Saravanan Subramanian , Heng-Fu Bu , Pauline M. Chou , Xiao Wang , Hua Geng , Suhail Akhtar , Chao Du , Stephanie C. Tan , Justin Eze Ideozu , Aasrita Tulluri , Yuxiang Sun , Wen-Xing Ding , Isabelle G. De Plaen , Xiao-Di Tan
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The 3-days-old neonatal 3xTg-iAP<sup>cIEC</sup> mice and their littermate controls were subcutaneously (s.c.) challenged with a single dose of Dox. Intestinal tissues were processed at different time points to examine scattered crypt IEC apoptosis-mediated NEC development. Gene knockout technology, antibody-mediated cell depletion, and antibiotic-facilitated Gram-positive bacteria depletion were used to study mechanisms.</p></div><div><h3>Results</h3><p>Treatment of 3xTg-iAP<sup>cIEC</sup> mouse pups with Dox induces scattered crypt IEC apoptosis followed by crypt inflammation and excessive villous necrosis resembling NEC. This progression correlated with elevated <em>Ifng</em>, <em>Rip3</em>, CD8<sup>+</sup> T cells, and Gram-positive bacteria in the ileum. Mechanistically, IFN-γ and RIP3-activated signals mediate the effect of scattered crypt IEC apoptosis on the induction of intestinal crypt inflammation and villous necrosis. Meanwhile, pathophysiological events of CD8<sup>+</sup> T cell infiltration and dysbiosis with Gram-positive bacteria primarily contribute to excessive villous inflammation and necrosis. Notably, blocking any of these events protects against NEC development in 3xTg-iAP<sup>cIEC</sup> mouse pups, underlining their central roles in NEC pathogenesis.</p></div><div><h3>Conclusions</h3><p>Scattered crypt IEC apoptosis induces NEC in mouse pups via IFN-γ, RIP3, CD8<sup>+</sup> T cells, and Gram-positive bacteria-mediated comprehensive pathophysiological events. 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引用次数: 0
摘要
背景与目的：坏死性小肠结肠炎（NEC）是一种危及生命的疾病，主要影响早产儿的回肠。肠上皮细胞（IEC）凋亡是 NEC 的发病机制之一。然而，分散的隐窝 IEC 细胞凋亡如何导致 NEC 以及绒毛上皮过度坏死仍不清楚：方法：利用多西环素（Dox）诱导的 tetO-rtTA 系统和绒毛-cre 技术，诱导 IEC 特异性过表达 Fasl 转基因，建立了一种新型三转基因小鼠模型，即 3xTg-iAPcIEC（隐窝 IEC 诱导性凋亡表型）。对出生三天的新生 3xTg-iAPcIEC 小鼠及其同窝对照小鼠皮下注射单剂量 Dox。在不同的时间点处理肠组织，以检测散在隐窝 IEC 凋亡介导的 NEC 发生。基因敲除技术、抗体介导的细胞耗竭和抗生素促进的革兰氏阳性菌耗竭被用于研究机制：结果：用 Dox 处理 3xTg-iAPcIEC 小鼠幼崽会诱发零星的隐窝 IEC 凋亡，随后出现隐窝炎症和类似 NEC 的绒毛过度坏死。这种进展与回肠中 Ifng、Rip3、CD8+ T 细胞和革兰氏阳性细菌的升高有关。从机制上讲，IFN-γ 和 RIP3 激活的信号介导了分散的隐窝 IEC 凋亡对肠隐窝炎症和绒毛坏死的诱导作用。同时，CD8+ T 细胞浸润和革兰氏阳性菌菌群失调等病理生理事件也是导致绒毛过度炎症和坏死的主要原因。值得注意的是，阻断这些事件中的任何一个都能防止 3xTg-iAPcIEC 小鼠幼崽发生 NEC，从而强调了它们在 NEC 发病机制中的核心作用：散发性隐窝 IEC 细胞凋亡通过 IFN-γ、RIP3、CD8+ T 细胞和革兰氏阳性细菌介导的综合病理生理事件诱导小鼠幼崽发生 NEC。我们的发现可能会促进对 NEC 的预防和治疗。
Necrotizing enterocolitis (NEC) is a life-threatening disease affecting mostly the ileum of preemies. Intestinal epithelial cell (IEC) apoptosis contributes to NEC pathogenesis. However, how scattered crypt IEC apoptosis leads to NEC with excessive villus epithelial necrosis remains unclear.
Methods
A novel triple-transgenic mouse model, namely, 3xTg-iAPcIEC (inducible apoptosis phenotype in crypt-IEC), was developed to induce IEC-specific overexpression of Fasl transgene using doxycycline (Dox)-inducible tetO-rtTA system and villin-cre technology. The 3-days-old neonatal 3xTg-iAPcIEC mice and their littermate controls were subcutaneously (s.c.) challenged with a single dose of Dox. Intestinal tissues were processed at different time points to examine scattered crypt IEC apoptosis-mediated NEC development. Gene knockout technology, antibody-mediated cell depletion, and antibiotic-facilitated Gram-positive bacteria depletion were used to study mechanisms.
Results
Treatment of 3xTg-iAPcIEC mouse pups with Dox induces scattered crypt IEC apoptosis followed by crypt inflammation and excessive villous necrosis resembling NEC. This progression correlated with elevated Ifng, Rip3, CD8+ T cells, and Gram-positive bacteria in the ileum. Mechanistically, IFN-γ and RIP3-activated signals mediate the effect of scattered crypt IEC apoptosis on the induction of intestinal crypt inflammation and villous necrosis. Meanwhile, pathophysiological events of CD8+ T cell infiltration and dysbiosis with Gram-positive bacteria primarily contribute to excessive villous inflammation and necrosis. Notably, blocking any of these events protects against NEC development in 3xTg-iAPcIEC mouse pups, underlining their central roles in NEC pathogenesis.
Conclusions
Scattered crypt IEC apoptosis induces NEC in mouse pups via IFN-γ, RIP3, CD8+ T cells, and Gram-positive bacteria-mediated comprehensive pathophysiological events. Our findings may advance knowledge in the prevention and treatment of NEC.
期刊介绍：
"Cell and Molecular Gastroenterology and Hepatology (CMGH)" is a journal dedicated to advancing the understanding of digestive biology through impactful research that spans the spectrum of normal gastrointestinal, hepatic, and pancreatic functions, as well as their pathologies. The journal's mission is to publish high-quality, hypothesis-driven studies that offer mechanistic novelty and are methodologically robust, covering a wide range of themes in gastroenterology, hepatology, and pancreatology.
CMGH reports on the latest scientific advances in cell biology, immunology, physiology, microbiology, genetics, and neurobiology related to gastrointestinal, hepatobiliary, and pancreatic health and disease. The research published in CMGH is designed to address significant questions in the field, utilizing a variety of experimental approaches, including in vitro models, patient-derived tissues or cells, and animal models. This multifaceted approach enables the journal to contribute to both fundamental discoveries and their translation into clinical applications, ultimately aiming to improve patient care and treatment outcomes in digestive health.
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