ORIGINAL RESEARCH PAPER NOD2 induces autophagy to control AIEC bacteria infectiveness in intestinal epithelial cells Anna Negroni 1 • Eleonora Colantoni 2 • Roberta Vitali 1 • Francesca Palone 2 • Maria Pierdomenico 2 • Manuela Costanzo 2 • Vincenzo Cesi 1 • Salvatore Cucchiara 2 • Laura Stronati 3 Received: 10 November 2015 / Revised: 13 May 2016 / Accepted: 16 June 2016 Ó Springer International Publishing 2016 Abstract Objective The importance of autophagy in mechanisms underlying inflammation has been highlighted. Down- stream 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 pedi- atric patients with inflammatory bowel disease (IBD). Methods A human epithelial colorectal adenocarcinoma (Caco2) cell line stably over-expressing NOD2 was pro- duced (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 TNFa, IL-8 and IL-1bmRNA 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. Results 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. Conclusions The NOD2-mediated autophagy induction is crucial to hold the intramucosal bacterial burden, espe- cially towards AIEC, and to limit the resulting inflammatory response. Autophagy is active in inflamed colonic tissues of IBD pediatric patients. Keywords Autophagy Á NOD2 Á AIEC Á Inflammatory bowel disease Á Inflammation Introduction Autophagy represents an evolutionarily highly conserved multi-step process of cellular self-digestion due to sequestration of excessive, damaged, or aged proteins and intracellular organelles in double-membranous vesicles of autophagosomes, terminally self-digested in lysosomes [1]. Autophagy is deeply involved in the regulation of cell development, differentiation, survival and senescence [2]. Moreover, it also affects the innate immunity, by inducing the inflammatory signaling leading to the removal of intracellular bacteria such as Salmonella typhimurium, Listeria monocytogenes, and Mycobacterium tuberculosis [2, 3]. Autophagy is mainly activated by sensors of the innate immunity, as pattern recognition receptor signal- ing [1, 4]. In particular nucleotide-oligomerization- domain-2 (NOD2), the cytoplasmic bacterial sensor, with Responsible Editor: John Di Battista. & Laura Stronati laura.stronati@uniroma1.it 1 Department of Radiation Biology and Human Health, ENEA, Rome, Italy 2 Department of Pediatrics and Infantile Neuropsychiatry, Pediatric Gastroenterology and Liver Unit, Sapienza University of Rome, Rome, Italy 3 Department of Cellular Biotechnology and Hematology, Sapienza University of Rome, Rome, Italy Inflamm. Res. DOI 10.1007/s00011-016-0964-8 Inflammation Research 123