Prostaglandin I2 signaling restrains Treg cell ST2 expression by repressing β-catenin in allergic airway inflammation

Abstract

Background: T regulatory (Treg) cells dampen immune activation. Treg cells downregulate the type 2 response to innocuous environmental antigens that produce allergic airway inflammation; however, ST2-positive Treg cells promote allergic airway inflammation. Prostaglandin I₂ (PGI₂), which signals through the G protein-coupled receptor IP, promotes Treg cell function in an ovalbumin-based model of allergic airway inflammation, suggesting a role for PGI₂ signaling through the IP receptor augmenting β-catenin activity in Treg cells.

Objective: We sought to define the mechanisms responsible for PGI₂'s promotion of Treg cell function in the context of an environmental allergen.

Methods: Treg cell-specific IP-deficient mice, Treg cell fate-tracking IP-deficient mice, and Treg cell-specific IP- and β-catenin-deficient mice were exposed to an Alternaria alternata extract sensitization and challenge model. Bronchoalveolar lavage fluid was evaluated for cell number, cell differential, and cytokines by ELISA. Lungs were evaluated by flow cytometry and histopathology.

Results: Utilizing Treg cell-specific IP-deficient mice, we found that loss of PGI₂ signaling impaired Treg cell-suppressive function in response to A alternata; specifically, we found enhanced type 2 cytokine production, eosinophil infiltration, vascular remodeling, and numbers of ST2-positive Treg cells compared to controls. We found that dual IP and β-catenin deficiency in Treg cells prevented the enhanced type 2 response and the further increase in ST2-positive Treg cells via prevention of an increase in GATA3 expression in response to A alternata.

Conclusions: Together, these data further support the importance of PGI₂ signaling within Treg cells to their support functionality and demonstrate that PGI₂ prevents Treg cell dysfunction through downregulation of β-catenin.

Keywords: Alternaria alternata; ST2; Treg cell; inflammation; prostaglandin I(2); β-catenin.

Figure 1:. Loss of PGI₂ signaling within Treg promotes type 2 inflammation in allergic airway inflammation.

Treg specific IP deficient mice (Foxp3^YFPcrexIP^flox mice) were subjected to Alt sensitization and challenge. A) Sensitization and challenge with Alternaria Alternata (Alt) schematic. B) Total IL-13 within the BAL (n=6–14) C) Total number of cells within the bronchoalveolar lavage fluid (BAL) (n=6–14) D) Total number of eosinophils within the BAL (n=6–14). E) Total number of lymphocytes in the BAL (n=6–14) Data are represented as mean ± SEM. Statistical significance was determined by 2-way ANOVA. *P < 0.05, **P < 0.01, ***P< 0.001, ****P < 0.0001.

Figure 2:. Loss of PGI₂ signaling within Treg restrains type 2 inflammation and vascular remodeling in allergic airway inflammation.

Treg specific IP deficient mice (Foxp3^YFPcrexIP^flox mice) were subjected to Alt sensitization and challenge. Lungs were scored by a pathologist who was blinded to the groups. A) Peribronchial inflammation score (n=5–10) B) Vascular remodeling adjacent to small airways score (n=5–10). C) Intra-acinar vascular remodeling score (n=5–10) D) Representative elastin-stained images for each group. Data are represented as mean ± SEM. Statistical significance was determined by 2-way ANOVA. *P < 0.05, ****P < 0.0001.

Figure 3:. Loss of PGI₂ signaling within Treg augments ST2+ Treg responses in allergic airway inflammation.

Treg specific IP deficient mice (Foxp3^YFPcrexIP^flox mice) were subjected to Alt sensitization and challenge. Lungs were analyzed by flow cytometry. A) Representative flow cytometric analysis (n=6–14), the first row shows Foxp3+CD25+ Treg and Foxp3−CD4+ T cells. The second row shows ST2+ Treg within the Foxp3+CD25+ Treg gate. The third row shows GATA3 as a histogram within Foxp3+CD25+ Treg. B) Total Treg (n=6–14) C) Total ST2+ Treg (n=6–14) D) GATA3 MFI (n=6–14). E) Representative flow cytometric analysis showing GATA3 expression within Foxp3−CD4+ T cells from the two Alt-challenged groups. F) GATA3 MFI within Foxp3−CD4+ T cells (n=6–14). Data are represented as mean ± SEM. Statistical significance was determined by 2-way ANOVA. *P < 0.05, ***P < 0.001, ****P < 0.0001.

