Human milk oligosaccharides promote intestinal epithelium regeneration independent of the microbiota during necrotizing enterocolitis.

PURPOSE: Necrotizing enterocolitis (NEC) is a severe intestinal disease primarily affecting premature infants, marked by impaired epithelial regeneration. Breastfed infants are less susceptible to NEC than formula-fed ones, and human milk oligosaccharides (HMO) found in breast milk have prebiotic properties that can protect against NEC. However, it is unclear how HMOs influence intestinal epithelium regeneration in relation to the gut microbiota. METHODS: Broad-spectrum antibiotics were administered to pregnant dams to reduce the microbiota in offspring. NEC was induced through administration of hyperosmolar formula, lipopolysaccharide, and hypoxia from postnatal days (p) 5-9. Intestinal epithelial organoids were derived from p9 mice. HMOs were isolated from human donor breast milk and then solubilized in the formula for each feed or culture media for organoids. RESULTS: HMOs did not alter the microbiota profile in the presence of a normal or reduced microbiota. In the reduced microbiota, HMO treatment decreased NEC intestinal injury, and increased proliferation and stem cell activity. Additionally, in the complete absence of the microbiota, HMOs stimulated intestinal organoid growth. CONCLUSION: This study demonstrates that HMOs promoted intestinal epithelial regeneration independent of the gut microbiota. These findings provide further insight into the various benefits HMOs may have in the protection against NEC.

Amniotic fluid stem cell administration can prevent epithelial injury from necrotizing enterocolitis.

BACKGROUND: Stem cell therapy has been proven to rescue intestinal injury and stimulate intestinal regeneration in necrotizing enterocolitis (NEC). Specifically, stem cells derived from amniotic fluid (AFSCs) and mesenchymal stem cells (MSCs) derived from bone marrow have shown promising results in the treatment of experimental NEC. This study aims to examine the effects of AFSCs and MSCs on the prevention of intestinal injury during experimental NEC. METHODS: Supernatants from AFSC and MSC cultures were collected to perform proteomic analysis. Prior to NEC induction, mice received intraperitoneal injections of phosphate-buffered saline (PBS), 2 × 106 AFSCs, or 2 × 106 MSCs. RESULTS: We found that AFSCs grew faster than MSCs. Proteomic analysis indicated that AFSCs are primarily involved in cell development and growth, while MSCs are involved in immune regulation. Administering AFSCs before NEC induction decreased NEC severity and mucosal inflammation. Intestinal proliferation and endogenous stem cell activation were increased after AFSC administration. However, administering MSCs before NEC induction had no beneficial effects. CONCLUSIONS: This study demonstrated that AFSCs and MSCs have different protein release profiles. AFSCs can potentially be used as a preventative strategy for neonates at risk of NEC, while MSCs cannot be used. IMPACT: AFSCs and MSCs have distinct protein secretory profiles, and AFSCs are primarily involved in cell development and growth, while MSCs are involved in immune regulation. AFSCs are unique in transiently enhancing healthy intestinal epithelial cell growth, which offers protection against the development of experimental NEC. The prevention of NEC via the administration of AFSCs should be evaluated in infants at great risk of developing NEC or in infants with early signs of NEC.

Lactoferrin Reduces Necrotizing Enterocolitis Severity by Upregulating Intestinal Epithelial Proliferation.

