Erythromycin Regulates Cigarette Smoke-Induced Proinflammatory Mediator Release Through Sirtuin 1-Nuclear Factor κB Axis in Macrophages and Mice Lungs.

BACKGROUND: Macrolides have anti-inflammatory and antioxidative stress function, but their pharmacological regulation remains unclear. Sirtuin 1 (SIRT1) is redox-sensitive protein belongs to class III histone/protein deacetylases, SIRT1 regulates the acetylation/expression of nuclear factor κB (NF-κB) and is involved in the airway inflammation of chronic obstructive pulmonary disease. OBJECTIVES: The present study was designed to examine the effects of erythromycin (EM) on the SIRT1-NF-κB axis and NF-κB-dependent proinflammatory cytokines. METHODS: Human macrophages were preincubated with EM and then treated with cigarette smoke extract (CSE). The mice were treated by injecting drugs to gastric with EM before cigarette smoke exposure. Reactive oxygen species (ROS) released by treated human macrophages were detected using flow cytometry. The expression of SIRT1 and NF-κB was analyzed by western blotting. SIRT1 and the RelA/p65 subunits of NF-κB interaction were detected by coimmunoprecipitation. We found that EM suppressed CSE-induced ROS released in human macrophages, which coincided with increases in SIRT1 protein expression in the macrophages and lungs of mice, resulting in suppressed -NF-κB acetylation and expression correlated with a reduction of inflammatory mediators. CONCLUSION: These findings suggest that EM increased SIRT1, leading to acetylation/expression of NF-κB, and thereby decreasing cigarette smoke-driven NF-κB-dependent proinflammatory cytokine.

Enhanced activation of circulating plasmacytoid dendritic cells in patients with Chronic Obstructive Pulmonary Disease and experimental smoking-induced emphysema.

Plasmacytoid dendritic cells (pDCs) are key cells bridging the innate with adaptive immunity. However, the phenotypic characteristics of circulating pDCs and its role in smoking related-Chronic Obstructive Pulmonary Disease (COPD) remain largely unknown. The aim of this study was analyzed the phenotype of circulating pDCs and the expression of IFN-γ producing CD8+T cells and IL-17-producing CD8+T cells in patients with COPD by using multi-colour flow cytometry. The cytokine profiles in peripheral blood from all subjects were measured by ELISA. The influence of cigarette smoke on pDCs was evaluated in an experimental mouse model of emphysema. Circulating pDCs in patients with COPD and in mice exposed to cigarette smoke expressed high levels of co-stimulatory molecules CD40 or CD86 accompanied by exaggerated IFN-γ producing CD8+T cells and IL-17-producing CD8+T cells. In vitro, cigarette smoke directly promoted pDCs maturation and release of IFN-α, IL-6 and IL-12, subsequently inducing differentiation of IFN-γ producing CD8+T cells and IL-17-producing CD8+T cells from mouse naïve CD8+T cells. These data suggested that circulating pDCs display an enhanced activation phenotype in patients with COPD and in experimental smoking mouse model of emphysema, which might contribute to exaggerated IFN-γ producing CD8+T and IL-17-producing CD8+T cell-mediated immune responses.

Neutrophil extracellular traps induced by cigarette smoke activate plasmacytoid dendritic cells.

BACKGROUND: Neutrophil extracellular traps (NETs) represent a distinct strategy by which neutrophils trap, confine and eliminate invading microorganisms. Emerging evidence suggests that NETs exert a deleterious effect to the host in the absence of microbial stimuli. However, the role of NETs in smoking-related lung diseases remains to be elucidated. OBJECTIVES: To evaluate the formation of NETs in the context of chronic inflammation induced by cigarette smoking and explore its potential role in an experimental mouse model of emphysema. METHODS: The formation and degradation of NETs in cigarette smoke exposed mice was assessed with a fluorescence microscope. The potential influences of NETs on plasmacytoiddendritic cells were also investigated. RESULTS: NETs were more prone to formation by polymorphonuclearneutrophils but defective in degradation in cigarette smoke exposed mice. Cigarette smoke extract (CSE) served as an important facilitator that triggered neutrophils to undergo NETosis in vitro. Furthermore, CSE-induced NETs were capable of driving plasmacytoiddendritic cell maturation and activation, thereby initiating a T-cell-mediated immune response. CONCLUSIONS: NETs may represent a critical connection between innate and adaptive immune responses under conditions of chronic inflammation induced by cigarette smoke exposure.

Dendritic cells induce Tc1 cell differentiation via the CD40/CD40L pathway in mice after exposure to cigarette smoke.

Dendritic cells and CD8(+) T cells participate in the pathology of chronic obstructive pulmonary disease, including emphysema, but little is known of the involvement of the CD40/CD40L pathway. We investigated the role of the CD40/CD40L pathway in Tc1 cell differentiation induced by dendritic cells in a mouse model of emphysema, and in vitro. C57BL/6J wild-type and CD40(-/-) mice were exposed to cigarette smoke (CS) or not (control), for 24 wk. In vitro experiments involved wild-type and CD40(-/-) dendritic cells treated with CS extract (CSE) or not. Compared with the control groups, the CS mice (both wild type and CD40(-/-)) had a greater percentage of lung dendritic cells and higher levels of major histocompatability complex (MHC) class I molecules and costimulatory molecules CD40 and CD80. Relative to the CS CD40(-/-) mice, the CS wild type showed greater signs of lung damage and Tc1 cell differentiation. In vitro, the CSE-treated wild-type cells evidenced more cytokine release (IL-12/p70) and Tc1 cell differentiation than did the CSE-treated CD40(-/-) cells. Exposure to cigarette smoke increases the percentage of lung dendritic cells and promotes Tc1 cell differentiation via the CD40/CD40L pathway. Blocking the CD40/CD40L pathway may suppress development of emphysema in mice exposed to cigarette smoke.

