The Role Of IL-17Producing Th17Cells In Bleomycin-induced Pulmonary Fibrosis

[Objective]Pulmonary fibrosis is a refractory interstitial lung disease with high mortality, characterized by impaired alveolar wall (alveolitis), abnormal accumulation of fibroblasts/myofibroblasts, excessive deposition of collagen and other extracellular matrix, and abnormal tissue repair resulting in loss of lung function. The causes and mechanisms of pulmonary fibrosis are not clear, but the pulmonary fibrosis caused by irreversible pulmonary dysfunction and respiratory failure results in a very serious and fatal results. The clinical trial confirmed that drugs including hormones and non-hormone immunosuppressants, anti-fibrosis, anti-cytokine and immune regulation are no clear effect on the treatment of pulmonary fibrosis, and Lung transplantation is the only effective method of treatment of end-stage pulmonary fibrosis.The pathogenesis of pulmonary fibrosis has been a hot and difficult point in domestic and international medical research. Previous studies of the pathogenesis of pulmonary fibrosis are mainly concentrated upon regulating the proliferation, activation and differentiation of fibroblasts secreting collagen. Recent studies have revealed that a variety of inflammatory mediators (TNF,IL-1(3,CXCL1,MIP-2,PDGF and TGF-β1, etc.), oxidative stress and anti-inflammatory mediators (PGE2, COX-2) play an important role in pulmonary fibrosis.In the past few years, the chemokine,chemokine receptor,chemokine receptor signaling,integrin α3β1, WNT1induced signaling protein1(WISPs-1) and PI3K-AKT-of S6K1signal pathway of the Wnt-β cateninas were regarded as the new interventional targets of anti-pulmonary fibrosis treatment and research. The present study confirmed T lymphocytes play an important role in the formation of pulmonary fibrosis, the current research focuses on the imbalance of Thl/Th2cells and regulatory T cells.Recent study found that a new T cells sub-group is different from Thl, Th2and Treg cells,and do not express IL-4or IFN-y, but high-level secretion of IL-17, which was named as Thl7cells. Th17cells play an effector functions through the secretion of IL-17A, IL-17F, IL-21, IL-22, IL-6,TNF-y and other cytokines in autoimmune diseases, infections and other diseases. Th17cells and IL-17A of their secretion are considered as the association with the pulmonary fibrosis, but the role of IL-17A was reported less in the pathogenesis of pulmonary fibrosis.In order to study the mechanism of pulmonary fibrosis, We speculate that Th17cells and IL-17may play an important role in the pathogenesis of pulmonary fibrosis. To prove this hypothesis, this study intends to do from the following aspects:①the role of IL-17producing Th17cells in bleomycin-induced pulmonary fibrosis;②the role of Th17differentiation in pulmonary fibrosis;③the effects of interleukin-17(IL-17) on the proliferation, transformation and collagen synthesis of the lung fibroblasts in vitro;④the role of IL-17on siRNA-Actl transfection of the lung fibroblasts and NF-κB pathway in vitro.[Methods]1. The role of IL-17producing Th17cells in bleomycin-induced pulmonary fibrosis:Forty male C57BL/6mice were randomized as normal control group (N group) and bleomycin-induced pulmonary fibrosis model group (BLM group) with20in each. Mice were administered with bleomycin intratracheally at a dose of5mg/kg in BLM group and intratracheally administered with0.9%saline of the same dose in N group. Then they were sacrificed on days3,7,14and28, respectively. The pathomorphology in lung tissue were evaluated by Hemotoxylin and eosin (H&E) and Masson staining, the hydroxyproline in lung tissue were measured by biochemistry. IL-17A in lung tissues was measured by semi-quantitative RT-PCR and immunohistochemisty. The number of CD4+IL-17+T cells in the spleen was measured by flow cytometry. 2. The role of Th17differentiation in pulmonary fibrosis:Pulmonary fibrosis was induced in C57BL/6male mice by intratracheal instillation of bleomycin (5mg.kg-1), which were randomly divided into control, bleomycin, and ATRA groups. During the experiments, mice in the ATRA group were injected intraperitoneally with0.5mg of ATRA powder dissolved in0.1mL DMSO. Injections were repeated3times weekly. Five mice in each group were sacrificed on day28after intratracheal instillation. Hemotoxylin and eosin (H&E) and Masson staining were used for pathological examination, and hydroxyproline in lung tissue was measured. Interleukin (IL)-17A protein expression was observed in lung with immunohistochemistry. The expression of IL-17A,IL-10, IL-6, and transforming growth factor (TGF)-β mRNAs were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Th17and Tregs expression in spleen lymphocytes were measured by flow cytometry.3. The effects of interleukin-17(IL-17) on the proliferation, transformation and collagen synthesis of the lung fibroblasts in vitro:Bleomycin (5mg/kg) was administered intratracheally in50μl saline to male C57BL/6mice. On days3,7,14,28after bleomycin treatment, the animals were sacrificed and the lungs were removed intact (n=5, in