A probiotic-derived protein regulates epigenetic programming in intestinal epithelial cells for long-term prevention of colitis

益生菌衍生的蛋白质调节肠上皮细胞的表观遗传编程，以长期预防结肠炎

• 批准号: 10386935
• 负责人: FANG YAN
• 金额: $ 44.15万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10386935
• 关键词: Address; Adult; Bacteria; Binding; CD4 Positive T Lymphocytes; Cell Line; Cell division; Cell physiology; Child; Chromatin; Clinical Trials; Colitis; Complex; Data; Deposition; Dimerization; Early Intervention; Ecosystem; Enhancers; Epidermal Growth Factor Receptor; Epigenetic Process; Epithelial; Epithelial Cells; Exposure to; Foundations; Funding; Gene Expression; Gene Proteins; Genes; Health; Health Promotion; Histone H3; Histones; Human; Incidence; Individual; Infant; Inflammation; Inflammatory Bowel Diseases; Inherited; Intervention; Intestines; Lactobacillus casei rhamnosus; Lamina Propria; Life; Longevity; Lysine; Mediating; Methyltransferase; Modeling; Mus; Natural regeneration; Neonatal; Outcome; Predisposition; Prevention; Prevention strategy; Prevention therapy; Probiotics; Production; Proteins; Public Health; Regulatory T-Lymphocyte; Reporting; Research; Research Support; Signal Transduction; Supplementation; Testing; Tight Junctions; Transactivation; Transcriptional Activation; Transforming Growth Factor beta; Translational Research; Up-Regulation; Work; antibody inhibitor; autoinflammatory; dimer; dysbiosis; epigenetic memory; gut inflammation; gut microbiome; gut microbiota; high risk; histone methyltransferase; histone modification; imprint; intestinal epithelium; intestinal homeostasis; knock-down; microbiome; mouse model; neonatal period; neonate; neutralizing antibody; novel; novel therapeutic intervention; novel therapeutics; postnatal development; preservation; prevent; programs; promoter; receptor; response; secretory protein; stem cells; transcription factor

PROJECT SUMMARY Dysbiosis in infants and children is associated with increased susceptibility to inflammatory bowel disease (IBD) in adults. However, the mechanisms whereby the gut microbiota colonization in early life confers health outcomes throughout the lifespan remain unclear. Studies from the previous funding period isolated and cloned a Lactobacillus rhamnosus GG (LGG)-derived secretory protein, p40, and demonstrated that neonatal p40 supplementation prevents colitis in adult mice. Our preliminary studies discovered that p40 interacts with two transcriptional factors, Mga and Max, to regulate expression of Setd1β gene, which encodes a methyltransferase for catalyzing mono and trimethylation of histone 3-lysine 4 (H3K4me1/3). We identified TGFβ as a potential target of p40-up-regulated Setd1β. IECs are rapidly renewed and continuously regenerated from intestinal stem cells (ISCs). We found that p40 modulates H3K4m31/3 in ISCs in early stage, and p40 treatment in neonates, but not adult mice, stimulates sustained increase in TGFβ gene expression in IECs. TGFβ has been shown to have multiple functions against inflammation. Thus, we hypothesize that p40 up-regulates Setd1β gene expression in IECs through increasing Mga:Max dimerization. Supplementation with p40 in early life stimulates Setd1β-dependent H3K4me1/3 deposition at the TGFβ locus in ISCs, which is inherited by IECs to enable the sustained increase in TGFβ production, and subsequently prevention of intestinal inflammation in adulthood. In Aim 1, we will determine whether p40-stimulated Setd1β gene expression and H3K4me1/3 are required to drive the increase in expressing TGFβ gene in IECs, and elucidate whether the interaction between p40 and the Mga:Max dimers mediates up-regulation of Setd1β production. In Aim 2, we will identify the temporal window of p40 exposure in early life that causes the sustained increase in TGFβ production in IECs, and define whether p40-regulated sustained increase in TGFβ gene expression in IECs requires the increase of Setd1β gene expression inISCs in early life. Human and mouse enteroids and colonoids and 2D cultures and cell lines with silencing Setd1β or Mga genes, and mouse models of constitutive and inducible Setd1β gene knock down in IECs or ISCs will be utilized for these two aims. In Aim 3, we will determine whether the sustained increase in TGFβ production after p40 supplementation in early life is required to prevent colitis in adult mice. We will use neutralizing antibodies and inhibitors to block TGFβ function in mice with induced and spontaneously developed colitis. We will also determine whether sustained increase in TGFβ production enhances Tregs induction in the intestine and protective epithelial responses for the prevention of colitis in adulthood. This aim will be tested in mice with TGFβ receptor II deletion in CD4+ T cells and Smad4 deletion in IECs. Together, our proposed research will elucidate a novel mechanism underlying the consequences of long-lasting effects of p40 on prevention of colitis, and lay the foundation for developing early intervention with p40 as a novel therapy for prevention of IBD in adults.

