Ward M. Richardson

Reciprocal Expression and Signaling of TLR4 and TLR9 in the Pathogenesis and Treatment of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a common and often fatal inflammatory disorder affecting preterm infants that develops upon interaction of indigenous bacteria with the premature intestine. We now demonstrate that the developing mouse intestine shows reciprocal patterns of expression of TLR4 and TLR9, the receptor for bacterial DNA (CpG-DNA). Using a novel ultrasound-guided in utero injection system, we administered LPS directly into the stomachs of early and late gestation fetuses to induce TLR4 signaling and demonstrated that TLR4-mediated signaling within the developing intestine follows its expression pattern. Murine and human NEC were associated with increased intestinal TLR4 and decreased TLR9 expression, suggesting that reciprocal TLR4 and TLR9 signaling may occur in the pathogenesis of NEC. Enteral administration of adenovirus expressing mutant TLR4 to neonatal mice reduced the severity of NEC and increased TLR9 expression within the intestine. Activation of TLR9 with CpG-DNA inhibited LPS-mediated TLR4 signaling in enterocytes in a mechanism dependent upon the inhibitory molecule IRAK-M. Strikingly, TLR9 activation with CpG-DNA significantly reduced NEC severity, whereas TLR9-deficient mice exhibited increased NEC severity. Thus, the reciprocal nature of TLR4 and TLR9 signaling within the neonatal intestine plays a role in the development of NEC and provides novel therapeutic approaches to this disease.

Toll-Like Receptor-4 Inhibits Enterocyte Proliferation via Impaired β-Catenin Signaling in Necrotizing Enterocolitis

BACKGROUND & AIMS Necrotizing enterocolitis (NEC), the leading cause of gastrointestinal death from gastrointestinal disease in preterm infants, is characterized by exaggerated TLR4 signaling and decreased enterocyte proliferation through unknown mechanisms. Given the importance of beta-catenin in regulating proliferation of many cell types, we hypothesize that TLR4 impairs enterocyte proliferation in NEC via impaired beta-catenin signaling. METHODS Enterocyte proliferation was detected in IEC-6 cells or in ileum or colon from wild-type, TLR4-mutant, or TLR4(-/-) mice after induction of NEC or endotoxemia. beta-Catenin signaling was assessed by cell fractionation or immunoconfocal microscopy to detect its nuclear translocation. Activation and inhibition of beta-catenin were achieved via cDNA or small interfering RNA, respectively. TLR4 in the intestinal mucosa was inhibited with adenoviruses expressing dominant-negative TLR4. RESULTS TLR4 activation significantly impaired enterocyte proliferation in the ileum but not colon in newborn but not adult mice and in IEC-6 enterocytes. beta-Catenin activation reversed these effects in vitro. To determine the mechanisms involved, TLR4 activation phosphorylated the upstream inhibitory kinase GSK3beta, causing beta-catenin degradation. NEC in both mouse and humans was associated with decreased beta-catenin and increased mucosal GSK3beta expression. Strikingly, the inhibition of enterocyte beta-catenin signaling in NEC could be reversed, and enterocyte proliferation restored, through adenoviral-mediated inhibition of TLR4 signaling in the small intestinal mucosa. CONCLUSION We now report a novel pathway linking TLR4 with inhibition of beta-catenin signaling via GSK3beta activation, leading to reduced enterocyte proliferation in vitro and in vivo. These data provide additional insights into the pathogenesis of diseases of intestinal inflammation such as NEC.

No Longer an Innocent Bystander: Epithelial Toll-Like Receptor Signaling in the Development of Mucosal Inflammation

Diseases of mucosal inflammation represent important causes of morbidity and mortality, and have led to Intense research efforts to understand the factors that lead to their development. It is well accepted that a breakdown of the normally impermeant epithelial barrier of the intestine, the lung, and the kidney is associated with the development of inflammatory disease in these organs, yet significant controversy exists as to how this breakdown actually occurs, and how such a breakdown may lead to inflammation. In this regard, much work has focused upon the role of the epithelium as an “innocent bystander,” a target of a leukocyte-mediated inflammatory cascade that leads to its destruction in the mucosal inflammatory process. However, recent evidence from a variety of laboratories indicates that the epithelium is not merely a passive component in the steps that lead to mucosal inflammation, but is a central participant in the process. In addressing this controversy, we and others have determined that epithelial cells express Toll-like receptors (TLRs) of the innate immune system, and that activation of TLRs by endogenous and exogenous ligands may play a central role in determining the balance between a state of “mucosal homeostasis,” as is required for optimal organ function, and “mucosal injury,” leading to mucosal inflammation and barrier breakdown. In particular, activation of TLRs within intestinal epithelial cells leads to the development of cellular injury and impairment in mucosal repair in the pathogenesis of intestinal inflammation, while activation of TLRs in the lung and kidney may participate in the development of pneumonitis and nephritis respectively. Recent work in support of these concepts is extensively reviewed, while essential areas of further study that are required to determine the significance of epithelial TLR signaling during states of health and disease are outlined.

