Cigarette Smoke Induction of Interleukin-27/WSX-1 Regulates the Differentiation of Th1 and Th17 Cells in a Smoking Mouse Model of Emphysema

Abstract

IFN-γ-producing CD4⁺ T (Th1) cells and IL-17-producing CD4⁺ T (Th17) cells play a critical role in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, the immune regulation between Th1 and Th17 cells remains unclear. Previous studies have demonstrated that interleukin-27 (IL-27)/WSX-1 exerted pro- or anti-inflammatory effects in many acute inflammatory diseases by modulating T cell-mediated immune response, but little was known about its role in chronic inflammatory disease, especially in smoking-related lung diseases. Considering IL-27 is an important regulator in T lymphocytes immune responses and was found markedly increased in patients with COPD, we hypothesized that IL-27/WSX-1 may exert immuno-regulatory effects on the differentiation of Th1 and Th17 cells in smoking-related COPD. In this study, we aimed to evaluate the expression of IL-27 in patients with COPD and explore the role of IL-27/WSX-1 on Th1 and Th17 cells differentiation in a smoking mouse model of emphysema. We found that elevated expression of IL-27 was associated with increased proportion of Th1 cells and Th17 cells in patients with COPD and demonstrated parallel findings in cigarette smoke-exposed mice. In addition, cigarette smoke exposure upregulated the expression of IL-27R (WSX-1) by naive CD4⁺ T cells in mice. In vitro, IL-27 significantly augmented the secretion of IFN-γ by naive CD4⁺ T cells via a T-bet, p-STAT1, and p-STAT3-dependent manner, but inhibited the production of IL-17 by a ROR-γt and p-STAT1-dependent way. Furthermore, anti-IL27 treatment dramatically decreased the expression of IFN-γ-producing CD4⁺ T cells in cigarette smoke-exposed mice. These findings proposed that IL-27 has functions for promoting the expression of Th1 cells but inhibiting the expression of Th17 cells in vitro and IL-27 neutralization-attenuated Th1-mediated inflammation in vivo, suggesting targeting IL-27/WSX-1 may provide a new therapeutic approach for smoking-related COPD.

Keywords: COPD; IL-27/WSX-1; Th1 cells; Th17 cells; cigarette smoke exposure.

Figure 1

Elevated expression serum levels of IL-27 in patients with COPD. (A) IL-27 protein concentrations in serum of non-smokers, healthy smokers, and COPD patients. Data are expressed as means ± SD. Horizontal bars indicate means. The comparisons were determined by one-way ANOVA. *p < 0.05 compared with one another among three groups. (B) Correlation between serum IL-27 and FEV1 (% pred) in COPD patients. Correlations between two parameters were determined by Spearman’s rank correlation test.

Figure 2

Increased expression of Th1 and Th17 cells in peripheral blood of COPD patients. (A) Lymphocytes were identified based on their characteristic properties shown in the FSC and SSC, and CD4⁺ T cells were gated from lymphocytes. The representative flow cytometric dot plots of Th1 and Th17 cells in peripheral blood of non-smokers, healthy smokers, and COPD patients. FSC, forward scatter cytometry; SSC, sides scatter cytometry. (B) Comparisons of percentages of Th1 cells (C) and Th17 cells in non-smokers, healthy smokers, and COPD patients. Data are expressed as means ± SD. Horizontal bars indicate means. The comparisons were determined by one-way ANOVA (*p < 0.05). (D) Correlation between serum IL-27 and Th1 (E) and Th17 cells in COPD patients. Correlations between two parameters were determined by Spearman’s rank correlation test.

Figure 3

Cigarette smoke exposure (CSE) induced alveolar destruction and airspace enlargement in mice. Representative photomicrographs of hematoxylin and eosin-stained lung tissue of air-control and cigarette smoke-exposed mice at 24 weeks (magnification, ×200). (A) Air-control mice (B) cigarette smoke-exposed mice. The arrows in (A) indicate the normal alveolar and the lymphocytes in alveolar septa in air-control mice. The arrows in (B) represent the enlargement of the air spaces the destruction of the alveolar architecture accompanied by the infiltration of lymphocytes. (C) Comparisons of Lm values in air-control and cigarette smoke-exposed mice. n = 10 animals/group; data are expressed as means ± SD. The comparisons were determined by Student’s t-test.

Figure 4

Elevated expression levels of IL-27 in cigarette smoke-exposed mice. (A) IL-27 protein concentrations in serum, lungs of air-control and cigarette smoke-exposed mice. (B) IL-27 p28 mRNA (C) and EBI3 mRNA expression in peripheral blood, spleens, and lungs of air-control and cigarette smoke-exposed mice. Data are expressed as means ± SD. The comparisons were determined by Student’s t-test (*p < 0.05).

