Tissue-specific inducible IL-33 expression elicits features of eosinophilic esophagitis

Abstract

Background: IL-33 is a type 2 inflammatory cytokine that is elevated in the esophageal epithelium of eosinophilic esophagitis (EoE) subjects. We previously developed a mouse model of EoE dependent on constitutive overexpression of IL-33 from the esophageal epithelium (EoE33).

Objective: Our objective was to develop an inducible, IL-33-dependent model of EoE and examine induction of EoE-associated pathology.

Methods: We utilized a tetracycline-inducible system to express IL-33 in the esophagus by generating 2 transgenic mice. The first (iSophagus) expresses a reverse tetracycline transactivator from the esophageal epithelium. The second (TRE33) features a tetracycline response element driving expression of IL-33. When crossed, these mice generate an inducible model of EoE (iEoE33). Mice were administered doxycycline-infused chow for up to 2 weeks. Cytokines were assessed by ELISA or bead-based multiplex analysis. T cells were assessed by flow cytometry. Pathology was assessed by histology and immunohistochemistry for IL-33, eosinophil peroxidase, CD4, and Ki-67. iEoE33 was treated with steroids and crossed with IL-13^-/- mice.

Results: Doxycycline-treated iEoE33 mice demonstrated expression of IL-33 in the esophageal epithelium, and esophageal pathology including eosinophilia, CD4⁺ cell infiltrate, basal zone hyperplasia, and dilated intercellular spaces. These findings became pronounced on day 7 of induction, were accompanied by weight loss and esophageal thickening, and were steroid responsive and IL-13 dependent.

Conclusion: Inducible IL-33 expression in the esophageal epithelium elicited features pathognomonic of EoE. iEoE33 enables investigation of EoE disease mechanisms as well as initiation, progression, and resolution.

Keywords: IL-33; eosinophil; eosinophilic esophagitis; transgene; type 2 inflammation.

Figure 1:. iEoE33 mouse model.

(A) The Epstein-Barr virus-derived promoter, ED-L2, was used to drive expression of reverse tetracycline transactivator (rtTA) from the esophageal epithelium. (B) A tetracycline response element (TRE) was used to drive expression of secreted and active IL-33 (sa-IL-33). (C) Immunocytochemical staining of IL-33 (red) on transfected 293T cells with expression of a reverse tetracycline transactivator (pTet) and either saIL-33 (pTRE-sa-IL-33) or empty vector (pTRE) ± doxycycline (dox). (D) ELISA of eosinophil (eos)-derived IL-13 following in vitro culture with conditioned media (CM) from cell cultures shown in panel C (1–4). (E) An inducible model of EoE (iEoE33) was generated by crossing iSophagus (ED-L2-rtTA) with TRE33 (TRE-sa-IL-33) mice. In the presence of dox, expression of secreted and active IL-33 is induced in the esophageal epithelium. (F) IL-33 expression by ELISA at day 5 of induction with different concentrations of doxycycline. (G) Representative IL-33 immunohistochemistry staining (brown) of esophageal cross sections from wild-type (WT) and iEoE33 mice ± 2 weeks dox treatment. (H) IL-33 ELISA of whole tissue homogenates from WT and iEoE33 mice. (I) Esophageal diameter measurements from WT and iEoE33 mice. (J) Change in body weight measurements from WT and iEoE33 mice. n = 3–20 mice per group. Data are shown as mean ± SEM. Two-way ANOVA with a Tukey’s multiple comparison test (D, H, I, J). Student’s t-test (F) *p<0.05, **p<0.01, ****p<0.0001 compared to all groups. Scale bar = 200 μm.

Figure 2:. Doxycycline-treated iEoE33 mice exhibited features pathognomonic of eosinophilic esophagitis.

(A) Representative hematoxylin & eosin (H&E), eosinophil peroxidase (EPX (red)), CD4 (brown), and Ki-67 (brown) staining of esophageal cross sections from wild-type (WT) and iEoE33 mice ± 2 weeks doxycycline (dox) treatment. (B) H&E-stained esophageal biopsies from WT - dox and iEoE33 + dox mice were assessed using the EoE Histologic Scoring System (EoEHSS). Mean EoEHSS component and composite scores are shown. EI, eosinophil infiltration, EA; eosinophil abscess, SL; eosinophil surface layering, DIS; dilated intercellular spaces; BZH; basal zone hyperplasia, and LPF; lamina propria fibrosis. H&E-stained esophageal biopsies from WT - dox and iEoE33 + dox mice were assessed for peak eosinophils per high-power field (eos/hpf) in the epithelium and stroma. (C) Flow cytometry plot and quantification of esophageal Th2-type CD4⁺ T cells in WT - dox and iEoE33 + dox mice. Frequencies shown as a percentage of live, CD45⁺, CD3⁺, CD4⁺ population. (D) Esophageal type 2 cytokine levels in WT - dox and iEoE33 + dox mice. n = 4–6 mice per group. Data are shown as mean ± SEM. Student’s t-test or Mann-Whitney test depending on data distribution. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bar = 200 μm. Inset scale bar = 100 μm. ND – not detected.

