目的:坏死性小肠结肠炎(NEC)是一种威胁生命的疾病,主要影响早产儿的回肠。肠上皮细胞(IEC)凋亡参与了NEC的发病机制。然而,分散的隐窝IEC凋亡如何导致NEC和过度的绒毛上皮坏死仍不清楚。
方法:一种新型的三转基因小鼠模型,即,3xTg-iAPcIEC(隐窝-IEC中的诱导型凋亡表型),开发了使用多西环素(Dox)可诱导的tetO-rtTA系统和villin-cre技术诱导Fasl转基因的IECs特异性过表达。用单剂量的Dox皮下(s.c.)攻击三天大的新生3xTg-iAPcIEC小鼠及其同窝对照。在不同时间点处理肠组织以检查分散的隐窝IEC凋亡介导的NEC发育。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用于研究机制。
结果:用Dox治疗3xTg-iAPcIEC小鼠幼崽可诱导分散的隐窝IEC凋亡,随后是隐窝炎症和过度的绒毛坏死,类似于NEC。这种进展与Ifng升高相关,Rip3,CD8+T细胞,和回肠中的革兰氏阳性菌。机械上,IFN-γ和RIP3激活的信号介导零散隐窝IEC凋亡对肠道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细菌菌群失调的病理生理事件主要导致过度绒毛炎症和坏死。值得注意的是,阻止任何这些事件可以防止3xTg-iAPcIEC小鼠幼崽的NEC发展,强调它们在NEC发病机制中的核心作用。
结论:分散的隐窝IEC凋亡通过IFN-γ诱导小鼠幼崽的NEC,RIP3,CD8+T细胞,和革兰氏阳性菌介导的综合病理生理事件。我们的发现可能会提高NEC预防和治疗的知识。
OBJECTIVE: 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.