Enterocyte Cell Line Research Articles

LY2928057, An Antibody Targeting Ferroportin, Is a Potent Inhibitor Of Hepcidin Activity and Increases Iron Mobilization In Normal Cynomolgus Monkeys

Hepcidin, a 25-amino acid peptide hormone which is primarily synthesized and secreted from the liver, is a key regulator of iron homeostasis. It regulates dietary iron absorption, plasma iron concentrations, and tissue iron distribution through interactions with ferroportin, the only known mammalian cellular iron exporter. Hepcidin induces the internalization and subsequent degradation of ferroportin. The reduction in iron release caused by the loss of ferroportin, combined with the continuing demand for iron by erythropoietic precursors, results in a decrease in circulating iron levels. Dysregulation of the hepcidin-ferroportin axis contributes to the pathogenesis of different anemias. Decreased synthesis of hepcidin may cause systemic iron overload in iron-loading anemias such as beta-thalassemia; whereas overproduction of hepcidin may contribute to the development of anemia in inflammatory disorders, malignancies, and chronic kidney disease. LY2928057 is a novel humanized IgG4 monoclonal antibody that binds to human ferroportin with a high affinity, blocks the binding of human hepcidin to ferroportin, and is a potent inhibitor of hepcidin activity in a recombinant ferroportin expressing HEK 293 cell-based assay. In addition, this antibody was able to significantly inhibit hepcidin-induced increase in ferritin levels using Caco-2 cells, a human enterocyte cell line that naturally expresses ferroportin. LY2928057 does not block the efflux of iron from ferroportin, nor does this antibody cause the internalization of this transporter in vitro. Administration of LY2928057 results in a dose dependent increase in serum iron and hepcidin in normal cynomolgus monkeys. LY2928057 may provide therapeutic benefit for patients with hepcidin-related anemia by stabilizing ferroportin located on the cell surface, thus restoring iron export and erythropoiesis. LY2928057 is currently in clinical evaluation. Disclosures:Witcher:Eli Lilly and Company: Employment, Equity Ownership. Leung:Eli Lilly and Company: Employment, Equity Ownership. Hill:Eli Lilly and Company: Employment, Equity Ownership. De Rosa:Eli Lilly and Company: Employment, Equity Ownership. Xu:Eli Lilly and Company: Employment, Equity Ownership. Manetta:Eli Lilly and Company: Employment, Equity Ownership. Wroblewski:Eli Lilly and Company: Employment, Equity Ownership. Benschop:Eli Lilly and Company: Employment, Equity Ownership.

FimH adhesin from host unrestricted Salmonella Enteritidis binds to different glycoprotein ligands expressed by enterocytes from sheep, pig and cattle than FimH adhesins from host restricted Salmonella Abortus-ovis, Salmonella Choleraesuis and Salmonella Dublin

Adhesion to gut tissues and colonization of the alimentary tract, two important stages in the pathogenesis of Salmonella, are mediated by FimH adhesin of type 1 fimbriae. It was suggested that minor differences in the structure of FimH are most likely associated with differences in adhesion specificities, and may determine the tropism of various Salmonella serovars to different species and tissues. We investigated this hypothesis by comparing the binding properties of FimH proteins from three Salmonella enterica serovars with limited (Choleraesuis, Dublin) or restricted (Abortusovis) host ranges to FimH from broad host range S. Enteritidis and mannose inactive FimH from S. Gallinarum. Although all active variants of FimH protein were able to bind mannose-rich glycoproteins (RNase B, HRP and Man-BSA) with comparable affinity measured by surface plasmon resonance, there were significant differences in the binding profiles of the FimH proteins from host restricted serovars and host unrestricted serovar Enteritidis, to glycoproteins from enterocyte cell lines established in vitro and derived from sheep, pig and cattle. When low-binding FimH adhesin from S. Enteritidis was subjected to Western blot analysis, it bound to surface membrane protein of about 130 kDa, and high-binding FimH adhesins from S. Abortusovis, S. Choleraesuis and S. Dublin bound to surface membrane protein of about 55 kDa present in each cell line. Differential binding of FimH proteins from host-restricted and broad-host-range Salmonella to intestinal receptors was confirmed using mutant FimH adhesins obtained by site-directed mutagenesis. It was found that the low-binding variant of FimH from S. Choleraesuis with mutation Leu57Pro lost the ability to bind protein band of 55 kDa, but instead interacted with glycoprotein of about 130 kDa. On the other hand, the high-binding variant of FimH adhesin from S. Enteritids with mutation Asn101Ser did not bind to its receptor of 130 kDa, but instead it interacted with glycoprotein ligand of 55 kDa. These results suggest that FimH adhesins of type 1 fimbriae are one of the factors responsible for different host-specificities of these Salmonella serovars.

