Involvement Of Interleukin-17-Producing CD₄⁺T Helper Cells In Mice With Cigarette Smoke-Induced Emphysema

CHAPTER I Expression and activity of interleukin-17-producing CD₄⁺T helper cells in mice with cigarette smoke-induced emphysemaObjective To evaluate the expression of Th17 in a cigarette smoke-induced mice model of emphysema. and to explore the role of Th17 cells in lung inflammation and emphysema of mice.Methods 40 male Balb/c mice were randomly divided into four groups,including control group C12, control group C24, smoke-exposure group S12 and smoke-exposure group S24, 10 mice each group. Morphological changes were evaluated by mean linear intercepts (Lm) and destructive index (DI). IFN-γand TNF-αlevels in bronchoalveolar lavage fluid (BALF) and lungs were tested by ELISA. The proportion of CD₄⁺IL-17⁺ Th17 cells in peripheral blood and lungs of mice was determined by flow cytometry. The mRNA expressions of RORγt and IL-17 in peripheral blood and lungs were measured by real-time PCR.Results Lm and DI were significantly higher in S12 and S24 groups [(39.19±3.51)μm,(46.87±7.16)μm], [(39.13±1.57),(45.16±3.13)] than that in C12 group [(32.60±3.21)μm,(28.23±1.62)] and C24 [(32.38±3.73)μm, (28.86±2.07)], all P<0.05. The IFN-γand TNF-αlevels in BALF of S12 and S24 groups [( 129.7±22.2 ) ng/L, ( 251.1±62.4 )ng/L],[( 17.35±1.60 ) ng/L,( 36.35±1.43 ) ng/L] were higher than those in C12 and C24 groups [(85.8±26.8)ng/L,(88.9±11.5)ng/L], [(6.41±0.90)ng/L,(5.85±0.92)ng/L], The IFN-γand TNF-αlevels in lungs of S12 and S24 groups [(1124.3±174.4)ng/L(,1342.7±206.1)ng/L],([77.2±13.7)ng/L(,101.7±19.0)ng/L ([129.7±22.2)ng/L,(251.1±62.4)ng/L] were higher than those in C12 and C24 groups [(946.2±81.9)ng/L,(1027.2±188.3)ng/L],[(62.1±16.1)ng/L,(64.4±15.1)ng/L],all P<0.05. The percentage of Th17 cells in lungs of S12 and S24 groups [(3.27±1.12)%,(7.19±2.24)%] , [(1.96±0.61)%,( 3.82±1.26)%] was significantly increased as compared with that of C12 and C24 groups [(1.80±0.75)%,(1.99±0.59)%], all P<0.05. And the percentage of Th17 cells in peripheral blood of S12 and S24 groups [(1.96±0.61)%,( 3.82±1.26)%] was also significantly higher than those in C12 and C24 groups [(0.90±0.37)%,(0.97±0.32)%], all P<0.05..In peripheral blood and lungs,the mRNA levels of RORγt and IL-17 in S12 and S24 groups were higher than in C12 and C24 groups, all P<0.05. Moreover, the frequency of Th17 cells had a positive correlation with Lm and DI.Conclusions Cigarette smoke increased the expression and activity of Th17 in peripheral blood and lungs of mice. Th17 may be involved in the amplified and persistent inflammation. CHAPTER II Mechanisms and dynamics of interleukin-17-producing CD₄⁺T helper cells in mice with cigarette smoke-induced emphysemaObjective To evaluate the expression of Th17 cells in a cigarette smoke-induced mice model of emphysema. To explore the probable mechanisms about how Th17 cells were elevated in lungs of mice.Methods 40 male Balb/c mice were randomly divided into four groups: control group for 12 weeks (C12),control group for 24 weeks (C24),smoke- exposure group for 12 weeks (S12) and smoke-exposure group for 24 weeks (S24). Morphological changes were evaluated by mean linear intercepts (Lm) and destructive index (DI). The percentages of CD₄⁺IL-17⁺T(Th17), CD₄⁺IFN-γ⁺T(Th1), CD₄⁺IL-17⁺IFN-γ⁺T(Th17/Th1), CD₄⁺IL-17⁺CCR6⁺T and CCR6⁺ Th17 were determined by four colors flow cytometry, the levels of IL-1β, IL-6, IL-23, TGF-β, IFN-γand CCL20 were tested by ELISA.Results Lm and DI were significantly higher in S12 and S24 groups [(39.19±3.51)μm,(46.87±7.16)μm], [(39.13±1.57),(45.16±3.13)] than that in C12 groups [(32.60±3.21)μm,(28.23±1.62)] and C24 [(32.38±3.73)μm, (28.86±2.07)], all P<0.05. The percentages of Th17,Th17/Th1 and Th1 in the lungs of S12 and S24 groups[(3.27±1.12),(7.19±2.24)],[(0.61±0.30),(1.82±0.52)] and [(10.02±3.68),(26.21±6.04)] were significantly increased than that in C12 and C24 groups[(1.80±0.75),(1.99±0.59)],[(0.27±0.12),(0.28±0.11)] and [(3.75±1.72),(4.16±1.32)], all P<0.01. The frequency of Th17,Th17/Th1 and Th1 cells had a positive correlation with Lm and DI, all P<0.01. The percentages of CD₄⁺IL-17⁺CCR6⁺T cells and CCR6⁺ Th17 cells were significantly elevated in