Increased epithelial gaps in the small intestines of patients with inflammatory bowel disease: density matters

doi:10.1016/j.gie.2011.01.018

Gastrointestinal Endoscopy

Volume 73, Issue 6, June 2011, Pages 1174-1180

https://doi.org/10.1016/j.gie.2011.01.018 Get rights and content

Background

Epithelial gaps created by shedding of epithelial cells in the small intestine can be visualized by using confocal laser endomicroscopy (CLE). The density of epithelial gaps in the small bowels of patients with inflammatory bowel disease (IBD) and controls without IBD is unknown.

Objective

To determine whether the epithelial gap density in patients with IBD is different from that in controls.

Design

Prospective, controlled, cohort study.

Setting

A tertiary-care referral center.

Patients

This study involved patients with IBD and control patients without IBD undergoing colonoscopy.

Intervention

Probe-based CLE (pCLE) was used to image the terminal ileum.

Main Outcome Measurements

The primary outcome of the study was gap density, defined as the total number of gaps per 1000 cells counted in adequately imaged villi by using pCLE. The pCLE images were blindly reviewed, and the number of epithelial gaps and cells were manually counted. The secondary outcomes were correlation of gap density with disease activity, location, and severity of clinical disease.

Results

There were 30 controls and 28 patients with IBD. Of the patients with IBD, 16 had Crohn's disease, and 12 had ulcerative colitis. The median epithelial gap densities for controls and patients with IBD were 18 and 61 gaps/1000 cells, respectively (P < .001). Gap density did not correlate with disease activity. Patients with ulcerative pan-colitis tended toward gap densities lower than those of patients with limited colitis (32 versus 97 gaps/1000 cells, P = .06). Patients with IBD with severe clinical disease also had lower median gap densities (37 vs 90 gaps/1000 cells, P = .04).

Limitations

A single-center study.

Conclusion

The epithelial gap density was significantly increased in patients with IBD compared with controls. (Clinical trial registration number: NCT00988273.)

Section snippets

Patients

This is a prospective, controlled, cohort study registered at ClinicalTrial.Gov (NCT00988273). The study protocol was reviewed and approved by the Human Ethics Research Review Board at the University of Alberta. The study group consisted of patients either previously diagnosed or suspected to have a diagnosis of IBD. The control group consisted of patients undergoing colonoscopy for non-IBD-related indications such as colon cancer screening, symptoms of irritable bowel syndrome, and rectal

Results

The baseline patient characteristics of the IBD and control groups are shown in Table 1. Patients with IBD were significantly younger than controls, with a mean (± SD) age of 40.3 ± 14.1 years versus 53.0 ± 14.7 years (P = .001). Analysis was performed on 27 of 28 patients with IBD, because pCLE could not be carried out in one patient with Crohn's disease because of the inability to intubate the terminal ileum. We imaged only endoscopically normal-appearing mucosa by using pCLE. A

Discussion

In this study, we showed that patients with IBD had significantly higher epithelial gap densities in the terminal ileum compared with controls who did not have IBD. The findings of our study are consistent with our hypothesis that patients with IBD have increased epithelial cell shedding in the small intestine, which can be measured by gap density by using pCLE. In our secondary analysis, we found that patients with ulcerative pancolitis tended to have gap densities lower than those with

Acknowledgment

We would like to thank Dr Jon Meddings and Dr Jerold Turner for their helpful suggestions with the design of our study.

