BACKGROUND & AIMS Little is known about factors that regulate intestinal epithelial differentiation; microbial recognition receptors such as Toll-like receptor (TLR)4 might be involved. We investigated whether intestinal TLR4 regulates epithelial differentiation and is involved in development of necrotizing enterocolitis (NEC) of the immature intestine. METHODS Mice with conditional disruption of TLR4 in the intestinal epithelium and TLR4 knockout (TLR4−/−) mice were generated by breeding TLR4loxp/loxp mice with villin-cre and Ella-cre, respectively. Enterocytes that did not express or overexpressed TLR4 were created by lentiviral or adenoviral transduction. Intestinal organoids were cultured on tissue matrices. Bile acids were measured by colorimetric assays, and microbial composition was determined by 16S pyrosequencing. NEC was induced in 7- to 10-day-old mice by induction of hypoxia twice daily for 4 days. RESULTS TLR4−/− mice and mice with enterocyte-specific deletion of TLR4 were protected from NEC; epithelial differentiation into goblet cells was increased via suppressed Notch signaling in the small intestinal epithelium. TLR4 also regulates differentiation of goblet cells in intestinal organoid and enterocyte cell cultures; differentiation was increased on deletion of TLR4 and restored when TLR4 was expressed ectopically. TLR4 signaling via Notch was increased in intestinal tissue samples from patients with NEC, and numbers of goblet cells were reduced. 16S pyrosequencing revealed that wild-type and TLR4-deficient mice had similar microbial profiles; increased numbers of goblet cells were observed in mice given antibiotics. TLR4 deficiency reduced levels of luminal bile acids in vivo, and addition of bile acids to TLR4-deficient cell cultures prevented differentiation of goblet cells. CONCLUSIONS TLR4 signaling and Notch are increased in intestinal tissues of patients with NEC and required for induction of NEC in mice. TLR4 prevents goblet cell differentiation, independently of the microbiota. Bile acids might initiate goblet cell development.

Breast milk is the most effective strategy to protect infants against necrotizing enterocolitis (NEC), a devastating disease which is characterized by severe intestinal necrosis. Previous studies have demonstrated that the lipopolysaccharide receptor toll-like receptor 4 (TLR4) plays a critical role in NEC development via deleterious effects on mucosal injury and repair. We now hypothesize that breast milk protects against NEC by inhibiting TLR4 within the intestinal epithelium, and sought to determine the mechanisms involved. Breast milk protected against NEC and reduced TLR4 signaling in wild-type neonatal mice, but not in mice lacking the epidermal growth factor receptor (EGFR), while selective removal of EGF from breast milk reduced its protective properties, indicating that breast milk inhibits NEC and attenuates TLR4 signaling via EGF/EGFR activation. Over-expression of TLR4 in the intestinal epithelium reversed the protective effects of breast milk. The protective effects of breast milk occurred via inhibition of enterocyte apoptosis and restoration of enterocyte proliferation. Importantly, in IEC-6 enterocytes, breast milk inhibited TLR4 signaling via inhibition of GSK3β. Taken together, these findings offer mechanistic insights into the protective role for breast milk in NEC, and support a link between growth factor and innate immune receptors in NEC pathogenesis.

Background: Factors that regulate intestinal stem cell (ISC) proliferation and apoptosis are unknown. Results: Toll-like receptor 4 (TLR4) is expressed on ISCs and regulates their proliferation and apoptosis, which is critical in the pathogenesis of necrotizing enterocolitis (NEC). Conclusion: TLR4 regulates ISC proliferation and apoptosis. Significance: This is the first study showing that ISC regulation by microbial receptors contributes to NEC pathogenesis.

Necrotizing enterocolitis (NEC) is a devastating disease of premature infants characterized by severe intestinal necrosis and for which breast milk represents the most effective protective strategy. Previous studies have revealed a critical role for the lipopolysaccharide receptor toll-like receptor 4 (TLR4) in NEC development through its induction of mucosal injury, yet the reasons for which intestinal ischemia in NEC occurs in the first place remain unknown. We hypothesize that TLR4 signaling within the endothelium plays an essential role in NEC development by regulating perfusion to the small intestine via the vasodilatory molecule endothelial nitric oxide synthase (eNOS). Using a unique mouse system in which we selectively deleted TLR4 from the endothelium, we now show that endothelial TLR4 activation is required for NEC development and that endothelial TLR4 activation impairs intestinal perfusion without effects on other organs and reduces eNOS expression via activation of myeloid differentiation primary response gene 88. NEC severity was significantly increased in eNOS −/− mice and decreased upon administration of the phosphodiesterase inhibitor sildenafil, which augments eNOS function. Strikingly, compared with formula, human and mouse breast milk were enriched in sodium nitrate-a precursor for enteral generation of nitrite and nitric oxide-and repletion of formula with sodium nitrate/nitrite restored intestinal perfusion, reversed the deleterious effects of endothelial TLR4 signaling, and reduced NEC severity. These data identify that endothelial TLR4 critically regulates intestinal perfusion leading to NEC and reveal that the protective properties of breast milk involve enhanced intestinal microcirculatory integrity via augmentation of nitrate-nitrite-NO signaling.neonatal inflammation | prematurity | infant formula | neonatal nutrition | sepsis N ecrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in the premature infant and is gradually increasing in frequency (1). The defining pathological feature of NEC is the presence of patchy areas of ischemia and necrosis of the small and large intestine (2). Although prematurity is the leading risk factor for NEC development, breast milk administration has been identified as the most important protective strategy (3). Importantly, the mechanisms that lead to the acute development of intestinal necrosis in the premature intestine and factors within breast milk that may prevent NEC remain largely unexplored.In seeking to understand the underlying biological mechanisms that lead to NEC, we and others have identified a critical role for the innate immune receptor toll-like receptor 4 (TLR4) in NEC pathogenesis, because mice deficient in TLR4 showed reduced mucosal inflammation and reduced intestinal necrosis in experimental NEC (4, 5). Microcirculatory perfusion of the premature intestine is primarily regulated by the vasodilator nitric oxide (NO), which is generated through the activity of endothelial NO synthase (eNOS) (6). ...

