Erika C. Claud

16S rRNA gene-based analysis of fecal microbiota from preterm infants with and without necrotizing enterocolitis

Neonatal necrotizing enterocolitis (NEC) is an inflammatory intestinal disorder affecting preterm infants. Intestinal bacteria have an important function; however no causative pathogen has been identified. The purpose of this study was to determine if there are differences in microbial patterns that may be critical to the development of this disease. Fecal samples from 20 preterm infants, 10 with NEC and 10 matched controls (including 4 twin pairs) were obtained from patients in a single site level III neonatal intensive care unit. Bacterial DNA from individual fecal samples was PCR-amplified and subjected to terminal restriction fragment length polymorphism analysis and library sequencing of the 16S rRNA gene to characterize diversity and structure of the enteric microbiota. The distribution of samples from NEC patients distinctly clustered separately from controls. Intestinal bacterial colonization in all preterm infants was notable for low diversity. Patients with NEC had even less diversity, an increase in abundance of Gammaproteobacteria, a decrease in other bacteria species, and had received a higher mean number of previous days of antibiotics. Our results suggest that NEC is associated with severe lack of microbiota diversity that may accentuate the impact of single dominant microorganisms favored by empiric and widespread use of antibiotics.

Hypothesis: inappropriate colonization of the premature intestine can cause neonatal necrotizing enterocolitis

Neonatal necrotizing enterocolitis (NEC) is a major cause of morbidity in preterm infants. We hypothesize that the intestinal injury in this disease is a consequence of synergy among three of the major risk factors for NEC: prematurity, enteral feeding, and bacterial colonization. Together these factors result in an exaggerated inflammatory response, leading to ischemic bowel necrosis. Human milk may decrease the incidence of NEC by decreasing pathogenic bacterial colonization, promoting growth of nonpathogenic flora, promoting maturation of the intestinal barrier, and ameliorating the proinflammatory response.— Claud, E. C., Walker, W. A. Hypothesis: inappropriate colonization of the premature intestine can cause neonatal necrotizing enterocolitis. The FASEB J. 15, 1398–1403 (2001)

The Mechanism of Excessive Intestinal Inflammation in Necrotizing Enterocolitis: An Immature Innate Immune Response

Necrotizing enterocolitis (NEC) is a devastating neonatal intestinal inflammatory disease, occurring primarily in premature infants, causing significant morbidity and mortality. The pathogenesis of NEC is associated with an excessive inflammatory IL-8 response. In this study, we hypothesized that this excessive inflammatory response is related to an immature expression of innate immune response genes. To address this hypothesis, intestinal RNA expression analysis of innate immune response genes was performed after laser capture microdissection of resected ileal epithelium from fetuses, NEC patients and children and confirmed in ex vivo human intestinal xenografts. Changes in mRNA levels of toll-like receptors (TLR)-2 and -4, their signaling molecules and transcription factors (MyD88, TRAF-6 and NFκB1) and negative regulators (SIGIRR, IRAK-M, A-20 and TOLLIP) and the effector IL-8 were characterized by qRT-PCR. The expression of TLR2, TLR4, MyD88, TRAF-6, NFκB1 and IL-8 mRNA was increased while SIGIRR, IRAK-M, A-20 and TOLLIP mRNA were decreased in fetal vs. mature human enterocytes and further altered in NEC enterocytes. Similar changes in mRNA expression were observed in immature, but not mature, human intestinal xenografts. Confirmation of gene expression was also validated with selective protein measurements and with suggested evidence that immature TRL4 enterocyte surface expression was internalized in mature enterocytes. Cortisone, an intestinal maturation factor, treatment corrected the mRNA differences only in the immature intestinal xenograft. Using specific siRNA to attenuate expression of primary fetal enterocyte cultures, both TOLLIP and A-20 were confirmed to be important when knocked down by exhibiting the same excessive inflammatory response seen in the NEC intestine. We conclude that the excessive inflammatory response of the immature intestine, a hallmark of NEC, is due to a developmental immaturity in innate immune response genes.

Modulation of human intestinal epithelial cell IL-8 secretion by human milk factors.

Necrotizing enterocolitis (NEC) seems to result from the inflammatory response of an immature intestine. Human milk is protective against NEC via an unknown mechanism. We hypothesized that specific factors found in human milk would decrease stimulated IL-8 secretion in intestinal epithelial cells. HT29-cl19A and Caco2 cells were compared with the fetal human primary intestinal epithelial cell line H4 and temperature-sensitive conditionally immortalized fetal human intestinal (tsFHI) cells. Cells were pretreated with transforming growth factor-β (TGF-β), erythropoietin (Epo), IL-10, or epidermal growth factor (EGF) at physiologic concentrations before stimulation with tumor necrosis factor-α (TNF-α) or IL-1β, and then IL-8 was measured by ELISA. The fetal cells produced significantly more IL-8 when stimulated by TNF-α or IL-1β. There were also differences in the pattern of alteration of IL-8 secretion by human milk factors. In HT29-cl19A cells, IL-10 inhibited TNF-α-stimulated IL-8 secretion by 52%, and EGF increased secretion by 144%. In H4 cells, TGF-β1 and Epo inhibited TNF-α-stimulated IL-8 secretion to control levels, and EGF increased secretion by 29%. IL-1β-stimulated IL-8 secretion was inhibited 25% by TGF-β1 in Caco2 cells and in H4 cells was inhibited by TGF-β1, Epo, and TGF-β2. TsFHI cells confirmed H4 cell results. Fetal human enterocytes have an exaggerated IL-8 secretion in response to TNF-α and IL-1β. TGF-β and Epo decrease this stimulated IL-8 secretion, which may partially explain the protective effect of human milk in NEC.

