The nitric oxide synthase 2 pathway is targeted by both pro- and anti-inflammatory treatments in the immature human intestine.
OBJECTIVE
NO synthase 2 (NOS2) was recently identified as one the most overexpressed genes in intestinal samples of premature infants with necrotizing enterocolitis (NEC). NOS2 is widely implicated in the processes of epithelial cell injury/apoptosis and host immune defense but its specific role in inflammation of the immature human intestinal mucosa remains unclear. Interestingly, factors that prevent NEC such as epidermal growth factor (EGF) attenuate the inflammatory response in the mid-gestation human small intestine using serum-free organ culture while drugs that are associated with NEC occurrence such as the non-steroidal anti-inflammatory drug, indomethacin (INDO), exert multiple detrimental effects on the immature human intestine. In this study we investigate the potential role of NOS2 in modulating the gut inflammatory response under protective and stressful conditions by determining the expression profile of NOS2 and its downstream pathways in the immature intestine.


METHODS
Gene expression profiles of cultured mid-gestation human intestinal explants were investigated in the absence or presence of a physiological concentration of EGF (50 ng/ml) or 1 μM INDO for 48 h using Illumina whole genome microarrays, Ingenuity Pathway Analysis software and quantitative PCR to investigate the expression of NOS2 and NOS2-pathway related genes.


RESULTS
In the immature intestine, NOS2 expression was found to be increased by EGF and repressed by INDO. Bioinformatic analysis identified differentially regulated pathways where NOS2 is known to play an important role including citrulline/arginine metabolism, epithelial cell junctions and oxidative stress. At the individual gene level, we identified many differentially expressed genes of the citrulline/arginine metabolism pathway such as ARG1, ARG2, GLS, OAT and OTC in response to EGF and INDO. Gene expression of tight junction components such as CLDN1, CLDN2, CLDN7 and OCN and of antioxidant markers such as DUOX2, GPX2, SOD2 were also found to be differentially modulated by EGF and INDO.


CONCLUSIONS
These results suggest that the protective effect of EGF and the deleterious influence of INDO on the immature intestine could be mediated via regulation of NOS2. Pathways downstream of NOS2 involved with these effects include metabolism linked to NO production, epithelial barrier permeability and antioxidant expression. These results suggest that NOS2 is a likely regulator of the inflammatory response in the immature human gut and may provide a mechanistic basis for the protective effect of EGF and the deleterious effects of INDO.
Research Consortium on Child Intestinal Inflammation, Division of Neonatology, Department of Pediatrics, University of Ottawa, Ottawa, Canada.
Emanuela
E
Ferretti
Research Consortium on Child Intestinal Inflammation, Department of Anatomy and Cell Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Canada.
Eric
E
Tremblay
Research Consortium on Child Intestinal Inflammation, Department of Anatomy and Cell Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Canada.
Marie-Pier
MP
Thibault
Research Consortium on Child Intestinal Inflammation, Department of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, Canada.
David
D
Grynspan
Research Consortium on Child Intestinal Inflammation, Division of Gastroenterology, Hepatology and Nutrition, Hospital for Sick Children and Department of Pediatrics, University of Toronto, Toronto, Canada.
Karolina M
KM
Burghardt
Research Consortium on Child Intestinal Inflammation, Department of Surgery, University of Ottawa, Ottawa, Canada.
Marcos
M
Bettolli
Research Consortium on Child Intestinal Inflammation, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Canada.
Corentin
C
Babakissa
Research Consortium on Child Intestinal Inflammation, Department of Nutrition, CHU Sainte-Justine, Université de Montréal, Montréal, Canada.
Emile
E
Levy
Research Consortium on Child Intestinal Inflammation, Department of Anatomy and Cell Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Canada. Electronic address: jean-francois.beaulieu@usherbrooke.ca.
Jean-François
JF
Beaulieu
D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D001120:Arginine / Q000378:metabolism; D002956:Citrulline / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D033042:Fetal Research; D005765:Gastrointestinal Agents / Q000494:pharmacology; D006801:Humans; D007082:Ileum / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D007213:Indomethacin / Q000494:pharmacology; D052247:Nitric Oxide Synthase Type II / Q000032:analysis / Q000378:metabolism*; D009924:Organ Culture Techniques; D015398:Signal Transduction / Q000187:drug effects*
D000893:Anti-Inflammatory Agents; D005765:Gastrointestinal Agents; D002956:Citrulline; D004815:Epidermal Growth Factor; D001120:Arginine; D052247:Nitric Oxide Synthase Type II; D007213:Indomethacin
Anti-oxidant; Epithelial barrier; Epithelial cells; Inflammation; Intestinal mucosa; NO synthase 2