Introduction: Enterobacter Sakazakii (ES) is a rare and virulent pathogen that has been associated with outbreaks of necrotizing enterocolitis (NEC) in neonates. Our previous studies have shown that ES expressing outer membrane protein A (OmpA) induces NEC in neonatal rats by triggering enterocyte apoptosis. Intestinal dendritic cells (DCs) play a key role in luminal antigen sampling and alert other leukocytes in the lamina propria to the presence of pathogenic bacteria. However the role of DCs and their interaction with bacteria has not been explored in NEC. In this study, we investigate the role of DCs in ES-induced disruption of the intestinal epithelial barrier; the first step in the pathogenesis of NEC.

Methods: To mimic the presence of DCs at the basement membrane of the intestine, double layers of CaCo2 cells and bone marrow-derived DCs (BMDCs) were developed using trans-wells. CaCo2 cells were seeded in the upper side of the trans well chamber while BMDCs were seeded at the bottom of the porous membrane and cultured for 24 hours. In addition, BMDCs were cultured for 24 hours with OmpA+ ES or OmpA- ES, and the supernatants were collected. The cytokine levels in these supernatants were measured using ELISA. The permeability of the double layer after treatment with OmpA+ or OmpA- ES was measured using trans-epithelial electrical resistance (TEER) as well as HRP leakage at one-hour time intervals up to 4 hours. These measurements were compared to the permeability of CaCo2 monolayers treated only with the bacteria as well as the monolayers pretreated with BMDC/ES co cultures supernatants and subsequently treated with ES.

Results: Presence of BMDCs in the double layer model exacerbates the effects of ES on barrier permeability as demonstrated by a faster decrease in the TEERs as well as HRP leakage (Figures 1, 2). Similarly the decrease in TEER is greater when the monolayers are pretreated with OmpA+, but not OmpA- DC/ES co-culture supernatants. Absence of OmpA protein dampens the effect of ES on the double layers as well as pretreated monolayers. The bacterial lysate does not show the same effect on the monolayers as the co-culture supernatants. Interaction of ES with BMDCs suppresses the production of pro-inflammatory cytokines (TNF? and IL-6), but increases the levels of anti-inflammatory cytokines (IL-10 and TGF-?). Preliminary data show that addition of anti-TGF-ß Antibody in the co-cultures protects barrier function.

Conclusions: Interaction of ES with DCs causes disruption of the epithelial barrier by inducing cytokine production, which exacerbates the damage to the tight junctions caused by ES. Presence of OmpA is necessary for the deleterious effects of ES on intestinal epithelial barrier permeability.