CD₄(+)Foxp3(+) regulatory T cells in inflammation and emphysema after smoking cessation in rats

Abstract

To evaluate the expression of Treg in a cigarette smoke-induced rat model of emphysema and after smoking cessation in the rats. Fifty male Wistar rats were randomly divided into control group 1 (12 weeks), control group 2 (24 weeks), smoke-exposure group 1 (12 weeks), smoke-exposure group 2 (24 weeks) and smoking cessation group, with 10 rats in each group. Alveolar airspace enlargement was observed by hematoxylin-eosin (HE) staining. IL-8 and TNF-α levels in bronchoalveolar lavage fluid (BALF) were tested by ELISA. The proportion of CD₄(+)Foxp3(+) Treg in peripheral blood and lungs of rats was determined by flow cytometry. The mRNA expression of Foxp3 was measured by real-time PCR. Comparisons of the data between different groups were performed using one-way ANOVA, and SNK and Games-Howell test was used for comparison between 2 groups. The mean linear intercept (MLI) in smoke-exposure group 1 and group 2 [(64.9 ± 5.3) µm, (77.9 ± 11.5) µm] was higher than those in the control group 1 and group 2 [(39.0 ± 3.8) µm, (40.3 ± 2.7) µm], all P < 0.01. Compared with smoke-exposure group 2, the MLI in smoking cessation group (71.5 ± 5.8) µm showed a lower value (P < 0.01), but still higher than that in smoke-exposure group 1 (P < 0.01). The IL-8 and TNF-α levels in BALF of smoke-exposure group 1 and group 2 [(68 ± 17) ng/L, (85 ± 16) ng/L], [(14.1 ± 1.8) ng/L, (20.1 ± 8.7) ng/L] were higher than those in control group 1 and group 2 [(44 ± 8) ng/L, (43 ± 9) ng/L], [(6.3 ± 2.3) ng/L, (5.8 ± 1.6) ng/L], all P < 0.05. The IL-8 and TNF-α levels were not statistically different between in smoking cessation group (56 ± 6) ng/L, (14.7 ± 4.7) ng/L and smoke-exposure group 1. The percentage of Treg in the lungs of smoke-exposure group 1 and group 2 [(6.6 ± 0.8)%, (5.3 ± 0.9)%] was significantly decreased as compared to control group 1 and group 2 [(9.0 ± 1.0)%, (9.6 ± 0.9)%], all P < 0.01. The percentage of Treg in lungs was not statistically different between smoke-exposure group 1 and smoking cessation group (7.2 ± 0.6)%. In peripheral blood, there was no significant difference between groups in the percentage of Treg. In the lung, Foxp3 mRNA expression in smoke-exposure group 1 and group 2 [(17 ± 7), (9 ± 7)] was less than that in control group 1 and group 2 [(39 ± 6), (42 ± 7)], all P < 0.01. The Foxp3 mRNA expression was not statistically different between smoke-exposure group 1 and smoking cessation group (21 ± 9). No significant differences in peripheral blood Foxp3 mRNA expression was found between groups. Decreased Treg was present in lungs of cigarette smoke-induced model of emphysema despite 12 weeks' smoking cessation, suggesting that down-regulation of Treg may be involved in the amplified and persistent inflammation after smoking cessation.