INTRODUCTION: Necrotizing enterocolitis (NEC) is a devastating intestinal illness in premature infants characterized by severe intestinal inflammation. Despite medical interventions, NEC mortality remains alarmingly high, which necessitates improved therapies. Lactoferrin is among the most abundant proteins in human milk and has important immunomodulatory functions. While previous studies have indicated protective effects of lactoferrin against neonatal sepsis and NEC, the underlying mechanism remains unclear. We hypothesize that lactoferrin downregulates inflammation and upregulates proliferation in intestinal epithelium during NEC injury. MATERIALS AND METHODS: NEC was induced by hypoxia, gavage feeding of hyperosmolar formula and lipopolysaccharide between postnatal day P5 and P9 (n = 8). Breastfed mice were used as control (n = 7). Lactoferrin (0.3 g/kg/day) was administered once daily by gavage from P6 to P8 in both NEC (NEC + Lac; n = 9) and control mice (Cont + Lac; n = 5). Distal ileum was harvested on P9 and analyzed for disease severity, inflammation, and proliferation. Groups were compared using one-way ANOVA and t-test appropriately; p < 0.05 was considered significant. RESULTS: Compared to NEC group, lactoferrin-treated NEC mice had reduced disease severity, reduced inflammation markers IL-6 and TNF-α expression and increased intestinal stem cell marker Lgr5 + expression. Lactoferrin-treated NEC mice exhibited increased nuclear ß-catenin, indicating upregulated Wnt pathway, and increased Ki67 positivity, suggesting enhanced proliferation. Furthermore, lactoferrin administration to control mice did not affect intestinal inflammation as well as Lgr5 + stem cell expression and epithelial proliferation. This supports the safety of lactoferrin administration and indicates that the beneficial effects of lactoferrin are present when intestinal injury such as NEC is present. CONCLUSION: Lactoferrin administration reduces the intestinal injury in experimental NEC by downregulating inflammation and upregulating cell proliferation. This beneficial effect of lactoferrin in stimulating cell proliferation is mediated by the Wnt pathway. This experimental study provides insights on the mechanism of action of lactoferrin in NEC and the role of lactoferrin in enteral feeding.

Impaired Wnt/ß-catenin pathway leads to dysfunction of intestinal regeneration during necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating neonatal disease characterized by acute intestinal injury. Intestinal stem cell (ISC) renewal is required for gut regeneration in response to acute injury. The Wnt/ß-catenin pathway is essential for intestinal renewal and ISC maintenance. We found that ISC expression, Wnt activity and intestinal regeneration were all decreased in both mice with experimental NEC and in infants with acute active NEC. Moreover, intestinal organoids derived from NEC-injured intestine of both mice and humans failed to maintain proliferation and presented more differentiation. Administration of Wnt7b reversed these changes and promoted growth of intestinal organoids. Additionally, administration of exogenous Wnt7b rescued intestinal injury, restored ISC, and reestablished intestinal epithelial homeostasis in mice with NEC. Our findings demonstrate that during NEC, Wnt/ß-catenin signaling is decreased, ISC activity is impaired, and intestinal regeneration is defective. Administration of Wnt resulted in the maintenance of intestinal epithelial homeostasis and avoidance of NEC intestinal injury.

Quantification and Visualization of Neutrophil Extracellular Traps (NETs) from Murine Bone Marrow-Derived Neutrophils.

Neutrophils are some of the first leukocytes to respond to inflammatory stimuli. Once recruited, these cells are equipped with an assortment of proteolytic enzymes and antimicrobial factors that disarm and degrade pathogens. Neutrophils employ a highly novel mechanism to contain and trap bacteria in the local inflammatory microenvironment, termed neutrophil extracellular traps (NETs). During NET formation, neutrophils eject weblike structures of chromatin, which captures and immobilizes invading pathogens. In this chapter, we describe protocols to isolate bone marrow-derived neutrophils from mice. We further describe in vitro methods to spectrophotometrically quantify, immunolabel, and visualize NET structures.

Mucosa-Associated Ileal Microbiota in New-Onset Pediatric Crohn's Disease.