Th17 cells and IL-17 promote the skin and lung inflammation and fibrosis process in a bleomycin-induced murine model of systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) is characterised by fibrosis of the skin and internal organs, such as the lungs. Enhanced Th17 responses are associated with skin fibrosis in patients with SSc, however, whether they are associated with lung fibrosis has not been clarified. This study aimed to investigate the potential association of Th17 responses with the skin and pulmonary fibrosis as well as the potential mechanisms in a mouse bleomycin (BLM) model of SSc. METHODS: BALB/c mice were injected subcutaneously with phosphate buffered saline (PBS) (control) or BLM for 28 days and the skin and pulmonary inflammation and fibrosis were characterized by histology. The percentages of circulating, skin and pulmonary infiltrating Th17 cells and the contents of collagen in mice were analysed. The levels of RORγt, IL-17A, IL-6 and TGF-ß1 mRNA transcripts in the skin and lungs were determined by quantitative RTPCR and the levels of serum IL-17A, IL-6 and TGF-ß1 were determined by ELISA. Furthermore, the effect of rIL-17A on the proliferation of pulmonary fibroblasts and their cytokine expression was analysed. The potential association of Th17 responses with the severity of skin and lung fibrosis was analysed. RESULTS: In comparison with the control mice, significantly increased skin and pulmonary inflammation and fibrosis and higher levels of hydroxyproline were detected in the BLM mice. Significantly higher frequency of circulating, skin and lung infiltrating Th17 cells and higher levels of serum, skin and lung IL-17A, TGF-ß1, IL-6 and RORγt were detected in the BLM mice. The concentrations of serum IL-17A were correlated positively with the percentages of Th17 cells and the contents of skin hydroxyproline in the BLM mice. The levels of IL-17A expression were positively correlated with the skin and lung inflammatory scores as well as the skin fibrosis in the BLM mice. In addition, IL-17A significantly enhanced pulmonary fibroblast proliferation and their type I collagen, TGF-ß and IL-6 expression in vitro, which were attenuated by treatment with anti-IL-17A. CONCLUSIONS: Our results indicate that Th17 cells participate in the pathogenesis of skin and lung fibrosis by enhancing fibroblast proliferation and cytokine production in a mouse BLM model of SSc.

Combination of erythromycin and dexamethasone improves corticosteroid sensitivity induced by CSE through inhibiting PI3K-δ/Akt pathway and increasing GR expression.

Corticosteroid insensitivity, which is induced by cigarette smoke extract (CSE), is a significant barrier when treating chronic obstructive pulmonary disease (COPD). Erythromycin (EM) has been shown to have an anti-inflammatory role in some chronic airway inflammatory diseases, particularly diffuse panbronchiolitis and cystic fibrosis. Here, we explored whether the combination therapy of EM and dexamethasone (Dex) reverses corticosteroid insensitivity and investigated the molecular mechanism by which this occurs. We demonstrated that the combination of EM and Dex restored corticosteroid sensitivity in peripheral blood mononuclear cells (PBMCs) from COPD patients and U937 cells after CSE exposure. Moreover, pretreatment with 10, 50, or 100 µg/ml EM reversed the HDAC2 protein reduction induced by CSE exposure in a dose-dependent manner. U937 cells exposed to CSE show a reduction in histone deacetylase (HDAC) activity, which was potently reversed by EM or combination treatment. Although 10 and 17.5% CSE increased phosphorylated Akt (PAkt) expression in a concentration-dependent manner, preapplication of EM and the combination treatment in particular blocked this PAkt increase. Total Akt levels were unaffected by CSE or EM treatments. Furthermore, the combination treatment enhanced glucocorticoid receptor (GR)α expression. Our results demonstrate that the combination therapy of EM and Dex can restore corticosteroid sensitivity through inhibition of the PI3K-δ/Akt pathway and enhancing GRα expression.

IL-21 induction of CD4+ T cell differentiation into Th17 cells contributes to bleomycin-induced fibrosis in mice.

Systemic sclerosis (SSc) is a connective tissue disease characterized by fibrosis of the skin and internal organs. Th17 cells and interleukin-17 (also called IL-17A) have been found to be increased in peripheral blood and skin in patients with SSc. IL-21 is a potent inducer of Th17 differentiation that is produced by activated T cells, and whose relationship with Th17 cells in SSc is unclear. Here, using a bleomycin (BLM)-induced mouse model of skin fibrosis, we detected the frequency of CD4+/IL-17+ (Th17) cells, CD4+/IL-21+ T cells and IL-21+ Th17 cells in peripheral blood, skin and lungs, as well as the serum content of IL-17A and IL-21. In addition, we assessed the differentiation of CD4+ T cells cultured from these mice into Th17 cells in response to treatment with IL-21. Compared with the control mice, Th17 cell counts and IL-17A levels were significantly increased and correlated with inflammatory and fibrotic indices in the skin and lungs of the BLM-induced fibrosis mice. Moreover, serum levels of CD4+/IL-21+ T cells, IL-21+ Th17 cells, and IL-21 were significantly increased in these mice, and correlated positively with serum levels of Th17 cells. In vitro experiments showed that IL-21 treated CD4+ T cells derived from BLM-induced mice differentiated into Th17 cells. Our results indicate that Th17 cells and IL-17A contributes to inflammatory and fibrotic processes in the skin and lungs in a BLM-induced mouse model of SSc. Moreover, the expansion of the Th17 cell population may be subsequent to IL-21 promotion of the differentiation of CD4+ T cells in these mice.

