Invade Intestinal Epithelial Cells Research Articles

Differential miRNA-Gene Expression in M Cells in Response to Crohn's Disease-Associated AIEC.

Adherent-invasive Escherichia coli (AIEC), which abnormally colonize the ileal mucosa of Crohn’s disease (CD) patients, are able to invade intestinal epithelial cells (IECs) and translocate through M cells overlying Peyer’s patches. The levels of microRNA (miRNA) and gene expression in IECs and M cells upon AIEC infection have not been investigated. Here, we used human intestinal epithelial Caco-2 monolayers and an in vitro M-cell model of AIEC translocation to analyze comprehensive miRNA and gene profiling under basal condition and upon infection with the reference AIEC LF82 strain. Our results showed that AIEC LF82 translocated through M cells but not Caco-2 monolayers. Both differential gene expression and miRNA profile in M cells compared to Caco-2 cells were obtained. In addition, AIEC infection induces changes in gene and miRNA profiles in both Caco-2 and M cells. In silico analysis showed that certain genes dysregulated upon AIEC infection were potential targets of AIEC-dysregulated miRNAs, suggesting a miRNA-mediated regulation of gene expression during AIEC infection in Caco-2, as well as M cells. This study facilitates the discovery of M cell-specific and AIEC response-specific gene-miRNA signature and enhances the molecular understanding of M cell biology under basal condition and in response to infection with CD-associated AIEC.

Exosomes transfer miRNAs from cell-to-cell to inhibit autophagy during infection with Crohn’s disease-associated adherent-invasive E. coli

ABSTRACT Adherent-invasive E. coli (AIEC), which abnormally colonize the intestinal mucosa of Crohn’s disease (CD) patients, are able to adhere to and invade intestinal epithelial cells (IECs), survive and replicate within macrophages and induce a pro-inflammatory response. AIEC infection of IECs induces secretion of exosomes that increase AIEC replication in exosome-receiving IECs and macrophages. Here, we investigated the mechanism underlying the increased AIEC replication in cells receiving exosomes from AIEC-infected cells. Exosomes released by uninfected human intestinal epithelial T84 cells (Exo-uninfected) or by T84 cells infected with the clinical AIEC LF82 strain (Exo-LF82), the nonpathogenic E. coli K12 strain (Exo-K12) or the commensal E. coli HS strain (Exo-HS) were purified and used to stimulate T84 cells. Stimulation of T84 cells with Exo-LF82 inhibited autophagy compared with Exo-uninfected, Exo-K12 and Exo-HS. qRT-PCR analysis revealed increased levels of miR-30c and miR-130a in Exo-LF82 compared to Exo-uninfected, Exo-K12 and Exo-HS. These miRNAs were transferred via exosomes to recipient cells, in which they targeted and inhibited ATG5 and ATG16L1 expression and thereby autophagy response, thus favoring AIEC intracellular replication. Inhibition of these miRNAs in exosome-donor cells infected with AIEC LF82 abolished the increase in miR-30c and miR-130a levels in the released Exo-LF82 and in Exo-LF82-receiving cells, thus suppressing the inhibitory effect of Exo-LF82 on ATG5 and ATG16L1 expression and on autophagy-mediated AIEC clearance in Exo-LF82-receiving cells. Our study shows that upon AIEC infection, IECs secrete exosomes that can transfer specific miRNAs to recipient IECs, inhibiting autophagy-mediated clearance of intracellular AIEC.

Adherent-Invasive E. coli: Update on the Lifestyle of a Troublemaker in Crohn's Disease.

