G. J. Ares1,3, C. Yuan3, D. Wood3, C. J. Hunter2,3 1University Of Illinois At Chicago,Department Of Surgery,Chicago, IL, USA 2Ann & Robert H. Lurie Children’s Hospital Of Chicago,Division Of Pediatric Surgery,Chicago, IL, USA 3Northwestern University,Department Of Pediatrics,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. Despite decades of research, its pathophysiology remains poorly understood. Enteroids are 3-dimensional epithelial organoids derived from intestinal stem cells (ISC). These novel structures allow for the study of complex physiologic interactions, with closer resemblance to the gut microenvironment than single cell tissue culture. Tight junctions (TJ) are paracellular protein complexes essential in regulating intestinal barrier structure. Claudin 2 is a pore forming TJ protein that regulates permeability. We hypothesized that Claudin 2 will be overexpressed in human enteroids exposed to lipopolysaccharide (LPS)-induced experimental NEC.
Methods:
After IRB approval, human intestinal fragments were obtained from patients undergoing bowel resection for NEC vs other conditions (controls). ISC were harvested by isolation of intestinal crypts and incubation in Matrigel with human ISC culture media for 5 days. Enteroids were exposed to LPS for 24 hours in an in vitro model of experimental NEC and compared to untreated controls. Claudin 2 was analyzed by immunofluorescence mean fluorescent intensity (MFI) in human tissue samples as well as in enteroids. Data was analyzed with student’s T-test.
Results:
Immunofluorescent microscopy (IF) demonstrated co-localization of Claudin 2 and actin in both control and +LPS enteroid groups at the expected, intercellular portion of the cell membrane. An increase of Claudin 2 expression was identified in +LPS enteroids vs controls (MFI=17677±1672 vs 7664±831 (p<0.0001), respectively). Likewise, there as increased Claudin 2 expression in humans with NEC vs controls (MFI=687±38 vs 1525±126 (p<0.0001)). In both humans and enteroids, IF revealed internalization of Claudin 2 from the cell membrane (controls) to that of a spiculated intracellular pattern in human and experimental NEC (+LPS) (Figure 1).
Conclusion:
In conclusion, human ISC-derived LPS-treated enteroids are a novel in vitro model of NEC. Both an increase in Claudin 2 expression and internalization are found in LPS-treated enteroids as well as in humans with NEC. The change in this pore-forming TJ protein may be responsible for the increased intestinal permeability seen in NEC. Further research in TJ proteins and enteroid models may delineate the pathophysiology of NEC that leads to the breakdown of barrier integrity and identify therapeutic targets.