Persistence of Th17/Tc17 cell expression upon smoking cessation in mice with cigarette smoke-induced emphysema.

Th17 and Tc17 cells may be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD), a disease caused predominantly by cigarette smoking. Smoking cessation is the only intervention in the management of COPD. However, even after cessation, the airway inflammation may be present. In the current study, mice were exposed to room air or cigarette smoke for 24 weeks or 24 weeks followed by 12 weeks of cessation. Morphological changes were evaluated by mean linear intercepts (Lm) and destructive index (DI). The frequencies of CD8(+)IL-17(+)(Tc17) and CD4(+)IL-17(+)(Th17) cells, the mRNA levels of ROR gamma and IL-17, and the levels of IL-8, TNF-alpha, and IFN-gamma in lungs or bronchoalveolar lavage fluid of mice were assayed. Here we demonstrated that alveolar enlargement and destruction induced by cigarette smoke exposure were irreversible and that cigarette smokeenhanced these T-cell subsets, and related cytokines were not significantly reduced after smoking cessation. In addition, the frequencies of Th17 and Tc17 cells in lungs of smoke-exposed mice and cessation mice were positively correlated with emphysematous lesions. More important, the frequencies of Tc17 cells were much higher than Th17 cells, and there was a significantly positive correlation between Th17 and Tc17. These results suggested that Th17/Tc17 infiltration in lungs may play a critical role in sustaining lung inflammation in emphysema. Blocking the abnormally increased numbers of Tc17 and Th17 cells may be a reasonable therapeutic strategy for emphysema.

Th17 cell enhances CD8 T-cell cytotoxicity via IL-21 production in emphysema mice.

Emphysema is a T-cell mediated autoimmune disease caused predominantly by cigarette smoking. Th17 cells and related cytokines may contribute to this disorder. However, the possible implication of Th17 cells in regulating inflammatory response in emphysema remains to be elucidated. In the current study, we tested the protein levels of IL-17 and IL-21 in peripheral blood and lung tissues from cigarette-smoke- (CS-) exposed mice and air-exposed mice, analyzed the frequencies of CD4(+)IL-17(+)(Th17) cells, IL-21(+)Th17 cells, and CD8(+)IL-21R(+) T cells in peripheral blood and lung tissues of mice, and their relationship with emphysematous lesions, and explored the impact of IL-21 on cytotoxic CD8(+) T cells function in vitro. It was found that the frequencies of Th17, IL-21(+)Th17, and CD8(+)IL-21R(+) T cells and the levels of IL-17 and IL-21 of CS-exposed mice were much higher than those of the air-exposed mice and correlated with emphysematous lesions. Additionally, the number of IL-21(+)Th17 cells positively correlated with the number of CD8(+)IL-21R(+) T cells. The in vitro experiments showed that IL-21 significantly augmented the secretion of perforin and granzyme B in CD8(+) T cells from CS-exposed mice. These data indirectly provide evidence that Th17 cells could be involved in the control of the local and system inflammatory response in emphysema by regulating CD8(+) cytotoxic T-cell function.

Erythromycin enhances CD4+Foxp3+ regulatory T-cell responses in a rat model of smoke-induced lung inflammation.

Heavy smoking can induce airway inflammation and emphysema. Macrolides can modulate inflammation and effector T-cell response in the lungs. However, there is no information on whether erythromycin can modulate regulatory T-cell (Treg) response. This study is aimed at examining the impact of erythromycin on Treg response in the lungs in a rat model of smoking-induced emphysema. Male Wistar rats were exposed to normal air or cigarette smoking daily for 12 weeks and treated by gavage with 100 mg/kg of erythromycin or saline daily beginning at the forth week for nine weeks. The lung inflammation and the numbers of inflammatory infiltrates in bronchoalveolar lavage fluid (BALF) were characterized. The frequency, the number of Tregs, and the levels of Foxp3 expression in the lungs and IL-8, IL-35, and TNF-α in BALF were determined by flow cytometry, RT-PCR and ELISA, respectively. Treatment with erythromycin reduced smoking-induced inflammatory infiltrates, the levels of IL-8 and TNF-α in the BALF and lung damages but increased the numbers of CD4+Foxp3+ Tregs and the levels of Foxp3 transcription in the lungs, accompanied by increased levels of IL-35 in the BALF of rats. Our novel data indicated that erythromycin enhanced Treg responses, associated with the inhibition of smoking-induced inflammation in the lungs of rats.

[Expression and significance of Th17 cells and related cytokines in a murine model of systemic sclerosis].

