells (ILCs), whose development requires signaling through the IL-2 receptor (IL-2R) common gamma chain and IL-7R alpha chain. Distinct subsets of ILCs are capable of producing similar sets of characteristic effector cytokines as produced by Th cells. Therefore, they are classified into type 1 innate lymphoid cells (ILC1s) that produce IFNg, type 2 innate lymphoid cells (ILC2s) that produce IL-5 and IL-13, and type 3 innate lymphoid cells (ILC3s) that produce IL-17 and IL-22. The ILCs also express one or two or even three of the master regulators T-bet, GATA3 and RORgt, in a single cell level, and these factors are critical for the development and functions of ILC subsets. Within the ILC3s all of which express RORgt, there are two subsets -- CCR6+ (mainly lymphoid tissue inducers, LTis) and CCR6- ILC3s -- with the latter having the potential to develop into NKp46+ ILC3s that express both RORgt and T-bet. CCR6+ and NKp46+ ILC3s seem to have distinct biological functions and develop from different precursors. Like Th cells, ILCs are important for protective immune responses to infections and are responsible for the pathogenesis of many inflammatory diseases. Some ILCs such as LTis are critical for lymphoid organ development. We have previously reported that T-bet and GATA3 are dynamically expressed by Foxp3-expressing regulatory T cells and such dynamic expression is critical for maintaining immune tolerance (Nature Immunology, 16: 197-206, 2015). However, lymphocytes from the secondary lymphoid organs of mice with GATA-3 single deletion specifically in Tregs were grossly normal in steady state. During the past year, we found that mice with Treg-specific Gata3 deletion exhibit a spontaneous Th2 and Th17 immunopathology in the lungs and in the intestine. The lungs of mice with GATA-3-null Treg cells exhibit eosinophilia and neutrophilia, which corresponds with Th2- and Th17-mediated inflammation. We also found that GATA-3 was expressed at different levels in Tregs cells depending on their activation status and tissue location. When we sub-optimally express GATA-3 in GATA-3-deficient Tregs, the Th2-mediated immunopathology was resolved and the lungs of these mice displayed a normal number of eosinophils. However, these mice had elevated levels Th17 cells and neutrophils. Therefore, we hypothesize that GATA-3 expressed at different levels in Tregs regulates several functional properties of these cells - while basal levels of GATA3 are required for controlling Th2 responses, optimal levels of GATA-3 expression are needed for restraining Th17 responses. Currently, we are investigating the molecular mechanism through which quantitative expression of GATA-3 may qualitatively regulate Treg functions. We also generated a ZsGreen-T2A-GATA3 knock-in mice with conditional knockout potential through Crispr/Cas9 technology to facilitate this study. Preliminary results suggest that this new reporter faithfully reflects GATA3 expression and it can be used to separate lymphocytes that express different levels of GATA3. For ILC studies, we have previously reported that GATA3 plays an essential role in the development of all IL-7Ra-expressing ILCs but not conventional NK cells (Immunity, 40: 378-88, 2014). We