each group). The expression of IL-17ARmRNA in lung tissue was detected by RT-PCR, At14days following bleomycin administration, the pulmonary primary fibroblasts were isolated, cultured and identified by Laser scanning confocal microscope. IL-17co-cultured with lung fibroblasts were observed for24h,48h and72h, the role of different concentrations IL-17on the proliferation of lung fibroblasts were detected by Tetrazolium colorimetric (MTT) and the best stimulus concentration was ascertained. a-SMA in myofibroblasts was detected by Immunofluorescence, RT-PCR and Western Blotting, and RT-PCR and Western blotting were employed to examine the mRNA and protein expressions of types I and III collagen in pulmonary fibroblasts.4. The role of IL-17on siRNA-Actl transfection of the lung fibroblasts and NF-κB pathway in vitro:the lung fibroblasts with siRNA of Actl were transfected by lipofectamine, the optimum concentration of transfection and interference time were determined, the Actl interference efficiency was identified by PCR and western blotting. Experimens were divided into three groups,TL-17-siRNA-Actl group,+IL-17-siRNA-Act1group,+IL-17+siRNA-Actl group. The proliferation effect of IL-17on lung fibroblasts and lung fibroblasts transfected with siRNA-Actl were measured by MTT, Myofibroblast marker a-SMA were measured by immunofluorescence and Western Blotting, the expression of I and type III collagen were detected by Western Blotting in different groups, lung fibroblasts or lung fibroblasts transferred with siRNA-Actl were co-cultured with IL-17,at0min,15min,30min and60min,the expression of P65,IκB and ActlmRNA were detected by fluorescence quantitative PCR, the expression of P65,p-p65,IκB, p-IκB and Actl protein were detected by western blotting.[Results]1. The role of IL-17producing CD4+T cells in bleomycin-induced pulmonary fibrosis:①the model of bleomycin-induced pulmonary fibrosis were constructed successfully, the peak of alveolitis was at14d by HE staining,and the most severity of pulmonary fibrosis was at28d by Masson staining and hydroxyproline content;②IL-17A was located in the terminal bronchioles, respiratory bronchioles and alveolar epithelia;③IL-17A began to increase on day7, reached the peak on day14and then began to decrease on day28, which was all higher than in normal group;④The pattern of changed number of CD4+IL-17+T/CD4+Tcells from spleen lymphocytes was the same as the amount of IL-17A expression2. The role of Th17differentiation in pulmonary fibrosis:①ATRA can inhibit IL-17+CD4+T cell differentiation and promote the differentiation of CD4+CD25+Tregs, and inhibited the expression of IL-17protein and mRNA,decreased content, reducing hydroxyproline content, which alleviated the pulmonary fibrosis induced by bleomycin;②ATRA can inhibit the expression of TGF-beta, IL-6mRNA expression in pulmonary fibrosis in mic,but had no obvious effect on the expression of IL-10mRNA. 3. The effects of interleukin-17(IL-17) on the proliferation, transformation and collagen synthesis of the lung fibroblasts in vitro:①IL-17AR mRNA was higher in bleomycin induced pulmonary fibrosis, and reached the peak at14d;②IL-17can significantly promote lung fibroblast proliferation, transformation, and collagen synthesis.4. The role of IL-17on siRNA-Act1transfection of the lung fibroblasts and NF-κB pathway in vitro:①25pmol concentration and cultured48h of siRNA-Act1transferred into lung fibroblasts were the optimal dose and time point;②the Act1protein on lung fibroblasts was inhibited by siRNA-Act1transfections;③the role of proliferation, transformation, and collagen synthesis were inhibited for IL-17on siRNA-Act1transferred into lung fibroblasts;④IL-17stimulation on siRNA-Act1non-transfer lung fibroblasts was associated with a significant increase in the level of phosphorylation of P65and IκB, Act1protein expression were decreased on siRNA-Act1transferred into lung fibroblasts, the roles IL-17activating phosphorylation of P65and IκB were inhibited.[Conclusions]1. IL-17producing Th17plays a role in bleomycin-induced pulmonary fibrosis.2. IL-17involed in pulmonary fibrosis may be associated with the increasing expression of TGF-β and IL-6mRNA, which promote Th17differentiation and inhibit the differentiation of CD4+CD25+Tregs.3. All-trans retinoic acid through inhibition of TGF-β and IL-6expression regulated the differentiation of Th17and Treg cells, and reduced expression of IL-17to alleviate the degree of pulmonary fibrosis.4. IL-17may promote the proliferation, transformation and synthesis of collagen in lung fibroblasts.5. The expression of Act1protein was decreased, and the roles of proliferation, transformation and synthesis of collagen for IL-17were inhibited on siRNA-Act1transfection into lung fibroblasts.6. IL-17influenced the function of lung fibroblasts through promoting phosphorylation of P65and IκB, activating NF-κB pathway. 7. IL-17plays a role in lung fibroblasts through combining the Actl protein and activating NF-κB pathway. The expression of Actl protein was interfered, NF-κB pathway was inhibited, the role of IL-17on lung fibroblasts was reduced.