OBJECTIVE: To study the expression and significance of Th17 cells and related cytokines in the peripheral blood, skin and lung in a murine model of systemic sclerosis (SSc). METHODS: Twenty female BALB/c mice were randomly divided into 2 groups, including a control group and a bleomycin(BLM) -injected-4-week group (SSc group). Pathological changes of the skin and lung were detected. The proportion of CD4(+)IL-17(+)Th17 cells in the peripheral blood, skin and lung of the mice was determined by flow cytometry. The mRNA expressions of RORγt, IL-17A, and IL-6 of the skin and lung were evaluated by real-time PCR. Enzyme linked immunosorbent assay was used to measure the levels of IL-17 and IL-6 in the serum. RESULTS: Dermal inflammation and the score of PF were significantly increased in the SSc group as compared with the control group (2.60±0.84 vs. 0.40±0.52, 2.80± 1.81 vs.0.60±0.70). Hydroxyproline(HYP) contents of the skin and lung were obviously increased in the SSc group than in the control group [(3.17±1.74) mg/g vs. (1.45±0.40) mg/g,(0.53±0.14) mg/g vs. (0.38±0.16) mg/g], all P<0.05. The percentage of Th17 cells in the peripheral blood, skin and lung of the SSc group were significantly increased as compared with the control group [(2.07±0.89)% vs. (1.02±0.32)%,(5.80±2.02)% vs. (1.64±0.58)%,(5.24±2.43)% vs. (1.92±0.98)%,P <0.01]. Compared with the control group, the mRNA levels of IL-17A, RORγt, IL-6 in the skin and lung of the SSc group were higher. The levels of IL-17, IL-6 of the SSc group in the serum were significantly increased, all P<0.05. The frequency of Th17 cells, and the levels of IL-17 and IL-6 in the blood had a positive correlation with dermal and pulmonary inflammation, fibrosis and HYP contents of the skin and lung, The frequency of Th17, IL-17 and IL-6 in the skin and lung had, respectively, a positive correlation with dermal and pulmonary inflammation, HYP contents of the skin and lung, all P<0.01. CONCLUSION: Th17 cells were significantly increased in the peripheral blood, skin and lung of a murine model of SSc, and had an intimate relationship with inflammation and fibrosis of the skin and lung, and involved the pathogenesis of SSc through producing IL-17, IL-6.

[Expression of interleukin-21 in the lungs of mice with emphysema and its effects on the differentiation of CD4+ T cell].

OBJECTIVE: To evaluate the expression of interleukin (IL)-21 in a cigarette smoke-induced mice model of emphysema and explore its effects on the differentiation of CD4(+)T cell. METHODS: Twenty male Balb/c mice were randomly divided into two groups: control group and smoke-exposed group. Morphological changes were evaluated by mean linear intercepts and alveolar destructive index. The proportion of CD4(+)IL-21R(+)T cells in lungs of mice was determined by flow cytometry. And the levels of IL-21 in lungs of mice were analyzed by enzyme-linked immunosorbent assay (ELISA). Fresh lung mononuclear cells were isolated from the smoke-exposed group and divided further into two sub-groups: blank sub-group and co-culture sub-group. Two sub-groups were cultured in medium with or without IL-21 for 24 h and 48 h. The proportions of Th1 and Th17 cells in cell culture medium were determined by flow cytometry. RESULTS: Mean linear intercepts and alveolar destructive index in the smoke-exposed group ((48.6 ± 4.8) µm and 44.9 ± 2.8) were significantly higher than the control group ((32.4 ± 4.0) µm and 28.1 ± 2.1, both P < 0.05). In lungs, the percentage of CD4(+)IL-21R(+)T cells in the smoke-exposed group (4.1% ± 1.5%) significantly increased than that in the control group (1.4% ± 0.4%) (P < 0.05). The levels of IL-21 in lung of the smoke-exposed group ((851 ± 28) ng/L) were higher than those in the control group ((415 ± 39) ng/L, P < 0.05). In lungs, the levels of IL-21 had positive correlations with mean linear intercepts and alveolar destructive index (r = 0.892 and 0.955, both P < 0.05). The percentages of Th1 and Th17 cells in cell culture medium of the co-culture sub-group for 24 h and 48 h significantly increased versus those in the blank group (all P < 0.05). CONCLUSION: IL-21 may participate in the occurrence and development of emphysema through the induced differentiation of CD4(+)T cells and the promotion of Th1 and Th17-cell responses in lungs.

[Impact of treatment with low dose roxithromycin on stable bronchiectasis].

OBJECTIVE: To investigate the impact of treatment with low dose roxithromycin on clinical symptoms and CT scores in patients with stable bronchiectasis. METHODS: Fifty patients with bronchiectasis in stable condition were randomly assigned to a control group and a treatment group. Patients in the control group received ambroxol hydrochloride tablet 90 mg 3 times a day. Patients in the treatment group received roxithromycin disperse tablet 0.15 g every day and ambroxol hydrochloride tablet 90 mg 3 times a day. The course of treatment lasted for 6 months. Quality of life was assessed using St. George's respiratory questionnaire (SGRQ). The British Medical Research Council (MRC) dyspnea scale was used to assess the degree of dyspnea. The score for CT evaluation of the thorax, quality of life and SGRQ were performed for all patients before and after the treatment. RESULTS: After 6 months, the scores for quality of life (48 ± 13) were lower compared to that (58 ± 15) before treatment in the control group; however, the scores for bronchial wall thickening of bronchiectasis (1.8 ± 0.5) were higher than that (1.8 ± 0.4) before study. The scores for the extent of bronchiectasis (2.7 ± 1.6), the bronchial wall thickening of bronchiectasis (1.3 ± 0.4) and the global CT score (6.7 ± 2.5) were reduced after treatment as compared to those before treatment [(4.8 ± 2.3), (1.8 ± 0.5), (9.5 ± 3.3)] in the treatment group, (all P < 0.01). The degree of dyspnea (1.3 ± 0.4) and quality of life (42 ± 12) were lower than those before treatment [(1.89 ± 0.45), (56 ± 15)] in the treatment group. Furthermore, the scores for extent of bronchiectasis (2.7 ± 1.6), the scores for the bronchial wall thickening of bronchiectasis (1.3 ± 0.4) and the global CT score (6.7 ± 2.5) in the treatment group were significantly improved as compared with those [(4.8 ± 2.0), (1.8 ± 0.5), (9.7 ± 3.6)] in the control group respectively after treatment. At the same time, the degree of dyspnea (1.3 ± 0.4) in the treatment group was significantly improved as compared with that (1.7 ± 0.4) in the control group after treatment. CONCLUSIONS: The scores for the bronchial wall thickening of bronchiectasis were increased in patients with stable bronchiectasis. Low dose roxithromycin combined with ambroxol hydrochloride significantly improved degree of dyspnea, reduced scores for extent of bronchiectasis, scores for the bronchial wall thickening of bronchiectasis and the global CT score as compared to treatment with ambroxol hydrochloride alone in patients with bronchiectasis in stable condition.

