IL‐17, GAL‐10, and TGF‐β are promising noninvasive biomarkers of pediatric eosinophilic esophagitis

Eosinophilic esophagitis (EoE) is a chronic/remittent allergic disorder that significantly impacts the quality of life (QoL) of affected children because of the absence of validated non-invasive tools and the need to periodically monitor the response to long-term treatments with esophagogastroduodenoscopy (EGD).1 It was widely demonstrated that EoE symptoms often persist despite the resolution of esophageal eosinophilia, suggesting that other inflammatory mechanisms or cell mediators may contribute to EoE pathogenesis.1 Several studies identified potential biomarkers that could be useful in EoE management. Still, they were limited by a non-randomized study design, a small sample size, outdated diagnostic criteria, and incomplete disease activity assessment that does not consider symptoms, endoscopic, and histologic findings.2 Therefore, no potential noninvasive biomarkers have been validated and integrated into guidelines and routine clinical practice. Despite several attempts and progress, there is a clinical need for validated noninvasive or minimally invasive biomarkers to be used as surrogates of tissue eosinophils. This study aimed to identify potential noninvasive biomarkers for EoE diagnosis and monitoring, assessing disease activity with the new proposed set of outcome measures (Pediatric Eosinophilic Esophagitis Symptom Scores [PEESS® v.2.0], Endoscopic Reference Score [EREFS], and the peak of eosinophils [PEC]) for improving the data quality of trials and observational studies (COREOS).3 Materials and methods are reported in the online repository. Twenty-one healthy controls and 21 EoE patients (14 [67%] males) were enrolled. Patient and control characteristics were reported in Table S1 (online repository). At enrollment and follow-up, most of the inflammatory, tissue, vascular, and eosinophil biomarkers were not significantly related to disease activity in univariate (Table S2) and multivariate analysis (Table 1). At enrollment, interleukin (IL)-10 was slightly correlated with EoE symptom severity in multivariate analysis (coefficient 0.49, 95% CI 0.08–0.89; p = .02) (Table 1). In the multivariate analysis, IL-17 values were strongly and significantly predictive of clinical [(coefficient 347.01, 95% CI 93.45–600.58; p = .01), (coefficient 428.86, 95% CI 198.77–658.95; p = .0001)] and endoscopic ([HR 5.91 × 1010, 95% CI 0.22–1.58 × 1020; p = .07]) active disease. Biomarkers that were significant at baseline and at follow-up were included in a prognostic model, confirming that IL-17 is predictive of disease activity (Table S3). To address whether a noninvasive biomarker could screen for EoE, we assessed serum biomarker differences between EoE and control patients. Mean values of galectin (GAL)-10 (1.17 ± 0.44 ng/mL vs. 0.91 ± 0.35 ng/mL) and transforming growth factor (TGF)- β (56,176.61 ± 26,251.29 pg/mL vs. 25,997.67 ± 6611.68 pg/mL) were significantly increased in EoE patients compared to healthy controls (p = .02 and p = .0001, respectively) (Figure 1A). For the other biomarkers, we did not find any statistically significant differences (Table S4). ROC curves were constructed to investigate the utility of GAL-10 and TGF- β for EoE diagnosis. The AUC for TGF- β values was 0.92 (sensitivity 0.84 and specificity 1.0), whereas it was 0.67 for GAL-10 (sensitivity 0.75 and specificity 0.57) (Figure 1B). In this prospective and explorative study, we substantially identified three promising noninvasive biomarkers for EoE diagnosis and surveillance using a panel of inflammatory, tissue, vascular, and eosinophil-derived markers. IL-17 values predicted clinically, endoscopically, and histologically active disease. IL-17 is a pro-inflammatory cytokine involved in several autoimmune diseases and allergic disorders, including asthma. IL-17 acts by recruiting neutrophils (but not eosinophils), activating innate immune cells, promoting B-cell functions, and facilitating the production of other pro-inflammatory cytokines (IL-1, IL-6, TNF- α , TGF- β ), chemokines, and prostaglandins.4 The role of IL-17 and Th-17 cells in EoE is still unknown. Sindher et al. demonstrated that Th-17 cells were involved in EoE pathogenesis. The expression of IL-17 was age-related, with higher levels in adults with active EoE compared to children with the same condition.5 Lianto et al. also postulated a potential mechanism in which the dysfunctional regulation of Th-17 cells triggers the unregulated inflammatory response and recruitment of innate immune cells into the esophagus.5 In the asthma pathogenetic model, increased IL-17 production is associated with a "Th2-low" endotype and a more severe disease.4 Similar to asthma, IL-17 might play a role in tissue remodeling, myofibroblast differentiation, esophageal dysmotility, and mechanisms of steroid resistance. In the case–control comparison, GAL-10 and TGF- β values were significantly increased in EoE patients compared to healthy controls. GAL-10 is a lectin family member and is the main component of the Charcot–Leyden crystals localized in the eosinophil cytoplasm. Recently, GAL-10 was identified as a surrogate biomarker for several allergic diseases, alone or in combination with other biomarkers.6 Luminal