Steven J. McElroy

Tumor necrosis factor receptor 1-dependent depletion of mucus in immature small intestine: a potential role in neonatal necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants. NEC is believed to occur when intestinal bacteria invade the intestinal epithelial layer, causing subsequent inflammation and tissue necrosis. Mucins are produced and secreted by epithelial goblet cells as a key component of the innate immune system and barrier function of the intestinal tract that help protect against bacterial invasion. To better understand the role of mucins in NEC, we quantified the number of mucus-containing small intestinal goblet cells present in infants with NEC and found they had significantly fewer goblet cells and Paneth cells compared with controls. To test whether inflammation has a developmentally dependent effect on intestinal goblet cells, TNF-α was injected into mice at various stages of intestinal development. TNF-α caused a loss of mucus-containing goblet cells only in immature mice and induced Muc2 and Muc3 mRNA upregulation only in mature ileum. Only minimal changes were seen in apoptosis and in expression of markers of goblet cell differentiation. TNF-α increased small intestinal mucus secretion and goblet cell hypersensitivity to prostaglandin E2 (PGE(2)), a known mucus secretagogue produced by macrophages. These TNF-α-induced changes in mucus mRNA levels required TNF receptor 2 (TNFR2), whereas TNF-α-induced loss of mucus-positive goblet cells required TNFR1. Our findings of developmentally dependent TNF-α-induced alterations on intestinal mucus may help explain why NEC is predominantly found in premature infants, and TNF-α-induced alterations of the intestinal innate immune system and barrier functions may play a role in the pathogenesis of NEC itself.

Paneth cells and necrotizing enterocolitis: a novel hypothesis for disease pathogenesis.

Current models of necrotizing enterocolitis (NEC) propose that intraluminal microbes destroy intestinal mucosa and activate an inflammatory cascade that ends in necrosis. We suggest an alternate hypothesis wherein NEC is caused by injury to Paneth cells (PCs) in the intestinal crypts. PCs are specialized epithelia that protect intestinal stem cells from pathogens, stimulate stem cell differentiation, shape the intestinal microbiota, and assist in repairing the gut. Our novel model of NEC uses neonatal mice and ablates PCs followed by enteral infection. We contrast this model with other animal examples of NEC and the clinical disease. Selective destruction of PCs using dithizone likely releases tumor necrosis factor-α and other inflammatory mediators. We propose that this event produces inflammation in the submucosa, generates platelet-activating factor, and induces a coagulopathy. The role of PCs in NEC is consistent with the onset of disease in preterm infants after a period of PC-related maturation, the central role of PCs in crypt-related homeostasis, the anatomic location of pneumatosis intestinalis close to the crypts, and the proximity of PCs to occluded blood vessels that cause coagulation necrosis of the intestinal villi. We offer this hypothesis to promote new thoughts about how NEC occurs and its potential prevention.

Transactivation of EGFR by LPS induces COX-2 expression in enterocytes

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal morbidity and mortality in preterm infants. NEC is characterized by an exaggerated inflammatory response to bacterial flora leading to bowel necrosis. Bacterial lipopolysaccharide (LPS) mediates inflammation through TLR4 activation and is a key molecule in the pathogenesis of NEC. However, LPS also induces cyclooxygenase-2 (COX-2), which promotes intestinal barrier restitution through stimulation of intestinal cell survival, proliferation, and migration. Epidermal growth factor receptor (EGFR) activation prevents experimental NEC and may play a critical role in LPS-stimulated COX-2 production. We hypothesized that EGFR is required for LPS induction of COX-2 expression. Our data show that inhibiting EGFR kinase activity blocks LPS-induced COX-2 expression in small intestinal epithelial cells. LPS induction of COX-2 requires Src-family kinase signaling while LPS transactivation of EGFR requires matrix metalloprotease (MMP) activity. EGFR tyrosine kinase inhibitors block LPS stimulation of mitogen-activated protein kinase ERK, suggesting an important role of the MAPK/ERK pathway in EGFR-mediated COX-2 expression. LPS stimulates proliferation of IEC-6 cells, but this stimulation is inhibited with either the EGFR kinase inhibitor AG1478, or the selective COX-2 inhibitor Celecoxib. Taken together, these data show that EGFR plays an important role in LPS-induction of COX-2 expression in enterocytes, which may be one mechanism for EGF in inhibition of NEC.

