Weishu Zhu

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.

Conditioned medium from Bifidobacteria infantis protects against Cronobacter sakazakii-induced intestinal inflammation in newborn mice

Necrotizing enterocolitis (NEC) is associated with a high morbidity and mortality in very low birth weight infants. Several hypotheses regarding the pathogenesis of NEC have been proposed but to date no effective treatment is available. Previous studies suggest that probiotic supplementation is protective. We recently reported that probiotic (Bifidobacterium infantis) conditioned medium (PCM) has an anti-inflammatory effect in cultured fetal human intestinal cells (H4) and fetal intestine explants. In this study, we tested in vivo whether PCM protects neonatal mice from developing intestinal inflammation induced by exposure to Cronobacter sakazakii (C. sakazakii), an opportunistic pathogen associated with NEC. We found that infected neonatal mice had a significantly lower body weight than control groups. Infection led to ileal tissue damage including villous rupture, disruption of epithelial cell alignment, intestinal inflammation, apoptotic cell loss, and decreased mucus production. Pretreatment with PCM prevented infection caused decrease in body weight, attenuated enterocyte apoptotic cell death, mitigated reduced mucin production, and maintained ileal structure. Infected ileum expressed reduced levels of IκBα, which could be restored upon pretreatment with PCM. We also observed a nuclear translocation of NF-κB p65 in H4 cells exposed to C. sakazakii, which was prevented in PCM-pretreated cells. Finally, treatment of neonatal mice with PCM prior to infection sustained the capacity of ileal epithelial proliferation. This study suggests that an active component(s) released into the culture medium by B. infantis may prevent ileal damage by a pathogen linked to NEC.

Toll-like receptor-4 in human and mouse colonic epithelium is developmentally regulated: a possible role in necrotizing enterocolitis

Background:Necrotizing enterocolitis (NEC) is an immature intestinal condition resulting in devastating intestinal inflammation due to unknown mechanisms. Evidence has suggested that intestinal maturation attenuates the severity of NEC and Toll-like receptor 4 (TLR4) has been suggested to play a critical role in its pathogenesis. We investigated whether maturational effects of TLR4 expression in immature colon might contribute to the development of NEC.Methods:TLR4 colonocyte expression was detected by immunofluorescence confocal microscopy. Interleukin-6 (IL-6) levels were assayed by an enzyme-linked immunosorbent assay (ELISA).Results:TLR4 expression was high in fetal colonic epithelium in human and mouse, with earlier gestation having a higher surface/cytoplasm distribution. TLR4 remained high in mouse postnatal day 1 but the surface/cytoplasm distribution was reduced. TLR4 decreased in amount and then was expressed in crypts in the mature human and mouse colon. Hydrocortisone (HC) reduced the surface/cytoplasm distribution of TLR4 in human fetal colon. Elevated IL-6 levels in immature colon after lipopolysaccharide were attenuated by HC in human and mouse.Conclusion:Expression, localization, and signaling of TLR4 in colonic epithelium may be developmentally regulated. HC may accelerate the TLR developmental pathway change to an adult type, which may account for its impact on TLR4 signaling.

Secretions of Bifidobacterium infantis and Lactobacillus acidophilus Protect Intestinal Epithelial Barrier Function

