02.04 Inflammatory Gatekeeper A20 Regulates Intestinal Motility in Necrotizing Enterocolitis in Mice.

J. W. Duess¹, D. Scheese¹, K. Tsuboi¹, Z. Raouf¹, M. Sampah¹, C. Tragesser¹, D. Klerk¹, H. Moore¹, S. Williams-McLeod¹, W. B. Fulton¹, T. Prindle¹, S. Wang¹, M. Wang¹, P. Lu¹, C. P. Sodhi¹, D. J. Hackam^{1  1}Johns Hopkins University School Of Medicine, Pediatric Surgery, Baltimore, MD, USA

Introduction: We have previously shown that impaired gastrointestinal dysmotility is a predisposing factor to the development of necrotizing enterocolitis (NEC), explaining in part the early ileus seen in patients with this disease. Importantly, the pathogenesis of impaired motility in NEC remains unknown, impaired signaling in the enteric glia was determined to play a role. The inflammatory gatekeeper A20 has been shown to dampen inflammation in a variety of cells, suggesting a possible role in the enteric glia. We now hypothesize that A20 exerts an inflammatory gatekeeper role in the enteric glia where it could regulate the development of ileus in the pathogenesis of NEC. 

Methods: We first generated mice lacking A20 in the enteric glia by crossing A20^loxp mice with Sox10^creERT mice, where Sox10^cre is activated in the enteric glia at the time of tamoxifen injection. Acute abdominal inflammation was induced at 3-weeks of age in C57Bl/6 and A20^?Sox10 mice by intra-peritoneal injection with gram negative bacterial lipopolysaccharide (LPS, 1mg/kg, 6h). A20 expression in the intestinal mucosa in wildtype mice was assessed by qRT-PCR. NEC was induced in newborn C57Bl/6 and A20^?Sox10 mice through four days of formular gavage with stool from an infant with severe NEC and intermittent hypoxia. Intestinal motility was measured by oral administration of fluorescent tracer FITC-dextran/30-min before sacrifice. Then, the serial sections from stomach to colon were divided into 1-cm sections to measure motility as a function of the Geometric center (GC). The terminal ileum was harvested to measure pro-inflammatory cytokine (TNF) expression and intestinal histology.  

Results: A20 was successfully depleted from the enteric glia by immunostaining and RT-PCR. A20^?Sox10 mice exposed to LPS (1mg/kg) had an extremely distended intestine, severe diarrhea, and lethal mortality. LPS treatment A20^?Sox10 mice induced severe intestinal dysmotility (p<0.01) as manifest by impaired passage of dye. Abdominal inflammation by LPS injection resulted in significant sepsis in A20^?Sox10 mice as revealed by extreme lethargy and high levels of the proinflammatory mediator Tnf in the intestinal mucosa vs. wild-type mice (p<0.001). A20 expression in the intestinal mucosa was significantly reduced, suggesting a loss of A20 may precede NEC development. Strikingly, and in support of this possibility, mice lacking A20 from Sox10 expressing cells developed severe dysmotility and NEC as revealed by significant mucosal injury, weight loss, and elevated expression of Tnf, confirming that A20 expression on the enteric glia regulates intestinal motility in the pathogenesis of NEC.

Conclusion: We now reveal that A20 regulates intestinal motility in the pathogenesis of NEC. These findings suggest that strategies to maintain A20 expression or signaling on the enteric glia may promote normal intestinal motility and reduce the incidence of NEC.