[Changes of CD(4)(+)Foxp3(+) regulatory T cells and CD(4)(+)IL-17(+)T cells in cigarette smoke-exposed rats].

OBJECTIVE: To evaluate the changes of CD(4)(+)IL-17(+) T (Th17) and CD(4)(+)Foxp3(+) regulatory T (Treg) cells in peripheral blood and bronchoalveolar lavage fluid (BALF), and therefore to explore the role of Th17 and Treg in cigarette smoke-induced airway inflammation/COPD in rats. METHODS: Forty male Wistar rats were randomly divided into 4 groups: a 12 wk smoke-exposure group, a 24 wk smoke-exposure group, a 12 wk control group and a 24 wk control group (n = 10 each). Cells in BALF were collected and analyzed by absolute and differential cell counts. IL-17 and IL-6 levels in serum and BALF were tested by enzyme linked immunosorbent assay (ELISA). The proportion of CD(4)(+)IL-17(+) T and CD(4)(+)Foxp3(+) Treg in peripheral blood and BALF were determined by flow cytometry. The mRNA expressions of IL-17 and Foxp3 were measured by real-time PCR. Comparisons of the data between different groups were performed using one-way ANOVA, and SNK and Games-Howell test were used for comparison between 2 groups. RESULTS: Levels of IL-17 were remarkable increased in the 12 wk smoke-exposure group and the 24 wk smoke-exposure group in serum [(52.6 ± 1.8) ng/L, (75.4 ± 6.0) ng/L] and BALF [(78.1 ± 5.8) ng/L, (95.0 ± 6.8) ng/L] compared with the 12 wk control group [(40.0 ± 3.2)ng/L, (54.5 ± 4.6) ng/L] and the 24 wk control group [(36.7 ± 3.2) ng/L, (53.9 ± 3.7) ng/L], all P < 0.05. IL-6 in serum was significantly increased in the 24 wk smoke-exposure group [(31.4 ± 2.1) ng/L] compared with the 24 wk control group [(11.5 ± 0.5) ng/L], and it was increased in the 12 wk and the 24 wk smoke-exposure group [(33.3 ± 2.3) ng/L, (44.6 ± 3.0) ng/L] compared with the 12 wk and the 24 wk control group [(15.6 ± 1.8) ng/L, (18.0 ± 1.9) ng/L] in BALF. Ratio of Th17 was higher in the 12 wk and the 24 wk smoke-exposure groups in peripheral blood [(1.81 ± 0.19)%, (3.74 ± 0.55)%] and BALF [(7.84 ± 0.28)%, (8.01 ± 0.39)%] compared with the12 wk [(0.97 ± 0.08)%, (5.64 ± 0.54)%] and the 24 wk control group [(1.08 ± 0.10)%, (5.95 ± 0.48)%]. Ratio of Treg in BALF was higher in the smoke-exposure groups [(8.81 ± 0.49)%, (11.98 ± 0.72)%] compared with the control groups [(4.34 ± 0.28)%, (5.21 ± 0.42)%]. The level of IL-17 mRNA was increased in the 12 wk and the 24 wk smoke-exposure group in peripheral blood (25.7 ± 2.0, 33.9 ± 1.5) and in BALF (22.2 ± 1.8, 34.7 ± 4.2) compared with the 12 wk (11.3 ± 2.6, 11.6 ± 2.4) and the 24 wk (11.1 ± 2.0, 13.5 ± 3.4) control groups. Foxp3 mRNA was increased in the smoke-exposure groups (24.4 ± 2.7, 30.3 ± 2.7) compared with the control groups (12.7 ± 2.7, 14.6 ± 3.8). Th17 in smoke-exposure groups was positively correlated with counts of total cells and macrophages (r = 0.512, 0.543, all P < 0.05). CONCLUSIONS: An elevated expression of Th17 and Treg cells and an increase of inflammatory cytokines were evident in airway inflammation of cigarette smoke-exposed rats, suggesting that Treg was involved in the immunological regulation and Th17 was associated with the persistent inflammation in cigarette smoke-induced airway inflammation in rats.

[The expression and mechanisms of interleukin-17 in CD(8)(+) T cells of mice with cigarette smoke-induced emphysema].

