Proton pump inhibitors decrease eotaxin-3 expression in the proximal esophagus of children with esophageal eosinophilia

Abstract

Objective: Besides reducing gastric acid secretion, proton pump inhibitors (PPIs) suppress Th2-cytokine-stimulated expression of an eosinophil chemoattractant (eotaxin-3) by esophageal epithelial cells through acid-independent, anti-inflammatory mechanisms. To explore acid-inhibitory and acid-independent, anti-inflammatory PPI effects in reducing esophageal eosinophilia, we studied eotaxin-3 expression by the proximal and distal esophagus of children with esophageal eosinophilia before and after PPI therapy. In vitro, we studied acid and bile salt effects on IL-13-stimulated eotaxin-3 expression by esophageal epithelial cells.

Design: Among 264 children with esophageal eosinophilia seen at a tertiary pediatric hospital from 2008 through 2012, we identified 10 with esophageal biopsies before and after PPI treatment alone. We correlated epithelial cell eotaxin-3 immunostaining with eosinophil numbers in those biopsies. In vitro, we measured eotaxin-3 protein secretion by esophageal squamous cells stimulated with IL-13 and exposed to acid and/or bile salt media, with or without omeprazole.

Results: There was strong correlation between peak eosinophil numbers and peak eotaxin-3-positive epithelial cell numbers in esophageal biopsies. Eotaxin-3 expression decreased significantly with PPIs only in the proximal esophagus. In esophageal cells, exposure to acid-bile salt medium significantly suppressed IL-13-induced eotaxin-3 secretion; omeprazole added to the acid-bile salt medium further suppressed that eotaxin-3 secretion, but not as profoundly as at pH-neutral conditions.

Conclusion: In children with esophageal eosinophilia, PPIs significantly decrease eotaxin-3 expression in the proximal but not the distal esophagus. In esophageal squamous cells, acid and bile salts decrease Th2 cytokine-stimulated eotaxin-3 secretion profoundly, possibly explaining the disparate PPI effects on the proximal and distal esophagus. In the distal esophagus, where acid reflux is greatest, a PPI-induced reduction in acid reflux (an effect that could increase eotaxin-3 secretion induced by Th2 cytokines) might mask the acid-independent, anti-inflammatory PPI effect of decreasing cytokine-stimulated eotaxin-3 secretion.

Figure 1. Flow diagram of study subject selection.

Figure 2. Highest peak eosinophil count at any esophageal level.

Highest pre- and post-PPI treatment peak eosinophil counts in (A) all PPI-treated cases, (B) PPI responders, and (C) PPI non-responders. Red bars represent means.

Figure 3. Eotaxin-3 immunostaining of esophageal biopsy specimens.

(A) Normal squamous epithelium shows no eotaxin-3 immunostaining. (B) Eotaxin-3 labels epithelial cells with variable intensity in biopsies with esophageal eosinophilia. (C) High power magnification (1000x) identifies a granular cytoplasmic staining that is perinuclear. (D) Eosinophils also showed eotaxin-3 immunostaining, but were readily identified by their morphological features and excluded from quantification. Arrows indicate eosinophils with eotaxin-3 labeling. The isolated arrowhead in the bottom left indicates a squamous epithelial cell with weak perinuclear labeling.

Figure 4. Highest peak number of eotaxin-3-positive epithelial cells at any esophageal level and correlation with highest peak eosinophil count.

Highest pre- and post-PPI treatment peak number of eotaxin-3-positive epithelial cells in (A) all PPI-treated cases, (B) PPI responders, and (C) PPI non-responders. Red bars represent means. (D) Correlation between highest peak eosinophil count and highest peak number of eotaxin-3-positive epithelial cells.

Figure 5. Peak number of eotaxi-3-positive epithelial cells at each esophageal level and correlation with peak eosinophil count.

Pre- and post-PPI treatment peak number of eotaxin-3-positive epithelial cells in the (A) proximal, (B) mid, and (C) distal esophagus. Light blue lines represent PPI Responders. Black lines represent PPI Non-responders. Red bars represent means. Correlations between peak eosinophil count and peak number of eotaxin-3-positive epithelial cells are shown pre- and post-PPI treatment in the (D) proximal, (E) mid, and (F) distal esophagus.

Figure 6. Acid and bile salt effects and PPI anti-inflammatory effects on IL-13-induced eotaxin-3 protein secretion in EoE1-T cells.