Cell invasion and survival of Shiga toxin-producing Escherichia coli within cultured human intestinal epithelial cells

Shiga toxin-producing Escherichia coli (STEC) cause severe human infections and their virulence abilities are not fully understood. Cattle are a key reservoir, and the terminal rectum is the principal site of bacterial carriage. Most STEC possess a pathogenicity island termed the locus of enterocyte effacement (LEE). Nonetheless, LEE-negative STEC have been associated with disease. We found that invasion of LEE-positive and LEE-negative strains was higher for human enterocytic cell lines and for undifferentiated Caco-2 cells. Intracellular bacteria could be detected as early as 5 min after infection and transmission electron microscopy showed bacteria within membrane-bound vacuoles. STEC invasion depended on actin microfilaments and protein kinases. Scanning electron microscopy revealed that bacterial entry was not associated with membrane ruffling. Absence of macropinocytosis or actin rearrangement at the entry points suggests a zipper-like entry mechanism. Disruption of the tight junction by EGTA enhanced invasion of Caco-2 monolayers, and bacterial invasion mostly proceeded through the basolateral pole of enterocytes. STEC persisted within Caco-2 cells for up to 96 h without cell death and bacterial viability increased after 48 h, suggesting intracellular multiplication. The relatively harmless intracellular localization of STEC can be an efficient strategy to prevent its elimination from the bovine intestinal tract.

Tu2013 Characterization of Epithelial Cell Loss Due to Cryptosporidium Parvum Infection in a Non-Transformed Porcine Jejunal Enterocyte (IPEC-J2) Cell Line

Tu2013 Characterization of Epithelial Cell Loss Due to Cryptosporidium Parvum Infection in a Non-Transformed Porcine Jejunal Enterocyte (IPEC-J2) Cell Line

Selective inhibition of the gliadin-specific, cell-mediated immune response by transamidation with microbial transglutaminase

CD is an immune-mediated enteropathy caused by the ingestion of wheat gluten. The modification of gluten by intestinal tTGase plays a crucial role in CD pathogenesis. In this study, we observed that extensive transamidation of wheat flour with K-C2H5 by mTGase yielded spf and K-gliadins fractions. By Western blot, we found that these modifications were associated with strongly reduced immune cross-reactivity. With the use of DQ8 tg mice as a model of gluten sensitivity, we observed a dramatic reduction in IFNγ production in gliadin-specific spleen cells challenged with spf and K-gliadins in vitro (n=12; median values: 813 vs. 29 and 99; control vs. spf and K-gliadins, P=0.012 for spf, and P=0.003 for K-gliadins). For spf, we also observed an increase in the IL-10/IFNγ protein ratio (n=12; median values: 0.3 vs. 4.7; control vs. spf, P=0.005). In intestinal biopsies from CD patients challenged in vitro with gliadins (n=10), we demonstrated further that K-gliadins dramatically reduced the levels of antigen-specific IFNγ mRNA in all specimens responsive to native gliadins (four of 10; P<0.05). As cytotoxic effects have been described for gliadins, we also studied GST and caspase-3 activities using the enterocytic Caco-2 cell line. We found that neither activities were modified by flour transamidation. Our results indicate that K-C2H5 cross-linking via mTGase specifically affects gliadin immunogenicity, reversing the inducible inflammatory response in models of gluten sensitivity without affecting other aspects of the biological activity of gliadins.

