O2173: QSOX1 Expression is Decreased in Necrotizing Enterocolitis: A Potential Mechanism for Disrupted Barrier Function in Premature Infants with Necrotizing Enterocolitis

Background: Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality for premature infants, in part, from intestinal barrier dysfunction. This barrier contains a viscous mucous layer that protects from pathogens and toxins. Mucous is comprised of mucin, a glycoprotein secreted by goblet cells with sialic acid glycans added by sialyltransferases (STs). QSOX1 has been implicated in crosslinking the STs that add sialic acid glycans and thus plays an essential role in barrier function. To date, the role of QSOX1 in NEC is unknown. We hypothesized that QSOX1 expression would be diminished in NEC which may contribute to its pathogenesis due to an abnormal mucous layer in the premature gut.

Methods: NEC was induced in neonatal rat pups for 4 days via formula feeding, hypoxia, and oral lipopolysaccharide. Breastfed (BF) pups were used as healthy controls. Alcian blue-PAS (AB-PAS) histological stain was performed to identify changes in mucin production and goblet cells in NEC (n=4). Ileal tissue (n=3) was immunostained for QSOX1 and Trefoil Factor 3 (TFF3; goblet cell marker) to quantify differences in QSOX1 expression and to show colocalization with TFF3+ goblet cells. To assess the function of QSOX1, we assessed precursors to sialylated glycans with wheat germ agglutin (WGA) lectin that binds N-acetylgalactosamine and end products of QSOX1 with sambucus nigra agglutinin (SNA) lectin for sialic acid (n=3). If QSOX1 is functioning, SNA will be more prevalent with low WGA staining, while WGA will be increased with low SNA staining with compromised QSOX1 function.

Results: By AB-PAS staining, the number of goblet cells was unchanged in NEC compared to BF pups. However, goblet cell distribution was shifted towards the crypt instead of the villus. QSOX1 was identified in both NEC and BF pups and colocalized with TFF3 goblet cells (Figure 1). NEC pups had significantly fewer QSOX1 positive cells than BF pups (3.44±1.6 vs. 33.0±5.5 cells/high power field [HPF]; p< 0.05). SNA was decreased (4.9±1.3 vs. 21.9±3.2 cells/HPF; p< 0.05) and WGA increased (26.8±8.3 vs. 5.8±1.8 cells/HPF; p< 0.05) in NEC compared to BF pups (Figure 2).

Conclusion: QSOX1, a critical component of proper synthesis of mucin and thus the intestinal barrier, is reduced in experimental NEC. QSOX1 colocalizes with TFF3+ goblet cells in BF and NEC. The distribution of goblet cells was altered in NEC suggesting altered mucin production and maturation of the protective mucous layer. The decrease of sialic acid in NEC suggests a decrease in mature, fully functional, mucin in NEC. While further study is needed, decreased QSOX1 expression, and thus ST function and proper sialyation of mucin, may contribute to impaired mucosal function contributing to NEC disease. Future work will expand on this hypothesis and investigate the ontogeny of QSOX1 expression in embryonic gut development.