Proton pump inhibitor effect on esophageal protein signature of eosinophilic esophagitis, prediction, and evaluation of treatment response.

BACKGROUND: Recently, we have identified a dysregulated protein signature in the esophageal epithelium of eosinophilic esophagitis (EoE) patients including proteins associated with inflammation and epithelial barrier function; however, the effect of proton pump inhibitor (PPI) treatment on this signature is unknown. Herein, we used a proteomic approach to investigate: (1) whether PPI treatment alters the esophageal epithelium protein profile observed in EoE patients and (2) whether the protein signature at baseline predicts PPI response. METHODS: We evaluated the protein signature of esophageal biopsies using a cohort of adult EoE (n = 25) patients and healthy controls (C) (n = 10). In EoE patients, esophageal biopsies were taken before (pre) and after (post) an 8-week PPI treatment, determining the histologic response. Eosinophil count PostPPI was used to classify the patients: ≥15 eosinophils/hpf as non-responders (non-responder) and < 15 eosinophils/hpf as responders (R). Protein signature was determined and differentially accumulated proteins were characterized to identify altered biological processes and signaling pathways. RESULTS: Comparative analysis of differentially accumulated proteins between groups revealed common signatures between three groups of patients with inflammation (responder-PrePPI, non-responder-PrePPI, and non-responder-PostPPI) and without inflammation (controls and responder-PostPPI). PPI therapy almost reversed the EoE specific esophageal protein signature, which is enriched in pathways associated with inflammation and epithelial barrier function, in responder-PostPPI. Furthermore, we identified a set of candidate proteins to differentiate responder-PrePPI and non-responder-PrePPI EoE patients before treatment. CONCLUSION: These findings provide evidence that PPI therapy reverses the alterations in esophageal inflammatory and epithelial proteins characterizing EoE, thereby providing new insights into the mechanism of PPI clinical response. Interestingly, our results also suggest that PPI response could be predicted at baseline in EoE.

Circulating immunome fingerprint in eosinophilic esophagitis is associated with clinical response to proton pump inhibitor treatment.

Objectives: The aim of the study was to characterize the circulating immunome of patients with EoE before and after proton pump inhibitor (PPI) treatment in order to identify potential non-invasive biomarkers of treatment response. Methods: PBMCs from 19 healthy controls and 24 EoE patients were studied using a 39-plex spectral cytometry panel. The plasmacytoid dendritic cell (pDC) population was differentially characterized by spectral cytometry analysis and immunofluorescence assays in esophageal biopsies from 7 healthy controls and 13 EoE patients. Results: Interestingly, EoE patients at baseline had lower levels of circulating pDC compared with controls. Before treatment, patients with EoE who responded to PPI therapy had higher levels of circulating pDC and classical monocytes, compared with non-responders. Moreover, following PPI therapy pDC levels were increased in all EoE patients, while normal levels were only restored in PPI-responding patients. Finally, circulating pDC levels inversely correlated with peak eosinophil count and pDC count in esophageal biopsies. The number of tissue pDCs significantly increased during active EoE, being even higher in non-responder patients when compared to responder patients pre-PPI. pDC levels decreased after PPI intake, being further restored almost to control levels in responder patients post-PPI. Conclusions: We hereby describe a unique immune fingerprint of EoE patients at diagnosis. Moreover, circulating pDC may be also used as a novel non-invasive biomarker to predict subsequent response to PPI treatment.

Impact of STAT6 Variants on the Response to Proton Pump Inhibitors and Comorbidities in Patients with Eosinophilic Esophagitis.

Proton pump inhibitors (PPIs) are the first-line drug for eosinophilic esophagitis (EoE), although it is estimated that there is a lack of histological remission in 50% of patients. This research aimed to identify pharmacogenetic biomarkers predictive of PPI effectiveness and to study their association with disease features. Peak eosinophil count (PEC) and the endoscopic reference score (EREFS) were determined before and after an eight-week PPI course in 28 EoE patients. The impact of the signal transducer and activator of transcription 6 (STAT6), CYP2C19, CYP3A4, CYP3A5, and ABCB1 genetic variations on baseline PEC and EREFS, their reduction and histological response, and on EoE symptoms and comorbidities was analyzed. PEC reduction was higher in omeprazole-treated patients (92.5%) compared to other PPIs (57.9%, p = 0.003). STAT6 rs12368672 (g.18453G>C) G/G genotype showed higher baseline PEC values compared to G/C and C/C genotypes (83.2 vs. 52.9, p = 0.027). EREFS reduction in STAT6 rs12368672 G/G and G/C genotypes was higher than in the C/C genotype (36.7% vs. -75.0% p = 0.011). However, significance was lost after Bonferroni correction. Heartburn incidence was higher in STAT6 rs167769 (g.27148G>A) G/G patients compared to G/A (54.55% vs. 11.77%, p = 0.030). STAT6 rs12368672G>C and rs167769G>A variants might have a relevant impact on EoE status and PPI response. Further research is warranted to clarify the clinical relevance of these variants.

ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin.

Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.

A New Method to Evaluate Lower Esophageal Distension Capacity in Eosinophilic Esophagitis by Using Functional Lumen Imaging Probe (EndoFLIP™).

Endoluminal functional lumen impedance planimetry (EndoFLIPTM) has become the gold standard to evaluate esophageal distensibility, although the study itself and its analysis present challenges. We propose here a new method to assess lower esophageal distension capacity that overcomes several limitations of prior approaches, including incomplete and corrupted EndoFLIPTM recordings. Esophageal distension capacity was evaluated with a 16-channel EndoFLIPTM in 10 controls and 14 patients with eosinophilic esophagitis (EoE). Controls were evaluated once. EoE patients were evaluated at baseline and after at least six weeks of treatment with orodispersible budesonide tablets, 1 mg bd. Balloon volumes were increased by 5 mL stepwise, either reaching a maximum volume of 60 mL or a maximum balloon pressure of 60 mmHg. Recordings were analyzed with a homemade R script. The mean esophageal diameter at 60 mL, D (60 mL), was calculated or extrapolated depending on whether the 60 mL volume was reached. By fitting a Michaelis-Menten curve across all measured diameters throughout all constant volume steps, the mean D (60 mL) was estimated. For control subjects, the mean ± SD value of D (60 mL) was 17.08 ± 1.69 mm, and for EoE patients at baseline, D (60 mL) was 14.51 ± 2.68 mm. After six weeks of treatment of EoE patients, D (60 mL) significantly increased to 16.22 ± 1.86 mm (paired Wilcoxon signed test: p = 0.0052), although the values for control subjects were not reached. The estimated mean esophageal diameter at 60 mL is a good proxy for esophageal distension capacity, which correlates with clinical outcomes in EoE. The method presented in this study overcomes difficulties encountered during the standard measurement protocol, allowing the analysis of recordings from incomplete and corrupted registries.

Esophageal biomechanics assessed by impedance planimetry (EndoFLIPTM) in healthy subjects and in patients with eosinophilic esophagitis. Normality values.

BACKGROUND: active eosinophilic esophagitis is associated with esophageal caliber, distensibility and motility changes that may be reversed with treatment. OBJECTIVES: to study esophageal diameter, distensibility and contractility in healthy subjects compared to patients with eosinophilic esophagitis, both before and after treatment. METHODS: a quasi-experimental study, EndoFLIP™, was used to analyze the esophageal body and esophago-gastric junction (EGJ) in all three groups, and a program was designed to obtain esophageal diameter, distensibility and contractility values. RESULTS: ten healthy volunteers (24-61 years, six men) and nine patients with eosinophilic esophagitis (21-52 years, seven men) were included. The esophagogastric junction distensibility index was 5.07 mm2/Hg in the control subjects, 2.40 mm2/Hg in the subjects with eosinophilic esophagitis before treatment and 2.46 mm2/Hg after treatment. The distensibility plateau was 20.02 mm, 15.43 mm and 17.41 mm, respectively, and the diameter was 21.90 mm, 17.73 mm and 18.30 mm, showing significant differences (p < 0.05), except between control subjects and patients after treatment (p = 0.079). Repetitive antegrade contractions developed in 90 % of control subjects, 66.7 % of eosinophilic esophagitis patients before treatment and 88.9 % of the latter after treatment (p > 0.05). CONCLUSIONS: esophago-gastric junction distensibility index, distensibility plateau and diameter values were higher in controls than in patients, although six weeks of treatment seems a short period to observe significant changes in esophageal biomechanics. Repetitive antegrade contractions are the predominant pattern in healthy subjects and eosinophilic esophagitis. We provide normality values for esophageal biomechanics, measured by impedance planimetry in our setting.

Proteomic analysis of the esophageal epithelium reveals key features of eosinophilic esophagitis pathophysiology.

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic non-IgE-mediated allergic disease of the esophagus. An unbiased proteomics approach was performed to investigate pathophysiological changes in esophageal epithelium. Additionally, an RNAseq-based transcriptomic analysis in paired samples was also carried out. METHODS: Total proteins were purified from esophageal endoscopic biopsies in a cohort of adult EoE patients (n = 25) and healthy esophagus controls (n = 10). Differentially accumulated (DA) proteins in EoE patients compared to control tissues were characterized to identify altered biological processes and signaling pathways. Results were also compared with a quantitative proteome dataset of the human esophageal mucosa. Next, results were contrasted with those obtained after RNAseq analysis in paired samples. Finally, we matched up protein expression with two EoE-specific mRNA panels (EDP and Eso-EoE panel). RESULTS: A total of 1667 proteins were identified, of which 363 were DA in EoE. RNA sequencing in paired samples identified 1993 differentially expressed (DE) genes. Total RNA and protein levels positively correlated, especially in DE mRNA-proteins pairs. Pathway analysis of these proteins in EoE showed alterations in immune and inflammatory responses for the upregulated proteins, and in epithelial differentiation, cornification and keratinization in those downregulated. Interestingly, a set of DA proteins, including eosinophil-related and secreted proteins, were not detected at the mRNA level. Protein expression positively correlated with EDP and Eso-EoE, and corresponded with the most abundant proteins of the human esophageal proteome. CONCLUSIONS: We unraveled for the first time key proteomic features involved in EoE pathogenesis. An integrative analysis of transcriptomic and proteomic datasets provides a deeper insight than transcriptomic alone into understanding complex disease mechanisms.