Objectives: The secreted metabolites of probiotics are cytoprotective to intestinal epithelium and have been shown to attenuate inflammation and reduce gut permeability. The present study was designed to determine the protective effects of probiotic conditioned media (PCM) from Bifidobacterium infantis (BCM) and Lactobacillus acidophilus (LCM) on interleukin (IL)-1&bgr;–induced intestinal barrier compromise. Methods: The epithelial barrier was determined by measuring the transepithelial electrical resistance (TER) across a Caco-2 cell monolayer using a Transwell model. The paracellular permeability was determined by fluorescein isothiocyanate–labeled dextran flux. The expression of tight junction (TJ) proteins and nuclear factor-kappa B (NF-&kgr;B) p65 were determined using Western blot and the distribution of NF-&kgr;B p65 was determined by immunofluorescence staining. Results: BCM and LCM induced a dose-dependent increase in Caco-2 TER after 4 and 24 hours of incubation (P < 0.05). The maximal increase of Caco-2 TER occurred at 4 hours of treatment with a PCM concentration of 15%. Preincubation with BCM and LCM for 4 hours significantly prevented the decrease of Caco-2 TER induced by 24 hours of stimulation with 10 ng/mL IL-1&bgr;. BCM and LCM decreased paracellular permeability in both stimulated and unstimulated Caco-2 monolayers (P < 0.05). IL-1&bgr; stimulation decreased occludin expression and increased claudin-1 expression in Caco-2 cells (P < 0.05), which was prevented in cells treated with BCM or LCM. The changes of claudin-1 expression in H4 cells were similar to Caco-2 cells in response to PCM treatment and IL-1&bgr; stimulation; however, a similar response in occludin was not demonstrated. The IL-1&bgr;–induced nuclear translocation of NF-&kgr;B p65 in Caco-2 cells was prevented by pretreatment with both PCMs. Conclusions: BCM and LCM protected the intestinal barrier against IL-1&bgr; stimulation by normalizing the protein expression of occludin and claudin-1 and preventing IL-1&bgr;–induced NF-&kgr;B activation in Caco-2 cells, which may be partly responsible for the preservation of intestinal permeability.

Differential Expression of the Activator Protein 1 Transcription Factor Regulates Interleukin-1ß Induction of Interleukin 6 in the Developing Enterocyte

The innate immune response is characterized by activation of transcription factors, nuclear factor kappa B and activator protein-1 and their downstream targets, the pro-inflammatory cytokines including interleukin 1β and interleukin 6. Normal development of this response in the intestine is critical to survival of the human neonate and delays can cause the onset of devastating inflammatory diseases such as necrotizing enterocolitis. Previous studies have addressed the role of nuclear factor kappa B in the development of the innate immune response in the enterocyte, however despite its central role in the control of multiple pro-inflammatory cytokine genes, little is known on the role of Activator Protein 1 in this response in the enterocyte. Here we show that the canonical Activator Protein 1 members, cJun and cFos and their upstream kinases JNK and p38 play an essential role in the regulation of interleukin 6 in the immature enterocyte. Our data supports a model whereby the cFos/cJun heterodimer and the more potent cJun homodimer downstream of JNK are replaced by less efficient JunD containing dimers, contributing to the decreased responsiveness to interleukin 1β and decreased interleukin 6 secretion observed in the mature enterocyte. The tissue specific expression of JunB in colonocytes and colon derived tissues together with its ability to repress Interleukin-1β induction of an Interleukin-6 gene reporter in the NCM-460 colonocyte suggests that induction of JunB containing dimers may offer an attractive therapeutic strategy for the control of IL-6 secretion during inflammatory episodes in this area of the intestine

The symbiotic bacterial surface factor polysaccharide A on Bacteroides fragilis inhibits IL-1β-induced inflammation in human fetal enterocytes via toll receptors 2 and 4

Colonizing bacteria interacting with the immature, unlike the mature, human intestine favors inflammation over immune homeostasis. As a result, ten percent of premature infants under 1500 grams weight develop an inflammatory necrosis of the intestine after birth, e.g., necrotizing enterocolitis (NEC). NEC is a major health problem in this population causing extensive morbidity and mortality and an enormous expenditure of health care dollars. NEC can be prevented by giving preterm infants their mother’s expressed breast milk or ingesting selective probiotic organisms. Vaginally delivered, breast fed newborns develop health promoting bacteria (“pioneer” bacteria) which preferentially stimulate intestinal host defense and anti-inflammation. One such “pioneer” organism is Bacteroides fragilis with a polysaccharide (PSA) on its capsule. B. fragilis has been shown developmentally in intestinal lymphocytes and dendritic cells to produce a balanced T-helper cell (TH1/TH2) response and to reduce intestinal inflammation by activity through the TLR2 receptor stimulating IL-10 which inhibits IL-17 causing inflammation. No studies have been done on the role of B. fragilis PSA on fetal enterocytes and its increased inflammation. Accordingly, using human and mouse fetal intestinal models, we have shown that B. fragilis with PSA and PSA alone inhibits IL-1β-induced IL-8 inflammation in fetal and NEC intestine. We have also begun to define the mechanism for this unique inflammation noted in fetal intestine. We have shown that B. fragilis PSA anti-inflammation requires both the TLR2 and TLR4 receptor and is in part mediated by the AP1 transcription factor (TLR2) which is developmentally regulated. These observations may help to devise future preventative treatments of premature infants against NEC.