Figure 5

Increased expression of IL-27R (WSX-1) on effector and naïve CD4⁺ T cells in cigarette smoke-exposed mice. (A) The representative flow cytometric dot plots of IL-27R (WSX-1) on total CD4⁺ T cells. The expression of IL-27R (WSX-1) on (B) CD44^highCD62L⁻effector CD4⁺ T cells (C) and CD44^lowCD62L⁺naïve CD4⁺ T cells in mice of air-control or cigarette smoke exposed. The comparison of IL-27R (WSX-1) expression on (D) effector CD4⁺ T cells (E) and naïve CD4⁺ T cells in peripheral blood, spleens, and lungs of air-control or cigarette smoke-exposed mice. NS, no difference. Data are expressed as means ± SD. The comparisons were determined by Student’s t-test (**p < 0.001).

Figure 6

Phenotypic characteristics of CD4⁺ T cells in cigarette smoke-exposed mice. (A) The representative flow cytometric dot plots of naïve (CD44^lowCD62L⁺), memory (CD44^highCD62L⁺), and effector (CD44^highCD62L⁻) CD4⁺ T cells in peripheral blood, spleens, and lungs of air-control and cigarette smoke-exposed mice. (B) Comparisons of percentages of naïve CD4⁺ T cells (C) and effector CD4⁺ T cells (D) memory CD4⁺ T cells in air-control and cigarette smoke-exposed mice. Data are expressed as means ± SD. The comparisons were determined by Student’s t-test (*p < 0.01, **p < 0.001).

Figure 7

Increased proportions of Th1 and Th17 cells in cigarette smoke-exposed mice. (A) Lymphocytes were identified based on their characteristic properties shown in the FSC and SSC, and CD4⁺ T cells were gated from lymphocytes. The representative flow cytometric dot plots of Th1 and Th17 cells in peripheral blood, spleens, and lungs of air-control and cigarette smoke-exposed mice. (B) Comparisons of percentages of Th1 cells (C) and Th17 cells (D) IFN-γ⁺IL-17⁺CD4⁺ T cells in air-control and cigarette smoke-exposed mice. Data are expressed as means ± SD. The comparisons were determined by Student’s t-test (*p < 0.01, **p < 0.001). (E) Correlation between Lm and Th1 (F) and Th17 cells in lungs of cigarette smoke-exposed mice. Correlations between two parameters were determined by Spearman’s rank correlation test.

Figure 8

IL-27 promoted the Th1 but inhibited the Th17 cells differentiation in vitro. (A) Naive CD4⁺ T cells from spleen of wild-type mice were cultured under Th1 and Th17 conditions in the presence or absence different concentrations of IL-27. The representative flow cytometric dot plots of Th1 and Th17 are showing from five independent experiments. (B) Comparisons of percentages of Th1 and Th17 cells in each group. The comparisons were determined by one-way ANOVA (*p < 0.05). (C,D) The effects of different concentrations of IL-27 on the cells under Th1 and Th17 conditions. (E,F) The effects of IL-27 on the cells after 6 days stimulated in Th1 and Th17 condition.

Figure 9

Effects of IL-27 on expression of T-bet and ROR-γt during the differentiation of Th1 and Th17 cells. Naive CD4⁺ T cells from spleen of wild-type mice were cultured under Th1 and Th17 conditions in the presence or absence different concentrations of IL-27. (A,B) The representative flow cytometric histogram-plot of T-bet or ROR-γt are showing from five independent experiments. (C–F) Comparisons percentages of T-bet and ROR-γt in each group. The comparisons were determined by one-way ANOVA (*p < 0.05).

Figure 10

Signal transductions involved in differentiation Th1 and Th17 cells. Naive CD4⁺ T cells from spleens of wild-type mice were cultured under Th1 and Th17 conditions. Three days later, the medium was supplemented with different concentrations of IL-27 and the cells were stimulated for 30 min and intracellular stained with p-STAT1, p-STAT3. (A,B) The representative expression of p-STAT1, p-STAT3 in cells under Th1 and Th17 conditions in the presence or absence of IL-27. (C–F) Comparisons of percentages of p-STAT1, p-STAT3 in each group. The comparisons were determined by one-way ANOVA (*p < 0.05).

Figure 11

Anti-IL-27 treatment decreased the proportions of effector CD4⁺ T cells and downregulated the expression of Th1 cells in cigarette smoke-exposed mice. Mouse was treated by tail vein injection with anti-mouse IL-27 p28 functional grade purified mAbs twice a week (10 μg in 200 μl PBS per time) in the course of cigarette smoke exposure. (A) Comparisons of Lm values in cigarette smoke-exposed mice treated with anti-IL-27 antibody and in mice treated with PBS. (B) Comparisons of percentages of naive CD4⁺ T cells (C) and effector CD4⁺ T cells (D) and memory CD4⁺ T cells (E) and Th1 cells (F) and Th17 cells in cigarette smoke-exposed mice treated with anti-IL-27 antibody and mice treated with PBS (each n = 8). Data are expressed as means ± SD. The comparisons were determined by Student’s t-test (*p < 0.05, **p < 0.001).