01.18 The Proinflammatory Cytokine TNF-a Aggravates Intestinal Injury in TLR4-deficient mice

M. Sampah¹, C. P. Sodhi¹, M. L. Kovler¹, A. J. Gonzalez Salazar¹, M. R. Ladd¹, H. Jia¹, P. Lu¹, Y. Yamaguchi¹, W. Fulton¹, T. Prindle¹, S. Wang¹, D. J. Hackam^{1  1}The Johns Hopkins University School Of Medicine,Division Of General Pediatric Surgery,Baltimore, MD, USA

Introduction: Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in premature infants. Our lab and others have shown that NEC is mediated by excessive activation of Toll-like receptor 4 (TLR4), which is upregulated in the premature intestinal epithelium. While mice who are genetically deficient in TLR4 are protected from NEC, less is known about the downstream consequences of TLR4 activation that lead to intestinal injury in this disease. Among the many injurious cytokines produced in response to TLR4 activation in the premature intestine, TNF-alpha is one of the strongest pro-inflammatory effectors. Therefore, to better understand the downstream effects of TLR4 activation in the intestine, we sought to determine if mice lacking TLR4 were protected from intestinal injury caused by exposure to TNF-alpha.

Methods: NEC was induced in 7d old wild-type (TLR4^WT) and TLR4 knockout (TLR4^-/-) mice (equal number male and female) through four days of gavage-fed formula mixed with NEC bacteria and repeated exposure to brief periods of hypoxia. NEC severity was assessed histologically using a validated NEC severity score and through measurement of gene expression of TNF-alpha by qRT-PCR of intestinal segments. To further understand the ensuing effects of TLR4 activation, juvenile TLR4^WT and TLR4^-/- mice were treated intraperitoneally with either murine TNF-alpha (5ug) or saline as controls (5 mice per group). After six hours, mice were sacrificed and ileal tissue was collected to assess for expression of the pro-inflammatory cytokines and necrotoptic markers including TNF-alpha, Lipocalin-2 and IL-1beta by quantitative RT-PCR.

Results:TLR4^WT mice subjected to experimental NEC showed significantly higher histologic severity scores and TNF-alpha expression when compared to breast fed controls. In contrast, mice genetically deficient in TLR4 were protected from experimental NEC and did not have increased TNF-alpha expression. In contrast, following direct intraperitoneal administration of TNF-alpha, TLR4^−/− mice were not protected from intestinal inflammation, and actually had elevated gene expression of Lipocalin-2 when compared to TLR4^WT mice.

Conclusion:These findings demonstrate that while excessive TLR4 signaling in the premature intestinal epithelium is a requisite component in the pathogenesis of NEC, TLR4 deficiency is not protective against the subsequent inflammatory response that proceeds. Therefore, while TLR4 inhibition remains an important therapeutic target for NEC, mediating the downstream inflammatory response to TLR4 signaling in the premature intestine – including the possibility of direct TNF-alpha inhibition – offers novel potential approaches to treating this devastating disease.