Enhanced E. coli LF82 Translocation through the Follicle-associated Epithelium in Crohn’s Disease is Dependent on Long Polar Fimbriae and CEACAM6 expression, and Increases Paracellular Permeability

Abstract Background and Aims Patients with Crohn’s disease [CD] harbour an increased number of adherent-invasive E. coli [AIEC]. The strain LF82, identified in the ileal mucosa of CD patients, has been extensively studied for pathogenic mechanisms. However, understanding of the interaction of LF82 with the intestinal mucosa of CD patients is lacking. Methods Here, we investigated the importance of long polar fimbriae [LPF] type 1 pili and the carcinoembryonic antigen-related cell-adhesion molecule 6 [CEACAM6] for translocation of LF82 in an in vitro model of follicle-associated epithelium [FAE], and in the FAE and villus epithelium [VE] of patients with CD and controls, using Ussing chambers. Results Significantly greater LF82 passage occurred in the FAE model compared with in the VE Caco-2cl1 mono-culture. Moreover, bacterial translocation was inhibited by either LPF disruption or pre-incubation with anti-CEACAM6 antibody. Tissue mounted in Ussing chambers showed significantly higher LF82 passage in FAE from patients with CD compared with control FAE, that was diminished in LF82 lacking LPF and by blocking host CEACAM6. Interestingly, addition of LF82 to the CD FAE tissues significantly increased paracellular permeability [of 51Chromium-EDTA] compared with baseline, and the increase was inhibited by anti-CEACAM6. Immunofluorescence and immunoblots showed higher expression of CEACAM6 in FAE of patients with CD compared with in FAE from controls. Conclusions These data suggest that the FAE of CD patients is a site of vulnerability for invasion by LF82 via a mechanism that requires both bacterial LPF and host CEACAM6. Further, LF82 has the ability to increase paracellular passage through the FAE of patients with CD. These data can help define novel therapeutic targets in CD for the prevention of clinical recurrence.

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Antagonism of Adherent Invasive E. coli LF82 With Human α-defensin 5 in the Follicle-associated Epithelium of Patients With Ileal Crohn’s Disease

Inflammatory Bowel Diseases ◽

10.1093/ibd/izaa315 ◽

2020 ◽

Author(s):

Lina Y Alkaissi ◽

Martin E Winberg ◽

Stéphanie DS Heil ◽

Staffan Haapaniemi ◽

Pär Myrelid ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Ex Vivo ◽

Ussing Chambers ◽

E Coli ◽

Follicle Associated Epithelium ◽

Vitro Model ◽

Set Up

Abstract Background The first visible signs of Crohn’s disease (CD) are microscopic erosions over the follicle-associated epithelium (FAE). The aim of the study was to investigate the effects of human α-defensin 5 (HD5) on adherent-invasive Escherichia coli LF82 translocation and HD5 secretion after LF82 exposure in an in vitro model of human FAE and in human FAE ex vivo. Methods An in vitro FAE-model was set up by the coculture of Raji B cells and Caco-2-cl1 cells. Ileal FAE from patients with CD and controls were mounted in Ussing chambers. The effect of HD5 on LF82 translocation was studied by LF82 exposure to the cells or tissues with or without incubation with HD5. The HD5 secretion was measured in human FAE exposed to LF82 or Salmonella typhimurium. The HD5 levels were evaluated by immunofluorescence, immunoblotting, and ELISA. Results There was an increased LF82 translocation across the FAE-model compared with Caco-2-cl1 (P < 0.05). Incubation of cell/tissues with HD5 before LF82 exposure reduced bacterial passage in both models. Human FAE showed increased LF82 translocation in CD compared with controls and attenuated passage after incubation with sublethal HD5 in both CD and controls (P < 0.05). LF82 exposure resulted in a lower HD5 secretion in CD FAE compared with controls (P < 0.05), whereas Salmonella exposure caused equal secretion on CD and controls. There were significantly lower HD5 levels in CD tissues compared with controls. Conclusions Sublethal HD5 reduces the ability of LF82 to translocate through FAE. The HD5 is secreted less in CD in response to LF82, despite a normal response to Salmonella. This further implicates the integrated role of antimicrobial factors and barrier function in CD pathogenesis.

