Tamas Jilling

The Roles of Bacteria and TLR4 in Rat and Murine Models of Necrotizing Enterocolitis

Bacteria are thought to contribute to the pathogenesis of necrotizing enterocolitis (NEC), but it is unknown whether their interaction with the epithelium can participate in the initiation of mucosal injury or they can act only following translocation across a damaged intestinal barrier. Our aims were to determine whether bacteria and intestinal epithelial TLR4 play roles in a well-established neonatal rat model and a novel neonatal murine model of NEC. Neonatal rats, C57BL/6J, C3HeB/FeJ (TLR4 wild type), and C3H/HeJ (TLR4 mutant) mice were delivered by Cesarean section and were subjected to formula feeding and cold asphyxia stress or were delivered naturally and were mother-fed. NEC incidence was evaluated by histological scoring, and gene expression was quantified using quantitative real-time PCR from cDNA generated from intestinal total RNA or from RNA obtained by laser capture microdissection. Spontaneous feeding catheter colonization or supplementation of cultured bacterial isolates to formula increased the incidence of experimental NEC. During the first 72 h of life, i.e., the time frame of NEC development in this model, intestinal TLR4 mRNA gradually decreases in mother-fed but increases in formula feeding and cold asphyxia stress, correlating with induced inducible NO synthase. TLR4, inducible NO synthase, and inflammatory cytokine induction occurred in the intestinal epithelium but not in the submucosa. NEC incidence was diminished in C3H/HeJ mice, compared with C3HeB/FeJ mice. In summary, bacteria and TLR4 play significant roles in experimental NEC, likely via an interaction of intraluminal bacteria and aberrantly overexpressed TLR4 in enterocytes.

New concepts in necrotizing enterocolitis

Necrotizing enterocolitis is an overwhelming gastrointestinal emergency that primarily afflicts premature infants born weighing less than 1500 g. Despite years of investigation, the etiology remains unclear, and accepted prevention and treatment strategies are lacking. Studies published over the last year have provided new insight into several aspects of this complex disease. In this review, novel information is presented on (1) the epidemiology; (2) methods of early diagnosis, such as abdominal magnetic resonance imaging; (3) the importance of risk factors, including assessment of feeding strategies and role of bacterial colonization; (4) the pathophysiology, highlighting experimental and clinical trials evaluating the role of inflammatory mediators and growth factors on the disease; (5) preventive strategies, such as anaerobic bacterial supplementation; and (6) surgical interventions, including peritoneal drainage. Understanding some of these important aspects of necrotizing enterocolitis may help improve the outlook of patients with this dreaded disease. Although the incidence of neonatal necrotizing enterocolitis (NEC) and the mortality stemming from this disease have not significantly improved over the last 30 years, there is exciting new information that may significantly improve the outlook of patients with this overwhelming intestinal emergency in the near future.

Preterm Fetal Hypoxia-Ischemia Causes Hypertonia and Motor Deficits in the Neonatal Rabbit: A Model for Human Cerebral Palsy?

Prenatal hypoxia-ischemia to the developing brain has been strongly implicated in the subsequent development of the hypertonic motor deficits of cerebral palsy (CP) in premature and full-term infants who present with neonatal encephalopathy. Despite the enormous impact of CP, there is no animal model that reproduces the hypertonia and motor disturbances of this disorder. We report a rabbit model of in utero placental insufficiency, in which hypertonia is accompanied by marked abnormalities in motor control. Preterm fetuses (67-70% gestation) were subjected to sustained global hypoxia. The dams survived and gave spontaneous birth. At postnatal day 1, the pups that survived were subjected to a battery of neurobehavioral tests developed specifically for these animals, and the tests were videotaped and scored in a masked manner. Newborn pups of hypoxic groups displayed significant impairment in multiple tests of spontaneous locomotion, reflex motor activity, and the coordination of suck and swallow. Increased tone of the limbs at rest and with active flexion and extension were observed in the survivors of the preterm insult. Histopathological studies identified a distinct pattern of acute injury to subcortical motor pathways that involved the basal ganglia and thalamus. Persistent injury to the caudate putamen and thalamus at P1 was significantly correlated with hypertonic motor deficits in the hypoxic group. Antenatal hypoxia-ischemia at preterm gestation results in hypertonia and abnormalities in motor control. These findings provide a unique behavioral model to define mechanisms and sequelae of perinatal brain injury from antenatal hypoxia-ischemia.

Intestinal epithelial apoptosis initiates gross bowel necrosis in an experimental rat model of neonatal necrotizing enterocolitis.