BACKGROUND: The composition of the intestinal microbiome seems relevant to the pathogenesis of Crohn's disease (CD), with differences in both diversity and composition of the gut microbiota in patients with CD compared with healthy individuals. However, there are still conflicting reports on the importance of various bacterial taxa in the pathogenesis of CD. The aim of this study was to characterize the composition of mucosa-associated intestinal microbiota in newly diagnosed pediatric patients with CD. METHODS: Mucosa-associated bacteria were identified from ileal biopsy specimens obtained at colonoscopy of 10 patients with either ileal or ileocolonic new-onset CD and 15 controls without mucosal inflammation. Microbial composition was performed by profiling the 16S rDNA V6 region using Illumina sequencing. Samples were analyzed for differences in alpha/beta diversity and also for differentially abundant taxa. RESULTS: Alpha diversity did not differ between the controls and CD cases or between CD subjects with localized ileal disease compared with those with more extensive disease. Controls also did not clearly separate from patients with CD by principal coordinate analyses; however, 117 operational taxonomic units were found to be differentially abundant between the two groups. In particular, numerous operational taxonomic units associated with Faecalibacterium prausnitzii species were increased in children with CD. CONCLUSIONS: These findings contribute to emerging evidence regarding dysbiosis in pediatric CD, and provide additional evidence challenging the protective role of F. prausnitzii in CD.

Phenotypic and genotypic characterisation of biallelic mismatch repair deficiency (BMMR-D) syndrome.

Lynch syndrome, the most common inherited colorectal cancer syndrome in adults, is an autosomal dominant condition caused by heterozygous germ-line mutations in DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. Inheriting biallelic (homozygous) mutations in any of the MMR genes results in a different clinical syndrome termed biallelic mismatch repair deficiency (BMMR-D) that is characterised by gastrointestinal tumours, skin lesions, brain tumours and haematologic malignancies. This recently described and under-recognised syndrome can present with adenomatous polyps leading to early-onset small bowel and colorectal adenocarcinoma. An important clue in the family history that suggests underling BMMR-D is consanguinity. Interestingly, pedigrees of BMMR-D patients typically show a paucity of Lynch syndrome cancers and most parents are unaffected. Therefore, a family history of cancers is often non-contributory. Detection of BMMR-D can lead to more appropriate genetic counselling and the implementation of targeted surveillance protocols to achieve earlier tumour detection that will allow surgical resection. This review describes an approach for diagnosis and management of these patients and their families.

GLP-1R Agonists Modulate Enteric Immune Responses Through the Intestinal Intraepithelial Lymphocyte GLP-1R.

Obesity and diabetes are characterized by increased inflammation reflecting disordered control of innate immunity. We reveal a local intestinal intraepithelial lymphocyte (IEL)-GLP-1 receptor (GLP-1R) signaling network that controls mucosal immune responses. Glp1r expression was enriched in intestinal IEL preparations and copurified with markers of Tαß and Tγδ IELs, the two main subsets of intestinal IELs. Exendin-4 increased cAMP accumulation in purified IELs and reduced the production of cytokines from activated IELs but not from splenocytes ex vivo. These actions were mimicked by forskolin, absent in IELs from Glp1r(-/-) mice, and attenuated by the GLP-1R agonist exendin (9-39) consistent with a GLP-1R-dependent mechanism of action. Furthermore, Glp1r(-/-) mice exhibited dysregulated intestinal gene expression, an abnormal representation of microbial species in feces, and enhanced sensitivity to intestinal injury following administration of dextran sodium sulfate. Bone marrow transplantation using wild-type C57BL/6 donors normalized expression of multiple genes regulating immune function and epithelial integrity in Glp1r(-/-) recipient mice, whereas acute exendin-4 administration robustly induced the expression of genes encoding cytokines and chemokines in normal and injured intestine. Taken together, these findings define a local enteroendocrine-IEL axis linking energy availability, host microbial responses, and mucosal integrity to the control of innate immunity.

Vitamin D deficiency predisposes to adherent-invasive Escherichia coli-induced barrier dysfunction and experimental colonic injury.

