D. F. Nino1, S. Sho3, C. P. Sodhi1, M. Good2, C. J. Luke2, G. A. Silverman2, D. J. Hackam1 1Johns Hopkins University School Of Medicine,Pediatric Surgery,Baltimore, MD, USA 2Children’s Hospital Of Pittsburgh Of UPMC,Newborn Medicine,Pittsburgh, PA, USA 3University Of California – Los Angeles,Surgery,Los Angeles, CA, USA
Introduction: Necrotizing enterocolitis (NEC) is the most frequent and lethal pathology that afflicts the gastrointestinal tract of premature infants. Our lab has previously demonstrated the critical role that the innate immune receptor for lipopolysaccharide (LPS), toll-like receptor 4 (TLR4), and subsequent exaggerated inflammatory signaling play in the pathogenesis of NEC and intestinal necrosis. Intracellular serpin B3 has been identified as a pro-survival factor with a novel role in necrosis inhibition. Given the characteristic necrosis observed in NEC, we hypothesized that TLR4 signaling in the gut plays a role in the regulation of intracellular serpin expression.
Methods: Under IRB approval, intestinal samples were obtained from patients diagnosed with NEC and evaluated for the expression of serpin B3 by qRT-PCR and immunohistochemistry (IHC). The role of TLR4 was determined using a model of endotoxemia (LPS 1mg/kg IP 3 h) or experimental NEC in neonatal (7 – 12 day old) C57Bl/6, TLR4 KO or mice lacking TLR4 in the intestinal epithelium (TLR4ΔIEC). NEC was induced by feeding formula supplemented with bacteria isolated from human NEC via gavage five times/day and exposure to hypoxia (5%O2, 95%N2) for 10 min in a hypoxic chamber twice daily for 4 days. TLR4 and serpin B3 deficient enterocytes (IEC-6 cells) were generated using lentivirus-TLR4-shRNA or lentivirus-serpin B3-shRNA, respectively. The role of serpin B3 in the inflammatory response of enterocytes challenged with LPS was evaluated by IL-6, iNOS and IL-1b mRNA expression determined by qRT-PCR.
Results: Human and murine NEC intestinal samples display a significantly increased expression of serpin B3 by mRNA (58 fold and 6 fold, respectively) and evidenced by IHC. These findings were determined to be TLR4-dependent, since serpin B3 expression was significantly lower in TLR4-deficient enterocytes compared to wild-type controls (0.16 vs. 8 fold relative to actin) when challenged with endotoxin. These results are further supported by the fact that endotoxemia leads to upregulation of serpin B3 in wild-type mice (2 fold) but have no effect on TLR4 deficient mice. Moreover, TLR4ΔIEC mice were protected from developing NEC and had decreased levels of serpin B3. Strikingly, when compared to wild-type IEC-6 cells exposed to LPS, serpin B3-deficient cells were found to have increased expression of the inflammatory mediators iNOS (4 fold), IL-6 (2 fold) and IL-1b (3 fold).
Conclusion: Intracellular serpin B3 levels are significantly increased in the intestine of both human and murine NEC. Normal TLR4 expression in the intestinal epithelium is required for serpin B3 expression. Our findings suggest a potential role for TLR4 regulation of serpin B3, a molecular modulator of intestinal inflammation and necrosis, hallmarks of this devastating disease.