The histopathology of necrotizing enterocolitis (NEC) is characterized by destruction of the mucosal layer in initial stages and by transmural necrosis of the intestinal wall in advanced stages of the disease. To test the hypothesis that enhanced epithelial apoptosis is an initial event underlying the gross histologic changes, we analyzed epithelial apoptosis and tissue morphology in an animal model of NEC and evaluated the effect of caspase inhibition on the incidence of experimental NEC in this model. Apoptosis was analyzed with terminal deoxynucleotidyltransferase-mediated dUTP-FITC nick end labeling (TUNEL) staining in intestinal sections and by measuring caspase 3 activity from intestinal lysates of neonatal rats subjected to formula feeding and cold/asphyxia stress (FFCAS) and from mother-fed (MF) controls. Morphologic evaluation was based on hematoxylin and eosin staining of intestinal sections. FFCAS resulted in histologic changes consistent with NEC, which were absent from MF animals. FFCAS was also associated with a significantly increased rate of nuclear DNA fragmentation in the small intestinal epithelium compared with MF. Elevated tissue caspase 3 activity confirmed the presence of apoptosis in samples with increased DNA fragmentation. Analysis of the coincidence of morphologic damage and apoptosis in corresponding tissue sections indicated that apoptosis precedes gross morphologic changes in this model. Furthermore, supplementation of formula with 8 boc-aspartyl(OMe)-fluoromethylketone, a pan-caspase inhibitor, significantly reduced the incidences of apoptosis and experimental NEC. These findings indicate that in neonatal rats FFCAS induces epithelial apoptosis that serves as an underlying cause for subsequent gross tissue necrosis.

Polyunsaturated fatty acid supplementation alters proinflammatory gene expression and reduces the incidence of necrotizing enterocolitis in a neonatal rat model.

Although supplementation of preterm formula with polyunsaturated fatty acids (PUFA) has been shown to reduce the incidence of necrotizing enterocolitis (NEC) in animal models and clinical trials, the mechanisms remain elusive. We hypothesized that the protective effect of PUFA on NEC may be due to the ability of PUFA to suppress Toll-like receptor (TLR) 4 and platelet-activating factor receptor (PAFR) gene expression (molecules that are important in the pathogenesis of NEC) in epithelial cells. To investigate the efficacy of different PUFA preparations on NEC in a neonatal rat model, we compared the incidence of NEC among the four PUFA supplemented groups—A: arachidonic acid and docosahexaenoic acid (AA+DHA), B: egg phospholipids (EP), C: DHA, and D: control without PUFA. PUFA supplementation reduced the incidence of NEC and inhibited intestinal PAFR and TLR4 gene expression compared with the controls. To validate the in vivo observations, IEC-6 cells were exposed to PAF after pretreatment with AA or DHA. Both AA and DHA supplementation blocked PAF-induced TLR4 and PAFR mRNA expression in these enterocytes. These results suggest that PUFA modulates gene expression of key factors involved in experimental NEC pathogenesis. These effects might in part explain the protective effect of PUFA on neonatal NEC.

Effect of Polyunsaturated Fatty Acid (PUFA) Supplementation on Intestinal Inflammation and Necrotizing Enterocolitis (NEC) in a Neonatal Rat Model

Inasmuch as long-chain polyunsaturated fatty acids (PUFA, metabolites of the essential n-3 and n-6 fatty acids) are known to modulate inflammation, we hypothesized that supplementation of formula with these compounds would prevent necrotizing enterocolitis (NEC) and intestinal inflammation in our neonatal rat model. Newborn rats were stressed with asphyxia and formula feeding, and randomly assigned to control formula, control with PUFA supplementation, and PUFA with nucleotides. Animals were followed for 72–96 h and assessed for death, gross and histologic NEC, intestinal apoptosis, endotoxemia, and intestinal mRNA synthesis of phospholipase A2-II (rate-limiting enzyme for platelet activating factor production), platelet activating factor receptor, and inducible nitric oxide synthase. We found that PUFA reduced the incidence of death and NEC compared with the other groups (NEC 8 of 24 versus 17 of 24 control and 13 of 23 PUFA + nucleotides, p < 0.05). Furthermore, PUFA reduced plasma endotoxemia at 48 h (25 ± 4 EU/mL versus 276 ± 39 EU/mL in control and 170 ± 28 EU/mL in PUFA + nucleotide), intestinal phospholipase A2-II expression at 24 h, and platelet activating factor receptor expression at 48 h. Formula supplementation had no effect on apoptosis of intestinal epithelium or intestinal inducible nitric oxide synthase expression. Addition of nucleotides with PUFA abrogated the beneficial effects of PUFA on intestinal inflammation. We conclude that PUFA reduces the incidence of NEC and intestinal inflammation in a neonatal rat model.

