G. J. Ares1,2, D. R. Wood1, C. Y. Yuan1, C. J. Hunter1 1Northwestern University,Pediatrics,Chicago, IL, USA 2University Of Illinois At Chicago,General Surgery,Chicago, IL, USA
Introduction:
Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in neonates, affecting 5-10% of patients in the neonatal intensive care unit (NICU). NEC is mostly seen in premature infants, but its pathophysiology remains unknown. Tight junction (TJ) proteins are paracellular complexes in intestinal villi important in maintenance of protective intestinal barrier. Claudin-2 (C2) is an intercellular pore forming TJ protein that regulates permeability. We hypothesize that Claudin-2 expression levels are upregulated in human NEC, as well as in an experimental model of bacteria-induced NEC.
Methods:
Caco-2 cells were exposed to LPS in an in vitro model of NEC in a 10 day time course experiment with samples taken on days 0, 1, 3, 5, 7, and 10 along with untreated controls per day. An in vivo model of rat pups subjected to hypoxia twice per day and fed clean formula (control) vs bacteria + formula (NEC). Cells were fragmented into cytosol, membrane, nucleus, and cytoskeleton compartments. C2 was analyzed by immunofluorescence mean fluorescent intensity (MFI), western blot, and quantitative PCR in Caco-2 cells, rat intestinal segments, and human intestines from patients with NEC vs humans without NEC requiring bowel resection. Western blots were standardized to loading controls. Data was analyzed with student’s T-test.
Results:
Staining showed increased expression of C2 in humans with NEC vs control (MFI=710±24 vs 1530±98 (p<0.0005), respectively) and in rats with experimental NEC vs control (MFI= 514±22 vs 706±21 (p<0.0005), respectively). There appears to be a change in localization of C2 from cytosol to membrane in IF for Caco-2 cells. Western blot and qPCR confirmed 2-fold increase in C2 expression in the cellular NEC model vs control over time, with an increased proportion of C2 in the membrane compartment. In rats, western blot showed increased C2 expression in NEC vs control, and a greater than 2-fold increase on qPCR (p<0.001). Figure 1 shows western blot for C2 in Caco-2 cells exposed to LPS (L) vs control group (C) and the measured immunoblot density of LPS group relative to Control per day.
Conclusion:
In conclusion, human cells and experimental cellular and rat models both showed increased expression of Claudin-2 TJ protein in the intestinal epithelium with NEC. The change in expression of this pore forming protein may play an important role in the breakdown of intestinal barrier integrity in NEC. Changes seen in localization may elucidate the mechanism by which this breakdown occurs. Further research in TJ proteins could help define the pathophysiology of NEC and provide targets for therapy.
