Transcriptional Modulation of Intestinal Innate Defense/Inflammation Genes by Preterm Infant Microbiota in a Humanized Gnotobiotic Mouse Model

Background and Aims: It is known that postnatal functional maturation of the small intestine is facilitated by microbial colonization of the gut. Preterm infants exhibit defects in gut maturation, weak innate immunity against intestinal infection and increased susceptibility to inflammatory disorders, all of which may be related to the inappropriate microbial colonization of their immature intestines. The earliest microbes to colonize the preterm infant gut encounter a naïve, immature intestine. Thus this earliest microbiota potentially has the greatest opportunity to fundamentally influence intestinal development and immune function. The aim of this study was to characterize the effect of early microbial colonization on global gene expression in the distal small intestine during postnatal gut development.Methods: Gnotobiotic mouse models with experimental colonization by early (prior to two weeks of life) intestinal microbiota from preterm human infants were utilized. Microarray analysis was used to assess global gene expression in the intestinal epithelium.Results and Conclusion: Multiple intestinal genes involved in metabolism, cell cycle regulation, cell-cell or cell-extracellular matrix communication, and immune function are developmental- and intestinal microbiota- regulated. Using a humanized gnotobiotic mouse model, we demonstrate that certain early preterm infant microbiota from prior to 2 weeks of life specifically induce increased NF-κB activation and a phenotype of increased inflammation whereas other preterm microbiota specifically induce decreased NF-κB activation. These fundamental differences correlate with altered clinical outcomes and suggest the existence of optimal early microbial communities to improve health outcomes.</p

Tight junction CLDN2 gene is a direct target of the vitamin D receptor

The breakdown of the intestinal barrier is a common manifestation of many diseases. Recent evidence suggests that vitamin D and its receptor VDR may regulate intestinal barrier function. Claudin-2 is a tight junction protein that mediates paracellular water transport in intestinal epithelia, rendering them "leaky". Using whole body VDR(-/-) mice, intestinal epithelial VDR conditional knockout (VDR(ΔIEC)) mice, and cultured human intestinal epithelial cells, we demonstrate here that the CLDN2 gene is a direct target of the transcription factor VDR. The Caudal-Related Homeobox (Cdx) protein family is a group of the transcription factor proteins which bind to DNA to regulate the expression of genes. Our data showed that VDR-enhances Claudin-2 promoter activity in a Cdx1 binding site-dependent manner. We further identify a functional vitamin D response element (VDRE) 5΄-AGATAACAAAGGTCA-3΄ in the Cdx1 site of the Claudin-2 promoter. It is a VDRE required for the regulation of Claudin-2 by vitamin D. Absence of VDR decreased Claudin-2 expression by abolishing VDR/promoter binding. In vivo, VDR deletion in intestinal epithelial cells led to significant decreased Claudin-2 in VDR(-/-) and VDR(ΔIEC) mice. The current study reveals an important and novel mechanism for VDR by regulation of epithelial barriers.status: publishe

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

Effects of Intestinal Microbiota on Brain Development in Humanized Gnotobiotic Mice

Poor growth in the Neonatal Intensive Care Unit is associated with an increased risk for poor neurodevelopmental outcomes for preterm infants, however the mechanism is unclear. The microbiome has increasingly been recognized as a modifiable environmental factor to influence host development. Here we explore the hypothesis that the microbiome influences both growth phenotype and brain development. A germ free mouse transfaunation model was used to examine the effects of preterm infant microbiotas known to induce either high growth or low growth phenotypes on postnatal brain development. The microbiome which induced the low growth phenotype was associated with decreases in the neuronal markers NeuN and neurofilament-L as well as the myelination marker MBP when compared to the microbiome associated with the high growth phenotype. Additionally, poor growth phenotype-associated microbiota was associated with increased neuroinflammation marked by increased Nos1, as well as alteration in IGF-1 pathway including decreased circulating and brain IGF-1, decreased circulating IGFBP3, and increased Igfbp3 brain mRNA expression. This study suggests that growth-associated microbiota can influence early neuron and oligodendrocyte development and that this effect may be mediated by effects on neuroinflammation and circulating IGF-1

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 play a key role; however no causative pathogen has been identified. The purpose of this study was to determine if there are differences in microbial patterns which may be critical to the development of this disease. Fecal samples from twenty preterm infants, ten with NEC and ten matched controls (including four twin pairs) were obtained from patients in a single site Level III neonatal intensive care unit. Bacterial DNA from individual fecal samples were 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 which may accentuate the impact of single dominant microorganisms favored by empiric and wide-spread use of antibiotics

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

Axin1 Prevents Salmonella Invasiveness and Inflammatory Response in Intestinal Epithelial Cells

Axin1 and its homolog Axin2 are scaffold proteins essential for regulating Wnt signaling. Axin-dependent regulation of Wnt is important for various developmental processes and human diseases. However, the involvement of Axin1 and Axin2 in host defense and inflammation remains to be determined.Here, we report that Axin1, but not Axin2, plays an essential role in host-pathogen interaction mediated by the Wnt pathway. Pathogenic Salmonella colonization greatly reduces the level of Axin1 in intestinal epithelial cells. This reduction is regulated at the posttranslational level in early onset of the bacterial infection. Further analysis reveals that the DIX domain and Ser614 of Axin1 are necessary for the Salmonella-mediated modulation through ubiquitination and SUMOylation.Axin1 apparently has a preventive effect on bacterial invasiveness and inflammatory response during the early stages of infection. The results suggest a distinct biological function of Axin1 and Axin2 in infectious disease and intestinal inflammation while they are functionally equivalent in developmental settings

Gut Microbiome–Brain Axis as an Explanation for the Risk of Poor Neurodevelopment Outcome in Preterm Infants with Necrotizing Enterocolitis

Necrotizing Enterocolitis (NEC) is characterized by an inflammation of intestinal tissue that primarily affects premature infants. It is the most common and devastating gastrointestinal morbidity of prematurity, but beyond intestinal morbidity, this condition has also been associated with an increased risk of neurodevelopmental delays that persist beyond infancy. Prematurity, enteral feeding, bacterial colonization, and prolonged exposure to antibiotics are all risk factors that predispose preterm infants to NEC. Interestingly, these factors are all also associated with the gut microbiome. However, whether or not there is a connection between the microbiome and the risk of neurodevelopmental delays in infants after NEC is still an emerging area of research. Furthermore, how microbes in the gut could impact a distant organ such as the brain is also poorly understood. In this review, we discuss the current understanding of NEC and the role of the gut microbiome–brain axis in neurodevelopmental outcomes after NEC. Understanding the potential role of the microbiome in neurodevelopmental outcomes is important as the microbiome is modifiable and thus offers the hope of improved therapeutic options. We highlight the progress and limitations in this field. Insights into the gut microbiome–brain axis may offer potential therapeutic approaches to improve the long-term outcomes of premature infants