Genetic and epigenetic contributions to the neonatal Th2 bias

描述(申请人提供)：新生儿的免疫反应通常由抗炎Th2细胞因子的主要产生来定义，这种情况被称为新生儿Th2偏向。这种细胞因子的分泌模式被认为有助于幼年动物对感染的易感性，并导致Th2介导的疾病的发展，如哮喘。因此，旺盛的Th2功能是早期免疫的关键定义特征。了解这种现象是如何发生的，对于我们理解适应性免疫系统的个体发育是至关重要的。新生儿的Th2偏向反应部分是由于快速、高水平的Th2细胞因子的产生。我们已经证明Th2细胞因子IL-4和IL-13都是由新激活的小鼠新生儿CD4+细胞早期和大量地产生的。在成人Th2细胞中，协调表达高水平的Th2细胞因子需要一个称为保守非编码区1(CNS-1)的基因间调控区。因此，CNS-1基因元件可能对新生儿早期、强劲的Th2细胞因子产生起重要作用。事实上，在个体发育的早期，这种DNA元素以一种相对允许的表观遗传状态存在。我们发现，与幼稚的成体细胞不同，CNS-1在幼稚的胎儿和新生儿T细胞中的CpG残基上处于低甲基化状态。这种低甲基化状态与新生儿CD4+细胞快速、高水平产生Th2细胞因子密切相关。CNS-1的甲基化在出生后迅速发生，在出生第一周结束时达到成年水平。随着发育的增加，CNS-1的甲基化增加与Th2细胞因子快速表达的进行性沉默有关。综上所述，这些观察表明，CNS-1可能在定义新生儿Th2偏向方面发挥核心作用。我们的长期目标是了解基因和表观遗传学对早期生命Th2主导状态的贡献。在这个提案中，我们将重点放在Th2细胞因子基因座上定义明确的CNS-1区域。我们的初步数据得出了两个明确的假设。具体目标1将测试这样一种想法，即CNS-1区域的物理存在是实现新生儿Th2细胞因子过度生产所必需的。这将通过比较野生型和CNS-1缺陷型新生儿Th2基因座和Th2细胞因子产生的表观遗传状态来实现。具体目标2将检验这样一个前提，即胎儿和新生儿生命中CNS-1的低甲基化是产生新生儿Th2偏倚的关键。这将通过创造强制的转基因小鼠来进行测试 CNS-1在整个发育过程中的甲基化。含有CNS-1靶向的锌指蛋白与从头DNA甲基转移酶DNMT3A的催化域偶联的构建体将被创建~这些构建体的表达将局限于T系细胞。表观遗传学 将在转基因小鼠中评估Th2基因座的状态和Th2功能。这些研究将首次剖析Th2细胞因子基因座在个体发育早期的遗传和表观遗传调控。这些研究的结果最终将导致旨在通过缓解早期Th2主导状态来改善儿科健康的干预策略。

DESCRIPTION (provided by applicant): Immune responses in neonates are often defined by dominant production of anti-inflammatory Th2 cytokines, a condition referred to as the neonatal Th2 bias. This pattern of cytokine secretion is thought to contribute to the susceptibility of young animals to infection and to the development of Th2-mediated diseases, such as asthma. Thus, vigorous Th2 function is a key defining feature of immunity in early life. Learning how this arises is of central importance for our understanding of the ontogeny of the adaptive immune system. The Th2 biased responses of neonates are due, in part, to rapid, high-level Th2 cytokine production. We have demonstrated that the Th2 cytokines IL-4 and IL-13 are both produced early and in copious amounts by newly activated murine neonatal CD4+ cells. In adult Th2 cells, coordinate expression of high levels of Th2 cytokines requires an intergenic regulatory region called conserved non-coding region 1 (CNS-1). Therefore, the CNS-1 genetic element may contribute importantly to the early, robust Th2 cytokine production in neonates. Indeed, this DNA element exists in a relatively permissive epigenetic state in early ontogeny. We have found that, unlike in naive adult cells, CNS-1 is hypomethylated at CpG residues in naive fetal and neonatal T lineage cells. This hypomethylated state is strongly linked to rapid, high level Th2 cytokine production by neonatal CD4+ cells. Methylation of CNS-1 occurs rapidly post birth, with adult levels of methylation being attained by the end of the first week o life. Increasing methylation of CNS-1 with increasing development is associated with the progressive silencing of rapid Th2 cytokine gene expression. Together, these observations indicate that CNS-1 may play a centrally important role in defining the neonatal Th2 bias. Our long-term goals are to understand the genetic and epigenetic contributions to the Th2 dominant state of early life. In this proposal, we will focus on the well-defined CNS-1 region in the Th2 cytokine locus. Our preliminary data lead to two clear hypotheses. Specific Aim 1 will test the idea that the physical presence of the CNS-1 region is required to achieve Th2 cytokine overproduction in neonates. This will be achieved by comparing the epigenetic state of the Th2 locus and Th2 cytokine production in wild-type and CNS-1-deficient neonates. Specific Aim 2 will examine the premise that hypomethylation of CNS-1 in fetal and neonatal life is essential for generating the neonatal Th2 bias. This will be tested by creating transgenic mice with forced methylation of CNS-1 throughout development. Constructs containing a CNS-1-targeted zinc finger protein coupled to the catalytic domain of the de novo DNA methyltransferase Dnmt3a will be created~ expression of the constructs will be confined to T lineage cells. The epigenetic state of the Th2 locus and Th2 function will be assessed in the transgenic mice. These studies will be the first to dissect genetic and epigenetic regulation at the Th2 cytokine locus in early ontogeny. Results from these studies will ultimately lead to interventional strategies aimed at improving pediatric health by mitigating the Th2 dominant state in early life.