Translocation of bacteria across the intestinal barrier is important in the pathogenesis of systemic sepsis, although the mechanisms by which bacterial translocation occurs remain largely unknown. We hypothesized that bacterial translocation across the intact barrier occurs after internalization of the bacteria by enterocytes in a process resembling phagocytosis and that TLR4 is required for this process. We now show that FcgammaRIIa-transfected enterocytes can internalize IgG-opsonized erythrocytes into actin-rich cups, confirming that these enterocytes have the molecular machinery required for phagocytosis. We further show that enterocytes can internalize Escherichia coli into phagosomes, that the bacteria remain viable intracellularly, and that TLR4 is required for this process to occur. TLR4 signaling was found to be necessary and sufficient for phagocytosis by epithelial cells, because IEC-6 intestinal epithelial cells were able to internalize LPS-coated, but not uncoated, latex particles and because MD2/TLR4-transfected human endothelial kidney (HEK)-293 cells acquired the capacity to internalize E. coli, whereas nontransfected HEK-293 cells and HEK-293 cells transfected with dominant-negative TLR4 bearing a P712H mutation did not. LPS did not induce membrane ruffling or macropinocytosis in enterocytes, excluding their role in bacterial internalization. Strikingly, the internalization of Gram-negative bacteria into enterocytes in vivo and the translocation of bacteria across the intestinal epithelium to mesenteric lymph nodes were significantly greater in wild-type mice as compared with mice having mutations in TLR4. These data suggest a novel mechanism by which bacterial translocation occurs and suggest a critical role for TLR4 in the phagocytosis of bacteria by enterocytes in this process.
BACKGROUND Traumatic brain injury (TBI) is the leading cause of morbidity and mortality in the pediatric population. Microglia and infiltrating monocyte-derived macrophages are crucial immune cells that modulate the neuroinflammatory response following TBI. Using C34, a novel pharmacologic toll-like receptor 4 inhibitor, we investigated the intricate interactions between these cells in a murine TBI model. METHODS A murine controlled cortical impact model was used, and the results were analyzed on postinjury days 1, 7, 28, and 35. The experimental groups are as follows: (1) sham C57BL/6 wild-type (WT), (2) TBI WT, (3) sham WT + C34, and (4) TBI WT + C34. Quantitative real-time polymerase chain reaction was used to quantify gene expression associated with microglial activation, apoptotic pathways, and type 1 interferon pathway. Flow cytometry was used to isolate microglia and infiltrating monocytes. Brain lesion volumes were assessed using magnetic resonance imaging. Last, neurocognitive outcomes were evaluated using the Morris Water Maze test. Student's t test and one-way analysis of variance were used for statistical analysis with significance achieved when p < 0.05. RESULTS Toll-like receptor 4 inhibition leads to improved neurological sequela post-TBI, possibly because of an increase in infiltrating anti-inflammatory monocytes and a decrease in IFN regulatory factor 7 during acute inflammation, followed by a reduction in apoptosis and M2 microglial expression during chronic inflammation. CONCLUSION Toll-like receptor 4 inhibition with C34 skews infiltrating monocytes toward an anti-inflammatory phenotype, leading to enhanced neurocognitive outcomes. Moreover, although M2 microglia have been consistently shown as inducers of neuroprotection, our results clearly demonstrate their detrimental role during the chronic phases of healing post-TBI.
Prior to birth, the neonate has limited exposure to pathogens. The transition from the intra-uterine to the postnatal environment initiates a series of complex interactions between the newborn host and a variety of potential pathogens that persist over the first few weeks of life. This transition is particularly complex in the case of the premature and very low birth weight infant, who may be susceptible to many disorders as a result of an immature and underdeveloped immune system. Chief amongst these disorders is necrotizing enterocolitis (NEC), an acute inflammatory disorder that leads to necrosis of the intestine, and which can affect multiple systems and have the potential to result in long term effects if the infant is to survive. Here, we examine what is known about the interplay of the immune system with the maternal uterine environment, microbes, nutritional and other factors in the pathogenesis of neonatal pathologies such as NEC, while also taking into consideration the effects on the long-term health of affected children.
Background: The response to tissue injury requires the symphonious interaction of immune cells, keratinocytes, fibroblasts, and endothelial cells, which unite to regenerate the damaged epithelium. Recent insights have elucidated the cellular and molecular mechanisms required for wound healing and have raised the prospect of novel therapeutic targets. Methods: Review of the pertinent literature. Results: The initial inflammatory response leads to the influx of macrophages and neutrophils, which release cytokines, growth factors, and nitric oxide, and induce nearby keratinocytes to migrate across the wounded epithelium. This process, known as re-epithelialization, requires integrin-mediated activation of Rho-GTPases. The subsequent influx of fibroblasts and endothelial cells results in the production of tissue stroma and formation of new blood vessels, which lead to the generation of functional tissue. Importantly, disease states associated with impaired or excessive wound healing can be attributed to defects in these responses, providing a rationale for the use of evidence-based biological therapies. Conclusion: The elucidation of the cellular and biochemical response to wound healing is essential for an understanding to the treatment of clinical conditions during which impaired healing is encountered.
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.