further reported that despite its low expression level in mature ILC3s, GATA3 has important functions in regulating homeostasis, further maturation and functions of distinct ILC3 subsets (Nat. Immunol., 17:169-78, 2016). Through RNA-Seq analysis of ILC3 subsets (namely CCR6+ and NKp46+ ILC3) isolated from a novel T-bet-ZsGreen/RORgt-E2-Crimson dual-reporter mouse strain that we have generated, we identified hundreds of CCR6+ and NKp46+ ILC3 lineage specific genes. During the past year, we find that GATA3 may serve as a switch in determining the development of CCR6+ LTi cells versus other ILC lineages. Lymphoid tissue inducer (LTi) population is the founding member of ILCs, however, recent study has shown that these cells are not derived from a PLZF-expressing ILC common progenitor that generates other ILCs. The transcription factor(s) determining the fate of non-LTi progenitor versus LTi progenitor are unknown. Our unpublished new data indicate that GATA3 is absolutely required for the generation of PLZF-expressing non-LTi progenitors, which express high level of GATA3, but not for the generation of RORgammat-expressing LTi progenitors consistent with low levels of GATA3 expression in these progenitors. Nevertheless, low level of GATA3 expression by LTi progenitors is critical for the generation of functional LTi cells. Thus, quantitative expression of GATA3 functionally determines the fates and functions of distinct ILC progenitors. We have also completed studies on other important transcription factors during the past year. We found that the transcription factor Bhlhe40 is required for optimal IFN-g production in Th1 cells through a mechanism independent of T-bet regulation. Bhlhe40 also represses IL-10 production by Th1 cells. Mice with conditional deletion of Bhlhe40 in T cells succumbed to Toxoplasma gondii infection and blockage of IL-10 signaling during infection rescued these mice from death. Thus, our results demonstrate that transcription factor Bhlhe40 is a molecular switch for determining the fate of inflammatory and anti-inflammatory Th1 cells. We also found that the transcription factor B cell lymphoma 11b (Bcl11b), a previously unknown component of the GATA3 transcriptional complex, is critically involved in GATA3-mediated gene regulation. Bcl11b binds to GATA3 through protein-protein interaction and they co-localize at many important cis-regulatory elements in Th2 cells. The expression of type 2 cytokines, including IL-4, IL-5 and IL-13, is up-regulated in Bcl11b-deficient Th2 cells both in vitro and in vivo; such up-regulation is completely GATA3-dependent. Genome-wide analyses of Bcl11b- and GATA3-mediated gene regulation (from RNA-Seq) and co-binding pattern (from ChIP-Seq) suggest that GATA3/Bcl11b complex is involved in limiting Th2 gene expression, as well as in inhibiting non-Th2 gene expression. Thus, Bcl11b controls both GATA3-mediated gene activation and repression. In collaboration with Dr. Keji Zhao's lab at the NHLBI, we reported that conditional deletion of histone demethylases UTX and JMJD3 by CD4-Cre leads to near complete loss of liver NKT cells, while conventional T cells are less affected (Cell Biosci. 7:25, 2017). Therefore, NKT cell development is sensitive to proper regulation of H3K27 methylation. The H3K27me3 demethylase enzymes, in particular UTX, promote NKT cell development, and are required for effective NKT function.

