In this study, we investigated cytokine expression levels in non-EE and EE patients in the esophageal mucosa and the blood. We identified that new cytokines not previously associated with EE, such as IL1F9 and CCL23, were upregulated in EE compared to NL patients. While patchiness is an issue in EE diagnosis, we found that only 8.7% of active EE samples had an eotaxin-3 level that overlapped with NL samples using only one biopsy sample per patient. We found that mRNA levels of the Th2 cytokines IL13, IL5, and eotaxin-3 correlated with each other but that IL4 did not correlate with IL13 or eotaxin-3 levels. Notably, the allergic status was an important confounder as IL4 and IL5 mRNA were increased in allergic EE patients. Except for the eosinophil level, none of the clinical parameters analyzed (therapy, allergic status, gender) was able to explain inter-patient variability of eotaxin-3 and IL-13 levels in active EE patients. The establishment of a scoring panel based on plasma levels, including 8 cytokines, was able to predict diagnosis with 79% positive predictive value, 68% negative predictive value, 83% specificity, and 61% sensitivity in this population of patients referred for endoscopy.
Herein, we analyzed cytokine levels of nearly 300 patients and assessed the overlap among cytokine levels. To our knowledge, this is the first time that such a large EE cohort has been studied for molecular parameters. Using real-time PCR, we demonstrated with 89% sensitivity that eotaxin-3 mRNA expression in EE patients is increased compared to control patients. Previous histopathological studies indicate that a minimum of 5 biopsies are required to achieve 100% sensitivity for diagnosis of EE with one biopsy only achieving 55% sensitivity;(23
) our results were obtained using only one RNA sample per patient, suggesting that molecular diagnosis is a relatively promising and sensitive method for disease diagnosis.
Cytokine correlations reveal the concerted expression of IL13, IL5,
mRNA and suggest expression in the same cell type, such as a Th2 cell producing IL-13 and IL-5. We have recently shown that IL-13 specifically induces eotaxin-3 in esophageal epithelial cells,(15
) and a recent study by Prussin et al. has emphasized the presence of unique food antigen-specific, IL-5-positive Th2 cells in patients with eosinophil-associated gastrointestinal disorders (EGID) compared with patients with food anaphylaxis.(11
) The implications of IL-5 and IL-13 in EE have also been demonstrated in murine EE models.(8
) While IL5RA
mRNA was upregulated in active EE patients, its low expression level may explain why it did not correlate with eosinophil levels. Finally, IL4
are dysregulated in allergic EE patients compared to non-allergic EE patients, and these increases may reflect the systemic allergic history of the patients rather than the local activity of the disease as reflected by eotaxin-3 and IL-13 expression levels.
Yamazaki et al. have shown that common food and environmental allergens induce increased production of IL-13 and IL-5 by peripheral blood mononuclear cells after stimulation with aeroallergens or food allergens in EE patients compared to healthy individuals.(25
) In the present study of patients referred for endoscopy, the establishment of a plasma scoring panel composed of 8 cytokines was able to predict diagnosis of EE with 79% positive predictive value, 68% negative predictive value, 83% specificity, and 61% sensitivity. While evidencing relatively high scores, our results also indicated that patients with an allergic history, who are challenging to diagnose, may result in false-positive occurrences. Additionally, the PPV is reflective of our population (potential EE patients) that was composed of about 50% non-EE and 50% EE in our cohort. In the general population, where the prevalence of EE is lower, the PPV would thus be lowered. While the cytokine dysregulation was not reproduced in the prospective study, specificity and sensitivity were relatively high due to the high threshold levels chosen, which were set above the maximum level observed in the non–EE group.
It is tempting to speculate on the potential roles of the cytokines that were significantly modified in EE compared to NL. CXCL14 downregulation has also been shown in squamous head and neck cancer and has an anti-proliferative role on epithelial cells. It is possible that specific EGFR tyrosine kinase inhibitor, which restores CXCL14 expression in head and neck squamous cell carcinoma,(26
) contributes to a decrease in esophageal epithelial cell proliferation in EE patients. In contrast, CCL23
mRNA is increased in EE and has been shown to be induced following STAT-6 activation:(28
) CCL23 is involved in endothelial cell proliferation, a feature that may contribute to the papillae elongation observed in EE. We have also identified dysregulation of novel cytokines and receptors in EE that deserve further consideration. Additionally, we noted marked changes in IL-1 family related molecules with upregulation of IL1B
and IL-1 related family member 6 and downregulation of the inhibitory receptor (IL1RA
) and IL-1 related family member 9. Thus, we propose that EE involves coordinate pro-inflammatory signals triggered by IL-1-related molecules, implying the importance of post IL-1-receptor signaling (e.g. NFkB). Indeed, the EE transcriptome has evidence for activation of this pathway via overexpression of IL8
, and TNFAIP6
In conclusion, we have extended our prior knowledge of the molecular pathogenesis of EE by identifying esophageal overexpression of a panel of chemokines and cytokines additional to the previously reported IL13 and eotaxin-3. Although our screening array encoded for 84 relevant mRNAs, only ~20% were dysregulated in EE. Notably, we identify a strong correlation between IL13, IL5, and eotaxin-3, but not IL4 mRNA levels, consistent with the presence of an IL-13-producing Th2 cell population. Using molecular analysis of only eotaxin-3 in a large cohort of patients, ~ 90% sensitivity for diagnosis is obtained. Furthermore, blood levels of the simplistic panel of 8 cytokines reached moderate specificity and sensitivity regardless of the global increase of these cytokines in the different groups of patients; atopy was a confounder for systemic cytokine levels. Taken together, evidence is presented that IL13 and IL5 associate with eosinophil and eotaxin-3 levels, indicating the key role of adaptive Th2 immunity in regulating eotaxin-3-driven esophageal eosinophilia in the absence of a consistent systemic change in cytokines.