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The leaky gut is often referred to altered permeability with enhanced paracellular diffusion of luminal contents and moreover, related to epithelial gaps or breaches resulting from an impaired epithelial cell turnover to replenish cells after cellular damage or apoptosis [31–34]. In contrast to that, the leaky gut model is supported by the observation of endoscopic analysis in IBD patients showing structural defects (microerosions) of the injured gut barrier and correlated mucosal wound healing [35–37]. Modelling these excessive epithelial damages was realized by the leaky gut model and the introduction of a sub-confluent Caco-2 layer.
Complex in vitro models of human immune cells and intestinal mucosa may have a translation-assisting role in the assessment of anti-inflammatory compounds. Chronic inflammation of the gastrointestinal tract is a hallmark of inflammatory bowel diseases (IBD). In both IBD entities, Crohn's disease and ulcerative colitis, impaired immune cell activation and dysfunctional epithelial barrier are the common pathophysiology. Current therapeutic approaches are targeting single immune modulator molecules to stop disease progression and reduce adverse effects. Such molecular targets can be difficult to assess in experimental animal models of colitis, due to the disease complexity and species differences. Previously, a co-culture model based on human epithelial cells and monocytes arranged in a physiological microenvironment was used to mimic inflamed mucosa for toxicological and permeability studies. The leaky gut model described here, a co-culture of Caco-2, THP-1 and MUTZ-3 cells, was used to mimic IBD-related pathophysiology and for combined investigations of permeability and target engagement of two Janus kinase (JAK) inhibitors, tofacitinib (TOFA) and a JAK1-targeting siRNA nanomedicine. The co-culture just before reaching confluency of the epithelium was used to mimic the compromised intestinal barrier. Delivery efficacy and target engagement against JAK1 was quantified via downstream analysis of STAT1 protein phosphorylation after IFN-γ stimulation. Compared to a tight barrier, the leaky gut model showed 92 ± 5% confluence, a barrier function below 200 Ω*cm², and enhanced immune response to bacteria-derived lipopolysaccharides. By confocal microscopy we observed an increased accumulation of siJAK1-nanoparticles within the sub-confluent regions leading to uptake into immune cells near the epithelium. A concentration-dependent downregulation of JAK/STAT pathway was observed for siJAK1-nanoparticles (10 ± 12% to 16 ± 12%), whereas TOFA inhibition was 86 ± 2%, compared to untreated cells.
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It was shown that the epithelial gap density of the terminal ileum is significantly higher that that of controls without IBD, in both CD and ulcerative colitis (UC) patients. Interestingly, gap density did not correlate with disease activity, hence further studies are needed to better understand the potential significance of these obervations in IBD [30]. Since cell shedding is a physiological regenerative phenomenon, the qualitative observation of epithelial gaps may not be sufficient to distinguish physiological versus pathological gut permeability.
Intestinal permeability is getting more and more attention in gastrointestinal research. Although well recognized, its exact role in health and disease is yet to be defined. There are many methods of quantifying intestinal permeability, but most of them fail to deliver tangible information about the morphological integrity of the intestinal barrier. In this review we aim to describe imaging options for the assessment of intestinal barrier integrity and their potential relevance for clinical practice. Our focus is on confocal laser endomicroscopy, which is at this time the only method for visualizing not only functional but also morphological aspects of the gut barrier in vivo.
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TNF is implicated in disruption of intestinal epithelial barrier integrity by altering tight junctions and inducing cell death [11,32–34]. Loss of epithelial barrier and increased permeability has been demonstrated in patients with CD and associated with higher risk of recurrent flares [35,36]. TNF can trigger either inflammatory or apoptotic cell response via TNFR1 and TNFR2, depending on the balance between pro- and anti-apoptotic factors inside as well as outside the cell [9].
Crohn's disease (CD) is chronic inflammatory bowel disease with different phenotypic characteristics influencing disease prognosis and therapeutic strategies. The aim of this pilot study was to analyze selected inflammatory and apoptotic markers in non-inflamed and inflamed samples of ileal mucosa of non-stricturing/non-penetrating (NS/NP) and stricturing (S) CD mucosal phenotypes in order to characterize their distinct profiles.