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Invasive Ability of an Escherichia coliStrain Isolated from the Ileal Mucosa of a Patient with Crohn’s Disease

Infection and Immunity ◽

10.1128/iai.67.9.4499-4509.1999 ◽

1999 ◽

Vol 67 (9) ◽

pp. 4499-4509 ◽

Cited By ~ 278

Author(s):

Jerome Boudeau ◽

Anne-Lise Glasser ◽

Estelle Masseret ◽

Bernard Joly ◽

Arlette Darfeuille-Michaud

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Host Cell ◽

Bacterial Invasion ◽

Intestinal Epithelial ◽

Invasive Ability ◽

Ileal Mucosa ◽

Gastrointestinal Infections ◽

E Coli ◽

Host Cell Membranes

ABSTRACT Crohn’s disease (CD) is an inflammatory bowel disease in whichEscherichia coli strains have been suspected of being involved. We demonstrated previously that ileal lesions of CD are colonized by E. coli strains able to adhere to intestinal Caco-2 cells but devoid of the virulence genes so far described in the pathogenic E. coli strains involved in gastrointestinal infections. In the present study we compared the invasive ability of one of these strains isolated from an ileal biopsy of a patient with CD, strain LF82, with that of reference enteroinvasive (EIEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enteraggregative (EAggEC), enterohemorrhagic (EHEC), and diffusely adhering (DAEC)E. coli strains. Gentamicin protection assays showed thatE. coli LF82 was able to efficiently invade HEp-2 cells. Its invasive level was not significantly different from that of EIEC and EPEC strains (P > 0.5) but significantly higher than that of ETEC (P < 0.03), EHEC (P < 0.005), EAggEC (P < 0.004) and DAEC (P < 0.02) strains. Strain LF82 also demonstrated efficient ability to invade intestinal epithelial cultured Caco-2, Intestine-407, and HCT-8 cells. Electron microscopy examination of infected HEp-2 cells revealed the presence of numerous intracellular bacteria located in vacuoles or free in the host cell cytoplasm. In addition, the interaction of strain LF82 with epithelial cells was associated with the elongation of microvillar extensions that extruded from the host cell membranes and engulfed the bacteria. This internalization mechanism strongly resembles Salmonella- orShigella-induced macropinocytosis. The use of cytochalasin D and colchicine showed that the uptake of strain LF82 by HEp-2 cells was mediated by both an actin microfilament-dependent mechanism and microtubule involvement. In addition, strain LF82 survived for at least 24 h in HEp-2 and Intestine-407 cells and efficiently replicated intracellularly in HEp-2 cells. PCR and hybridization experiments did not reveal the presence of any of the genetic determinants encoding EIEC, EPEC, or ETEC proteins involved in bacterial invasion. Thus, these findings show that LF82, which colonized the ileal mucosa of a patient with CD, is a true invasive E. coli strain and suggest the existence of a new potentially pathogenic group of E. coli, which we propose be designated adherent-invasive E. coli.

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DOP06 Non-invasive identification of adherent-invasive E. coli in patients with Crohn’s disease

Journal of Crohn s and Colitis ◽

10.1093/ecco-jcc/jjz203.045 ◽

2020 ◽

Vol 14 (Supplement_1) ◽

pp. S044-S045 ◽

Cited By ~ 1

Author(s):