Intestinal epithelial vitamin D receptor deletion leads to defective autophagy in colitis

Objective Vitamin D and the vitamin D receptor (VDR) appear to be important immunological regulators of inflammatory bowel diseases (IBD). Defective autophagy has also been implicated in IBD, where interestingly, polymorphisms of genes such as ATG16L1 have been associated with increased risk. Although vitamin D, the microbiome and autophagy are all involved in pathogenesis of IBD, it remains unclear whether these processes are related or function independently. Design We investigated the effects and mechanisms of intestinal epithelial VDR in healthy and inflamed states using cell culture models, a conditional VDR knockout mouse model (VDRΔIEC), colitis models and human samples. Results Absence of intestinal epithelial VDR affects microbial assemblage and increases susceptibility to dextran sulfate sodium-induced colitis. Intestinal epithelial VDR downregulates expressions of ATG16L1 and lysozyme, and impairs antimicrobial function of Paneth cells. Gain and loss-of-function assays showed that VDR levels regulate ATG16L1 and lysozyme at the transcriptional and translational levels. Moreover, low levels of intestinal epithelial VDR correlated with reduced ATG16L1 and representation by intestinal Bacteroides in patients with IBD. Administration of the butyrate (a fermentation product of gut microbes) increases intestinal VDR expression and suppresses inflammation in a colitis model. Conclusions Our study demonstrates fundamental relationship between VDR, autophagy and gut microbial assemblage that is essential for maintaining intestinal homeostasis, but also in contributing to the pathophysiology of IBD. These insights can be leveraged to define therapeutic targets for restoring VDR expression and function.

Bacterial community structure and functional contributions to emergence of health or necrotizing enterocolitis in preterm infants

BackgroundPreterm infants represent a unique patient population that is born functionally immature and must accomplish development under the influence of a hospital environment. Neonatal necrotizing enterocolitis (NEC) is an inflammatory intestinal disorder affecting preterm infants. The purpose of this study was to evaluate the progression of intestinal microbiota community development between preterm infants who remained healthy compared to preterm infants who developed NEC.ResultsWeekly fecal samples from ten preterm infants, five with NEC and five matched healthy controls were obtained. Bacterial DNA from individual fecal samples was subjected to sequencing of 16S rRNA-based inventories using the 454 GS-FLX platform. Fecal samples from control infants demonstrated a temporal pattern in their microbiota, which converged toward that of a healthy full term breast-fed infant. Microbiota development in NEC patients diverged from controls beginning three weeks prior to diagnosis. Shotgun metagenomic sequencing was performed to identify functional differences in the respective microbiota of fecal samples from a set of twins in which one twin developed NEC and one did not. The majority of the differentially abundant genes in the NEC patient were associated with carbohydrate metabolism and mapped to members of the family Enterobacteriaceae. This may indicate an adaptation of the community to an altered profile of substrate availability for specific members as a first step towards the development of NEC. We propose that the microbial communities as a whole may metabolize milk differently, resulting in differential substrate availability for specific microbial groups. Additional differentially represented gene sets of interest were related to antibiotic resistance and vitamin biosynthesis.ConclusionsOur results suggest that there is a temporal component to microbiome development in healthy preterm infants. Thus, bacteriotherapy for the treatment or prevention of NEC must consider this temporal component of the microbial community in addition to its taxonomic composition and functional content.

Bacterial Colonization, Probiotics, and Necrotizing Enterocolitis

The intestinal microflora has a significant role in intestinal health and gut function. The neonatal population is unique in that intestinal colonization is not established and is known to be influenced by delivery method, feeding, gestational age, and medical interventions. The preterm infant is particularly sensitive to colonization patterns as inherent intestinal defense mechanisms are immature and immature intestinal epithelial cells are known to have exaggerated inflammatory responses to both commensal and pathogenic bacteria. These responses contribute to the development of neonatal necrotizing enterocolitis in this patient population. As certain bacteria are known to influence intestinal maturation and down-regulate intestinal inflammation, it has been suggested that influencing the intestinal flora of preterm infants may be beneficial. Clinical studies indicate that probiotic therapy may decrease the incidence of necrotizing enterocolitis and studies are ongoing to elucidate the mechanism of action of different probiotic organisms. Although concerns remain and further study is necessary, probiotics are a plausible means of optimizing intestinal colonization and influencing outcome of these vulnerable infants.

Salmonella type III effector AvrA stabilizes cell tight junctions to inhibit inflammation in intestinal epithelial cells.