Besides genetic polymorphisms and environmental factors, the intestinal microbiota is an important factor in the etiology of Crohn’s disease (CD). Among microbiota alterations, a particular pathotype of Escherichia coli involved in the pathogenesis of CD abnormally colonizes the intestinal mucosa of patients: the adherent-invasive Escherichia coli (AIEC) pathobiont bacteria, which have the abilities to adhere to and to invade intestinal epithelial cells (IECs), as well as to survive and replicate within macrophages. AIEC have been the subject of many studies in recent years to unveil some genes linked to AIEC virulence and to understand the impact of AIEC infection on the gut and consequently their involvement in CD. In this review, we describe the lifestyle of AIEC bacteria within the intestine, from the interaction with intestinal epithelial and immune cells with an emphasis on environmental and genetic factors favoring their implantation, to their lifestyle in the intestinal lumen. Finally, we discuss AIEC-targeting strategies such as the use of FimH antagonists, bacteriophages, or antibiotics, which could constitute therapeutic options to prevent and limit AIEC colonization in CD patients.

Crohn's Disease-Associated Adherent-Invasive Escherichia coli Manipulate Host Autophagy by Impairing SUMOylation.

The intestinal mucosa of Crohn’s disease (CD) patients is abnormally colonized with adherent-invasive Escherichia coli (AIEC) that are able to adhere to and to invade intestinal epithelial cells (IECs), to survive in macrophages, and to induce a pro-inflammatory response. AIEC persist in the intestine, and induce inflammation in CEABAC10 transgenic mice expressing human CAECAM6, the receptor for AIEC. SUMOylation is a eukaryotic-reversible post-translational modification, in which SUMO, an ubiquitin-like polypeptide, is covalently linked to target proteins. Here, we investigated the role of SUMOylation in host responses to AIEC infection. We found that infection with the AIEC LF82 reference strain markedly decreased the levels of SUMO-conjugated proteins in human intestinal epithelial T84 cells. This was also observed in IECs from LF82-infected CEABAC10 transgenic mice. LF82-induced deSUMOylation in IECs was due in part to increased level of microRNA (miR)-18, which targets PIAS3 mRNA encoding a protein involved in SUMOylation. Over-expression of SUMOs in T84 cells induced autophagy, leading to a significant decrease in the number of intracellular LF82. Consistently, a decreased expression of UBC9, a protein necessary for SUMOylation, was accompanied with a decrease of LF82-induced autophagy, increasing bacterial intracellular proliferation and inflammation. Finally, the inhibition of miR-18 significantly decreased the number of intracellular LF82. In conclusion, our results suggest that AIEC inhibits the autophagy response to replicate intracellularly by manipulating host SUMOylation.

The IbeA invasin of adherent-invasive Escherichia coli mediates interaction with intestinal epithelia and macrophages.

Adherent-invasive Escherichia coli (AIEC) pathogroup isolates are a group of isolates from the intestinal mucosa of Crohn's disease patients that can invade intestinal epithelial cells (IECs) or macrophages and survive and/or replicate within. We have identified the ibeA gene in the genome of AIEC strain NRG857c and report the contribution of IbeA to the interaction of AIEC with IECs and macrophages and colonization of the mouse intestine. An ibeA deletion mutant strain (NRG857cΔibeA) was constructed, and the in vitro effect on AIEC adhesion and invasion of nonpolarized and polarized Caco-2 cells, the adhesion and transcytosis of M-like cells, the intracellular survival in THP-1 macrophages, and the contribution to intestinal colonization of the CD-1 murine model of infection were evaluated. A significant reduction in invasion was observed with the ibeA mutant in Caco-2 and M-like cells, whereas adhesion was not affected. Complementation of the mutant reestablished Caco-2 invasive phenotype to wild-type levels. Reduction in invasion did not significantly affect transcytosis through M-like cells at early time points. The absence of ibeA significantly affected AIEC intramacrophage survival up to 24 h postinfection. No significant changes associated with IbeA were found in AIEC colonization across the murine gastrointestinal tract, but a slight reduction of gamma interferon was observed in the ceca of mice infected with the ibeA mutant. In addition, a decrease in the pathology scores was observed in the ilea and ceca of mice infected with the ibeA mutant. Our data support the function of IbeA in the AIEC invasion process, macrophage survival, and inflammatory response in the murine intestine.