Epidermal growth factor receptor inhibits colitis-associated cancer in mice

Inflammatory bowel disease (IBD) is a chronic illness caused by complex interactions between genetic and environmental factors that propagate inflammation and damage to the gastrointestinal epithelium. This state of chronic inflammation increases the risk for development of colitis-associated cancer in IBD patients. Thus, the development of targeted therapeutics that can disrupt the cycle of inflammation and epithelial injury is highly attractive. However, such biological therapies, including those targeting epidermal growth factor receptor pathways, pose a risk of increasing cancer rates. Using two mouse models of colitis-associated cancer, we found that epidermal growth factor receptor inactivation accelerated the incidence and progression of colorectal tumors. By modulating inflammation and epithelial regeneration, epidermal growth factor receptor optimized the response to chronic inflammation and limited subsequent tumorigenesis. These findings provide important insights into the pathogenesis of colitis-associated cancer and suggest that epidermal growth factor-based therapies for IBD may reduce long-term cancer risk.

Paneth cell ablation in the presence of Klebsiella pneumoniae induces necrotizing enterocolitis (NEC)-like injury in the small intestine of immature mice

SUMMARY Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants. During NEC pathogenesis, bacteria are able to penetrate innate immune defenses and invade the intestinal epithelial layer, causing subsequent inflammation and tissue necrosis. Normally, Paneth cells appear in the intestinal crypts during the first trimester of human pregnancy. Paneth cells constitute a major component of the innate immune system by producing multiple antimicrobial peptides and proinflammatory mediators. To better understand the possible role of Paneth cell disruption in NEC, we quantified the number of Paneth cells present in infants with NEC and found that they were significantly decreased compared with age-matched controls. We were able to model this loss in the intestine of postnatal day (P)14-P16 (immature) mice by treating them with the zinc chelator dithizone. Intestines from dithizone-treated animals retained approximately half the number of Paneth cells compared with controls. Furthermore, by combining dithizone treatment with exposure to Klebsiella pneumoniae, we were able to induce intestinal injury and inflammatory induction that resembles human NEC. Additionally, this novel Paneth cell ablation model produces NEC-like pathology that is consistent with other currently used animal models, but this technique is simpler to use, can be used in older animals that have been dam fed, and represents a novel line of investigation to study NEC pathogenesis and treatment.

TNFR1 Promotes Tumor Necrosis Factor-mediated Mouse Colon Epithelial Cell Survival through RAF Activation of NF-κB

Tumor necrosis factor (TNF) is a therapeutic target in the treatment of inflammatory bowel disease; however, the exact role of TNF signaling in the colon epithelium remains unclear. We demonstrate that TNF activation of TNF receptor (R)1 stimulates both pro- and anti-apoptotic signaling pathways in the colon epithelium; however, TNFR1 protects against colon epithelial cell apoptosis following TNF exposure. To investigate anti-apoptotic signaling pathways downstream of TNFR1, we generated an intestinal epithelium-specific Raf knock-out mouse and identified Raf kinase as a key regulator of colon epithelial cell survival in response to TNF. Surprisingly, Raf promotes NF-κB p65 phosphorylation, independent of MEK signaling, to support cell survival. Taken together, these data demonstrate a novel pathway in which Raf promotes colon epithelial cell survival through NF-κB downstream of TNFR1 activation. Thus, further understanding of colon epithelial cell-specific TNFR signaling may result in the identification of new targets for inflammatory bowel disease treatment and define novel mediators of colitis-associated cancer.

