Amniotic fluid inhibits Toll-like receptor 4 signaling in the fetal and neonatal intestinal epithelium

Abstract

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...

Endothelial TLR4 activation impairs intestinal microcirculatory perfusion in necrotizing enterocolitis via eNOS–NO–nitrite signaling

“…In further support of this model, the microcirculatory perfusion of remote organs, including the kidney and pancreas, were unaffected in experimental NEC, indicating the importance of the local effects of bacterial translocation on impairing perfusion of the nearest (i.e., intestinal) vascular bed. The present study may also serve to further explain the unique susceptibility of the premature infant for the development of NEC, given the observation by ourselves and others that TLR4 expression within the premature intestine is significantly elevated compared with that in full-term infants (9,19,20), which we now show to be deficient in eNOS (Fig. 3).…”

Toll-like Receptor 4-mediated Endoplasmic Reticulum Stress in Intestinal Crypts Induces Necrotizing Enterocolitis

“…Fetuses were delivered by cesarean section 24 h later. The total injected volume was 5 l. In control experiments, the spontaneous fetal demise rate was under 20%, as in our prior publications (10,22).…”

“…In Utero Microinjection of the Fetal Intestine-Backscatter ultrasound-guided microinjection to deliver reagents directly into the fetal gut was performed as we recently described (10). At embryonic day 17.5, the uterus of a pregnant C57BL/6 mouse was exposed by laparotomy, and a fenestrated dish was placed over the uterus.…”

“…For instance, necrotizing enterocolitis (NEC) 3 is a debilitating and often life-threatening disease of premature infants whose pathogenesis remains incompletely understood and that has been attributed in part to an acquired loss of stem cells within the premature gut (2, 3), resulting in a profound inability to heal the damaged intestinal mucosa (4 -7). In seeking to understand how intestinal stem cell loss may occur in NEC, several groups, including our own, have identified that NEC occurs in the setting of exaggerated activation of the LPS receptor, namely Toll-like receptor 4 (TLR4), on intestinal epithelial cells (5,8), because mice lacking TLR4 within the intestinal epithelium are protected from NEC development (9), whereas patients and humans with NEC show elevated intestinal TLR4 expression and signaling (10). In seeking to understand the precise mechanisms by which TLR4 activation in the gut can lead to NEC, we have recently identified that TLR4 is expressed at high levels on Lgr5-positive intestinal stem cells (11), that TLR4 regulates intestinal epithelial differ-entiation (9), and that exaggerated activation of TLR4 leads to apoptosis of intestinal stem cells through the up-regulation of PUMA (p53 upregulated modulator of apoptosis) (11).…”

Transforming growth factor-β2 and endotoxin interact to regulate homeostasis via interleukin-8 levels in the immature intestine

“…On the other hand, a reduced risk of NEC and reduced levels of inflammatory mediators are observed in preterm pigs fed donor human milk, bovine colostrum, and amniotic fluid (16,36,37). These enteral diets have greater levels of bioactive components, such as TGF-␤2 and epidermal growth factor (10,17), which may reduce NEC, at least in the more artificial models of NEC in mice (10,19). Identification of key bioactive milk components responsible for NEC protection in preterm pigs is important to understand the mechanisms of NEC prevention and to develop optimal infant formulas for preterm infants.…”