Analysis of the σE regulon in Crohn's disease-associated Escherichia coli revealed involvement of the waaWVL operon in biofilm formation.

Ileal lesions of patients with Crohn's disease are colonized by adherent-invasive Escherichia coli (AIEC), which is able to adhere to and to invade intestinal epithelial cells (IEC), to replicate within macrophages, and to form biofilms on the surface of the intestinal mucosa. Previous analyses indicated the involvement of the σ(E) pathway in AIEC-IEC interaction, as well as in biofilm formation, with σ(E) pathway inhibition leading to an impaired ability of AIEC to colonize the intestinal mucosa and to form biofilms. The aim of this study was to characterize the σ(E) regulon of AIEC strain LF82 in order to identify members involved in AIEC phenotypes. Using comparative in silico analysis of the σ(E) regulon, we identified the waaWVL operon as a new member of the σ(E) regulon in reference AIEC strain LF82. We determined that the waaWVL operon is involved in AIEC lipopolysaccharide structure and composition, and the waaWVL operon was found to be essential for AIEC strains to produce biofilm and to colonize the intestinal mucosa. An increased prevalence of adherent-invasive Escherichia coli (AIEC) bacteria was previously observed in the intestinal mucosa of Crohn's disease (CD) patients, and clinical observations revealed bacterial biofilms associated with the mucosa of CD patients. Here, analysis of the σ(E) regulon in AIEC and commensal E. coli identified 12 genes controlled by σ(E) only in AIEC. Among them, WaaWVL factors were found to play an essential role in biofilm formation and mucosal colonization by AIEC. In addition to identifying molecular tools that revealed a pathogenic population of E. coli colonizing the mucosa of CD patients, these results indicate that targeting the waaWVL operon could be a potent therapeutic strategy to interfere with the ability of AIEC to form biofilms and to colonize the gut mucosa.

436e Crohn's Disease-Associated Adherent-Invasive Escherichia coli Suppress Autophagy Response to Replicate Intracellularly by Regulating Host MicroRNA Expression

Background and Aims: The dysregulation of microRNAs (miRNAs, miR) in Crohn's disease (CD) was observed, suggesting a role for miRNAs as contributors to CD susceptibility. We previously showed that the adherent-invasive E. coli (AIEC), which abnormally colonize the intestinal mucosa of CD patients, are able to adhere to and invade intestinal epithelial cells (IECs). Here, we elucidated the mechanism by which AIEC infection modulates expression of miRNAs to suppress the host defense response. Methods: Mature miRNA expression was quantified by quantitative RT-PCR. Direct binding of miRNAs to the 3'-untranslated regions of their mRNA targets was determined by luciferase assay. Binding of NFκB to miRNA gene promoter was assessed by Chromatin immunoprecipitation assay. Autophagic activity was assessed by Western blot analysis and immunofluorescent labelling of LC3. Intracellular bacterial number was determined by invasion assay and confocal microscopy. In vivo transfection of miRNAs or anti-miRNAs was performed using a mouse ileal loop model. Results: AIEC infection activated NF-kB pathway, driving the transcription of miR-30c and miR130a genes. The AIEC-induced miR-30c and miR-130a upregulation in turn resulted in a decrease in ATG5 and ATG16L1 expression and inhibited autophagic activity, leading to abnormal intracellular persistence of AIEC and enhanced pro-inflammatory response. A reverse correlation between expression of miR-30c and miR-130a and that of ATG5 and ATG16L1 in CD ileal biopsies was observed, supporting the pathological relevance of the in vitro data. Inhibition of miR-30c and miR-130a in vitro and in vivo effectively blocked the AIEC-induced defect in autophagy, suppressing AIEC intracellular replication and AIECinduced inflammation. Conclusions: We show here that AIEC infection suppresses autophagy response to replicate within host cells by regulating miRNA expression. Our study strongly reinforces the increasing evidence showing autophagy deficiency as a molecular pathway underlying the pathogenesis of CD. This raises autophagy-targeting miRNAs as promising therapeutic targets to inhibit the intracellular replication of CD-associated bacteria and the consequent bacterial-induced inflammation.