(A) Acid and/or bile salt effects on IL-13-induced eotaxin-3 protein secretion. Note the profound suppression of eotaxin-3 protein secretion with exposures to acid alone, bile salts alone, or the combination of acid and bile salts. Data are mean ± SEM of 2 experiments. ****P<0.0001 compared to IL-13 alone (pH 7.2 with no bile). (B) Combined effects of acid-bile salt medium and omeprazole (OME) on IL-13-induced eotaxin-3 protein secretion. Data are mean ± SEM of 2 experiments. ****P<0.0001 compared to IL-13 alone (pH 7.2, no bile, no OME). ⁺⁺P<0.01and ⁺⁺⁺⁺P<0.0001. (C) Graph depicts the magnitude of the decrease in IL-13-stimulated eotaxin-3 secretion achieved by omeprazole for each acid-bile salt exposure condition.

Figure 7. Acid and bile salt effects and PPI anti-inflammatory effects on IL-13-induced eotaxin-3 protein secretion in EoE2-T cells.

(A) Combined effects of acid-bile salt medium and omeprazole (OME) on eotaxin-3 protein secretion in EoE2-T cells stimulated with 100 ng/ml of IL-13. Data are mean ± SEM of 2 experiments. **P<0.01, ***P<0.001, and ****P<0.0001 compared to IL-13 alone (pH 7.2, no bile, no OME). ⁺⁺P<0.01 and ⁺⁺⁺P<0.001. (B) Graph depicts the magnitude of the decrease in IL-13-stimulated eotaxin-3 secretion achieved by omeprazole for each acid-bile salt exposure condition in Figure 7A. (C) Combined effects of acid-bile salt medium and omeprazole on eotaxin-3 protein secretion in EoE2-T cells stimulated with 1 ng/ml of IL-13. Data are mean ± SEM of 2 experiments. ****P<0.0001 compared to IL-13 alone (pH 7.2, no bile, no OME). ⁺⁺P<0.01 and ⁺⁺⁺P<0.001. (D) Graph depicts the magnitude of the decrease in IL-13-stimulated eotaxin-3 secretion achieved by omeprazole for each acid-bile salt exposure condition in Figure 7C.

Figure 8. Acid and bile salt effects and PPI anti-inflammatory effects on IL-13-induced eotaxin-3 protein secretion in GERD cells.

(A) Combined effects of acid-bile salt medium and omeprazole (OME) on IL-13-induced eotaxin-3 protein secretion in NES-G4T. Data are mean ± SEM of 2 experiments. ****P<0.0001 compared to IL-13 alone (pH 7.2, no bile, no OME). ⁺⁺P<0.01, ⁺⁺⁺P<0.001, ⁺⁺⁺⁺P<0.0001. (B) Graph depicts the magnitude of the decrease in IL-13-stimulated eotaxin-3 secretion achieved by omeprazole for each acid-bile salt exposure condition in NES-G4T. (C) Combined effects of acid-bile salt medium and omeprazole on IL-13-induced eotaxin-3 protein secretion in NES-B10T. Data are mean ± SEM of 2 experiments. ****P<0.0001 compared to IL-13 alone (pH 7.2, no bile, no OME). ⁺⁺P<0.01 and ⁺⁺⁺P<0.001. (D) Graph depicts the magnitude of the decrease in IL-13-stimulated eotaxin-3 secretion achieved by omeprazole for each acid-bile salt exposure condition in NES-B10T.

Figure 9. Conceptual summary of in vitro findings.

(A) In patients with Th2-cytokine driven esophageal eosinophilia who have little or no reflux, epithelial cell expression of eotaxin-3 will be high in both the proximal and distal esophagus. (B) When patients without reflux are given PPIs, epithelial cells in both the proximal and distal esophagus can respond to the acid-independent, anti-inflammatory effects, which decrease eotaxin-3 expression in both locations. (C) For patients who have Th2-cytokine driven esophageal eosinophilia and gastroesophageal reflux, eotaxin-3 expression in the distal esophagus might be suppressed by reflux, while eotaxin-3 expression remains high in the proximal esophagus where reflux exposure is minimal. (D) When patients with reflux are treated with PPIs, the proximal esophagus responds to the acid-independent, anti-inflammatory PPI effects in suppressing eotaxin-3. In the distal esophagus, however, refluxed acid already might be suppressing eotaxin-3 expression. PPI antisecretory effects could increase cytokine-stimulated eotaxin-3 expression by decreasing acid reflux, while PPI anti-inflammatory effects could decrease eotaxin-3 expression. Thus, PPI treatment might have little net effect on eotaxin-3 expression in the distal esophagus.