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

Abstract

Introduction: Environmental 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.

Methods: We 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.

Results: CLE 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.

Conclusions: Confocal 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.

Fig 1

Confocal laser endomicroscopy images of (A) normal villous epithelium showing black, fluorescein-free lumen (arrow); (B) a plume of fluorescein (arrow) seen against a dark lumen; (C) fluorescein filling the lumen (thin arrow), a microerosion (thick arrow) and a breach at a villus tip (hollow arrow); (D) single cell defect with fluorescein plume (arrow), (E) example of a multiple cell defect, a small microerosion where two cells are seen to have detached from the basement membrane (arrow), and (F) total breakdown of epithelial contiguity (arrows).

Fig 2

Corresponding confocal laser endomicroscopy images (A, D), claudin 4 immunostains (B, E) and histology (C, F) from two individuals (A-C and D-F). In A, three plumes (arrows) are seen; in B claudin 4 immunostaining is disorganised at the site of cell shedding, and in C an epithelial defect is seen with a breach of the basement membrane (arrow). In D, a microerosion (thin arrow) and plume (thick arrow) are associated; in E claudin 4 immunoreactivity is disorganised along track of cell loss at a villus tip (arrow) with exposure of the basement membrane; and the corresponding histology image is shown in F.

Fig 3

A-D Four images from the same participant showing complete loss of epithelial integrity even though the mucosa appeared normal at endoscopy. In A, a loss of epithelial continuity is apparent at several points (arrows) in a CLE image. In B, funnels of claudin 4 are visible (arrows) at the point of cell shedding from the epithelium [32], but non-detaching epithelium displays less immunoreactivity. In C, complete epithelial detachment is apparent. In D, increased claudin 4 immunoreactivity is seen in the detaching epithelium compared to the intact epithelium, with at least one prominent funnel (arrow).

Fig 4

Lipopolysaccharide concentrations (Endotoxin Units/ml) in plasma were correlated with: (A) cell shedding (β = 0.83; P = 0.035 in the multivariate model); (B) GLP-2 (β = -0.13; P = 0.007 in the multivariate model); and (C) villus surface area:volume ratio estimated from morphometry of intestinal biopsies. In (C) the data points are shown together with the curve and 95% confidence limits from a fractional polynomial regression model which was significant with 2 degrees (P = 0.007 and P = 0.025). (D) Polynomial fractional regression plot as in (C) but showing the relationship between zinc uptake and surface area:volume ratio.