We now define a novel mechanism by which TLR4 is regulated in the newborn intestinal epithelium that has important implications in the pathogenesis of necrotizing enterocolitis, a disease that is characterized by exaggerated TLR4 signaling within the intestinal mucosa (3
). Specifically, we identify that the induction of Hsp70 leads to a reduction in TLR4-induced signaling in enterocytes as measured by a reduction in NFkB activation, cytokine induction and apoptosis, and that induction of Hsp70 either pharmacologically or genetically leads to a reduction in TLR4 signaling and a marked inhibition in the severity of NEC. The current results identify a novel pathway that links cytoplasmic Hsp70 induction with TLR4 regulation, and demonstrate that impaired Hsp70 expression or function may in part underlie the causes of this devastating disease. These findings represent a novel departure from current thinking in the field, by revealing that future treatments for NEC may involve not only non-specific immunological approaches such as the elimination of microbial pathogens or the administration of particular feeding regimens (43
), but rather, may involve the pharmacologic induction of an intracellular chaperone such as Hsp70 to limit disease progression through inhibitory effects on the innate immune receptor TLR4.
One of the most important findings of the current study involves the proposed mechanism of action of Hsp70 in limiting TLR4 signaling within enterocytes. As a molecular chaperone, Hsp70 can associate with co-chaperone proteins through an EEVD motif in its C-terminus (28
). As shown in , we now demonstrate that the inhibition of TLR4 signaling in response to Hsp70 induction required this EEVD-binding motif (30
), as the introduction of a mutant lacking this domain prevented the association between TLR4 and Hsp70, and also reversed the protection of Hsp70 induction on TLR4 signaling (). This mechanism of action for Hsp70 is in agreement with recent work of Chow et al, who showed that Hsp70 mutants with a functional EEVD motif but lacking N-terminus ATPase activity were still capable of protecting L929 fibroblasts from apoptosis induced by pro-inflammatory cytokines(44
). We also determined that the association between Hsp70 and TLR4 results in enhanced ubiquitination and degradation of TLR4, a process that we have now determined to require the co-chaperone CHIP (). CHIP has not previously been linked to TLR4 signaling, and we further reveal that mutations in both its docking (K30A) and ubiquitination domains (H260Q) prevented the protective effects of Hsp70 induction on TLR4. These findings clarify the mechanism by which CHIP acts to mediate the inhibitory effects of Hsp70 on TLR4 signaling and are in agreement with the known function of CHIP in regulating the activity of other Hsp70 targets through ubiquitination (45
), yet represent the first direct link of CHIP to an intestinal inflammatory disease. It is noteworthy that in the original description of the CHIP-deficient mouse, attention was drawn to the intestinal phenotype that was observed when mice were subjected to a brief hyperthermic stress, characterized by friability of the small intestine with marked apoptosis of the intestinal epithelium (47
), although potential CHIP targets that could mediate this effect on the small intestine during stress were not identified. It is tempting to now speculate that CHIP may play a central role in the maintenance of intestinal homeostasis in part by preventing the unbridled activation of immune targets of CHIP such as TLR4.
It should be noted that the current findings in which cytoplasmic Hsp70 serves to curtail the signaling of TLR4 within the intestinal epithelium lie in distinction to a growing and somewhat controversial body of work concerning the extracellular role of Hsp70 and other heat shock proteins in activating the innate immune system via TLR4 (48
). In this regard, Retzlaff et al showed that the exogenous administration of Hsp70 could increase IL-1, IL-6 and TNF in cultured macrophages (54
), while Wheeler et al have shown that the extracellular exposure of Hsp70 to neutrophils from wild-type mice leads to the release of IL-8, yet this effect is not observed in neutrophils from C3H/HeJ mice that have inhibitory mutations in TLR4 (55
). While very exciting, such studies have recently been called into question by concerns that the observed effects might actually result not from the heat shock proteins themselves, but rather from contaminants such as LPS which could inadvertently be present within the protein preparations, or which could be bound specifically to the heat shock proteins (56
). For example, Wallin et al (57
), Bausinger et al (58
) and Gao and Tsan (59
) have shown that the activation of immune cells previously attributed to Hsp70 were lost when highly purified (i.e. contaminant free) recombinant proteins were used, although these results have been recently and convincingly rebutted in a review article on this topic (60
). In contrast to studies in the field of extracellular Hsp70 biology, the novelty and importance of the current findings lie in the newly discovered link between TLR4 and Hsp70 within the enterocyte both in vitro and in vivo, and the potential etiological relevance to the development of NEC. And while they represent an extension of the classic role of Hsp70 in modulating the fate of cytoplasmic proteins, the relevance – if any – to the body of literature surrounding the fate of Hsp70 outside the cells is unknown.