New Insights into the Pathogenesis and Treatment of Necrotizing Enterocolitis: Toll-like Receptors and Beyond

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 (TLRs) 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 who are affected by this devastating disorder.

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

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.

Nucleotide-binding Oligomerization Domain-2 Inhibits Toll Like Receptor-4 Signaling in the Intestinal Epithelium

BACKGROUND & AIMS Factors that regulate enterocyte apoptosis in necrotizing enterocolitis (NEC) remain incompletely understood, although Toll-like receptor-4 (TLR4) signaling in enterocytes plays a major role. Nucleotide-binding oligomerization domain-2 (NOD2) is an immune receptor that regulates other branches of the immune system, although its effects on TLR4 in enterocytes and its role in NEC remain unknown. We now hypothesize that activation of NOD2 in the newborn intestine inhibits TLR4, and that failure of NOD2 signaling leads to NEC through increased TLR4-mediated enterocyte apoptosis. METHODS The effects of NOD2 on enterocyte TLR4 signaling and intestinal injury and repair were assessed in enterocytes lacking TLR4 or NOD2, in mice with intestinal-specific wild-type or dominant-negative TLR4 or NOD2, and in mice with NEC. A protein array was performed on NOD2-activated enterocytes to identify novel effector molecules involved. RESULTS TLR4 activation caused apoptosis in newborn but not adult small intestine or colon, and its intestinal expression was influenced by NOD2. NOD2 activation inhibited TLR4 in enterocytes, but not macrophages, and reversed the effects of TLR4 on intestinal mucosal injury and repair. Protection from TLR4-induced enterocyte apoptosis by NOD2 required a novel pathway linking NOD2 with the apoptosis mediator second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low PI (SMAC-DIABLO), both in vitro and in vivo. Strikingly, activation of NOD2 reduced SMAC-DIABLO expression, attenuated the extent of enterocyte apoptosis, and reduced the severity of NEC. CONCLUSIONS These findings reveal a novel inhibitory interaction between TLR4 and NOD2 signaling in enterocytes leading to the regulation of enterocyte apoptosis and suggest a therapeutic role for NOD2 in the protection of intestinal diseases such as NEC.

A Critical Role for TLR4 Induction of Autophagy in the Regulation of Enterocyte Migration and the Pathogenesis of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) develops in response to elevated TLR4 signaling in the newborn intestinal epithelium and is characterized by TLR4-mediated inhibition of enterocyte migration and reduced mucosal healing. The downstream processes by which TLR4 impairs mucosal healing remain incompletely understood. In other systems, TLR4 induces autophagy, an adaptive response to cellular stress. We now hypothesize that TLR4 induces autophagy in enterocytes and that TLR4-induced autophagy plays a critical role in NEC development. Using mice selectively lacking TLR4 in enterocytes (TLR4ΔIEC) and in TLR4-deficient cultured enterocytes, we now show that TLR4 activation induces autophagy in enterocytes. Immature mouse and human intestine showed increased expression of autophagy genes compared with full-term controls, and NEC development in both mouse and human was associated with increased enterocyte autophagy. Importantly, using mice in which we selectively deleted the autophagy gene ATG7 from the intestinal epithelium (ATG7ΔIEC), the induction of autophagy was determined to be required for and not merely a consequence of NEC, because ATG7ΔIEC mice were protected from NEC development. In defining the mechanisms involved, TLR4-induced autophagy led to impaired enterocyte migration both in vitro and in vivo, which in cultured enterocytes required the induction of RhoA-mediated stress fibers. These findings depart from current dogma in the field by identifying a unique effect of TLR4-induced autophagy within the intestinal epithelium in the pathogenesis of NEC and identify that the negative consequences of autophagy on enterocyte migration play an essential role in its development.