OBJECTIVE: To evaluate the expression of Tc17 in a cigarette smoke-induced mice model of emphysema. To explore the probable mechanisms about how Tc17 cells to elevate in lungs of mice. METHODS: Forty male Balb/c mice were randomly divided into four groups, including control group (12 weeks, C12), control group (24 weeks, C24), smoke-exposure group (12 weeks, S12) and smoke-exposure group (24 weeks, S24), 10 mice each group, Emphysema of mice was observed by HE pigmentation. Morphological changes were evaluated by mean linear intercepts (Lm) and destructive index (DI). The proportion of CD(8)(+)IL-17(+)Tc17, CD(8)(+)IL-17(+) CC chemokine receptor type 6 (CCR6)(+) and 6CCR6(+)Tc17 cells in lungs of mice was determined by flow cytometry. The mRNA expressions of retinoid-related orphan nuclear receptor (RORγt) and IL-17 were evaluated by real-time PCR. The levels of IL-1ß, IL-6, IL-23, transforming growth factor ß (TGFß) and CC chemokine ligand 20(CCL20) were tested by ELISA. Correlations among these indexes were analyzed. RESULTS: Lm and DI were significantly higher in S12 and S24 than in C12 and C24, S24 in particular (t value 4.378 - 15.188, all P < 0.05). The percentages of Tc17 in S12 and S24 [(9.28 ± 1.12)%, (13.13 ± 3.56)%] was significantly increased as compared with that in C12 and C24 [(2.40 ± 0.60)%, (2.64 ± 0.96)%], S24 in particular. The mRNA levels of RORγt and IL-17 in S12 and S24 were higher than in C12 and C24, S12 and S24 in particular. There was significant difference (all P < 0.05). The frequency of Tc17 cells had a positive correlation with Lm and DI (r value were 0.734 and 0.884 respectively, P < 0.01). The percentages of CD(8)(+)IL-17(+)CCR6(+)T cells and CCR6(+)Tc17 were significantly elevated in S12 and S24 compared to C12 and C24, S24 in particular (all P < 0.05). There was positive correlation between Tc17 cell ratio and CCL20 levels (r = 0.899, P < 0.01). The levels of IL-1ß, IL-6, IL-23 and TGFß in S12 and S24 were significantly increased as compared with that in C12 and C24. There was significant difference (all P < 0.05). Meanwhile, the frequency of Tc17 cells had a positive correlation with IL-1ß, IL-6, IL-23, and TGFß. CONCLUSIONS: An up-regulation of proportions Tc17 in lungs of cigarette smoke-induced emphysema mice were detected. The CCR6/CCL20 axis and the increased IL-1ß, IL-6, IL-23 and TGFß probably contributed to this up-regulation.

[The clinical characteristics of intra-acinar pulmonary artery inflammation and its effect on clinical parameters in smokers with normal lung function and patients with chronic obstructive pulmonary disease].

OBJECTIVE: To study the pathological characteristics of intra-acinar pulmonary artery inflammation and its correlation with smoking index and disease progression in smokers with normal lung function and smokers with chronic obstructive pulmonary disease (COPD). METHODS: Patients requiring lung resection for peripheral lung cancer were divided into group A (nonsmokers with normal lung function, n = 10), group B (smokers with normal lung function, n = 13), and group C (smokers with stable COPD, n = 10). The lung tissue far away from tumor were resected to compare the pathological changes of intra-acinar pulmonary arteries and infiltration level of inflammatory cell in pulmonary non-muscularized arteries (NMA), pulmonary partially muscularized arteries (PMA) and muscularized arteries (MA) among the three groups. The correlation analysis was made among infiltration level, smoking index, percentage of predicted value of forced expiratory volume in one second (FEV(1)%Pred), six-minute-walk distance (6MWD) and BODE index. RESULTS: (1) Both group B and group C showed the intima and media thickness of MA was significantly higher, the lumen area of MA was narrower and the proportion of MA was higher, and collagenous fiber of MA adventitial proliferated and area increased in group C (P < 0.05 or P < 0.01). (2) In group B and group C, the percentage of the intra-acinar pulmonary arteries that contained leukocytes, T lymphocytes, CD(8)(+)T lymphocytes and the number of these positive cells infiltrating the intra-acinar pulmonary arteries were increased, especially an increased number of CD(8)(+)T lymphocytes infiltrating in the arterial adventitia as compared with group A, moreover there were significant difference between group C and group B (P < 0.05 or P < 0.01). In group B and group C, the degree of these positive cells infiltrating NMA, PMA and MA presented a decreasing sequence (P < 0.05 or P < 0.01). Among the intima, media and adventitia of MA, the infiltration of these positive cells was the highest in the adventitia. Among group A, group B and group C, infiltration degree of CD(4)(+)T lymphocyte, B lymphocyte, macrophage and neutrophil demonstrated no significant difference, also among NMA, PMA and MA (P > 0.05). (3) The number of leukocytes, T lymphocytes, CD(8)(+)T lymphocytes infiltrating MA showed a positive correlation with the thickness of MA (r = 0.563, 0.627, 0.589, P < 0.01, respectively) and smoking index (r = 0.551, 0.665, 0.600, P < 0.01, respectively), moreover the degree of these cells infiltrating MA demonstrated negative correlation with FEV(1)%Pred (r = -0.763, -0.703, -0.767, P < 0.01, respectively). Also infiltrating degree of T lymphocytes and CD(8)(+)T lymphocytes was positively correlated with BODE (r = 0.390, 0.476, P < 0.05, respectively). Furthermore the infiltrating degree of CD(8)(+)T lymphocytes had negative correlation with 6MWD (r = -0.356, P < 0.05). CONCLUSIONS: (1) Pulmonary arterial inflammation appears in smokers with normal lung function and smokers with COPD patients. It involves in all types of intra-acinar pulmonary arteries especially NMA and infiltrates whole layer of MA with a characteristic of CD(8)(+)T lymphocytes infiltrating in the adventitia of intra-acinar pulmonary arteries. (2) Pulmonary inflammation is closely correlated to cigarette smoking and clinical parameters such as BODE index, FEV(1)% pred and 6MWD. It is one of the key factors affecting the progression of COPD.

