Aplicabilidad de los microarns y otros mediadores (proteicos y lipídicos) como biomarcadores en la esofagitis eosinofílica. Utilidad para el diagnóstico y valoración de la respuesta al tratamiento

Abstract

Eosinophilic esophagitis (EoE) is a chronic, immune-based disease of the esophagus, characterized clinically by symptoms of esophageal dysfunction and histologically by an inflammatory infiltrate with a predominance of eosinophils. Since upper endoscopy (EGD) with biopsy is the only diagnostic and follow-up method for these patients at present, it is important to determine molecular markers that correlate with esophageal inflammation and can predict response to treatment. Proton pump inhibitors (PPIs) are a first-line treatment in EoE, with a high rate of clinical-histological remission, although so far no predictive factors for response have been identified. The main objective of the present study is to determine the profile of mediators (proteins and lipids) and microRNAs in the esophageal mucosa and serum of pediatric patients with EoE at diagnosis and after PPI treatment, in order to find molecular markers of inflammatory activity in these patients and possible predictive factors of response to PPI treatment. Methods: We conducted a prospective observational case-control study with pediatric patients aged 1-15 years from two hospitals. The cases selected were those diagnosed with EoE according to the latest European consensus guidelines. They were treated with esomeprazole 1mg/kg/dose twice daily for 8-12 weeks and a second endoscopy with biopsies was performed. Histological remission was defined as isolation of <15 eosinophils/ high power field (HPF) in all biopsies obtained. Controls were pediatric patients, with no previous diagnosis of EoE, who underwent an EGD for gastrointestinal symptoms, with a macroand microscopically normal esophagus. In esophageal biopsies taken proximally and distally, the expression of proinflammatory genes was determined after RNA extraction and subsequent qRT-PCR (semiquantitative real-time PCR). The protein expression of these genes was determined by western blot. On the other hand, massive sequencing of RNA extracted from esophageal biopsies of a group of cases with EoE pre and post PPI treatment was performed to determine the miRNAs differentially expressed in these patients, and the results were subsequently validated in the rest of the patients by qRT-PCR. Likewise, a peripheral blood sample was extracted from all patients during the EGD and the expression of different molecules (cytokines, lipid mediators, eosinophil-derived proteins) was determined by ELISA and the Luminex multiplex immunoassay system. The expression of miRNAs in the serum of patients was also evaluated by qRT-PCR. Results: Forty-three patients with EoE and thirty-nine controls were included in the study. In cases, overexpression of proinflammatory genes (CCL26, IL-5, IL-13, POSTN, MBP) and downregulation of a single gene, filaggrin (FLG), was observed. This gene overexpression was significantly decreased in patients responding to PPI treatment (PPI-R), although without reaching the values of healthy controls. In contrast, FLG expression increased in these patients. Gene expression was corroborated by protein determination. No differences were detected at baseline in gene expression between PPI responders and non-responders. No differences were observed in gene expression determined in serum after PPI treatment or between cases and controls, with the exception of IL-1β overexpression in patients with EoE. The esophageal miRNA profile was analyzed in patients with EoE and controls, showing overexpression of four miRNAs that regulate proinflammatory pathways (miR-21-3p, miR7-5p, miR-223-3p and miR-664a-3p) and decreased expression of miR-375-3p (with protective effect on epithelial cells) in subjects with EoE. PPI-R patients presented basally higher expression of all detected miRNAs and these levels were restored after PPI treatment. The changes observed in the esophagus did not correlate with serum. Conclusions: The findings of this work show the restoration of gene and protein expression after PPI treatment in EoE, corroborating its anti-inflammatory and protective effect on the epithelial barrier. These molecular changes are not reflected systemically. On the other hand, the present study has described a profile of esophageal miRNAs whose expression levels discriminate PPI-R and PPI-NR at baseline and could constitute a tool for predicting the response to PPIs in these patients.