Eosinophilic esophagitis auxiliary diagnosis based on a peptide ligand to eosinophil cationic protein in esophageal mucus of pediatric patients

Abstract

Eosinophilic esophagitis (EoE) is a chronic inflammatory condition of the esophagus characterized by increased number of eosinophils. Currently, EoE diagnosis is based on endoscopic procedures for histopathological examination, eosinophils' counting and, often, in clinical practice, the challenge is the differentiation between EoE and gastroesophageal reflux disease (GERD). Our aim was to develop novel peptide ligand to Eosinophil cationic protein (ECP) present in EoE biopsies of patients with potential to be used for detection. We performed a comparative proteomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) of esophageal biopsies from pediatric patients with eosinophilic esophagitis, gastroesophageal reflux disease and control individuals. Then, phage display technology was used to select peptides against specific up-regulated protein from EoE patients. Twelve phage clones were selected after three biopanning rounds, and the best phage clone reactivity was evaluated by phage-ELISA assay using esophageal mucus samples from 94 pediatric patients. Mass spectrometry showed that eosinophil cationic protein (ECP) was one of the most up-regulated proteins in EoE patients, which is an eosinophil granule protein usually deposited on tissues to mediate remodeling, but in excess may cause fibrosis and hypertrophy, especially in allergic responses. A highly reactive ECP-ligand peptide (E5) was able to distinguish reactive mucus of EoE patients from GERD and the control individuals by Phage-ELISA, achieving a sensitivity of 84.62%, and a specificity of 82.72%. This is the first study that successfully demonstrated an antibody-like peptide targeting ECP at the esophagus mucus as a useful auxilliary tool for EoE diagnosis with a significant association with atopic disorders and dysphagia.ClinicalTrials.gov no.: NCT03069573.

Conflict of interest statement

The authors declare no competing interests.

© 2022. The Author(s).

Figures

Figure 1

A scheme of the biopanning experiment. Phage library was incubated with Eosinophilic Cationic Protein (ECP) coated on surface. Unbound phages were washed and phages bound to ECP were eluted and amplified for next round of biopanning. After the 3rd round the eluate was plated and individual clones were amplified. Then, the most reactive phage clones were selected by Phage-ELISA for sequencing analysis.

Figure 2

Biopanning, enrichment of phage library and perfomance of the phage clones selected by phage display. (A) The phage titer after each biopanning round, (B) Three rounds of biopanning against ECP were performed, and the phage titer (pfu) after each round was measured. Numbers on bars are the enrichment fold of the phage titer over the first round, (C) amino acid sequences and frequency of the selected peptides, (D) reactivity obtained though the interaction of the sequenced phage clones, Irrelevant phage clone and ECP.

Figure 3

Detection of ECP using Phage-ELISA and molecular docking between the recombinant human Eosinophil Cationic Protein (ECP) and E5 peptide. (A) Increasing concentrations of ECP were pre-coated on plates and then incubated with the selected peptides E3, E5 and Irrelevant phage clone and then, incubated with anti-M13 antibody. Antibody was detected by HRP-conjugated anti-IgG and the enzyme substrate. Optical density (OD) was measured at a wavelength of 450 nm. Data represent mean OD ± standard deviation of assays performed in duplicates. (B) The full cartoon structure of ECP (wheat) coupled with E5 (hot pink), (C) top view of interaction surface topography revealing a binding pocket favoring the best affinity, (D) identification of E5 binding residues and interaction sites onto ECP. ECP binding residues are shown in orange. Yellow dashed lines represents polar contacts.

Figure 4

Phage-ELISA on mucus from patients. (A) Reactivity Index (RI) from the Phage-ELISA performed for the 94 patients classified in 3 groups: C (control), GERD (gastroesophageal reflux disease), and EoE (eosinophilic esophagitis). A diagnosis cut-off Phage-ELISA = 0.2993 at RI = 1 (datashed line) was derived from later, larger-scale studies by receiver operating characteristic (ROC) analysis, (B) ROC curve based on 0.2293 cut-off, with an area under the curve (AUC) of 0.84 and Se 84.62%, Sp 82.72%, LR + 4.896 and (*) p < 0.0001, (C) for the 94 patientes a linear correlation was performed between reactivity index (RI) and PEC (Eos/hpf). In blue circles (control and GERD) and in red circles (EoE), Spearman r and p value are shown in the figure, (D) For the 13 EoE patients a linear correlation was performed between reactivity index (RI) and PEC (Eos/hpf), Spearman r and p value are shown in the figure.