Immunoregulatory Protein Profiles of Necrotizing Enterocolitis versus Spontaneous Intestinal Perforation in Preterm Infants

Necrotizing enterocolitis (NEC) and spontaneous intestinal perforation (SIP) are the most common acute surgical emergencies associated with high morbidity and mortality in preterm infants. We aimed to compare the profiles of immunoregulatory proteins and identify novel mediators in plasma of NEC and SIP infants. We also investigated the expression of target genes in resected intestinal tissues and an enterocyte cell line. Using Cytokine Antibody Array assay, we reported the first comparative profiles of immunoregulatory proteins in plasma of NEC and SIP infants, and showed that dysregulated proteins belonged to functionally diversified categories, including pro- and anti-inflammation, angiogenesis, cell growth, wound healing, anti-apoptosis, cell adhesion and extracellular matrix reorganization. Validation by ELISA confirmed significantly higher concentrations of interleukin (IL)-6, angiopoietin (Ang)-2, soluble type II interleukin-1 receptor (sIL-1RII), and soluble urokinase-type plasminogen activator receptor (suPAR) in NEC infants compared with gestational age-matched control, and a lower level of an epidermal growth factor receptor, secreted form of receptor tyrosine-protein kinase ErbB3 (sErbB3), compared with SIP infants. mRNA expressions of IL1-RII and uPAR were up-regulated in resected bowel tissues from NEC infants, indicating that immunoregulation also occurred at the cellular level. In FHs-74 Int cells, Ang-2, IL1-RII and uPAR mRNA expressions were significantly induced by the combined treatment with lipopolysaccharide (LPS) and platelet activating factor (PAF). Our study provided plasmatic signatures of immunoregulatory proteins in NEC and SIP infants, and demonstrated involvement of multiple functional pathways. The magnitude of changes in these proteins was significantly more extensive in NEC infants, reflecting the different nature of injury and/or severity of inflammation. We speculate that dysregulation of IL-6, Ang-2, IL-1RII and uPAR occurred at both systemic and cellular levels, and probably mediated via LPS and endogeneous PAF signals. Such exaggerated immunologic responses may account for the high morbidity and mortality in NEC compared with SIP patients.

Interferon-γ Activates Transglutaminase 2 via a Phosphatidylinositol-3-Kinase-Dependent Pathway: Implications for Celiac Sprue Therapy

The mechanism for activation of extracellular transglutaminase 2 (TG2) in the small intestine remains a fundamental mystery in our understanding of celiac sprue pathogenesis. Using the T84 human enterocytic cell line, we show that interferon-γ (IFN-γ), the predominant cytokine secreted by gluten-reactive T cells in the celiac intestine, activates extracellular TG2 in a dose-dependent manner. IFN-γ mediated activation of TG2 requires phosphatidylinositol-3-kinase (PI3K) activity, but is uninfluenced by a number of other kinases reported to be active in T84 cells. Pharmacological inhibition of PI3K in the presence of IFN-γ prevents TG2 activation as well as the previously characterized increase in transepithelial permeability. Our findings therefore establish PI3K as an attractive target for celiac sprue therapy, a possibility that is underscored by the encouraging safety profiles of several PI3K inhibitors undergoing human clinical trials.

