Endomicroscopic and Transcriptomic Analysis of Impaired Barrier Function and Malabsorption in Environmental Enteropathy

Abstract

IntroductionEnvironmental enteropathy (EE) is associated with growth failure, micronutrient malabsorption and impaired responses to oral vaccines. We set out to define cellular mechanisms of impaired barrier function in EE and explore protective mechanisms.MethodsWe studied 49 adults with environmental enteropathy in Lusaka, Zambia using confocal laser endomicroscopy (CLE); histology, immunohistochemistry and mRNA sequencing of small intestinal biopsies; and correlated these with plasma lipopolysaccharide (LPS) and a zinc uptake test.ResultsCLE images (median 134 for each study) showed virtually ubiquitous small intestinal damage. Epithelial defects, imaged by histology and claudin 4 immunostaining, were predominantly seen at the tips of villi and corresponded with leakage imaged in vivo by CLE. In multivariate analysis, circulating log-transformed LPS was correlated with cell shedding events (β = 0.83; P = 0.035) and with serum glucagon-like peptide-2 (β = -0.13; P = 0.007). Zinc uptake from a test dose of 25mg was attenuated in 30/47 (64%) individuals and in multivariate analysis was reduced by HIV, but positively correlated with GLP-2 (β = 2.72; P = 0.03). There was a U-shaped relationship between circulating LPS and villus surface area. Transcriptomic analysis identified 23 differentially expressed genes in severe enteropathy, including protective peptides and proteins.ConclusionsConfocal endomicroscopy, claudin 4 immunostaining and histology identify epithelial defects which are probably sites of bacterial translocation, in the presence of which increased epithelial surface area increases the burden of translocation. GLP 2 and other protective peptides may play an important role in mucosal protection in EE.

The mucosal barrier at a glance

“…Although this discussion has focused on inflammatory bowel disease and graft-versus-host disease, which has been studied extensively, other examples of disease-associated gut barrier loss abound, including those that occur within the contexts of intestinal infection (Halliez et al, 2016;In et al, 2016;Zolotarevsky et al, 2002), irritable bowel syndrome (Bertiaux-Vandaële et al, 2011;Martínez et al, 2013;Wu et al, 2016), celiac disease (Schumann et al, 2012;Setty et al, 2015;Szakál et al, 2010) and environmental enteric dysfunction (Kelly et al, 2016;Vinetz et al, 2016;Yu et al, 2016). Although studied to a lesser extent, barrier defects also occur in pulmonary, renal and dermatologic diseases.…”

The potential role of optical biopsy in the study and diagnosis of environmental enteric dysfunction

“…For patients with IBS 60,61 the leakage of intravenous fluorescein into the lumen has been visualized in real time 61 , indicating that endomicroscopy can be used to monitor dynamic, cellular level changes in gut function and permeability. In a study of EED in 41 Zambian adults using the Pentax ISC-1000 endo microscope 4 , only 8 (20%) exhibited no luminal leakage of fluorescein and only 2 (5%) showed no fluorescein plumes. By comparison, in a healthy population, 80% showed no fluorescein leakage or plumes (P < 0.0001) 62 .…”

“…By comparison, in a healthy population, 80% showed no fluorescein leakage or plumes (P < 0.0001) 62 . Endomicroscopy was also used to quantify single and multiple cell defects, cell shedding events, apoptotic cells and epithelial breakdown lesions in patients with severe and minimal EED, and these features were corre lated to the expression of 23 genes that were found to be differentially expressed in intestinal epithelium 4 .…”

“…HD and NBI endoscopy and endomicroscopy both have high TRLs, being fully commercialized and widely deployed, and could be adopted for use in EED immediately. Both have the potential to provide vital new information on the structural changes that occur in the gut as a result of EED, and endo microscopy has already been applied to the study of both gut structure and permeability in adults with EED 4 . Endoscopy and endomicroscopy could be applied in the short term to small-scale aetiological investigations, most practicably in cases where children are already undergoing sedation for other procedures (given the invasiveness of the proposed measurements).…”

“…Although it is by definition asymptomatic, EED in children is strongly associated with growth stunting and poor developmental outcomes [1][2][3][4] . The syndrome manifests as a global disturbance of gut function, leading to impaired intestinal barrier function and poor uptake of nutrients [2][3][4] . Environmental origins are suspected, but the aetiology is still not well understood and the most effective route to prevention and treatment remains unclear 5,6 .…”