Thymic stromal lymphopoietin-elicited basophil responses promote eosinophilic esophagitis

Abstract

Eosinophilic esophagitis (EoE) is a food allergy-associated inflammatory disease characterized by esophageal eosinophilia. Current management strategies for EoE are nonspecific, and thus there is a need to identify specific immunological pathways that could be targeted to treat this disease. EoE is associated with polymorphisms in the gene that encodes thymic stromal lymphopoietin (TSLP), a cytokine that promotes allergic inflammation, but how TSLP might contribute to EoE disease pathogenesis has been unclear. Here, we describe a new mouse model of EoE-like disease that developed independently of IgE, but was dependent on TSLP and basophils, as targeting TSLP or basophils during the sensitization phase limited disease. Notably, therapeutic TSLP neutralization or basophil depletion also ameliorated established EoE-like disease. In human subjects with EoE, we observed elevated TSLP expression and exaggerated basophil responses in esophageal biopsies, and a gain-of-function TSLP polymorphism was associated with increased basophil responses in patients with EoE. Together, these data suggest that the TSLP-basophil axis contributes to the pathogenesis of EoE and could be therapeutically targeted to treat this disease.

Figure 1

Experimental mouse model of EoE-like disease. (a) Schematic of sensitization and intragastric (i.g.) challenge in WT BALB/c mice. (b) TSLP expression in supernatants of overnight-cultured skin (ears) measured by ELISA. Data depicted are from one experiment (EtOH + OVA, n = 3; MC903, n = 3; MC903 + OVA, n = 4), and are representative of three independent experiments. (c) Histological sections (H & E staining) from the esophagus. Arrows identify tissue-infiltrating eosinophils. Scale bar: 25 μm. (d) Number of eosinophils per hpf in the esophagus. (e) Representative flow cytometry plots showing frequencies of eosinophils in esophageal tissues. Data depicted in (c–e) are from one experiment (EtOH + OVA, n = 3; MC903, n = 3; MC903 + OVA, n = 4), and are representative of three or more independent experiments. (f) Frequencies of eosinophils in esophageal tissues as measured by flow cytometry. Data depicted are from three pooled experiments (EtOH + OVA, n = 7; MC903, n = 8; MC903 + OVA, n = 11). (g) Immunofluorescent staining for eosinophils (Siglec-F-specific mAb, red) in esophageal tissues. Counterstaining with DAPI (blue). Scale bar: 25 μm. Data are representative of two controls and three EoE-like disease samples. (h) Representative electron microscopy (EM) image of an eosinophil in the esophagus of control mice with intact granules with electron dense cores (left panel) or degranulating eosinophils in MC903 + OVA treated mice (right panel), showing loss of electron density in granule cores (red arrow), granule extrusion channels (blue), and loss of granule contents (purple). Scale bar: 2 μm. (i) mRNA expression levels of T_H2 cytokines (Il4, Il5, Il13), the basophil-specific protease Mcpt8, and Tslp in the esophagus. Data depicted are from one experiment (EtOH + OVA, n = 3; MC903, n = 3; MC903 + OVA, n = 4), and are representative of three independent experiments. (j) Representative images of esophagi, with incidence of impaction. Arrows identify impacted food. Data depicted are from two pooled experiments (EtOH + OVA, n = 7, MC903 + OVA, n = 9). All parameters were assessed 12 h post-final oral antigen challenge. Data depicted in (a–i) are from mice challenged twice with OVA, and data depicted in (j) are from mice challenged repeatedly with OVA. Results are shown as mean ± sem, and a non-parametric, one-way Kruskal-Wallis ANOVA with Dunn’s post-hoc testing was used to determine significance. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Figure 2

