Rahul J. Anand

Emerging paradigm: toll-like receptor 4-sentinel for the detection of tissue damage.

ABSTRACT The systemic inflammatory response syndrome initiated by infection shares many features in common with the trauma-induced systemic response. The toll-like receptors (TLRs) stand at the interface of innate immune activation in the settings of both infection and sterile injury by responding to a variety of microbial and endogenous ligands alike. Recently, a body of literature has evolved describing a key role for TLRs in acute injury using rodent models of hemorrhagic shock, ischemia and reperfusion, tissue trauma and wound repair, and various toxic exposures. This review will detail the observations implicating a TLR family member, TLR4, as a key component of the initial injury response.

The role of the intestinal barrier in the pathogenesis of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in neonates and is increasing in frequency because of recent advances in neonatal care. NEC develops in a stressed preterm infant in the setting of intestinal barrier disruption, systemic inflammation, and leads to, multisystem organ failure. The intestinal barrier lies at the interface between microbes within the intestinal lumen and the immune system of the host, and has both immunological and mechanical components. These components serve to protect the host from invading pathogens and, at the same time, provide a surface area for nutrient absorption. Factors that lead to impairments in the function of the intestinal barrier may predispose the host to the invasion of gut-derived microbes and to the development of systemic inflammatory disease. This process, termed bacterial translocation, may be compounded during instances in which the mechanisms that regulate the repair of the intestinal barrier are disrupted. Bacterial translocation is of particular concern to the newborn patient, in which immaturity of the mechanical barrier and incomplete development of the host immune system combine to render the host at particular risk for the development of intestinal inflammation. This review will serve to provide an overview of recent evidence regarding the components of the intestinal barrier, and the mechanisms by which disruptions in barrier function may contribute to the pathogenesis of NEC.

The role of epithelial Toll-like receptor signaling in the pathogenesis of intestinal inflammation

Emerging evidence suggests that the innate immune system, comprised of Toll‐like receptors (TLRs) and their associated molecules, plays a pivotal role in the regulation of intestinal inflammation and in the response to invading pathogens. Although TLRs are thought to have predominantly beneficial effects in pathogen recognition and bacterial clearance by leukocytes, their dysregulation and unique signaling effects within intestinal epithelia in the setting of inflammation may have devastating consequences. For instance, activation of TLR4 in enterocytes leads to an inhibition of enterocyte migration and proliferation as well as the induction of enterocyte apoptosis—factors that would be expected to promote intestinal injury while inhibiting intestinal repair. TLR signaling has been shown to be abnormal in several intestinal inflammatory diseases, including Crohn’s disease, ulcerative colitis, and necrotizing enterocolitis. This review serves to examine the evidence regarding the patterns of expression and signaling of TLRs in the intestinal mucosa at basal levels and during physiologic stressors to gain insights into the pathogenesis of intestinal inflammation. We conclude that the data reviewed suggest that epithelial TLR signaling—acting in concert with TLR signaling by leukocytes—participates in the development of intestinal inflammation. We further conclude that the evidence reviewed provides a rationale for the development of novel, epithelial‐specific, TLR‐based agents in the management of diseases of intestinal inflammation.

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.

Hypoxia causes an increase in phagocytosis by macrophages in a HIF-1α-dependent manner

Phagocytosis is the process by which microbial pathogens are engulfed by macrophages and neutrophils and represents the first line of defense against bacterial infection. The importance of phagocytosis for bacterial clearance is of particular relevance to systemic inflammatory diseases, which are associated with the development of hypoxia, yet the precise effects of hypoxia on phagocytosis remain largely unexplored. We now hypothesize that hypoxia inhibits phagocytosis in macrophages and sought to determine the mechanisms involved. Despite our initial prediction, hypoxia significantly increased the phagocytosis rate of particles in vitro by RAW264.7 and primary peritoneal macrophages and increased phagocytosis of labeled bacteria in vivo by hypoxic mice compared with normoxic controls. In understanding the mechanisms involved, hypoxia caused no changes in RhoA‐GTPase signaling but increased the phosphorylation of p38‐MAPK significantly. Inhibition of p38 reversed the effects of hypoxia on phagocytosis, suggesting a role for p38 in the hypoxic regulation of phagocytosis. Hypoxia also significantly increased the expression of hypoxia‐inducible factor‐1α (HIF‐1α) in macrophages, which was reversed after p38 inhibition, suggesting a link between p38 activation and HIF‐1α expression. It is striking that small interfering RNA knockdown of HIF‐1α reversed the effects of hypoxia on phagocytosis, and overexpression of HIF‐1α caused a surprising increase in phagocytosis compared with nontransfected controls, demonstrating a specific role for HIF‐1α in the regulation of phagocytosis. These data indicate that hypoxia enhances phagocytosis in macrophages in a HIF‐1α‐dependent manner and shed light on an important role for HIF‐1α in host defense.