436g M-CSF Deficient Op/Op Mice Are Protected Against the Development of Diabetic Gastroparesis in Streptozotocin-Induced Diabetes

Background and Aims: The dysregulation of microRNAs (miRNAs, miR) in Crohn's disease (CD) was observed, suggesting a role for miRNAs as contributors to CD susceptibility. We previously showed that the adherent-invasive E. coli (AIEC), which abnormally colonize the intestinal mucosa of CD patients, are able to adhere to and invade intestinal epithelial cells (IECs). Here, we elucidated the mechanism by which AIEC infection modulates expression of miRNAs to suppress the host defense response. Methods: Mature miRNA expression was quantified by quantitative RT-PCR. Direct binding of miRNAs to the 3'-untranslated regions of their mRNA targets was determined by luciferase assay. Binding of NFκB to miRNA gene promoter was assessed by Chromatin immunoprecipitation assay. Autophagic activity was assessed by Western blot analysis and immunofluorescent labelling of LC3. Intracellular bacterial number was determined by invasion assay and confocal microscopy. In vivo transfection of miRNAs or anti-miRNAs was performed using a mouse ileal loop model. Results: AIEC infection activated NF-kB pathway, driving the transcription of miR-30c and miR130a genes. The AIEC-induced miR-30c and miR-130a upregulation in turn resulted in a decrease in ATG5 and ATG16L1 expression and inhibited autophagic activity, leading to abnormal intracellular persistence of AIEC and enhanced pro-inflammatory response. A reverse correlation between expression of miR-30c and miR-130a and that of ATG5 and ATG16L1 in CD ileal biopsies was observed, supporting the pathological relevance of the in vitro data. Inhibition of miR-30c and miR-130a in vitro and in vivo effectively blocked the AIEC-induced defect in autophagy, suppressing AIEC intracellular replication and AIECinduced inflammation. Conclusions: We show here that AIEC infection suppresses autophagy response to replicate within host cells by regulating miRNA expression. Our study strongly reinforces the increasing evidence showing autophagy deficiency as a molecular pathway underlying the pathogenesis of CD. This raises autophagy-targeting miRNAs as promising therapeutic targets to inhibit the intracellular replication of CD-associated bacteria and the consequent bacterial-induced inflammation.

436f Human Gut Microbiota Modulates Gut Serotonergic Pathway and Motility

Background and Aims: The dysregulation of microRNAs (miRNAs, miR) in Crohn's disease (CD) was observed, suggesting a role for miRNAs as contributors to CD susceptibility. We previously showed that the adherent-invasive E. coli (AIEC), which abnormally colonize the intestinal mucosa of CD patients, are able to adhere to and invade intestinal epithelial cells (IECs). Here, we elucidated the mechanism by which AIEC infection modulates expression of miRNAs to suppress the host defense response. Methods: Mature miRNA expression was quantified by quantitative RT-PCR. Direct binding of miRNAs to the 3'-untranslated regions of their mRNA targets was determined by luciferase assay. Binding of NFκB to miRNA gene promoter was assessed by Chromatin immunoprecipitation assay. Autophagic activity was assessed by Western blot analysis and immunofluorescent labelling of LC3. Intracellular bacterial number was determined by invasion assay and confocal microscopy. In vivo transfection of miRNAs or anti-miRNAs was performed using a mouse ileal loop model. Results: AIEC infection activated NF-kB pathway, driving the transcription of miR-30c and miR130a genes. The AIEC-induced miR-30c and miR-130a upregulation in turn resulted in a decrease in ATG5 and ATG16L1 expression and inhibited autophagic activity, leading to abnormal intracellular persistence of AIEC and enhanced pro-inflammatory response. A reverse correlation between expression of miR-30c and miR-130a and that of ATG5 and ATG16L1 in CD ileal biopsies was observed, supporting the pathological relevance of the in vitro data. Inhibition of miR-30c and miR-130a in vitro and in vivo effectively blocked the AIEC-induced defect in autophagy, suppressing AIEC intracellular replication and AIECinduced inflammation. Conclusions: We show here that AIEC infection suppresses autophagy response to replicate within host cells by regulating miRNA expression. Our study strongly reinforces the increasing evidence showing autophagy deficiency as a molecular pathway underlying the pathogenesis of CD. This raises autophagy-targeting miRNAs as promising therapeutic targets to inhibit the intracellular replication of CD-associated bacteria and the consequent bacterial-induced inflammation.