BACKGROUND: Adherent-invasive Escherichia coli (AIEC) colonization has been strongly implicated in the pathogenesis of Crohn's disease. Environmental triggers such as vitamin D deficiency have emerged as key factors in the pathogenesis of inflammatory bowel diseases. The aim of this study was to investigate the effects of 1,25(OH)2D3 on AIEC infection-induced changes in vivo and in vitro. METHODS: Barrier function was assessed in polarized epithelial Caco-2-bbe cells grown in medium with or without vitamin D and challenged with AIEC strain LF82. Weaned C57BL/6 mice were fed either a vitamin D-sufficient or -deficient diet for 5 weeks and then infected with AIEC, in the absence and presence of low-dose dextran sodium sulphate. Disease severity was assessed by histological analysis and in vivo intestinal permeability assay. Presence of invasive bacteria was assessed by transmission electron microscopy. RESULTS: Caco-2-bbe cells incubated with 1,25(OH)2D3 were protected against AIEC-induced disruption of transepithelial electrical resistance and tight-junction protein redistribution. Vitamin D-deficient C57BL/6 mice given a course of 2% dextran sodium sulphate exhibited pronounced epithelial barrier dysfunction, were more susceptible to AIEC colonization, and showed exacerbated colonic injury. Transmission electron microscopy of colonic tissue from infected mice demonstrated invasion of AIEC and fecal microbiome analysis revealed shifts in microbial communities. CONCLUSIONS: These data show that vitamin D is able to mitigate the deleterious effects of AIEC on the intestinal mucosa, by maintaining intestinal epithelial barrier homeostasis and preserving tight-junction architecture. This study highlights the association between vitamin D status, dysbiosis, and Crohn's disease.

Transforming growth factor-ß1 protects against intestinal epithelial barrier dysfunction caused by hypoxia-reoxygenation.

Intestinal epithelia regulate barrier integrity when challenged by inflammation, oxidative stress, and microbes. Transforming growth factor-ß1 (TGF-ß1) is a cytokine with known beneficial effects on intestinal epithelia, including barrier enhancement, after exposure to proinflammatory cytokines and infectious agents. The aim of this study was to determine whether TGF-ß1 directly protects intestinal epithelia during hypoxia-reoxygenation (HR). Intestinal epithelial monolayers (T84, Caco-2) were exposed to either hypoxia (1% O2, 1 h) or oxidative stress (hydrogen peroxide, 1 mM), followed by normoxic atmosphere for different time points in the absence and presence of varying concentrations of TGF-ß1. Transepithelial electrical resistance (TER) assessed barrier function, with RNA extracted for reverse transcription polymerase chain reaction analysis of GPx-1, HIF-1, heme-oxygenase-1 (HO-1), and NOX-1. In some experiments, intestinal epithelia were exposed to enterohemorrhagic Escherichia coli (EHEC) O157:H7 during the reoxygenation period and TER recorded 7 h after the infectious challenge. Hypoxia-reoxygenation significantly decreased TER in intestinal epithelia compared with normoxic controls. Transforming growth factor-ß1 pretreatment ameliorated HR-induced epithelial barrier dysfunction in T84 (at 1 - 3 h) and Caco-2 (1 h) monolayers. Transforming growth factor-ß1 preserved barrier integrity for up to 16 h after challenge with hydrogen peroxide. In TGF-ß1-treated epithelial monolayers, only HO-1 mRNA significantly increased after HR (P < 0.05 vs. normoxic controls). The EHEC-induced epithelial barrier dysfunction was significantly worsened by intestinal HR (P < 0.05 vs. normoxia-EHEC-infected cells), but this was not protected by TGF-ß1 pretreatment. Transforming growth factor-ß1 preserves loss of epithelial barrier integrity caused by the stress of HR via a mechanism that may involve the upregulation of HO-1 transcription. Targeted treatment with TGF-ß could lead to novel therapies in enteric diseases characterized by HR injury.

Probiotics normalize the gut-brain-microbiota axis in immunodeficient mice.

The gut-brain-microbiota axis is increasingly recognized as an important regulator of intestinal physiology. Exposure to psychological stress causes activation of the hypothalamic-pituitary-adrenal (HPA) axis and causes altered intestinal barrier function, intestinal dysbiosis, and behavioral changes. The primary aim of this study was to determine whether the effects of psychological stress on intestinal physiology and behavior, including anxiety and memory, are mediated by the adaptive immune system. Furthermore, we wanted to determine whether treatment with probiotics would normalize these effects. Here we demonstrate that B and T cell-deficient Rag1(-/-) mice displayed altered baseline behaviors, including memory and anxiety, accompanied by an overactive HPA axis, increased intestinal secretory state, dysbiosis, and decreased hippocampal c-Fos expression. Both local (intestinal physiology and microbiota) and central (behavioral and hippocampal c-Fos) changes were normalized by pretreatment with probiotics, indicating an overall benefit on health conferred by changes in the microbiota, independent of lymphocytes. Taken together, these findings indicate a role for adaptive immune cells in maintaining normal intestinal and brain health in mice and show that probiotics can overcome this immune-mediated deficit in the gut-brain-microbiota axis.