A Buisson ◽

E Vazeille ◽

X Hébuterne ◽

M Fumery ◽

B Pariente ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Unmet Need ◽

Inhibition Test ◽

Invasive Ability ◽

Ileal Mucosa ◽

Multi Centre Study ◽

E Coli ◽

Non Invasive ◽

Adhesion Inhibition

Abstract Background Medications limiting the adhesion of ‘adherent and invasive E. coli’ (AIEC) represent potential strategies to treat Crohn’s disease (CD). However, the ileal AIEC identification is a time-consuming procedure, and the number of AIEC strains which colonise ileal CD mucosa remains unknown. There is an unmet need for non-invasive biomarkers to identify patients colonised by AIEC. We aimed to evaluate non-invasive biomarker of ileal AIEC colonisation in patients with CD. Methods This prospective and multi-centre study included CD patients requiring ileocoloscopy. Saliva, serum, stools and ileal biopsies were collected. Abundance and global invasive ability of ileal or faecal E. coli were performed. Isolated E. coli were characterised as AIEC or non-AIEC on I407 epithelial cells and THP1 macrophages. The ERIC-PCR profiles of ileal E. coli were performed. Ileal E. coli/CEACAM6 interaction was analysed by a yeast aggregation test and T84 assays (CEACAM6 protein expression, adhesion inhibition test with D-mannose). Quantification of serum anti-E. coli and ileal or salivary CEACAM6 was realised by ELISA. Results Overall, 102 CD patients were enrolled in this study and 25.8% of them exhibited ileal AIEC colonisation (AIEC+). The abundance and global invasive ability of ileal mucosa-associated E. coli were higher in AIEC+ CD patients compared with CD patients without AIEC (AIEC−) (p = 0.0065 and p = 0.0007, respectively). There was no difference between faecal abundance and invasive ability of E. coli between AIEC+ and AIEC− patients. The ERIC-PCR profiles of ileal E. coli showed that CD AIEC+ were for 78% of them colonised by not more than 2 clonal AIEC strains. In addition, AIEC were able to interact with CEACAM6 by binding D-mannose residues and to induce CEACAM6 expression in T84 cells (p = 0.0009 and p = 0.0185, vs. non-AIEC; respectively). This was also supported by adhesion inhibition test. Serum anti-E. coli level was higher for CD AIEC+ (vs. CD AIEC-). Ileal CEACAM6 level were positively correlated with abundance of ileal associated E. coli in AIEC+ patients (r = 0.4000; p = 0.0362) and with salivary CEACAM6 level (r = 0.4690; p < 0.0001). The non-invasive biomarker ‘serum anti-E.coli/salivary CEACAM6’ index was higher for CD AIEC+ (p = 0.0174; vs. CD AIEC-). A cut-off value < 1.34 × 10−6 eliminated the presence of ileal AIEC with a high negative predictive value (90% CI95% [69%–95%]). Conclusion Our study reported that identification of faecal AIEC cannot replace identification of AIEC from ileal biopsies, most of AIEC infection are mono or bi-clonal (≤ 2 strains) and that non-invasive biomarker such as ‘serum anti-E.coli/salivary CEACAM6’ index could be helpful to screen CD patients for AIEC infection.

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P681 Higher in vitro mucin degradation, but no increased paracellular permeability by faecal water from Crohn’s disease patients

Journal of Crohn s and Colitis ◽

10.1093/ecco-jcc/jjab076.801 ◽

2021 ◽

Vol 15 (Supplement_1) ◽

pp. S601-S601

Author(s):

H Becker ◽

N Kameli ◽

A Rustichelli ◽

H Britt ◽

F Stassen ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Barrier Function ◽

Disease Onset ◽

Paracellular Permeability ◽

Epithelial Barrier ◽

Rrna Gene ◽

Relative Abundances ◽

The Impact

Abstract Background Crohn’s disease (CD) is a chronic inflammatory gastro-intestinal condition with a variable disease course. Impaired intestinal integrity and microbial dysbiosis are associated with disease onset and exacerbations. We hypothesized that a perturbed microbial activity in CD patients may contribute to the impaired barrier function. Therefore, this study aimed to examine the impact of faecal bacterial products of active CD patients, CD patients in remission, and healthy controls (HC) on mucin degradation and intestinal epithelial barrier function in vitro. Methods Six HC and twelve CD patients were included. Faecal samples were obtained within one week prior to endoscopy processed within 6 hours after collection. Disease activity was determined by the short endoscopic score for CD (SES-CD). Faecal water (FW) and bacterial membrane vesicles (MVs) were applied on mucin agar to determine mucin degradation. Further, differentiated Caco-2 cell monolayers were exposed to FW and MVs to assess transepithelial electrical resistance (TEER) and paracellular junction stability using permeation of fluorescein isothiocyanate-labelled dextran of 4 kDa. Relative abundances of faecal bacterial genera were evaluated by 16S rRNA gene amplicon sequencing. Results FW-induced mucin degradation was higher in CD samples as compared to HC (p<0.01), but was not linked to specific bacterial relative abundances. FW resulted in 78–87% decrease of TEER in three of the remissive (p<0.001) but not the active CD or HC samples. The decrease of TEER was not linked to increased paracellular permeability. MVs did not induce mucin degradation or epithelial barrier disruption. Conclusion The higher mucin degradation capacity of CD patient-derived FW might indicate contributions of microbial products to CD pathophysiology and warrants further investigation. Moreover, the altered epithelial resistance in some individuals is not due to paracellular alterations.