[Mechanisms and dynamics of Th17 cells in mice with cigarette smoke-induced emphysema].

OBJECTIVE: To evaluate the expression of Th17 cell in a cigarette smoke-induced mice model of emphysema and explore the probable mechanisms of its elevation. METHODS: Forty male Balb/c mice were randomly divided into 4 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)(n = 10 each). Morphological changes were evaluated by mean linear intercepts (Lm) and destructive index (DI). The percentages of Th17, Th1, Th17/Th1, CD4(+)IL-17(+)CCR6(+)T and CCR6(+)Th17 cells were determined by tetra-color flow cytometry while the levels of interleukin (IL)-1ß, IL-6, IL-23, transforming growth factor (TGF)-ß, interferon (IFN)-γ and CC chemokine ligand (CCL)-20 assayed by enzyme-linked immunosorbent assay (ELISA). RESULTS: The values of Lm [(39 ± 4) µm, (47 ± 7) µm] and DI [(39.1 ± 1.6), (45.2 ± 3.1)] were significantly higher in S12 and S24 than those in C12 [(33 ± 3) µm, (28.2 ± 1.6)] and C24 [(32 ± 4) µm, (28.9 ± 2.1)], particularly in C24 (all P < 0.05). The percentages of Th17 cell [(3.27 ± 1.12), (7.19 ± 2.24)], Th17/Th1 cell [(0.61 ± 0.30), (1.82 ± 0.52)] and Th1 cell [(10.02 ± 3.68), (26.21 ± 6.04)] in the lungs of S12 and S24 significantly increased than those in C12 [(1.80 ± 0.75), (0.27 ± 0.12), (3.75 ± 1.72)] and C24 [(1.99 ± 0.59), (0.28 ± 0.11), (4.16 ± 1.32)], particularly in C24 (all P < 0.01). The percentages of Th17, Th17/Th1 and Th1 cells in the lungs of S12 and S24 had a positive correlation with Lm and DI (all P < 0.01). The percentages of CD4(+)IL-17(+)CCR-6(+)T cell [(0.69 ± 0.34), (1.11 ± 0.48)] and CCR6(+)Th17 cell [(12.23 ± 2.13), (18.65 ± 1.17)] were significantly elevated in S12 and S24 compared to those in C12 [(0.22 ± 0.18), (6.55 ± 2.13)] and C24 [(0.25 ± 0.17), (7.29 ± 1.57)], particularly in C24 (all P < 0.05). Furthermore, a positive correlation between CCR6(+)Th17 cell and emphysematous lesions was also found (all P < 0.05). The levels of IL-1ß, IL-6, IL-23, TGF-ß, IFN-γ and CCL20 significantly increased in S12 and S24 as compared with those of C12 and C24 (all P < 0.05). Meanwhile, the percentage of Th17 cell had a positive correlation with IL-1ß, IL-6, IL-23, TGF-ß, IFN-γ and CCL20. CONCLUSION: There is an up-regulated expression of Th17 in lungs of cigarette smoke-induced emphysema mice. The CCR6/CCL20 axis and the elevated levels of IL-1ß, IL-6, IL-23, TGF-ß and IFN-γ may be related with the above effect.

[Management of benign tracheal stenosis by intubation dilatation under flexible bronchoscopic guidance].

OBJECTIVE: To evaluate the efficacy and safety of intubation dilatation under flexible bronchoscopic guidance in the management of benign tracheal stenosis. METHODS: A retrospective analysis of the clinical data was performed for 12 patients with benign tracheal stenosis from March 2010 to August 2011. There were 5 males and 7 females with a mean age of 37 ± 11 years old (range: 27 - 65). They were treated by intubation dilatation with different sizes under bronchoscopic guidance. And balloon dilatation was also performed for left or right main stem bronchial stenosis. And metal stents were implanted if necessary. Airway diameter, dyspnea index, complications and blood gas analysis were evaluated in all patients. And the forced expiratory volume in one second (FEV(1)) was tested in 9 cases before and after the treatments of intubation dilation, balloon dilation and other interventions. RESULTS: One to five attempts of intubation dilation were required to achieve satisfactory dilatation. There was immediate postoperative relief of dyspnea for all 12 cases. And PaO2 and SaO2 rose markedly, but PaCO2 declined after intervention. The effective rate of intubation dilation was 100%. The average airway diameter increased from (5.7 ± 1.2) to (12.2 ± 2.1) mm and FEV(1) improved from (0.67 ± 0.13) to (1.73 ± 0.37) L (P < 0.01). CONCLUSION: The minimally invasive management of benign tracheal stenosis with intubation dilatation is both safe and efficacious.

[Effect of interleukin-17-producing CD4+ T helper lymphocytes on cigarette smoke-induced lung inflammation and emphysema in mice].