TSLP-TSLPR interactions are critical for the pathogenesis of EoE-like disease. (a) Histological sections (H & E staining) from the esophagus of BALB/c Tslpr^+/+ or BALB/c Tslp^−/− mice. Arrows identify tissue-infiltrating eosinophils. Scale bar: 25 μm. (b) Number of eosinophils per hpf in the esophagus. (c) Representative flow cytometry plots showing frequencies of eosinophils in esophageal tissues. Data depicted in (a–c) are from one experiment (Tslpr^+/+ EtOH + OVA, n = 3; Tslpr^+/+ MC903 + OVA, n = 5; Tslpr^−/− EtOH + OVA, n = 3; Tslpr^−/− MC903 + OVA, n = 5), and are representative of three independent experiments. (d) Frequencies of eosinophils in esophageal tissues as measured by flow cytometry. Data depicted are from three pooled experiments (Tslpr^+/+ EtOH + OVA, n = 5; Tslpr^+/+ MC903 + OVA, n = 11; Tslpr^−/− EtOH + OVA, n = 5; Tslpr^−/− MC903 + OVA, n = 12). (e) Histological sections (H & E staining) from the esophagus of WT BALB/c mice treated with an isotype control or TSLP-specific mAb. Arrows identify tissue-infiltrating eosinophils. Scale bar: 50 μm. (f) Number of eosinophils per hpf in the esophagus. (g) Representative flow cytometry plots showing frequencies of eosinophils in esophageal tissues. Data depicted in (e–g) are from one experiment (EtOH + OVA + IgG, n = 3; MC903 + OVA + IgG, n = 3; MC903 + OVA + anti-TSLP mAb, n = 3), and are representative of three independent experiments. (h) Frequencies of eosinophils in esophageal tissues as measured by flow cytometry. Data depicted are from three pooled experiments (EtOH + OVA +IgG, n = 5; MC903 + OVA + IgG, n = 9; MC903 + OVA + anti-TSLP mAb, n = 10). All parameters were assessed 12 h post-final oral antigen challenge. Data depicted are from mice challenged twice with OVA. Results are shown as mean ± sem, and a non–parametric, one-way Kruskal-Wallis ANOVA with Dunn’s post-hoc testing or a non-parametric, two-way ANOVA with Bonferroni post-hoc testing were used to determine significance. ns: not significant. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Figure 3

EoE-like disease development is independent of IgE. (a) OVA-specific serum IgE levels from BALB/c Tslpr^+/+ and Tslpr^−/− mice. Data depicted are from one experiment (EtOH + OVA Tslpr^+/+, n = 3; MC903 + OVA Tslpr^+/+, n = 4; EtOH + OVA Tslpr^−/−, n = 3; MC903 + OVA Tslpr^−/−, n = 4), and are representative of three or more independent experiments. (b) Histological sections (H & E staining) from the esophagus of BALB/c Igh-7^+/+ and BALB/c Igh-7^−/− mice. Arrows identify tissue-infiltrating eosinophils. Scale bar: 25 μm. (c) Number of eosinophils per hpf. (d) Representative flow cytometry plots showing frequencies of eosinophils in esophageal tissues. Data depicted in (b–d) are from one experiment (EtOH + OVA Igh-7^+/+ n = 3; MC903 + OVA Igh-7^+/+ n = 3; EtOH + OVA Igh-7^−/− n = 3; MC903 + OVA Igh-7^−/− n = 4), and are representative of three or more independent experiments. (f) Representative EM image of an eosinophil in the esophagus of control Igh-7^+/+ mice with intact granules with electron dense cores (left panel) or degranulating eosinophils in MC903 + OVA treated Igh-7^+/+ (middle panel) or Igh-7^−/− (right panel) mice in various stages of degranulation, with loss of electron density in granule cores (red arrow), formation of granule extrusion channels (blue), complete loss of granule contents (green), and formation of lipid vesicles (yellow). Scale bar: 2 μm. (g) mRNA expression levels of T_H2 cytokines in the esophagus. Data depicted are from one experiment (EtOH + OVA Igh-7^+/+ n = 3; MC903 + OVA Igh-7^+/+ n = 3; EtOH + OVA Igh-7^−/− n = 3; MC903 + OVA Igh-7^−/− n = 3), and are representative of two independent experiments. All parameters were assessed 12 h post-final oral antigen challenge. Data depicted are from mice challenged twice with OVA. Results are shown as mean ± sem, and a non-parametric, two-way ANOVA with Bonferroni post-hoc testing was used to determine significance. **, P ≤ 0.01; ***, P ≤ 0.001.