S12 and S24 groups compared to the controls,all P<0.05. In addition, a positive correlation between CCR6⁺ Th17 cells and emphysematous lesions was also found, P<0.05. The levels of IL-1β,IL-6,IL-23,TGF-β,IFN-γand CCL20 in S12 and S24 groups were significantly increased as compared with that of C12 and C24 groups,all P<0.05. Meanwhile, The frequency of Th17 cells had a positive correlation with IL-1β,IL-6,IL-23,TGF-β,IFN-γand CCL20.Conclusions An up-regulated expression of Th17 in lungs of cigarette smoke-induced emphysema mice is detected. The CCR6/CCL20 axis and the increased IL-1β,IL-6,IL-23,TGF-βand IFN-γbe related with this up-regulation. CHAPTER III Effect of interleukin-17-producing CD₄⁺T helper cells on cytotoxic effector function in CD⁸ T cells of mice with cigarette smoke-induced emphysemaObjective To evaluate the expression of IL-21 in Th17 cells and study its influence on CD⁸ T cells of mice with cigarette smoke-induced emphysema. To explore the mechanisms about how Th17 cells to enlarge inflammatory reaction of mice.Methods 40 male Balb/c mice were randomly divided into four groups: control group for 12 weeks (C12),control group for 24 weeks (C24),smoke- exposure group for 12 weeks (S12) and smoke-exposure group for 24 weeks (S24). Morphological changes were evaluated by mean linear intercepts (Lm) and destructive index (DI). The proportion of CD₄⁺IL-17⁺IL-21+ and CD⁸+IL-21R⁺ T cells in peripheral blood and lungs of mice was determined by flow cytometry. CD⁸+ T cells from spleencytes were isolated, and were cultured in medium with or without mouse rIL-21 for 3days. The proportion of CD⁸+perforin+ and CD⁸+granzyme B+ T cells in cell curture medium was determined by flow cytometry. The levels of perforin and granzyme B in supernatants from cytokine-stimulated cells were analyzed by ELISA.Results Lm and DI were significantly higher in S12 and S24 groups [(39.19±3.51)μm, (46.87±7.16)μm], [(39.13±1.57), (45.16±3.13)] than in C12 and C24 groups [(32.60±3.21)μm, (32.38±3.73)μm], [(28.23±1.62), (28.86±2.07)], all P<0.05. In peripheral blood, the percentage of CD₄⁺IL-17⁺IL-21+T cells was significantly increased in S12 and S24 groups [( 0.18±0.04)%,( 0.53±0.22)%] compared to controls [(0.06±0.03)%, (0.08±0.03)%],all P<0.05. Meanwhile,compare with that of controls [(0.59±0.21)%,( 0.61±0.18)%], the percentage of CD⁸+IL-21R⁺ T cells was also significantly increased in S12 and S24 groups[(2.35±0.75)%,(3.14±1.35)%],all P<0.05, but there were no differences among smoke-exposure groups (P>0.05). In lungs, the percentage of CD₄⁺IL-17⁺IL-21+T cells was significantly increased in S12 and S24 groups[(0.19±0.04)%, (0.55±0.24)%] compared to controls [(0.07±0.03)%, (0.08±0.03)%],all P<0.05. Meanwhile,compare with that of controls[(1.22±0.31), (1.34±0.18)], the percentage of CD⁸+IL-21R⁺ T cells was also significantly increased in S12 and S24 groups[(2.94±1.26), (4.12±2.26)], but there were no differences among smoke-exposure groups,P>0.05. Moreover, the frequency of CD₄⁺IL-17⁺IL-21+T cells and CD⁸+IL-21R⁺ T cells in peripheral blood and lungs had a positive correlation with Lm and DI,all P<0.05. The percentage of CD⁸+perforin+T cells and CD⁸+granzyme B+ T cells in cell curture medium of C24IL-21+ and S24IL-21+ groups[(58.68±7.87), (76.06±5.19)] and [(42.70±5.72),(71.13±4.97)] was significantly increased as compared with that in C24IL-21- group [(23.28±3.41),(20.27±3.35)] and S24IL-21-group[(29.19±6.81),(25.36±5.78)],all P<0.05. The levels of perforin and granzyme B in supernatants of C24IL-21+ and S24IL-21+groups [(17.54±1.80)ng/L,(24.47±2.61) ng/L],[(1.43±0.18) ng/L,(1.92±0.21) ng/L] were significantly increased as compared with that of C24IL-21-group [(5.23±2.02)ng/L,(0.84±0.07)ng/L] and S24IL-21-group[(9.24±1.12) ng/L, (1.35±0.20)ng/L],all P<0.05. In each group, the level of perforin was significantly higher than that of granzyme B, all P<0.05.Conclusions An up-regulation of proportions CD₄⁺IL-17⁺IL-21+ and CD⁸+IL-21R⁺ T cells in peripheral blood and lungs of cigarette smoke-induced emphysema mice were detected. The IL-21/IL-21R pathway may be involved in the Th17-induced the lung inflammation and tissue destructive. And it may provide a novel therapeutic strategy for COPD.