Figure 4:. PGI₂ signaling restrains ST2 expression on Treg limiting their conversion to IL-13 expressing Treg in a model of allergic airway inflammation.

ST2 expression on Treg and IL-13+ Treg within mouse lungs evaluated by flow cytometry. A) Representative flow cytometric analysis (n=4–12), the first row shows expression of GFP and YFP in CD3+CD4+ T cells. The second row shows expression of ST2 on the GFP+YFP+ Treg subset from row 1. The third row shows expression of IL-13 on the GFP+YFP+ Treg subset from row 1. The fourth row shows IL-13 expression on the ST2+GFP+YFP+ Treg subset from row 2. B) Total Treg (n=4–12) C) Total number of ST2+Treg (n=4–12). D) ST2 MFI on GFP+YFP+ Treg E) Total number of IL-13+Treg (n=4–12) F) Total number of IL-13+ST2+Treg (n=4–12) Data are represented as mean ± SEM. Statistical significance was determined by 2-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5:. PGI₂ signaling restrains ST2 expression on Ex Treg, thus limiting their conversion to IL-13 expressing Treg in a model of allergic airway inflammation.

ST2 expression on Ex Treg and IL-13+ Ex Treg within mouse lung evaluated by flow cytometry. A) Representative flow cytometric analysis (n=4–12), the first row shows expression of GFP and YFP in CD3+CD4+ T cells. The second row shows expression of ST2 on the GFP−YFP+ Ex-Treg subset from row 1. The third row shows expression of IL-13 on the GFP−YFP+ Ex-Treg subset from row 1. The fourth row shows IL-13 expression on the ST2+GFP−YFP+ Ex-Treg subset from row 2. B) Total Ex Treg (n=4–12) C) Total number of ST2+Ex Treg (n=4–12). D) ST2 MFI of YFP+ Ex Treg E) Total number of IL-13+Ex Treg (n=4–12) F) Total number of IL-13+ST2+Ex Treg (n=4–12) Data are represented as mean ± SEM. Statistical significance was determined by 2-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 6:. PGI₂ signaling restrains ST2 expression on Treg at baseline.

ST2 expression on Treg from mouse spleen evaluated by flow cytometry. A) Representative flow cytometric analysis (n=20) B) Percentage of ST2+Treg (n=20) Data are represented as mean ± SEM. Statistical significance was determined by t-test. *P < 0.05.

Figure 7:. Loss of β-catenin signaling in addition to PGI₂ signaling within Treg prevents enhanced type 2 inflammation.

Treg specific IP deficient mice (Foxp3^YFPcrexIP^flox mice) and Treg specific IP and Beta Catenin deficient mice (Foxp3^YFPcrexIP^floxxCtnnb1^flox were subjected to Alt sensitization and challenge. BAL was collected and analyzed by ELISA and for cell counts and differentials. A) Total IL-13 in the BAL (n=5–10) B) Total numbers of cells within the BAL (n=5–10) C) Total eosinophils in the BAL (n=5–10) Data are represented as mean ± SEM. Statistical significance was determined by 2-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 8:. Loss of β-catenin signaling in addition to PGI₂ signaling prevented the increase in Treg GATA-3 expression thereby restraining ST2+ Treg expansion in allergic airway inflammation.

Treg specific IP deficient mice (Foxp3^YFPcrexIP^flox mice) and Treg specific IP and Beta Catenin deficient mice (Foxp3^YFPcrexIP^floxxCtnnb1^flox were subjected to Alt sensitization and challenge. Lungs were analyzed by flow cytometry. A) Total lung Tregs (n=5–10) B) Total lung ST2+ Tregs (n=5–10). C) Treg GATA-3 mean fluorescence intensity (MFI) representative figure and quantification (n=2–5, one representative experiment shown). Data are represented as mean ± SEM. Statistical significance was determined by 2-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.