The importance of pro-inflammatory signaling in neonatal necrotizing enterocolitis.

Despite modern medical advances, necrotizing enterocolitis (NEC) remains a significant cause of morbidity and mortality in neonatal intensive care units, affecting 10% of premature neonates born weighing less than 1500 g. Although many advances have been made in the understanding of NEC, the etiology and pathophysiology remain incompletely understood, and treatment is limited to supportive care. In recent years, many studies have evaluated the inflammatory cascade that is central to the disease process, and research is ongoing into strategies to prevent and/or ameliorate neonatal NEC. In this review, we examine the key points in the signaling pathways involved in NEC, and potential strategies for prevention and treatment of this dreaded disease.

Adenosine regulation of alveolar fluid clearance

Adenosine is a purine nucleoside that regulates cell function through G protein-coupled receptors that activate or inhibit adenylyl cyclase. Based on the understanding that cAMP regulates alveolar epithelial active Na+ transport, we hypothesized that adenosine and its receptors have the potential to regulate alveolar ion transport and airspace fluid content. Herein, we report that type 1 (A1R), 2a (A2aR), 2b (A2bR), and 3 (A3R) adenosine receptors are present in rat and mouse lungs and alveolar type 1 and 2 epithelial cells (AT1 and AT2). Rat AT2 cells generated and produced cAMP in response to adenosine, and micromolar concentrations of adenosine were measured in bronchoalveolar lavage fluid from mice. Ussing chamber studies of rat AT2 cells indicated that adenosine affects ion transport through engagement of A1R, A2aR, and/or A3R through a mechanism that increases CFTR and amiloride-sensitive channel function. Intratracheal instillation of low concentrations of adenosine (≤10−8M) or either A2aR- or A3R-specific agonists increased alveolar fluid clearance (AFC), whereas physiologic concentrations of adenosine (≥10−6M) reduced AFC in mice and rats via an A1R-dependent pathway. Instillation of a CFTR inhibitor (CFTRinh-172) attenuated adenosine-mediated down-regulation of AFC, suggesting that adenosine causes Cl− efflux by means of CFTR. These studies report a role for adenosine in regulation of alveolar ion transport and fluid clearance. These findings suggest that physiologic concentrations of adenosine allow the alveolar epithelium to counterbalance active Na+ absorption with Cl− efflux through engagement of the A1R and raise the possibility that adenosine receptor ligands can be used to treat pulmonary edema.

Inhibition of Nuclear Factor-κB Ameliorates Bowel Injury and Prolongs Survival in a Neonatal Rat Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a major cause of morbidity and death in premature infants. NEC is associated with increased levels of pro-inflammatory cytokines in plasma and tissues that are regulated by the transcription factor nuclear factor-κB (NF-κB). It remains unknown, however, whether NF-κB mediates injury in neonatal NEC. We therefore examined the activation status of NF-κB perinatally in the small intestine and in a neonatal rat model of NEC. We found that intestinal NF-κB is strongly activated at birth and, in dam-fed newborn rats, is down-regulated within a day. In contrast, NF-κB remains strongly activated at both d 1 and d 2 in stressed animals, and this is accompanied by a significant decrease in the levels of the endogenous NF-κB inhibitor protein IκBα and IκBβ at d 2. To determine the importance of elevated NF-κB activity in intestinal injury in NEC, we administered the NEMO-binding domain (NBD) peptide that selectively inhibits the critical upstream IκB kinase (IKK). NBD but not a control peptide decreased mortality and bowel injury in this model, supporting the hypothesis that bowel injury in NEC results from elevated NF-κB activity. Our findings therefore lead us to conclude that selective NF-κB inhibition represents a promising therapeutic strategy for NEC.

The biogenesis, traffic, and function of the cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride channel that is encoded by the gene that is defective in cystic fibrosis. This ion channel resides at the luminal surfaces and in endosomes of epithelial cells that line the airways, intestine, and a variety of exocrine glands. In this article we discuss current hypotheses regarding how CFTR functions as a regulated ion channel and how CF mutations lead to disease. We also evaluate the emerging notion that CFTR is a multifunctional protein that is capable of regulating epithelial physiology at several levels, including the modulation of other ion channels and the regulation of intracellular membrane traffic. Elucidating the various functions of CFTR should contribute to our understanding of the pathology in cystic fibrosis, the most common lethal genetic disorder among Caucasians.