Interleukin-15 expression is increased in human eosinophilic esophagitis and mediates pathogenesis in mice

Abstract

Background & aims: Quantitative microarray analyses have shown increased expression of interleukin-15 (IL-15) messenger RNA in the esophagus of patients with eosinophilic esophagitis (EoE), a recently recognized allergic disorder with poorly understood pathogenesis.

Methods: Quantitative polymerase chain reaction and enzyme-linked immunosorbent assay analyses were performed to examine protein and transcript levels in tissue samples from patients with EoE. Tissues from IL-15Ra-deficient and wild-type (control) mice were also examined. Tissue eosinophilia was determined by immunostaining for major basic protein and flow cytometry for cell-surface receptors.

Results: Quantitative polymerase chain reaction analyses showed that levels of IL-15 and its receptor IL-15Ra were increased approximately 6- and approximately 10-fold, respectively, in tissues from patients with EoE and approximately 3- and approximately 4-fold, respectively, in mice with allergen-induced EoE. A >2-fold increase in serum IL-15 protein levels was also detected in human EoE samples compared with those from healthy individuals. Human IL-15 messenger RNA levels correlated with esophageal eosinophilia (P < .001). IL-15Ra-deficient mice were protected from allergen-induced esophageal eosinophilia compared with controls (P < .001), even though similar levels of airway eosinophilia were observed in all mice. IL-15 activated STAT5 and CD4(+) T cells to produce cytokines that act on eosinophils. Incubation of primary esophageal epithelial cells from mice and humans with IL-15 caused a dose-dependent increase in the mRNA expression and protein levels of eotaxin-1, -2, and -3.

Conclusions: IL-15 mediates in the pathogenesis of EoE. IL-15 activates CD4(+) T cells to produce cytokines that act on eosinophils.

Figure 1. Induced expression of IL-15 and IL-15Rα transcript in the esophagus of experimental and human EoE

Esophageal mRNA expression of IL-15 and IL-15Rα was measured in non-EoE and EoE patient esophageal biopsies performing quantitative real-time PCR analysis. The relative expression compared to respective controls are shown, n=14–16 esophageal biopsies/group (A–B) and saline- and allergen-challenged mice, n = 12 mice/group (C–D). The data is expressed as mean ± SD.

Figure 2. Analysis of esophageal eosinophilia and IL-15 in non-EoE, active EoE, and treated EoE patients

Correlation between the maximum eosinophil number/hpf and IL-15 mRNA expression in human EoE is shown (A). P -value and r were calculated using Spearman correlation test. IL-15 mRNA expression in non-EoE individuals, active EoE patients (> 24 eosinophils/hpf), patients on elemental diet, and patients responsive and not responsive to Flovent are shown (B). Serum IL-15 protein levels in non-EoE individuals, active EoE patients (> 24 eosinophils/hpf), patients on elemental diet, and patients treated and responsive to Flovent are shown (C). Immunoreactivity of IL-15 was tested on esophageal biopsies on non-EoE and EoE patients by performing IHC. No IL-15-positive cells were detected in non-EOE patient esophageal biopsies, marked with arrows, original magnification 10x [D, (a)], and inset magnification in 400x [D, (b)]. A number of infiltrating cells were detected positive for IL-15 in the esophageal biopsies of EoE patient, original magnification 10x [D, (c)] and inset magnification in 400x [D, (d)]. P-values were calculated using Kruskal–Wallis test followed by Dunn’s multiple comparison tests. Diet-R (diet treatment responder), FP-R (Flovent responders) and FP-NR (Flovent non-responders).

Figure 3. IL-15Rα-deficient mice are protected from allergen-induced EoE

Mice were intranasally challenged three times a week for 3 weeks with Aspergillus fumigatus (Asp) extract or saline as per the protocol shown (A). Bronchoalveolar lavage fluid was collected and analyzed 18–20 hrs after the last intranasal saline or allergen exposure, and BALF eosinophil numbers were counted and are shown (B). Eosinophils in the esophagus were counted by performing morphometric analysis and are expressed as eosinophils/mm² (C). Data are expressed as mean ± S.D.; n = 12 mice/group. NS (non significant).