Proton pump inhibitor therapy reverses endoscopic features of fibrosis in eosinophilic esophagitis.

BACKGROUND: Long-standing inflammation leads to esophageal remodeling with stricture formation in patients with eosinophilic esophagitis (EoE). The ability of proton pump inhibitors (PPI) to reverse endoscopic features of fibrosis is still unknown. OBJECTIVE: To investigate the effect of a short course of PPI treatment in reducing endoscopic findings indicative of esophageal fibrosis in EoE patients. METHODS: Cross-sectional analysis of the EoE CONNECT registry. Patients who received PPI to induce EoE remission were evaluated. Endoscopic features were graded using the EoE Endoscopic Reference Score (EREFS), with rings and strictures indicating fibrosis. Results were compared to those from patients treated with swallowed topic corticosteroids (STC). RESULTS: Clinico-histological remission was achieved in 83/166 adult patients treated with PPI (50%) and in 65/79 (82%) treated with STC; among responders, 60 (36%) and 57 (72%) patients respectively achieved deep histological remission (<5 eosinophils/hpf). At baseline, mean±SD EREFS was lower in patients treated with PPI compared to those who received STC (p < 0.001). Short term treatment significantly reduced EREFS scores in patients treated either with PPI or STC as well as rings and strictures. Among patients treated with PPI, deep histological remission (<5 eosinophils/hpf) provided further reduction in total EREFS score. CONCLUSION: Effective PPI therapy for EoE significantly reduced endoscopic esophageal fibrosis in the short term.

Naturally Occurring and Engineered Alphaviruses Sensitive to Double-Stranded-RNA-Activated Protein Kinase Show Restricted Translation in Mammalian Cells, Increased Sensitivity to Interferon, and Marked Oncotropism.

Alphaviruses are insect-borne viruses that alternate between replication in mosquitoes and vertebrate species. Adaptation of some alphaviruses to vertebrate hosts has involved the acquisition of an RNA structure (downstream loop [DLP]) in viral subgenomic mRNAs that confers translational resistance to protein kinase (PKR)-mediated eIF2α phosphorylation. Here, we found that, in addition to promoting eIF2-independent translation of viral subgenomic mRNAs, presence of the DLP structure also increased the resistance of alphavirus to type I interferon (IFN). Aura virus (AURAV), an ecologically isolated relative of Sindbis virus (SV) that is poorly adapted to replication in vertebrate cells, displayed a nonfunctional DLP structure and dramatic sensitivity to type I IFN. Our data suggest that an increased resistance to IFN emerged during translational adaptation of alphavirus mRNA to vertebrate hosts, reinforcing the role that double-stranded RNA (dsRNA)-activated protein kinase (PKR) plays as both a constitutive and IFN-induced antiviral effector. Interestingly, a mutant SV lacking the DLP structure (SV-ΔDLP) and AURAV both showed a marked oncotropism for certain tumor cell lines that have defects in PKR expression and/or activation. AURAV selectively replicated in and killed some cell lines derived from human hepatocarcinoma (HCC) that lacked PKR response to infection or poly(I·C) transfection. The oncolytic activities of SV-ΔDLP and AURAV were also confirmed using tumor xenografts in mice, showing tumor regression activities comparable to wild-type SV. Our data show that translation of alphavirus subgenomic mRNAs plays a central role in IFN susceptibility and cell tropism, suggesting an unanticipated oncolytic potential that some naive arboviruses may have in virotherapy.IMPORTANCE Interferons (IFNs) induce the expression of a number of antiviral genes that protect the cells of vertebrates against viruses and other microbes. The susceptibility of cells to viruses greatly depends on the level and activity of these antiviral effectors but also on the ability of viruses to counteract this antiviral response. Here, we found that the level of one of the main IFN effectors in the cell, the dsRNA-activated protein kinase (PKR), greatly determines the permissiveness of cells to alphaviruses that lack mechanisms to counteract its activation. These naive viruses also showed a hypersensitivity to IFN, suggesting that acquisition of IFN resistance (even partial) has probably been involved in expanding the host range of alphaviruses in the past. Interestingly, some of these naive viruses showed a marked oncotropism for some tumor cell lines derived from human hepatocarcinoma (HCC), opening the possibility of their use in oncolytic therapy to treat human tumors.