The fetal intestinal mucosa is characterized by elevated Toll-like receptor 4 (TLR4) expression, which can lead to the development of necrotizing enterocolitis (NEC)-a devastating inflammatory disease of the premature intestine-upon exposure to microbes. To define endogenous strategies that could reduce TLR4 signaling, we hypothesized that amniotic fluid can inhibit TLR4 signaling within the fetal intestine and attenuate experimental NEC, and we sought to determine the mechanisms involved. We show here that microinjection of amniotic fluid into the fetal (embryonic day 18.5) gastrointestinal tract reduced LPS-mediated signaling within the fetal intestinal mucosa. Amniotic fluid is abundant in EGF, which we show is required for its inhibitory effects on TLR4 signaling via peroxisome proliferator-activated receptor, because inhibition of EGF receptor (EGFR) with cetuximab or EGF-depleted amniotic fluid blocked the inhibitory effects of amniotic fluid on TLR4, whereas amniotic fluid did not prevent TLR4 signaling in EGFR-or peroxisome proliferator-activated receptor γ-deficient enterocytes or in mice deficient in intestinal epithelial EGFR, and purified EGF attenuated the exaggerated intestinal mucosal TLR4 signaling in wild-type mice. Moreover, amniotic fluid-mediated TLR4 inhibition reduced the severity of NEC in mice through EGFR activation. Strikingly, NEC development in both mice and humans was associated with reduced EGFR expression that was restored upon the administration of amniotic fluid in mice or recovery from NEC in humans, suggesting that a lack of amniotic fluid-mediated EGFR signaling could predispose to NEC. These findings may explain the unique susceptibility of premature infants to the development of NEC and offer therapeutic approaches to this devastating disease. N ecrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in premature infants (1). Although the underlying etiology of NEC remains incompletely understood, recent studies have identified a critical role for the LPS receptor, Toll-like receptor 4 (TLR4) in its pathogenesis. TLR4 activation within the intestinal epithelium leads to increased mucosal injury through accelerated enterocyte apoptosis as well as reduced healing through impaired intestinal restitution and proliferation (2), and mice lacking TLR4 (2, 3) show reduced NEC severity as the result of reduced injury and enhanced healing (4). These findings suggest that NEC develops in part in response to exaggerated TLR4 signaling in the intestinal mucosa and, by extension, that strategies may exist within the intestine that can limit TLR4 signaling and the propensity for NEC development. Given that the premature intestine is bathed in amniotic fluid throughout its development and that an abrupt lack of exposure to amniotic fluid is a natural consequence of early delivery, we hypothesized that amniotic fluid may exert a restraining influence on TLR4 signaling and that the absence of the anti-TLR4 signaling effects of the amniotic fluid places the p...

Background: Cellular cues that regulate intestinal stem cell (ISC) apoptosis are unknown. Results: Toll-like-receptor 4 (TLR4) activation on ISCs induces endoplasmic reticulum (ER) stress, leading to ISC apoptosis and necrotizing enterocolitis (NEC).

Many inflammatory diseases may be linked to pathologically elevated signaling via the receptor for lipopolysaccharide (LPS), toll-like receptor 4 (TLR4). There has thus been great interest in the discovery of TLR4 inhibitors as potential anti-inflammatory agents. Recently, the structure of TLR4 bound to the inhibitor E5564 was solved, raising the possibility that novel TLR4 inhibitors that target the E5564-binding domain could be designed. We utilized a similarity search algorithm in conjunction with a limited screening approach of small molecule libraries to identify compounds that bind to the E5564 site and inhibit TLR4. Our lead compound, C34, is a 2-acetamidopyranoside (MW 389) with the formula C17H27NO9, which inhibited TLR4 in enterocytes and macrophages in vitro, and reduced systemic inflammation in mouse models of endotoxemia and necrotizing enterocolitis. Molecular docking of C34 to the hydrophobic internal pocket of the TLR4 co-receptor MD-2 demonstrated a tight fit, embedding the pyran ring deep inside the pocket. Strikingly, C34 inhibited LPS signaling ex-vivo in human ileum that was resected from infants with necrotizing enterocolitis. These findings identify C34 and the β-anomeric cyclohexyl analog C35 as novel leads for small molecule TLR4 inhibitors that have potential therapeutic benefit for TLR4-mediated inflammatory diseases.

Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in the preterm infant. The dismal results of current treatment for NEC highlight the urgent need for greater understanding of the pathogenesis of this disease, and the importance of discovering novel, molecular-specific therapies for it. Current dogma indicates that NEC development reflects an abnormal response by the premature infant to the microbial flora that colonizes the gastrointestinal tract, although the mechanisms that mediate these abnormal bacterial-enterocyte interactions, and the reasons for the particularly increased susceptibility of the premature infant to the development of NEC remain incompletely explained. Recent evidence has shed light on an emerging role for the Toll like receptors (TLR's) of the innate immune system as central players in the pathways that signal in response to enteric bacteria resulting in the development of NEC. We now review recent advances in the field of NEC and identify several exciting potential avenues for novel treatments by focusing on abnormal TLR4 signaling in the premature intestine in the pathogenesis of NEC. In so doing, we seek to offer new hope to the patients and their families that are affected by this devastating disorder.