A Role for Connexin43 in Macrophage Phagocytosis and Host Survival after Bacterial Peritoneal Infection

The pathways that lead to the internalization of pathogens via phagocytosis remain incompletely understood. We now demonstrate a previously unrecognized role for the gap junction protein connexin43 (Cx43) in the regulation of phagocytosis by macrophages and in the host response to bacterial infection of the peritoneal cavity. Primary and cultured macrophages were found to express Cx43, which localized to the phagosome upon the internalization of IgG-opsonized particles. The inhibition of Cx43 using small interfering RNA or by obtaining macrophages from Cx43 heterozygous or knockout mice resulted in significantly impaired phagocytosis, while transfection of Cx43 into Fc-receptor expressing HeLa cells, which do not express endogenous Cx43, conferred the ability of these cells to undergo phagocytosis. Infection of macrophages with adenoviruses expressing wild-type Cx43 restored phagocytic ability in macrophages from Cx43 heterozygous or deficient mice, while infection with viruses that expressed mutant Cx43 had no effect. In understanding the mechanisms involved, Cx43 was required for RhoA-dependent actin cup formation under adherent particles, and transfection with constitutively active RhoA restored a phagocytic phenotype after Cx43 inactivation. Remarkably, mortality was significantly increased in a mouse model of bacterial peritonitis after Cx43 inhibition and in Cx43 heterozygous mice compared with untreated and wild-type counterparts. These findings reveal a novel role for Cx43 in the regulation of phagocytosis and rearrangement of the F-actin cytoskeleton, and they implicate Cx43 in the regulation of the host response to microbial infection.

Increased expression and internalization of the endotoxin coreceptor CD14 in enterocytes occur as an early event in the development of experimental necrotizing enterocolitis

BACKGROUNDnThe early signaling events in the development of necrotizing enterocolitis (NEC) remain undefined. We have recently shown that the endotoxin (lipopolysaccharide [LPS]) receptor toll-like receptor 4 (TLR4) on enterocytes is critical in the pathogenesis of experimental NEC. Given that the membrane receptor CD14 is known to facilitate the activation of TLR4, we now hypothesize that endotoxemia induces an early upregulation of CD14 in enterocytes and that this participates in the early intestinal inflammatory response in the development of NEC.nnnMETHODSnIEC-6 enterocytes were treated with LPS (50 microg/mL), and the subcellular localization of CD14 and TLR4 was assessed by confocal microscopy. C57/Bl6 or CD14-/- mice were treated with LPS (5 mg/kg), whereas experimental NEC was induced using a combination of gavage formula feeding and intermittent hypoxia. CD14 expression was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse transcriptase-polymerase chain reaction, and interleukin 6 was quantified by enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction.nnnRESULTSnExposure of IEC-6 enterocytes to LPS led to an initial, transient increase in CD14 expression. The early increase in CD14 expression was associated with internalization of CD14 to a perinuclear compartment where increased colocalization with TLR4 was noted. The in vivo significance of these findings is suggested as treatment of mice with LPS led to an early increase in CD14 expression in the intestinal mucosa, whereas the persistent endotoxemia of experimental NEC was associated with decreased CD14 expression within enterocytes.nnnCONCLUSIONSnLPS signaling in the enterocyte is marked by an early, transient increase in expression of CD14 and redistribution of the receptor. This process may contribute to the early activation of the intestinal inflammatory response that is observed in the development of NEC.

The role of gap junctions in health and disease.

I t has been said many times that “no man is an island.” Implied in this age-old statement is the realization that each of us is immeasurably affected by the activities of those around us. As a corollary, it goes without saying that each of our own individual activities may have tremendous effects on the activities and functions of society as a whole. And from the point of view of an individual cell—whether under basal conditions or during pathologic processes—the effects of surrounding cells are paramount. Much attention has been paid to the role of circulating substances, including cytokines and hormones, on individual cellular function. Of equal or perhaps even greater importance may be the role that direct cell-to-cell communication may have on the function of individual cells within tissue. This review discusses the basic mechanisms that regulate communication between adjoining cells and examines the effects of impaired communication on health and disease.