CD4 T 细胞在协调适应性免疫反应中发挥着核心作用。在特定细胞因子环境中通过 T 细胞受体 (TCR) 激活后，幼稚 CD4 T 细胞分化成不同的 T 辅助细胞 (Th) 谱系，包括产生干扰素 (IFN)-γ、白细胞介素 (IL) 的 Th1、Th2 和 Th17 细胞)-4 和 IL-17 分别作为其特征效应细胞因子。这些细胞对于针对各种微生物的不同类型的免疫是不可或缺的。对病原体的不当 Th 反应可能会导致宿主慢性感染和/或组织损伤。同样，无害的环境抗原或自身抗原对 Th1、Th17 或 Th2 细胞的不必要激活可能导致器官特异性自身免疫性疾病或过敏性炎症性疾病。 Th 细胞的激活、分化和扩增受到特定转录因子的严格调控。在谱系特异性转录因子中，T-bet、GATA3、RORgt 和 Foxp3 分别对 Th1、Th2、Th17 和 Treg 细胞的分化和功能具有决定性作用。这些转录因子被称为主调节因子。 Th 细胞的先天对应物是先天淋巴样细胞 (ILC)，其发育需要通过 IL-2 受体 (IL-2R) 共同伽马链和 IL-7R α 链进行信号传导。 ILC 的不同亚群能够产生与 Th 细胞相似的特征性效应细胞因子组。因此，它们被分为产生IFNg的1型先天淋巴细胞（ILC1s）、产生IL-5和IL-13的2型先天淋巴细胞（ILC2s）和产生IL-17的3型先天淋巴细胞（ILC3s）和IL-22。 ILC 还在单细胞水平上表达一种或两种甚至三种主调节因子 T-bet、GATA3 和 RORgt，这些因子对于 ILC 亚群的发育和功能至关重要。在所有表达 RORgt 的 ILC3 中，有两个子集——CCR6+（主要是淋巴组织诱导剂，LTis）和 CCR6- ILC3——后者有可能发育成同时表达 RORgt 和 T-bet 的 NKp46+ ILC3。 CCR6+ 和 NKp46+ ILC3 似乎具有不同的生物学功能，并且由不同的前体发育而来。与 Th 细胞一样，ILC 对于感染的保护性免疫反应很重要，并且是许多炎症性疾病的发病机制。一些 ILC（例如 LTis）对于淋巴器官的发育至关重要。 我们之前报道过表达 Foxp3 的调节性 T 细胞动态表达 T-bet 和 GATA3，这种动态表达对于维持免疫耐受至关重要（Nature Immunology, 16: 197-206, 2015）。然而，来自具有 GATA-3 单一缺失（特别是在 Tregs 中）的小鼠的次级淋巴器官的淋巴细胞在稳定状态下总体上是正常的。 在过去的一年里，我们发现 Treg 特异性 Gata3 缺失的小鼠在肺部和肠道中表现出自发的 Th2 和 Th17 免疫病理学。 GATA-3 缺失 Treg 细胞的小鼠肺部表现出嗜酸性粒细胞增多和中性粒细胞增多，这与 Th2 和 Th17 介导的炎症相对应。我们还发现 GATA-3 在 Tregs 细胞中的表达水平不同，具体取决于其激活状态和组织位置。当我们在 GATA-3 缺陷的 Tregs 中次优表达 GATA-3 时，Th2 介导的免疫病理学得到解决，这些小鼠的肺部显示出正常数量的嗜酸性粒细胞。然而，这些小鼠的 Th17 细胞和中性粒细胞水平升高。因此，我们假设 Tregs 中不同水平表达的 GATA-3 调节这些细胞的多种功能特性——控制 Th2 反应需要基础水平的 GATA3，而抑制 Th17 反应需要最佳水平的 GATA-3 表达。目前，我们正在研究GATA-3定量表达定性调节Treg功能的分子机制。我们还通过 Crispr/Cas9 技术生成了具有条件敲除潜力的 ZsGreen-T2A-GATA3 敲入小鼠以促进这项研究。初步结果表明，该新报告基因忠实地反映了GATA3的表达，可用于分离表达不同水平GATA3的淋巴细胞。 对于 ILC 研究，我们之前曾报道过 GATA3 在所有表达 IL-7Ra 的 ILC 的发育中发挥着重要作用，但在传统 NK 细胞中则不然 (Immunity, 40: 378-88, 2014)。我们进一步报道，尽管 GATA3 在成熟 ILC3 中的表达水平较低，但它在调节稳态、进一步成熟和不同 ILC3 子集的功能方面具有重要功能 (Nat.Immunol., 17:169-78, 2016)。通过对从我们生成的新型 T-bet-ZsGreen/RORgt-E2-Crimson 双报告小鼠品系中分离出的 ILC3 子集（即 CCR6+ 和 NKp46+ ILC3）进行 RNA 测序分析，我们鉴定了数百个 CCR6+ 和 NKp46+ ILC3 谱系特异性基因。 在过去的一年中，我们发现 GATA3 可能作为决定 CCR6+ LTi 细胞与其他 ILC 谱系发育的开关。淋巴组织诱导物 (LTi) 群体是 ILC 的创始成员，然而，最近的研究表明，这些细胞并非源自产生其他 ILC 的表达 PLZF 的 ILC 共同祖细胞。决定非 LTi 祖细胞与 LTi 祖细胞命运的转录因子尚不清楚。我们未发表的新数据表明，GATA3 对于产生表达高水平 GATA3 的 PLZF 表达非 LTi 祖细胞来说是绝对必需的，但对于产生与这些祖细胞中低水平 GATA3 表达一致的表达 RORgammat 的 LTi 祖细胞来说不是必需的。然而，LTi 祖细胞的低水平 GATA3 表达对于功能性 LTi 细胞的生成至关重要。因此，GATA3 的定量表达在功能上决定了不同 ILC 祖细胞的命运和功能。 去年我们还完成了其他重要转录因子的研究。我们发现转录因子 Bhlhe40 是 Th1 细胞中通过独立于 T-bet 调节的机制产生最佳 IFN-g 所必需的。 Bhlhe40 还抑制 Th1 细胞产生 IL-10。 T 细胞中条件性删除 Bhlhe40 的小鼠死于弓形虫感染，感染期间阻断 IL-10 信号传导使这些小鼠免于死亡。因此，我们的结果表明转录因子 Bhlhe40 是决定炎症和抗炎 Th1 细胞命运的分子开关。我们还发现转录因子 B 细胞淋巴瘤 11b (Bcl11b) 是 GATA3 转录复合物中以前未知的成分，它与 GATA3 介导的基因调控密切相关。 Bcl11b 通过蛋白质-蛋白质相互作用与 GATA3 结合，并且它们共定位于 Th2 细胞中许多重要的顺式调控元件。在 Bcl11b 缺陷的 Th2 细胞中，无论体外还是体内，2 型细胞因子（包括 IL-4、IL-5 和 IL-13）的表达均上调；这种上调完全依赖于 GATA3。 Bcl11b 和 GATA3 介导的基因调控（来自 RNA-Seq）和共结合模式（来自 ChIP-Seq）的全基因组分析表明，GATA3/Bcl11b 复合物参与限制 Th2 基因表达，以及抑制非-Th2基因表达。因此，Bcl11b 控制 GATA3 介导的基因激活和抑制。 我们与 NHLBI 的赵克吉博士实验室合作，报道了 CD4-Cre 有条件删除组蛋白去甲基酶 UTX 和 JMJD3 导致肝脏 NKT 细胞几乎完全丧失，而传统 T 细胞受影响较小 (Cell Biosci.7: 2017 年 25 日）。因此，NKT 细胞发育对 H3K27 甲基化的适当调节敏感。 H3K27me3 去甲基酶，特别是 UTX，可促进 NKT 细胞发育，并且是有效 NKT 功能所必需的。