A striking local esophageal cytokine expression profile in eosinophilic esophagitis

Abstract

Background: Eosinophilic esophagitis (EE) is an emerging worldwide disease that mimics gastroesophageal reflux disease.

Objective: Early studies have suggested that esophageal eosinophilia occurs in association with T(H)2 allergic responses, yet the local and systemic expression of relevant cytokines has not been well characterized.

Methods: A human inflammatory cytokine and receptor PCR array containing 84 genes followed by PCR validation and multiplex arrays were used to quantify cytokine mRNA in esophageal biopsies and blood samples.

Results: Esophageal transcripts of numerous chemokines (eg, chemokine [C-C motif] ligand [CCL] 1, CCL1, CCL23, CCL26 [eotaxin-3], chemokine [C-X-C motif] ligand [CXCL] 1, and CXCL2), cytokines (eg, IL13 and ATP-binding cassette, subfamily F, member 1), and cytokine receptors (eg, IL5 receptor, alpha) were induced at least 4-fold in individuals with EE. Analysis of esophageal biopsies (n = 288) revealed that eotaxin-3 mRNA level alone had 89% sensitivity for distinguishing individuals with and without EE. The presence of allergy was associated with significantly increased esophageal expression of IL4 and IL5 mRNA in patients with active EE. We identified 8 cytokines (IL-4, IL-13, IL-5, IL-6, IL-12p70, CD40 ligand, IL-1α, and IL-17) whose blood levels retrospectively distinguished 12 patients without EE from 13 patients with EE with 100% specificity and 100% sensitivity. When applied to a blind, prospectively recruited group of 36 patients, the cytokine panel scoring system had a 79% positive predictive value, 68% negative predictive value, 61% sensitivity, and 83% specificity for identifying EE.

Conclusion: Evidence is presented that IL13 and IL5 associate with eosinophil and eotaxin-3 levels, indicating the key role of adaptive T(H)2 immunity in regulating eotaxin-3-driven esophageal eosinophilia in the absence of a consistent systemic change in cytokines.

Figure 1. Cytokine mRNA levels in EE patients

Cytokine mRNA levels were quantified using real-time PCR in 11 NL and 11 EE patients. Expression levels were normalized to the housekeeping gene GAPDH and are expressed as a relative ratio. EE patients were subdivided based on the maximum eosinophil number in the biopsy into active (> 23 eosinophils/hpf), intermediate (partially treated EE with 1 ≤ 23 eosinophils/hpf), and inactive (0 eosinophils/hpf). p values were calculated using the Mann-Whitney test: *p < 0.05, **p < 0.005, and ***p < 0.0005.

Figure 2. Eotaxin-3, IL5, IL4, IL13 and IL5RA mRNA levels in EE patients

Levels were quantified using real-time PCR in a large cohort of NL, CE and EE patients. Expression levels were normalized to the housekeeping gene GAPDH and are expressed as a relative ratio. EE patients were subdivided based on the maximum eosinophil number in the biopsy into active (> 23 eosinophils/hpf), intermediate (partially treated EE with 1 ≤ 23 eosinophils/hpf), and inactive (0 eosinophils/hpf). p values were calculated using the Kruskal-Wallis test with a Dunn's Multiple Comparison Test: *p < 0.05, **p < 0.005, ***p < 0.0005.

Figure 3. IL4, IL5, IL13, IL5RA, and eotaxin-3 expression in active allergic and active non-allergic EE patients

IL4, IL5, IL13, IL15RA, and eotaxin-3 mRNA levels were quantified using real-time PCR in active EE patients (> 23 eosinophils/hpf) from the large cohort. Expression levels were normalized to the housekeeping gene GAPDH and are expressed as a relative ratio. p values were calculated using the Mann-Whitney test: *p < 0.05, **p < 0.005, ***p < 0.0005.

Figure 4. Correlation and linear regression between IL4, IL5, IL13, and eotaxin-3

IL4, IL5, IL13, and eotaxin-3 mRNA levels were quantified using real- time PCR in active EE patients. Spearman correlations r (italics) and p values were calculated to test correlation between the ranked cytokines levels (nonparametric). Linear regression curves are presented with the corresponding p value and r² (bold) on the curves to test if the levels of cytokine directly correlate with each other. A non-significant p value (ns) indicates that the slope of the curve is not significantly different from 0.

Figure 5. Plasma cytokine levels in NL and EE patients

Blood cytokine levels significantly differentially expressed in NL and EE patents are shown for IL-4, IL-5, IL-6, IL-8, IL-17, IL-13, IL-12p70, CD40L, and IL-1α (A). Blood cytokine levels in EE patients before and after therapy are shown (B). The p values were calculated using the Mann-Whitney test (A) or paired t test (B): *p < 0.05, **p < 0.005, ***p < 0.0005.