Short-chain fructo-oligosaccharide and inulin modulate inflammatory responses and microbial communities in Caco2-bbe cells and in a mouse model of intestinal injury.

BACKGROUND: Few studies have focused on the ability of prebiotics to prevent pathogen-induced cellular changes or alter the composition of the intestinal microbiota in complimentary relevant cell and animal models of inflammatory bowel disease. OBJECTIVE: The objective of this study was to determine if pretreatment with inulin and a short-chain fructo-oligosaccharide (sc-FOS) prevents enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection in Caco2-bbe epithelial cells and what effect 10% wt:v sc-FOS or inulin has on C57BL/6 mice under sham conditions or pretreatment with prebiotics before Citrobacter rodentium infection (10(8) colony-forming units). METHODS: Actin rearrangement and tight junction protein (zona occludin-1) were examined with immunofluorescence. Barrier function was assessed by a fluorescent probe and by measuring transepithelial electrical resistance (TER). Alterations in cytokine gene expression and microbiome were assessed with quantitative reverse transcriptase-polymerase chain reaction and fluorescence in situ hybridization. Short-chain fatty acids (SCFAs) were measured by GC. RESULTS: sc-FOS added to monolayers altered actin polymerization without affecting TER or permeability to a fluorescein isothiocyanate (FITC) probe, whereas inulin increased TER (P < 0.005) and altered actin arrangement without affecting FITC permeability. Neither prebiotic attenuated EHEC-induced decreases in barrier function. Prebiotics increased interleukin 10 (Il10) and transforming growth factor-ß (Tgfß) cytokine responses alone (P < 0.05) or with EHEC O157:H7 infection (P < 0.05) in vitro. Increases in tumor necrosis factor-α (Tnfα) (P < 0.05) and decreases in chemokine CXC motif ligand 8 (Cxcl8) (P < 0.05) expression were observed with prebiotic treatment prior to EHEC infection. No differences were noted in barrier function or cytokine responses in the absence or presence of C. rodentium in vivo. Alterations in microbiome were evident at 6 d and 10 d postinfection in treatment groups, but a change in C. rodentium load was not observed. Inulin and sc-FOS (P < 0.05) increased fecal SCFAs in the absence of infection. CONCLUSION: This study provides new insights as to how prebiotics act in complementary in vitro and in vivo models of intestinal injury.

Protein kinase C mediates enterohemorrhagic Escherichia coli O157:H7-induced attaching and effacing lesions.

Enterohemorrhagic Escherichia coli serotype O157:H7 causes outbreaks of diarrhea, hemorrhagic colitis, and the hemolytic-uremic syndrome. E. coli O157:H7 intimately attaches to epithelial cells, effaces microvilli, and recruits F-actin into pedestals to form attaching and effacing lesions. Lipid rafts serve as signal transduction platforms that mediate microbe-host interactions. The aims of this study were to determine if protein kinase C (PKC) is recruited to lipid rafts in response to E. coli O157:H7 infection and what role it plays in attaching and effacing lesion formation. HEp-2 and intestine 407 tissue culture epithelial cells were challenged with E. coli O157:H7, and cell protein extracts were then separated by buoyant density ultracentrifugation to isolate lipid rafts. Immunoblotting for PKC was performed, and localization in lipid rafts was confirmed with an anti-caveolin-1 antibody. Isoform-specific PKC small interfering RNA (siRNA) was used to determine the role of PKC in E. coli O157:H7-induced attaching and effacing lesions. In contrast to uninfected cells, PKC was recruited to lipid rafts in response to E. coli O157:H7. Metabolically active bacteria and cells with intact lipid rafts were necessary for the recruitment of PKC. PKC recruitment was independent of the intimin gene, type III secretion system, and the production of Shiga toxins. Inhibition studies, using myristoylated PKCζ pseudosubstrate, revealed that atypical PKC isoforms were activated in response to the pathogen. Pretreating cells with isoform-specific PKC siRNA showed that PKCζ plays a role in E. coli O157:H7-induced attaching and effacing lesions. We concluded that lipid rafts mediate atypical PKC signal transduction responses to E. coli O157:H7. These findings contribute further to the understanding of the complex array of microbe-eukaryotic cell interactions that occur in response to infection.

