Department of Radiation Biology and Human Health, ENEA, Rome, Italy.
Department of Pediatrics and Infantile Neuropsychiatry, Pediatric Gastroenterology and Liver Unit, Sapienza University of Rome, Rome, Italy.
Inflamm Res. 2016 Oct;65(10):803-13. doi: 10.1007/s00011-016-0964-8. Epub 2016 Jun 22.
The importance of autophagy in mechanisms underlying inflammation has been highlighted. Downstream effects of the bacterial sensor NOD2 include autophagy induction. Recently, a relationship between defects in autophagy and adherent/invasive Escherichia coli (AIEC) persistence has emerged. The present study aims at investigating the interplay between autophagy, NOD2 and AIEC bacteria and assessing the expression level of autophagic proteins in intestinal biopsies of pediatric patients with inflammatory bowel disease (IBD).
A human epithelial colorectal adenocarcinoma (Caco2) cell line stably over-expressing NOD2 was produced (Caco2NOD2). ATG16L1, LC3 and NOD2 levels were analysed in the Caco2 cell line and Caco2NOD2 after exposure to AIEC strains, by western blot and immunofluorescence. AIEC survival inside cells and TNFα, IL-8 and IL-1βmRNA expression were analysed by gentamicin protection assay and real time PCR. ATG16L1 and LC3 expression was analyzed in the inflamed ileum and colon of 28 patients with Crohn's disease (CD), 14 with ulcerative colitis (UC) and 23 controls by western blot.
AIEC infection increased ATG16L1 and LC3 in Caco2 cells. Exposure to AIEC strains increased LC3 and ATG16L1 in Caco2 overexpressing NOD2, more than in Caco2 wild type, while a decrease of AIEC survival rate and cytokine expression was observed in the same cell line. LC3 expression was increased in the inflamed colon of CD and UC children.
The NOD2-mediated autophagy induction is crucial to hold the intramucosal bacterial burden, especially towards AIEC, and to limit the resulting inflammatory response. Autophagy is active in inflamed colonic tissues of IBD pediatric patients.
自噬在炎症机制中的重要性已得到强调。细菌传感器 NOD2 的下游效应包括自噬诱导。最近，自噬缺陷与黏附/侵袭性大肠杆菌（AIEC）持续存在之间的关系已经出现。本研究旨在研究自噬、NOD2 和 AIEC 细菌之间的相互作用，并评估炎症性肠病（IBD）儿科患者肠活检中自噬蛋白的表达水平。
生产了人结肠直肠腺癌细胞系（Caco2）稳定过表达 NOD2 的细胞系（Caco2NOD2）。通过 Western blot 和免疫荧光法分析 Caco2 细胞系和 Caco2NOD2 暴露于 AIEC 菌株后 ATG16L1、LC3 和 NOD2 的水平。通过庆大霉素保护试验和实时 PCR 分析 AIEC 细胞内存活和 TNFα、IL-8 和 IL-1βmRNA 的表达。通过 Western blot 分析 28 例克罗恩病（CD）、14 例溃疡性结肠炎（UC）和 23 例对照儿童的炎症性回肠和结肠中 ATG16L1 和 LC3 的表达。
AIEC 感染增加了 Caco2 细胞中的 ATG16L1 和 LC3。暴露于 AIEC 菌株增加了 Caco2 过表达 NOD2 中的 LC3 和 ATG16L1，比 Caco2 野生型更多，同时观察到 AIEC 存活率和细胞因子表达降低。CD 和 UC 儿童的炎症性结肠中 LC3 表达增加。
NOD2 介导的自噬诱导对于控制黏膜内细菌负荷至关重要，特别是针对 AIEC，并且可以限制由此产生的炎症反应。自噬在 IBD 儿科患者的炎症性结肠组织中活跃。