P-glycoprotein induction by breast milk attenuates intestinal inflammation in experimental necrotizing enterocolitis

P-glycoprotein (Pgp), a product of the multi-drug resistance gene MDR1a, is a broad specificity efflux ATP cassette transmembrane transporter that is predominantly expressed in epithelial tissues. Because mdr1a−/− mice tend to develop spontaneous colitis in bacteria-dependent manner, Pgp is believed to have a role in protection of the intestinal epithelium from luminal bacteria. Here we demonstrate that levels of Pgp in the small intestine of newborn rodents dramatically increase during breastfeeding, but not during formula feeding (FF). In rats and mice, levels of intestinal Pgp peak on days 3–7 and 1–5 of breastfeeding, respectively. The mdr1a−/− neonatal mice subjected to FF, hypoxia, and hypothermia have significantly higher incidence and pathology, as well as significantly earlier onset of necrotizing enterocolitis (NEC) than congenic wild type mice. Breast-fed mdr1a−/− neonatal mice are also more susceptible to intestinal damage caused by the opportunistic pathogen Cronobacter sakazakii that has been associated with hospital outbreaks of NEC. Breast milk, but not formula, induces Pgp expression in enterocyte cell lines in a dose- and time-dependent manner. High levels of ectopically expressed Pgp protect epithelial cells in vitro from apoptosis induced by C. sakazakii. Taken together, these results show that breast milk-induced expression of Pgp may have a role in the protection of the neonatal intestinal epithelium from injury associated with nascent bacterial colonization.

Mother-to-child transmission of HTLV-1:in vitro study of HTLV-1 passage across a tight human epithelial barrier

Background Besides horizontal transmission, HTLV-1 is transmitted vertically mainly through prolonged breastfeeding. Maternal transmission via breast milk is the dominant mode of HTLV-1 spread in high endemic areas, and is inked to the presence of HTLV-1 infected cells (lymphocytes, epithelial cells..) in the milk. Infection in young childhood by such mean appears to be a major risk factor for development of ATL in adults. Materials and methods We developed an in vitro model of epithelial barrier (Caco-2 human enterocytic cell line) to assess the mode of passage of HTLV-1 through the digestive tract. Integrity of the barrier was checked by ultrastructural approach, measurement of the trans-epithelial resistance (TER), and diffusion of fluorescently labeled molecules. Results When enterocytes were co-cultured with HTLV-1infected lymphocytes, no structural modifications were detected in intercellular tight junctions. Moreover, the functional integrity of the epithelial barrier was maintained since no TER change was detected in the presence of infected lymphocytes, and passage of small fluorescent markers was unaffected. Although enterocytes were not found to be susceptible to HTLV-1 infection, free infectious HTLV-1 virions were detected in the basal compartment, and such a passage was temperaturedependent, suggesting a transcytotic mechanism of passage. When human dendritic cells were added to the basal compartment, they were found to be productively infected by HTLV-1 that had crossed the epithelial barrier.

Differentiation- and polarization-dependent zinc tolerance in Caco-2 cells

Enterocytes are feasibly confronted with enormous zinc concentrations especially as a result of oral zinc supplementation. In the present study, we investigated the mechanisms underlying the exceptional ability to withstand this usually toxic load using the enterocytic cell line Caco-2. By MTT test analysis, we compared zinc tolerance in undifferentiated Caco-2 cells (udCaco-2) and differentiated Caco-2 cells (dCaco-2). By RT-PCR, we compared the respective baseline expression levels of certain zinc transporters and metallothioneins (MTs) as well as the regulation of these components in response to high zinc concentrations. Moreover, using dCaco-2 cells cultured on porous membranes, we explored zinc tolerance in dependence of the side of zinc administration: apical versus basolateral. dCaco-2 cells tolerate significantly higher levels of zinc compared to udCaco-2 cells. This adaptation was accompanied by upregulated ZnT-1 and downregulated ZIP1 levels. The expression of metallothioneins MT1A and MT1X was also significantly downregulated during differentiation. Moreover, apparent from profound differences in zinc tolerance between apical and basolateral zinc application, polarization was concluded to have substantial impact on cellular zinc tolerance. The profound increase in zinc tolerance we found in differentiated enterocyte-type Caco-2 cells and in particular, the impact of polarization are likely to reflect the physiologically indispensable capability of enterocytes to cope with remarkable concentrations of intestinal zinc.

