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

项目总结 婴儿和儿童的微生物失调与炎症性肠易感性增加有关 成人疾病(IBD)。然而，肠道微生物群在早期生命中定植的机制 整个生命周期的健康结果仍不清楚。来自前一个资助期的研究孤立和 克隆了鼠李糖乳杆菌GG(LGG)来源的分泌蛋白p40，并证明了新生儿 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Setd1β基因的表达，该基因编码一个 催化组蛋白3-赖氨酸4(H3K4me1/3)单甲基化和三甲基化的甲基转移酶。我们确认了 转化生长因子β作为p40上调SET-1β的潜在靶点IEC快速且持续更新 从肠道干细胞(ISCs)再生。我们在早期发现p40在ISCs中调节H3K4m31/3， 和p40治疗对新生小鼠，而不是成年小鼠，刺激转化生长因子β基因表达持续增加。 IECS。转化生长因子β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IEC中Setd1β基因的表达。补充： 早期生命中的p40刺激胰岛素样干细胞转化生长因子ββ位点的setd1依赖的H3K4me1/3沉积，这是 由IECS继承，以使转化生长因子β产量持续增加，并随后防止 成年期的肠炎。在目标1中，我们将确定p40刺激的setd1β基因 转化生长因子β基因在血管内皮细胞中的表达增加需要表达和H3K4me1/3，并阐明 P40和MGA：MAX二聚体之间的相互作用是否介导了Setd1β产量的上调。在……里面 目的2，我们将确定早期生命中p40暴露的时间窗，它导致了 转化生长因子β在血管内皮细胞中的表达，并确定p40是否调节血管内皮细胞转化生长因子β基因表达的持续增加 IECS早期需要增加ISCs中Setd1β基因的表达。人和小鼠的肠类和 克隆和2D培养，沉默β或MGA基因的细胞系，以及构成的小鼠模型 而诱导型Setd1β基因在IECs或ISCs中的敲除将被用来达到这两个目的。在《目标3》中，我们将 确定早期补充p40后转化生长因子β的持续增加是否 预防成年小鼠结肠炎所需。我们将使用中和抗体和抑制剂来阻断转化生长因子β 对诱发和自发发展的结肠炎小鼠的作用。我们还将确定是否持续 转化生长因子β产量的增加增强肠道Tregs的诱导和保护上皮细胞的反应 成人结肠炎的预防。这一目的将在CD4+T细胞β受体II缺失的小鼠身上进行测试 IECS中的细胞和Smad4缺失。总之，我们提出的研究将阐明一种新的机制。 深入了解p40对预防结肠炎的长期影响的后果，并为 发展p40早期干预作为预防成人IBD的新疗法。

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.