Adherent-invasive Escherichia coli Biofilm Formation Assays

Patients with Crohn’s disease are abnormally colonized by adherent-invasive Escherichia coli (AIEC) bacteria (Chassaing and Darfeuille-Michaud, 2011). These bacteria are able to adhere to and invade intestinal epithelial cells (IEC), to replicate within macrophages, and were recently described to be able to form biofilms (Martinez-Medina et al., 2009; Chassaing and Darfeuille-Michaud, 2013). The reference strain of adherent-invasive E. coli is the strain LF82, associated with ileal Crohn’s disease (Darfeuille-Michaud et al., 1998).This protocol described basic steps of a biofilm formation assay on I) non-cell-treated polystyrene microtiter plates and on II) paraformaldehyde-fixed I-407 IEC monolayers.

The σ E Pathway Is Involved in Biofilm Formation by Crohn's Disease-Associated Adherent-Invasive Escherichia coli

Ileal lesions of patients with Crohn's disease are colonized by adherent-invasive Escherichia coli (AIEC) bacteria that are able to adhere to and invade intestinal epithelial cells (IEC), to replicate within macrophages, and to form biofilm. Clinical observations showed that bacterial biofilms were associated with the mucosa of inflammatory bowel disease patients. In the present study, we analyzed the relationship between AIEC colonization of the gut and the formation of biofilm, focusing on the involvement of the σ(E) pathway in the AIEC-IEC interaction. We observed that σ(E) pathway inhibition in AIEC reference strain LF82 led to an impaired ability to adhere to and invade IEC but also induced a large decrease in the abilities to colonize the intestinal mucosa and form biofilm. This indicates that targeting of the σ(E) pathway could be a very potent therapeutic strategy by which to interfere with the ability of AIEC to form biofilm on the gut mucosa of Crohn's disease patients.

Role of Meprins to Protect Ileal Mucosa of Crohn's Disease Patients from Colonization by Adherent-Invasive E. coli

Ileal lesions in Crohn's disease (CD) patients are colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to adhere to and invade intestinal epithelial cells (IEC), and to survive within macrophages. The interaction of AIEC with IEC depends on bacterial factors mainly type 1 pili, flagella, and outer membrane proteins. In humans, proteases can act as host defence mechanisms to counteract bacterial colonization. The protease meprin, composed of multimeric complexes of the two subunits alpha and beta, is abundantly expressed in IECs. Decreased levels of this protease correlate with the severity of the inflammation in patients with inflammatory bowel disease. The aim of the present study was to analyze the ability of meprin to modulate the interaction of AIEC with IECs. In patients with ileal CD we observed decreased levels of meprins, in particular that of meprin β. Dose-dependent inhibition of the abilities of AIEC strain LF82 to adhere to and invade intestinal epithelial T84 cells was observed when bacteria were pre-treated with both exogenous meprin α and meprin β. Dose-dependent proteolytic degradation of type 1 pili was observed in the presence of active meprins, but not with heat-inactivated meprins, and pretreatment of AIEC bacteria with meprins impaired their ability to bind mannosylated host receptors and led to decreased secretion of the pro-inflammatory cytokine IL-8 by infected T84 cells. Thus, decreased levels of protective meprins as observed in CD patients may contribute to increased AIEC colonization.