A Three-Dimensional Coculture of Enterocytes, Monocytes and Dendritic Cells To Model Inflamed Intestinal Mucosa in Vitro

While epithelial cell culture models (e.g., Caco-2 cell line) are widely used to assess the absorption of drug molecules across healthy intestinal mucosa, there are no suitable in vitro models of the intestinal barrier in the state of inflammation. Thus development of novel drugs and formulations for the treatment of inflammatory bowel disease is largely bound to animal models. We here report on the development of a complex in vitro model of the inflamed intestinal mucosa, starting with the selection of suitable enterocyte cell line and proinflammatory stimulus and progressing to the setup and characterization of a three-dimensional coculture of human intestinal epithelial cells and immunocompetent macrophages and dendritic cells. In the 3D setup, controlled inflammation can be induced allowing the mimicking of pathophysiological changes occurring in vivo in the inflamed intestine. Different combinations of proinflammatory stimuli (lipopolysaccharides from Escherichia coli and Salmonella typhimurium, interleukin-1β, interferon-γ) and intestinal epithelial cell lines (Caco-2, HT-29, T84) were evaluated, and only Caco-2 cells were responsive to stimulation, with interleukin-1β being the strongest stimulator. Caco-2 cells responded to the proinflammatory stimulus with a moderate upregulation of proinflammatory markers and a slight, but significant, decrease (20%) of transepithelial electrical resistance (TEER) indicating changes in the epithelial barrier properties. Setting up the coculture model, macrophages and dendritic cells derived from periphery blood monocytes were embedded in a collagen layer on a Transwell filter insert and Caco-2 cells were seeded atop. Even in the presence of immunocompetent cells Caco-2 cells formed a tight monolayer. Addition of IL-1β increased inflammatory cytokine response more strongly compared to Caco-2 single culture and stimulated immunocompetent cells proved to be highly active in sampling apically applied nanoparticles. Thus the 3D coculture provides additional complexity and information compared to the stimulated single cell model. The coculture system may serve as a valuable tool for developing drugs and formulations for the treatment of inflammatory bowel diseases, as well as for studying the interaction of xenobiotics and nanoparticles with the intestinal epithelial barrier in the state of inflammation.

Effect of osmotic stress and culture density on invasiveness of Listeria monocytogenes strains

The effect of osmotic stress on its capacity to invade the human enterocytic cell line HT-29 was studied in the early log through the stationary phase in 10 L. monocytogenes strains representing three genetically independent lineages. The results demonstrate that the transition of the bacteria from the log to the stationary phase results in a stepwise reduction of invasiveness. This effect was heterogeneous in the studied L. monocytogenes population, as the range of invasiveness reduction between the log and stationary phases varied from 10- to 380-fold depending on the strain. Ten-minute exposure to 0.3M NaCl was sufficient to generate invasiveness alteration. No significant change in invasiveness induction caused by osmotic stress was found between the different points of the log phase (OD600 0.4–1.2), being significantly different in the early log phase (OD600 0.2–0.3) and in the stationary phase after 18h of culture. The level of internalins and opuCA transcripts in response to osmotic stress did not correlate with invasiveness alteration in most L. monocytogenes strains.Prolongation of stress exposure to 1h and an increase in NaCl concentration from 0.3 to 1.8M had no significant effect on a further increase in invasiveness. Short exposure times and low NaCl concentrations were sufficient for the generation of maximal invasiveness response of L. monocytogenes. It appears that although stationary-phase bacteria exhibit lower invasiveness than log-phase bacteria, they have a greater capacity to enhance their pathogenicity in response to stress.