项目摘要 婴儿和儿童的生态失调与炎症性肠病易感性增加有关 成年人的IBD。然而，早期生命中肠道微生物群定植的机制 整个生命周期的健康结果仍不清楚。上一个供资期的研究孤立， 克隆了鼠李糖乳杆菌GG（LGG）衍生的分泌蛋白p40，并证明新生儿 补充p40可预防成年小鼠的结肠炎。我们的初步研究发现，p40与 两个转录因子Mga和Max调节Setd 1 β基因的表达，Setd 1 β基因编码 甲基转移酶，用于催化组蛋白3-赖氨酸4（H3 K4 me 1/3）的单和三甲基化。我们确定 TGFβ是p40上调Setd 1 β的潜在靶点IEC更新迅速， 肠干细胞（ISCs）。我们发现p40在早期调节ISCs中的H3 K4 m31/3， 在新生小鼠中，而不是在成年小鼠中，p40治疗刺激TGFβ基因表达的持续增加， IEC。TGFβ已被证明具有多种抗炎功能。因此，我们假设p40 通过增加Mga：Max二聚化上调肠上皮细胞中Setd 1 β基因表达。补充 生命早期的p40刺激ISCs中TGFβ基因座上Setd 1 β依赖性H3 K4 me 1/3沉积， 由IEC遗传，使TGFβ产生的持续增加，并随后预防 成年期肠道炎症。目的1：检测p40刺激的Setd 1 β基因是否能在体外表达， 表达和H3 K4 me 1/3是驱动IEC中TGFβ基因表达增加所必需的，并阐明了 p40和Mga：Max二聚体之间的相互作用是否介导Setd 1 β产生的上调。在 目的2，我们将确定生命早期p40暴露的时间窗口，导致持续增加的 TGFβ在IEC中的表达，并确定p40调节的TGFβ基因表达是否持续增加。 肠上皮细胞需要Setd 1 β基因在生命早期在ISCs中表达增加。人类和小鼠类肠细胞， 具有沉默Setd 1 β或Mga基因的类结肠和2D培养物和细胞系，以及组成性 而在IEC或ISC中可诱导的Setd 1 β基因敲低将用于这两个目的。在目标3中，我们 确定在生命早期补充p40后TGFβ产生的持续增加是否 预防成年小鼠结肠炎所必需的。我们将使用中和抗体和抑制剂来阻断TGFβ 功能与诱导和自发发展的结肠炎小鼠。我们还将确定是否持续 TGFβ产生的增加增强了肠中TGFAP的诱导和保护性上皮反应， 预防成年期结肠炎。这一目的将在CD 4 + T细胞中TGFβ受体II缺失的小鼠中进行测试。 细胞和IEC中Smad 4缺失。总之，我们提出的研究将阐明一种新的机制， p40对预防结肠炎的长期作用的潜在后果，并为 开发p40早期干预作为预防成人IBD的新疗法。