Figure 4. Macrophages and dendritic cells are increased following experimental EoE induction

The total esophageal cells from saline- and Aspergillus-challenged mice were analyzed for macrophages and dendritic cells using FACS analysis. The 7AAD⁻ CD45⁺ (pan marker) cells were gated (A) and CD11b/MHC class II double positive cells were analyzed for macrophages in saline- (B) and Aspergillus-challenged mice (C). Similarly, CD11c/MHC class II double positive cells were analyzed for dendritic cells in 7AAD⁻ CD45⁺ (pan marker) gated cells in saline- (D) and Aspergillus-challenged mice (E). Further, intracellular IL-15 was detected in MHCII-CD11b double positive cells (G) and MHCII-CD11c double positive cells (I) isolated from allergen-challenged mice. Some baseline IL-15-positive cells were also detected in control cells (F, H). These experiments are representative of three independent experiments performed in triplicate. Data are expressed as percent change in cell populations.

Figure 5. IL-15 treatment of purified murine CD4⁺T cells

FACS analysis was performed to examine IL-15 receptor on purified splenic CD4⁺ T cells. Approximately 30% of the purified population expressed IL-15-specfic receptor IL-15Rα (A). IL-15 dose dependently increased proliferation as observed by performing thymidine incorporation analysis as shown (B). Western blot analysis indicates that 100 ng/ml IL-15-induced STAT5 phosphorylation was observed between 15–60 min (C), and flow cytometric analysis indicates that only 20% of CD4⁺ T cells responded to 100 ng/ml of IL-15 for STAT5 phosphorylation at 30 min. The regular line in the histogram represents 100 ng/ml IL-15 treated, and the dotted line represents non-treated CD4⁺T cells (D–F). Data is representative of three independent experiments and are expressed as mean ± S.D.

Figure 6. CD4⁺ T cell and Th2 cytokine profiles following IL-15 exposure

A dose response analysis indicates that IL-15 priming to CD4⁺ T cells induces Th2 cytokine (IL-5 and IL-13) mRNA (A, B) and proteins (C, E). The IL-15-primed CD4⁺ T cells following stimulation with anti-CD3/anti-CD28 enhance the production of IL-5 and IL-13 (D, F). These are representative of three independent experiments performed in triplicate. Data are expressed as mean ± S.D.

Figure 7. Eotaxin gene induction in human and mouse PEEC following IL-15 treatment

The characteristics of isolated and cultured mouse and human PEEC was verified by immunostaining with mouse and human anti-cytokeratin antibody and tested by performing FACS analysis, mouse (A) and human (B). The cells were further examined for the expression of IL-15 receptor using IL-15Rα antibody against mouse and human and data is shown (C, D). Eosinophil-specific chemokines eotaxin-1, -2, and -3 expression following IL-15 stimulation in mouse and human PEEC was quantified using real-time PCR. Eotaxin-1 (E) eotaxin-2 (F) mRNA expression in mouse primary esophageal epithelial cells following 48 h of mIL-15 (0, 1, 10, 100 ng/ml) exposure is shown. Eotaxin-3 mRNA expression in human primary esophageal epithelial cells following 48 h of hIL-15 (0, 1, 10, 100 ng/ml) exposure is shown (G). These are representative of three independent experiments performed in triplicate. Data are expressed as mean ± S.D.

Figure 8. Diagrammatic proposed pathway representation of IL-15-induced EoE

Allergen-induced IL-15 producing macrophages (MC) and dendritic cells (DC) are increased in the esophagus in experimental EoE. IL-15 activates and proliferates specific populations of CD4⁺ T cells. IL-15-induced CD4+ T cells activation in certain conditions produces eosinophil-active cytokines IL-5 and IL-13 that are regulated by signal transducer and activator of transcription (STAT) 5. In addition, IL-15 induces eosinophil active chemokines (eotaxin) in the esophageal epithelial cells that attract eosinophils into the esophageal epithelial mucosa from the blood. The STAT5 mediated pathway of Th2 cytokine induction support previous findings that allergen-induced Th2 responses in EoE are dependent and independent to STAT6.