Tumor necrosis factor inhibits ligand-stimulated EGF receptor activation through a TNF receptor 1-dependent mechanism

Tumor necrosis factor (TNF) and epidermal growth factor (EGF) are key regulators in the intricate balance maintaining intestinal homeostasis. Previous work from our laboratory shows that TNF attenuates ligand-driven EGF receptor (EGFR) phosphorylation in intestinal epithelial cells. To identify the mechanisms underlying this effect, we examined EGFR phosphorylation in cells lacking individual TNF receptors. TNF attenuated EGF-stimulated EGFR phosphorylation in wild-type and TNFR2(-/-), but not TNFR1(-/-), mouse colon epithelial (MCE) cells. Reexpression of wild-type TNFR1 in TNFR1(-/-) MCE cells rescued TNF-induced EGFR inhibition, but expression of TNFR1 deletion mutant constructs lacking the death domain (DD) of TNFR1 did not, implicating this domain in EGFR downregulation. Blockade of p38 MAPK, but not MEK, activation of ERK rescued EGF-stimulated phosphorylation in the presence of TNF, consistent with the ability of TNFR1 to stimulate p38 phosphorylation. TNF promoted p38-dependent EGFR internalization in MCE cells, suggesting that desensitization is achieved by reducing receptor accessible to ligand. Taken together, these data indicate that TNF activates TNFR1 by DD- and p38-dependent mechanisms to promote EGFR internalization, with potential impact on EGF-induced proliferation and migration key processes that promote healing in inflammatory intestinal diseases.

Targeting IL-17A attenuates neonatal sepsis mortality induced by IL-18

Significance Infants born prematurely suffer the greatest incidence of and impact from sepsis among all age groups. Therapeutic interventions aimed at reducing morbidity and mortality in this vulnerable population have been unsuccessful. Interleukin (IL)-18 is a proinflammatory member of the IL-1 superfamily. Serum IL-18 concentrations in uninfected premature infants are increased as compared with healthy adults. We show that IL-18 in the setting of sepsis results in gut injury, a potentiation of the host’s inflammatory response, increased bacteremia, and mortality mediated by IL-1 receptor 1 (IL-1R1)–dependent IL-17A produced by γδT and myeloid cells. The discovery of this novel IL-18/IL-1R1/IL-17A axis brings new hope for therapeutic interventions that target downstream IL-17A and ultimately reduce the increased mortality from sepsis in this understudied population. Interleukin (IL)-18 is an important effector of innate and adaptive immunity, but its expression must also be tightly regulated because it can potentiate lethal systemic inflammation and death. Healthy and septic human neonates demonstrate elevated serum concentrations of IL-18 compared with adults. Thus, we determined the contribution of IL-18 to lethality and its mechanism in a murine model of neonatal sepsis. We find that IL-18–null neonatal mice are highly protected from polymicrobial sepsis, whereas replenishing IL-18 increased lethality to sepsis or endotoxemia. Increased lethality depended on IL-1 receptor 1 (IL-1R1) signaling but not adaptive immunity. In genome-wide analyses of blood mRNA from septic human neonates, expression of the IL-17 receptor emerged as a critical regulatory node. Indeed, IL-18 administration in sepsis increased IL-17A production by murine intestinal γδT cells as well as Ly6G+ myeloid cells, and blocking IL-17A reduced IL-18–potentiated mortality to both neonatal sepsis and endotoxemia. We conclude that IL-17A is a previously unrecognized effector of IL-18–mediated injury in neonatal sepsis and that disruption of the deleterious and tissue-destructive IL-18/IL-1/IL-17A axis represents a novel therapeutic approach to improve outcomes for human neonates with sepsis.

Correlation of abdominal rSO2 with superior mesenteric artery velocities in preterm infants.

