In the current studies, we describe a novel mechanism by which the interplay between two innate immune receptors in enterocytes, namely NOD2 and TLR4, drives the extent of intestinal injury in the pathogenesis of NEC. We demonstrate that NOD2 activation, as well as the expression of the NOD2 protein itself, limits the extent of TLR4 signaling in enterocytes. In seeking to identify the signaling pathways involve, we now uncover a novel pathway linking these innate immune receptors with the mitochondrial protein SMAC-diablo, which serves to regulate the extent by which NOD2 activation with MDP limits the extent of TLR4-induced intestinal injury and thus serve as a potential therapeutic agent for NEC. This is not only the first demonstration of the importance of SMAC-diablo signaling in enterocytes during inflammation, but also sheds light on a novel signaling effector mechanism linking TLR4 activation with cell death that can be constrained through the activity of NOD2.
Three lines of evidence indicate a role for impaired NOD2 signaling in the pathogenesis of NEC. First, the expression of NOD2 is reduced in NEC compared with controls. Second, inhibition of NOD2 in enterocytes increased NEC severity and increased enterocyte apoptosis. And third, the administration of MDP led to a reduction in enterocyte apoptosis and reduction in NEC severity. In each case, we believe that NOD2 signaling serves to restrain TLR4 signaling in enterocytes, leading to a reduction in the extent of apoptosis, and that the development of NEC represents a failure of this protection. This may in fact represent a general theme by which activation of NOD2 limits TLR4 signaling in other cells also, as has been demonstrated to occur in dendritic cells9
. And given that Crohn’s disease is characterized in certain cases by inhibitory mutations in NOD2, it is tempting to speculate that the signaling pathway that we now describe may have relevance to inflammatory bowel disease also.
In terms of seeking to explain the molecular mechanisms by which NOD2 activation limits TLR4 signaling in enterocytes, we were able to exclude various possibilities, and to shed light on others. For instance, although NOD2 activation did lead to the activation of the inflammasome in enterocytes, its activation was not required for the protection of TLR4-induced signaling. Further, NOD2 activation with MDP did not alter the expression of the predominant endogenous inhibitors of TLR4. This finding in enterocytes stands in distinction to the prior work by Watanabe et al who demonstrated that MDP activation of dendritic cells leads to increased IRF4 expression, and that IRF4 mediates the protective effects of MDP in experimental colitis9
. And given our novel observation that TLR4 expression is influenced by NOD2, we now suggest that the mechanisms by which activation of NOD2 inhibits TLR4 signaling involves both pre-translational and post-translational factors, and that such modifications lead to the activation of effector signaling pathways that directly affect barrier integrity, including those involving SMAC-diablo.
What is the potential teleological explanation for which NOD2 and TLR signaling would coexist and negatively interact in enterocytes? One possibility is that NOD2 activation may serve to activate the inflammasome in enterocytes and thus alert the host to the presence of invasion by mounting a pro-inflammatory response while concomitantly limiting the extent of TLR4-induced intestinal injury. However, unlike dendritic cells and macrophages, the extent of inflammasome activation by NOD2 in enterocytes would be expected to be relatively small19
. An alternative role for the interplay between NOD2 and TLR4 may be found in our observation that TLR4 leads to the internalization of bacteria by enterocytes, a process that may be necessary for antigen sampling and the education of the gut to the presence of various immunologically active peptides16
. Such intracellular antigens could then conceivably activate NOD2 and thereby restrain TLR4 signaling, effectively preventing further bacterial entry, while also limiting the effects of TLR4-induced intestinal injury. In a similar manner, we have recently shown that bacterial DNA can inhibit TLR4 signaling via activation of TLR9 – a close homolog of TLR4 that is present on endomembranes within enterocytes13
. We now propose that the extent of TLR4 signaling in the newborn intestine – and by extension the predisposition to NEC development in susceptible neonates – is regulated in large part through the combined interactions of TLR4, NOD2 and other innate immune receptors such as TLR9, and that the extent of these interactions is governed by a dynamic interplay between the microbial flora and the inflammatory microenvironment of the host.
In summary, the current work represents a novel mechanism linking NOD2 activation with the inhibition of TLR4 signaling in enterocytes leading to a reduction in apoptosis via SMAC-diablo. Further studies to explore the interplay between TLR4 and NOD2 activation may provide a useful framework in advancing our understanding of the role of the innate immune system in the maintenance of intestinal homeostasis during health, and how dysregulation of these receptors may lead to disease processes such as NEC.