Probiotic Lactobacillus rhamnosus inhibits the formation of neutrophil extracellular traps.

Neutrophil extracellular traps (NETs) are an essential component of the antimicrobial repertoire and represent an effective means by which neutrophils capture, contain, and kill microorganisms. However, the uncontrolled or excessive liberation of NETs also damages surrounding cells and can contribute to disease pathophysiology. Alterations in the gut microbiota, as well as the presence of local and systemic markers of inflammation, are strongly associated with the manifestation of a spectrum of intestinal disorders, including chronic inflammatory bowel disease. Although probiotics exert beneficial effects on gut homeostasis, their direct effect on neutrophils, which are abundant in the setting of intestinal inflammation, remains unclear. In this study, we investigated the effects of nonpathogenic, enteropathogenic, and probiotic bacteria on the dynamics of NET formation. Using murine bone marrow-derived neutrophils and the neutrophil-differentiated human myeloid cell line d.HL-60, we demonstrate for the first time, to our knowledge, that probiotic Lactobacillus rhamnosus strain GG inhibits both PMA- and Staphylococcus aureus-induced formation of NETs. Moreover, probiotic L. rhamnosus strain GG had potent antioxidative activity: dampening reactive oxygen species production and phagocytic capacity of the neutrophils while protecting against cell cytotoxicity. Within the milieu of the gut, this represents a novel mechanism by which probiotics can locally dampen innate immune responses and confer desensitization toward luminal Ags.

Quantification and visualization of neutrophil extracellular traps (NETs) from murine bone marrow-derived neutrophils.

Neutrophils are one of the first cells to respond to an inflammatory stimulus, and are equipped with an assortment of antimicrobial and proteolytic enzymes to disarm and degrade bacterial pathogens. A novel mechanism of bacterial trapping, termed neutrophil extracellular traps (NETs), was recently described whereby neutrophils were shown to cast out web-like structures of chromatin, capturing and immobilizing invading pathogens. Herein we describe protocols to isolate murine bone marrow-derived neutrophils, and spectrophotometrically quantify, immunolabel, and visualize NET structures in vitro.

An update on the use and investigation of probiotics in health and disease.

Probiotics are derived from traditional fermented foods, from beneficial commensals or from the environment. They act through diverse mechanisms affecting the composition or function of the commensal microbiota and by altering host epithelial and immunological responses. Certain probiotic interventions have shown promise in selected clinical conditions where aberrant microbiota have been reported, such as atopic dermatitis, necrotising enterocolitis, pouchitis and possibly irritable bowel syndrome. However, no studies have been conducted that can causally link clinical improvements to probiotic-induced microbiota changes. Whether a disease-prone microbiota pattern can be remodelled to a more robust, resilient and disease-free state by probiotic administration remains a key unanswered question. Progress in this area will be facilitated by: optimising strain, dose and product formulations, including protective commensal species; matching these formulations with selectively responsive subpopulations; and identifying ways to manipulate diet to modify bacterial profiles and metabolism.

Adherent-invasive Escherichia coli blocks interferon-γ-induced signal transducer and activator of transcription (STAT)-1 in human intestinal epithelial cells.

