Data_Sheet_1_Rapid Gut Adaptation to Preterm Birth Involves Feeding-Related DNA Methylation Reprogramming of Intestinal Genes in Pigs.xlsx

Following preterm birth, the immature gut function and immunology must rapidly adapt to cope with bacterial colonization and enteral milk feeding. We hypothesized that intestinal epigenetic changes are involved in the gut response to preterm birth and the first feeding. Using piglets as models for infants, preterm, and term pigs were fed total parenteral nutrition (TPN) or partial enteral feeding for 5 days, followed by exclusive enteral feeding with bovine milk until day 26 (weaning age). Intestinal structure, function, microbiome, DNA methylome, and gene expressions were compared between preterm and term pigs on days 0, 5, and 26 (n = 8 in each group). At birth, the intestine of preterm pigs showed villus atrophy and global hypermethylation, affecting genes related to the Wnt signaling pathway. Hypermethylation-associated lowered expression of lipopolysaccharide-binding protein and genes related to the Toll-like receptor 4 pathway were evident during the first 5 days of life, but most early methylation differences disappeared by day 26. Regardless, sucrase and maltase activities (adult-type brush border enzymes) remained reduced, and the gut microbiota altered (fewer Akkermansia, more Lachnoclostridia and Lactobacilli) until day 26 in preterm pigs. During the 0- to 5-day period, many new preterm–term methylation differences appeared, but mainly when no enteral feed was provided (TPN feeding). These methylation differences affected intestinal genes related to cell metabolism, including increased GCK (glucokinase) expression via promoter hypomethylation. In conclusion, the immature intestine has a remarkable capacity to adapt its gene methylation and expression after preterm birth, and only few preterm-related defects persisted until weaning. Early enteral feeding may be important to stimulate the methylation reprogramming of intestinal genes, allowing rapid intestinal adaptation to preterm birth.

早产发生后，未成熟的肠道功能与免疫系统必须快速适应，以应对细菌定植与肠内乳类喂养。本研究假设肠道表观遗传变化参与了肠道对早产及首次喂养的应答反应。
本研究以仔猪作为婴儿的模型动物，将早产仔猪与足月仔猪分别给予全肠外营养（total parenteral nutrition, TPN）或部分肠内喂养，持续5天，随后采用牛乳进行完全肠内喂养，直至第26天（断奶日龄）。分别在第0、5、26天对早产与足月仔猪的肠道结构、肠道功能、肠道微生物组、DNA甲基化组以及基因表达进行比较（每组n=8）。
出生时，早产仔猪的肠道表现出绒毛萎缩与全基因组高甲基化，该表观遗传变化影响了与Wnt信号通路相关的基因。在出生后的前5天，可观察到高甲基化介导的脂多糖结合蛋白以及与Toll样受体4通路相关基因的表达下调，但多数早期甲基化差异在第26天时消失。尽管如此，早产仔猪的蔗糖酶与麦芽糖酶（成年型刷状缘酶）活性仍处于较低水平，且肠道微生物组发生改变：阿克曼菌属丰度降低，拉氏梭菌类与乳杆菌属丰度升高，该状态持续至第26天。
在出生后0至5天期间，出现了大量新的早产与足月仔猪间的甲基化差异，但此类差异主要出现在未给予肠内喂养（即全肠外营养喂养）的组别中。这些甲基化差异影响了与细胞代谢相关的肠道基因，包括通过启动子低甲基化上调葡萄糖激酶（glucokinase, GCK）的表达。
综上，未成熟肠道在早产出生后具备显著的基因甲基化与表达重编程能力，仅少数早产相关缺陷持续至断奶阶段。早期肠内喂养可能对刺激肠道基因的甲基化重编程至关重要，从而帮助肠道快速适应早产环境。