ILC2 Cells Promote Th2 Cell Differentiation in AECOPD Through Activated Notch-GATA3 Signaling Pathway

Abstract

This study is to investigate the capacity of type 2 innate lymphoid cells (ILC2s) in regulating the Th2 type adaptive immune response of acute exacerbation of chronic obstructive pulmonary disease (AECOPD). The study enrolled healthy people, stable chronic obstructive pulmonary disease (COPD) patients, and AECOPD patients. Flow cytometry was used to detect Th2 and ILC2 cells in the peripheral blood. In addition, ILC2s from the peripheral blood of AECOPD patients were stimulated with PBS, IL-33, Jagged1, DAPT, IL-33+Jagged1, IL-33+DAPT, and IL-33+Jagged-1+DAP in vitro. The levels of cytokines in the culture supernatant were detected by ELISA and the culture supernatant was used to culture CD4 ⁺ T cells. The mRNA and protein levels of Notch1, hes1, GATA3, RORα, and NF-κB of ILC2s were detected by real-time PCR and Western blot. The proportion of Th2 and ILC2s was significantly increased in the peripheral blood of AECOPD patients, alone with the increased Notch1, hes1, and GATA3 mRNA levels. In vitro results showed that the mRNA and protein levels of Notch1, hes1, GATA3 and NF-κB were significantly increased after stimulation with Notch agonist, meanwhile, the level of type 2 cytokines were increased in the supernatant of cells stimulated with Notch agonist, and significantly promoted differentiation of Th2 cells in vitro. Disruption of Notch pathway weakened GATA3 expression and cytokine production, and ultimately affected the differentiation of Th2 cells. In conclusion, our results suggest that ILC2s can promote Th2 cell differentiation in AECOPD via activated Notch-GATA3 signal pathway.

Keywords: Notch-GATA3 pathway; Th2 polarized; acute exacerbation of chronic obstructive pulmonary disease; cell differentiation; type 2 innate lymphoid cell.

Figure 1

Th2 and ILC2 cells in different groups. Th2 cells and ILC2 cells in healthy control group, stable COPD group and AECOPD group were analyzed by flow cytometry. (A)Th2 (CD183⁻ CD196⁻) was labeled with specific antibodies and quantified in each groups. (B) Percentage of Th2 cells. (C) Uniform manifold approximation and projection (UMAP) analysis of ILC2 cells in peripheral blood among the three groups. (D) Percentage of ILC2s in three groups. Data are shown as mean ± SEM. n=50. ^*** P < 0 001, compared with the control group. ^### P < 0.001 compared with the stable COPD group.

Figure 2

Levels of ILC2 related transcription factors in different groups. The mRNA levels of (A) Notch1, (B) Hes1, (C) GATA3 and (D) RORα were tested by real-time PCR. Data were normalized to control group and represented as mean ± SEM. n=50. ^* P < 0.05, ^** P < 0.01 compared with control group.

Figure 3

The mRNA levels of Notch1, Hes1, RORα, GATA3 and NF-κB in different groups. The relative levels of Notch1 (A), Hes1 (B), RORα (C), GATA3 (D), and NF-κB (E) mRNA in the supernatant of PBS group, IL-33 stimulation group, Jagged 1 treatment group, DAPT treatment group, IL-33 + Jagged 1 treatment group, IL-33 + DAPT treatment group were determined by real-time PCR. Data are shown as mean ± SEM. n=10. ^* P < 0.05 compared with PBS group, ^#P < 0.05, compared with IL-33 group, ^▽ P < 0.05 compared with Jagged1 group, ^▼ P < 0.05 compared with DATP group, ^★ P < 0.05 compared with IL-33+Jagged1 group.

Figure 4

Protein levels of Notch1, Hes1, RORα, GATA3 and NF-κB in different groups. The protein expressions of Notch1, Hes1, RORα, GATA3 and NF-κB in the supernatant of PBS group, IL-33 stimulation group, Jagged 1 treatment group, DAPT treatment group, IL-33 + Jagged 1 treatment group, IL-33 + DAPT treatment group were determined by Western Blot. (A) Representative Western blot results of RORα, Notch1, Hes1, GATA3 and NF-κB in each treatment group. Expression of (B) Notch1, (C) Hes1, (D) RORα, (E) GATA3, and (F) NF-κB was measured by Western Blot relative to β-actin. Data are shown as mean ± SEM. n=10. ^*P < 0.05 compared with PBS group, ^# P < 0.05, compared with IL-33 group, ^▽ P < 0.05 compared with Jagged1 group, ^▼ P < 0.05 compared with DATP group, ^★ P < 0.05 compared with IL-33+Jagged1 group.

Figure 5

Cytokines after IL-33, Jagged 1 and DATP treatment. Level of (A) IL-4, (B) IL-5 and (C) IL-13 in the supernatant of PBS group, IL-33 stimulation group, Jagged 1 treatment group, DAPT treatment group, IL-33 + Jagged 1 treatment group, IL-33 + DAPT treatment group were detected by ELISA. Data are shown as mean ± SEM. n = 10. ^* P < 0.05 compared with PBS group, ^# P < 0.05 compared with IL-33 group, ^▽ P < 0.05 compared with Jagged1 group, ^▼ P < 0.05 compared with DATP group, ^★ P < 0.05 compared with IL-33+Jagged1 group.

Figure 6

Th2 cells after treatment in different groups. Level of Th2 cells after treatment with the supernatant of PBS group, IL-33 stimulation group, Jagged 1 treatment group, DAPT treatment group, IL-33 + Jagged 1 treatment group, IL-33 + DAPT treatment group was determined by flow cytometry. (A) Th2 (CD183⁻ CD196⁻) was labeled with specific antibodies and quantified in each treatment groups. (B) Percentage of Th2 cells. Data are shown as mean ± SEM. n=10.^* P < 0.05 compared with CD4⁺ T cell group.