The current findings in support of a role for Hsp70 in the protection from the development of NEC through the inhibition of TLR4 signaling in the small intestine may or may not apply to other diseases of intestinal inflammation including ulcerative colitis and Crohn's disease, in which TLR4 signaling may play a lesser or perhaps even opposite role. Although we (3
) and others (5
) have shown that the development of NEC requires TLR4 activation, it has been shown that TLR4 plays a protective role in experimental colitis (62
). Several reasons may account for this apparent discrepancy that have relevance to the current study. TLR4 activation leads to intestinal injury in a well-defined and physiologically relevant context, namely the newborn small intestine. In support of this concept, we have recently demonstrated that TLR4 activation with LPS leads to increased enterocyte apoptosis in the terminal ileum of newborn mice but not adult mice, and in the small intestine but not the newborn colon (7
). Further, reports that demonstrate a protective role for TLR4 in models of colitis have typically been based upon the use of global TLR4 knockout mice, in which TLR4 signaling is disrupted in enterocytes as well as T-cells and myeloid cells. We have recently shown that TLR4 signaling within the enterocyte itself is important for the induction of intestinal injury leading to NEC, using enterally administered adenoviral constructs that bear inhibitory mutations in TLR4 whose expression is largely favored within the small bowel mucosa (4
). It is therefore reasonable to conclude that the protective effects attributed to TLR4 signaling in the gut by previous authors may reflect in part the mitigating effects of TLR4 signaling on other cells. In support of this possibility, we note that Fukata and colleagues have recently shown in an elegant study using chimeric mice that TLR4 signaling in colonic epithelial cells worsened intestinal inflammation (64
). These findings argue that the effects of TLR4 in the development of intestinal inflammation are strongly influenced by a variety of factors, including the effector cells involved, developmental factors and involved region of the intestine. The precise effects of Hsp70 at these varying stages of development and within these different cell types remains to be explored in further detail, but are likely to provide important clues to the underlying causes of these diseases.
Based upon the current findings, we now propose a model by which Hsp70 limits TLR4 signaling and plays a key role in influencing the development of enterocyte apoptosis and the development of NEC (). Under healthy conditions, the relationship between the indigenous flora of the host and the baseline activation of TLR4 exists in homeostatic balance, which we now attribute in part to a constitutive role of Hsp70 in limiting the extent of TLR4 signaling by controlling its degradation through proteosomal pathways. The interaction between TLR4 and Hsp70 may occur within intracellular compartments such as the Golgi apparatus, where TLR4 signaling has been shown to occur within the enterocyte(65
). By contrast, under the conditions of stress that favor the development of NEC (increased LPS, hypoxia and prematurity), the exhaustion of Hsp70 signaling accompanied by the relative increase in TLR4 expression leads to exaggerated TLR4 activation and the development of the increased enterocyte apoptosis and pro-inflammatory cytokine expression in the newborn intestine that leads to NEC. It is also notable that the pharmacologic induction of Hsp70 can curtail TLR4 signaling and both prevent and treat experimental NEC.
Proposed model: Hsp70 regulates TLR4 signaling in enterocytes in the pathogenesis of NEC
In summary, we have now identified a novel pathway by which through Hsp70 that serves to limit TLR4 signaling in the intestinal epithelium, and moreover, have shown that factors that increase Hsp70 signaling can attenuate NEC severity through inhibition of TLR4. Such findings, we believe, offer new insights not only into the molecular requirements that lead to NEC development, but also to offer novel therapeutic approaches for this devastating disease.