Inhibition of intestinal glucose absorption by anti-diabetic medicinal plants derived from the James Bay Cree traditional pharmacopeia

Type II diabetes and obesity are major health problems worldwide and aboriginal peoples are particularly at risk. To address this problem in Canadian native populations who find modern pharmaceuticals culturally inappropriate, our team is testing the traditional pharmacopeia of the James Bay Cree for anti-diabetic and anti-obesity activities. More specifically, the aim of the present study was to define the effects of traditional plants on intestinal glucose absorption, an under-appreciated anti-hyperglycaemic and anti-obesity activity. Crude ethanol extracts of 17 Boreal forest medicinal plants were tested in vitro using the Caco-2 human enterocytic cell line and in vivo using an oral glucose tolerance test. Thirteen of seventeen extracts were observed to significantly inhibit uptake when administered simultaneously with (3)H-deoxyglucose. Inhibition was dose-dependent and, in a few cases, even surpassed that induced by a combination of the positive controls. To validate these effects in vivo, four plant extracts were administered by intragastric gavage at 250 mg/kg to normal rats simultaneously with a 3g/kg bolus of glucose. This resulted in a decrease in peak glycaemia by approximately 40% for two of them. Similarly, only 2 extracts reduced glucose transport after long term incubation and this could be related to reductions in the expression of SGLT-1 or GLUT-2 proteins. These findings indicate that competitive inhibition of intestinal glucose uptake can be achieved by crude extracts of medicinal plants. Such extracts could be taken with meals to control postprandial glycaemia and reduce caloric intake in high risk populations that are positively inclined towards traditional medicine.

Matrix Metalloproteinase-9 Functions as a Tumor Suppressor in Colitis-Associated Cancer

There is a well-documented association of matrix metalloproteinase-9 (MMP-9) and receptor Notch-1 overexpression in colon cancer. We recently showed that MMP-9 is also upregulated in colitis, where it modulates tissue damage and goblet cell differentiation via proteolytic cleavage of Notch-1. In this study, we investigated whether MMP-9 is critical for colitis-associated colon cancer (CAC). Mice that are wild type (WT) or MMP-9 nullizygous (MMP-9(-/-)) were used for in vivo studies and the human enterocyte cell line Caco2-BBE was used for in vitro studies. CAC was induced in mice using an established carcinogenesis protocol that involves exposure to azoxymethane followed by treatment with dextran sodium sulfate. MMP-9(-/-) mice exhibited increased susceptibility to CAC relative to WT mice. Elevations in tumor multiplicity, size, and mortality were associated with increased proliferation and decreased apoptosis. Tumors formed in MMP-9(-/-) mice exhibited expression of p21(WAF1/Cip1) and increased expression of beta-catenin relative to WT mice. In vitro studies of MMP-9 overexpression showed increased Notch-1 activation with a reciprocal decrease in beta-catenin. Notch and beta-catenin/Wnt signaling have crucial roles in determining differentiation and carcinogenesis in gut epithelia. Despite being a mediator of proinflammatory responses in colitis, MMP-9 plays a protective role and acts as a tumor suppressor in CAC by modulating Notch-1 activation, thereby resulting in activation of p21(WAF1/Cip1) and suppression of beta-catenin.

The absence of functional dectin-1 on enterocytes may serve to prevent intestinal damage

Enterocytes are exposed to antigens present in the intestinal lumen, like beta-glucans that are carbohydrate structures present not only in the cell wall of yeast and fungi but also in cereals. Beta-glucans are known for their immune modulating properties and we have earlier reported an increased immune response by enterocytes after addition of fecal water prepared from ileostomic contents obtained from participants consuming an oat beta-glucan diet versus a placebo diet. We hypothesized that our observation of immune stimulating effects by oat beta-glucan in enterocytes was mediated through the beta-glucan receptor dectin-1. Presence of dectin-1 in enterocytes was examined by reverse transcriptase PCR, western blot, and flow cytometry followed by an evaluation of the functional involvement of dectin-1 by using dectin-1 inhibitors during fecal water incubations. Reverse transcriptase PCR and western blot analysis showed dectin-1 presence in the INT407 and Caco-2 NF-kappaB reporter enterocyte cell lines. Moreover, human enterocytes isolated from ileum or colon biopsies also contained dectin-1 protein. However, dectin-1 expression could not be confirmed by flow cytometry in INT407 cells, suggesting that in these cell lines dectin-1 is not expressed at the extracellular membrane. Furthermore, dectin-1 inhibitors did not suppress the beta-glucan containing fecal water-induced IL-8 production by INT407 cells and NF-kappaB transactivation by Caco-2 NF-kappaB reporter cells. INT407 and Caco-2 NF-kappaB reporter cells seem to express no functional dectin-1. The absence of this pattern recognition receptor may function to protect the intestine against inflammatory damage, as the dectin-1 ligand beta-glucan is largely present in the intestinal lumen.