Figure 3:. iEoE33 induction kinetics.

(A) Hematoxylin & eosin (H&E), IL-33, eosinophil peroxidase (EPX), CD4, and Ki-67 staining of esophageal cross sections from doxycycline (dox)-treated iEoE33 mice over time. (B) H&E-stained esophageal biopsies from iEoE33 + dox mice were assessed using the EoE Histologic Scoring System (EoEHSS) for grade and stage. Mean EoEHSS component scores and composite scores are shown. EI, eosinophil infiltration, EA; eosinophil abscess, SL; eosinophil surface layering, DIS; dilated intercellular spaces; BZH; basal zone hyperplasia, and LPF; lamina propria fibrosis. Interepithelial and stromal eosinophils/hpf, CD4+ cells/section, and Ki-67+ nuclei/mm basement membrane were counted. (C) Esophageal diameter measurements from WT - dox and iEoE33 + dox mice. (D) Change in body weight measurements from WT - dox and iEoE33 + dox mice, repeated measures ANOVA showed significant decreases in body weight in iEoE33 + dox mice; F(1;8) = 34.85; p=0.0004. (E) IL-33 ELISA and (F) IL-13 ELISA of whole tissue homogenates from WT - dox and iEoE33 + dox mice. n = 4–5 mice per group. Data are shown as mean ± SEM. Student’s t-test or Mann-Whitney test depending on data distribution. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bar = 200 μm. ns – not significant. ND – not detected.

Figure 4:. Induced iEoE33 mice were responsive to steroid treatment.

(A) Representative hematoxylin & eosin (H&E), eosinophil peroxidase (EPX (red)), CD4 (brown), and Ki-67 (brown) staining of esophageal cross sections from doxycycline (dox) treated wild-type (WT) and iEoE33 mice that were concurrently treated with saline (sal) or dexamethasone (dex) for 2 weeks. (B) H&E-stained esophageal biopsies from dox-treated WT and iEoE33 mice also treated with sal or dex were assessed using the EoE Histologic Scoring System (EoEHSS). Esophageal cross sections were scored for both degree (grade) and extent (stage) of pathology. Mean EoEHSS component scores and composite scores are shown. EI, eosinophil infiltration, EA; eosinophil abscess, SL; eosinophil surface layering, DIS; dilated intercellular spaces; BZH; basal zone hyperplasia, and LPF; lamina propria fibrosis. H&E-stained esophageal cross sections were assessed for peak eosinophils per high-power field (eos/hpf) in the epithelium and stroma. (C) Esophageal diameter measurements from dox-treated WT and iEoE33 mice also treated with sal or dex. (D) IL-13 ELISA from whole esophagus homogenates. n = 3 – 8 mice per group. Data are shown as mean ± SEM. Two-way ANOVA with a Tukey’s multiple comparison test. **p<0.01 compared to all groups. Scale bar = 200 μm. ND – not detected.

Figure 5:. iEoE33 pathologies were dependent on IL-13.

(A) Representative hematoxylin & eosin (H&E), eosinophil peroxidase [EPX (red)], CD4 (brown), and Ki-67 (brown) staining of esophageal cross sections from 2 weeks doxycycline (dox)-treated wild-type (WT), IL-13^−/−, iEoE33, and iEoE33 x IL-13^−/− mice. (B) H&E-stained esophageal biopsies from dox-treated WT, IL-13^−/−, iEoE33, and iEoE33 x IL-13^−/− mice were assessed using the EoE Histologic Scoring System (EoEHSS). Esophageal cross sections were scored for both degree (grade) and extent (stage) of pathology. Mean EoEHSS component scores and composite scores are shown. EI, eosinophil infiltration, EA; eosinophil abscess, SL; eosinophil surface layering, DIS; dilated intercellular spaces; BZH; basal zone hyperplasia, and LPF; lamina propria fibrosis. H&E-stained esophageal cross sections were assessed for peak eosinophils per high-power field (eos/hpf) in the epithelium and stroma. (C) Esophageal diameter measurements from dox-treated WT, IL-13^−/−, iEoE33, and iEoE33 x IL-13^−/− mice. (D) IL-13 ELISA from whole esophagus homogenates. n = 3 – 9 mice per group. Data are shown as mean ± SEM. Two-way ANOVA with a Tukey’s multiple comparison test. ***p<0.001, ****p<0.0001 compared to all groups. Scale bar = 200 μm. ND – not detected.