From twenty CD patients (9 NS/NP, 11 S) paired non-inflamed and inflamed ileal biopsies were collected and used for analysis of cytokine (TNF and IL6) and apoptotic (Bcl2, Bax, Fas and FasL) genes' expression levels by real-time PCR, while NFκB transcriptional potency was assessed by electromobility gel shift assay.
Our results demonstrated significant upregulation of TNF and IL6 in inflamed area of both NS/NP (p = 0.03, p = 0.01) and S phenotypes (p = 0.04, p = 0.04), respectively. However, TNF increase was more prominent in NS/NP compared to S inflamed mucosa (p = 0.02). Also, level of proapoptotic Bax was significantly higher in NS/NP compared to S inflamed mucosa (p = 0.01). Opposing transcription potency of NFκB has been detected between two phenotypes: being decreased in NS/NP (p = 0.07) and increased in S (p = 0.1) inflamed compared to non-inflamed mucosa, demonstrating trend towards statistical significance.
We found that two distinct CD phenotypes have specific molecular signatures. Obtained results could direct improvement of current and development of new therapeutic strategies based on more specific molecular stratification of CD patients.
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The intestinal tract is the largest barrier between a person and the environment. In this role, the intestinal tract is responsible not only for absorbing essential dietary nutrients, but also for protecting the host from a variety of ingested toxins and microbes. The intestinal barrier system is composed of a mucus layer, intestinal epithelial cells (IECs), tight junctions (TJs), immune cells, and a gut microbiota, which are all susceptible to external factors such as dietary fats. When components of this barrier system are disrupted, intestinal permeability to luminal contents increases, which is implicated in intestinal pathologies such as inflammatory bowel disease, necrotizing enterocolitis, and celiac disease. Currently, there is mounting evidence that consumption of excess dietary fats can enhance intestinal permeability differentially. For example, dietary fat modulates the expression and distribution of TJs, stimulates a shift to barrier-disrupting hydrophobic bile acids, and even induces IEC oxidative stress and apoptosis. In addition, a high-fat diet (HFD) enhances intestinal permeability directly by stimulating proinflammatory signaling cascades and indirectly via increasing barrier-disrupting cytokines [TNFα, interleukin (IL) 1B, IL6, and interferon γ (IFNγ)] and decreasing barrier-forming cytokines (IL10, IL17, and IL22). Finally, an HFD negatively modulates the intestinal mucus composition and enriches the gut microflora with barrier-disrupting species. Although further research is necessary to understand the precise role HFDs play in intestinal permeability, current data suggest a stronger link between diet and intestinal disease than was first thought to exist. Therefore, this review seeks to highlight the various ways an HFD disrupts the gut barrier system and its many implications in human health.
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Confocal laser endomicroscopy (CLE) is a novel endoscopic imaging modality allowing in vivo characterization of microscopic tissue details during ongoing endoscopy in real time.15 In patients with irritable bowel disease (IBD), CLE enables the detection of either inflammatory16−18 or dysplastic19 histopathologic mucosal changes. In patients with CD, this technique can distinguish patients from controls and active versus quiescent disease even in areas of macroscopically uneventful mucosa.20
Assessment of prognostic factors in patients with Crohn’s disease (CD) is of pivotal importance for early intervention and “treat-to-target” strategies. Confocal laser endomicroscopy (CLE) enables on-demand in vivo characterization of mucosal inflammatory and architectural changes during endoscopy. We prospectively assessed the value of CLE for prediction of clinical outcome parameters in CD.
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: DISCLOSURE: This work was supported in part by a grant to J. Liu from the Canadian Institutes of Health Research/Canadian Association of Gastroenterology and a grant to R. Fedorak from the Canada Foundation for Innovation. No other financial relationships relevant to this publication were disclosed.

: If you would like to chat with an author of this article, you may contact Dr Liu at [email protected].

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Original articleClinical endoscopyIncreased epithelial gaps in the small intestines of patients with inflammatory bowel disease: density matters

Background

Objective

Design

Setting

Patients

Intervention

Main Outcome Measurements

Results

Limitations

Conclusion

Section snippets

Patients

Results

Discussion

Acknowledgment

Gastroenterology

Gastroenterology

Lab Invest

Gastroenterology

Cell

Lancet

Gastroenterology

Am J Gastroenterol

Immunity

Insights into IBD pathogenesis

Curr Gastroenterol Rep

Original article
Clinical endoscopy
Increased epithelial gaps in the small intestines of patients with inflammatory bowel disease: density matters