Anti-inflammatory effects of Bifidobacterium longum subsp infantis secretions on fetal human enterocytes are mediated by TLR-4 receptors

The therapeutic and preventive application of probiotics for necrotizing enterocolitis (NEC) has been supported by more and more experimental and clinical evidence in which Toll-like receptor 4 (TLR-4) exerts a significant role. In immune cells, probiotics not only regulate the expression of TLR-4 but also use the TLR-4 to modulate the immune response. Probiotics may also use the TLR-4 in immature enterocytes for anti-inflammation. Here we demonstrate that probiotic conditioned media (PCM) from Bifidobacterium longum supp infantis but not isolated organisms attenuates interleukin-6 (IL-6) induction in response to IL-1β by using TLR-4 in a human fetal small intestinal epithelial cell line (H4 cells), human fetal small intestinal xenografts, mouse fetal small intestinal organ culture tissues, and primary NEC enterocytes. Furthermore, we show that PCM, using TLR-4, downregulates the mRNA expression of interleukin-1 receptor-associated kinase 2 (IRAK-2), a common adapter protein shared by IL-1β and TLR-4 signaling. PCM also reduces the phosphorylation of the activator-protein 1 (AP-1) transcription factors c-Jun and c-Fos in response to IL-1β stimulation in a TLR-4-dependent manner. This study suggests that PCM may use TLR-4 through IRAK-2 and via AP-1 to prevent IL-1β-induced IL-6 induction in immature enterocytes. Based on these observations, the combined use of probiotics and anti-TLR-4 therapy to prevent NEC may not be a good strategy.

Human Fetal-Derived Enterospheres Provide Insights on Intestinal Development and a Novel Model to Study Necrotizing Enterocolitis (NEC)

Background & Aims Untreated necrotizing enterocolitis (NEC) can lead to massive inflammation resulting in intestinal necrosis with a high mortality rate in preterm infants. Limited access to human samples and relevant experimental models have hampered progress in NEC pathogenesis. Earlier evidence has suggested that bacterial colonization of an immature and developing intestine can lead to an abnormally high inflammatory response to bacterial bioproducts. The aim of our study was to use human fetal organoids to gain insights into NEC pathogenesis. Methods RNA sequencing analysis was performed to compare patterns of gene expression in human fetal-derived enterospheres (FEnS) and adult-derived enterospheres (AEnS). Differentially expressed genes were analyzed using computational techniques for dimensional reduction, clustering, and gene set enrichment. Unsupervised cluster analysis, Gene Ontology, and gene pathway analysis were used to predict differences between gene expression of samples. Cell monolayers derived from FEnS and AEnS were evaluated for epithelium function and responsiveness to lipopolysaccharide and commensal bacteria. Results Based on gene expression patterns, FEnS clustered according to their developmental age in 2 distinct groups: early and late FEnS, with the latter more closely resembling AEnS. Genes involved in maturation, gut barrier function, and innate immunity were responsible for these differences. FEnS-derived monolayers exposed to either lipopolysaccharide or commensal Escherichia coli showed that late FEnS activated gene expression of key inflammatory cytokines, whereas early FEnS monolayers did not, owing to decreased expression of nuclear factor-κB–associated machinery. Conclusions Our results provide insights into processes underlying human intestinal development and support the use of FEnS as a relevant human preclinical model for NEC. Accession number of repository for expression data: GSE101531.