Objective:Near-infrared spectroscopy (NIRS) is used to monitor brain and kidney perfusion in at-risk premature and term neonates. Although NIRS holds potential for bedside monitoring of intestinal perfusion, there is insufficient evidence showing correlation with mesenteric blood flow. To determine if an association exists between abdominal regional oxygen saturation (A-rSO2) and mesenteric blood flow, we compared changes in A-rSO2 to changes in blood flow velocity in the superior mesenteric artery (SMA) before and after feedings in very-low birthweight infants.Study Design:A-rSO2 was continuously monitored midline below the umbilicus for 3 days in 18 stable 25 to 31 week bolus-fed infants (median BW 1203 g, median age 5 days). We compared change in SMA velocity from immediately before to 10 min and 60 to 120 min after feeding with change in A-rSO2 over the same time. Spearman’s rank correlation was used to ascertain if a significant association existed.Result:Change in A-rSO2 was significantly associated with change in systolic, diastolic, and mean SMA velocity from fasting to 60 to 120 min after feeding (P=0.016, 0.021, 0.010) and from 10 min after a feed to 60 to 120 min after feeding (P=0.009, 0.035, 0.032).Conclusion:In very preterm infants, A-rSO2 reflects blood flow in the SMA and can provide non-invasive continuous monitoring of intestinal perfusion. Further studies are indicated to determine the sensitivity of NIRS to detect early intestinal pathology in this population.

Elective High-Frequency Oscillatory Ventilation versus Conventional Ventilation for Acute Pulmonary Dysfunction in Preterm Infants

BACKGROUND Respiratory failure due to lung immaturity is a major cause of mortality in preterm infants. Although the use of intermittent positive pressure ventilation in neonates with respiratory failure saves lives, its use is associated with lung injury and chronic lung disease (CLD). A newer form of ventilation called high-frequency oscillatory ventilation (HFOV) has been shown to result in less lung injury in experimental studies. OBJECTIVES The objective of this review is to determine the effect of the elective use of HFOV as compared to conventional ventilation (CV) on the incidence of CLD, mortality and other complications associated with prematurity and assisted ventilation in preterm infants who are mechanically ventilated for respiratory distress syndrome (RDS). SEARCH METHODS Searches were made of the Oxford Database of Perinatal Trials, MEDLINE, EMBASE, previous reviews including cross-references, abstracts, conferences and symposia proceedings, expert informants, journal hand searching by the Cochrane Collaboration, mainly in the English language. The search was updated in January 2009. SELECTION CRITERIA Randomized controlled trials comparing HFOV and CV in preterm or low birth weight infants with pulmonary dysfunction, mainly due to RDS, who required assisted ventilation. Randomization and commencement of treatment needed to be as soon as possible after the start of CV and usually in the first 12 h of life. DATA COLLECTION AND ANALYSIS The methodological quality of each trial was independently reviewed by the various authors. The standard effect measures are relative risk (RR) and risk difference (RD). From 1/RD the number needed to treat to produce one outcome were calculated. For all measures of effect, 95% confidence intervals were used. In subgroup analyses the 99% CIs are also given for summary RRs in the text. Meta-analysis was performed using a fixed effect model. Where heterogeneity was over 50%, the random effects RR is also given. MAIN RESULTS Seventeen eligible studies of 3,652 infants were included. Meta-analysis comparing HFOV with CV revealed no evidence of effect on mortality at 28-30 days of age or at approximately term equivalent age. These results were consistent across studies and in subgroup analyses. The effect of HFOV on CLD in survivors at term equivalent gestational age was inconsistent across studies and the reduction was of borderline significance overall. The effect was similar in trials with a high lung volume strategy for HFOV targeting at very low FiO(2) and trials with a high lung volume strategy with somewhat higher or unspecified target FiO(2). Subgroups of trials showed a significant reduction in CLD with HFOV when no surfactant was used, when piston oscillators were used for HFOV, when lung protective strategies for CV were not used, when randomization occurred at two to six hours of age, and when inspiratory:expiratory ratio of 1:2 was used for HFOV. In the meta-analysis of all trials, pulmonary air leaks occurred more frequently in the HFOV group. In some studies, short-term neurological morbidity with HFOV was found, but this effect was not statistically significant overall. The subgroup of two trials not using a high-volume strategy with HFOV found increased rates of grade 3 or 4 intraventricular hemorrhage and of periventricular leukomalacia. An adverse effect of HFOV on long-term neurodevelopment was found in one large trial but not in the five other trials that reported this outcome. The rate of retinopathy of prematurity is reduced overall in the HFOV group.