Salmonella Typhimurium is a major cause of human gastroenteritis. The Salmonella type III secretory system secretes virulence proteins, called effectors. Effectors are responsible for the alteration of tight junction (TJ) structure and function in intestinal epithelial cells. AvrA is a newly described bacterial effector found in Salmonella. We report here that AvrA expression stabilizes cell permeability and tight junctions in intestinal epithelial cells. Cells colonized with an AvrA-deficient bacterial strain (AvrA−) displayed decreased cell permeability, disruption of TJs, and an increased inflammatory response. Western blot data showed that TJ proteins, such as ZO-1, claudin-1, decreased after AvrA- colonization for only 1 hour. In contrast, cells colonized with AvrA-sufficient bacteria maintained cell permeability with stabilized TJ structure. This difference was confirmed in vivo. Fluorescent tracer studies showed increased fluorescence in the blood of mice infected with AvrA- compared to those infected with the AvrA-sufficient strains. AvrA- disrupted TJ structure and function and increased inflammation in vivo, compared to the AvrA- sufficient strain. Additionally, AvrA overexpression increased TJ protein expression when transfected into colonic epithelial cells. An intriguing aspect of this study is that AvrA stabilized TJs, even though the other TTSS proteins, SopB, SopE, and SopE2, are known to disrupt TJs. AvrA may play a role in stabilizing TJs and balancing the opposing action of other bacterial effectors. Our findings indicate an important role for the bacterial effector AvrA in regulation of intestinal epithelial cell TJs during inflammation. The role of AvrA represents a highly refined bacterial strategy that helps the bacteria survive in the host and dampen the inflammatory response.

Bacteria-Free Solution Derived from Lactobacillus plantarum Inhibits Multiple NF-KappaB Pathways and Inhibits Proteasome Function

Background: Bacteria play a role in inflammatory bowel disease and other forms of intestinal inflammation. Although much attention has focused on the search for a pathogen or inciting inflammatory bacteria, another possibility is a lack of beneficial bacteria that normally confer anti‐inflammatory properties in the gut. The purpose of this study was to determine whether normal commensal bacteria could inhibit inflammatory pathways important in intestinal inflammation. Methods: Conditioned media from Lactobacillus plantarum (Lp‐CM) and other gut bacteria was used to treat intestinal epithelial cell (YAMC) and macrophage (RAW 264.7) or primary culture murine dendritic cells. NF‐&kgr;B was activated through TNF‐Receptor, MyD88‐dependent and ‐independent pathways and effects of Lp‐CM on the NF‐&kgr;B pathway were assessed. NF‐&kgr;B binding activity was measured using ELISA and EMSA. 1&kgr;B expression was assessed by Western blot analysis, and proteasome activity determined using fluorescence‐based proteasome assays. MCP‐1 release was determined by ELISA. Results: Lp‐CM inhibited NF‐&kgr;B binding activity, degradation of I&kgr;B&agr; and the chymotrypsin‐like activity of the proteasome. Moreover, Lp‐CM directly inhibited the activity of purified mouse proteasomes. This effect was specific, since conditioned media from other bacteria had no inhibitory effect. Unlike other proteasome inhibitors, Lp‐CM was not toxic in cell death assays. Lp‐CM inhibited MCP‐1 release in all cell types tested. Conclusions: These studies confirm, and provide a mechanism for, the anti‐inflammatory effects of the probiotic and commensal bacterium Lactobacillus plantarum. The use of bacteria‐free Lp‐CM provides a novel strategy for treatment of intestinal inflammation which would eliminate the risk of bacteremia reported with conventional probiotics. Inflamm Bowel Dis 2009

Erythropoietin Protects Intestinal Epithelial Barrier Function and Lowers the Incidence of Experimental Neonatal Necrotizing Enterocolitis

The impermeant nature of the intestinal barrier is maintained by tight junctions (TJs) formed between adjacent intestinal epithelial cells. Disruption of TJs and loss of barrier function are associated with a number of gastrointestinal diseases, including neonatal necrotizing enterocolitis (NEC), the leading cause of death from gastrointestinal diseases in preterm infants. Human milk is protective against NEC, and the human milk factor erythropoietin (Epo) has been shown to protect endothelial cell-cell and blood-brain barriers. We hypothesized that Epo may also protect intestinal epithelial barriers, thereby lowering the incidence of NEC. Our data demonstrate that Epo protects enterocyte barrier function by supporting expression of the TJ protein ZO-1. As immaturity is a key factor in NEC, Epo regulation of ZO-1 in the human fetal immature H4 intestinal epithelial cell line was examined and demonstrated Epo-stimulated ZO-1 expression in a dose-dependent manner through the PI3K/Akt pathway. In a rat NEC model, oral administration of Epo lowered the incidence of NEC from 45 to 23% with statistical significance. In addition, Epo treatment protected intestinal barrier function and prevented loss of ZO-1 at the TJs in vivo. These effects were associated with elevated Akt phosphorylation in the intestine. This study reveals a novel role of Epo in the regulation of intestinal epithelial TJs and barrier function and suggests the possible use of enteral Epo as a therapeutic agent for gut diseases.