The development of animal models for the study of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is the leading cause of death and long-term disability from gastrointestinal disease in preterm infants, and is characterized by acute and chronic intestinal inflammation that may lead to systemic sepsis and multi-system organ failure. NEC typically develops in the preterm infant after the administration of tube feeds, although it may occasionally be seen in full-term babies. Despite extensive clinical experience in the management of patients with NEC, the underlying cellular and molecular mechanisms leading to its development remain incompletely understood. Several animal models have therefore been developed in a variety of species in order to study the pathogenesis of NEC and to develop more effective treatment strategies. This review seeks to examine the pros and cons of animal models that have been developed in the study of NEC over the past 30 years. It will highlight the various strengths and weaknesses of experimental approaches that have been used, and discuss potential directions for the development of such models for the future.

Extracellular High Mobility Group Box-1 (HMGB1) Inhibits Enterocyte Migration via Activation of Toll-like Receptor-4 and Increased Cell-Matrix Adhesiveness

Toll-like receptor-4 (TLR4) is the receptor for bacterial lipopolysaccharide, yet it may also respond to a variety of endogenous molecules. Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in newborn infants and is characterized by intestinal mucosal destruction and impaired enterocyte migration due to increased TLR4 signaling on enterocytes. The endogenous ligands for TLR4 that lead to impaired enterocyte migration remain unknown. High mobility group box-1 (HMGB1) is a DNA-binding protein that is released from injured cells during inflammation. We thus hypothesize that extracellular HMGB1 inhibits enterocyte migration via activation of TLR4 and sought to define the pathways involved. We now demonstrate that murine and human NEC are associated with increased intestinal HMGB1 expression, that serum HMGB1 is increased in murine NEC, and that HMGB1 inhibits enterocyte migration in vitro and in vivo in a TLR4-dependent manner. This finding was unique to enterocytes as HMGB1 enhanced migration of inflammatory cells in vitro and in vivo. In seeking to understand the mechanisms involved, TLR4-dependent HMGB1 signaling increased RhoA activation in enterocytes, increased phosphorylation of focal adhesion kinase, and increased phosphorylation of cofilin, resulting in increased stress fibers and focal adhesions. Using single cell force traction microscopy, the net effect of HMGB1 signaling was a TLR4-dependent increase in cell force adhesion, accounting for the impaired enterocyte migration. These findings demonstrate a novel pathway by which TLR4 activation by HMGB1 delays mucosal repair and suggest a novel potential therapeutic target in the amelioration of intestinal inflammatory diseases like NEC.

Interferon-γ inhibits enterocyte migration by reversibly displacing connexin43 from lipid rafts

Necrotizing enterocolitis (NEC) is associated with the release of interferon-gamma (IFN) by enterocytes and delayed intestinal restitution. Our laboratory has recently demonstrated that IFN inhibits enterocyte migration by impairing enterocyte gap junctions, intercellular channels that are composed of connexin43 (Cx43) monomers and that are required for enterocyte migration to occur. The mechanisms by which IFN inhibits gap junctions are incompletely understood. Lipid rafts are cholesterol-sphingolipid-rich microdomains of the plasma membrane that play a central role in the trafficking and signaling of various proteins. We now hypothesize that Cx43 is present on enterocyte lipid rafts and that IFN inhibits enterocyte migration by displacing Cx43 from lipid rafts in enterocytes. We now confirm our previous observations that intestinal restitution is impaired in NEC and demonstrate that Cx43 is present on lipid rafts in IEC-6 enterocytes. We show that lipid rafts are required for enterocyte migration, that IFN displaces Cx43 from lipid rafts, and that the phorbol ester phorbol 12-myristate 13-acetate (PMA) restores Cx43 to lipid rafts after treatment with IFN in a protein kinase C-dependent manner. IFN also reversibly decreased the phosphorylation of Cx43 on lipid rafts, which was restored by PMA. Strikingly, restoration of Cx43 to lipid rafts by PMA or by transfection of enterocytes with adenoviruses expressing wild-type Cx43 but not mutant Cx43 is associated with the restoration of enterocyte migration after IFN treatment. Taken together, these findings suggest an important role for lipid raft-Cx43 interactions in the regulation of enterocyte migration during exposure to IFN, such as NEC.