TLR-mediated recognition of conserved microbial structures and ligand-induced cellular activation was first discovered on professional immune cells such as macrophages and dendritic cells that reside in largely sterile tissue environments (27
). The situation on epithelial surfaces, however, is markedly different. The intestinal tract is exposed to a variety of environmental microbial ligands and is colonized by a dense, complex, and highly dynamic microbial flora that contributes to the intestinal physiology by substrate degradation and synthesis of essential nutrients (28
). The composition of the microbial flora is complex and provides most of the identified innate immune receptor ligands such as LPS at high concentrations. Thus, innate immune recognition within the gastrointestinal tract must be tightly regulated to avoid inappropriate stimulation.
The vast majority of previous studies on TLR-mediated recognition by epithelial cells have been performed using established cell lines under cell culture conditions and resulted in a heterogenous picture of TLR expression and ligand susceptibility (29
). In contrast, the use of mouse chimera with myeloid cell–restricted TLR expression indicated an important contribution of lung and bladder epithelial cells for the TLR4-mediated host microbe interaction (30
). In the present study, we performed a detailed in vivo characterization of primary IECs within their complex natural environment by using laser microdissection, immunohistology, as well as ex vivo assays with isolated epithelial cells. LPS stimulation of fetal IECs in vitro readily resulted in intracellular cell signaling, transcriptional activation, as well as chemokine synthesis and secretion. However, LPS susceptibility of IECs was lost after birth, indicating perinatal induction of negative regulatory control mechanisms of epithelial TLR signaling. This unique adaptive phenotype of epithelial cells with a postnatal acquisition of TLR resistance is further illustrated by the strikingly different situation found in intestinal macrophages. Intestinal macrophages exhibited a constitutive, age-independent nonresponsive phenotype that appears to be causally related to their tissue localization. Therefore, the observed postnatal activation represents a previously unrecognized epithelium-specific adaptive process that might play an important role to facilitate postnatal microbial colonization and establishment of a stable homeostasis on intestinal surfaces.
Consistent with the LPS-susceptible phenotype of fetal IECs, exposure to the nonsterile environment after birth led to activation of the intestinal epithelium. However, the rapid, strong, and transient response observed in vivo within 2 h after birth was unexpected. In fact, nuclear translocation of the NF-κB subunit p65 was observed already at 60 min after birth, indicating stimulation in close context with the delivery process. Analysis of neonates born by Caesarean section suggested an exogenous stimulus. LPS measurements in newborn intestinal tissue, the stimulatory effect of oral LPS exposure in Caesarean-born neonates, as well as the absence of epithelial activation in TLR4-deficient mice finally identified LPS as the major microbial stimulus for postnatal epithelial activation. The high concentrations of biologically active endotoxin in intestinal and fecal samples of adult animals illustrated the minute volumes of ingested contaminated material required to evoke stimulation. However, although clearly the predominant stimulus, endotoxin might act in synergism with other microbial compounds in vivo. It is tempting to speculate that the observed postnatal epithelial activation might also occur on other epithelial body surfaces and could be involved in the up-regulation of host defense molecules to prepare for subsequent microbial exposure.
The activation of IECs after birth showed a self-limited time course with MIP-2 mRNA levels returning to baseline within only 4–6 h after delivery and no detectable proinflammatory tissue infiltration. Thus, the observed epithelial stimulation induced a potent negative regulatory mechanism that caused rapid termination of cellular activation and induced unresponsiveness to subsequent stimulation. Posttranscriptional down-regulation of IRAK-1 was detected selectively in primary IECs shortly after birth, and in vitro studies using differentiated intestinal epithelial m-ICcl2
cells suggested that posttranscriptional depletion of IRAK-1 might significantly contribute to the observed LPS nonresponsive phenotype of primary postnatal IECs. IRAK-1 together with IRAK-4 binds to the TLR/adaptor molecule complex and transmits the signal to the TNF receptor–associated factor 6 to facilitate NF-κB activation (32
). The important role of IRAK-1 for IL-1 receptor/TLR-mediated signaling has been demonstrated using IRAK-1–deficient mice (24
). Interestingly, earlier work also revealed an important role of IRAK-1 for TNF receptor signaling (27
). Although IRAK-1 down-regulation upon LPS stimulation has previously been noted in macrophages, our study is the first report to identify IRAK-1 depletion in epithelial cells as a mechanism of TLR hyporesponsiveness in vivo (20
A recent study demonstrated that large amounts of LPS given orally induced intestinal pathology with significant mortality in fetal and newborn rats, but not in older animals (34
). LPS susceptibility of fetal IECs has also been observed in humans (35
). Thus, postnatal epithelial activation might also occur in humans, and dysfunction of negative regulation and tolerance acquisition, e.g., in undifferentiated enterocytes, or increased stimulation by enhanced stimulus concentration might lead to the development of enteric inflammation. A corresponding clinical picture is seen in patients with necrotizing enterocolitis (NEC), a devastating inflammatory disease of the intestine frequently seen in premature newborns. NEC is characterized by enteric cytokine production, polymorphonuclear cell (PMN) activation, tissue infiltration, ischemic hemorrhage, and high mortality (37
). Microbial colonization, enteral feeding, hyperresponsiveness of immature enterocytes, and uncontrolled cytokine production have been proposed to contribute to the pathogenesis of this disease, although its exact etiology has remained unclear (38
). Our data suggest that dysregulation of the observed postnatal proinflammatory epithelial activation might participate in the pathogenesis of NEC.
In conclusion, this study provides conclusive evidence that primary IECs express TLR4 and are able to respond to LPS in vivo. It is the first to describe spontaneous postnatal epithelial cell activation induced by exogenous endotoxin and identifies posttranscriptional down-regulation of IRAK-1 as a negative regulatory mechanism of TLR4 signaling in IECs in vivo. This adaptive process might be crucial to facilitate postnatal microbial colonization and subsequent development of the astonishingly stable, lifelong symbiosis. The finding that epithelial unresponsiveness of postnatal IECs is not intrinsically determined, but acquired, illustrates the requirement for effective negative regulatory mechanisms of innate immune recognition by IECs to prevent inflammatory disease. In addition, the newly discovered epithelial activation shortly after birth might pose a significant risk for individuals who are unable to mount an adequate negative regulatory control. The further elucidation of the described tolerance acquisition process and characterization of all regulatory mechanisms involved will certainly help to reveal a more detailed picture of innate immune recognition within the gastrointestinal tract and its possible role in the pathogenesis of inflammatory diseases.