concentrations of several eosinophil biomarkers, including GAL-10, have been evaluated using the esophageal string test and significantly correlated with esophageal inflammation and distensibility.7-9 Lingblom et al. found that peripheral eosinophils from children with EoE had higher levels of GAL-10 mRNA than adults, highlighting that eosinophil molecular patterns differ between children and adults.10 In this context, GAL-10 might be a promising diagnostic biomarker for pediatric patients, although more extensive studies should be performed to confirm its specificity and sensitivity. TGF-β is a multifunctional cytokine and is considered the primary mediator of fibrosis.2 In EoE, TGF-β stimulates myofibroblast differentiation, promotes the synthesis of extracellular matrix proteins, and induces epithelial-mesenchymal transition, leading to esophageal subepithelial fibrosis.9 TGF-β is also implied in epithelial barrier dysfunction and impaired esophageal smooth muscle cell contraction.2 In a recent study from Poland, statistically significantly higher concentrations of TGF-β were demonstrated in adults with EoE compared to controls and correlated with treatment response.2 In our study, TGF-β levels are significantly elevated in EoE cases compared to controls and showed a high specificity and sensitivity, suggesting that tissue remodeling phenomena are probably active in affected patients regardless of disease activity. The strengths of this study are the prospective design and the application to a cohort of children and adolescents who are the patient categories most afflicted by the disease burden and the need for several EGDs.1 Besides, we applied—for the first time—a validated set of measures to analyze clinical, histological, and endoscopic findings that were correlated to serum biomarkers. Therefore, this study shows the scientific standards recently approved and required to standardize the results of observational studies in EoE. AUR-ROC analysis also confirmed TGF-β as a reliable diagnostic biomarker. Finally, we decided to evaluate serum biomarkers with a procedure (blood sample) that is less invasive, easier to collect, not expensive, and more rapid than other methods, including string testing or tissue cytokine expression. Some limitations need to be highlighted. Firstly, this is a pilot single-center study with a small number of patients enrolled; thus, pooling data across other pediatric centers may be helpful to confirm and reinforce our results. The panel of serum biomarkers assessed is limited to some—but not all—inflammatory, tissue-remodeling, vascular mediators, and eosinophil proteins. Healthy patients represented the control group; therefore, to confirm the application of GAL-10 and TGF-β as diagnostic markers, we are prospective enrolling children and adolescents who underwent EGD for gastrointestinal symptoms and received a definitive diagnosis of gastroesophageal reflux disease or non-EoE esophagitis as a control group. The results of this explorative study are promising and open new future scenarios in EoE diagnosis and surveillance. IL-17 might be considered a new marker of EoE activity, severity, and poor treatment response. Besides, as reported in the asthma model, high expression of IL-17 might define or correspond to a severe and difficult-to-treat EoE phenotype. More extensive studies are needed to investigate the pathogenetic role of IL-17 and confirm its utility as a noninvasive biomarker of disease activity. The case–control comparison found that GAL-10 and TGF-β values were significantly higher in children with EoE than in controls. They might help identify patients with EoE, thus improving the diagnosis of this chronic condition, which is still burdened by clinically relevant diagnostic delay and low QoL.1 Notably, TGF-β values showed excellent and interesting specificity and sensitivity in identifying patients with EoE. However, this study is the first step towards more extensive studies to confirm the results and attempt the identification of noninvasive biomarkers, which are an urgent need in pediatric EoE diagnosis and management. Martina Votto: Methodology; conceptualization; writing – original draft. Matteo Naso: Writing – original draft. Mara De Amici: Methodology. Riccardo Castagnoli: Writing – review and editing. Annalisa De Silvestri: Data curation; formal analysis. Maria De Filippo: Writing – review and editing. Giorgia Testa: Methodology. Gian Luigi Marseglia: Supervision; writing – review and editing. Amelia Licari: Supervision; writing – review and editing. This study is a part of the no-profit research project "National, multicenter, retrospective, prospective study to evaluate pediatric gastrointestinal eosinophilic disorders"—GOLDEN (Gastrointestinal eOsinophiLic Disorders pEdiatric patieNts) study (protocol number 0003241/22). Open access funding provided by BIBLIOSAN. The authors declare that they did not receive funding for this study. The authors declare no conflicts of interest. This study was conducted according to the Helsinki Declaration. All patients provided written informed consent. The Ethical Committee of Fondazione IRCCS Policlinico San Matteo approved this study (protocol number 3241/22). Table S1.–S4. Data S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.