Bronchial epithelial cells of young and old mice directly regulate the differentiation of Th2 and Th17

Abstract

To determine whether or not house dust mite (HDM) and HDM+lipopolysaccharide (LPS) exposure causes a difference in T-cell subsets from young and old mice. The bronchial epithelial cells (BECs) from young and old mice were divided into three groups (PBS (control), HDM, and HDM+LPS). CD4⁺ naive T cells from the spleen and lymph nodes were collected after 24 h of co-culture with BECs. The number of Th2 and Th17 cells was elevated in the HDM and HDM+LPS groups compared with the control group; these responses were exacerbated when exposed to HDM+LPS. The number of HDM- and HDM+LPS-specific Th2/Th17 cells in young mice was higher than old mice; however, the Th2:Th17 cell ratio was greater in young mice, whereas the Th17:Th2 cell ratio was greater in old mice. The expression of GATA-3 and RORc was increased in the HDM+LPS and HDM groups compared with the PBS group and exhibited most in HDM+LPS group. The expression of HDM+LPS-specific GATA-3 in young mice was higher, while the expression of HDM+LPS-specific RORc in old mice was higher. Murine BECs directly regulated CD4⁺ naive T-cell differentiation under allergen exposure.

Keywords: Th17 cells; Th2 cells; asthma; epithelial cells.

Figure 1. Culture of BECs in vitro on the seventh day

The positive rate was assessed by flow cytometry and immunofluorescence using epithelial-specific cytokeratin 19 antibody. (A) The positive rate of primary culture of old BECs by flow cytometry. (B) The positive rate of primary culture of young BECs by immunofluorescence.

Figure 2. BECs from young mice regulate CD4⁺ naive T-cell differentiation in vitro

(A) After irritation with 100 μg/ml of HDM, 100 μg/ml of HDM + 100 ng LPS, or PBS for 8  h and co-culture with CD4⁺ naive T cells for 24 h, the rate of Th2 cells increased in the HDM and HDM+LPS groups, but had the greatest increase in the HDM+LPS group. (B) After irritation with 100 μg/ml of HDM, 100 μg/ml of HDM + 100 ng/ml LPS, or PBS for 8  h and co-culture with CD4⁺ naive T cells for 24 h, the rate of Th17 cells increased in the HDM and HDM + LPS groups, but had the greatest increase in the HDM + LPS group (*, P<0.05; **, P<0.01).

Figure 3. The number of Th2 and Th17 cells in young and old groups

After 100 μg/ml of HDM + 100 ng/ml of LPS or PBS was given to BECs for 8 h, BECs and CD4⁺ naive T cells from young mice or BECs from old mice and CD4⁺ naive T cells from young mice were co-cultured for 24 h. T cells were collected and Th2 and Th17 cells were measured by flow cytometry (*, P<0.05; **, P<0.01).

Figure 4. The number of Th2 and Th17 cells in the old group to HDM or HDM + LPS-specific exposure to BECs

A total of 100 μg/ml of HDM + 100 ng/ml of LPS or PBS was given to BECs for 8 h. BECs from old mice and CD4⁺ naive T cells from young mice were co-cultured for 24 h. T cells were collected and Th2 and Th17 cells were measured by flow cytometry. The increased number of Th2 and Th17 cells were compared (*, P<0.05; **, P<0.01).

Figure 5. The number of Th2 and Th17 cell in the young group to HDM or HDM+LPS-specific exposure to BECs

A total of 100 μg/ml of HDM + 100 ng/ml of LPS or PBS was given to BECs for 8 h. BECs and CD4⁺ naive T cells from young mice were co-cultured for 24 h. T cells were collected and Th2 and Th17 cells were measured by flow cytometry. The increased number of Th2 and Th17 cells were compared (*, P<0.05; **, P<0.01).

Figure 6. GATA-3 and RORc protein expression in HDM, HDM+LPS, and PBS groups

GATA-3 and RORc protein expression in HDM, HDM+LPS, and PBS groups. A total of 100 μg/ml of HDM, 100 μg/ml of HDM + 100 ng/ml of LPS or PBS was given to BECs for 8 h. BECs and CD4⁺ naive T cells both from young mice were co-cultured for 24 h. T cells were collected and the total protein was extracted. The expression of GATA-3 and RORc was examined from the above groups by Western blotting. RORc and GATA-3 protein expression was normalized to GADPH (*, P<0.05; **, P<0.01).

Figure 7. GATA-3 and RORc protein expression in young and old groups to HDM or HDM+LPS-specific exposure to BECs

The young group contained BECs and CD4⁺ naive T cells from young mice (A) and the old group contained BECs from old mice and CD4⁺ naive T cells from young mice (B). A total of 100 μg/ml of HDM, 100 μg/ml of HDM + 100 ng/ml of LPS or PBS was given to BECs for 8 h. BECs and CD4⁺ naive T cells were co-cultured for 24 h. T cells were collected and the total protein was extracted. The expression of GATA-3 and RORc was examined from the above groups by Western blotting. RORc and GATA-3 protein expression were normalized to GADPH (*, P<0.05; **, P<0.01).