Adherent-invasive Escherichia coli (AIEC) is a pathogen isolated from the ileum of patients with Crohn disease. IFNγ is a key mediator of immunity, which regulates inflammatory responses to microbial infections. Previously, we showed enterohemorrhagic E. coli prevents STAT1 activation. The aim of this study was to determine whether activation of STAT1 by IFNγ was prevented by AIEC infection, and to define the mechanisms used. Human epithelial cells were infected with three different AIEC strains or other pathogenic and commensal E. coli strains. Following infection, cells were stimulated with IFNγ, and STAT1 activation was monitored by immunoblotting. Our data show that live AIEC with active protein synthesis machinery is able to prevent IFNγ-mediated STAT1 phosphorylation, and that a secreted factor may be involved. We conclude that the suppression of epithelial cell STAT1 signal transduction by AIEC strains isolated from patients with Crohn disease represents a novel mechanism by which the pathogen evades host immune responses to the infection.

Up-regulation of Annexin-A1 and lipoxin A(4) in individuals with ulcerative colitis may promote mucosal homeostasis.

BACKGROUND: One of the characteristics of an active episode of ulcerative colitis (UC) is the intense mucosal infiltration of leukocytes. The pro-resolution mediators Annexin-A1 (AnxA1) and lipoxin A(4) (LXA(4)) exert counter-regulatory effects on leukocyte recruitment, however to date, the dual/cumulative effects of these formyl peptide receptor-2 (FPR2/ALX) agonists in the context of human intestinal diseases are unclear. To define the contribution of these mediators, we measured their expression in biopsies from individuals with UC. METHODS: Colonic mucosal biopsies were collected from two broad patient groups: healthy volunteers without ('Ctrl' n  = 20) or with a prior history of UC ('hx of UC' n = 5); individuals with UC experiencing active disease ('active' n = 8), or in medically-induced remission ('remission' n = 16). We assessed the mucosal expression of LXA(4), AnxA1, and the FPR2/ALX receptor in each patient group using a combination of fluorescence microscopy, biochemical and molecular analyses. RESULTS: Mucosal expression of LXA(4) was elevated exclusively in biopsies from individuals in remission (3-fold, P<0.05 vs. Ctrl). Moreover, in this same group we observed an upregulation of AnxA1 protein expression (2.5-fold increase vs. Ctrl, P<.01), concurrent with an increased level of macrophage infiltration, and an elevation in FPR2/ALX mRNA (7-fold increase vs. Ctrl, P<.05). Importantly, AnxA1 expression was not limited to cells infiltrating the lamina propria but was also detected in epithelial cells lining the intestinal crypts. CONCLUSIONS: Our results demonstrate a specific up-regulation of this pro-resolution circuit in individuals in remission from UC, and suggest a significant role for LXA(4) and AnxA1 in promoting mucosal homeostasis.

Enterohemorrhagic Escherichia coli O157:H7 Shiga toxins inhibit gamma interferon-mediated cellular activation.

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a food-borne pathogen that causes significant morbidity and mortality in developing and industrialized nations. EHEC infection of host epithelial cells is capable of inhibiting the gamma interferon (IFN-γ) proinflammatory pathway through the inhibition of Stat-1 phosphorylation, which is important for host defense against microbial pathogens. The aim of this study was to determine the bacterial factors involved in the inhibition of Stat-1 tyrosine phosphorylation. Human HEp-2 and Caco-2 epithelial cells were challenged directly with either EHEC or bacterial culture supernatants and stimulated with IFN-γ, and then the protein extracts were analyzed by immunoblotting. The data showed that IFN-γ-mediated Stat-1 tyrosine phosphorylation was inhibited by EHEC secreted proteins. Using two-dimensional difference gel electrophoresis, EHEC Shiga toxins were identified as candidate inhibitory factors. EHEC Shiga toxin mutants were then generated and complemented in trans, and mutant culture supernatant was supplemented with purified Stx to confirm their ability to subvert IFN-γ-mediated cell activation. We conclude that while other factors are likely involved in the suppression of IFN-γ-mediated Stat-1 tyrosine phosphorylation, E. coli-derived Shiga toxins represent a novel mechanism by which EHEC evades the host immune system.