ABSTRACT

A stressor can induce resilience in another, different stressor, a phenomenon known as cross-tolerance. To learn if cross-tolerance is governed by epigenetic regulation, we used embryonic heat conditioning (EHC) in chicks, during the development of the hypothalamus, to increase the immunization response. Indeed, EHC induced a lifelong systemic antibody response to immunization, in addition to reduced hypothalamic IL6 inflammatory expression following LPS challenge. Since the outcome of EHC was long-term cross-tolerance with the immune system, we studied possible epigenetic mechanisms. We first analysed the methylation and hydroxymethylation patterns of IL6. We found reduced hydroxymethylation on IL6 intron 1 in the EHC group, a segment enriched with CpGs and NFkB-binding sites. Luciferase assay in cell lines expressing NFkB showed that IL6 intron 1 is indeed an enhancer. ChiP in the same segment against NFkB in the hypothalamus presented reduced binding to IL6 intron 1 in the EHC group, before and during LPS challenge. In parallel, EHC chicks’ IL6 intron 1 presented increased H3K27me3, a repressive translational modification mediated by EZH2. This histone modification occurred during embryonic conditioning and persisted later in life. Moreover, we showed reduced expression of miR-26a, which inhibits EZH2 transcription, during conditioning along with increased EZH2 expression. We demonstrate that stress cross-tolerance, which was indicated by EHC-induced inflammatory resilience and displayed by attenuated inflammatory expression of IL6, is regulated by different epigenetic layers.

摘要

压力源可以在另一个不同的压力源中诱导恢复力，这种现象称为交叉耐受。为了了解交叉耐受性是否受表观遗传调控，我们在下丘脑发育期间对雏鸡使用胚胎热调节 (EHC) 来增加免疫反应。事实上，除了LPS 攻击后下丘脑IL6炎症表达降低之外，EHC 还诱导了对免疫的终生全身抗体反应。由于 EHC 的结果是与免疫系统的长期交叉耐受性，我们研究了可能的表观遗传机制。我们首先分析了IL6的甲基化和羟甲基化模式。我们发现IL6上的羟甲基化减少EHC 组中的内含子 1，富含 CpG 和 NFkB 结合位点的片段。表达 NFkB 的细胞系中的荧光素酶测定表明IL6内含子 1 确实是增强子。在 LPS 攻击之前和期间，下丘脑中针对 NFkB 的同一节段中的 ChiP 与EHC 组中的IL6内含子 1 的结合减少。同时，EHC 小鸡的IL6内含子 1 呈现增加的 H3K27me3，这是一种由 EZH2 介导的抑制性翻译修饰。这种组蛋白修饰发生在胚胎调节期间并在以后的生活中持续存在。此外，我们发现在调节过程中抑制EZH2转录的 miR-26a 的表达减少，同时EZH2增加表达。我们证明了压力交叉耐受性，这由 EHC 诱导的炎症恢复力所表明，并由IL6的炎症表达减弱所显示，受不同的表观遗传层的调节。