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

PLoS ONE ◽

10.1371/journal.pone.0021199 ◽

2011 ◽

Vol 6 (6) ◽

pp. e21199 ◽

Cited By ~ 29

Author(s):

Emilie Vazeille ◽

Marie-Agnès Bringer ◽

Aurélie Gardarin ◽

Christophe Chambon ◽

Christoph Becker-Pauly ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Ileal Mucosa ◽

E Coli

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Mechanism of glucocorticoid regulation of the intestinal tight junction barrier

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00252.2006 ◽

2007 ◽

Vol 292 (2) ◽

pp. G590-G598 ◽

Cited By ~ 64

Author(s):

Michel A. Boivin ◽

Dongmei Ye ◽

John C. Kennedy ◽

Rana Al-Sadi ◽

Chris Shepela ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Protein Expression ◽

Tight Junction ◽

Barrier Function ◽

In Vitro Model ◽

Intestinal Epithelial ◽

Tnf Α ◽

Vitro Model

A defective intestinal epithelial tight junction (TJ) barrier has been proposed as an important pathogenic factor contributing to the intestinal inflammation of Crohn's disease. Glucocorticoids are first-line therapeutic agents for the treatment of moderate to severe Crohn's disease. Glucocorticoid treatment has been shown to induce retightening of the intestinal TJ barrier defect in Crohn's disease patients. However, the mechanisms that mediate the glucocorticoid therapeutic action on intestinal TJ barrier function remain unknown. The aim of this study was to elucidate the mechanism of glucocorticoid modulation of the intestinal epithelial TJ barrier using an in vitro model system. Filter-grown Caco-2 intestinal epithelial cells were used as an in vitro model to examine the effects of glucocorticoids on basal intestinal epithelial TJ barrier function and on TNF-α-induced disruption of the TJ barrier. Glucocorticoids (prednisolone and dexamethasone) did not have a significant effect on baseline Caco-2 TJ barrier function but prevented the TNF-α-induced increase in Caco-2 TJ permeability. The glucocorticoid protective effect against the TNF-α-induced increase in Caco-2 TJ permeability required activation of the glucocorticoid receptor (GR) complex. The activation of the GR complex resulted in GR complex binding to the glucocorticoid response element (GRE) site on DNA and activation of a GR-responsive promoter. Glucocorticoids inhibited the TNF-α-induced increase in myosin light chain kinase (MLCK) protein expression, a key process mediating the TNF-α increase in intestinal TJ permeability. The glucocorticoid inhibition of the TNF-α-induced increase in MLCK protein expression was due to the binding of the GR complex to a GRE binding site on the MLCK promoter region suppressing the TNF-α-induced activation. Glucocorticoids inhibit the TNF-α-induced increase in Caco-2 TJ permeability. The prednisolone protective action was mediated by binding of activated GR complex to the GRE site on the MLCK promoter, suppressing the TNF-α-induced increase in MLCK gene activity, protein expression, and subsequent opening of the intestinal TJ barrier.

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A β-Glucan-Based Dietary Fiber Reduces Mast Cell-Induced Hyperpermeability in Ileum From Patients With Crohn’s Disease and Control Subjects

Inflammatory Bowel Diseases ◽

10.1093/ibd/izx002 ◽

2017 ◽

Vol 24 (1) ◽

pp. 166-178 ◽

Cited By ~ 15

Author(s):

John-Peter Ganda Mall ◽

Maite Casado-Bedmar ◽

Martin E Winberg ◽

Robert J Brummer ◽

Ida Schoultz ◽

...