Figure 4

Basophils mediate the pathogenesis of EoE-like disease. (a) Schematic of in vivo basophil depletion strategy. C57BL/6 (Baso-DTR^neg) or Baso-DTR^pos mice in which the DTR is exclusively expressed on basophils were treated with DT during the course of epicutaneous sensitization. (b) Histological sections (H & E staining) from the esophagus. Arrows identify tissue-infiltrating eosinophils. Scale bar: 25 μm. (c) Number of eosinophils per hpf in the esophagus. (d) Representative flow cytometry plots showing frequencies of eosinophils in esophageal tissues. Data depicted in (b–d) are from one experiment (Baso-DTR^neg EtOH + OVA, n = 3; Baso-DTR^neg MC903 + OVA, n = 3; Baso-DTR^pos MC903 + OVA, n = 4), and are representative of three independent experiments. (e) Frequencies of eosinophils in esophageal tissues as measured by flow cytometry. Data depicted are from three pooled experiments (Baso-DTR^neg EtOH + OVA, n = 7; Baso-DTR^neg MC903 + OVA, n = 10; Baso-DTR^pos MC903 + OVA, n = 11). (f) Schematic of in vivo basophil depletion strategy using CD200R3-specific mAb in WT BALB/c mice. (g) Histological sections (H & E staining) from the esophagus. Arrows identify tissue-infiltrating eosinophils. Scale bar: 25 μm. (h) Number of eosinophils per hpf in the esophagus. (i) Representative flow cytometry plots showing frequencies of eosinophils in esophageal tissues. Data depicted in (g–i) are from one experiment (EtOH + OVA + IgG, n = 3; MC903 + OVA + IgG, n = 3; EtOH + OVA + anti-CD200R3 mAb, n = 3; MC903 + OVA + anti-CD200R3 mAb, n = 4), and are representative of three independent experiments. (j) Frequencies of eosinophils in esophageal tissues as measured by flow cytometry. Data depicted are from three pooled experiments (EtOH + OVA + IgG, n = 8; MC903 + OVA + IgG, n = 9; EtOH + OVA + anti-CD200R3 mAb, n = 8; MC903 + OVA + anti-CD200R3 mAb, n = 10). All parameters were assessed 12 h post-final oral antigen challenge. Data depicted are from mice challenged twice with OVA. Results are shown as mean ± sem, and a non-parametric, two-tailed Mann-Whitney t-test or a non-parametric, one-way Kruskal-Wallis ANOVA with Dunn’s post-hoc testing were used to determine significance. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Figure 5

Neutralization of TSLP or depletion of basophils ameliorates established EoE-like disease. (a) Schematic of treatment with TSLP-specific mAb in WT BALB/c mice in established EoE-like disease. (b) Histological sections (H & E staining) from the esophagus. Arrows identify tissue-infiltrating eosinophils. Scale bar: 25 μm. (c) Frequencies of CD45⁺ cells in esophageal tissues as measured by flow cytometry. (d) Representative flow cytometry plots showing frequencies and total numbers of eosinophils in esophageal tissues. Data depicted in (b–d) are from one experiment (MC903 + OVA + IgG, n = 5; MC903 + OVA + anti-TSLP mAb, n = 5), and are representative of three independent experiments. (e) Schematic of CD200R3-specific mAb basophil-depletion treatment in WT BALB/c mice in established EoE-like disease. (f) Histological sections (H & E staining) from the esophagus. Arrows identify tissue-infiltrating eosinophils. Scale bar: 25 μm. (g) Frequencies of CD45⁺ cells in esophageal tissues as measured by flow cytometry. (h) Representative flow cytometry plots showing frequencies and total numbers of eosinophils in esophageal tissues. Data depicted in (f–h) are from one experiment (MC903 + OVA + IgG, n = 4; MC903 + OVA + anti-CD200R3 mAb, n = 5), and are representative of three independent experiments. (i) Quantified incidence of food impaction. All parameters were assessed 12 h post-final oral antigen challenge. Data depicted are from mice challenged repeatedly with OVA. Results are shown as mean ± sem, and a non-parametric, two-tailed Mann-Whitney t-test was used to determine significance. *, P ≤ 0.05; **, P ≤ 0.01.

Figure 6

The TSLP-basophil axis is active in human subjects with EoE. (a) Number of eosinophils per hpf in esophageal biopsy tissue sections were quantified for pediatric control subjects (n = 19) and subjects with active EoE (n = 16) or inactive EoE (n = 15). (b) Relative expression of TSLP in esophageal biopsies of pediatric control subjects (n = 8) and subjects with active EoE (n = 25) or inactive EoE (n = 10). (c) Immunohistochemical staining for TSLP (red) in an esophageal biopsy. Data are representative of 5 subjects with active EoE. Scale bar: 100 μm. (d) Basophils were identified by flow cytometry in esophageal biopsies from pediatric subjects with active EoE (plots are representative of 19 control subjects and 16 subjects with active EoE). (e) Frequencies of basophils in the lineage negative (lin⁻) compartment (see Methods) in esophageal biopsies from pediatric control subjects (n = 19) and subjects with active EoE (n = 16) or inactive EoE (n = 15). (f) Correlation of frequencies of basophils in pediatric esophageal biopsies and the number of eosinophils per hpf observed histologically (n = 50) (Spearman r = 0.6638). (g) Frequencies of basophils in the PBMCs of pediatric subjects with EoE that were homozygous (n = 26) or heterozygous for the TSLP^risk polymorphism (n = 26), or that lacked the TSLP^risk polymorphism (n = 9) were identified by flow cytometry. All data are shown as mean ± sem, and a non-parametric, two-tailed Mann-Whitney t-test or a non-parametric, one-way Kruskal-Wallis ANOVA with Dunn’s post-hoc testing were used to determine significance. Correlation analysis was performed using a non-parametric Spearman correlation (sensitivity analyses were performed), and a linear regression of the data is displayed. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.