Dietary (1→3), (1→4)-β-d-glucans from oat activate nuclear factor-κB in intestinal leukocytes and enterocytes from mice

Dietary components, like beta-glucans, can modulate the intestinal immune response. We previously showed that fecal water enriched with oat beta-glucan stimulated the cytokine-induced immune response of enterocytes. It is, however, unclear whether beta-glucans activate nuclear factor-kappaB (NF-kappaB) pathways in the intestine in vivo and if so, whether enterocytes, intestinal leukocytes, or both respond to beta-glucans. We evaluated the effects of an oral gavage of 3 mg dietary oat (1-->3), (1-->4)-beta-D-glucans that was administered twice daily during 3.5 days on intestinal NF-kappaB transactivation and subsequent cytokine production of intestinal leukocytes and enterocytes in 16 NF-kappaB reporter mice. We hypothesized that oat beta-glucan activates the central immune transcription factor NF-kappaB and increased cytokine secretion, as we previously reported immune stimulating effects by oat beta-glucan. We found that mice that were administered oat beta-glucans (n = 8) showed an increased intestinal NF-kappaB transactivation in leukocytes (P = .021) and enterocytes (P = .012), particularly in the proximal part of the small intestine (ileum), as compared to placebo mice (n = 8). Surprisingly, NF-kappaB was not activated in the colon. Furthermore, the level of interleukin 12 was increased in intestinal lysates from all compartments, whereas the concentration of interferon gamma was decreased in the proximal small intestine (P = .046). Finally, tumor necrosis factor alpha showed a trend toward a reduced production in the colon (P = .057). Because we have earlier shown that human enterocyte cell lines do not express the beta-glucan receptor dectin-1 in vitro, we now conclude that after consumption, dietary oat beta-glucans most likely firstly activate the intestinal leukocytes, which in turn increases cellular activation of enterocytes.

The bacterial signal indole increases epithelial-cell tight-junction resistance and attenuates indicators of inflammation

Interkingdom signaling is established in the gastrointestinal tract in that human hormones trigger responses in bacteria; here, we show that the corollary is true, that a specific bacterial signal, indole, is recognized as a beneficial signal in intestinal epithelial cells. Our prior work has shown that indole, secreted by commensal Escherichia coli and detected in human feces, reduces pathogenic E. coli chemotaxis, motility, and attachment to epithelial cells. However, the effect of indole on intestinal epithelial cells is not known. Because intestinal epithelial cells are likely to be exposed continuously to indole, we hypothesized that indole may be beneficial for these cells, and investigated changes in gene expression with the human enterocyte cell line HCT-8 upon exposure to indole. Exposure to physiologically relevant amounts of indole increased expression of genes involved in strengthening the mucosal barrier and mucin production, which were consistent with an increase in the transepithelial resistance of HCT-8 cells. Indole also decreased TNF-alpha-mediated activation of NF-kappaB, expression of the proinflammatory chemokine IL-8, and the attachment of pathogenic E. coli to HCT-8 cells, as well as increased expression of the antiinflammatory cytokine IL-10. The changes in transepithelial resistance and NF-kappaB activation were specific to indole: other indole-like molecules did not elicit a similar response. Our results are similar to those observed with probiotic strains and suggest that indole could be important in the intestinal epithelial cells response to gastrointestinal tract pathogens.

