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 婴儿和儿童的营养不良与炎症性肠的易感性增加有关 成人疾病（IBD）。但是，早期肠道菌群殖民化的机制赋予了 整个生命周期的健康结果尚不清楚。前一个资金期的研究孤立， 克隆了rhamnosus lamnosus gg（LGG）衍生的秘密蛋白P40的克隆，并证明了新生儿 补充P40可防止成年小鼠的结肠炎。我们的初步研究发现P40与 MGA和MAX的两个转录因子，用于调节SetD1β基因的表达，该基因编码A 甲基转移酶用于催化单声道和组蛋白3-赖氨酸4（H3K4ME1/3）的三甲基化。我们确定了 TGFβ是P40-UP调节的SETD1β的潜在靶标。 IEC迅速更新和连续 从肠道干细胞（ISC）再生。我们发现P40在早期ISC中调节H3K4M31/3， 在新生儿（而不是成年小鼠）中的P40治疗刺激TGFβ基因表达持续增加 IEC。 TGFβ已显示具有针对炎症的多个功能。那我们假设p40 通过增加MGA：最大二聚化来上调IEC中的SETD1β基因表达。补充 早期生命中的p40刺激了ISC中TGFβ基因座的SETD1β依赖性H3K4ME1/3沉积， IEC遗传以使TGFβ产生的持续增加以及随后的预防 成年时肠炎。在AIM 1中，我们将确定P40刺激的SETD1β基因是否 需要表达和H3K4ME1/3才能驱动IEC中表达TGFβ基因的增加并阐明 P40与MGA：最大二聚体之间的相互作用是否介导SETD1β产生的上调。在 AIM 2，我们将确定早期生命中P40暴露的临时窗口，这会导致持续增加 IEC中的TGFβ产生，并确定P40调节的TGFβ基因表达是否持续增加 IEC需要在早期生命中增加setD1β基因表达。人类和小鼠肠内 结石和2D培养物和带有沉默的SETD1β或MGA基因的细胞系，以及一致性的小鼠模型 这两个目标将使用诱导的setD1β基因敲低IEC或ISC。在AIM 3中，我们将 确定早期补充p40后TGFβ产生的持续增加是否为 需要预防成年小鼠结肠炎。我们将使用中和抗体和抑制剂来阻断TGFβ 在具有诱导和赞助的结肠炎的小鼠中功能。我们还将确定是否持续 TGFβ产生的增加增强了肠道的Treg诱导和受保护的上皮反应的诱导 预防成年后结肠炎。该目标将在CD4+ T中的TGFβ受体II缺失的小鼠中测试 IEC中的细胞和SMAD4删除。我们提出的研究将共同​​阐明一种新的机制 p40对预防结肠炎的持久影响的后果是基础的，并为 用P40进行早期干预，作为一种新型疗法，用于预防成人IBD。