48 IL-6 is Required for Microbiome-Mediated and Innate Immune-Dependent Recruitment of Neutrophils and Macrophages and is Necessary for Mucosal Repair After Intestinal Injury

Background: Current evidence indicates that inflammatory bowel diseases (IBD) may be caused in part by aberrant immune responses to commensal intestinal microbes including Bacteroides thetaiotaomicron (B. theta). Healthy, germ-free HLA-B27 transgenic (Tg) rats develop chronic colitis when colonized with complex gut commensal bacteria or Bacteroides vulgatus whereas non-transgenic (nTg) rats remain disease-free. However, the role of B. theta, a well-characterized anaerobic commensal bacterium, in causing disease in Tg rats is unknown. Moreover, while host immune responses to the gut microbiota have been extensively studied, relatively little is known about how microbes respond to host inflammation. Hypothesis: B. theta monoassociated Tg rats develop chronic, immune-mediated colitis that is associated with transcriptional responses in the bacteria that initiate and perpetuate disease. Methods: Four Tg and five nTg rats were monoassociated with a human isolate of B. theta (VPI-5482) for six weeks. Colonic inflammation was quantified by blinded histological scoring and real-time RT-PCR assays of pro-inflammatory cytokines. Cecal bacterial concentrations were measured by quantitative plating on BHI agar. Whole genome transcriptional profiling of B. theta recovered from the cecum was performed using custom GeneChips and data analyzed using DChip, Significance Analysis of Microarrays, and Gene Set Enrichment Analysis (GSEA) software. Results: B. theta monoassociated Tg rats had significantly more colonic inflammation and increased colonic levels of pro-inflammatory cytokine mRNAs than nTg controls (Table). Transcriptional profiles of cecal B. theta were significantly different in Tg vs. nTg rats (329 transcripts representing 44 KEGG canonical pathways). GSEA revealed that the GO molecular function of receptor activity, which is comprised mainly of genes that encode nutrient binding proteins, was significantly enriched with genes upregulated in B. theta from Tg rats [GSEA false discovery rate (FDR)=0.148]. KEGG canonical pathways of ribosome (FDR=0.048), oxidative phosphorylation (FDR=0.098), pyrimidine metabolism (FDR=0.081), purine metabolism (FDR=0.197), peptidoglycan biosynthesis (FDR=0.184), and metabolism (FDR=0.191) were significantly enriched with genes downregulated in B. theta from Tg rats. Numbers of viable bacteria/gram cecal contents in Tg vs. nTg rats were not significantly different. Conclusions: A well-characterized human isolate of B. theta induces mild colitis in HLA-B27 Tg rats and colitis is associated with changes in the expression of microbial metabolic and nutrient binding pathways, but no difference in concentrations of viable cecal bacteria. Mechanistic studies of differentially expressed B. theta genes may reveal novel pathways that initiate and/or perpetuate IBD.

563 Colonization-Dependent Colonic Fucosylation is Mediated by Gram Negative Bacteria via Activation of a Novel Fucosylated TLR4

Adult colonic mucosa is highly fucosylated relative to immature mucosa, and this fucosylation is dependent upon bacterial colonization. Toll-like receptor-4 (TLR4) was hypothesized to mediate this colonization-dependent fucosylation. Germ-free (GF) and bacteria-depleted (BD) mice were compared with conventional and mutant mice with regard to induction of epithelial fut2 mRNA levels and α1,2-fucosyltransferase (FUT2) activity. Induction of these activities by recolonization were compared with induction by LPS (TLR4 ligand) andmonocolonization by bacteriodes and E. coli strains. Cultured human erythroleukemia (Hel) cells were used to study fucosylated TLR4 In Vitro. Colonization-dependent induction of fucosylation occurs only in TLR2-/and wild-type mice but not in TLR4-/and MyD88-/mice, suggesting TLR4-MyD88 dependent signaling is important for fucosylation. TLR4 is fucosylated in the colonocytes of only GF and BD mice. LPS activate fut2 mRNA and FUT2 activity in the absence of bacterial colonization. Fucose-utilizing Bacteroides fragilis induce colonic fucosylation, but not a fucose-non-utilizing mutant. In Hel cells, which express fucosylated TLR4 activates fut2 mRNA and fucosylation. TLR4-specific siRNA inhibits this activation, and fucosidase treatment abolished TLR4-dependent activation of fut2 mRNA, confirming the central role of fucosylated-TLR4 in activating fut2 and cell surface fucosylation. Fucosylated-TLR4 expressed on uncolonized colonocytes mediates bacterially-induced activation of fut2 mRNA and FUT2 activity without activating inflammation, and is a necessary initial step in the communication between the colonizing bacteria and the host epithelium.