OBJECTIVE: To evaluate the expression and the role of Th17 in cigarette smoke-induced lung inflammation and emphysema in mice. METHODS: Forty male BALB/c mice were randomly divided into 4 groups, including a control group C12, a control group C24, a smoke-exposure 12 week group (S12) and a smoke-exposure 24 week group S24 (n = 10 each). Morphological changes were evaluated by mean linear intercepts and destructive index (DI). The proportion of CD(4)(+)IL-17(+)Th17, CD(4)(+)IFN-γ(+)Th1, CD(4)(+)IL-17(+)IFN-γ(+)T(Th17/Th1), CD(8)(+)IFN-γ(+)Tc1, CD(8)(+)IL-21R(+) and CD(4)(+)IL-17(+)IL-21(+) T cells in lungs of mice was determined by flow cytometry. The mRNA expressions of RORγt and IL-17 were evaluated by real-time PCR. RESULTS: Mean linear intercepts and DI were significantly higher in S12 and S24 groups [(39 ± 4) µm, (47 ± 7) µm], (39.1 ± 1.6, 45.2 ± 3.1) as compared to C12 [(32 ± 4) µm, 28.2 ± 1.6] and C24 groups [(33 ± 3) µm, 28.9 ± 2.1], all P < 0.05. The percentage of Th17 of S12 and S24 groups [(3.3 ± 1.1)%, (7.2 ± 2.2)%] was significantly increased as compared with that of C12 and C24 groups [(1.8 ± 0.8)%, (2.0 ± 0.6)%], all P < 0.05. The mRNA levels of RORγt [(25 ± 4), (35 ± 3)] and IL-17 [(26 ± 3), (36 ± 3)] in S12 and S24 groups were higher than in C12 [(10 ± 5), (13 ± 5)] and C24 groups [(11 ± 7), (8 ± 6)], all P < 0.05. The percentage of Th1, Th17/Th1 and Tc1 cells of S12 and S24 groups [(10.0 ± 3.7)%, (26.2 ± 6.0)%], [(0.61 ± 0.30)%, (1.82 ± 0.52)%], [(17.0 ± 4.5)%, (26.8 ± 8.5)%] was significantly increased as compared with that of C12 [(3.8 ± 1.7)%, (0.27 ± 0.17)%, (4.8 ± 1.9)%] and C24 groups [(4.2 ± 1.3)%, (0.28 ± 0.11)%, (5.2 ± 1.0)%], all P < 0.05. Moreover, the frequency of Th17 cells had a positive correlation with Th1, Tc1 cells and emphysematous lesions (r = 0.519 - 0.797, all P < 0.01). In addition, a positive correlation between Th17/Th1 cells and emphysematous lesions was also found (r = 0.742, 0.802, all P < 0.01). The percentage of CD(4)(+)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, as compared with that of the controls [(1.22 ± 0.31), (1.34 ± 0.18)], the percentage of CD(8)(+)IL-21R(+) T cells was also 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). The frequency of CD(4)(+)IL-17(+)IL-21(+) T cells had a positive correlation with Th1, Tc1 cells and emphysematous lesions (r = 0.694 - 0.754, all P < 0.05). And the frequency of CD(8)(+)IL-21R(+) T cells also had a positive correlation with emphysematous lesions (r = 0.516, 0.725, all P < 0.05). CONCLUSIONS: Cigarette smoke increased the expression and the activity of Th17 in mice. Th17 may play a potential (active) role in the development of lung inflammation through IL-21/IL-21R pathway.

[Comparative study of protein markers in serum and bronchoalveolar lavage fluid from patients with lung cancer by surface-enhanced laser desorption ionization time of flight mass spectrometry].

OBJECTIVE: To detect the protein markers in serum and bronchoalveolar lavage fluid (BALF) of the patients with lung cancer by surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS) technology, and to explore if they can be used as markers for the diagnosis of lung cancer. METHODS: SELDI-TOF-MS technology and protein chips weak cation exchange (WCX-2 chip) were used to detect the protein mass spectrum in serum and BALF of 35 patients with lung cancer and 18 cases of benign pulmonary diseases. The different protein markers were analyzed by Biomarker Pattern Software and the initial diagnosis models were set up. The diagnosis models were verified further by blind screen to confirm the efficacy of diagnosis. RESULTS: Five protein peaks in the sera of the patients with lung cancer were significantly higher (P < 0.05). The protein peak with a mass/charge ratio (M/Z) of 5639 was selected to establish the classification tree model. The sensitivity of diagnosis was 80% (28/35) and the specificity was 78% (14/18). The results verified by blind screen showed a sensitivity of 85% (17/20), a specificity of 90% (9/10), a crude accuracy (CA) of 87% (26/30) and Youden's index (γ) of 0.7. Eight protein peaks in the BALF of the patients with lung cancer were significantly higher (P < 0.05). The different protein peaks with M/Z of 7976 and 11 809 respectively were selected to establish the classification tree model. The sensitivity of diagnosis was 86% (30/35) and the specificity was 72% (13/18). The results verified by blind screen showed a sensitivity of 90% (18/20), a specificity of 90% (9/10), a CA of 90% (27/30) and γ of 0.8. There was a complementary role in combination of differential proteins in serum and BALF and the sensitivity, specificity and accuracy of diagnosis for lung cancer were 100% by parallel test. CONCLUSIONS: The SELDI-TOF-MS technology can screen out the differential protein markers in serum and BALF of the patients with lung cancer, which show high sensitivity and specificity as tumor markers. The differential proteins in the BALF may be more promising for clinical application.