Cryptosporidium parvumInfection Rapidly Induces a Protective Innate Immune Response Involving Type I Interferon

Type II interferon (IFN), IFN-gamma, is important in innate immunity to the intestinal protozoan parasite Cryptosporidium species, which infects epithelial cells (enterocytes). This investigation is, to our knowledge, the first to characterize the role of type I IFN in innate immunity to this parasite. Pretreatment of human or murine enterocyte cell lines with IFN-alpha/beta inhibited parasite development, and we identified that a key mechanism of cytokine action was to prevent parasite invasion of enterocytes. IFN-alpha/beta was rapidly expressed by infected murine enterocytes and also by bone marrow-derived dendritic cells that were exposed to live parasites. Treatment of neonatal severe combined immunodeficiency mice with anti-IFN-alpha/beta neutralizing antibodies before infection increased oocyst reproduction, as measured at the peak of infection, and parasite numbers in gut epithelium were also increased 2 days after infection. The latter observation correlated with strong intestinal expression of both IFN-alpha and IFN-beta messenger RNA within 24 h after infection. Treatment with anti-IFN-alpha/beta, however, did not reduce early expression of IFN-gamma. These findings identify a novel early innate host response against Cryptosporidium parvum involving IFN-alpha/beta.

Genotoxicity of a freshwater cyanotoxin, cylindrospermopsin, in two human cell lines: Caco‐2 and HepaRG

Cylindrospermopsin (CYN), a cyanotoxin produced by certain freshwater cyanobacteria, causes human intoxications and animal mortalities. CYN is a potent inhibitor of protein- and glutathione-synthesis. Preliminary evidence for in vivo tumor initiation has been found in mice but the mechanism remains unclear. Several in vitro and in vivo studies demonstrate that CYN is genotoxic and requires metabolic activation. In the present study, the genotoxicity of CYN was assessed in human hepatocyte and enterocyte cell lines, which are models for CYN target organs. The cytokinesis-block micronucleus assay was conducted on liver-derived HepaRG cells and colon-derived Caco-2 cells. Each cell-type was exposed to CYN in both the differentiated and the undifferentiated states, and both with and without the cytochrome P450 inhibitor, ketoconazole, to determine the involvement of metabolism in CYN genotoxicity. CYN increased the frequency of micronuclei in binucleated cells (MNBNC) in both Caco-2 and HepaRG cells. Moreover, ketoconazole reduced both the genotoxicity and cytotoxicity caused by CYN. Our results confirm the involvement of metabolic activation of CYN in mediating its toxicity and suggest that CYN is progenotoxic.

Dysregulation of interferon-γ-mediated signalling pathway in intestinal epithelial cells byCryptosporidium parvuminfection

The apicomplexan parasite Cryptosporidium parvum, the agent of cryptosporidiosis, primarily infects and reproduces in enterocytes. Interferon (IFN)-gamma is important for early control of the infection and acts directly on enterocytes to inhibit parasite development, although complete inhibition is not obtained. Addressing this latter observation, an investigation was made of the modulatory effect of C. parvum infection on IFN-gamma-dependent enterocyte gene expression. Initial studies showed that IFN-gamma-induced expression of indoleamine 2, 3 dioxygenase (IDO) mRNA and protein in CMT-93 cells was abrogated by C. parvum infection. Infection also inhibited IDO expression by the human enterocyte cell lines HT29 and Caco-2. Expression of IFN-gamma-inducible genes important in the development of immune responses, including major histocompatibility complex class II and CIITA, was also inhibited by the parasite. Investigating a possible mechanism for these findings, it was shown that infection caused depletion of STAT1alpha protein, a key transcription factor in IFN-gamma signalling. These findings indicate C. parvum interferes with IFN-gamma-dependent gene expression in enterocytes and suggest this activity could be a novel immuno-evasive strategy employed by the parasite.