Keyword(s):

Mast Cell ◽

Crohn’S Disease ◽

Crohn's Disease ◽

Intestinal Barrier ◽

Intestinal Barrier Function ◽

Barrier Dysfunction ◽

Ussing Chambers ◽

Beneficial Effects ◽

And Control

Abstract Background Administration of β-glucan has shown immune-enhancing effects. Our aim was to investigate whether β-glucan could attenuate mast cell (MC)-induced hyperpermeability in follicle-associated epithelium (FAE) and villus epithelium (VE) of patients with Crohn’s disease (CD) and in noninflammatory bowel disease (IBD)-controls. Further, we studied mechanisms of β-glucan uptake and effects on MCs in vitro. Methods Segments of FAE and VE from 8 CD patients and 9 controls were mounted in Ussing chambers. Effects of the MC-degranulator compound 48/80 (C48/80) and yeast-derived β-1,3/1,6 glucan on hyperpermeability were investigated. Translocation of β-glucan and colocalization with immune cells were studied by immunofluorescence. Caco-2-cl1- and FAE-cultures were used to investigate β-glucan-uptake using endocytosis inhibitors and HMC-1.1 to study effects on MCs. Results β-glucan significantly attenuated MC-induced paracellular hyperpermeability in CD and controls. Transcellular hyperpermeability was only significantly attenuated in VE. Baseline paracellular permeability was higher in FAE than VE in both groups, P<0.05, and exhibited a more pronounced effect by C48/80 and β-glucan P<0.05. No difference was observed between CD and controls. In vitro studies showed increased passage, P<0.05, of β-glucan through FAE-culture compared to Caco-2-cl1. Passage was mildly attenuated by the inhibitor methyl-β-cyclodextrin. HMC-1.1 experiments showed a trend to decreasing MC-degranulation and levels of TNF-α but not IL-6 by β-glucan. Immunofluorescence revealed more β-glucan-uptake and higher percentage of macrophages and dendritic cells close to β-glucan in VE of CD compared to controls. Conclusions We demonstrated beneficial effects of β-glucan on intestinal barrier function and increased β-glucan-passage through FAE model. Our results provide important and novel knowledge on possible applications of β-glucan in health disorders and diseases characterized by intestinal barrier dysfunction.

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Filtered supernatant of lactobacillus GG (LGG) prevents Crohn's disease (CD) mucosa adherent-E. coli induced alterations in paracellular permeability

Gastroenterology ◽

10.1016/s0016-5085(03)82408-3 ◽

2003 ◽

Vol 124 (4) ◽

pp. A476

Author(s):

Zahangir Khaled ◽

Stefano Guandalini ◽

Barbara S. Kirschner ◽

Barbara Hendrickson

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Paracellular Permeability ◽

E Coli ◽

Lactobacillus Gg

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Enteroids Generated from Patients with Severe Inflammation in Crohn’s Disease Maintain Alterations of Junctional Proteins

Journal of Crohn s and Colitis ◽

10.1093/ecco-jcc/jjaa085 ◽

2020 ◽

Vol 14 (10) ◽

pp. 1473-1487 ◽

Cited By ~ 2

Author(s):

Michael Meir ◽

Jonas Salm ◽

Christina Fey ◽

Matthias Schweinlin ◽

Catherine Kollmann ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Tight Junction ◽

In Vitro Model ◽

Transcriptional Level ◽

Mrna Levels ◽

Severe Inflammation ◽

Junctional Proteins ◽

Vitro Model

Abstract Background The mechanisms underlying loss of intestinal epithelial barrier [IEB] function in Crohn’s disease [CD] are poorly understood. We tested whether human enteroids generated from isolated intestinal crypts of CD patients serve as an appropriate in vitro model to analyse changes of IEB proteins observed in patients’ specimens. Methods Gut samples from CD patients and healthy individuals who underwent surgery were collected. Enteroids were generated from intestinal crypts and analyses of junctional proteins in comparison to full wall samples were performed. Results Histopathology confirmed the presence of CD and the extent of inflammation in intestinal full wall sections. As revealed by immunostaining and Western blot analysis, profound changes in expression patterns of tight junction, adherens junction and desmosomal proteins were observed in full wall specimens when CD was present. Unexpectedly, when enteroids were generated from specimens of CD patients with severe inflammation, alterations of most tight junction proteins and the majority of changes in desmosomal proteins but not E-cadherin were maintained under culture conditions. Importantly, these changes were maintained without any additional stimulation of cytokines. Interestingly, qRT-PCR demonstrated that mRNA levels of junctional proteins were not different when enteroids from CD patients were compared to enteroids from healthy controls. Conclusions These data indicate that enteroids generated from patients with severe inflammation in CD maintain some characteristics of intestinal barrier protein changes on a post-transcriptional level. The enteroid in vitro model represents an appropriate tool to gain further cellular and molecular insights into the pathogenesis of barrier dysfunction in CD.

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